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1
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Gu J, He Y, He C, Zhang Q, Huang Q, Bai S, Wang R, You Q, Wang L. Advances in the structures, mechanisms and targeting of molecular chaperones. Signal Transduct Target Ther 2025; 10:84. [PMID: 40069202 PMCID: PMC11897415 DOI: 10.1038/s41392-025-02166-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2024] [Revised: 11/25/2024] [Accepted: 01/15/2025] [Indexed: 03/15/2025] Open
Abstract
Molecular chaperones, a class of complex client regulatory systems, play significant roles in the prevention of protein misfolding and abnormal aggregation, the modulation of protein homeostasis, and the protection of cells from damage under constantly changing environmental conditions. As the understanding of the biological mechanisms of molecular chaperones has increased, their link with the occurrence and progression of disease has suggested that these proteins are promising targets for therapeutic intervention, drawing intensive interest. Here, we review recent advances in determining the structures of molecular chaperones and heat shock protein 90 (HSP90) chaperone system complexes. We also describe the features of molecular chaperones and shed light on the complicated regulatory mechanism that operates through interactions with various co-chaperones in molecular chaperone cycles. In addition, how molecular chaperones affect diseases by regulating pathogenic proteins has been thoroughly analyzed. Furthermore, we focus on molecular chaperones to systematically discuss recent clinical advances and various drug design strategies in the preclinical stage. Recent studies have identified a variety of novel regulatory strategies targeting molecular chaperone systems with compounds that act through different mechanisms from those of traditional inhibitors. Therefore, as more novel design strategies are developed, targeting molecular chaperones will significantly contribute to the discovery of new potential drugs.
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Affiliation(s)
- Jinying Gu
- State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, China
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, China
| | - Yanyi He
- State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, China
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, China
| | - Chenxi He
- State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, China
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, China
| | - Qiuyue Zhang
- State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, China
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, China
| | - Qifei Huang
- State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, China
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, China
| | - Shangjun Bai
- State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, China
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, China
| | - Ruoning Wang
- School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China.
- Jiangsu Provincial TCM Engineering Technology Research Center of Highly Efficient Drug Delivery Systems (DDSs), Nanjing, China.
| | - Qidong You
- State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, China.
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, China.
| | - Lei Wang
- State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, China.
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, China.
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2
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Oz S, Keren-Raifman T, Sharon T, Subramaniam S, Pallien T, Katz M, Tsemakhovich V, Sholokh A, Watad B, Tripathy DR, Sasson G, Chomsky-Hecht O, Vysochek L, Schulz-Christian M, Fecher-Trost C, Zühlke K, Bertinetti D, Herberg FW, Flockerzi V, Hirsch JA, Klussmann E, Weiss S, Dascal N. Tripartite interactions of PKA catalytic subunit and C-terminal domains of cardiac Ca 2+ channel may modulate its β-adrenergic regulation. BMC Biol 2024; 22:276. [PMID: 39609812 PMCID: PMC11603854 DOI: 10.1186/s12915-024-02076-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2023] [Accepted: 11/21/2024] [Indexed: 11/30/2024] Open
Abstract
BACKGROUND The β-adrenergic augmentation of cardiac contraction, by increasing the conductivity of L-type voltage-gated CaV1.2 channels, is of great physiological and pathophysiological importance. Stimulation of β-adrenergic receptors (βAR) activates protein kinase A (PKA) through separation of regulatory (PKAR) from catalytic (PKAC) subunits. Free PKAC phosphorylates the inhibitory protein Rad, leading to increased Ca2+ influx. In cardiomyocytes, the core subunit of CaV1.2, CaV1.2α1, exists in two forms: full-length or truncated (lacking the distal C-terminus (dCT)). Signaling efficiency is believed to emanate from protein interactions within multimolecular complexes, such as anchoring PKA (via PKAR) to CaV1.2α1 by A-kinase anchoring proteins (AKAPs). However, AKAPs are inessential for βAR regulation of CaV1.2 in heterologous models, and their role in cardiomyocytes also remains unclear. RESULTS We show that PKAC interacts with CaV1.2α1 in heart and a heterologous model, independently of Rad, PKAR, or AKAPs. Studies with peptide array assays and purified recombinant proteins demonstrate direct binding of PKAC to two domains in CaV1.2α1-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Data indicate both partial competition and possible simultaneous interaction of PCRD and DCRD with PKAC. The βAR regulation of CaV1.2α1 lacking dCT (which harbors DCRD) was preserved, but subtly altered, in a heterologous model, the Xenopus oocyte. CONCLUSIONS We discover direct interactions between PKAC and two domains in CaV1.2α1. We propose that these tripartite interactions, if present in vivo, may participate in organizing the multimolecular signaling complex and fine-tuning the βAR effect in cardiomyocytes.
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Affiliation(s)
- Shimrit Oz
- School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University, 6997601, Tel Aviv, Israel
- Department of Neuroscience, Faculty of Medicine, The Ruth and Bruce Rappaport, Haifa, 3109601, Israel
| | - Tal Keren-Raifman
- School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University, 6997601, Tel Aviv, Israel
| | - Tom Sharon
- School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University, 6997601, Tel Aviv, Israel
| | - Suraj Subramaniam
- School of Neurobiology, Biochemistry and Biophysics, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 6997601, Israel
| | - Tamara Pallien
- Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany
| | - Moshe Katz
- School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University, 6997601, Tel Aviv, Israel
| | - Vladimir Tsemakhovich
- School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University, 6997601, Tel Aviv, Israel
| | - Anastasiia Sholokh
- Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany
| | - Baraa Watad
- School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University, 6997601, Tel Aviv, Israel
| | - Debi Ranjan Tripathy
- School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University, 6997601, Tel Aviv, Israel
- School of Neurobiology, Biochemistry and Biophysics, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 6997601, Israel
- National Forensic Science University, Radhanagar, Agartala, Tripura, 799001, India
| | - Giorgia Sasson
- School of Neurobiology, Biochemistry and Biophysics, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 6997601, Israel
| | - Orna Chomsky-Hecht
- School of Neurobiology, Biochemistry and Biophysics, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 6997601, Israel
| | - Leonid Vysochek
- Heart Center, Sheba Medical Center, Ramat Gan, 5262000, Israel
| | - Maike Schulz-Christian
- Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany
| | - Claudia Fecher-Trost
- Experimentelle Und Klinische Pharmakologie & Toxikologie, Universität Des Saarlandes, Homburg, 66421, Germany
| | - Kerstin Zühlke
- Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany
| | - Daniela Bertinetti
- Department of Biochemistry, University of Kassel, Heinrich-Plett-Str. 40, Kassel, 34132, Germany
| | - Friedrich W Herberg
- Department of Biochemistry, University of Kassel, Heinrich-Plett-Str. 40, Kassel, 34132, Germany
| | - Veit Flockerzi
- Experimentelle Und Klinische Pharmakologie & Toxikologie, Universität Des Saarlandes, Homburg, 66421, Germany
| | - Joel A Hirsch
- School of Neurobiology, Biochemistry and Biophysics, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 6997601, Israel
| | - Enno Klussmann
- Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany
- DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Germany
| | - Sharon Weiss
- School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University, 6997601, Tel Aviv, Israel.
| | - Nathan Dascal
- School of Medicine, Faculty of Medical and Health Sciences, Tel Aviv University, 6997601, Tel Aviv, Israel.
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3
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Kumar M, Mehan S, Kumar A, Sharma T, Khan Z, Tiwari A, Das Gupta G, Narula AS. Therapeutic efficacy of Genistein in activation of neuronal AC/cAMP/CREB/PKA and mitochondrial ETC-Complex pathways in experimental model of autism: Evidence from CSF, blood plasma and brain analysis. Brain Res 2024; 1846:149251. [PMID: 39384128 DOI: 10.1016/j.brainres.2024.149251] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2024] [Revised: 09/10/2024] [Accepted: 09/22/2024] [Indexed: 10/11/2024]
Abstract
Autism is a complex neurodevelopmental condition characterized by repetitive behaviors, impaired social communication, and various associated conditions such as depression and anxiety. Its multifactorial etiology includes genetic, environmental, dietary, and gastrointestinal contributions. Pathologically, Autism is linked to mitochondrial dysfunction, oxidative stress, neuroinflammation, and neurotransmitter imbalances involving GABA, glutamate, dopamine, and oxytocin. Propionic acid (PRPA) is a short-chain fatty acid produced by gut bacteria, influencing central nervous system functions. Elevated PRPA levels can exacerbate Autism-related symptoms by disrupting metabolic processes and crossing the blood-brain barrier. Our research investigates the neuroprotective potential of Genistein (GNT), an isoflavone compound with known benefits in neuropsychiatric and neurodegenerative disorders, through modulation of the AC/cAMP/CREB/PKA signaling pathway and mitochondrial ETC complex (I-IV) function. In silico analyses revealed GNT's high affinity for these targets. Subsequent in vitro and in vivo experiments using a PRPA-induced rat model of autism demonstrated that GNT (40 and 80 mg/kg., orally) significantly improves locomotion, neuromuscular coordination, and cognitive functions in PRPA-treated rodents. Behavioral assessments showed reduced immobility in the forced swim test, enhanced Morris water maze performance, and restored regular locomotor activity. On a molecular level, GNT restored levels of key signaling molecules (AC, cAMP, CREB, PKA) and mitochondrial complexes (I-V), disrupted by PRPA exposure. Additionally, GNT reduced neuroinflammation and apoptosis, normalized neurotransmitter levels, and improved the complete blood count profile. Histopathological analyses confirmed that GNT ameliorated PRPA-induced brain injuries, restored normal brain morphology, reduced demyelination, and promoted neurogenesis. The study supports GNT's potential in autism treatment by modulating neural pathways, reducing inflammation, and restoring neurotransmitter balance.
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Affiliation(s)
- Manjeet Kumar
- Division of Neuroscience, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India; Affiliated to IK Gujral Punjab Technical University, Jalandhar, Punjab 144603, India
| | - Sidharth Mehan
- Division of Neuroscience, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India; Affiliated to IK Gujral Punjab Technical University, Jalandhar, Punjab 144603, India.
| | - Aakash Kumar
- Division of Neuroscience, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India; Affiliated to IK Gujral Punjab Technical University, Jalandhar, Punjab 144603, India
| | - Tarun Sharma
- Division of Neuroscience, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India; Affiliated to IK Gujral Punjab Technical University, Jalandhar, Punjab 144603, India
| | - Zuber Khan
- Division of Neuroscience, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India; Affiliated to IK Gujral Punjab Technical University, Jalandhar, Punjab 144603, India
| | - Aarti Tiwari
- Division of Neuroscience, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India; Affiliated to IK Gujral Punjab Technical University, Jalandhar, Punjab 144603, India
| | - Ghanshyam Das Gupta
- Affiliated to IK Gujral Punjab Technical University, Jalandhar, Punjab 144603, India; Department of Pharmaceutics, ISF College of Pharmacy, Moga, Punjab, India
| | - Acharan S Narula
- Narula Research, LLC, 107 Boulder Bluff, Chapel Hill, NC 27516, USA
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4
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Lee G, Muir TW. Distinct phases of cellular signaling revealed by time-resolved protein synthesis. Nat Chem Biol 2024; 20:1353-1360. [PMID: 38977789 DOI: 10.1038/s41589-024-01677-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2023] [Accepted: 06/12/2024] [Indexed: 07/10/2024]
Abstract
The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here we deploy proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from premade parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions breakpoint cluster region-Abelson (BCR-ABL) and DNAJ-PKAc where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as new therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.
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Affiliation(s)
- Gihoon Lee
- Department of Chemistry, Princeton University, Princeton, NJ, USA
| | - Tom W Muir
- Department of Chemistry, Princeton University, Princeton, NJ, USA.
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5
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Whittaker M, Bendzunas GN, Shirani M, LeClair TJ, Shebl B, Dill TC, Coffino P, Simon SM, Kennedy EJ. Targeted Degradation of Protein Kinase A via a Stapled Peptide PROTAC. ACS Chem Biol 2024; 19:1888-1895. [PMID: 39137166 PMCID: PMC11420944 DOI: 10.1021/acschembio.4c00237] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2024] [Revised: 08/05/2024] [Accepted: 08/12/2024] [Indexed: 08/15/2024]
Abstract
Proteolysis-targeting chimeras (PROTACs) are bifunctional molecules that bind and recruit an E3 ubiquitin ligase to a targeted protein of interest, often through the utilization of a small molecule inhibitor. To expand the possible range of kinase targets that can be degraded by PROTACs, we sought to develop a PROTAC utilizing a hydrocarbon-stapled peptide as the targeting agent to bind the surface of a target protein of interest. In this study, we describe the development of a proteolysis-targeting chimera, dubbed Stapled Inhibitor Peptide - PROTAC or StIP-TAC, linking a hydrocarbon-stapled peptide with an E3 ligase ligand for targeted degradation of Protein Kinase A (PKA). This StIP-TAC molecule stimulated E3-mediated protein degradation of PKA, and this effect could be reversed by the addition of the proteasomal inhibitor MG-132. Further, StIP-TAC treatment led to a significant reduction in PKA substrate phosphorylation. Since many protein targets of interest lack structural features that make them amenable to small molecule targeting, development of StIP-TACs may broaden the potential range of protein targets using a PROTAC-mediated proteasomal degradation approach.
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Affiliation(s)
- Matthew
K. Whittaker
- Department
of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia 30602, United States
| | - George N. Bendzunas
- Department
of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia 30602, United States
| | - Mahsa Shirani
- Laboratory
of Cellular Biophysics, The Rockefeller
University, New York, New York 10065, United States
| | - Timothy J. LeClair
- Department
of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia 30602, United States
| | - Bassem Shebl
- Laboratory
of Cellular Biophysics, The Rockefeller
University, New York, New York 10065, United States
| | - Taylor C. Dill
- Department
of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia 30602, United States
| | - Philip Coffino
- Laboratory
of Cellular Biophysics, The Rockefeller
University, New York, New York 10065, United States
| | - Sanford M. Simon
- Laboratory
of Cellular Biophysics, The Rockefeller
University, New York, New York 10065, United States
| | - Eileen J. Kennedy
- Department
of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia 30602, United States
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6
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Krishnan SR, Bung N, Srinivasan R, Roy A. Target-specific novel molecules with their recipe: Incorporating synthesizability in the design process. J Mol Graph Model 2024; 129:108734. [PMID: 38442440 DOI: 10.1016/j.jmgm.2024.108734] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2023] [Revised: 02/14/2024] [Accepted: 02/15/2024] [Indexed: 03/07/2024]
Abstract
Application of Artificial intelligence (AI) in drug discovery has led to several success stories in recent times. While traditional methods mostly relied upon screening large chemical libraries for early-stage drug-design, de novo design can help identify novel target-specific molecules by sampling from a much larger chemical space. Although this has increased the possibility of finding diverse and novel molecules from previously unexplored chemical space, this has also posed a great challenge for medicinal chemists to synthesize at least some of the de novo designed novel molecules for experimental validation. To address this challenge, in this work, we propose a novel forward synthesis-based generative AI method, which is used to explore the synthesizable chemical space. The method uses a structure-based drug design framework, where the target protein structure and a target-specific seed fragment from co-crystal structures can be the initial inputs. A random fragment from a purchasable fragment library can also be the input if a target-specific fragment is unavailable. Then a template-based forward synthesis route prediction and molecule generation is performed in parallel using the Monte Carlo Tree Search (MCTS) method where, the subsequent fragments for molecule growth can again be obtained from a purchasable fragment library. The rewards for each iteration of MCTS are computed using a drug-target affinity (DTA) model based on the docking pose of the generated reaction intermediates at the binding site of the target protein of interest. With the help of the proposed method, it is now possible to overcome one of the major obstacles posed to the AI-based drug design approaches through the ability of the method to design novel target-specific synthesizable molecules.
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Affiliation(s)
| | - Navneet Bung
- TCS Research (Life Sciences Division), Tata Consultancy Services Limited, Hyderabad, 500081, India
| | - Rajgopal Srinivasan
- TCS Research (Life Sciences Division), Tata Consultancy Services Limited, Hyderabad, 500081, India
| | - Arijit Roy
- TCS Research (Life Sciences Division), Tata Consultancy Services Limited, Hyderabad, 500081, India.
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Lauer SM, Omar MH, Golkowski MG, Kenerson HL, Lee KS, Pascual BC, Lim HC, Forbush K, Smith FD, Gordan JD, Ong SE, Yeung RS, Scott JD. Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma. Cell Rep 2024; 43:113678. [PMID: 38236773 PMCID: PMC10964278 DOI: 10.1016/j.celrep.2024.113678] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2023] [Revised: 11/23/2023] [Accepted: 01/02/2024] [Indexed: 01/30/2024] Open
Abstract
The DNAJ-PKAc fusion kinase is a defining feature of fibrolamellar carcinoma (FLC). FLC tumors are notoriously resistant to standard chemotherapies, with aberrant kinase activity assumed to be a contributing factor. By combining proximity proteomics, biochemical analyses, and live-cell photoactivation microscopy, we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates, including proteins involved in translation and the anti-apoptotic factor Bcl-2-associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Tissue samples from patients with FLC exhibit increased levels of BAG2 in primary and metastatic tumors. Furthermore, drug studies implicate the DNAJ-PKAc/Hsp70/BAG2 axis in potentiating chemotherapeutic resistance. We find that the Bcl-2 inhibitor navitoclax enhances sensitivity to etoposide-induced apoptosis in cells expressing DNAJ-PKAc. Thus, our work indicates BAG2 as a marker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.
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Affiliation(s)
- Sophia M Lauer
- Department of Pharmacology, University of Washington Medical Center, Seattle, WA 98195, USA
| | - Mitchell H Omar
- Department of Pharmacology, University of Washington Medical Center, Seattle, WA 98195, USA
| | - Martin G Golkowski
- Department of Pharmacology, University of Washington Medical Center, Seattle, WA 98195, USA
| | - Heidi L Kenerson
- Department of Surgery, University of Washington Medical Center, Seattle, WA 98195, USA
| | - Kyung-Soon Lee
- Department of Pharmacology, University of Washington Medical Center, Seattle, WA 98195, USA
| | - Bryan C Pascual
- Division of Hematology and Oncology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Huat C Lim
- Division of Hematology and Oncology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Katherine Forbush
- Department of Pharmacology, University of Washington Medical Center, Seattle, WA 98195, USA
| | - F Donelson Smith
- Department of Pharmacology, University of Washington Medical Center, Seattle, WA 98195, USA
| | - John D Gordan
- Division of Hematology and Oncology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Shao-En Ong
- Department of Pharmacology, University of Washington Medical Center, Seattle, WA 98195, USA
| | - Raymond S Yeung
- Department of Surgery, University of Washington Medical Center, Seattle, WA 98195, USA
| | - John D Scott
- Department of Pharmacology, University of Washington Medical Center, Seattle, WA 98195, USA.
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8
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Leiting JL, Hernandez MC, Bergquist JR, Yonkus JA, Abdelrahman AM, Torbenson MS, Tran NH, Halfdanarson TR, Graham RP, Smoot RL, Truty MJ. Therapeutic Efficacy of Temsirolimus in a Patient-derived Model of Metastatic Fibrolamellar Hepatocellular Carcinoma. In Vivo 2023; 37:1940-1950. [PMID: 37652480 PMCID: PMC10500502 DOI: 10.21873/invivo.13290] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2023] [Revised: 06/14/2023] [Accepted: 06/28/2023] [Indexed: 09/02/2023]
Abstract
BACKGROUND/AIM Fibrolamellar hepatocellular carcinoma (FLHCC) is a rare tumor presenting in younger patients without chronic liver disease. Up to 80-100% develop recurrent disease, necessitating additional surgery or systemic treatment. Systemic options and pre-clinical treatment studies are lacking. We previously described patient-derived xenograft (PDX) development, allowing for pre-clinical studies. Herein, we develop FLHCC PDX models and utilize these to define tumor characteristics and determine the efficacy of systemic agents. MATERIALS AND METHODS Primary and lymph node metastatic tumor tissues were obtained at the time of FLHCC resection in two patients. Tumor lysates were screened for protein upregulation. Cell lines were generated from metastatic and primary tumor tissue. The viability of the cell lines was assessed after treatment with temsirolimus, gemcitabine/oxaliplatin, and FOLFIRINOX. Two PDX models were developed from metastatic tissue. For in vivo studies, tumor-bearing mice were treated with temsirolimus, FOLFIRINOX, and Gemcitabine/oxaliplatin. RESULTS PDX models were successfully generated from metastatic FLHCC, which closely recapitulated the original tumor. Upregulation of mTOR was seen in metastatic tissue compared to primary tumors. Cell lines from metastatic tissue demonstrated significant sensitivity to temsirolimus. In vivo testing of PDX models demonstrated a significant response to single-agent temsirolimus with minimal toxicity. CONCLUSION Herein, we demonstrate the feasibility of developing PDX models that closely recapitulate FLHCC. Upregulation of mTOR was seen in metastatic tissue compared to primary tissue. The efficacy of mTOR inhibition with temsirolimus treatment suggests that the upregulation of the mTOR pathway may be a significant mechanism for growth in metastatic lesions and a potential target for therapeutics.
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Affiliation(s)
| | | | | | | | | | | | - Nguyen H Tran
- Department of Medical Oncology, Mayo Clinic, Rochester, MN, U.S.A
| | | | | | - Rory L Smoot
- Department of Surgery, Mayo Clinic, Rochester, MN, U.S.A
| | - Mark J Truty
- Department of Surgery, Mayo Clinic, Rochester, MN, U.S.A.;
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9
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Islam Z, Diane A, Khattab N, Dehbi M, Thornalley P, Kolatkar PR. DNAJB3 attenuates ER stress through direct interaction with AKT. PLoS One 2023; 18:e0290340. [PMID: 37594932 PMCID: PMC10437922 DOI: 10.1371/journal.pone.0290340] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2023] [Accepted: 08/03/2023] [Indexed: 08/20/2023] Open
Abstract
Metabolic stress involved in several dysregulation disorders such as type 2 diabetes mellitus (T2DM) results in down regulation of several heat shock proteins (HSPs) including DNAJB3. This down regulation of HSPs is associated with insulin resistance (IR) and interventions which induce the heat shock response (HSR) help to increase the insulin sensitivity. Metabolic stress leads to changes in signaling pathways through increased activation of both c-jun N-terminal kinase-1 (JNK1) and the inhibitor of κB inflammatory kinase (IKKβ) which in turn leads to inactivation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2). DNAJB3 interacts with both JNK1 and IKKβ kinases to mitigate metabolic stress. In addition DNAJB3 also activates the PI3K-PKB/AKT pathway through increased phosphorylation of AKT1 and its substrate AS160, a Rab GTPase-activating protein, which results in mobilization of GLUT4 transporter protein and improved glucose uptake. We show through pull down that AK T1 is an interacting partner of DNAJB3, further confirmed by isothermal titration calorimetry (ITC) which quantified the avidity of AKT1 for DNAJB3. The binding interface was identified by combining protein modelling with docking of the AKT1-DNAJB3 complex. DNAJB3 is localized in the cytoplasm and ER, where it interacts directly with AKT1 and mobilizes AS160 for glucose transport. Inhibition of AKT1 resulted in loss of GLUT4 translocation activity mediated by DNAJB3 and also abolished the protective effect of DNAJB3 on tunicamycin-induced ER stress. Taken together, our findings provide evidence for a direct protein-protein interaction between DNAJB3 and AKT1 upon which DNAJB3 alleviates ER stress and promotes GLUT4 translocation.
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Affiliation(s)
- Zeyaul Islam
- Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Abdoulaye Diane
- Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Namat Khattab
- Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Mohammed Dehbi
- Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Paul Thornalley
- Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
| | - Prasanna R. Kolatkar
- Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar
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10
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Lee G, Muir TW. Distinct phases of cellular signaling revealed by time-resolved protein synthesis. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.07.10.548208. [PMID: 37503273 PMCID: PMC10369872 DOI: 10.1101/2023.07.10.548208] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/29/2023]
Abstract
The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here, we deploy a proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from pre-made parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions BCR/ABL and DNAJB1/PRKACA where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as novel therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.
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Affiliation(s)
- Gihoon Lee
- Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
| | - Tom W. Muir
- Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
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11
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Lauer SM, Omar MH, Golkowski MG, Kenerson HL, Pascual BC, Forbush K, Smith FD, Gordan J, Ong SE, Yeung RS, Scott JD. Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.06.28.546958. [PMID: 37425703 PMCID: PMC10327129 DOI: 10.1101/2023.06.28.546958] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/11/2023]
Abstract
The DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolamellar carcinoma (FLC). A single lesion on chromosome 19 generates this mutant kinase by creating a fused gene encoding the chaperonin binding domain of Hsp40 (DNAJ) in frame with the catalytic core of protein kinase A (PKAc). FLC tumors are notoriously resistant to standard chemotherapies. Aberrant kinase activity is assumed to be a contributing factor. Yet recruitment of binding partners, such as the chaperone Hsp70, implies that the scaffolding function of DNAJ- PKAc may also underlie pathogenesis. By combining proximity proteomics with biochemical analyses and photoactivation live-cell imaging we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates. One validated DNAJ-PKAc target is the Bcl-2 associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Immunoblot and immunohistochemical analyses of FLC patient samples correlate increased levels of BAG2 with advanced disease and metastatic recurrences. BAG2 is linked to Bcl-2, an anti-apoptotic factor that delays cell death. Pharmacological approaches tested if the DNAJ- PKAc/Hsp70/BAG2 axis contributes to chemotherapeutic resistance in AML12 DNAJ-PKAc hepatocyte cell lines using the DNA damaging agent etoposide and the Bcl-2 inhibitor navitoclax. Wildtype AML12 cells were susceptible to each drug alone and in combination. In contrast, AML12 DNAJ-PKAc cells were moderately affected by etoposide, resistant to navitoclax, but markedly susceptible to the drug combination. These studies implicate BAG2 as a biomarker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.
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12
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Rüland L, Andreatta F, Massalini S, Chuva de Sousa Lopes S, Clevers H, Hendriks D, Artegiani B. Organoid models of fibrolamellar carcinoma mutations reveal hepatocyte transdifferentiation through cooperative BAP1 and PRKAR2A loss. Nat Commun 2023; 14:2377. [PMID: 37137901 PMCID: PMC10156813 DOI: 10.1038/s41467-023-37951-6] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2022] [Accepted: 04/06/2023] [Indexed: 05/05/2023] Open
Abstract
Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A. Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1-mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACAfus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC.
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Affiliation(s)
- Laura Rüland
- The Princess Maxima Center for Pediatric Oncology, Utrecht, The Netherlands
| | | | - Simone Massalini
- The Princess Maxima Center for Pediatric Oncology, Utrecht, The Netherlands
| | | | - Hans Clevers
- The Princess Maxima Center for Pediatric Oncology, Utrecht, The Netherlands
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands
- Oncode Institute, Utrecht, The Netherlands
- University Medical Center Utrecht, Utrecht, The Netherlands
- Pharma, Research and Early Development (pRED) of F. Hoffmann-La Roche Ltd, Basel, Switzerland
| | - Delilah Hendriks
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands.
- Oncode Institute, Utrecht, The Netherlands.
| | - Benedetta Artegiani
- The Princess Maxima Center for Pediatric Oncology, Utrecht, The Netherlands.
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13
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Wilson BAP, Li N, Martinez Fiesco JA, Dalilian M, Wang D, Smith EA, Wamiru A, Shah R, Goncharova EI, Beutler JA, Grkovic T, Zhang P, O’Keefe BR. Biochemical Discovery, Intracellular Evaluation, and Crystallographic Characterization of Synthetic and Natural Product Adenosine 3',5'-Cyclic Monophosphate-Dependent Protein Kinase A (PKA) Inhibitors. ACS Pharmacol Transl Sci 2023; 6:633-650. [PMID: 37082750 PMCID: PMC10111623 DOI: 10.1021/acsptsci.3c00010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2023] [Indexed: 04/22/2023]
Abstract
The recent demonstration that adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) plays an oncogenic role in a number of important cancers has led to a renaissance in drug development interest targeting this kinase. We therefore have established a suite of biochemical, cell-based, and structural biology assays for identifying and evaluating new pharmacophores for PKA inhibition. This discovery process started with a 384-well high-throughput screen of more than 200,000 substances, including fractionated natural product extracts. Identified active compounds were further prioritized in biochemical, biophysical, and cell-based assays. Priority lead compounds were assessed in detail to fully characterize several previously unrecognized PKA pharmacophores including the generation of new X-ray crystallography structures demonstrating unique interactions between PKA and bound inhibitor molecules.
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Affiliation(s)
- Brice A. P. Wilson
- Molecular
Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
| | - Ning Li
- Center
for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
| | - Juliana A. Martinez Fiesco
- Center
for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
| | - Masoumeh Dalilian
- Molecular
Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
- Basic
Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States
| | - Dongdong Wang
- Molecular
Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
| | - Emily A. Smith
- Molecular
Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
- Basic
Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States
| | - Antony Wamiru
- Molecular
Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
- Basic
Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States
| | - Rohan Shah
- Molecular
Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
| | - Ekaterina I. Goncharova
- Molecular
Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
- Advanced
Biomedical Computational Science, Frederick
National Laboratory for Cancer Research, Frederick, Maryland 21702, United States
| | - John A. Beutler
- Molecular
Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
| | - Tanja Grkovic
- Molecular
Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
- Natural
Products Branch, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, National Cancer
Institute, Frederick, Maryland 21702, United States
| | - Ping Zhang
- Center
for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
| | - Barry R. O’Keefe
- Molecular
Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
- Natural
Products Branch, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, National Cancer
Institute, Frederick, Maryland 21702, United States
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14
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Olivieri C, Walker C, Manu V, Porcelli F, Taylor SS, Bernlohr DA, Veglia G. An NMR portrait of functional and dysfunctional allosteric cooperativity in cAMP-dependent protein kinase A. FEBS Lett 2023; 597:1055-1072. [PMID: 36892429 PMCID: PMC11334100 DOI: 10.1002/1873-3468.14610] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2023] [Revised: 02/16/2023] [Accepted: 02/17/2023] [Indexed: 03/10/2023]
Abstract
The cAMP-dependent protein kinase A (PKA) is the archetypical eukaryotic kinase. The catalytic subunit (PKA-C) structure is highly conserved among the AGC-kinase family. PKA-C is a bilobal enzyme with a dynamic N-lobe, harbouring the Adenosine-5'-triphosphate (ATP) binding site and a more rigid helical C-lobe. The substrate-binding groove resides at the interface of the two lobes. A distinct feature of PKA-C is the positive binding cooperativity between nucleotide and substrate. Several PKA-C mutations lead to the development of adenocarcinomas, myxomas, and other rare forms of liver tumours. Nuclear magnetic resonance (NMR) spectroscopy shows that these mutations disrupt the allosteric communication between the two lobes, causing a drastic decrease in binding cooperativity. The loss of cooperativity correlates with changes in substrate fidelity and reduced kinase affinity for the endogenous protein kinase inhibitor (PKI). The similarity between PKI and the inhibitory sequence of the kinase regulatory subunits suggests that the overall mechanism of regulation of the kinase may be disrupted. We surmise that a reduced or obliterated cooperativity may constitute a common trait for both orthosteric and allosteric mutations of PKA-C that may lead to dysregulation and disease.
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Affiliation(s)
- Cristina Olivieri
- Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Caitlin Walker
- Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - V.S. Manu
- Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Fernando Porcelli
- Department for Innovation in Biological, Agrofood and Forest Systems, University of Tuscia, 01100 Viterbo, Italy
| | - Susan S. Taylor
- Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093
- Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093
| | - David A. Bernlohr
- Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Gianluigi Veglia
- Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
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15
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Schalm SS, O’Hearn E, Wilson K, LaBranche TP, Silva G, Zhang Z, DiPietro L, Bifulco N, Woessner R, Stransky N, Sappal D, Campbell R, Lobbardi R, Palmer M, Kim J, Ye C, Dorsch M, Lengauer C, Guzi T, Kadambi V, Garner A, Hoeflich KP. Evaluation of Protein Kinase cAMP-Activated Catalytic Subunit Alpha as a Therapeutic Target for Fibrolamellar Carcinoma. GASTRO HEP ADVANCES 2022; 2:307-321. [PMID: 39132655 PMCID: PMC11307690 DOI: 10.1016/j.gastha.2022.11.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Accepted: 11/02/2022] [Indexed: 08/13/2024]
Abstract
Background and Aims Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. Methods FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as previously. Results Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment vs control (P = .003 and P = .0005, respectively). Conclusion We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.
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Affiliation(s)
| | - Erin O’Hearn
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | - Kevin Wilson
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | | | - Grace Silva
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | - Zhuo Zhang
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | | | - Neil Bifulco
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | | | | | - Darshan Sappal
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | | | - Riadh Lobbardi
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | - Michael Palmer
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | - Joseph Kim
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | - Chaoyang Ye
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | - Marion Dorsch
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | | | - Timothy Guzi
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | - Vivek Kadambi
- Blueprint Medicines Corporation, Cambridge, Massachusetts
| | - Andrew Garner
- Blueprint Medicines Corporation, Cambridge, Massachusetts
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16
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Abdelhamed W, El-Kassas M. Fibrolamellar hepatocellular carcinoma: A rare but unpleasant event. World J Gastrointest Oncol 2022; 14:1103-1114. [PMID: 35949219 PMCID: PMC9244987 DOI: 10.4251/wjgo.v14.i6.1103] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/15/2022] [Revised: 03/19/2022] [Accepted: 05/08/2022] [Indexed: 02/06/2023] Open
Abstract
Fibrolamellar carcinoma (FLC) is a rare variant of hepatocellular carcinoma (HCC), comprising 1%–9% of all HCCs. FLC is a poorly understood malignancy, which seems to be more prevalent in young patients with no underlying liver diseases. The term “fibrolamellar” is derived from thick fibrous collagen bands surrounding the tumor cells. Unlike HCC, cirrhosis and viral hepatitis infection are not predisposing to FLC, and it is not associated with elevations in serum alpha-fetoprotein. FLC patients often present with vague abdominal pain, nausea, malaise, and weight loss. Most cases present are at an advanced stage at the time of initial diagnosis. However, curative treatment options can still be offered to up to 70% of patients. Surgery (resection/liver transplantation) is the mainstay of treatment and the only potentially curative option. FLCs have been less chemo-responsive than the conventional HCC, however, in advanced cases, multimodality treatments can be effective. Recent advances in molecular studies of FLC have found a unique DNAJB1–PRKACA fusion transcript in most of the cases studied. The review aims to describe clinical characteristics, diagnostic methods, and therapeutic modalities for this rare tumor to raise awareness among clinicians and surgeons.
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Affiliation(s)
- Walaa Abdelhamed
- Department of Endemic Medicine, Sohag University, Sohag 14322, Egypt
| | - Mohamed El-Kassas
- Department of Endemic Medicine, Faculty of Medicine, Helwan University, Cairo 11795, Egypt
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17
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Dinh TA, Utria AF, Barry KC, Ma R, Abou-Alfa GK, Gordan JD, Jaffee EM, Scott JD, Zucman-Rossi J, O’Neill AF, Furth ME, Sethupathy P. A framework for fibrolamellar carcinoma research and clinical trials. Nat Rev Gastroenterol Hepatol 2022; 19:328-342. [PMID: 35190728 PMCID: PMC9516439 DOI: 10.1038/s41575-022-00580-3] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 01/13/2022] [Indexed: 12/17/2022]
Abstract
Fibrolamellar carcinoma (FLC), a rare, lethal hepatic cancer, occurs primarily in adolescents and young adults. Unlike hepatocellular carcinoma, FLC has no known association with viral, metabolic or chemical agents that cause cirrhosis. Currently, surgical resection is the only treatment demonstrated to achieve cure, and no standard of care exists for systemic therapy. Progress in FLC research illuminates a transition from an obscure cancer to one for which an interactive community seems poised to uncover fundamental mechanisms and initiate translation towards novel therapies. In this Roadmap, we review advances since the seminal discovery in 2014 that nearly all FLC tumours express a signature oncogene (DNAJB1-PRKACA) encoding a fusion protein (DNAJ-PKAc) in which the J-domain of a heat shock protein 40 (HSP40) co-chaperone replaces an amino-terminal segment of the catalytic subunit of the cyclic AMP-dependent protein kinase (PKA). Important gains include increased understanding of oncogenic pathways driven by DNAJ-PKAc; identification of potential therapeutic targets; development of research models; elucidation of immune mechanisms with potential for the development of immunotherapies; and completion of the first multicentre clinical trials of targeted therapy for FLC. In each of these key areas we propose a Roadmap for future progress.
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Affiliation(s)
- Timothy A. Dinh
- Medical Scientist Training Program, University of North Carolina, Chapel Hill, NC, USA.,Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA.,These authors contributed equally: Timothy A. Dinh, Alan F. Utria, Kevin C. Barry
| | - Alan F. Utria
- Department of Surgery, University of Washington, Seattle, WA, USA.,These authors contributed equally: Timothy A. Dinh, Alan F. Utria, Kevin C. Barry
| | - Kevin C. Barry
- Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.,These authors contributed equally: Timothy A. Dinh, Alan F. Utria, Kevin C. Barry
| | - Rosanna Ma
- Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA
| | - Ghassan K. Abou-Alfa
- Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,Department of Medicine, Weill Medical College at Cornell University, New York, NY, USA
| | - John D. Gordan
- Gastrointestinal oncology, University of California at San Francisco Comprehensive Cancer Center, San Francisco, CA, USA
| | - Elizabeth M. Jaffee
- Department of oncology, Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA
| | - John D. Scott
- Department of Pharmacology, University of Washington, Seattle, WA, USA
| | - Jessica Zucman-Rossi
- Centre de Recherche des Cordeliers, Sorbonne université, Inserm, Université de Paris, Functional Genomics of Solid Tumors, Paris, France
| | - Allison F. O’Neill
- Department of Paediatric Hematology/oncology, Dana-Farber Cancer Institute, Harvard University, Boston, MA, USA
| | - Mark E. Furth
- Fibrolamellar Cancer Foundation, Greenwich, CT, USA.,;
| | - Praveen Sethupathy
- Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA.,;
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18
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Regulatory spine RS3 residue of protein kinases: a lipophilic bystander or a decisive element in the small-molecule kinase inhibitor binding? Biochem Soc Trans 2022; 50:633-648. [PMID: 35226061 PMCID: PMC9022976 DOI: 10.1042/bst20210837] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2021] [Revised: 02/15/2022] [Accepted: 02/17/2022] [Indexed: 11/30/2022]
Abstract
In recent years, protein kinases have been one of the most pursued drug targets. These determined efforts have resulted in ever increasing numbers of small-molecule kinase inhibitors reaching to the market, offering novel treatment options for patients with distinct diseases. One essential component related to the activation and normal functionality of a protein kinase is the regulatory spine (R-spine). The R-spine is formed of four conserved residues named as RS1–RS4. One of these residues, RS3, located in the C-terminal part of αC-helix, is usually accessible for the inhibitors from the ATP-binding cavity as its side chain is lining the hydrophobic back pocket in many protein kinases. Although the role of RS3 has been well acknowledged in protein kinase function, this residue has not been actively considered in inhibitor design, even though many small-molecule kinase inhibitors display interactions to this residue. In this minireview, we will cover the current knowledge of RS3, its relationship with the gatekeeper, and the role of RS3 in kinase inhibitor interactions. Finally, we comment on the future perspectives how this residue could be utilized in the kinase inhibitor design.
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19
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Trisciuzzi D, Siragusa L, Baroni M, Autiero I, Nicolotti O, Cruciani G. Getting Insights into Structural and Energetic Properties of Reciprocal Peptide-Protein Interactions. J Chem Inf Model 2022; 62:1113-1125. [PMID: 35148095 DOI: 10.1021/acs.jcim.1c01343] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Peptide-protein interactions play a key role for many cellular and metabolic processes involved in the onset of largely spread diseases such as cancer and neurodegenerative pathologies. Despite the progress in the structural characterization of peptide-protein interfaces, the in-depth knowledge of the molecular details behind their interactions is still a daunting task. Here, we present the first comprehensive in silico morphological and energetic study of peptide binding sites by focusing on both peptide and protein standpoints. Starting from the PixelDB database, a nonredundant benchmark collection of high-quality 3D crystallographic structures of peptide-protein complexes, a classification analysis of the most representative categories based on the nature of each cocrystallized peptide has been carried out. Several interpretable geometrical and energetic descriptors have been computed both from peptide and target protein sides in the attempt to unveil physicochemical and structural causative correlations. Finally, we investigated the most frequent peptide-protein residue pairs at the binding interface and made extensive energetic analyses, based on GRID MIFs, with the aim to study the peptide affinity-enhancing interactions to be further exploited in rational drug design strategies.
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Affiliation(s)
- Daniela Trisciuzzi
- Department of Pharmacy, Pharmaceutical Sciences, Università degli Studi di Bari "Aldo Moro", 70125 Bari, Italy.,Molecular Horizon s.r.l., Via Montelino, 30, 06084 Bettona (PG), Italy
| | - Lydia Siragusa
- Molecular Horizon s.r.l., Via Montelino, 30, 06084 Bettona (PG), Italy.,Molecular Discovery Ltd., Kinetic Business Centre, Theobald Street, Elstree, Borehamwood, Hertfordshire WD6 4PJ, United Kingdom
| | - Massimo Baroni
- Molecular Discovery Ltd., Kinetic Business Centre, Theobald Street, Elstree, Borehamwood, Hertfordshire WD6 4PJ, United Kingdom
| | - Ida Autiero
- Molecular Horizon s.r.l., Via Montelino, 30, 06084 Bettona (PG), Italy.,National Research Council, Institute of Biostructures and Bioimaging, 80138 Naples, Italy
| | - Orazio Nicolotti
- Department of Pharmacy, Pharmaceutical Sciences, Università degli Studi di Bari "Aldo Moro", 70125 Bari, Italy
| | - Gabriele Cruciani
- Department of Chemistry, Biology and Biotechnology, Università degli Studi di Perugia, via Elce di Sotto, 8, 06123 Perugia (PG), Italy
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20
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Bolger GB. The cAMP-signaling cancers: Clinically-divergent disorders with a common central pathway. Front Endocrinol (Lausanne) 2022; 13:1024423. [PMID: 36313756 PMCID: PMC9612118 DOI: 10.3389/fendo.2022.1024423] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/21/2022] [Accepted: 09/27/2022] [Indexed: 12/01/2022] Open
Abstract
The cAMP-signaling cancers, which are defined by functionally-significant somatic mutations in one or more elements of the cAMP signaling pathway, have an unexpectedly wide range of cell origins, clinical manifestations, and potential therapeutic options. Mutations in at least 9 cAMP signaling pathway genes (TSHR, GPR101, GNAS, PDE8B, PDE11A, PRKARA1, PRKACA, PRKACB, and CREB) have been identified as driver mutations in human cancer. Although all cAMP-signaling pathway cancers are driven by mutation(s) that impinge on a single signaling pathway, the ultimate tumor phenotype reflects interactions between five critical variables: (1) the precise gene(s) that undergo mutation in each specific tumor type; (2) the effects of specific allele(s) in any given gene; (3) mutations in modifier genes (mutational "context"); (4) the tissue-specific expression of various cAMP signaling pathway elements in the tumor stem cell; and (5) and the precise biochemical regulation of the pathway components in tumor cells. These varying oncogenic mechanisms reveal novel and important targets for drug discovery. There is considerable diversity in the "druggability" of cAMP-signaling components, with some elements (GPCRs, cAMP-specific phosphodiesterases and kinases) appearing to be prime drug candidates, while other elements (transcription factors, protein-protein interactions) are currently refractory to robust drug-development efforts. Further refinement of the precise driver mutations in individual tumors will be essential for directing priorities in drug discovery efforts that target these mutations.
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21
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Qiao K, Jiang Y, Hu T, Li S, Gui W. Prokaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assay. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 2021; 228:113023. [PMID: 34839138 DOI: 10.1016/j.ecoenv.2021.113023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/03/2021] [Revised: 11/16/2021] [Accepted: 11/20/2021] [Indexed: 06/13/2023]
Abstract
The extensively studied cAMP-dependent protein kinase A (PKA) is involved in the regulation of critical cell processes, including metabolism, gene expression, and cell proliferation. Therefore, PKA has been viewed increasingly as potential target for variety of drugs and environmental endocrine disruptors. Consequentially, the preparation of PKA protein became an important initial step for the subsequent exploration of PKA's character in endocrine disrupting effects of pesticides. To investigate PKA protein, which is potential to be the environmental endocrine toxicity target of triazole fungicides, a strategy to heterologously express protein kinase A catalytic alpha subunit of human (hPKAcα) and zebrafish (zPKAcα) in Escherichia coli (E. coli) BL21(DE3) host cells was demonstrated. After optimizing conditions and protein purification, we successfully obtained enzymatically active hPKAcα and zPKAcα. Western blot analysis indicated that the recombinant hPKAcα and zPKAcα still retained their characteristic antigenicity and binding activity, while in vitro kinase activity assays revealed that the recombinant hPKAcα and zPKAcα maintained enzyme activity. By in silico methods including homology modelling and molecular docking, the affinity of ligands and the models of hPKAcα and zPKAcα were further tested. The present study offered a valuable method to achieve the prokaryotic expression of a eukaryotic protein kinase and laid a foundation to facilitate further investigation of toxicological target of triazole pesticides.
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Affiliation(s)
- Kun Qiao
- Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China; Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou 310058, PR China
| | - Yao Jiang
- Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China
| | - Tiantian Hu
- Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China
| | - Shuying Li
- Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China; Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, Zhejiang University, Hangzhou 310058, PR China; Key Laboratory of Biology of Crop Pathogens and Insect Pests of Zhejiang Province, Zhejiang, Hangzhou 310058, PR China.
| | - Wenjun Gui
- Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China; Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, Zhejiang University, Hangzhou 310058, PR China; Key Laboratory of Biology of Crop Pathogens and Insect Pests of Zhejiang Province, Zhejiang, Hangzhou 310058, PR China
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22
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Ramms DJ, Raimondi F, Arang N, Herberg FW, Taylor SS, Gutkind JS. G αs-Protein Kinase A (PKA) Pathway Signalopathies: The Emerging Genetic Landscape and Therapeutic Potential of Human Diseases Driven by Aberrant G αs-PKA Signaling. Pharmacol Rev 2021; 73:155-197. [PMID: 34663687 PMCID: PMC11060502 DOI: 10.1124/pharmrev.120.000269] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Many of the fundamental concepts of signal transduction and kinase activity are attributed to the discovery and crystallization of cAMP-dependent protein kinase, or protein kinase A. PKA is one of the best-studied kinases in human biology, with emphasis in biochemistry and biophysics, all the way to metabolism, hormone action, and gene expression regulation. It is surprising, however, that our understanding of PKA's role in disease is largely underappreciated. Although genetic mutations in the PKA holoenzyme are known to cause diseases such as Carney complex, Cushing syndrome, and acrodysostosis, the story largely stops there. With the recent explosion of genomic medicine, we can finally appreciate the broader role of the Gαs-PKA pathway in disease, with contributions from aberrant functioning G proteins and G protein-coupled receptors, as well as multiple alterations in other pathway components and negative regulators. Together, these represent a broad family of diseases we term the Gαs-PKA pathway signalopathies. The Gαs-PKA pathway signalopathies encompass diseases caused by germline, postzygotic, and somatic mutations in the Gαs-PKA pathway, with largely endocrine and neoplastic phenotypes. Here, we present a signaling-centric review of Gαs-PKA-driven pathophysiology and integrate computational and structural analysis to identify mutational themes commonly exploited by the Gαs-PKA pathway signalopathies. Major mutational themes include hotspot activating mutations in Gαs, encoded by GNAS, and mutations that destabilize the PKA holoenzyme. With this review, we hope to incite further study and ultimately the development of new therapeutic strategies in the treatment of a wide range of human diseases. SIGNIFICANCE STATEMENT: Little recognition is given to the causative role of Gαs-PKA pathway dysregulation in disease, with effects ranging from infectious disease, endocrine syndromes, and many cancers, yet these disparate diseases can all be understood by common genetic themes and biochemical signaling connections. By highlighting these common pathogenic mechanisms and bridging multiple disciplines, important progress can be made toward therapeutic advances in treating Gαs-PKA pathway-driven disease.
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Affiliation(s)
- Dana J Ramms
- Department of Pharmacology (D.J.R., N.A., J.S.G.), Department of Chemistry and Biochemistry (S.S.T.), and Moores Cancer Center (D.J.R., N.A., J.S.G.), University of California, San Diego, La Jolla, California; Laboratorio di Biologia Bio@SNS, Scuola Normale Superiore, Pisa, Italy (F.R.); and Department of Biochemistry, University of Kassel, Kassel, Germany (F.W.H.)
| | - Francesco Raimondi
- Department of Pharmacology (D.J.R., N.A., J.S.G.), Department of Chemistry and Biochemistry (S.S.T.), and Moores Cancer Center (D.J.R., N.A., J.S.G.), University of California, San Diego, La Jolla, California; Laboratorio di Biologia Bio@SNS, Scuola Normale Superiore, Pisa, Italy (F.R.); and Department of Biochemistry, University of Kassel, Kassel, Germany (F.W.H.)
| | - Nadia Arang
- Department of Pharmacology (D.J.R., N.A., J.S.G.), Department of Chemistry and Biochemistry (S.S.T.), and Moores Cancer Center (D.J.R., N.A., J.S.G.), University of California, San Diego, La Jolla, California; Laboratorio di Biologia Bio@SNS, Scuola Normale Superiore, Pisa, Italy (F.R.); and Department of Biochemistry, University of Kassel, Kassel, Germany (F.W.H.)
| | - Friedrich W Herberg
- Department of Pharmacology (D.J.R., N.A., J.S.G.), Department of Chemistry and Biochemistry (S.S.T.), and Moores Cancer Center (D.J.R., N.A., J.S.G.), University of California, San Diego, La Jolla, California; Laboratorio di Biologia Bio@SNS, Scuola Normale Superiore, Pisa, Italy (F.R.); and Department of Biochemistry, University of Kassel, Kassel, Germany (F.W.H.)
| | - Susan S Taylor
- Department of Pharmacology (D.J.R., N.A., J.S.G.), Department of Chemistry and Biochemistry (S.S.T.), and Moores Cancer Center (D.J.R., N.A., J.S.G.), University of California, San Diego, La Jolla, California; Laboratorio di Biologia Bio@SNS, Scuola Normale Superiore, Pisa, Italy (F.R.); and Department of Biochemistry, University of Kassel, Kassel, Germany (F.W.H.)
| | - J Silvio Gutkind
- Department of Pharmacology (D.J.R., N.A., J.S.G.), Department of Chemistry and Biochemistry (S.S.T.), and Moores Cancer Center (D.J.R., N.A., J.S.G.), University of California, San Diego, La Jolla, California; Laboratorio di Biologia Bio@SNS, Scuola Normale Superiore, Pisa, Italy (F.R.); and Department of Biochemistry, University of Kassel, Kassel, Germany (F.W.H.)
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23
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Liquid-liquid phase separation: a principal organizer of the cell's biochemical activity architecture. Trends Pharmacol Sci 2021; 42:845-856. [PMID: 34373114 DOI: 10.1016/j.tips.2021.07.003] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2021] [Revised: 06/28/2021] [Accepted: 07/13/2021] [Indexed: 12/25/2022]
Abstract
Numerous processes occur simultaneously in the cell both for normal function and in response to changes in the environment. The ability of cells to segregate biochemical reactions into separate compartments is essential to ensure specificity and efficiency in cellular processes. The discovery of liquid-liquid phase separation as a mechanism of compartmentalization has revised our thinking regarding the intracellular organization of molecular pathways such as signal transduction. Here, we highlight recent studies that advance our understanding of how phase separation impacts the organization of biochemical processes, with a particular focus on the tools used to study the functional impact of phase separation. In addition, we offer some of our perspectives on the pathological consequences of dysregulated phase separation in biochemical pathways.
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24
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Walker C, Wang Y, Olivieri C, V S M, Gao J, Bernlohr DA, Calebiro D, Taylor SS, Veglia G. Is Disrupted Nucleotide-Substrate Cooperativity a Common Trait for Cushing's Syndrome Driving Mutations of Protein Kinase A? J Mol Biol 2021; 433:167123. [PMID: 34224748 DOI: 10.1016/j.jmb.2021.167123] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Revised: 06/24/2021] [Accepted: 06/27/2021] [Indexed: 12/14/2022]
Abstract
Somatic mutations in the PRKACA gene encoding the catalytic α subunit of protein kinase A (PKA-C) are responsible for cortisol-producing adrenocortical adenomas. These benign neoplasms contribute to the development of Cushing's syndrome. The majority of these mutations occur at the interface between the two lobes of PKA-C and interfere with the enzyme's ability to recognize substrates and regulatory (R) subunits, leading to aberrant phosphorylation patterns and activation. Rarely, patients with similar phenotypes carry an allosteric mutation, E31V, located at the C-terminal end of the αA-helix and adjacent to the αC-helix, but structurally distinct from the PKA-C/R subunit interface mutations. Using a combination of solution NMR, thermodynamics, kinetic assays, and molecular dynamics simulations, we show that the E31V allosteric mutation disrupts central communication nodes between the N- and C- lobes of the enzyme as well as nucleotide-substrate binding cooperativity, a hallmark for kinases' substrate fidelity and regulation. For both orthosteric (L205R and W196R) and allosteric (E31V) Cushing's syndrome mutants, the loss of binding cooperativity is proportional to the density of the intramolecular allosteric network. This structure-activity relationship suggests a possible common mechanism for Cushing's syndrome driving mutations in which decreased nucleotide/substrate binding cooperativity is linked to loss in substrate fidelity and dysfunctional regulation.
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Affiliation(s)
- Caitlin Walker
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Yingjie Wang
- Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA; Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen 518055, China
| | - Cristina Olivieri
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Manu V S
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Jiali Gao
- Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA; Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen 518055, China
| | - David A Bernlohr
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Davide Calebiro
- Institute of Metabolism and Systems Research, University of Birmingham, B15 2TT Birmingham, UK; Centre of Membrane Proteins and Receptors, Universities of Birmingham and Nottingham, B15 2TT Birmingham, UK
| | - Susan S Taylor
- Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA; Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA
| | - Gianluigi Veglia
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA; Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
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25
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PKA Cβ: a forgotten catalytic subunit of cAMP-dependent protein kinase opens new windows for PKA signaling and disease pathologies. Biochem J 2021; 478:2101-2119. [PMID: 34115095 DOI: 10.1042/bcj20200867] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2021] [Revised: 04/30/2021] [Accepted: 05/05/2021] [Indexed: 12/17/2022]
Abstract
3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits are encoded by the two major genes PRKACA and PRKACB, respectively. The PRKACA gene encodes two known splice variants, the ubiquitously expressed Cα1 and the sperm-specifically expressed Cα2. In contrast, the PRKACB gene encodes several splice variants expressed in a highly cell and tissue-specific manner. The Cβ proteins are called Cβ1, Cβ2, Cβ3, Cβ4 and so-called abc variants of Cβ3 and Cβ4. Whereas Cβ1 is ubiquitously expressed, Cβ2 is enriched in immune cells and the Cβ3, Cβ4 and their abc variants are solely expressed in neuronal cells. All Cα and Cβ splice variants share a kinase-conserved catalytic core and a C-terminal tail encoded by exons 2 through 10 in the PRKACA and PRKACB genes, respectively. All Cα and Cβ splice variants with the exception of Cα1 and Cβ1 are hyper-variable at the N-terminus. Here, we will discuss how the PRKACA and PRKACB genes have developed as paralogs that encode distinct and functionally non-redundant proteins. The fact that Cα and Cβ splice variant mutations are associated with numerous diseases further opens new windows for PKA-induced disease pathologies.
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26
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Gaji RY, Sharp AK, Brown AM. Protein kinases in Toxoplasma gondii. Int J Parasitol 2021; 51:415-429. [PMID: 33581139 PMCID: PMC11065138 DOI: 10.1016/j.ijpara.2020.11.006] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2020] [Revised: 11/10/2020] [Accepted: 11/15/2020] [Indexed: 02/06/2023]
Abstract
Toxoplasma gondii is an obligatory intracellular pathogen that causes life threatening illness in immunodeficient individuals, miscarriage in pregnant woman, and blindness in newborn children. Similar to any other eukaryotic cell, protein kinases play critical and essential roles in the Toxoplasma life cycle. Accordingly, many studies have focused on identifying and defining the mechanism of function of these signalling proteins with a long-term goal to develop anti-Toxoplasma therapeutics. In this review, we briefly discuss classification and key components of the catalytic domain which are critical for functioning of kinases, with a focus on domains, families, and groups of kinases within Toxoplasma. More importantly, this article provides a comprehensive, current overview of research on kinase groups in Toxoplasma including the established eukaryotic AGC, CAMK, CK1, CMGC, STE, TKL families and the apicomplexan-specific FIKK, ROPK and WNG family of kinases. This work provides an overview and discusses current knowledge on Toxoplasma kinases including their localization, function, signalling network and role in acute and chronic pathogenesis, with a view towards the future in probing kinases as viable drug targets.
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Affiliation(s)
- Rajshekhar Y Gaji
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech University, Blacksburg, VA, USA; Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA.
| | - Amanda K Sharp
- Interdisciplinary Program in Genetics, Bioinformatics, and Computational Biology, Virginia Tech, Blacksburg, VA, USA
| | - Anne M Brown
- Department of Biochemistry, Virginia Tech, Blacksburg, VA, USA; University Libraries, Virginia Tech, Blacksburg, VA, USA
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27
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Regulation of Cardiac PKA Signaling by cAMP and Oxidants. Antioxidants (Basel) 2021; 10:antiox10050663. [PMID: 33923287 PMCID: PMC8146537 DOI: 10.3390/antiox10050663] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2021] [Revised: 04/16/2021] [Accepted: 04/20/2021] [Indexed: 12/31/2022] Open
Abstract
Pathologies, such as cancer, inflammatory and cardiac diseases are commonly associated with long-term increased production and release of reactive oxygen species referred to as oxidative stress. Thereby, protein oxidation conveys protein dysfunction and contributes to disease progression. Importantly, trials to scavenge oxidants by systemic antioxidant therapy failed. This observation supports the notion that oxidants are indispensable physiological signaling molecules that induce oxidative post-translational modifications in target proteins. In cardiac myocytes, the main driver of cardiac contractility is the activation of the β-adrenoceptor-signaling cascade leading to increased cellular cAMP production and activation of its main effector, the cAMP-dependent protein kinase (PKA). PKA-mediated phosphorylation of substrate proteins that are involved in excitation-contraction coupling are responsible for the observed positive inotropic and lusitropic effects. PKA-actions are counteracted by cellular protein phosphatases (PP) that dephosphorylate substrate proteins and thus allow the termination of PKA-signaling. Both, kinase and phosphatase are redox-sensitive and susceptible to oxidation on critical cysteine residues. Thereby, oxidation of the regulatory PKA and PP subunits is considered to regulate subcellular kinase and phosphatase localization, while intradisulfide formation of the catalytic subunits negatively impacts on catalytic activity with direct consequences on substrate (de)phosphorylation and cardiac contractile function. This review article attempts to incorporate the current perception of the functionally relevant regulation of cardiac contractility by classical cAMP-dependent signaling with the contribution of oxidant modification.
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28
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Olivieri C, Walker C, Karamafrooz A, Wang Y, Manu VS, Porcelli F, Blumenthal DK, Thomas DD, Bernlohr DA, Simon SM, Taylor SS, Veglia G. Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A. Commun Biol 2021; 4:321. [PMID: 33692454 PMCID: PMC7946884 DOI: 10.1038/s42003-021-01819-6] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Accepted: 01/29/2021] [Indexed: 02/08/2023] Open
Abstract
An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera's allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.
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Affiliation(s)
- Cristina Olivieri
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA
| | - Caitlin Walker
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA
| | - Adak Karamafrooz
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA
| | - Yingjie Wang
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA
- Chemistry, University of Minnesota, Minneapolis, MN, USA
- Shenzhen Bay Laboratory, Shenzhen, China
| | - V S Manu
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA
| | - Fernando Porcelli
- DIBAF - University of Tuscia - Largo dell' Università, Viterbo, Italy
| | - Donald K Blumenthal
- Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT, USA
| | - David D Thomas
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA
| | - David A Bernlohr
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA
| | - Sanford M Simon
- Laboratory of Cellular Biophysics, Rockefeller University, New York, NY, USA
| | - Susan S Taylor
- Department of Chemistry and Biochemistry and Pharmacology, University of California at San Diego, La Jolla, CA, USA
| | - Gianluigi Veglia
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA.
- Chemistry, University of Minnesota, Minneapolis, MN, USA.
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29
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Postler TS. A most versatile kinase: The catalytic subunit of PKA in T-cell biology. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2021; 361:301-318. [PMID: 34074497 DOI: 10.1016/bs.ircmb.2021.01.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The cAMP-dependent protein kinase, more commonly referred to as protein kinase A (PKA), is one of the most-studied enzymes in biology. PKA is ubiquitously expressed in mammalian cells, can be activated in response to a plethora of biological stimuli, and phosphorylates more than 250 known substrates. Indeed, PKA is of central importance to a wide range of organismal processes, including energy homeostasis, memory formation and immunity. It serves as the primary effector of the second-messenger molecule 3',5'-cyclic adenosine monophosphate (cAMP), which is believed to have mostly inhibitory effects on the adaptive immune response. In particular, elevated levels of intracellular cAMP inhibit the activation of conventional T cells by limiting signal transduction through the T-cell receptor and altering gene expression, primarily in a PKA-dependent manner. Regulatory T cells have been shown to increase the cAMP levels in adjacent T cells by direct and indirect means, but the role of cAMP within regulatory T cells themselves remains incompletely understood. Paradoxically, cAMP has been implicated in promoting T-cell activation as well, adding another functional dimension beyond its established immunosuppressive effects. Furthermore, PKA can phosphorylate the NF-κB subunit p65, a transcription factor that is essential for T-cell activation, independently of cAMP. This phosphorylation of p65 drastically enhances NF-κB-dependent transcription and thus is likely to facilitate immune activation. How these immunosuppressive and immune-activating properties of PKA balance in vivo remains to be elucidated. This review provides a brief overview of PKA regulation, its ability to affect NF-κB activation, and its diverse functions in T-cell biology.
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Affiliation(s)
- Thomas S Postler
- Department of Microbiology & Immunology, Vagelos College of Physicians & Surgeons, Columbia University Irving Medical Center, New York, NY, United States.
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30
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Lu TW, Aoto PC, Weng JH, Nielsen C, Cash JN, Hall J, Zhang P, Simon SM, Cianfrocco MA, Taylor SS. Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma. PLoS Biol 2020; 18:e3001018. [PMID: 33370777 PMCID: PMC7793292 DOI: 10.1371/journal.pbio.3001018] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2020] [Revised: 01/08/2021] [Accepted: 12/18/2020] [Indexed: 12/31/2022] Open
Abstract
When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.
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Affiliation(s)
- Tsan-Wen Lu
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, United States of America
| | - Phillip C. Aoto
- Department of Pharmacology, University of California, San Diego, La Jolla, California, United States of America
| | - Jui-Hung Weng
- Department of Pharmacology, University of California, San Diego, La Jolla, California, United States of America
| | - Cole Nielsen
- Department of Pharmacology, University of California, San Diego, La Jolla, California, United States of America
| | - Jennifer N. Cash
- Life Sciences Institute, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, United States of America
| | - James Hall
- Department of Pharmacology, University of California, San Diego, La Jolla, California, United States of America
| | - Ping Zhang
- Department of Pharmacology, University of California, San Diego, La Jolla, California, United States of America
| | - Sanford M. Simon
- Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York, United States of America
| | - Michael A. Cianfrocco
- Life Sciences Institute, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, United States of America
| | - Susan S. Taylor
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, United States of America
- Department of Pharmacology, University of California, San Diego, La Jolla, California, United States of America
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31
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Zhang JZ, Lu TW, Stolerman LM, Tenner B, Yang JR, Zhang JF, Falcke M, Rangamani P, Taylor SS, Mehta S, Zhang J. Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling. Cell 2020; 182:1531-1544.e15. [PMID: 32846158 PMCID: PMC7502557 DOI: 10.1016/j.cell.2020.07.043] [Citation(s) in RCA: 201] [Impact Index Per Article: 40.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2019] [Revised: 03/30/2020] [Accepted: 07/30/2020] [Indexed: 12/25/2022]
Abstract
The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.
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Affiliation(s)
- Jason Z Zhang
- Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA; Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA
| | - Tsan-Wen Lu
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA
| | - Lucas M Stolerman
- Department of Mechanical and Aerospace Engineering, University of California, San Diego, La Jolla, CA 92093, USA
| | - Brian Tenner
- Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA
| | - Jessica R Yang
- Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Jin-Fan Zhang
- Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA; Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA
| | - Martin Falcke
- Mathematical Cell Physiology, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany; Department of Physics, Humboldt University, 12489 Berlin, Germany
| | - Padmini Rangamani
- Department of Mechanical and Aerospace Engineering, University of California, San Diego, La Jolla, CA 92093, USA
| | - Susan S Taylor
- Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA; Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA
| | - Sohum Mehta
- Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA
| | - Jin Zhang
- Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA; Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA; Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA; Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
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32
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Wu Y, Li Y, Liu J, Chen M, Li W, Chen Y, Xu M. The diagnostic value of extracellular protein kinase A (ECPKA) in serum for gastric and colorectal cancer. Transl Cancer Res 2020; 9:3870-3878. [PMID: 35117754 PMCID: PMC8797401 DOI: 10.21037/tcr-20-764] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2020] [Accepted: 04/24/2020] [Indexed: 01/26/2023]
Abstract
Background For the biomarkers of cancer, many chromosomal and genetic alterations have been examined as possible. However, some tumors do not display a clear molecular and genetic signature. While there are some cellular processes regulated by second messenger intracellular pathways indeed involved in carcinogenesis. The first intracellular second messenger was described as cyclic adenosine 3',5'-monophosphate (cAMP), and cAMP-dependent protein kinase (PKA) play a crucial role in several biological processes; The dysregulation of PKA-mediated signaling in several types of cancer should be investigated. More interesting, the alpha catalytic subunit of PKA (PKACα) could be secreted into the conditioned medium by different types of cancer cells, and it also existed in the serum of some cancer patients, defined as extracellular protein kinase A (ECPKA). Methods The levels of serum PKACα from healthy people, gastric cancer and colon cancer patients were detected by ELISA kits. Western blotting was used to detect the expression of PKACα in cancer tissue and the adjacent mucosa. Mann-Whitney test was applied to analyze the patients’ characteristics and serum PKACα. ROC analyses were performed to further evaluate the utility of PKACα in cancer diagnosis. The correlation of serum PKACα and T stage, age, and tumor markers were analyzed by Spearman rank and Pearson correlation analysis, respectively. Results There were significant differences of PKACα in serum between the volunteers and the gastric cancers (P<0.01), but not the colorectal cancers (P>0.05). ROC analyses evaluated the utility of PKACα for gastric cancer with 61.90% sensitivities and 87.50% specificities. The serum PKACα was correlated with tumor marker CA50, while there was no significant difference of PKACα expression between the gastric/colorectal cancer tissue and the adjacent mucosa. Conclusions The above results implied that PKACα levels might be a potential biomarker for the early screening of gastric cancers. Moreover, further research is still needed to investigate the role of secreted PKACα and the regulatory mechanism in tumor progression.
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Affiliation(s)
- Yan Wu
- Department of Physiology, School of Medicine, Jiangsu University, Zhenjiang, China
| | - Yafang Li
- Department of Physiology, School of Medicine, Jiangsu University, Zhenjiang, China
| | - Junqiang Liu
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, China
| | - Mengjiao Chen
- Department of Physiology, School of Medicine, Jiangsu University, Zhenjiang, China
| | - Wei Li
- The Fourth Affiliated Hospital of Jiangsu University, Zhenjiang, China
| | - Yongchang Chen
- Department of Physiology, School of Medicine, Jiangsu University, Zhenjiang, China
| | - Min Xu
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, China
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Shin H, Oh S, Kang D, Choi Y. Protein Quantification and Imaging by Surface-Enhanced Raman Spectroscopy and Similarity Analysis. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2020; 7:1903638. [PMID: 32537409 PMCID: PMC7284192 DOI: 10.1002/advs.201903638] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/15/2019] [Revised: 02/07/2020] [Accepted: 03/09/2020] [Indexed: 05/29/2023]
Abstract
Protein quantification techniques such as immunoassays have been improved considerably, but they have several limitations, including time-consuming procedures, low sensitivity, and extrinsic detection. Because direct surface-enhanced Raman spectroscopy (SERS) can detect intrinsic signals of proteins, it can be used as an effective detection method. However, owing to the complexity and reliability of SERS signals, SERS is rarely adopted for quantification without a purified target protein. This study reports an efficient and effective direct SERS-based immunoassay (SERSIA) technique for protein quantification and imaging. SERSIA relies on the uniform coating of gold nanoparticles (GNPs) on a target-protein-immobilized substrate by simple centrifugation. As centrifugation induces close contact between the GNPs and target proteins, the intrinsic signals of the target protein can be detected. For quantification, the protein levels in a cell lysate are estimated using similarity analysis between antibody-only and protein-conjugated samples. This method reliably estimates the protein level at a sub-picomolar detection limit. Furthermore, this method enables quantitative imaging of immobilized protein at a micrometer range. Because this technique is fast, sensitive, and requires only one type of antibody, this approach can be a useful method to detect proteins in biological samples.
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Affiliation(s)
- Hyunku Shin
- Department of Bio‐convergence EngineeringKorea UniversitySeoul02841Republic of Korea
| | - Seunghyun Oh
- School of Biomedical EngineeringKorea UniversitySeoul02841Republic of Korea
| | - Daehyeon Kang
- School of Biomedical EngineeringKorea UniversitySeoul02841Republic of Korea
| | - Yeonho Choi
- Department of Bio‐convergence EngineeringKorea UniversitySeoul02841Republic of Korea
- School of Biomedical EngineeringKorea UniversitySeoul02841Republic of Korea
- Department of BioengineeringKorea UniversitySeoul02841Republic of Korea
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34
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Olivieri C, Wang Y, Li GC, V S M, Kim J, Stultz BR, Neibergall M, Porcelli F, Muretta JM, Thomas DDT, Gao J, Blumenthal DK, Taylor SS, Veglia G. Multi-state recognition pathway of the intrinsically disordered protein kinase inhibitor by protein kinase A. eLife 2020; 9:e55607. [PMID: 32338601 PMCID: PMC7234811 DOI: 10.7554/elife.55607] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2020] [Accepted: 04/27/2020] [Indexed: 12/17/2022] Open
Abstract
In the nucleus, the spatiotemporal regulation of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is orchestrated by an intrinsically disordered protein kinase inhibitor, PKI, which recruits the CRM1/RanGTP nuclear exporting complex. How the PKA-C/PKI complex assembles and recognizes CRM1/RanGTP is not well understood. Using NMR, SAXS, fluorescence, metadynamics, and Markov model analysis, we determined the multi-state recognition pathway for PKI. After a fast binding step in which PKA-C selects PKI's most competent conformations, PKI folds upon binding through a slow conformational rearrangement within the enzyme's binding pocket. The high-affinity and pseudo-substrate regions of PKI become more structured and the transient interactions with the kinase augment the helical content of the nuclear export sequence, which is then poised to recruit the CRM1/RanGTP complex for nuclear translocation. The multistate binding mechanism featured by PKA-C/PKI complex represents a paradigm on how disordered, ancillary proteins (or protein domains) are able to operate multiple functions such as inhibiting the kinase while recruiting other regulatory proteins for nuclear export.
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Affiliation(s)
- Cristina Olivieri
- Department of Biochemistry, Molecular Biology, and Biophysics, University of MinnesotaMinneapolisUnited States
| | - Yingjie Wang
- Department of Chemistry and Supercomputing Institute, University of MinnesotaMinneapolisUnited States
- Shenzhen Bay LaboratoryShenzhenChina
| | - Geoffrey C Li
- Department of Chemistry and Supercomputing Institute, University of MinnesotaMinneapolisUnited States
| | - Manu V S
- Department of Biochemistry, Molecular Biology, and Biophysics, University of MinnesotaMinneapolisUnited States
| | - Jonggul Kim
- Department of Chemistry and Supercomputing Institute, University of MinnesotaMinneapolisUnited States
| | | | | | | | - Joseph M Muretta
- Department of Biochemistry, Molecular Biology, and Biophysics, University of MinnesotaMinneapolisUnited States
| | - David DT Thomas
- Department of Biochemistry, Molecular Biology, and Biophysics, University of MinnesotaMinneapolisUnited States
| | - Jiali Gao
- Department of Chemistry and Supercomputing Institute, University of MinnesotaMinneapolisUnited States
- Laboratory of Computational Chemistry and Drug Design, Peking University Shenzhen Graduate SchoolShenzhenChina
| | - Donald K Blumenthal
- Department of Pharmacology and Toxicology, University of UtahSalt Lake CityUnited States
| | - Susan S Taylor
- Department of Chemistry and Biochemistry and Pharmacology, University of California, San DiegoLa JollaUnited States
| | - Gianluigi Veglia
- Department of Biochemistry, Molecular Biology, and Biophysics, University of MinnesotaMinneapolisUnited States
- Department of Chemistry and Supercomputing Institute, University of MinnesotaMinneapolisUnited States
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35
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Liu Q, Liang C, Zhou L. Structural and functional analysis of the Hsp70/Hsp40 chaperone system. Protein Sci 2019; 29:378-390. [PMID: 31509306 DOI: 10.1002/pro.3725] [Citation(s) in RCA: 85] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2019] [Revised: 08/29/2019] [Accepted: 09/03/2019] [Indexed: 12/22/2022]
Abstract
As one of the most abundant and highly conserved molecular chaperones, the 70-kDa heat shock proteins (Hsp70s) play a key role in maintaining cellular protein homeostasis (proteostasis), one of the most fundamental tasks for every living organism. In this role, Hsp70s are inextricably linked to many human diseases, most notably cancers and neurodegenerative diseases, and are increasingly recognized as important drug targets for developing novel therapeutics for these diseases. Hsp40s are a class of essential and universal partners for Hsp70s in almost all aspects of proteostasis. Thus, Hsp70s and Hsp40s together constitute one of the most important chaperone systems across all kingdoms of life. In recent years, we have witnessed significant progress in understanding the molecular mechanism of this chaperone system through structural and functional analysis. This review will focus on this recent progress, mainly from a structural perspective.
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Affiliation(s)
- Qinglian Liu
- Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia
| | - Ce Liang
- Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia
| | - Lei Zhou
- Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia
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36
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Ahrari S, Mogharrab N, Navapour L. Structure and dynamics of inactive and active MARK4: conformational switching through the activation process. J Biomol Struct Dyn 2019; 38:2468-2481. [DOI: 10.1080/07391102.2019.1655479] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Affiliation(s)
- Sajjad Ahrari
- Biophysics and Computational Biology Laboratory (BCBL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran
| | - Navid Mogharrab
- Biophysics and Computational Biology Laboratory (BCBL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran
| | - Leila Navapour
- Biophysics and Computational Biology Laboratory (BCBL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran
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37
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Riehle KJ, Kenerson HL, Riggle KM, Turnham R, Sullivan K, Bauer R, Scott JD, Yeung RS. Neurotensin as a source of cyclic AMP and co-mitogen in fibrolamellar hepatocellular carcinoma. Oncotarget 2019; 10:5092-5102. [PMID: 31489118 PMCID: PMC6707953 DOI: 10.18632/oncotarget.27149] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2019] [Accepted: 07/28/2019] [Indexed: 12/20/2022] Open
Abstract
Fibrolamellar hepatocellular carcinomas (FL-HCCs) possess a unique mutation that encodes a chimeric form of protein kinase A (DNAJ-PKAc), which includes a chaperonin binding domain. DNAJ-PKAc retains most of the biochemical properties of the native enzyme, however, and activity remains dependent on cAMP. We thus speculated that a persistent source of cAMP is necessary to promote FL-HCC carcinogenesis, and that neurotensin (NTS) may drive cAMP production in this setting, given that NS serum and tumor levels are elevated in many patients with FL-HCC. We examined expression of NTS pathway components in human FL-HCCs and paired normal livers, and determined the role of NTS in driving proliferation in tumor slice cultures. Cultured hepatocytes were used to determine interactions between NTS and other proliferative pathways, and to determine the effects of NTS on cAMP production and PKA activity. We found that the NTS pathway is up-regulated in human FL-HCCs, and that NTS activates cAMP and PKA in hepatocytes. NTS increases proliferation in the presence of epidermal growth factor (EGF), and NTS-induced proliferation is dependent on NTSR1 and the EGFR/MEK pathway. We conclude that NTS serves as a co-mitogen in FL-HCC, and provides a source of cAMP to facilitate ongoing activation of DNAJ-PKAc.
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Affiliation(s)
| | | | - Kevin M. Riggle
- Department of Surgery, University of Washington, Seattle, WA, USA
| | - Rigney Turnham
- Department of Pharmacology, University of Washington, Seattle, WA, USA
| | - Kevin Sullivan
- Department of Surgery, University of Washington, Seattle, WA, USA
| | - Renay Bauer
- Department of Surgery, University of Washington, Seattle, WA, USA
| | - John D. Scott
- Department of Pharmacology, University of Washington, Seattle, WA, USA
| | - Raymond S. Yeung
- Department of Surgery, University of Washington, Seattle, WA, USA
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38
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Walker C, Wang Y, Olivieri C, Karamafrooz A, Casby J, Bathon K, Calebiro D, Gao J, Bernlohr DA, Taylor SS, Veglia G. Cushing's syndrome driver mutation disrupts protein kinase A allosteric network, altering both regulation and substrate specificity. SCIENCE ADVANCES 2019; 5:eaaw9298. [PMID: 31489371 PMCID: PMC6713507 DOI: 10.1126/sciadv.aaw9298] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/06/2019] [Accepted: 07/16/2019] [Indexed: 05/05/2023]
Abstract
Genetic alterations in the PRKACA gene coding for the catalytic α subunit of the cAMP-dependent protein kinase A (PKA-C) are linked to cortisol-secreting adrenocortical adenomas, resulting in Cushing's syndrome. Among those, a single mutation (L205R) has been found in up to 67% of patients. Because the x-ray structures of the wild-type and mutant kinases are essentially identical, the mechanism explaining aberrant function of this mutant remains under active debate. Using NMR spectroscopy, thermodynamics, kinetic assays, and molecular dynamics simulations, we found that this single mutation causes global changes in the enzyme, disrupting the intramolecular allosteric network and eliciting losses in nucleotide/pseudo-substrate binding cooperativity. Remarkably, by rewiring its internal allosteric network, PKA-CL205R is able to bind and phosphorylate non-canonical substrates, explaining its changes in substrate specificity. Both the lack of regulation and change in substrate specificity reveal the complex role of this mutated kinase in the formation of cortisol-secreting adrenocortical adenomas.
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Affiliation(s)
- Caitlin Walker
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Yingjie Wang
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
- Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
| | - Cristina Olivieri
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Adak Karamafrooz
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Jordan Casby
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Kerstin Bathon
- Institute for Pharmacology and Toxicology, University of Würzburg, 97078 Würzburg, Germany
| | - Davide Calebiro
- Institute of Metabolism and Systems Research, University of Birmingham, Birmingham B15 2TT, UK
- Centre of Membrane Proteins and Receptors, University of Birmingham, Birmingham B15 2TT, UK
| | - Jiali Gao
- Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
- Shenzhen Bay Laboratory and Laboratory of Computational Chemistry and Drug Design, Peking University Graduate School, Shenzhen 518055, China
| | - David A. Bernlohr
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Susan S. Taylor
- Departments of Chemistry and Biochemistry and Pharmacology, University of California San Diego, La Jolla, CA 92093, USA
| | - Gianluigi Veglia
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
- Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
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39
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Ranganathan S, Cheung J, Cassidy M, Ginter C, Pata JD, McDonough KA. Novel structural features drive DNA binding properties of Cmr, a CRP family protein in TB complex mycobacteria. Nucleic Acids Res 2019; 46:403-420. [PMID: 29165665 PMCID: PMC5758884 DOI: 10.1093/nar/gkx1148] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2017] [Accepted: 11/13/2017] [Indexed: 11/16/2022] Open
Abstract
Mycobacterium tuberculosis (Mtb) encodes two CRP/FNR family transcription factors (TF) that contribute to virulence, Cmr (Rv1675c) and CRPMt (Rv3676). Prior studies identified distinct chromosomal binding profiles for each TF despite their recognizing overlapping DNA motifs. The present study shows that Cmr binding specificity is determined by discriminator nucleotides at motif positions 4 and 13. X-ray crystallography and targeted mutational analyses identified an arginine-rich loop that expands Cmr’s DNA interactions beyond the classical helix-turn-helix contacts common to all CRP/FNR family members and facilitates binding to imperfect DNA sequences. Cmr binding to DNA results in a pronounced asymmetric bending of the DNA and its high level of cooperativity is consistent with DNA-facilitated dimerization. A unique N-terminal extension inserts between the DNA binding and dimerization domains, partially occluding the site where the canonical cAMP binding pocket is found. However, an unstructured region of this N-terminus may help modulate Cmr activity in response to cellular signals. Cmr’s multiple levels of DNA interaction likely enhance its ability to integrate diverse gene regulatory signals, while its novel structural features establish Cmr as an atypical CRP/FNR family member.
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Affiliation(s)
- Sridevi Ranganathan
- Department of Biomedical Sciences, School of Public Health, University at Albany, SUNY, Albany, NY 12201, USA
| | - Jonah Cheung
- New York Structural Biology Center, New York, NY 10027, USA
| | | | | | - Janice D Pata
- Department of Biomedical Sciences, School of Public Health, University at Albany, SUNY, Albany, NY 12201, USA.,Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, PO Box 22002, Albany, NY 12201-2002, USA
| | - Kathleen A McDonough
- Department of Biomedical Sciences, School of Public Health, University at Albany, SUNY, Albany, NY 12201, USA.,Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, PO Box 22002, Albany, NY 12201-2002, USA
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40
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Rinaldi L, Delle Donne R, Catalanotti B, Torres-Quesada O, Enzler F, Moraca F, Nisticò R, Chiuso F, Piccinin S, Bachmann V, Lindner HH, Garbi C, Scorziello A, Russo NA, Synofzik M, Stelzl U, Annunziato L, Stefan E, Feliciello A. Feedback inhibition of cAMP effector signaling by a chaperone-assisted ubiquitin system. Nat Commun 2019; 10:2572. [PMID: 31189917 PMCID: PMC6561907 DOI: 10.1038/s41467-019-10037-y] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2018] [Accepted: 04/12/2019] [Indexed: 02/07/2023] Open
Abstract
Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone-bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities.
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Affiliation(s)
- Laura Rinaldi
- Department of Molecular Medicine and Medical Biotechnologies, University Federico II, 80131, Naples, Italy
| | - Rossella Delle Donne
- Department of Molecular Medicine and Medical Biotechnologies, University Federico II, 80131, Naples, Italy
| | - Bruno Catalanotti
- Department of Pharmacy, University Federico II, 80131, Naples, Italy
| | - Omar Torres-Quesada
- Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, A-6020, Innsbruck, Austria
| | - Florian Enzler
- Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, A-6020, Innsbruck, Austria
| | - Federica Moraca
- Department of Chemical Sciences, University Federico II, 80131, Naples, Italy
| | - Robert Nisticò
- European Brain Research Institute, Rita Levi-Montalcini Foundation and Department of Biology, University Tor Vergata, 00143, Rome, Italy
| | - Francesco Chiuso
- Department of Molecular Medicine and Medical Biotechnologies, University Federico II, 80131, Naples, Italy
| | - Sonia Piccinin
- European Brain Research Institute, Rita Levi-Montalcini Foundation and Department of Biology, University Tor Vergata, 00143, Rome, Italy
| | - Verena Bachmann
- Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, A-6020, Innsbruck, Austria
| | - Herbert H Lindner
- Division of Clinical Biochemistry, Biocenter Medical University of Innsbruck, Innrain 80-82, A-6020, Innsbruck, Austria
| | - Corrado Garbi
- Department of Molecular Medicine and Medical Biotechnologies, University Federico II, 80131, Naples, Italy
| | - Antonella Scorziello
- Department of Neuroscience, Reproductive and Odontostomatological Sciences, University Federico II, 80131, Naples, Italy
| | | | - Matthis Synofzik
- Department of Neurodegeneration, Hertie Institute for Clinical Brain Research (HIH), University of Tübingen and German Center for Neurodegenerative Diseases (DZNE), 72076, Tübingen, Germany
| | - Ulrich Stelzl
- Institute of Pharmaceutical Sciences, University of Graz and BioTechMed-Graz, 8010, Graz, Austria
| | | | - Eduard Stefan
- Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, A-6020, Innsbruck, Austria
| | - Antonio Feliciello
- Department of Molecular Medicine and Medical Biotechnologies, University Federico II, 80131, Naples, Italy.
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41
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Cao B, Lu TW, Martinez Fiesco JA, Tomasini M, Fan L, Simon SM, Taylor SS, Zhang P. Structures of the PKA RIα Holoenzyme with the FLHCC Driver J-PKAcα or Wild-Type PKAcα. Structure 2019; 27:816-828.e4. [PMID: 30905674 PMCID: PMC6506387 DOI: 10.1016/j.str.2019.03.001] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2018] [Revised: 01/11/2019] [Accepted: 02/28/2019] [Indexed: 01/07/2023]
Abstract
Fibrolamellar hepatocellular carcinoma (FLHCC) is driven by J-PKAcα, a kinase fusion chimera of the J domain of DnaJB1 with PKAcα, the catalytic subunit of protein kinase A (PKA). Here we report the crystal structures of the chimeric fusion RIα2:J-PKAcα2 holoenzyme formed by J-PKAcα and the PKA regulatory (R) subunit RIα, and the wild-type (WT) RIα2:PKAcα2 holoenzyme. The chimeric and WT RIα holoenzymes have quaternary structures different from the previously solved WT RIβ and RIIβ holoenzymes. The WT RIα holoenzyme showed the same configuration as the chimeric RIα2:J-PKAcα2 holoenzyme and a distinct second conformation. The J domains are positioned away from the symmetrical interface between the two RIα:J-PKAcα heterodimers in the chimeric fusion holoenzyme and are highly dynamic. The structural and dynamic features of these holoenzymes enhance our understanding of the fusion chimera protein J-PKAcα that drives FLHCC as well as the isoform specificity of PKA.
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Affiliation(s)
- Baohua Cao
- Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA
| | - Tsan-Wen Lu
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA
| | - Juliana A Martinez Fiesco
- Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA
| | - Michael Tomasini
- Laboratory of Cellular Biophysics, The Rockefeller University, New York, NY, USA
| | - Lixin Fan
- Small-Angle X-ray Scattering Core Facility, Center for Cancer Research of the National Cancer Institute, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA
| | - Sanford M Simon
- Laboratory of Cellular Biophysics, The Rockefeller University, New York, NY, USA
| | - Susan S Taylor
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA; Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA
| | - Ping Zhang
- Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA.
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42
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Turnham RE, Smith FD, Kenerson HL, Omar MH, Golkowski M, Garcia I, Bauer R, Lau HT, Sullivan KM, Langeberg LK, Ong SE, Riehle KJ, Yeung RS, Scott JD. An acquired scaffolding function of the DNAJ-PKAc fusion contributes to oncogenic signaling in fibrolamellar carcinoma. eLife 2019; 8:44187. [PMID: 31063128 PMCID: PMC6533061 DOI: 10.7554/elife.44187] [Citation(s) in RCA: 48] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2018] [Accepted: 05/05/2019] [Indexed: 12/22/2022] Open
Abstract
Fibrolamellar carcinoma (FLC) is a rare liver cancer. FLCs uniquely produce DNAJ-PKAc, a chimeric enzyme consisting of a chaperonin-binding domain fused to the Cα subunit of protein kinase A. Biochemical analyses of clinical samples reveal that a unique property of this fusion enzyme is the ability to recruit heat shock protein 70 (Hsp70). This cellular chaperonin is frequently up-regulated in cancers. Gene-editing of mouse hepatocytes generated disease-relevant AML12DNAJ-PKAc cell lines. Further analyses indicate that the proto-oncogene A-kinase anchoring protein-Lbc is up-regulated in FLC and functions to cluster DNAJ-PKAc/Hsp70 sub-complexes with a RAF-MEK-ERK kinase module. Drug screening reveals Hsp70 and MEK inhibitor combinations that selectively block proliferation of AML12DNAJ-PKAc cells. Phosphoproteomic profiling demonstrates that DNAJ-PKAc biases the signaling landscape toward ERK activation and engages downstream kinase cascades. Thus, the oncogenic action of DNAJ-PKAc involves an acquired scaffolding function that permits recruitment of Hsp70 and mobilization of local ERK signaling. Fibrolamellar carcinoma (or FLC for short) is a rare type of liver cancer that affects teenagers and young adults. FLC tumors are often resistant to standard radiotherapy or chemotherapy treatments. The only way to treat FLC is to remove tumors by surgery. However, often the tumors come back after initial treatment and spread to other locations. Therefore, there is a genuine need to improve the treatment options available to FLC patients. The tumor cells of FLC patients contain a genetic defect that fuses together two genes, which produce proteins called DNAJ and PKAc. Normally, DNAJ helps other proteins in the cell to fold into their correct shapes, while PKAc is an enzyme that can control how cells communicate. However, it is not clear what the abnormal DNAJ-PKAc fusion protein does, or how it causes FLC. Turnham, Smith et al. have now used gene editing to make mouse liver cells that mimic the human FLC mutation. Biochemical experiments on these cells showed that the DNAJ-PKAc protein brings together unique combinations of enzymes that drive uncontrolled cell growth. Analyzing cells taken from tumors in FLC patients confirmed that these enzymes are also activated in the human disease. Turnham, Smith et al. also found that combinations of drugs that simultaneously target the DNAJ-PKAc protein and the recruited enzymes slowed down the growth of FLC cells. More experiments are now needed to test these drug combinations on human FLC cells or in mice.
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Affiliation(s)
- Rigney E Turnham
- Department of Pharmacology, University of Washington Medical Center, Seattle, United States
| | - F Donelson Smith
- Department of Pharmacology, University of Washington Medical Center, Seattle, United States
| | - Heidi L Kenerson
- Department of Surgery, University of Washington Medical Center, Seattle, United States
| | - Mitchell H Omar
- Department of Pharmacology, University of Washington Medical Center, Seattle, United States
| | - Martin Golkowski
- Department of Pharmacology, University of Washington Medical Center, Seattle, United States
| | - Irvin Garcia
- Department of Pharmacology, University of Washington Medical Center, Seattle, United States
| | - Renay Bauer
- Department of Surgery, University of Washington Medical Center, Seattle, United States
| | - Ho-Tak Lau
- Department of Pharmacology, University of Washington Medical Center, Seattle, United States
| | - Kevin M Sullivan
- Department of Surgery, University of Washington Medical Center, Seattle, United States
| | - Lorene K Langeberg
- Department of Pharmacology, University of Washington Medical Center, Seattle, United States
| | - Shao-En Ong
- Department of Pharmacology, University of Washington Medical Center, Seattle, United States
| | - Kimberly J Riehle
- Department of Surgery, University of Washington Medical Center, Seattle, United States
| | - Raymond S Yeung
- Department of Surgery, University of Washington Medical Center, Seattle, United States
| | - John D Scott
- Department of Pharmacology, University of Washington Medical Center, Seattle, United States
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43
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Szimler T, Gráczer É, Györffy D, Végh B, Szilágyi A, Hajdú I, Závodszky P, Vas M. New type of interaction between the SARAH domain of the tumour suppressor RASSF1A and its mitotic kinase Aurora A. Sci Rep 2019; 9:5550. [PMID: 30944388 PMCID: PMC6447619 DOI: 10.1038/s41598-019-41972-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2019] [Accepted: 03/08/2019] [Indexed: 12/11/2022] Open
Abstract
The tumour suppressor protein RASSF1A is phosphorylated by Aurora A kinase, thereby impairing its tumour suppressor function. Consequently, inhibiting the interaction between Aurora A and RASSF1A may be used for anti-tumour therapy. We used recombinant variants of RASSF1A to map the sites of interaction with Aurora A. The phosphorylation kinetics of three truncated RASSF1A variants has been analysed. Compared to the RASSF1A form lacking the 120 residue long N-terminal part, the Km value of the phosphorylation is increased from 10 to 45 μM upon additional deletion of the C-terminal SARAH domain. On the other hand, deletion of the flexible loop (Δ177-197) that precedes the phosphorylation site/s (T202/S203) results in a reduction of the kcat value from about 40 to 7 min-1. Direct physical interaction between the isolated SARAH domain and Aurora A was revealed by SPR. These data demonstrate that the SARAH domain of RASSF1A is involved in the binding to Aurora A kinase. Structural modelling confirms that a novel complex is feasible between the SARAH domain and the kinase domain of Aurora A. In addition, a regulatory role of the loop in the catalytic phosphorylation reaction has been demonstrated both experimentally and by structural modelling.
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Affiliation(s)
- T Szimler
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117, Budapest, Hungary
| | - É Gráczer
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117, Budapest, Hungary
| | - D Györffy
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117, Budapest, Hungary
| | - B Végh
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117, Budapest, Hungary
- ELTE NAP Neuroimmunology Research Group, Department of Biochemistry, Institute of Biology, Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117, Budapest, Hungary
| | - A Szilágyi
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117, Budapest, Hungary
| | - I Hajdú
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117, Budapest, Hungary.
| | - P Závodszky
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117, Budapest, Hungary
| | - M Vas
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117, Budapest, Hungary.
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44
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Averill AM, Rehman HT, Charles JW, Dinh TA, Danyal K, Verschraegen CF, Stein GS, Dostmann WR, Ramsey JE. Inhibition of the chimeric DnaJ-PKAc enzyme by endogenous inhibitor proteins. J Cell Biochem 2019; 120:13783-13791. [PMID: 30938854 DOI: 10.1002/jcb.28651] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2018] [Accepted: 01/16/2019] [Indexed: 12/22/2022]
Abstract
The chimeric DnaJ-PKAc enzymeresulting from an approximately 400-kb deletion of chromosome 19 is a primary contributor to the oncogenic transformation that occurs in fibrolamellar hepatocellular carcinoma, also called fibrolamellar carcinoma (FLC). This oncogenic deletion juxtaposes exon 1 of the DNAJB1 heat shock protein gene with exon 2 of the PRKACA gene encoding the protein kinase A catalytic subunit, resulting in DnaJ-PKAc fusion under the transcriptional control of the DNAJB1 promoter. The expression of DnaJ-PKAc is approximately 10 times that of wild-type (wt) PKAc catalytic subunits, causing elevated and dysregulated kinase activity that contributes to oncogenic transformation. In normal cells, PKAc activity is regulated by a group of endogenous proteins, termed protein kinase inhibitors (PKI) that competitively inhibit PKAc and assist with the nuclear export of the enzyme. Currently, it is scarcely known whether interactions with PKI are perturbed in DnaJ-PKAc. In this report, we survey existing data sets to assess the expression levels of the various PKI isoforms that exist in humans to identify those that are candidates to encounter DnaJ-PKAc in both normal liver and FLC tumors. We then compare inhibition profiles of wtPKAc and DnaJ-PKAc against PKI and demonstrate that extensive structural homology in the active site clefts of the two enzymes confers similar kinase activities and inhibition by full-length PKI and PKI-derived peptides.
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Affiliation(s)
- April M Averill
- Department of Microbiology and Molecular Genetics, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Hibba Tul Rehman
- Division of Hematology and Oncology, Department of Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont.,University of Vermont Cancer Center, Burlington, Vermont
| | - Joseph W Charles
- Department of Pharmacology, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Timothy A Dinh
- Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York.,Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
| | - Karamatullah Danyal
- Department of Pathology, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Claire F Verschraegen
- Division of Medical Oncology, The Ohio State Comprehensive Cancer Center, Columbus, Ohio
| | - Gary S Stein
- University of Vermont Cancer Center, Burlington, Vermont.,Department of Biochemistry,, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Wolfgang R Dostmann
- Department of Pharmacology, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Jon E Ramsey
- University of Vermont Cancer Center, Burlington, Vermont.,Department of Biochemistry,, Larner College of Medicine, University of Vermont, Burlington, Vermont
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45
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Luxenburger A, Schmidt D, Ianes C, Pichlo C, Krüger M, von Drathen T, Brunstein E, Gainsford GJ, Baumann U, Knippschild U, Peifer C. Design, Synthesis and Biological Evaluation of Isoxazole-Based CK1 Inhibitors Modified with Chiral Pyrrolidine Scaffolds. Molecules 2019; 24:E873. [PMID: 30832206 PMCID: PMC6429214 DOI: 10.3390/molecules24050873] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Revised: 02/23/2019] [Accepted: 02/27/2019] [Indexed: 01/27/2023] Open
Abstract
In this study, we report on the modification of a 3,4-diaryl-isoxazole-based CK1 inhibitor with chiral pyrrolidine scaffolds to develop potent and selective CK1 inhibitors. The pharmacophore of the lead structure was extended towards the ribose pocket of the adenosine triphosphate (ATP) binding site driven by structure-based drug design. For an upscale compatible multigram synthesis of the functionalized pyrrolidine scaffolds, we used a chiral pool synthetic route starting from methionine. Biological evaluation of key compounds in kinase and cellular assays revealed significant effects of the scaffolds towards activity and selectivity, however, the absolute configuration of the chiral moieties only exhibited a limited effect on inhibitory activity. X-ray crystallographic analysis of ligand-CK1δ complexes confirmed the expected binding mode of the 3,4-diaryl-isoxazole inhibitors. Surprisingly, the original compounds underwent spontaneous Pictet-Spengler cyclization with traces of formaldehyde during the co-crystallization process to form highly potent new ligands. Our data suggests chiral "ribose-like" pyrrolidine scaffolds have interesting potential for modifications of pharmacologically active compounds.
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Affiliation(s)
- Andreas Luxenburger
- Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Rd, Lower Hutt 5040, New Zealand.
| | - Dorian Schmidt
- Institute of Pharmacy, Christian-Albrechts-University of Kiel, Gutenbergstraße 76, D-24116 Kiel, Germany.
| | - Chiara Ianes
- Department of General and Visceral Surgery, Ulm University Hospital, Albert-Einstein-Allee 23, D-89081 Ulm, Germany.
| | - Christian Pichlo
- Institute of Biochemistry, University of Cologne, Zuelpicher Str. 47, D-50674 Cologne, Germany.
| | - Marc Krüger
- Department of General and Visceral Surgery, Ulm University Hospital, Albert-Einstein-Allee 23, D-89081 Ulm, Germany.
| | - Thorsten von Drathen
- Institute of Pharmacy, Christian-Albrechts-University of Kiel, Gutenbergstraße 76, D-24116 Kiel, Germany.
| | - Elena Brunstein
- Institute of Biochemistry, University of Cologne, Zuelpicher Str. 47, D-50674 Cologne, Germany.
| | - Graeme J Gainsford
- Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Rd, Lower Hutt 5040, New Zealand.
| | - Ulrich Baumann
- Institute of Biochemistry, University of Cologne, Zuelpicher Str. 47, D-50674 Cologne, Germany.
| | - Uwe Knippschild
- Department of General and Visceral Surgery, Ulm University Hospital, Albert-Einstein-Allee 23, D-89081 Ulm, Germany.
| | - Christian Peifer
- Institute of Pharmacy, Christian-Albrechts-University of Kiel, Gutenbergstraße 76, D-24116 Kiel, Germany.
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46
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Dinh TA, Jewell ML, Kanke M, Francisco A, Sritharan R, Turnham RE, Lee S, Kastenhuber ER, Wauthier E, Guy CD, Yeung RS, Lowe SW, Reid LM, Scott JD, Diehl AM, Sethupathy P. MicroRNA-375 Suppresses the Growth and Invasion of Fibrolamellar Carcinoma. Cell Mol Gastroenterol Hepatol 2019; 7:803-817. [PMID: 30763770 PMCID: PMC6468197 DOI: 10.1016/j.jcmgh.2019.01.008] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/18/2018] [Revised: 01/31/2019] [Accepted: 01/31/2019] [Indexed: 01/08/2023]
Abstract
BACKGROUND & AIMS Fibrolamellar carcinoma (FLC) is a rare liver cancer that primarily affects adolescents and young adults. It is characterized by a heterozygous approximately 400-kb deletion on chromosome 19 that results in a unique fusion between DnaJ heat shock protein family member B1 (DNAJB1) and the alpha catalytic subunit of protein kinase A (PRKACA). The role of microRNAs (miRNAs) in FLC remains unclear. We identified dysregulated miRNAs in FLC and investigated whether dysregulation of 1 key miRNA contributes to FLC pathogenesis. METHODS We analyzed small RNA sequencing (smRNA-seq) data from The Cancer Genome Atlas to identify dysregulated miRNAs in primary FLC tumors and validated the findings in 3 independent FLC cohorts. smRNA-seq also was performed on a FLC patient-derived xenograft model as well as purified cell populations of the liver to determine whether key miRNA changes were tumor cell-intrinsic. We then used clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) technology and transposon-mediated gene transfer in mice to determine if the presence of DNAJB1-PRKACA is sufficient to suppress miR-375 expression. Finally, we established a new FLC cell line and performed colony formation and scratch wound assays to determine the functional consequences of miR-375 overexpression. RESULTS We identified miR-375 as the most dysregulated miRNA in primary FLC tumors (27-fold down-regulation; P = .009). miR-375 expression also was decreased significantly in a FLC patient-derived xenograft model compared to 4 different cell populations of the liver. Introduction of DNAJB1-PRKACA by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 engineering and transposon-mediated somatic gene transfer in mice was sufficient to induce significant loss of miR-375 expression (P < .05). Overexpression of miR-375 in FLC cells inhibited Hippo signaling pathway proteins, including yes-associated protein 1 and connective tissue growth factor, and suppressed cell proliferation and migration (P < .05). CONCLUSIONS We identified miR-375 as the most down-regulated miRNA in FLC tumors and showed that overexpression of miR-375 mitigated tumor cell growth and invasive potential. These findings open a potentially new molecular therapeutic approach. Further studies are necessary to determine how DNAJB1-PRKACA suppresses miR-375 expression and whether miR-375 has additional important targets in this tumor. Transcript profiling: GEO accession numbers: GSE114974 and GSE125602.
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Affiliation(s)
- Timothy A Dinh
- Curriculum in Genetics and Molecular Biology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York
| | - Mark L Jewell
- Department of Medicine, School of Medicine, Duke University, Durham, North Carolina
| | - Matt Kanke
- Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York
| | - Adam Francisco
- Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York
| | - Ramja Sritharan
- Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York
| | - Rigney E Turnham
- Department of Pharmacology, School of Medicine, University of Washington, Seattle, Washington
| | - Seona Lee
- Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York
| | - Edward R Kastenhuber
- Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York
| | - Eliane Wauthier
- Department of Cell Biology and Physiology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
| | - Cynthia D Guy
- Department of Pathology, School of Medicine, Duke University, Durham, North Carolina
| | - Raymond S Yeung
- Department of Surgery, University of Washington, Seattle, Washington
| | - Scott W Lowe
- Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York
| | - Lola M Reid
- Department of Cell Biology and Physiology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
| | - John D Scott
- Department of Pharmacology, School of Medicine, University of Washington, Seattle, Washington
| | - Anna M Diehl
- Department of Medicine, School of Medicine, Duke University, Durham, North Carolina.
| | - Praveen Sethupathy
- Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York.
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47
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Kastenhuber ER, Craig J, Ramsey J, Sullivan KM, Sage J, de Oliveira S, Riehle KJ, Scott JD, Gordan JD, Bardeesy N, Abou-Alfa GK. Road map for fibrolamellar carcinoma: progress and goals of a diversified approach. J Hepatocell Carcinoma 2019; 6:41-48. [PMID: 30951568 PMCID: PMC6362920 DOI: 10.2147/jhc.s194764] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Fibrolamellar carcinoma is a rare liver cancer, which primarily afflicts adolescents and young adults worldwide and is frequently lethal. Given the rarity of this disease, patient recruitment for clinical trials remains a challenge. In November 2017, the Second Fibrolamellar Cancer Foundation Scientific Summit (Stamford, CT, USA) provided an opportunity for investigators to discuss recent advances in the characterization of the disease and its surrounding liver and immune context. The Fibrolamellar Cancer Foundation has thus set out a road map to identify and test therapeutic targets in the most efficient possible manner.
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Affiliation(s)
- Edward R Kastenhuber
- Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA, .,Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - John Craig
- Fibrolamellar Cancer Foundation, Greenwich, CT, USA
| | - Jon Ramsey
- Department of Biochemistry, University of Vermont Cancer Center, Burlington, VT, USA
| | - Kevin M Sullivan
- Northwest Liver Research Program, University of Washington, Seattle, WA, USA
| | - Julien Sage
- Department of Pediatrics, Stanford University, Stanford, CA, USA.,Department of Genetics, Stanford University, Stanford, CA, USA
| | - Sofia de Oliveira
- Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, USA
| | - Kimberly J Riehle
- Northwest Liver Research Program, University of Washington, Seattle, WA, USA
| | - John D Scott
- Northwest Liver Research Program, University of Washington, Seattle, WA, USA
| | - John D Gordan
- Department of Medicine, University of California, San Francisco, San Francisco, CA, USA
| | - Nabeel Bardeesy
- Center for Cancer Research, Massachusetts General Hospital, Boston, MA, USA.,Department of Medicine, Harvard Medical School, Boston, MA, USA
| | - Ghassan K Abou-Alfa
- Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA, .,Department of Medicine, Weill Cornell School of Medicine, New York, NY, USA,
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48
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Weigand I. Pathogenesis of benign unilateral adrenocortical tumors: focus on cAMP/PKA pathway. MINERVA ENDOCRINOL 2018; 44:25-32. [PMID: 29963826 DOI: 10.23736/s0391-1977.18.02874-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Somatic mutations affecting genes in the cAMP/PKA (protein kinase A) signaling pathway have been described as causative for the pathogenesis of benign unilateral adrenocortical adenomas associated with cortisol over secretion. These include predominantly somatic mutations in the PRKACA gene which encodes the catalytic subunit α of PKA. In addition, mutations in the GNAS gene, coding for the stimulatory G protein α, have been observed in approximately 10% of cortisol producing adenomas (CPA). The mutations render PKA signaling constitutively active and are therefore involved in cortisol over secretion of these tumors. Despite the prominent role of the cAMP/PKA pathway in the pathogenesis of unilateral CPA, also mutations in the CTNNB1 gene, encoding β-catenin, were identified in CPA. However, mutations in β-catenin are not limited to CPA and are not associated with cortisol secretion since they were predominantly found in endocrine-inactive adenomas (EIA) and might hence contribute to tumorigenesis in adrenocortical tissues. In this review, recent findings in the pathogenesis of benign adrenocortical tumors with a particular focus on the cAMP/PKA signaling pathway are summarized.
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Affiliation(s)
- Isabel Weigand
- Unit of Endocrinology and Diabetes, Department of Internal Medicine I, University Hospital, University of Wuerzburg, Wuerzburg, Germany -
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49
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Knape MJ, Ballez M, Burghardt NC, Zimmermann B, Bertinetti D, Kornev AP, Herberg FW. Divalent metal ions control activity and inhibition of protein kinases. Metallomics 2018; 9:1576-1584. [PMID: 29043344 DOI: 10.1039/c7mt00204a] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Protein kinases are key enzymes in the regulation of eukaryotic signal transduction. As metalloenzymes they employ divalent cations for catalysis and regulation. We used the catalytic (C) subunit of cAMP-dependent protein kinase (PKA) as a model protein to investigate the role of a variety of physiologically or pathophysiologically relevant divalent metal ions in distinct steps within the catalytic cycle. It is established that divalent metal ions play a crucial role in co-binding of nucleotides and also assist in catalysis. Our studies reveal that besides the physiologically highly relevant magnesium, metals like zinc and manganese can assist in phosphoryl transfer, however, only a few support efficient substrate turnover (turnover catalysis). Those trace metals allow for substrate binding and phosphotransfer but hamper product release. We further established the unique role of magnesium as the common biologically relevant divalent metal ideally enabling (co-) substrate binding and orientation. Magnesium allows stable substrate binding and, on the other hand accelerates product release, thus being extremely efficient in turnover catalysis. We extended our studies to non-catalytic functions of protein kinases looking at pseudokinases, a subfamily of protein kinases inherently lacking critical residues for catalysis. Recently, pseudokinases have been linked to human diseases. Some pseudokinases are still capable of binding metal ions, yet have lost the ability to transfer the phosphoryl group from ATP to a given substrate. Here metal ions stabilize an active like, though catalytically unproductive conformation and are therefore crucial to maintain non-catalytic function. Finally, we demonstrate for the canonical kinase PKA that the trace metal manganese alone can stabilize protein kinases in an active like conformation allowing them to bind substrates even in the absence of nucleotides.
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Affiliation(s)
- Matthias J Knape
- Department of Biochemistry, University of Kassel, 34132 Kassel, Germany.
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50
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Sullivan KM, Kenerson HL, Pillarisetty VG, Riehle KJ, Yeung RS. Precision oncology in liver cancer. ANNALS OF TRANSLATIONAL MEDICINE 2018; 6:285. [PMID: 30105235 DOI: 10.21037/atm.2018.06.14] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
With the widespread adoption of molecular profiling in clinical oncology practice, many physicians are faced with making therapeutic decisions based upon isolated genomic alterations. For example, epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) are effective in EGFR-mutant non-small cell lung cancers (NSCLC) while anti-EGFR monoclonal antibodies are ineffective in Ras-mutant colorectal cancers. The matching of mutations with drugs aimed at their respective gene products represents the current state of "precision" oncology. Despite the great expectations of this approach, only a fraction of cancers responds to 'targeted' interventions, and many early responders will ultimately develop resistance to these agents. The underwhelming success of mutation-driven therapies across all cancer types is not due to an inability to detect genetic changes in tumors; rather a deficit in functional insight into the genomic alterations that give rise to each cancer. The Achilles heel of precision oncology thus remains the lack of a robust functional understanding of an individual cancer genome that then allows prediction of the best therapy and resultant outcome for that patient. Current practice focuses on one 'actionable' mutation at a time, while solid cancers typically possess many mutations that involve different cellular sub-populations within a tumor. No method or platform currently exists to guide the interpretation of these complex data, nor to accurately predict response to treatment. This problem is particularly germane to primary liver cancers (PLC), for which only a handful of targeted therapies have been introduced. Here, we will review strategies aimed at overcoming some of these challenges in precision oncology, using liver cancer as an example.
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Affiliation(s)
- Kevin M Sullivan
- Northwest Liver Research Program, Department of Surgery, University of Washington, Seattle, Washington, USA
| | - Heidi L Kenerson
- Northwest Liver Research Program, Department of Surgery, University of Washington, Seattle, Washington, USA
| | - Venu G Pillarisetty
- Northwest Liver Research Program, Department of Surgery, University of Washington, Seattle, Washington, USA
| | - Kimberly J Riehle
- Northwest Liver Research Program, Department of Surgery, University of Washington, Seattle, Washington, USA
| | - Raymond S Yeung
- Northwest Liver Research Program, Department of Surgery, University of Washington, Seattle, Washington, USA
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