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1
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Xu L, Jang H, Nussinov R. Allosteric modulation of NF1 GAP: Differential distributions of catalytically competent populations in loss-of-function and gain-of-function mutants. Protein Sci 2025; 34:e70042. [PMID: 39840811 PMCID: PMC11751910 DOI: 10.1002/pro.70042] [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: 10/01/2024] [Revised: 12/13/2024] [Accepted: 01/09/2025] [Indexed: 01/23/2025]
Abstract
Neurofibromin (NF1), a Ras GTPase-activating protein (GAP), catalyzes Ras-mediated GTP hydrolysis and thereby negatively regulates the Ras/MAPK pathway. NF1 mutations can cause neurofibromatosis type 1 manifesting tumors, and neurodevelopmental disorders. Exactly how the missense mutations in the GAP-related domain of NF1 (NF1GRD) allosterically impact NF1 GAP to promote these distinct pathologies is unclear. Especially tantalizing is the question of how same-domain, same-residue NF1GRD variants exhibit distinct clinical phenotypes. Guided by clinical data, we take up this dilemma. We sampled the conformational ensembles of NF1GRD in complex with GTP-bound K-Ras4B by performing molecular dynamics simulations. Our results show that mutations in NF1GRD retain the active conformation of K-Ras4B but with biased propensities of the catalytically competent populations of K-Ras4B-NF1GRD complex. In agreement with clinical depiction and experimental tagging, compared to the wild type, NF1GRD E1356A and E1356V mutants effectively act through loss-of-function and gain-of-function mechanisms, leading to neurofibromatosis and developmental disorders, respectively. Allosteric modulation of NF1GRD GAP activity through biasing the conformational ensembles in the different states is further demonstrated by the diminished GAP activity by NF1GRD isoform 2, further manifesting propensities of conformational ensembles as powerful predictors of protein function. Taken together, our work identifies a NF1GRD hotspot that could allosterically tune GAP function, suggests targeting Ras oncogenic mutations by restoring NF1 catalytic activity, and offers a molecular mechanism for NF1 phenotypes determined by their distinct conformational propensities.
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Affiliation(s)
- Liang Xu
- Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Cancer Innovation LaboratoryNational Cancer InstituteFrederickMarylandUSA
| | - Hyunbum Jang
- Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Cancer Innovation LaboratoryNational Cancer InstituteFrederickMarylandUSA
| | - Ruth Nussinov
- Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Cancer Innovation LaboratoryNational Cancer InstituteFrederickMarylandUSA
- Department of Human Molecular Genetics and Biochemistry, Sackler School of MedicineTel Aviv UniversityTel AvivIsrael
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2
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Brill J, Clarke B, Hong I, Huganir RL. Dissociation of SYNGAP1 Enzymatic and Structural Roles: Intrinsic Excitability and Seizure Susceptibility. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.14.633019. [PMID: 39868300 PMCID: PMC11761602 DOI: 10.1101/2025.01.14.633019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/28/2025]
Abstract
SYNGAP1 is a key Ras-GAP protein enriched at excitatory synapses, with mutations causing intellectual disability and epilepsy in humans. Recent studies have revealed that in addition to its role as a negative regulator of G-protein signaling through its GAP enzymatic activity, SYNGAP1 plays an important structural role through its interaction with post-synaptic density proteins. Here, we reveal that intrinsic excitability deficits and seizure phenotypes in heterozygous Syngap1 knockout (KO) mice are differentially dependent on Syngap1 GAP activity. Cortical excitatory neurons in heterozygous KO mice displayed reduced intrinsic excitability, including lower input resistance, and increased rheobase, a phenotype recapitulated in GAP-deficient Syngap1 mutants. However, seizure severity and susceptibility to pentylenetetrazol (PTZ)-induced seizures were significantly elevated in heterozygous KO mice but unaffected in GAP-deficient mutants, implicating the structural rather than enzymatic role of Syngap1 in seizure regulation. These findings highlight the complex interplay between SYNGAP1 structural and catalytic functions in neuronal physiology and disease.
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Affiliation(s)
- Julia Brill
- The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Blaise Clarke
- The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Ingie Hong
- The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Richard L. Huganir
- The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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3
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Mijanović L, Putar D, Mimica L, Klajn S, Filić V, Weber I. The IQGAP-related RasGAP IqgC regulates cell-substratum adhesion in Dictyostelium discoideum. Cell Mol Biol Lett 2025; 30:4. [PMID: 39789437 PMCID: PMC11720917 DOI: 10.1186/s11658-024-00678-3] [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: 05/06/2024] [Accepted: 12/11/2024] [Indexed: 01/12/2025] Open
Abstract
Proper adhesion of cells to their environment is essential for the normal functioning of single cells and multicellular organisms. To attach to the extracellular matrix (ECM), mammalian cells form integrin adhesion complexes consisting of many proteins that together link the ECM and the actin cytoskeleton. Similar to mammalian cells, the amoeboid cells of the protist Dictyostelium discoideum also use multiprotein adhesion complexes to control their attachment to the underlying surface. However, the exact composition of the multiprotein complexes and the signaling pathways involved in the regulation of adhesion in D. discoideum have not yet been elucidated. Here, we show that the IQGAP-related protein IqgC is important for normal attachment of D. discoideum cells to the substratum. Mutant iqgC-null cells have impaired adhesion, whereas overexpression of IqgC promotes directional migration. A RasGAP C-terminal (RGCt) domain of IqgC is sufficient for its localization in the ventral adhesion focal complexes, while RasGAP activity of a GAP-related domain (GRD) is additionally required for the proper function of IqgC in adhesion. We identify the small GTPase RapA as a novel direct IqgC interactor and show that IqgC participates in a RapA-regulated signaling pathway targeting the adhesion complexes that include talin A, myosin VII, and paxillin B. On the basis of our results, we propose that IqgC is a positive regulator of adhesion, responsible for the strengthening of ventral adhesion structures and for the temporal control of their subsequent degradation.
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Affiliation(s)
- Lucija Mijanović
- Department of Molecular Biology, Ruđer Bošković Institute, 10000, Zagreb, Croatia
| | - Darija Putar
- Department of Molecular Biology, Ruđer Bošković Institute, 10000, Zagreb, Croatia
| | - Lucija Mimica
- Department of Molecular Biology, Ruđer Bošković Institute, 10000, Zagreb, Croatia
| | - Sabina Klajn
- Department of Molecular Biology, Ruđer Bošković Institute, 10000, Zagreb, Croatia
| | - Vedrana Filić
- Department of Molecular Biology, Ruđer Bošković Institute, 10000, Zagreb, Croatia
| | - Igor Weber
- Department of Molecular Biology, Ruđer Bošković Institute, 10000, Zagreb, Croatia.
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4
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Cuevas-Navarro A, Pourfarjam Y, Hu F, Rodriguez DJ, Vides A, Sang B, Fan S, Goldgur Y, de Stanchina E, Lito P. Pharmacological restoration of GTP hydrolysis by mutant RAS. Nature 2025; 637:224-229. [PMID: 39476862 PMCID: PMC11666464 DOI: 10.1038/s41586-024-08283-2] [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: 04/23/2024] [Accepted: 10/24/2024] [Indexed: 12/06/2024]
Abstract
Approximately 3.4 million patients worldwide are diagnosed each year with cancers that have pathogenic mutations in one of three RAS proto-oncogenes (KRAS, NRAS and HRAS)1,2. These mutations impair the GTPase activity of RAS, leading to activation of downstream signalling and proliferation3-6. Long-standing efforts to restore the hydrolase activity of RAS mutants have been unsuccessful, extinguishing any consideration towards a viable therapeutic strategy7. Here we show that tri-complex inhibitors-that is, molecular glues with the ability to recruit cyclophilin A (CYPA) to the active state of RAS-have a dual mechanism of action: not only do they prevent activated RAS from binding to its effectors, but they also stimulate GTP hydrolysis. Drug-bound CYPA complexes modulate residues in the switch II motif of RAS to coordinate the nucleophilic attack on the γ-phosphate of GTP in a mutation-specific manner. RAS mutants that were most sensitive to stimulation of GTPase activity were more susceptible to treatment than mutants in which the hydrolysis could not be enhanced, suggesting that pharmacological stimulation of hydrolysis potentiates the therapeutic effects of tri-complex inhibitors for specific RAS mutants. This study lays the foundation for developing a class of therapeutics that inhibit cancer growth by stimulating mutant GTPase activity.
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Affiliation(s)
- Antonio Cuevas-Navarro
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Yasin Pourfarjam
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Feng Hu
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Diego J Rodriguez
- Tri-Institutional MD-PhD Program, Weill Cornell Medical College and Rockefeller University and Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Alberto Vides
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Ben Sang
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Shijie Fan
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Yehuda Goldgur
- Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Elisa de Stanchina
- Antitumor Assessment Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Piro Lito
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
- Tri-Institutional MD-PhD Program, Weill Cornell Medical College and Rockefeller University and Memorial Sloan Kettering Cancer Center, New York, NY, USA.
- Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
- Department of Medicine, Weill Cornell Medical College, New York, NY, USA.
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5
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Bandaru M, Sultana OF, Islam MA, Rainier A, Reddy PH. Rlip76 in ageing and Alzheimer's disease: Focus on oxidative stress and mitochondrial mechanisms. Ageing Res Rev 2025; 103:102600. [PMID: 39617058 DOI: 10.1016/j.arr.2024.102600] [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: 08/14/2024] [Revised: 11/12/2024] [Accepted: 11/25/2024] [Indexed: 12/13/2024]
Abstract
RLIP76 (Rlip), a stress-responsive protein, plays a multifaceted role in cellular function. This protein acts primarily as a glutathione-electrophile conjugate (GS-E) transporter, crucial for detoxifying hazardous compounds and converting them into mercapturic acids. RLIP76 also modulates cytoskeletal motility and membrane plasticity through its role in the Ral-signaling pathway, interacting with RalA and RalB, key small GTPases involved in growth and metastasis. Beyond its ATP-dependent transport functions in various tissues, RLIP76 also demonstrates GTPase Activating Protein (GAP) activity towards Rac1 and Cdc42, with a preference for Ral-GTP over Ral-GDP. Its functions span critical physiological processes including membrane dynamics, oxidative stress response, and mitochondrial dynamics. The protein's widespread expression and evolutionary conservation underscore its significance. Our lab discovered that Rlip interacts with Alzheimer's disease (AD) proteins, amyloid beta and phosphorylated and induce oxidative stress, mitochondrial dysfnction and synaptic damage in AD. Our in vitro studies revealed that overexpression of Rlip reduces mitochondrial abnormalities. Further, our in vivo studies (Rlip+/- mice) revealed that a partial reduction of Rlip in mice (Rlip+/-), leads to mitochondrial abnormalities, elevated oxidative stress, and cognitive deficits resembling late-onset AD, emphasizing the protein's crucial role in neuronal health and disease. Finally, we discuss the experimental cross-breedings of overexpression of mice Rlip TG/TG or Rlip + /- mice with Alzheimer's disease models - earlyonset 5XFAD, late-onset APPKI and Tau transgenic mice, providing new insights into RLIP76's role in AD progression and development. This review summarizes RLIP76's structure, function, and cellular pathways, highlighting its implications in AD and its potential as a therapeutic target.
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Affiliation(s)
- Madhuri Bandaru
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - Omme Fatema Sultana
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - Md Ariful Islam
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - Alvir Rainier
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - P Hemachandra Reddy
- Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; Nutritional Sciences Department, College of Human Sciences, Texas Tech University, Lubbock, TX 79409, United States; Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; Department of Neurology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA 5. Department of Public Health, Graduate School of Biomedical Sciences, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; Department of Speech, Language, and Hearing Sciences, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
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6
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Frey Y, Lungu C, Olayioye MA. Regulation and functions of the DLC family of RhoGAP proteins: Implications for development and cancer. Cell Signal 2025; 125:111505. [PMID: 39549821 DOI: 10.1016/j.cellsig.2024.111505] [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: 07/26/2024] [Revised: 10/18/2024] [Accepted: 11/05/2024] [Indexed: 11/18/2024]
Abstract
The DLC (Deleted in Liver Cancer) family of RhoGAP (Rho GTPase-activating) proteins has been extensively studied since the identification of the first family member nearly 30 years ago. Rho GTPase signaling is essential for various cellular processes, including cytoskeletal dynamics, cell migration, and proliferation. Members of the DLC family are key regulators of this signaling pathway, with well-established roles in development and carcinogenesis. Here, we provide a comprehensive review of research into DLC regulation and cellular functions over the last three decades. In particular, we summarize control mechanisms of DLC gene expression at both the transcriptional and post-transcriptional level. Additionally, recent advances in understanding the post-translational regulation of DLC proteins that allow for tuning of protein activity and localization are highlighted. This detailed overview will serve as resource for future studies aimed at further elucidating the complex regulatory mechanisms of DLC family proteins and exploring their potential as targets for therapeutic applications.
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Affiliation(s)
- Yannick Frey
- University of Stuttgart, Institute of Cell Biology and Immunology, Stuttgart, Germany; Medical University of Innsbruck, Institute of Pathophysiology, Innsbruck, Austria
| | - Cristiana Lungu
- University of Stuttgart, Institute of Cell Biology and Immunology, Stuttgart, Germany; University of Stuttgart, Stuttgart Research Center Systems Biology, Stuttgart, Germany
| | - Monilola A Olayioye
- University of Stuttgart, Institute of Cell Biology and Immunology, Stuttgart, Germany; University of Stuttgart, Stuttgart Research Center Systems Biology, Stuttgart, Germany.
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7
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Soubias O, Foley SL, Jian X, Jackson RA, Zhang Y, Rosenberg EM, Li J, Heinrich F, Johnson ME, Sodt AJ, Randazzo PA, Byrd RA. The PH domain in the ArfGAP ASAP1 drives catalytic activation through an unprecedented allosteric mechanism. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.12.20.629688. [PMID: 39763923 PMCID: PMC11702723 DOI: 10.1101/2024.12.20.629688] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/14/2025]
Abstract
ASAP1 is a multidomain Arf GTPase-activating protein (ArfGAP) that catalyzes GTP hydrolysis on the small GTPase Arf1 and is implicated in cancer progression. The PH domain of ASAP1 enhances its activity greater than 7 orders of magnitude but the underlying mechanisms remain poorly understood. Here, we combined Nuclear Magnetic Resonance (NMR), Molecular Dynamic (MD) simulations and mathematical modeling of functional data to build a comprehensive structural-mechanistic model of the complex of Arf1 and the ASAP1 PH domain on a membrane surface. Our results support a new conceptual model in which the PH domain contributes to efficient catalysis not only by membrane recruitment but by acting as a critical component of the catalytic interface, binding Arf·GTP and allosterically driving it towards the catalytic transition state. We discuss the biological implications of these results and how they may apply more broadly to poorly understood membrane-dependent regulatory mechanisms controlling catalysis of the ArfGAP superfamily as well as other peripheral membrane enzymes.
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Affiliation(s)
- Olivier Soubias
- Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA
| | - Samuel L. Foley
- Department of Biophysics, The Johns Hopkins University, Baltimore, MD, USA
| | - Xiaoying Jian
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Rebekah A. Jackson
- Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA
| | - Yue Zhang
- Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA
| | - Eric M. Rosenberg
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Jess Li
- Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA
| | - Frank Heinrich
- Department of Physics Carnegie Mellon University, Pittsburgh, PA, USA. NIST Center for Neutron Research, Gaithersburg, MD, USA
- Department of Physics Carnegie Mellon University, Pittsburgh, PA, USA. NIST Center for Neutron Research, Gaithersburg, MD, USA
| | | | - Alexander J. Sodt
- Unit of Membrane Chemical Physics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
| | - Paul A. Randazzo
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - R. Andrew Byrd
- Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA
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8
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Bergoug M, Mosrin C, Serrano A, Godin F, Doudeau M, Dundović I, Goffinont S, Normand T, Suskiewicz MJ, Vallée B, Bénédetti H. An Atypical Mechanism of SUMOylation of Neurofibromin SecPH Domain Provides New Insights into SUMOylation Site Selection. J Mol Biol 2024; 436:168768. [PMID: 39216515 DOI: 10.1016/j.jmb.2024.168768] [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: 01/30/2024] [Revised: 08/08/2024] [Accepted: 08/23/2024] [Indexed: 09/04/2024]
Abstract
Neurofibromin (Nf1) is a giant multidomain protein encoded by the tumour-suppressor gene NF1. NF1 is mutated in a common genetic disease, neurofibromatosis type I (NF1), and in various cancers. The protein has a Ras-GAP (GTPase activating protein) activity but is also connected to diverse signalling pathways through its SecPH domain, which interacts with lipids and different protein partners. We previously showed that Nf1 partially colocalized with the ProMyelocytic Leukemia (PML) protein in PML nuclear bodies, hotspots of SUMOylation, thereby suggesting the potential SUMOylation of Nf1. Here, we demonstrate that the full-length isoform 2 and a SecPH fragment of Nf1 are substrates of the SUMO pathway and identify a well-defined SUMOylation profile of SecPH with two main modified lysines. One of these sites, K1731, is highly conserved and surface-exposed. Despite the presence of an inverted SUMO consensus motif surrounding K1731, and a potential SUMO-interacting motif (SIM) within SecPH, we show that neither of these elements is necessary for K1731 SUMOylation, which is also independent of Ubc9 SUMOylation on K14. A 3D model of an interaction between SecPH and Ubc9 centred on K1731, combined with site-directed mutagenesis, identifies specific structural elements of SecPH required for K1731 SUMOylation, some of which are affected in reported NF1 pathogenic variants. This work provides a new example of SUMOylation dependent on the tertiary rather than primary protein structure surrounding the modified site, expanding our knowledge of mechanisms governing SUMOylation site selection.
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Affiliation(s)
- Mohammed Bergoug
- Centre de Biophysique Moléculaire, CNRS, UPR 4301, Affiliated to University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France
| | - Christine Mosrin
- Centre de Biophysique Moléculaire, CNRS, UPR 4301, Affiliated to University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France
| | - Amandine Serrano
- Centre de Biophysique Moléculaire, CNRS, UPR 4301, Affiliated to University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France
| | - Fabienne Godin
- Centre de Biophysique Moléculaire, CNRS, UPR 4301, Affiliated to University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France
| | - Michel Doudeau
- Centre de Biophysique Moléculaire, CNRS, UPR 4301, Affiliated to University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France
| | - Iva Dundović
- Centre de Biophysique Moléculaire, CNRS, UPR 4301, Affiliated to University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France
| | - Stephane Goffinont
- Centre de Biophysique Moléculaire, CNRS, UPR 4301, Affiliated to University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France
| | - Thierry Normand
- Centre de Biophysique Moléculaire, CNRS, UPR 4301, Affiliated to University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France
| | - Marcin J Suskiewicz
- Centre de Biophysique Moléculaire, CNRS, UPR 4301, Affiliated to University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France
| | - Béatrice Vallée
- Centre de Biophysique Moléculaire, CNRS, UPR 4301, Affiliated to University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France
| | - Hélène Bénédetti
- Centre de Biophysique Moléculaire, CNRS, UPR 4301, Affiliated to University of Orléans, Rue Charles Sadron, 45071 Orléans Cedex 2, France.
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9
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Hume E, Cossio ML, Vargas P, Cubillos MP, Maccioni A, Lay-Son G. Another face of RASA1: Report of familial germline variant in RASA1 with dysmorphic features. Am J Med Genet A 2024; 194:e63711. [PMID: 38934655 DOI: 10.1002/ajmg.a.63711] [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: 10/12/2023] [Revised: 03/07/2024] [Accepted: 05/05/2024] [Indexed: 06/28/2024]
Abstract
RASopathies encompass a diverse set of disorders affecting genes that encode proteins within the RAS-MAPK pathway. RASA1 mutations are the cause of an autosomal dominant disorder called capillary malformation-arteriovenous malformation type 1 (CM-AVM1). Unlike other RASopathies, facial dysmorphism has not been described in these patients. We phenotypically delineated a large family of individuals with multifocal fast-flow capillary malformations, severe lymphatic anomalies of perinatal onset, and dysmorphic features not previously described. Sequencing studies were performed on probands and related family members, confirming the segregation of dysmorphic features in affected members of a novel heterozygous variant in RASA1 (NM_002890.3:c.2366G>A, p.(Arg789Gln)). In this work, we broaden the phenotypic spectrum of CM-AVM type 1 and propose a new RASA1 variant as likely pathogenic.
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Affiliation(s)
- Esteban Hume
- Sección de Genética y Errores Congénitos del Metabolismo, División de Pediatría, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - María-Laura Cossio
- Department of Dermatology, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Paula Vargas
- Centro de Investigación e Innovación Materno Fetal, Complejo Asistencial Dr. Sótero del Río, Santiago, Chile
- División de Obstetricia y Ginecología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - María Paz Cubillos
- Servicio de Neonatología, Complejo Asistencial Dr. Sótero del Río, Santiago, Chile
| | - Andrea Maccioni
- Servicio de Neonatología, Complejo Asistencial Dr. Sótero del Río, Santiago, Chile
- Departamento de Neonatología, División de Pediatría, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Guillermo Lay-Son
- Sección de Genética y Errores Congénitos del Metabolismo, División de Pediatría, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
- Unidad de Genética, Complejo Asistencial Dr. Sótero del Río, Santiago, Chile
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10
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Cao P, Chen H, Zhang Y, Zhang Q, Shi M, Han H, Wang X, Jin L, Guo B, Hao R, Zhao X, Li Y, Gao C, Liu X, Wang Y, Yang A, Yang C, Si A, Li H, Song Q, He F, Zhou G. Genomic Amplification of TBC1D31 Promotes Hepatocellular Carcinoma Through Reducing the Rab22A-Mediated Endolysosomal Trafficking and Degradation of EGFR. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2405459. [PMID: 39206796 PMCID: PMC11516053 DOI: 10.1002/advs.202405459] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/19/2024] [Revised: 07/29/2024] [Indexed: 09/04/2024]
Abstract
Hepatocellular carcinomas (HCCs) are characterized by a vast spectrum of somatic copy number alterations (CNAs); however, their functional relevance is largely unknown. By performing a genome-wide survey on prognosis-associated focal CNAs in 814 HCC patients by an integrative computational framework based on transcriptomic data, genomic amplification is identified at 8q24.13 as a promising candidate. Further evidence is provided that the 8q24.13 amplification-driven overexpression of Rab GTPase activating protein TBC1D31 exacerbates HCC growth and metastasis both in vitro and in vivo through activating Epidermal growth factor receptor (EGFR) signaling. Mechanistically, TBC1D31 acts as a Rab GTPase activating protein to catalyze GTP hydrolysis for Rab22A and then reduces the Rab22A-mediated endolysosomal trafficking and degradation of EGFR. Notably, overexpression of TBC1D31 markedly increases the resistance of HCC cells to lenvatinib, whereas inhibition of the TBC1D31-EGFR axis can reverse this resistance phenotype. This study highlights that TBC1D31 at 8q24.13 is a new critical oncogene, uncovers a novel mechanism of EGFR activation in HCC, and proposes the potential strategies for treating HCC patients with TBC1D31 amplification or overexpression.
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Affiliation(s)
- Pengbo Cao
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | - Hongxia Chen
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | - Ying Zhang
- School of Life SciencesTsinghua UniversityBeijing100084China
| | - Qi Zhang
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
- University of South ChinaHengyang421001China
| | | | - Huihui Han
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | - Xiaowen Wang
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of LifeomicsBeijing102206China
| | - Liang Jin
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | - Bingqian Guo
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | | | - Xi Zhao
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | - Yuanfeng Li
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | - Chengming Gao
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | - Xinyi Liu
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | - Yahui Wang
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | - Aiqing Yang
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | - Chenning Yang
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
| | - Anfeng Si
- Jinling HospitalAffiliated Hospital of Medical SchoolNanjing UniversityNanjing210002China
| | - Hua Li
- Department of OncologyChengdu Military General HospitalChengdu610083China
| | - Qingfeng Song
- Affiliated Cancer Hospital of Guangxi Medical UniversityNanning530021China
| | - Fuchu He
- School of Life SciencesTsinghua UniversityBeijing100084China
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of LifeomicsBeijing102206China
| | - Gangqiao Zhou
- State Key Laboratory of Medical ProteomicsNational Center for Protein Sciences at BeijingBeijing Institute of Radiation MedicineBeijing100850China
- School of Life SciencesTsinghua UniversityBeijing100084China
- University of South ChinaHengyang421001China
- Guangxi Medical UniversityNanning530021China
- Hebei UniversityBaoding071000China
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11
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Katsanevaki D, Till SM, Buller-Peralta I, Nawaz MS, Louros SR, Kapgal V, Tiwari S, Walsh D, Anstey NJ, Petrović NG, Cormack A, Salazar-Sanchez V, Harris A, Farnworth-Rowson W, Sutherland A, Watson TC, Dimitrov S, Jackson AD, Arkell D, Biswal S, Dissanayake KN, Mizen LAM, Perentos N, Jones MW, Cousin MA, Booker SA, Osterweil EK, Chattarji S, Wyllie DJA, Gonzalez-Sulser A, Hardt O, Wood ER, Kind PC. Key roles of C2/GAP domains in SYNGAP1-related pathophysiology. Cell Rep 2024; 43:114733. [PMID: 39269903 DOI: 10.1016/j.celrep.2024.114733] [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: 01/18/2023] [Revised: 07/30/2024] [Accepted: 08/23/2024] [Indexed: 09/15/2024] Open
Abstract
Mutations in SYNGAP1 are a common genetic cause of intellectual disability (ID) and a risk factor for autism. SYNGAP1 encodes a synaptic GTPase-activating protein (GAP) that has both signaling and scaffolding roles. Most pathogenic variants of SYNGAP1 are predicted to result in haploinsufficiency. However, some affected individuals carry missense mutations in its calcium/lipid binding (C2) and GAP domains, suggesting that many clinical features result from loss of functions carried out by these domains. To test this hypothesis, we targeted the exons encoding the C2 and GAP domains of SYNGAP. Rats heterozygous for this deletion exhibit reduced exploration and fear extinction, altered social investigation, and spontaneous seizures-key phenotypes shared with Syngap heterozygous null rats. Together, these findings indicate that the reduction of SYNGAP C2/GAP domain function is a main feature of SYNGAP haploinsufficiency. This rat model provides an important system for the study of ID, autism, and epilepsy.
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Affiliation(s)
- Danai Katsanevaki
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Sally M Till
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK; Centre for Brain Development and Repair, Instem, Bangalore 560065, India
| | - Ingrid Buller-Peralta
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Mohammad Sarfaraz Nawaz
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Centre for Brain Development and Repair, Instem, Bangalore 560065, India
| | - Susana R Louros
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Vijayakumar Kapgal
- Centre for Brain Development and Repair, Instem, Bangalore 560065, India; The University of Transdisciplinary Health Sciences and Technology, Bangalore 560065, India
| | - Shashank Tiwari
- Centre for Brain Development and Repair, Instem, Bangalore 560065, India
| | - Darren Walsh
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Natasha J Anstey
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK; Centre for Brain Development and Repair, Instem, Bangalore 560065, India
| | - Nina G Petrović
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Alison Cormack
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Vanesa Salazar-Sanchez
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Anjanette Harris
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - William Farnworth-Rowson
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Andrew Sutherland
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Thomas C Watson
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Siyan Dimitrov
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Adam D Jackson
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK; Centre for Brain Development and Repair, Instem, Bangalore 560065, India
| | - Daisy Arkell
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | | | - Kosala N Dissanayake
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Lindsay A M Mizen
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Nikolas Perentos
- Department of Veterinary Medicine, University of Nicosia School of Veterinary Medicine, 2414 Nicosia, Cyprus
| | - Matt W Jones
- School of Physiology, Pharmacology, and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, BS8 1TD Bristol, UK
| | - Michael A Cousin
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK; Centre for Brain Development and Repair, Instem, Bangalore 560065, India
| | - Sam A Booker
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Emily K Osterweil
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Sumantra Chattarji
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK; Centre for Brain Development and Repair, Instem, Bangalore 560065, India
| | - David J A Wyllie
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK; Centre for Brain Development and Repair, Instem, Bangalore 560065, India
| | - Alfredo Gonzalez-Sulser
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK
| | - Oliver Hardt
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK; Centre for Brain Development and Repair, Instem, Bangalore 560065, India; Department of Psychology, McGill University, Montreal, QC H3A 1G1, Canada
| | - Emma R Wood
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK; Centre for Brain Development and Repair, Instem, Bangalore 560065, India
| | - Peter C Kind
- Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, EH8 9XD Edinburgh, UK; Patrick Wild Centre, University of Edinburgh, EH8 9XD Edinburgh, UK; Centre for Brain Development and Repair, Instem, Bangalore 560065, India.
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12
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Chippalkatti R, Parisi B, Kouzi F, Laurini C, Ben Fredj N, Abankwa DK. RAS isoform specific activities are disrupted by disease associated mutations during cell differentiation. Eur J Cell Biol 2024; 103:151425. [PMID: 38795504 DOI: 10.1016/j.ejcb.2024.151425] [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: 02/13/2024] [Revised: 05/02/2024] [Accepted: 05/21/2024] [Indexed: 05/28/2024] Open
Abstract
The RAS-MAPK-pathway is aberrantly regulated in cancer and developmental diseases called RASopathies. While typically the impact of Ras on the proliferation of various cancer cell lines is assessed, it is poorly established how Ras affects cellular differentiation. Here we implement the C2C12 myoblast cell line to systematically study the effect of Ras mutants and Ras-pathway drugs on differentiation. We first provide evidence that a minor pool of Pax7+ progenitors replenishes a major pool of transit amplifying cells that are ready to differentiate. Our data indicate that Ras isoforms have distinct roles in the differentiating culture, where K-Ras depletion increases and H-Ras depletion decreases terminal differentiation. This assay could therefore provide significant new insights into Ras biology and Ras-driven diseases. In line with this, we found that all oncogenic Ras mutants block terminal differentiation of transit amplifying cells. By contrast, RASopathy associated K-Ras variants were less able to block differentiation. Profiling of eight targeted Ras-pathway drugs on seven oncogenic Ras mutants revealed their allele-specific activities and distinct abilities to restore normal differentiation as compared to triggering cell death. In particular, the MEK-inhibitor trametinib could broadly restore differentiation, while the mTOR-inhibitor rapamycin broadly suppressed differentiation. We expect that this quantitative assessment of the impact of Ras-pathway mutants and drugs on cellular differentiation has great potential to complement cancer cell proliferation data.
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Affiliation(s)
- Rohan Chippalkatti
- Cancer Cell Biology and Drug Discovery group, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette 4362, Luxembourg
| | - Bianca Parisi
- Cancer Cell Biology and Drug Discovery group, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette 4362, Luxembourg
| | - Farah Kouzi
- Cancer Cell Biology and Drug Discovery group, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette 4362, Luxembourg
| | - Christina Laurini
- Cancer Cell Biology and Drug Discovery group, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette 4362, Luxembourg
| | - Nesrine Ben Fredj
- Cancer Cell Biology and Drug Discovery group, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette 4362, Luxembourg
| | - Daniel Kwaku Abankwa
- Cancer Cell Biology and Drug Discovery group, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette 4362, Luxembourg.
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13
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Warnock JL, Ball JA, Najmi SM, Henes M, Vazquez A, Koshnevis S, Wieden HJ, Conn GL, Ghalei H. Differential roles of putative arginine fingers of AAA + ATPases Rvb1 and Rvb2. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.13.593962. [PMID: 38798342 PMCID: PMC11118528 DOI: 10.1101/2024.05.13.593962] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2024]
Abstract
The evolutionarily conserved AAA+ ATPases Rvb1 and Rvb2 proteins form a heteromeric complex (Rvb1/2) required for assembly or remodeling of macromolecular complexes in essential cellular processes ranging from chromatin remodeling to ribosome biogenesis. Rvb1 and Rvb2 have a high degree of sequence and structural similarity, and both contain the classical features of ATPases of their clade, including an N-terminal AAA+ subdomain with the Walker A motif, an insertion domain that typically interacts with various binding partners, and a C-terminal AAA+ subdomain containing a Walker B motif, the Sensor I and II motifs, and an arginine finger. In this study, we find that despite the high degree of structural similarity, Rvb1 and Rvb2 have distinct active sites that impact their activities and regulation within the Rvb1/2 complex. Using a combination of biochemical and genetic approaches, we show that replacing the homologous arginine fingers of Rvb1 and Rvb2 with different amino acids not only has distinct effects on the catalytic activity of the complex, but also impacts cell growth, and the Rvb1/2 interactions with binding partners. Using molecular dynamics simulations, we find that changes near the active site of Rvb1 and Rvb2 cause long-range effects on the protein dynamics in the insertion domain, suggesting a molecular basis for how enzymatic activity within the catalytic site of ATP hydrolysis can be relayed to other domains of the Rvb1/2 complex to modulate its function. Further, we show the impact that the arginine finger variants have on snoRNP biogenesis and validate the findings from molecular dynamics simulations using a targeted genetic screen. Together, our results reveal new aspects of the regulation of the Rvb1/2 complex by identifying a relay of long-range molecular communication from the ATPase active site of the complex to the binding site of cofactors. Most importantly, our findings suggest that despite high similarity and cooperation within the same protein complex, the two proteins have evolved with unique properties critical for the regulation and function of the Rvb1/2 complex.
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Affiliation(s)
- Jennifer L. Warnock
- Emory University School of Medicine, Department of Biochemistry, Atlanta, Georgia, USA
| | - Jacob A. Ball
- Emory University School of Medicine, Department of Biochemistry, Atlanta, Georgia, USA
| | - Saman M. Najmi
- Emory University School of Medicine, Department of Biochemistry, Atlanta, Georgia, USA
| | - Mina Henes
- Emory University School of Medicine, Department of Biochemistry, Atlanta, Georgia, USA
- Graduate Program in Biochemistry, Cell & Developmental Biology (BCDB), Emory University, Atlanta, Georgia, USA
- Medical Scientist Training Program, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Amanda Vazquez
- Department of Microbiology, Faculty of Science, University of Manitoba, Manitoba, Canada
| | - Sohail Koshnevis
- Emory University School of Medicine, Department of Biochemistry, Atlanta, Georgia, USA
| | - Hans-Joachim Wieden
- Department of Microbiology, Faculty of Science, University of Manitoba, Manitoba, Canada
| | - Graeme L. Conn
- Emory University School of Medicine, Department of Biochemistry, Atlanta, Georgia, USA
| | - Homa Ghalei
- Emory University School of Medicine, Department of Biochemistry, Atlanta, Georgia, USA
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14
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Putar D, Čizmar A, Chao X, Šimić M, Šoštar M, Ćutić T, Mijanović L, Smolko A, Tu H, Cosson P, Weber I, Cai H, Filić V. IqgC is a potent regulator of macropinocytosis in the presence of NF1 and its loading to macropinosomes is dependent on RasG. Open Biol 2024; 14:230372. [PMID: 38263885 PMCID: PMC10806400 DOI: 10.1098/rsob.230372] [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: 10/09/2023] [Accepted: 11/26/2023] [Indexed: 01/25/2024] Open
Abstract
RasG is a major regulator of macropinocytosis in Dictyostelium discoideum. Its activity is under the control of an IQGAP-related protein, IqgC, which acts as a RasG-specific GAP (GTPase activating protein). IqgC colocalizes with the active Ras at the macropinosome membrane during its formation and for some time after the cup closure. However, the loss of IqgC induces only a minor enhancement of fluid uptake in axenic cells that already lack another RasGAP, NF1. Here, we show that IqgC plays an important role in the regulation of macropinocytosis in the presence of NF1 by restricting the size of macropinosomes. We further provide evidence that interaction with RasG is indispensable for the recruitment of IqgC to forming macropinocytic cups. We also demonstrate that IqgC interacts with another small GTPase from the Ras superfamily, Rab5A, but is not a GAP for Rab5A. Since mammalian Rab5 plays a key role in early endosome maturation, we hypothesized that IqgC could be involved in macropinosome maturation via its interaction with Rab5A. Although an excessive amount of Rab5A reduces the RasGAP activity of IqgC in vitro and correlates with IqgC dissociation from endosomes in vivo, the physiological significance of the Rab5A-IqgC interaction remains elusive.
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Affiliation(s)
- Darija Putar
- Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
| | - Anja Čizmar
- Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
| | - Xiaoting Chao
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101 Beijing, People's Republic of China
- College of Life Sciences, University of Chinese Academy of Sciences, 100049 Beijing, People's Republic of China
| | - Marija Šimić
- Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
| | - Marko Šoštar
- Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
| | - Tamara Ćutić
- Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
| | - Lucija Mijanović
- Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
| | - Ana Smolko
- Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
| | - Hui Tu
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101 Beijing, People's Republic of China
- College of Life Sciences, University of Chinese Academy of Sciences, 100049 Beijing, People's Republic of China
| | - Pierre Cosson
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland
| | - Igor Weber
- Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
| | - Huaqing Cai
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101 Beijing, People's Republic of China
- College of Life Sciences, University of Chinese Academy of Sciences, 100049 Beijing, People's Republic of China
| | - Vedrana Filić
- Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
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15
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Ivanova I, Shen K. Structures and Functions of the Human GATOR1 Complex. Subcell Biochem 2024; 104:269-294. [PMID: 38963491 PMCID: PMC11997690 DOI: 10.1007/978-3-031-58843-3_12] [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] [Indexed: 07/05/2024]
Abstract
Eukaryotic cells coordinate available nutrients with their growth through the mechanistic target of rapamycin complex 1 (mTORC1) pathway, in which numerous evolutionarily conserved protein complexes survey and transmit nutrient inputs toward mTORC1. mTORC1 integrates these inputs and activates downstream anabolic or catabolic programs that are in tune with cellular needs, effectively maintaining metabolic homeostasis. The GAP activity toward Rags-1 (GATOR1) protein complex is a critical negative regulator of the mTORC1 pathway and, in the absence of amino acid inputs, is activated to turn off mTORC1 signaling. GATOR1-mediated inhibition of mTORC1 signaling is tightly regulated by an ensemble of protein complexes that antagonize or promote its activity in response to the cellular nutrient environment. Structural, biochemical, and biophysical studies of the GATOR1 complex and its interactors have advanced our understanding of how it regulates cellular metabolism when amino acids are limited. Here, we review the current research with a focus on GATOR1 structure, its enzymatic mechanism, and the growing group of proteins that regulate its activity. Finally, we discuss the implication of GATOR1 dysregulation in physiology and human diseases.
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Affiliation(s)
- Ilina Ivanova
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Kuang Shen
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA.
- Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
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16
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Johansen KH, Golec DP, Okkenhaug K, Schwartzberg PL. Mind the GAP: RASA2 and RASA3 GTPase-activating proteins as gatekeepers of T cell activation and adhesion. Trends Immunol 2023; 44:917-931. [PMID: 37858490 PMCID: PMC10621891 DOI: 10.1016/j.it.2023.09.002] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2023] [Revised: 09/11/2023] [Accepted: 09/11/2023] [Indexed: 10/21/2023]
Abstract
Following stimulation, the T cell receptor (TCR) and its coreceptors integrate multiple intracellular signals to initiate T cell proliferation, migration, gene expression, and metabolism. Among these signaling molecules are the small GTPases RAS and RAP1, which induce MAPK pathways and cellular adhesion to activate downstream effector functions. Although many studies have helped to elucidate the signaling intermediates that mediate T cell activation, the molecules and pathways that keep naive T cells in check are less understood. Several recent studies provide evidence that RASA2 and RASA3, which are GAP1-family GTPase-activating proteins (GAPs) that inactivate RAS and RAP1, respectively, are crucial molecules that limit T cell activation and adhesion. In this review we describe recent data on the roles of RASA2 and RASA3 as gatekeepers of T cell activation and migration.
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Affiliation(s)
- Kristoffer H Johansen
- Cell Signaling and Immunity Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK; Section of Experimental and Translational Immunology, Department of Health Technology, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
| | - Dominic P Golec
- Cell Signaling and Immunity Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Klaus Okkenhaug
- Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
| | - Pamela L Schwartzberg
- Cell Signaling and Immunity Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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17
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Myers PJ, Lee SH, Lazzara MJ. An integrated mechanistic and data-driven computational model predicts cell responses to high- and low-affinity EGFR ligands. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.06.25.543329. [PMID: 37425852 PMCID: PMC10327094 DOI: 10.1101/2023.06.25.543329] [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 biophysical properties of ligand binding heavily influence the ability of receptors to specify cell fates. Understanding the rules by which ligand binding kinetics impact cell phenotype is challenging, however, because of the coupled information transfers that occur from receptors to downstream signaling effectors and from effectors to phenotypes. Here, we address that issue by developing an integrated mechanistic and data-driven computational modeling platform to predict cell responses to different ligands for the epidermal growth factor receptor (EGFR). Experimental data for model training and validation were generated using MCF7 human breast cancer cells treated with the high- and low-affinity ligands epidermal growth factor (EGF) and epiregulin (EREG), respectively. The integrated model captures the unintuitive, concentration-dependent abilities of EGF and EREG to drive signals and phenotypes differently, even at similar levels of receptor occupancy. For example, the model correctly predicts the dominance of EREG over EGF in driving a cell differentiation phenotype through AKT signaling at intermediate and saturating ligand concentrations and the ability of EGF and EREG to drive a broadly concentration-sensitive migration phenotype through cooperative ERK and AKT signaling. Parameter sensitivity analysis identifies EGFR endocytosis, which is differentially regulated by EGF and EREG, as one of the most important determinants of the alternative phenotypes driven by different ligands. The integrated model provides a new platform to predict how phenotypes are controlled by the earliest biophysical rate processes in signal transduction and may eventually be leveraged to understand receptor signaling system performance depends on cell context. One-sentence summary Integrated kinetic and data-driven EGFR signaling model identifies the specific signaling mechanisms that dictate cell responses to EGFR activation by different ligands.
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18
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Maggiolo AO, Mahajan S, Rees DC, Clemons WM. Intradimeric Walker A ATPases: Conserved Features of A Functionally Diverse Family. J Mol Biol 2023; 435:167965. [PMID: 37330285 DOI: 10.1016/j.jmb.2023.167965] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2022] [Accepted: 01/12/2023] [Indexed: 06/19/2023]
Abstract
Nucleoside-triphosphate hydrolases (NTPases) are a diverse, but essential group of enzymes found in all living organisms. NTPases that have a G-X-X-X-X-G-K-[S/T] consensus sequence (where X is any amino acid), known as the Walker A or P-loop motif, constitute a superfamily of P-loop NTPases. A subset of ATPases within this superfamily contains a modified Walker A motif, X-K-G-G-X-G-K-[S/T], wherein the first invariant lysine residue is essential to stimulate nucleotide hydrolysis. Although the proteins in this subset have vastly differing functions, ranging from electron transport during nitrogen fixation to targeting of integral membrane proteins to their correct membranes, they have evolved from a shared ancestor and have thus retained common structural features that affect their functions. These commonalities have only been disparately characterized in the context of their individual proteins systems, but have not been generally annotated as features that unite the members of this family. In this review, we report an analysis based on the sequences, structures, and functions of several members in this family that highlight their remarkable similarities. A principal feature of these proteins is their dependence on homodimerization. Since their functionalities are heavily influenced by changes that happen in conserved elements at the dimer interface, we refer to the members of this subclass as intradimeric Walker A ATPases.
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Affiliation(s)
- Ailiena O Maggiolo
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, United States
| | - Shivansh Mahajan
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, United States
| | - Douglas C Rees
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, United States.
| | - William M Clemons
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, United States.
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19
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Dinet C, Mignot T. Unorthodox regulation of the MglA Ras-like GTPase controlling polarity in Myxococcus xanthus. FEBS Lett 2023; 597:850-864. [PMID: 36520515 DOI: 10.1002/1873-3468.14565] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2022] [Revised: 12/09/2022] [Accepted: 12/12/2022] [Indexed: 12/23/2022]
Abstract
Motile cells have developed a large array of molecular machineries to actively change their direction of movement in response to spatial cues from their environment. In this process, small GTPases act as molecular switches and work in tandem with regulators and sensors of their guanine nucleotide status (GAP, GEF, GDI and effectors) to dynamically polarize the cell and regulate its motility. In this review, we focus on Myxococcus xanthus as a model organism to elucidate the function of an atypical small Ras GTPase system in the control of directed cell motility. M. xanthus cells direct their motility by reversing their direction of movement through a mechanism involving the redirection of the motility apparatus to the opposite cell pole. The reversal frequency of moving M. xanthus cells is controlled by modular and interconnected protein networks linking the chemosensory-like frizzy (Frz) pathway - that transmits environmental signals - to the downstream Ras-like Mgl polarity control system - that comprises the Ras-like MglA GTPase protein and its regulators. Here, we discuss how variations in the GTPase interactome landscape underlie single-cell decisions and consequently, multicellular patterns.
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Affiliation(s)
- Céline Dinet
- Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, CNRS-Aix-Marseille University, France
| | - Tâm Mignot
- Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, CNRS-Aix-Marseille University, France
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20
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GAP positions catalytic H-Ras residue Q61 for GTP hydrolysis in molecular dynamics simulations, complicating chemical rescue of Ras deactivation. Comput Biol Chem 2023; 104:107835. [PMID: 36893567 DOI: 10.1016/j.compbiolchem.2023.107835] [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: 10/26/2022] [Revised: 02/01/2023] [Accepted: 02/16/2023] [Indexed: 03/05/2023]
Abstract
Functional interaction of Ras signaling proteins with upstream, negative regulatory GTPase activating proteins (GAPs) represents a crucial step in cellular decision making related to growth and survival. Key components of the catalytic transition state for Ras deactivation by GAP-accelerated hydrolysis of Ras-bound guanosine triphosphate (GTP) are thought to include an arginine residue from the GAP (the arginine finger), a glutamine residue from Ras (Q61), and a water molecule that is likely coordinated by Q61 to engage in nucleophilic attack on GTP. Here, we use in-vitro fluorescence experiments to show that 0.1-100 mM concentrations of free arginine, imidazole, and other small nitrogenous molecule fail to accelerate GTP hydrolysis, even in the presence of the catalytic domain of a mutant GAP lacking its arginine finger (R1276A NF1). This result is surprising given that imidazole can chemically rescue enzyme activity in arginine-to-alanine mutant protein tyrosine kinases (PTKs) that share many active site components with Ras/GAP complexes. Complementary all-atom molecular dynamics (MD) simulations reveal that an arginine finger GAP mutant still functions to enhance Ras Q61-GTP interaction, though less extensively than wild-type GAP. This increased Q61-GTP proximity may promote more frequent fluctuations into configurations that enable GTP hydrolysis as a component of the mechanism by which GAPs accelerate Ras deactivation in the face of arginine finger mutations. The failure of small molecule analogs of arginine to chemically rescue catalytic deactivation of Ras is consistent with the idea that the influence of the GAP goes beyond the simple provision of its arginine finger. However, the failure of chemical rescue in the presence of R1276A NF1 suggests that the GAPs arginine finger is either unsusceptible to rescue due to exquisite positioning or that it is involved in complex multivalent interactions. Therefore, in the context of oncogenic Ras proteins with mutations at codons 12 or 13 that inhibit arginine finger penetration toward GTP, drug-based chemical rescue of GTP hydrolysis may have bifunctional chemical/geometric requirements that are more difficult to satisfy than those that result from arginine-to-alanine mutations in other enzymes for which chemical rescue has been demonstrated.
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21
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Stiegler AL, Vish KJ, Boggon TJ. Tandem engagement of phosphotyrosines by the dual SH2 domains of p120RasGAP. Structure 2022; 30:1603-1614.e5. [PMID: 36417908 PMCID: PMC9722645 DOI: 10.1016/j.str.2022.10.009] [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: 06/27/2022] [Revised: 09/22/2022] [Accepted: 10/27/2022] [Indexed: 11/23/2022]
Abstract
p120RasGAP is a multidomain GTPase-activating protein for Ras. The presence of two Src homology 2 domains in an SH2-SH3-SH2 module raises the possibility that p120RasGAP simultaneously binds dual phosphotyrosine residues in target proteins. One known binding partner with two proximal phosphotyrosines is p190RhoGAP, a GTPase-activating protein for Rho GTPases. Here, we present the crystal structure of the p120RasGAP SH2-SH3-SH2 module bound to a doubly tyrosine-phosphorylated p190RhoGAP peptide, revealing simultaneous phosphotyrosine recognition by the SH2 domains. The compact arrangement places the SH2 domains in close proximity resembling an SH2 domain tandem and exposed SH3 domain. Affinity measurements support synergistic binding, while solution scattering reveals that dual phosphotyrosine binding induces compaction of this region. Our studies reflect a binding mode that limits conformational flexibility within the SH2-SH3-SH2 cassette and relies on the spacing and sequence surrounding the two phosphotyrosines, potentially representing a selectivity mechanism for downstream signaling events.
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Affiliation(s)
- Amy L Stiegler
- Department of Pharmacology, Yale University, New Haven, CT, USA
| | - Kimberly J Vish
- Department of Pharmacology, Yale University, New Haven, CT, USA; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
| | - Titus J Boggon
- Department of Pharmacology, Yale University, New Haven, CT, USA; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA; Yale Cancer Center, Yale University, New Haven, CT, USA.
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22
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Chau JE, Vish KJ, Boggon TJ, Stiegler AL. SH3 domain regulation of RhoGAP activity: Crosstalk between p120RasGAP and DLC1 RhoGAP. Nat Commun 2022; 13:4788. [PMID: 35970859 PMCID: PMC9378701 DOI: 10.1038/s41467-022-32541-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Accepted: 08/04/2022] [Indexed: 11/10/2022] Open
Abstract
RhoGAP proteins are key regulators of Rho family GTPases and influence a variety of cellular processes, including cell migration, adhesion, and cytokinesis. These GTPase activating proteins (GAPs) downregulate Rho signaling by binding and enhancing the intrinsic GTPase activity of Rho proteins. Deleted in liver cancer 1 (DLC1) is a tumor suppressor and ubiquitously expressed RhoGAP protein; its activity is regulated in part by binding p120RasGAP, a GAP protein for the Ras GTPases. In this study, we report the co-crystal structure of the p120RasGAP SH3 domain bound directly to DLC1 RhoGAP, at a site partially overlapping the RhoA binding site and impinging on the catalytic arginine finger. We demonstrate biochemically that mutation of this interface relieves inhibition of RhoGAP activity by the SH3 domain. These results reveal the mechanism for inhibition of DLC1 RhoGAP activity by p120RasGAP and demonstrate the molecular basis for direct SH3 domain modulation of GAP activity.
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Affiliation(s)
- Jocelyn E Chau
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
| | - Kimberly J Vish
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
- Department of Pharmacology, Yale University, New Haven, CT, USA
| | - Titus J Boggon
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
- Department of Pharmacology, Yale University, New Haven, CT, USA
| | - Amy L Stiegler
- Department of Pharmacology, Yale University, New Haven, CT, USA.
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23
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CDC42-IQGAP Interactions Scrutinized: New Insights into the Binding Properties of the GAP-Related Domain. Int J Mol Sci 2022; 23:ijms23168842. [PMID: 36012107 PMCID: PMC9408373 DOI: 10.3390/ijms23168842] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 08/05/2022] [Accepted: 08/06/2022] [Indexed: 11/17/2022] Open
Abstract
The IQ motif-containing GTPase-activating protein (IQGAP) family composes of three highly-related and evolutionarily conserved paralogs (IQGAP1, IQGAP2 and IQGAP3), which fine tune as scaffolding proteins numerous fundamental cellular processes. IQGAP1 is described as an effector of CDC42, although its effector function yet re-mains unclear. Biophysical, biochemical and molecular dynamic simulation studies have proposed that IQGAP RASGAP-related domains (GRDs) bind to the switch regions and the insert helix of CDC42 in a GTP-dependent manner. Our kinetic and equilibrium studies have shown that IQGAP1 GRD binds, in contrast to its C-terminal 794 amino acids (called C794), CDC42 in a nucleotide-independent manner indicating a binding outside the switch regions. To resolve this discrepancy and move beyond the one-sided view of GRD, we carried out affinity measurements and a systematic mutational analysis of the interfacing residues between GRD and CDC42 based on the crystal structure of the IQGAP2 GRD-CDC42Q61L GTP complex. We determined a 100-fold lower affinity of the GRD1 of IQGAP1 and of GRD2 of IQGAP2 for CDC42 mGppNHp in comparison to C794/C795 proteins. Moreover, partial and major mutation of CDC42 switch regions substantially affected C794/C795 binding but only a little GRD1 and remarkably not at all the GRD2 binding. However, we clearly showed that GRD2 contributes to the overall affinity of C795 by using a 11 amino acid mutated GRD variant. Furthermore, the GRD1 binding to the CDC42 was abolished using specific point mutations within the insert helix of CDC42 clearly supporting the notion that CDC42 binding site(s) of IQGAP GRD lies outside the switch regions among others in the insert helix. Collectively, this study provides further evidence for a mechanistic framework model that is based on a multi-step binding process, in which IQGAP GRD might act as a ‘scaffolding domain’ by binding CDC42 irrespective of its nucleotide-bound forms, followed by other IQGAP domains downstream of GRD that act as an effector domain and is in charge for a GTP-dependent interaction with CDC42.
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24
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Binding of the M. tuberculosis EccC ATPase double hexameric ring to the EsxAB virulence factor is enhanced by ATP. Biochem J 2022; 479:1559-1579. [PMID: 35770799 DOI: 10.1042/bcj20210430] [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: 06/10/2021] [Revised: 06/29/2022] [Accepted: 06/30/2022] [Indexed: 11/17/2022]
Abstract
The EccC enzyme of M. tuberculosis ESX-1 secretion system is involved in EsxAB virulence factor secretion and offers an attractive target for antivirulence inhibitors development against M. tuberculosis. The EccCb1 polypeptide of the EccC enzyme contains two Ftsk/SpoIIIE type ATPase domains (D2 and D3) and binds to EsxAB factor at C-terminal region of the D3 domain. In current study, we have determined a low-resolution structure of EccCb1, and its mechanism involved in ATPase activity and EsxAB factor binding. Small-angle X-ray scattering data yielded a double hexameric ring structure of EccCb1 in solution and was further confirmed by SEC-MALS and dynamic light scattering. ATPase activity of wild-type, D2, and D3 mutants showed that D2-K90A and D3-K382A mutations led to a complete loss of enzyme activity. The full-length EccCb1 showed ~ 3.7-fold lower catalytic efficiency than D2 domain and ~1.7 fold lower than D3 domain. The EsxAB factor binds EccCb1 with Kd ~ 11.3±0.6 nM and its affinity is enhanced ~2 fold in presence of ATP+Mg2+. These data indicate the involvement of ATPase activity in EsxAB factor translocation. Molecular dynamics simulation on wild-type, ATP+Mg2+ and EsxAB+ATP+Mg2+ bound EccCb1 double-ring structure showed enhanced stability of enzyme upon ATP+Mg2+ and EsxAB binding. Overall, our study showed a low-resolution structure of EccCb1, and the mechanism involved in ATPase activity and EsxAB factor recognition, which can be targeted for the development of anti-virulence drugs against M. tuberculosis.
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25
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Smith KP, Lee W, Tonelli M, Lee Y, Light SH, Cornilescu G, Chakravarthy S. Solution structure and dynamics of the mitochondrial-targeted GTPase-activating protein (GAP) VopE by an integrated NMR/SAXS approach. Protein Sci 2022; 31:e4282. [PMID: 35137487 PMCID: PMC9047041 DOI: 10.1002/pro.4282] [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: 09/29/2021] [Revised: 01/17/2022] [Accepted: 01/27/2022] [Indexed: 11/11/2022]
Abstract
The bacterial pathogen Vibrio cholerae use a type III secretion system to inject effector proteins into a host cell. Recently, a putative Toxic GTPase Activating Protein (ToxGAP) called Vibrio outer protein E (VopE) was identified as a T3SS substrate and virulence factor that affected host mitochondrial dynamics and immune response. However, biophysical and structural characterization has been absent. Here, we describe solution NMR structure of the putative GTPase-activating protein (GAP) domain (73-204) of VopE. Using size exclusion chromatography coupled with small-angle x-ray scattering and residual dipolar coupling data, we restrained the MD process to efficiently determine the overall fold and improve the quality of the output calculated structures. Comparing the structure of VopE with other ToxGAP's revealed a similar overall fold with several features unique to VopE. Specifically, the "Bulge 1," α1 helix, and noteworthy "backside linker" elements on the N-terminus are dissimilar to the other ToxGAP's. By using NMR relaxation dispersion experiments, we demonstrate that these regions undergo motions on a > 6 s-1 timescale. Based on the disposition of these mobile regions relative to the putative catalytic arginine residue, we hypothesize that the protein may undergo structural changes to bind cognate GTPases.
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Affiliation(s)
- Kyle P. Smith
- Department of Cell & Developmental BiologyNorthwestern University ChicagoIllinoisUSA
- Xilio TherapeuticsWalthamMassachusettsUSA
| | - Woonghee Lee
- Department of ChemistryUniversity of Colorado‐DenverDenverColoradoUSA
| | - Marco Tonelli
- National Magnetic Resonance Facility at Madison, Department of BiochemistryUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
| | - Yeongjoon Lee
- Department of ChemistryUniversity of Colorado‐DenverDenverColoradoUSA
| | - Samuel H. Light
- Department of MicrobiologyUniversity of ChicagoChicagoIllinoisUSA
| | - Gabriel Cornilescu
- Advanced Technology Research Facility, Frederick National Laboratory for Cancer ResearchLeidos Biomedical Research, Inc., National Cancer Institute, National Institutes of HealthFrederickMarylandUSA
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26
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Simanshu DK, Morrison DK. A Structure is Worth a Thousand Words: New Insights for RAS and RAF Regulation. Cancer Discov 2022; 12:899-912. [PMID: 35046094 PMCID: PMC8983508 DOI: 10.1158/2159-8290.cd-21-1494] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2021] [Revised: 12/13/2021] [Accepted: 12/14/2021] [Indexed: 11/16/2022]
Abstract
The RAS GTPases are frequently mutated in human cancer, with KRAS being the predominant tumor driver. For many years, it has been known that the structure and function of RAS are integrally linked, as structural changes induced by GTP binding or mutational events determine the ability of RAS to interact with regulators and effectors. Recently, a wealth of information has emerged from structures of specific KRAS mutants and from structures of multiprotein complexes containing RAS and/or RAF, an essential effector of RAS. These structures provide key insights regarding RAS and RAF regulation as well as promising new strategies for therapeutic intervention. SIGNIFICANCE The RAS GTPases are major drivers of tumorigenesis, and for RAS proteins to exert their full oncogenic potential, they must interact with the RAF kinases to initiate ERK cascade signaling. Although binding to RAS is typically a prerequisite for RAF to become an activated kinase, determining the molecular mechanisms by which this interaction results in RAF activation has been a challenging task. A major advance in understanding this process and RAF regulation has come from recent structural studies of various RAS and RAF multiprotein signaling complexes, revealing new avenues for drug discovery.
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Affiliation(s)
- Dhirendra K. Simanshu
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, Maryland
| | - Deborah K. Morrison
- Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute, Frederick, Maryland
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27
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Hidalgo F, Nocka LM, Shah NH, Gorday K, Latorraca NR, Bandaru P, Templeton S, Lee D, Karandur D, Pelton JG, Marqusee S, Wemmer D, Kuriyan J. A saturation-mutagenesis analysis of the interplay between stability and activation in Ras. eLife 2022; 11:e76595. [PMID: 35272765 PMCID: PMC8916776 DOI: 10.7554/elife.76595] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2022] [Accepted: 01/25/2022] [Indexed: 12/31/2022] Open
Abstract
Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with the results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g. at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras - and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.
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Affiliation(s)
- Frank Hidalgo
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Howard Hughes Medical Institute, University of California, BerkeleyBerkeleyUnited States
- Department of Chemistry, University of California, BerkeleyBerkeleyUnited States
| | - Laura M Nocka
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Howard Hughes Medical Institute, University of California, BerkeleyBerkeleyUnited States
- Department of Chemistry, University of California, BerkeleyBerkeleyUnited States
| | - Neel H Shah
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Howard Hughes Medical Institute, University of California, BerkeleyBerkeleyUnited States
- Department of Molecular and Cell Biology, University of California, BerkeleyBerkeleyUnited States
- Department of Chemistry, Columbia UniversityNew YorkUnited States
| | - Kent Gorday
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Howard Hughes Medical Institute, University of California, BerkeleyBerkeleyUnited States
- Biophysics Graduate Group, University of California, BerkeleyBerkeleyUnited States
| | - Naomi R Latorraca
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Department of Molecular and Cell Biology, University of California, BerkeleyBerkeleyUnited States
| | - Pradeep Bandaru
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Howard Hughes Medical Institute, University of California, BerkeleyBerkeleyUnited States
- Department of Molecular and Cell Biology, University of California, BerkeleyBerkeleyUnited States
| | - Sage Templeton
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Howard Hughes Medical Institute, University of California, BerkeleyBerkeleyUnited States
| | - David Lee
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Howard Hughes Medical Institute, University of California, BerkeleyBerkeleyUnited States
| | - Deepti Karandur
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Howard Hughes Medical Institute, University of California, BerkeleyBerkeleyUnited States
- Department of Molecular and Cell Biology, University of California, BerkeleyBerkeleyUnited States
| | - Jeffrey G Pelton
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
| | - Susan Marqusee
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Department of Chemistry, University of California, BerkeleyBerkeleyUnited States
- Department of Molecular and Cell Biology, University of California, BerkeleyBerkeleyUnited States
| | - David Wemmer
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Department of Chemistry, University of California, BerkeleyBerkeleyUnited States
| | - John Kuriyan
- California Institute for Quantitative Biosciences (QB3), University of California, BerkeleyBerkeleyUnited States
- Howard Hughes Medical Institute, University of California, BerkeleyBerkeleyUnited States
- Department of Chemistry, University of California, BerkeleyBerkeleyUnited States
- Department of Molecular and Cell Biology, University of California, BerkeleyBerkeleyUnited States
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Abstract
Ring-shaped hexameric helicases are essential motor proteins that separate duplex nucleic acid strands for DNA replication, recombination, and transcriptional regulation. Two evolutionarily distinct lineages of these enzymes, predicated on RecA and AAA+ ATPase folds, have been identified and characterized to date. Hexameric helicases couple NTP hydrolysis with conformational changes that move nucleic acid substrates through a central pore in the enzyme. How hexameric helicases productively engage client DNA or RNA segments and use successive rounds of NTPase activity to power translocation and unwinding have been longstanding questions in the field. Recent structural and biophysical findings are beginning to reveal commonalities in NTP hydrolysis and substrate translocation by diverse hexameric helicase families. Here, we review these molecular mechanisms and highlight aspects of their function that are yet to be understood.
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29
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Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions. Int J Mol Sci 2021; 22:ijms222212493. [PMID: 34830380 PMCID: PMC8622166 DOI: 10.3390/ijms222212493] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2021] [Revised: 11/15/2021] [Accepted: 11/16/2021] [Indexed: 01/13/2023] Open
Abstract
Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.
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30
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Bandyopadhyay A, Saxena AK. Structural and ATPase activity analysis of nucleotide binding domain of Rv3870 enzyme of M. tuberculosis ESX-1 system. Int J Biol Macromol 2021; 189:879-889. [PMID: 34428493 DOI: 10.1016/j.ijbiomac.2021.08.130] [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: 02/10/2021] [Revised: 08/17/2021] [Accepted: 08/17/2021] [Indexed: 10/20/2022]
Abstract
The EccC enzyme of ESX-1 system contains (i) a membrane bound Rv3870 with single ATPase domain and (ii) a cytoplasmic Rv3871 with two ATPase domains and involved in secretion of ESAT6/CFP10 factor out of the cell. In current study, we have structurally and biochemically characterized the ATPase domain (442-747 residues) of Rv3870 enzyme. The ΔRv3870 eluted as oligomer (~813 kDa) from Superdex 200 (16/60) column, as identified based on molecular mass standard and dynamics light scattering. The SAXS analysis yielded a tetrameric ring envelope of ΔRv3870, quite consistent to dynamic light scattering data. The ΔRv3870 exhibited ATPase activity having kinetic parameters, Km ~ 100 ± 40 μM, kcat ~ 1.81 ± 0.27 min-1 and Vmax ~ 54.41 μM/min/mg. ATPase activity using nine ΔRv3870 mutants showed 70-91% decrease in catalytic efficiency of the enzyme. ΔRv3870 binds Rv3871 with KD ~ 484.0 ± 10.3 nM and its catalytic efficiency is enhanced ~6.7-fold in presence of Rv3871. CD data revealed the high TM ~ 82.2 ± 0.5 °C for ΔRv3870 and enhanced in presence of ATP + Mg2+, as observed in dynamics simulation on ΔRv3870 hexameric models. Overall, our structural and biochemical studies on ΔRv3870 have explained the mechanism, which will contribute in development of antivirulence inhibitors against M. tuberculosis.
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Affiliation(s)
- Arkita Bandyopadhyay
- Rm-403/440, Structural Biology Lab, School of Life Sciences, Jawaharlal Nehru University, New Delhi-67, India
| | - Ajay K Saxena
- Rm-403/440, Structural Biology Lab, School of Life Sciences, Jawaharlal Nehru University, New Delhi-67, India.
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31
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Long A, Liu H, Liu J, Daniel M, Bedwell DM, Korf B, Kesterson RA, Wallis D. Analysis of patient-specific NF1 variants leads to functional insights for Ras signaling that can impact personalized medicine. Hum Mutat 2021; 43:30-41. [PMID: 34694046 DOI: 10.1002/humu.24290] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2021] [Revised: 10/13/2021] [Accepted: 10/19/2021] [Indexed: 01/04/2023]
Abstract
We have created a panel of 29 NF1 variant complementary DNAs (cDNAs) representing missense variants, many with clinically relevant phenotypes, in-frame deletions, splice variants, and nonsense variants. We have determined the functional consequences of the variants, assessing their ability to produce mature neurofibromin and restore Ras signaling activity in NF1 null (-/-) cells. cDNAs demonstrate variant-specific differences in neurofibromin protein levels, suggesting that some variants lead to neurofibromatosis type 1 (NF1) gene or protein instability or enhanced degradation. When expressed at high levels, some variant proteins are still able to repress Ras activity, indicating that the NF1 phenotype may be due to low protein abundance. In contrast, other variant proteins are incapable of repressing Ras activity, indicating that some do not functionally engage Ras and stimulate GTPase activity. We observed that effects on protein abundance and Ras activity can be mutually exclusive. These assays allow us to categorize variants by functional effects, may help to classify variants of unknown significance, and may have future implications for more directed therapeutics.
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Affiliation(s)
- Ashlee Long
- Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Hui Liu
- Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Jian Liu
- Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Michael Daniel
- Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - David M Bedwell
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Bruce Korf
- Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Robert A Kesterson
- Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Deeann Wallis
- Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA
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32
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Mysore VP, Zhou ZW, Ambrogio C, Li L, Kapp JN, Lu C, Wang Q, Tucker MR, Okoro JJ, Nagy-Davidescu G, Bai X, Plückthun A, Jänne PA, Westover KD, Shan Y, Shaw DE. A structural model of a Ras-Raf signalosome. Nat Struct Mol Biol 2021; 28:847-857. [PMID: 34625747 PMCID: PMC8643099 DOI: 10.1038/s41594-021-00667-6] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Accepted: 08/25/2021] [Indexed: 01/29/2023]
Abstract
The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.
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Affiliation(s)
| | - Zhi-Wei Zhou
- Departments of Biochemistry and Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Chiara Ambrogio
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy
| | - Lianbo Li
- Departments of Biochemistry and Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Jonas N Kapp
- Department of Biochemistry, University of Zürich, Zürich, Switzerland
| | - Chunya Lu
- Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA
- Department of Respiratory Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Qi Wang
- D. E. Shaw Research, New York, NY, USA
| | | | - Jeffrey J Okoro
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
| | | | - Xiaochen Bai
- Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Andreas Plückthun
- Department of Biochemistry, University of Zürich, Zürich, Switzerland
| | - Pasi A Jänne
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
- Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, MA, USA
| | - Kenneth D Westover
- Departments of Biochemistry and Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | | | - David E Shaw
- D. E. Shaw Research, New York, NY, USA.
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
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33
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Mahdi S, Bezsonova I, Beuning PJ, Korzhnev DM. NMR resonance assignments for the nucleotide binding domains of the E. coli clamp loader complex γ subunit. BIOMOLECULAR NMR ASSIGNMENTS 2021; 15:281-285. [PMID: 33761093 PMCID: PMC8460709 DOI: 10.1007/s12104-021-10018-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/10/2021] [Accepted: 03/13/2021] [Indexed: 06/12/2023]
Abstract
The E. coli γ clamp loader is a pentameric complex of δ, δ' and three γ subunits that opens and loads β-clamp proteins onto DNA in an ATP-dependent process essential for efficient DNA replication. ATP binding to the γ subunits promotes conformational changes that enable the clamp loader to bind and open the ring-shaped β-clamp homodimer. Here we report the nearly complete backbone and side-chain 1H, 13C and 15N NMR resonance assignments of the 242-residue truncated γ subunit of the clamp loader complex, which includes the N-terminal mini (domain I) and lid (domain II) domains. This construct represents the nucleotide binding module in the clamp loader complex and provides a model system for studies of conformational rearrangements of the clamp loader induced by nucleotide binding.
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Affiliation(s)
- Sam Mahdi
- Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT, 06030, USA
| | - Irina Bezsonova
- Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT, 06030, USA
| | - Penny J Beuning
- Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, 02115, USA
| | - Dmitry M Korzhnev
- Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT, 06030, USA.
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34
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Ojeda-May P, Mushtaq AU, Rogne P, Verma A, Ovchinnikov V, Grundström C, Dulko-Smith B, Sauer UH, Wolf-Watz M, Nam K. Dynamic Connection between Enzymatic Catalysis and Collective Protein Motions. Biochemistry 2021; 60:2246-2258. [PMID: 34250801 PMCID: PMC8297476 DOI: 10.1021/acs.biochem.1c00221] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
![]()
Enzymes employ a wide range of protein motions to achieve efficient catalysis of
chemical reactions. While the role of collective protein motions in substrate binding,
product release, and regulation of enzymatic activity is generally understood, their
roles in catalytic steps per se remain uncertain. Here, molecular dynamics simulations,
enzyme kinetics, X-ray crystallography, and nuclear magnetic resonance spectroscopy are
combined to elucidate the catalytic mechanism of adenylate kinase and to delineate the
roles of catalytic residues in catalysis and the conformational change in the enzyme.
This study reveals that the motions in the active site, which occur on a time scale of
picoseconds to nanoseconds, link the catalytic reaction to the slow conformational
dynamics of the enzyme by modulating the free energy landscapes of subdomain motions. In
particular, substantial conformational rearrangement occurs in the active site following
the catalytic reaction. This rearrangement not only affects the reaction barrier but
also promotes a more open conformation of the enzyme after the reaction, which then
results in an accelerated opening of the enzyme compared to that of the reactant state.
The results illustrate a linkage between enzymatic catalysis and collective protein
motions, whereby the disparate time scales between the two processes are bridged by a
cascade of intermediate-scale motion of catalytic residues modulating the free energy
landscapes of the catalytic and conformational change processes.
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Affiliation(s)
- Pedro Ojeda-May
- Department of Chemistry, Umeå University, Umeå SE-90187, Sweden.,High Performance Computing Centre North (HPC2N), Umeå University, Umeå SE-90187, Sweden
| | | | - Per Rogne
- Department of Chemistry, Umeå University, Umeå SE-90187, Sweden
| | - Apoorv Verma
- Department of Chemistry, Umeå University, Umeå SE-90187, Sweden
| | - Victor Ovchinnikov
- Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
| | | | - Beata Dulko-Smith
- Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, Texas 76019, United States
| | - Uwe H Sauer
- Department of Chemistry, Umeå University, Umeå SE-90187, Sweden
| | | | - Kwangho Nam
- Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, Texas 76019, United States
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35
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Khrenova MG, Grigorenko BL, Nemukhin AV. Molecular Modeling Reveals the Mechanism of Ran-RanGAP-Catalyzed Guanosine Triphosphate Hydrolysis without an Arginine Finger. ACS Catal 2021. [DOI: 10.1021/acscatal.1c00582] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Affiliation(s)
- Maria G. Khrenova
- Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1/3, Moscow 119991, Russia
- Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences, Moscow 119071, Russia
| | - Bella L. Grigorenko
- Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1/3, Moscow 119991, Russia
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow 19334, Russia
| | - Alexander V. Nemukhin
- Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1/3, Moscow 119991, Russia
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow 19334, Russia
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36
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Khrenova MG, Bulavko ES, Mulashkin FD, Nemukhin AV. Mechanism of Guanosine Triphosphate Hydrolysis by the Visual Proteins Arl3-RP2: Free Energy Reaction Profiles Computed with Ab Initio Type QM/MM Potentials. Molecules 2021; 26:3998. [PMID: 34208932 PMCID: PMC8271468 DOI: 10.3390/molecules26133998] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2021] [Revised: 06/27/2021] [Accepted: 06/28/2021] [Indexed: 11/23/2022] Open
Abstract
We report the results of calculations of the Gibbs energy profiles of the guanosine triphosphate (GTP) hydrolysis by the Arl3-RP2 protein complex using molecular dynamics (MD) simulations with ab initio type QM/MM potentials. The chemical reaction of GTP hydrolysis to guanosine diphosphate (GDP) and inorganic phosphate (Pi) is catalyzed by GTPases, the enzymes, which are responsible for signal transduction in live cells. A small GTPase Arl3, catalyzing the GTP → GDP reaction in complex with the activating protein RP2, constitute an essential part of the human vision cycle. To simulate the reaction mechanism, a model system is constructed by motifs of the crystal structure of the Arl3-RP2 complexed with a substrate analog. After selection of reaction coordinates, energy profiles for elementary steps along the reaction pathway GTP + H2O → GDP + Pi are computed using the umbrella sampling and umbrella integration procedures. QM/MM MD calculations are carried out, interfacing the molecular dynamics program NAMD and the quantum chemistry program TeraChem. Ab initio type QM(DFT)/MM potentials are computed with atom-centered basis sets 6-31G** and two hybrid functionals (PBE0-D3 and ωB97x-D3) of the density functional theory, describing a large QM subsystem. Results of these simulations of the reaction mechanism are compared to those obtained with QM/MM calculations on the potential energy surface using a similar description of the QM part. We find that both approaches, QM/MM and QM/MM MD, support the mechanism of GTP hydrolysis by GTPases, according to which the catalytic glutamine side chain (Gln71, in this system) actively participates in the reaction. Both approaches distinguish two parts of the reaction: the cleavage of the phosphorus-oxygen bond in GTP coupled with the formation of Pi, and the enzyme regeneration. Newly performed QM/MM MD simulations confirmed the profile predicted in the QM/MM minimum energy calculations, called here the pathway-I, and corrected its relief at the first elementary step from the enzyme-substrate complex. The QM/MM MD simulations also revealed another mechanism at the part of enzyme regeneration leading to pathway-II. Pathway-II is more consistent with the experimental kinetic data of the wild-type complex Arl3-RP2, whereas pathway-I explains the role of the mutation Glu138Gly in RP2 slowing down the hydrolysis rate.
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Affiliation(s)
- Maria G. Khrenova
- Chemistry Department, M.V. Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia; (M.G.K.); (F.D.M.)
- Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences, 119071 Moscow, Russia
| | - Egor S. Bulavko
- Biology Department, M.V. Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia;
| | - Fedor D. Mulashkin
- Chemistry Department, M.V. Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia; (M.G.K.); (F.D.M.)
| | - Alexander V. Nemukhin
- Chemistry Department, M.V. Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia; (M.G.K.); (F.D.M.)
- N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygina 4, 119334 Moscow, Russia
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37
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Su MY, Fromm SA, Remis J, Toso DB, Hurley JH. Structural basis for the ARF GAP activity and specificity of the C9orf72 complex. Nat Commun 2021; 12:3786. [PMID: 34145292 PMCID: PMC8213707 DOI: 10.1038/s41467-021-24081-0] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2021] [Accepted: 05/27/2021] [Indexed: 02/05/2023] Open
Abstract
Mutation of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD), which is attributed to both a gain and loss of function. C9orf72 forms a complex with SMCR8 and WDR41, which was reported to have GTPase activating protein activity toward ARF proteins, RAB8A, and RAB11A. We determined the cryo-EM structure of ARF1-GDP-BeF3- bound to C9orf72:SMCR8:WDR41. The SMCR8longin and C9orf72longin domains form the binding pocket for ARF1. One face of the C9orf72longin domain holds ARF1 in place, while the SMCR8longin positions the catalytic finger Arg147 in the ARF1 active site. Mutations in interfacial residues of ARF1 and C9orf72 reduced or eliminated GAP activity. RAB8A GAP required ~10-fold higher concentrations of the C9orf72 complex than for ARF1. These data support a specific function for the C9orf72 complex as an ARF GAP. The structure also provides a model for the active forms of the longin domain GAPs of FLCN and NPRL2 that regulate the Rag GTPases of the mTORC1 pathway.
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Affiliation(s)
- Ming-Yuan Su
- School of Medicine, Southern University of Science and Technology, Shenzhen, China
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
- California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA
| | - Simon A Fromm
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
- California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA
- Imaging Centre, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Jonathan Remis
- California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA
| | - Daniel B Toso
- California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA
| | - James H Hurley
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
- California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA.
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38
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SRPassing Co-translational Targeting: The Role of the Signal Recognition Particle in Protein Targeting and mRNA Protection. Int J Mol Sci 2021; 22:ijms22126284. [PMID: 34208095 PMCID: PMC8230904 DOI: 10.3390/ijms22126284] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Revised: 06/02/2021] [Accepted: 06/05/2021] [Indexed: 01/13/2023] Open
Abstract
Signal recognition particle (SRP) is an RNA and protein complex that exists in all domains of life. It consists of one protein and one noncoding RNA in some bacteria. It is more complex in eukaryotes and consists of six proteins and one noncoding RNA in mammals. In the eukaryotic cytoplasm, SRP co-translationally targets proteins to the endoplasmic reticulum and prevents misfolding and aggregation of the secretory proteins in the cytoplasm. It was demonstrated recently that SRP also possesses an earlier unknown function, the protection of mRNAs of secretory proteins from degradation. In this review, we analyze the progress in studies of SRPs from different organisms, SRP biogenesis, its structure, and function in protein targeting and mRNA protection.
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39
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Soriano O, Alcón-Pérez M, Vicente-Manzanares M, Castellano E. The Crossroads between RAS and RHO Signaling Pathways in Cellular Transformation, Motility and Contraction. Genes (Basel) 2021; 12:genes12060819. [PMID: 34071831 PMCID: PMC8229961 DOI: 10.3390/genes12060819] [Citation(s) in RCA: 43] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2021] [Revised: 05/25/2021] [Accepted: 05/26/2021] [Indexed: 02/07/2023] Open
Abstract
Ras and Rho proteins are GTP-regulated molecular switches that control multiple signaling pathways in eukaryotic cells. Ras was among the first identified oncogenes, and it appears mutated in many forms of human cancer. It mainly promotes proliferation and survival through the MAPK pathway and the PI3K/AKT pathways, respectively. However, the myriad proteins close to the plasma membrane that activate or inhibit Ras make it a major regulator of many apparently unrelated pathways. On the other hand, Rho is weakly oncogenic by itself, but it critically regulates microfilament dynamics; that is, actin polymerization, disassembly and contraction. Polymerization is driven mainly by the Arp2/3 complex and formins, whereas contraction depends on myosin mini-filament assembly and activity. These two pathways intersect at numerous points: from Ras-dependent triggering of Rho activators, some of which act through PI3K, to mechanical feedback driven by actomyosin action. Here, we describe the main points of connection between the Ras and Rho pathways as they coordinately drive oncogenic transformation. We emphasize the biochemical crosstalk that drives actomyosin contraction driven by Ras in a Rho-dependent manner. We also describe possible routes of mechanical feedback through which myosin II activation may control Ras/Rho activation.
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Affiliation(s)
- Olga Soriano
- Tumor Biophysics Laboratory, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-University of Salamanca, 37007 Salamanca, Spain;
| | - Marta Alcón-Pérez
- Tumour-Stroma Signalling Laboratory, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-University of Salamanca, 37007 Salamanca, Spain;
| | - Miguel Vicente-Manzanares
- Tumor Biophysics Laboratory, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-University of Salamanca, 37007 Salamanca, Spain;
- Correspondence: (M.V.-M.); (E.C.)
| | - Esther Castellano
- Tumour-Stroma Signalling Laboratory, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-University of Salamanca, 37007 Salamanca, Spain;
- Correspondence: (M.V.-M.); (E.C.)
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40
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Rudack T, Teuber C, Scherlo M, Güldenhaupt J, Schartner J, Lübben M, Klare J, Gerwert K, Kötting C. The Ras dimer structure. Chem Sci 2021; 12:8178-8189. [PMID: 34194708 PMCID: PMC8208300 DOI: 10.1039/d1sc00957e] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2021] [Accepted: 04/29/2021] [Indexed: 12/31/2022] Open
Abstract
Oncogenic mutated Ras is a key player in cancer, but despite intense and expensive approaches its catalytic center seems undruggable. The Ras dimer interface is a possible alternative drug target. Dimerization at the membrane affects cell growth signal transduction. In vivo studies indicate that preventing dimerization of oncogenic mutated Ras inhibits uncontrolled cell growth. Conventional computational drug-screening approaches require a precise atomic dimer model as input to successfully access drug candidates. However, the proposed dimer structural models are controversial. Here, we provide a clear-cut experimentally validated N-Ras dimer structural model. We incorporated unnatural amino acids into Ras to enable the binding of labels at multiple positions via click chemistry. This labeling allowed the determination of multiple distances of the membrane-bound Ras-dimer measured by fluorescence and electron paramagnetic resonance spectroscopy. In combination with protein-protein docking and biomolecular simulations, we identified key residues for dimerization. Site-directed mutations of these residues prevent dimer formation in our experiments, proving our dimer model to be correct. The presented dimer structure enables computational drug-screening studies exploiting the Ras dimer interface as an alternative drug target.
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Affiliation(s)
- Till Rudack
- Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr University Bochum 44801 Bochum Germany
- Department of Biophysics, Ruhr University Bochum 44801 Bochum Germany
| | - Christian Teuber
- Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr University Bochum 44801 Bochum Germany
- Department of Biophysics, Ruhr University Bochum 44801 Bochum Germany
| | - Marvin Scherlo
- Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr University Bochum 44801 Bochum Germany
- Department of Biophysics, Ruhr University Bochum 44801 Bochum Germany
| | - Jörn Güldenhaupt
- Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr University Bochum 44801 Bochum Germany
- Department of Biophysics, Ruhr University Bochum 44801 Bochum Germany
| | - Jonas Schartner
- Department of Biophysics, Ruhr University Bochum 44801 Bochum Germany
| | - Mathias Lübben
- Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr University Bochum 44801 Bochum Germany
- Department of Biophysics, Ruhr University Bochum 44801 Bochum Germany
| | - Johann Klare
- Department of Physics, Osnabrück University 49074 Osnabrück Germany
| | - Klaus Gerwert
- Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr University Bochum 44801 Bochum Germany
- Department of Biophysics, Ruhr University Bochum 44801 Bochum Germany
| | - Carsten Kötting
- Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr University Bochum 44801 Bochum Germany
- Department of Biophysics, Ruhr University Bochum 44801 Bochum Germany
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41
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Duan X, Chen X, Wang K, Chen L, Glomb O, Johnsson N, Feng L, Zhou XQ, Bi E. Essential role of the endocytic site-associated protein Ecm25 in stress-induced cell elongation. Cell Rep 2021; 35:109122. [PMID: 34010635 PMCID: PMC8202958 DOI: 10.1016/j.celrep.2021.109122] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2020] [Revised: 02/16/2021] [Accepted: 04/22/2021] [Indexed: 11/27/2022] Open
Abstract
How cells adopt a different morphology to cope with stress is not well understood. Here, we show that budding yeast Ecm25 associates with polarized endocytic sites and interacts with the polarity regulator Cdc42 and several late-stage endocytic proteins via distinct regions, including an actin filament-binding motif. Deletion of ECM25 does not affect Cdc42 activity or cause any strong defects in fluid-phase and clathrin-mediated endocytosis but completely abolishes hydroxyurea-induced cell elongation. This phenotype is accompanied by depolarization of the spatiotemporally coupled exo-endocytosis in the bud cortex while maintaining the overall mother-bud polarity. These data suggest that Ecm25 provides an essential link between the polarization signal and the endocytic machinery to enable adaptive morphogenesis under stress conditions. How cells adopt a different morphology to cope with stress is not well understood. Duan et al. report that the budding yeast protein Ecm25 plays an essential role in stress-induced cell elongation by linking the polarity regulator Cdc42 to the late-stage endocytic machinery.
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Affiliation(s)
- Xudong Duan
- Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6058, USA; Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, 611130 Sichuan, China; Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China
| | - Xi Chen
- Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6058, USA
| | - Kangji Wang
- Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6058, USA
| | - Li Chen
- Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6058, USA
| | - Oliver Glomb
- Institut für Molekulare Genetik und Zellbiologie, Universität Ulm, 89081 Ulm, Germany
| | - Nils Johnsson
- Institut für Molekulare Genetik und Zellbiologie, Universität Ulm, 89081 Ulm, Germany
| | - Lin Feng
- Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, 611130 Sichuan, China
| | - Xiao-Qiu Zhou
- Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, 611130 Sichuan, China.
| | - Erfei Bi
- Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6058, USA.
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42
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Khrenova MG, Kulakova AM, Nemukhin AV. Light-Induced Change of Arginine Conformation Modulates the Rate of Adenosine Triphosphate to Cyclic Adenosine Monophosphate Conversion in the Optogenetic System Containing Photoactivated Adenylyl Cyclase. J Chem Inf Model 2021; 61:1215-1225. [PMID: 33677973 DOI: 10.1021/acs.jcim.0c01308] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
We report the first computational characterization of an optogenetic system composed of two photosensing BLUF (blue light sensor using flavin adenine dinucleotide) domains and two catalytic adenylyl cyclase (AC) domains. Conversion of adenosine triphosphate (ATP) to the reaction products, cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi), catalyzed by ACs initiated by excitation in photosensing domains has emerged in the focus of modern optogenetic applications because of the request in photoregulated enzymes that modulate cellular concentrations of signaling messengers. The photoactivated AC from the soil bacterium Beggiatoa sp. (bPAC) is an important model showing a considerable increase in the ATP to cAMP conversion rate in the catalytic domain after the illumination of the BLUF domain. The 1 μs classical molecular dynamics simulations reveal that the activation of the BLUF domain leading to tautomerization of Gln49 in the chromophore-binding pocket results in switching of the position of the side chain of Arg278 in the active site of AC. Allosteric signal transmission pathways between Gln49 from BLUF and Arg278 from AC were revealed by the dynamical network analysis. The Gibbs energy profiles of the ATP → cAMP + PPi reaction computed using QM(DFT(ωB97X-D3/6-31G**))/MM(CHARMM) molecular dynamics simulations for both Arg278 conformations in AC clarify the reaction mechanism. In the light-activated system, the corresponding arginine conformation stabilizes the pentacoordinated phosphorus of the α-phosphate group in the transition state, thus lowering the activation energy. Simulations of the bPAC system with the Tyr7Phe replacement in the BLUF demonstrate occurrence of both arginine conformations in an equal ratio, explaining the experimentally observed intermediate catalytic activity of the bPAC-Y7F variant as compared with the dark and light states of the wild-type bPAC.
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Affiliation(s)
- Maria G Khrenova
- Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russian Federation.,Bach Institute of Biochemistry, Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Moscow 119071 Russian Federation
| | - Anna M Kulakova
- Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russian Federation
| | - Alexander V Nemukhin
- Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russian Federation.,Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow 119334, Russian Federation
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Turn RE, Linnert J, Gigante ED, Wolfrum U, Caspary T, Kahn RA. Roles for ELMOD2 and Rootletin in ciliogenesis. Mol Biol Cell 2021; 32:800-822. [PMID: 33596093 PMCID: PMC8108518 DOI: 10.1091/mbc.e20-10-0635] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
ELMOD2 is a GTPase-activating protein with uniquely broad specificity for ARF family GTPases. We previously showed that it acts with ARL2 in mitochondrial fusion and microtubule stability and with ARF6 during cytokinesis. Mouse embryonic fibroblasts deleted for ELMOD2 also displayed changes in cilia-related processes including increased ciliation, multiciliation, ciliary morphology, ciliary signaling, centrin accumulation inside cilia, and loss of rootlets at centrosomes with loss of centrosome cohesion. Increasing ARL2 activity or overexpressing Rootletin reversed these defects, revealing close functional links between the three proteins. This was further supported by the findings that deletion of Rootletin yielded similar phenotypes, which were rescued upon increasing ARL2 activity but not ELMOD2 overexpression. Thus, we propose that ARL2, ELMOD2, and Rootletin all act in a common pathway that suppresses spurious ciliation and maintains centrosome cohesion. Screening a number of markers of steps in the ciliation pathway supports a model in which ELMOD2, Rootletin, and ARL2 act downstream of TTBK2 and upstream of CP110 to prevent spurious release of CP110 and to regulate ciliary vesicle docking. These data thus provide evidence supporting roles for ELMOD2, Rootletin, and ARL2 in the regulation of ciliary licensing.
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Affiliation(s)
- Rachel E Turn
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.,Biochemistry, Cell & Developmental Biology Graduate Program, Emory University, Atlanta, GA 30322
| | - Joshua Linnert
- Institut für Molekulare Physiologie, Johannes Gutenberg-Universität, Mainz 655099, Germany
| | - Eduardo D Gigante
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322.,Neuroscience Graduate Program, Emory University, Atlanta, GA 30322
| | - Uwe Wolfrum
- Institut für Molekulare Physiologie, Johannes Gutenberg-Universität, Mainz 655099, Germany
| | - Tamara Caspary
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322
| | - Richard A Kahn
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322
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Zheng H, Li L, Yu Z, Yuan Y, Zheng Q, Xie Q, Li G, Abubakar YS, Zhou J, Wang Z, Zheng W. FgSpa2 recruits FgMsb3, a Rab8 GAP, to the polarisome to regulate polarized trafficking, growth and pathogenicity in Fusarium graminearum. THE NEW PHYTOLOGIST 2021; 229:1665-1683. [PMID: 32978966 DOI: 10.1111/nph.16935] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/04/2020] [Accepted: 09/01/2020] [Indexed: 06/11/2023]
Abstract
In filamentous fungi, hyphal growth depends on the continuous delivery of vesicles to the growing tips. It is unclear how fast-growing hyphae coordinate simultaneous cell extension and expansion in the tip cells. We have functionally characterized 12 TBC (Tre-2/Bub2/Cdc16) domain-containing proteins in Fusarium graminearum. Among them, FgMsb3 is found to regulate hyphal tip expansion and to be required for pathogenicity. The regulatory mechanism of FgMsb3 has been further investigated by genetic, high-resolution microscopy and high-throughput co-immunoprecipitation strategies. The FgMsb3 protein localizes at the polarisome and the hyphal apical dome (HAD) where it acts as a GTPase-activating protein for FgRab8 which is required for apical secretion-mediated growth and pathogenicity. Deletion of FgMSB3 causes excessive polarized trafficking but blocks the fusion of FgSnc1-associated vesicles to the plasma membrane. Moreover, we establish that FgSpa2 interacts with FgMsb3, enabling FgMsb3 tethering to the polarisome. Loss of FgSpa2 or other polarisome components (FgBud6 and FgPea2) causes complete shifting of FgMsb3 to the HAD and this affects the polarized growth and pathogenicity of the fungus. In summary, we conclude that FgSpa2 regulates FgMsb3-FgRab8 cascade and this is crucial for creating a steady-state equilibrium that maintains continuous polarized growth and contributes to the pathogenicity of F. graminearum.
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Affiliation(s)
- Huawei Zheng
- Marine and Agricultural Biotechnology Laboratory, Institute of Oceanography, Minjiang University, Fuzhou, 350108, China
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Lingping Li
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Zhi Yu
- Marine and Agricultural Biotechnology Laboratory, Institute of Oceanography, Minjiang University, Fuzhou, 350108, China
- Fujian Province Key Laboratory of Pathogenic Fungi and Mycotoxins, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Yanping Yuan
- Fujian Province Key Laboratory of Pathogenic Fungi and Mycotoxins, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Qiaojia Zheng
- Marine and Agricultural Biotechnology Laboratory, Institute of Oceanography, Minjiang University, Fuzhou, 350108, China
| | - Qiurong Xie
- Fujian Key Laboratory of Integrative Medicine on Geriatric, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, 350122, China
| | - Guangpu Li
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA
| | - Yakubu Saddeeq Abubakar
- Department of Biochemistry, Faculty of Life Sciences, Ahmadu Bello University, Zaria, 810211, Nigeria
| | - Jie Zhou
- Fujian Province Key Laboratory of Pathogenic Fungi and Mycotoxins, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Zonghua Wang
- Marine and Agricultural Biotechnology Laboratory, Institute of Oceanography, Minjiang University, Fuzhou, 350108, China
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
- Fujian Province Key Laboratory of Pathogenic Fungi and Mycotoxins, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Wenhui Zheng
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
- Fujian Province Key Laboratory of Pathogenic Fungi and Mycotoxins, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
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Calebiro D, Koszegi Z, Lanoiselée Y, Miljus T, O'Brien S. G protein-coupled receptor-G protein interactions: a single-molecule perspective. Physiol Rev 2020; 101:857-906. [PMID: 33331229 DOI: 10.1152/physrev.00021.2020] [Citation(s) in RCA: 62] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
G protein-coupled receptors (GPCRs) regulate many cellular and physiological processes, responding to a diverse range of extracellular stimuli including hormones, neurotransmitters, odorants, and light. Decades of biochemical and pharmacological studies have provided fundamental insights into the mechanisms of GPCR signaling. Thanks to recent advances in structural biology, we now possess an atomistic understanding of receptor activation and G protein coupling. However, how GPCRs and G proteins interact in living cells to confer signaling efficiency and specificity remains insufficiently understood. The development of advanced optical methods, including single-molecule microscopy, has provided the means to study receptors and G proteins in living cells with unprecedented spatio-temporal resolution. The results of these studies reveal an unexpected level of complexity, whereby GPCRs undergo transient interactions among themselves as well as with G proteins and structural elements of the plasma membrane to form short-lived signaling nanodomains that likely confer both rapidity and specificity to GPCR signaling. These findings may provide new strategies to pharmaceutically modulate GPCR function, which might eventually pave the way to innovative drugs for common diseases such as diabetes or heart failure.
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Affiliation(s)
- Davide Calebiro
- Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, United Kingdom; Centre of Membrane Proteins and Receptors (COMPARE), Universities of Nottingham and Birmingham, Birmingham, United Kingdom
| | - Zsombor Koszegi
- Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, United Kingdom; Centre of Membrane Proteins and Receptors (COMPARE), Universities of Nottingham and Birmingham, Birmingham, United Kingdom
| | - Yann Lanoiselée
- Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, United Kingdom; Centre of Membrane Proteins and Receptors (COMPARE), Universities of Nottingham and Birmingham, Birmingham, United Kingdom
| | - Tamara Miljus
- Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, United Kingdom; Centre of Membrane Proteins and Receptors (COMPARE), Universities of Nottingham and Birmingham, Birmingham, United Kingdom
| | - Shannon O'Brien
- Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, United Kingdom; Centre of Membrane Proteins and Receptors (COMPARE), Universities of Nottingham and Birmingham, Birmingham, United Kingdom
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Natural Products Attenuating Biosynthesis, Processing, and Activity of Ras Oncoproteins: State of the Art and Future Perspectives. Biomolecules 2020; 10:biom10111535. [PMID: 33182807 PMCID: PMC7698260 DOI: 10.3390/biom10111535] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Revised: 11/03/2020] [Accepted: 11/08/2020] [Indexed: 02/07/2023] Open
Abstract
RAS genes encode signaling proteins, which, in mammalian cells, act as molecular switches regulating critical cellular processes as proliferation, growth, differentiation, survival, motility, and metabolism in response to specific stimuli. Deregulation of Ras functions has a high impact on human health: gain-of-function point mutations in RAS genes are found in some developmental disorders and thirty percent of all human cancers, including the deadliest. For this reason, the pathogenic Ras variants represent important clinical targets against which to develop novel, effective, and possibly selective pharmacological inhibitors. Natural products represent a virtually unlimited resource of structurally different compounds from which one could draw on for this purpose, given the improvements in isolation and screening of active molecules from complex sources. After a summary of Ras proteins molecular and regulatory features and Ras-dependent pathways relevant for drug development, we point out the most promising inhibitory approaches, the known druggable sites of wild-type and oncogenic Ras mutants, and describe the known natural compounds capable of attenuating Ras signaling. Finally, we highlight critical issues and perspectives for the future selection of potential Ras inhibitors from natural sources.
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Riccardi C, Perrone L, Napolitano F, Sampaolo S, Melone MAB. Understanding the Biological Activities of Vitamin D in Type 1 Neurofibromatosis: New Insights into Disease Pathogenesis and Therapeutic Design. Cancers (Basel) 2020; 12:E2965. [PMID: 33066259 PMCID: PMC7602022 DOI: 10.3390/cancers12102965] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Revised: 09/18/2020] [Accepted: 10/08/2020] [Indexed: 02/07/2023] Open
Abstract
Vitamin D is a fat-soluble steroid hormone playing a pivotal role in calcium and phosphate homeostasis as well as in bone health. Vitamin D levels are not exclusively dependent on food intake. Indeed, the endogenous production-occurring in the skin and dependent on sun exposure-contributes to the majority amount of vitamin D present in the body. Since vitamin D receptors (VDRs) are ubiquitous and drive the expression of hundreds of genes, the interest in vitamin D has tremendously grown and its role in different diseases has been extensively studied. Several investigations indicated that vitamin D action extends far beyond bone health and calcium metabolism, showing broad effects on a variety of critical illnesses, including cancer, infections, cardiovascular and autoimmune diseases. Epidemiological studies indicated that low circulating vitamin D levels inversely correlate with cutaneous manifestations and bone abnormalities, clinical hallmarks of neurofibromatosis type 1 (NF1). NF1 is an autosomal dominant tumour predisposition syndrome causing significant pain and morbidity, for which limited treatment options are available. In this context, vitamin D or its analogues have been used to treat both skin and bone lesions in NF1 patients, alone or combined with other therapeutic agents. Here we provide an overview of vitamin D, its characteristic nutritional properties relevant for health benefits and its role in NF1 disorder. We focus on preclinical and clinical studies that demonstrated the clinical correlation between vitamin D status and NF1 disease, thus providing important insights into disease pathogenesis and new opportunities for targeted therapy.
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Affiliation(s)
- Claudia Riccardi
- Department of Chemical Sciences, University of Naples Federico II, via Cintia 21, I-80126 Naples, Italy;
- Department of Advanced Medical and Surgical Sciences, 2nd Division of Neurology, Center for Rare Diseases and InterUniversity Center for Research in Neurosciences, University of Campania Luigi Vanvitelli, via Sergio Pansini 5, I-80131 Naples, Italy; (L.P.); (F.N.); (S.S.)
| | - Lorena Perrone
- Department of Advanced Medical and Surgical Sciences, 2nd Division of Neurology, Center for Rare Diseases and InterUniversity Center for Research in Neurosciences, University of Campania Luigi Vanvitelli, via Sergio Pansini 5, I-80131 Naples, Italy; (L.P.); (F.N.); (S.S.)
| | - Filomena Napolitano
- Department of Advanced Medical and Surgical Sciences, 2nd Division of Neurology, Center for Rare Diseases and InterUniversity Center for Research in Neurosciences, University of Campania Luigi Vanvitelli, via Sergio Pansini 5, I-80131 Naples, Italy; (L.P.); (F.N.); (S.S.)
| | - Simone Sampaolo
- Department of Advanced Medical and Surgical Sciences, 2nd Division of Neurology, Center for Rare Diseases and InterUniversity Center for Research in Neurosciences, University of Campania Luigi Vanvitelli, via Sergio Pansini 5, I-80131 Naples, Italy; (L.P.); (F.N.); (S.S.)
| | - Mariarosa Anna Beatrice Melone
- Department of Advanced Medical and Surgical Sciences, 2nd Division of Neurology, Center for Rare Diseases and InterUniversity Center for Research in Neurosciences, University of Campania Luigi Vanvitelli, via Sergio Pansini 5, I-80131 Naples, Italy; (L.P.); (F.N.); (S.S.)
- Sbarro Institute for Cancer Research and Molecular Medicine, Department of Biology, Temple University, BioLife Building (015-00), 1900 North 12th Street, Philadelphia, PA 19122-6078, USA
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Gasper R, Wittinghofer F. The Ras switch in structural and historical perspective. Biol Chem 2020; 401:143-163. [PMID: 31600136 DOI: 10.1515/hsz-2019-0330] [Citation(s) in RCA: 53] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Accepted: 09/23/2019] [Indexed: 12/22/2022]
Abstract
Since its discovery as an oncogene more than 40 years ago, Ras has been and still is in the focus of many academic and pharmaceutical labs around the world. A huge amount of work has accumulated on its biology. However, many questions about the role of the different Ras isoforms in health and disease still exist and a full understanding will require more intensive work in the future. Here we try to survey some of the structural findings in a historical perspective and how it has influenced our understanding of structure-function and mechanistic relationships of Ras and its interactions. The structures show that Ras is a stable molecular machine that uses the dynamics of its switch regions for the interaction with all regulators and effectors. This conformational flexibility has been used to create small molecule drug candidates against this important oncoprotein.
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Affiliation(s)
- Raphael Gasper
- Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Str. 11, D-44227 Dortmund, Germany
| | - Fred Wittinghofer
- Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Str. 11, D-44227 Dortmund, Germany
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Abstract
Activating KRAS mutations are present in 25% of human cancer. Although oncogenic Ras was deemed “undruggable” in the past, recent efforts led to the development of pharmacological inhibitors targeting the KRASG12C mutant, which have shown promise in early clinical trials. The development of allele-specific K-RasG12C inhibitors marked a new chapter in targeting oncogenic KRAS mutant in cancer. However, drug resistance against these new drugs will likely limit their efficacy in the clinic. Genome-wide approaches have been used to interrogate the mechanisms of resistance to K-RasG12C inhibitors, which would facilitate the development of therapeutics overcoming drug resistance. This article reviews the latest progress in resistance to K-RasG12C-targeted therapies and aims to provide insight in future research targeting drug resistance in cancer.
Clinical grade K-RasG12C inhibitor marks a new chapter in targeted drug discovery Resistance to K-RasG12C inhibitors is driven by intrinsic or acquired mechanisms Co-targeting vertical Ras signaling overcomes resistance to K-RasG12C inhibition Standard-of-care chemo- and immunotherapies synergize with K-RasG12C inhibition
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Affiliation(s)
- Delong Jiao
- Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA 17033, USA
| | - Shengyu Yang
- Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA 17033, USA
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50
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Yan W, Markegard E, Dharmaiah S, Urisman A, Drew M, Esposito D, Scheffzek K, Nissley DV, McCormick F, Simanshu DK. Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR. Cell Rep 2020; 32:107909. [PMID: 32697994 PMCID: PMC7437355 DOI: 10.1016/j.celrep.2020.107909] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Revised: 05/25/2020] [Accepted: 06/24/2020] [Indexed: 02/07/2023] Open
Abstract
Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.
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Affiliation(s)
- Wupeng Yan
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21701, USA
| | - Evan Markegard
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Srisathiyanarayanan Dharmaiah
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21701, USA
| | - Anatoly Urisman
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Matthew Drew
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21701, USA
| | - Dominic Esposito
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21701, USA
| | - Klaus Scheffzek
- Institute of Biological Chemistry, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria
| | - Dwight V Nissley
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21701, USA
| | - Frank McCormick
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21701, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA.
| | - Dhirendra K Simanshu
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21701, USA.
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