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1
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Church TR, Margolis SS. Mechanisms of ubiquitin-independent proteasomal degradation and their roles in age-related neurodegenerative disease. Front Cell Dev Biol 2025; 12:1531797. [PMID: 39990094 PMCID: PMC11842346 DOI: 10.3389/fcell.2024.1531797] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2024] [Accepted: 12/23/2024] [Indexed: 02/25/2025] Open
Abstract
Neurodegenerative diseases are characterized by the progressive breakdown of neuronal structure and function and the pathological accumulation of misfolded protein aggregates and toxic protein oligomers. A major contributor to the deterioration of neuronal physiology is the disruption of protein catabolic pathways mediated by the proteasome, a large protease complex responsible for most cellular protein degradation. Previously, it was believed that proteolysis by the proteasome required tagging of protein targets with polyubiquitin chains, a pathway called the ubiquitin-proteasome system (UPS). Because of this, most research on proteasomal roles in neurodegeneration has historically focused on the UPS. However, additional ubiquitin-independent pathways and their importance in neurodegeneration are increasingly recognized. In this review, we discuss the range of ubiquitin-independent proteasome pathways, focusing on substrate identification and targeting, regulatory molecules and adaptors, proteasome activators and alternative caps, and diverse proteasome complexes including the 20S proteasome, the neuronal membrane proteasome, the immunoproteasome, extracellular proteasomes, and hybrid proteasomes. These pathways are further discussed in the context of aging, oxidative stress, protein aggregation, and age-associated neurodegenerative diseases, with a special focus on Alzheimer's Disease, Huntington's Disease, and Parkinson's Disease. A mechanistic understanding of ubiquitin-independent proteasome function and regulation in neurodegeneration is critical for the development of therapies to treat these devastating conditions. This review summarizes the current state of ubiquitin-independent proteasome research in neurodegeneration.
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Affiliation(s)
- Taylor R. Church
- Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Seth S. Margolis
- Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
- Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
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2
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Marescal O, Cheeseman IM. 19S proteasome loss causes monopolar spindles through ubiquitin-independent KIF11 degradation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.08.632038. [PMID: 39829864 PMCID: PMC11741298 DOI: 10.1101/2025.01.08.632038] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/22/2025]
Abstract
To direct regulated protein degradation, the 26S proteasome recognizes ubiquitinated substrates through its 19S particle and then degrades them in the 20S enzymatic core. Despite this close interdependency between proteasome subunits, we demonstrate that knockouts from different proteasome subcomplexes result in distinct highly cellular phenotypes. In particular, depletion of 19S PSMD lid proteins, but not that of other proteasome subunits, prevents bipolar spindle assembly during mitosis, resulting in a mitotic arrest. We find that the monopolar spindle phenotype is caused by ubiquitin-independent proteasomal degradation of the motor protein KIF11 upon loss of 19S proteins. Thus, negative regulation of 20S-mediated proteasome degradation is essential for mitotic progression and 19S and 20S proteasome components can function independently outside of the canonical 26S structure. This work reveals a role for the proteasome in spindle formation and identifies the effects of ubiquitin-independent degradation on cell cycle control.
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Affiliation(s)
- Océane Marescal
- Whitehead Institute for Biomedical Research, Cambridge, MA 02142
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142
| | - Iain M. Cheeseman
- Whitehead Institute for Biomedical Research, Cambridge, MA 02142
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142
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3
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Alboreggia G, Udompholkul P, Rodriguez I, Blaha G, Pellecchia M. Targeted degradation of Pin1 by protein-destabilizing compounds. Proc Natl Acad Sci U S A 2024; 121:e2403330121. [PMID: 39531501 PMCID: PMC11588135 DOI: 10.1073/pnas.2403330121] [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: 02/19/2024] [Accepted: 09/04/2024] [Indexed: 11/16/2024] Open
Abstract
The concept of targeted protein degradation is at the forefront of modern drug discovery, which aims to eliminate disease-causing proteins using specific molecules. In this paper, we explored the idea to design protein degraders based on the section of ligands that cause protein destabilization, hence that facilitate the cellular breakdown of the target. Our studies present covalent agents targeting Pin1, a cis-trans prolyl isomerase that plays a crucial role in tumorigenesis. Our design strategy entailed iterative optimizations of agents for potency and Pin1 destabilization in vitro. Biophysical and cellular studies suggest that the agents may act like molecular crowbars, displacing protein-stabilizing interactions that open the structure for recognition by the proteasome degradation machinery. This approach resulted in a series of potent and effective Pin1 degraders with potential applications in target validation and in therapeutic development. We propose that our design strategy can identify molecular degraders without engineering bifunctional agents that artificially create interactions between a disease-causing protein and a ubiquitin ligase.
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Affiliation(s)
- Giulia Alboreggia
- Division of Biomedical Sciences, School of Medicine, University of California Riverside, Riverside, CA92521
| | - Parima Udompholkul
- Division of Biomedical Sciences, School of Medicine, University of California Riverside, Riverside, CA92521
| | - Isaac Rodriguez
- Department of Biochemistry, College of Natural and Agricultural Sciences, University of California Riverside, Riverside, CA92521
| | - Gregor Blaha
- Department of Biochemistry, College of Natural and Agricultural Sciences, University of California Riverside, Riverside, CA92521
| | - Maurizio Pellecchia
- Division of Biomedical Sciences, School of Medicine, University of California Riverside, Riverside, CA92521
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Hsieh J, Leong P, Yang Y, Liu Y, Liu G, Hung H. Protein degradation of antizyme depends on the N-terminal degrons. Protein Sci 2024; 33:e5199. [PMID: 39473024 PMCID: PMC11521938 DOI: 10.1002/pro.5199] [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: 03/12/2024] [Revised: 10/02/2024] [Accepted: 10/10/2024] [Indexed: 11/02/2024]
Abstract
Antizyme (AZ) is a regulatory protein that plays a crucial role in modulating the activity of ornithine decarboxylase (ODC), which is the initial and rate-limiting enzyme in the complex pathway of polyamine biosynthesis. AZ facilitates the swift degradation of ODC, thereby modulating the levels of cellular polyamines. This study unveils a new ubiquitin-independent mechanism for AZ degradation, emphasizing the essential role of N-terminal degrons. Contrary to traditional ubiquitin-dependent degradation, our findings reveal that AZ degradation is significantly influenced by its N-terminal region. By conducting a series of experiments, including in vitro degradation assays, cycloheximide chase experiments, differential scanning calorimetry, and measurement of cellular concentrations of polyamines, we demonstrate that N-terminal truncation significantly enhances AZ's stability and facilitates the reduction of polyamine levels by accelerating ODC degradation. The removal of the N-terminal portion of AZ results in a reduced degradation rate and enhanced thermal stability of the protein, leading to a more efficient inhibition of polyamine synthesis. These findings are corroborated by the analysis of AZ isoforms, AZ1, AZ2, and AZ3, which display differential degradation patterns based on the specific N-terminal segments. This substantiates a degradation mechanism driven by an intrinsically disordered N-terminal region acting as a degron, independent of lysine ubiquitination. These results underscore the significant regulatory function of the N-terminal domain in the activity of AZ and the maintenance of polyamine homeostasis.
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Affiliation(s)
- Ju‐Yi Hsieh
- Department of Life SciencesNational Chung Hsing UniversityTaichungTaiwan, ROC
- Institute of Medicine, School of MedicineChung Shan Medical UniversityTaichungTaiwan, ROC
| | - Pui‐Ying Leong
- Institute of Medicine, School of MedicineChung Shan Medical UniversityTaichungTaiwan, ROC
- Division of Allergy, Immunology and Rheumatology, Department of MedicineChung Shan Medical University HospitalTaichungTaiwan, ROC
| | - Yi‐Fang Yang
- Department of Life SciencesNational Chung Hsing UniversityTaichungTaiwan, ROC
- Doctoral Program in Tissue Engineering and Regenerative MedicineNational Chung Hsing UniversityTaichungTaiwan, ROC
| | - Yi‐Liang Liu
- Department of Life SciencesNational Chung Hsing UniversityTaichungTaiwan, ROC
| | - Guang‐Yaw Liu
- Institute of Medicine, School of MedicineChung Shan Medical UniversityTaichungTaiwan, ROC
- Division of Allergy, Immunology and Rheumatology, Department of MedicineChung Shan Medical University HospitalTaichungTaiwan, ROC
| | - Hui‐Chih Hung
- Department of Life SciencesNational Chung Hsing UniversityTaichungTaiwan, ROC
- iEGG and Animal Biotechnology CenterNational Chung Hsing UniversityTaichungTaiwan, ROC
- Advanced Plant and Food Crop Biotechnology CenterNational Chung Hsing UniversityTaichungTaiwan, ROC
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5
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Shilenok I, Kobzeva K, Deykin A, Pokrovsky V, Patrakhanov E, Bushueva O. Obesity and Environmental Risk Factors Significantly Modify the Association between Ischemic Stroke and the Hero Chaperone C19orf53. Life (Basel) 2024; 14:1158. [PMID: 39337941 PMCID: PMC11433390 DOI: 10.3390/life14091158] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2024] [Revised: 09/07/2024] [Accepted: 09/10/2024] [Indexed: 09/30/2024] Open
Abstract
The unique chaperone-like properties of C19orf53, discovered in 2020 as a "hero" protein, make it an intriguing subject for research in relation to ischemic stroke (IS). Our pilot study aimed to investigate whether C19orf53 SNPs are associated with IS. DNA samples from 2138 Russian subjects (947 IS and 1308 controls) were genotyped for 7 C19orf53 SNPs using probe-based PCR. Dominant (D), recessive (R), and log-additive (A) regression models in relation to the effect alleles (EA) were used to interpret associations. An increased risk of IS was associated with rs10104 (EA G; Pbonf(R) = 0.0009; Pbonf(A) = 0.0004), rs11666524 (EA A; Pbonf(R) = 0.003; Pbonf(A) = 0.02), rs346158 (EA C; Pbonf(R) = 0.006; Pbonf(A) = 0.045), and rs2277947 (EA A; Pbonf(R) = 0.002; Pbonf(A) = 0.01) in patients with obesity; with rs11666524 (EA A; Pbonf(R) = 0.02), rs346157 (EA G; Pbonf(R) = 0.036), rs346158 (EA C; Pbonf(R) = 0.005), and rs2277947 (EA A; Pbonf(R) = 0.02) in patients with low fruit and vegetable intake; and with rs10104 (EA G; Pbonf(R) = 0.03) and rs11666524 (EA A; Pbonf(R) = 0.048) in patients with low physical activity. In conclusion, our pilot study provides comprehensive genetic and bioinformatic evidence of the involvement of C19orf53 in IS risk.
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Affiliation(s)
- Irina Shilenok
- Laboratory of Genomic Research, Research Institute for Genetic and Molecular Epidemiology, Kursk State Medical University, 305041 Kursk, Russia
- Division of Neurology, Kursk Emergency Hospital, 305035 Kursk, Russia
| | - Ksenia Kobzeva
- Laboratory of Genomic Research, Research Institute for Genetic and Molecular Epidemiology, Kursk State Medical University, 305041 Kursk, Russia
| | - Alexey Deykin
- Laboratory of Genome Editing for Biomedicine and Animal Health, Belgorod State National Research University, 308015 Belgorod, Russia
- Department of Pharmacology and Clinical Pharmacology, Belgorod State National Research University, 308015 Belgorod, Russia
| | - Vladimir Pokrovsky
- Laboratory of Genetic Technologies and Gene Editing for Biomedicine and Veterinary Medicine, Belgorod State National Research University, 308015 Belgorod, Russia
| | - Evgeny Patrakhanov
- Laboratory of Genetic Technologies and Gene Editing for Biomedicine and Veterinary Medicine, Belgorod State National Research University, 308015 Belgorod, Russia
| | - Olga Bushueva
- Laboratory of Genomic Research, Research Institute for Genetic and Molecular Epidemiology, Kursk State Medical University, 305041 Kursk, Russia
- Department of Biology, Medical Genetics and Ecology, Kursk State Medical University, 305041 Kursk, Russia
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6
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Ma X, Wang X, Chen F, Zou W, Ren J, Xin L, He P, Liang J, Xu Z, Dong C, Lan K, Wu S, Zhou HB. Novel Acyl Thiourea-Based Hydrophobic Tagging Degraders Exert Potent Anti-Influenza Activity through Two Distinct Endonuclease Polymerase Acidic-Targeted Degradation Pathways. J Med Chem 2024; 67:8791-8816. [PMID: 38775356 DOI: 10.1021/acs.jmedchem.4c00131] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2024]
Abstract
The spread of the influenza virus has caused devastating pandemics and huge economic losses worldwide. Antiviral drugs with diverse action modes are urgently required to overcome the challenges of viral mutation and drug resistance, and targeted protein degradation strategies constitute excellent candidates for this purpose. Herein, the first degradation of the influenza virus polymerase acidic (PA) protein using small-molecule degraders developed by hydrophobic tagging (HyT) technology to effectively combat the influenza virus was reported. The SAR results revealed that compound 19b with Boc2-(L)-Lys demonstrated excellent inhibitory activity against A/WSN/33/H1N1 (EC50 = 0.015 μM) and amantadine-resistant strain (A/PR/8/H1N1), low cytotoxicity, high selectivity, substantial degradation ability, and good drug-like properties. Mechanistic studies demonstrated that the proteasome system and autophagic lysosome pathway were the potential drivers of these HyT degraders. Thus, this study provides a powerful tool for investigating the targeted degradation of influenza virus proteins and for antiviral drug development.
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Affiliation(s)
- Xiaoyu Ma
- Department of Hematology, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China
| | - Xueyun Wang
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China
| | - Feifei Chen
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China
| | - Wenting Zou
- Department of Hematology, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China
| | - Junrui Ren
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China
| | - Lilan Xin
- Department of Hematology, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China
| | - Pei He
- Department of Hematology, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China
| | - Jinsen Liang
- Department of Hematology, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China
| | - Zhichao Xu
- Department of Hematology, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China
| | - Chune Dong
- Department of Hematology, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China
| | - Ke Lan
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China
| | - Shuwen Wu
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China
| | - Hai-Bing Zhou
- Department of Hematology, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China
- Frontier Science Center for Immunology and Metabolism, State Key Laboratory of Virology, Provincial Key Laboratory of Developmentally Originated Disease, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE) and Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Wuhan University, Wuhan 430071, China
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7
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Kamada Y, Tateishi H, Nakayamada U, Hinata D, Iwasaki A, Zhu J, Fukuda R, Okiyoneda T. UBE3C Facilitates the ER-Associated and Peripheral Degradation of Misfolded CFTR. Cells 2023; 12:2741. [PMID: 38067172 PMCID: PMC10706245 DOI: 10.3390/cells12232741] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2023] [Revised: 11/24/2023] [Accepted: 11/28/2023] [Indexed: 12/18/2023] Open
Abstract
The ubiquitin E3 ligase UBE3C promotes the proteasomal degradation of cytosolic proteins and endoplasmic reticulum (ER) membrane proteins. UBE3C is proposed to function downstream of the RNF185/MBRL ER-associated degradation (ERAD) branch, contributing to the ERAD of select membrane proteins. Here, we report that UBE3C facilitates the ERAD of misfolded CFTR, even in the absence of both RNF185 and its functional ortholog RNF5 (RNF5/185). Unlike RNF5/185, UBE3C had a limited impact on the ubiquitination of misfolded CFTR. UBE3C knockdown (KD) resulted in an additional increase in the functional ∆F508-CFTR channels on the plasma membrane when combined with the RNF5/185 ablation, particularly in the presence of clinically used CFTR modulators. Interestingly, although UBE3C KD failed to attenuate the ERAD of insig-1, it reduced the ERAD of misfolded ∆Y490-ABCB1 and increased cell surface expression. UBE3C KD also stabilized the mature form of ∆F508-CFTR and increased the cell surface level of T70-CFTR, a class VI CFTR mutant. These results suggest that UBE3C plays a vital role in the ERAD of misfolded CFTR and ABCB1, even within the RNF5/185-independent ERAD pathway, and it may also be involved in maintaining the peripheral quality control of CFTR.
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Affiliation(s)
| | | | | | | | | | | | | | - Tsukasa Okiyoneda
- Department of Biomedical Sciences, School of Biological and Environmental Sciences, Kwansei Gakuin University, Hyogo 669-1330, Japan; (Y.K.); (H.T.); (U.N.); (D.H.); (A.I.); (J.Z.); (R.F.)
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8
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Bialek W, Collawn JF, Bartoszewski R. Ubiquitin-Dependent and Independent Proteasomal Degradation in Host-Pathogen Interactions. Molecules 2023; 28:6740. [PMID: 37764516 PMCID: PMC10536765 DOI: 10.3390/molecules28186740] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Revised: 09/18/2023] [Accepted: 09/19/2023] [Indexed: 09/29/2023] Open
Abstract
Ubiquitin, a small protein, is well known for tagging target proteins through a cascade of enzymatic reactions that lead to protein degradation. The ubiquitin tag, apart from its signaling role, is paramount in destabilizing the modified protein. Here, we explore the complex role of ubiquitin-mediated protein destabilization in the intricate proteolysis process by the 26S proteasome. In addition, the significance of the so-called ubiquitin-independent pathway and the role of the 20S proteasome are considered. Next, we discuss the ubiquitin-proteasome system's interplay with pathogenic microorganisms and how the microorganisms manipulate this system to establish infection by a range of elaborate pathways to evade or counteract host responses. Finally, we focus on the mechanisms that rely either on (i) hijacking the host and on delivering pathogenic E3 ligases and deubiquitinases that promote the degradation of host proteins, or (ii) counteracting host responses through the stabilization of pathogenic effector proteins.
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Affiliation(s)
- Wojciech Bialek
- Department of Biophysics, Faculty of Biotechnology, University of Wrocław, 50-383 Wrocław, Poland
| | - James F. Collawn
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35233, USA;
| | - Rafal Bartoszewski
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35233, USA;
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9
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Kamoshita K, Ishii KA, Tahira Y, Kikuchi A, Abuduwaili H, Tajima-Shirasaki N, Li Q, Takayama H, Matsumoto K, Takamura T. Insulin Suppresses Ubiquitination via the Deubiquitinating Enzyme Ubiquitin-Specific Protease 14, Independent of Proteasome Activity in H4IIEC3 Hepatocytes. J Pharmacol Exp Ther 2023; 385:5-16. [PMID: 36328485 DOI: 10.1124/jpet.122.001088] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2022] [Revised: 08/16/2022] [Accepted: 09/09/2022] [Indexed: 11/06/2022] Open
Abstract
Ubiquitin-proteasome dysfunction contributes to obesity-related metabolic disorders, such as diabetes and fatty liver disease. However, the regulation of ubiquitin-proteasome activity by insulin remains to be elucidated. Here, we show that prolonged insulin stimulation activates proteasome function even though it reduces the ubiquitinated proteins in H4IIEC3 hepatocytes. Looking for a pathway by which insulin inhibits ubiquitination, we found that hepatic expression of ubiquitin-specific protease 14 (USP14) was upregulated in the liver of patients with insulin resistance. Indeed, the USP14-specific inhibitor IU1 canceled the insulin-mediated reduction of ubiquitinated proteins. Furthermore, insulin-induced endoplasmic reticulum (ER) stress, which was canceled by IU1, suggesting that USP14 activity is involved in insulin-induced ER stress. Co-stimulation with insulin and IU1 for 2 hours upregulated the nuclear translocation of the lipogenic transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), upregulated the expression of the lipogenic gene, fatty acid synthase (Fasn), and repressed the gluconeogenic genes. In conclusion, insulin activates proteasome function even though it inhibits protein ubiquitination by activating USP14 in hepatocytes. USP14 activation by insulin inhibits mature SREBP-1c while upregulating ER stress and the expression of genes involved in gluconeogenesis. Further understanding mechanisms underlying the USP14 activation and its pleiotropic effects may lead to therapeutic development for obesity-associated metabolic disorders, such as diabetes and fatty liver disease. SIGNIFICANCE STATEMENT: This study shows that insulin stimulation inhibits ubiquitination by activating USP14, independent of its effect on proteasome activity in hepatocytes. USP14 also downregulates the nuclear translocation of the lipogenic transcription factor SREBP-1c and upregulates the expression of genes involved in gluconeogenesis. Since USP14 is upregulated in the liver of insulin-resistant patients, understanding mechanisms underlying the USP14 activation and its pleiotropic effects will help develop treatments for metabolic disorders such as diabetes and fatty liver.
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Affiliation(s)
- Kyoko Kamoshita
- Department of Endocrinology and Metabolism, Graduate School of Medical Sciences (K.K., K.A.I., Y.T., A.K., H.A., N.T.S., Q.L., H.T., T.T.); Department of Integrative Medicine for Longevity, Graduate School of Medical Sciences (K.A.I.); Life Sciences Division, Engineering and Technology Department (H.T.); and Division of Tumor Dynamics and Regulation, Cancer Research Institute (K.M.), Kanazawa University, Kanazawa, Ishikawa, Japan
| | - Kiyo-Aki Ishii
- Department of Endocrinology and Metabolism, Graduate School of Medical Sciences (K.K., K.A.I., Y.T., A.K., H.A., N.T.S., Q.L., H.T., T.T.); Department of Integrative Medicine for Longevity, Graduate School of Medical Sciences (K.A.I.); Life Sciences Division, Engineering and Technology Department (H.T.); and Division of Tumor Dynamics and Regulation, Cancer Research Institute (K.M.), Kanazawa University, Kanazawa, Ishikawa, Japan
| | - Yumiko Tahira
- Department of Endocrinology and Metabolism, Graduate School of Medical Sciences (K.K., K.A.I., Y.T., A.K., H.A., N.T.S., Q.L., H.T., T.T.); Department of Integrative Medicine for Longevity, Graduate School of Medical Sciences (K.A.I.); Life Sciences Division, Engineering and Technology Department (H.T.); and Division of Tumor Dynamics and Regulation, Cancer Research Institute (K.M.), Kanazawa University, Kanazawa, Ishikawa, Japan
| | - Akihiro Kikuchi
- Department of Endocrinology and Metabolism, Graduate School of Medical Sciences (K.K., K.A.I., Y.T., A.K., H.A., N.T.S., Q.L., H.T., T.T.); Department of Integrative Medicine for Longevity, Graduate School of Medical Sciences (K.A.I.); Life Sciences Division, Engineering and Technology Department (H.T.); and Division of Tumor Dynamics and Regulation, Cancer Research Institute (K.M.), Kanazawa University, Kanazawa, Ishikawa, Japan
| | - Halimulati Abuduwaili
- Department of Endocrinology and Metabolism, Graduate School of Medical Sciences (K.K., K.A.I., Y.T., A.K., H.A., N.T.S., Q.L., H.T., T.T.); Department of Integrative Medicine for Longevity, Graduate School of Medical Sciences (K.A.I.); Life Sciences Division, Engineering and Technology Department (H.T.); and Division of Tumor Dynamics and Regulation, Cancer Research Institute (K.M.), Kanazawa University, Kanazawa, Ishikawa, Japan
| | - Natsumi Tajima-Shirasaki
- Department of Endocrinology and Metabolism, Graduate School of Medical Sciences (K.K., K.A.I., Y.T., A.K., H.A., N.T.S., Q.L., H.T., T.T.); Department of Integrative Medicine for Longevity, Graduate School of Medical Sciences (K.A.I.); Life Sciences Division, Engineering and Technology Department (H.T.); and Division of Tumor Dynamics and Regulation, Cancer Research Institute (K.M.), Kanazawa University, Kanazawa, Ishikawa, Japan
| | - Qifang Li
- Department of Endocrinology and Metabolism, Graduate School of Medical Sciences (K.K., K.A.I., Y.T., A.K., H.A., N.T.S., Q.L., H.T., T.T.); Department of Integrative Medicine for Longevity, Graduate School of Medical Sciences (K.A.I.); Life Sciences Division, Engineering and Technology Department (H.T.); and Division of Tumor Dynamics and Regulation, Cancer Research Institute (K.M.), Kanazawa University, Kanazawa, Ishikawa, Japan
| | - Hiroaki Takayama
- Department of Endocrinology and Metabolism, Graduate School of Medical Sciences (K.K., K.A.I., Y.T., A.K., H.A., N.T.S., Q.L., H.T., T.T.); Department of Integrative Medicine for Longevity, Graduate School of Medical Sciences (K.A.I.); Life Sciences Division, Engineering and Technology Department (H.T.); and Division of Tumor Dynamics and Regulation, Cancer Research Institute (K.M.), Kanazawa University, Kanazawa, Ishikawa, Japan
| | - Kunio Matsumoto
- Department of Endocrinology and Metabolism, Graduate School of Medical Sciences (K.K., K.A.I., Y.T., A.K., H.A., N.T.S., Q.L., H.T., T.T.); Department of Integrative Medicine for Longevity, Graduate School of Medical Sciences (K.A.I.); Life Sciences Division, Engineering and Technology Department (H.T.); and Division of Tumor Dynamics and Regulation, Cancer Research Institute (K.M.), Kanazawa University, Kanazawa, Ishikawa, Japan
| | - Toshinari Takamura
- Department of Endocrinology and Metabolism, Graduate School of Medical Sciences (K.K., K.A.I., Y.T., A.K., H.A., N.T.S., Q.L., H.T., T.T.); Department of Integrative Medicine for Longevity, Graduate School of Medical Sciences (K.A.I.); Life Sciences Division, Engineering and Technology Department (H.T.); and Division of Tumor Dynamics and Regulation, Cancer Research Institute (K.M.), Kanazawa University, Kanazawa, Ishikawa, Japan
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10
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Wegner J, Hunkler C, Ciupka K, Hartmann G, Schlee M. Increased IKKϵ protein stability ensures efficient type I interferon responses in conditions of TBK1 deficiency. Front Immunol 2023; 14:1073608. [PMID: 36936901 PMCID: PMC10020501 DOI: 10.3389/fimmu.2023.1073608] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2022] [Accepted: 02/20/2023] [Indexed: 03/06/2023] Open
Abstract
TBK1 and IKKϵ are related, crucial kinases in antiviral immune signaling pathways downstream of cytosolic nucleic acid receptors such as cGAS and RIG-I-like receptors. Upon activation, they phosphorylate the transcription factors IRF3 and IRF7 and thereby initiate the expression of type I interferons and antiviral effectors. While point mutation-induced loss of TBK1 kinase activity results in clinical hyper-susceptibility to viral infections, a complete lack of TBK1 expression in humans is unexpectedly not associated with diminished antiviral responses. Here, we provide a mechanistic explanation for these so-far unexplained observations by showing that TBK1 controls the protein expression of its related kinase IKKϵ in human myeloid cells. Mechanistically, TBK1 constitutively diminishes the protein stability of IKKϵ independent of TBK1 kinase activity but dependent on its interaction with the scaffold protein TANK. In consequence, depletion of TBK1 protein but not mutation-induced kinase deficiency induces the upregulation of IKKϵ. Due to the functional redundancy of the kinases in cGAS-STING and RIG-I-like receptor signaling in human myeloid cells, enhanced IKKϵ expression can compensate for the loss of TBK1. We show that IKKϵ upregulation is crucial to ensure unmitigated type I interferon production in conditions of TBK1 deficiency: While the type I interferon response to Listeria monocytogenes infection is maintained upon TBK1 loss, it is strongly diminished in cells harboring a kinase-deficient TBK1 variant, in which IKKϵ is not upregulated. Many pathogens induce TBK1 degradation, suggesting that loss of TBK1-mediated destabilization of IKKϵ is a critical backup mechanism to prevent diminished interferon responses upon TBK1 depletion.
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11
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He YY, Zhou HF, Chen L, Wang YT, Xie WL, Xu ZZ, Xiong Y, Feng YQ, Liu GY, Li X, Liu J, Wu QP. The Fra-1: Novel role in regulating extensive immune cell states and affecting inflammatory diseases. Front Immunol 2022; 13:954744. [PMID: 36032067 PMCID: PMC9404335 DOI: 10.3389/fimmu.2022.954744] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2022] [Accepted: 07/22/2022] [Indexed: 11/13/2022] Open
Abstract
Fra-1(Fos-related antigen1), a member of transcription factor activator protein (AP-1), plays an important role in cell proliferation, apoptosis, differentiation, inflammation, oncogenesis and tumor metastasis. Accumulating evidence suggest that the malignancy and invasive ability of tumors can be significantly changed by directly targeting Fra-1. Besides, the effects of Fra-1 are gradually revealed in immune and inflammatory settings, such as arthritis, pneumonia, psoriasis and cardiovascular disease. These regulatory mechanisms that orchestrate immune and non-immune cells underlie Fra-1 as a potential therapeutic target for a variety of human diseases. In this review, we focus on the current knowledge of Fra-1 in immune system, highlighting its unique importance in regulating tissue homeostasis. In addition, we also discuss the possible critical intervention strategy in diseases, which also outline future research and development avenues.
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12
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Hsiao JC, Neugroschl AR, Chui AJ, Taabazuing CY, Griswold AR, Wang Q, Huang HC, Orth-He EL, Ball DP, Hiotis G, Bachovchin DA. A ubiquitin-independent proteasome pathway controls activation of the CARD8 inflammasome. J Biol Chem 2022; 298:102032. [PMID: 35580636 PMCID: PMC9213247 DOI: 10.1016/j.jbc.2022.102032] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2022] [Revised: 04/28/2022] [Accepted: 04/29/2022] [Indexed: 11/26/2022] Open
Abstract
CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes constitutive autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 inhibitors, including Val-boroPro, accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin modification, controls activation of the CARD8 inflammasome. In unstressed cells, we discovered that the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. However, in Val-boroPro-stressed cells, we show the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Taken together, these results show that the susceptibility of the CARD8 NT domain to 20S proteasome-mediated degradation controls inflammasome activation.
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Affiliation(s)
- Jeffrey C Hsiao
- Pharmacology Program of the Weill Cornell Graduate School of Medical Sciences, Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Atara R Neugroschl
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Ashley J Chui
- Tri-Institutional PhD Program, Weill Cornell Medical College, Rockefeller University and Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Cornelius Y Taabazuing
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Andrew R Griswold
- Tri-Institutional MD-PhD Program, Weill Cornell Medical College, Rockefeller University and Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Qinghui Wang
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Hsin-Che Huang
- Tri-Institutional PhD Program, Weill Cornell Medical College, Rockefeller University and Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Elizabeth L Orth-He
- Tri-Institutional PhD Program, Weill Cornell Medical College, Rockefeller University and Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Daniel P Ball
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Giorgos Hiotis
- Tri-Institutional PhD Program, Weill Cornell Medical College, Rockefeller University and Memorial Sloan Kettering Cancer Center, New York, New York, USA
| | - Daniel A Bachovchin
- Pharmacology Program of the Weill Cornell Graduate School of Medical Sciences, Memorial Sloan Kettering Cancer Center, New York, New York, USA; Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA; Tri-Institutional PhD Program, Weill Cornell Medical College, Rockefeller University and Memorial Sloan Kettering Cancer Center, New York, New York, USA.
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13
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Emmert ME, Aggarwal P, Shay-Winkler K, Lee SJ, Goh Q, Cornwall R. Sex-specific role of myostatin signaling in neonatal muscle growth, denervation atrophy, and neuromuscular contractures. eLife 2022; 11:81121. [PMID: 36314781 PMCID: PMC9873256 DOI: 10.7554/elife.81121] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2022] [Accepted: 10/31/2022] [Indexed: 01/27/2023] Open
Abstract
Neonatal brachial plexus injury (NBPI) causes disabling and incurable muscle contractures that result from impaired longitudinal growth of denervated muscles. This deficit in muscle growth is driven by increased proteasome-mediated protein degradation, suggesting a dysregulation of muscle proteostasis. The myostatin (MSTN) pathway, a prominent muscle-specific regulator of proteostasis, is a putative signaling mechanism by which neonatal denervation could impair longitudinal muscle growth, and thus a potential target to prevent NBPI-induced contractures. Through a mouse model of NBPI, our present study revealed that pharmacologic inhibition of MSTN signaling induces hypertrophy, restores longitudinal growth, and prevents contractures in denervated muscles of female but not male mice, despite inducing hypertrophy of normally innervated muscles in both sexes. Additionally, the MSTN-dependent impairment of longitudinal muscle growth after NBPI in female mice is associated with perturbation of 20S proteasome activity, but not through alterations in canonical MSTN signaling pathways. These findings reveal a sex dimorphism in the regulation of neonatal longitudinal muscle growth and contractures, thereby providing insights into contracture pathophysiology, identifying a potential muscle-specific therapeutic target for contracture prevention, and underscoring the importance of sex as a biological variable in the pathophysiology of neuromuscular disorders.
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Affiliation(s)
- Marianne E Emmert
- Department of Medical Sciences, University of Cincinnati College of MedicineCincinnatiUnited States
| | - Parul Aggarwal
- Division of Orthopaedic Surgery, Cincinnati Children’s Hospital Medical CenterCincinnatiUnited States
| | - Kritton Shay-Winkler
- Division of Orthopaedic Surgery, Cincinnati Children’s Hospital Medical CenterCincinnatiUnited States
| | - Se-Jin Lee
- The Jackson LaboratoryFarmingtonUnited States,Department of Genetics and Genome Sciences, University of Connecticut School of MedicineFarmingtonUnited States
| | - Qingnian Goh
- Division of Orthopaedic Surgery, Cincinnati Children’s Hospital Medical CenterCincinnatiUnited States,Department of Orthopaedic Surgery, University of Cincinnati College of MedicineCincinnatiUnited States
| | - Roger Cornwall
- Division of Orthopaedic Surgery, Cincinnati Children’s Hospital Medical CenterCincinnatiUnited States,Department of Orthopaedic Surgery, University of Cincinnati College of MedicineCincinnatiUnited States,Division of Developmental Biology, Cincinnati Children’s Hospital Medical CenterCincinnatiUnited States,Department of Pediatrics, University of Cincinnati College of MedicineCincinnatiUnited States
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14
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Ayala-Marin YM, Grant AH, Rodriguez G, Kirken RA. Quadruple and Truncated MEK3 Mutants Identified from Acute Lymphoblastic Leukemia Promote Degradation and Enhance Proliferation. Int J Mol Sci 2021; 22:12210. [PMID: 34830095 PMCID: PMC8618549 DOI: 10.3390/ijms222212210] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Revised: 11/06/2021] [Accepted: 11/08/2021] [Indexed: 11/17/2022] Open
Abstract
Compared to other ethnicities, Hispanic children incur the highest rates of leukemia, and most cases are diagnosed as Acute Lymphoblastic Leukemia (ALL). Despite improved treatment and survival for ALL, disproportionate health outcomes in Hispanics persist. Thus, it is essential to identify oncogenic mutations within this demographic to aid in the development of new strategies to diagnose and treat ALL. Using whole-exome sequencing, five single nucleotide polymorphisms within mitogen-activated protein kinase 3 (MAP2K3) were identified in an ALL cancer patient library from the U.S./Mexico border. MAP2K3 R26T and P11T are located near the substrate-binding site, while R65L and R67W localized to the kinase domain. Truncated-MAP2K3 mutant Q73* was also identified. Transfection in HEK293 cells showed that the quadruple-MEK3 mutant (4M-MEK3) impacted protein stability, inducing degradation and reducing expression. The expression of 4M-MEK3 could be rescued by cysteine/serine protease inhibition, and proteasomal degradation of truncated-MEK3 occurred in a ubiquitin-independent manner. MEK3 mutants displayed reduced auto-phosphorylation and enzymatic activity, as seen by decreases in p38 phosphorylation. Furthermore, uncoupling of the MEK3/p38 signaling pathway resulted in less suppressive activity on HEK293 cell viability. Thus, disruption of MEK3 activation may promote proliferative signals in ALL. These findings suggest that MEK3 represents a potential therapeutic target for treating ALL.
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Affiliation(s)
| | | | | | - Robert A. Kirken
- Border Biomedical Research Center, Department of Biological Sciences, The University of Texas at El Paso, El Paso, TX 79968, USA; (Y.M.A.-M.); (A.H.G.); (G.R.)
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15
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Boughey H, Jurga M, El-Khamisy SF. DNA Homeostasis and Senescence: Lessons from the Naked Mole Rat. Int J Mol Sci 2021; 22:ijms22116011. [PMID: 34199458 PMCID: PMC8199619 DOI: 10.3390/ijms22116011] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2021] [Revised: 05/28/2021] [Accepted: 05/31/2021] [Indexed: 12/13/2022] Open
Abstract
As we age, our bodies accrue damage in the form of DNA mutations. These mutations lead to the generation of sub-optimal proteins, resulting in inadequate cellular homeostasis and senescence. The build-up of senescent cells negatively affects the local cellular micro-environment and drives ageing associated disease, including neurodegeneration. Therefore, limiting the accumulation of DNA damage is essential for healthy neuronal populations. The naked mole rats (NMR) are from eastern Africa and can live for over three decades in chronically hypoxic environments. Despite their long lifespan, NMRs show little to no biological decline, neurodegeneration, or senescence. Here, we discuss molecular pathways and adaptations that NMRs employ to maintain genome integrity and combat the physiological and pathological decline in organismal function.
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Affiliation(s)
- Harvey Boughey
- The Healthy Lifespan Institute and the Institute of Neuroscience, University of Sheffield, Sheffield S10 2TN, UK;
| | - Mateusz Jurga
- The Institute of Cancer Therapeutics, University of Bradford, Bradford BD7 1DP, UK;
| | - Sherif F. El-Khamisy
- The Healthy Lifespan Institute and the Institute of Neuroscience, University of Sheffield, Sheffield S10 2TN, UK;
- The Institute of Cancer Therapeutics, University of Bradford, Bradford BD7 1DP, UK;
- Correspondence: ; Tel.: +44-(0)-114-2222-791; Fax: +44-(0)-114-222-2850
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16
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Stevens LM, Kim G, Koromila T, Steele JW, McGehee J, Stathopoulos A, Stein DS. Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster. PLoS Genet 2021; 17:e1009544. [PMID: 33999957 PMCID: PMC8158876 DOI: 10.1371/journal.pgen.1009544] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2020] [Revised: 05/27/2021] [Accepted: 04/12/2021] [Indexed: 12/12/2022] Open
Abstract
Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo. Much of what we know about biological processes has come from the analysis of mutants whose loss-of-function phenotypes provide insight into their normal functions. However, for genes that are required for viability and which have multiple functions in the life of a cell or organism one can only observe mutant phenotypes produced up to the time of death. Normal functions performed in wild-type individuals later than the time of death of mutants cannot be observed. In one approach to overcoming this limitation, a class of peptide degradation signals (degrons) have been developed, which when fused to proteins-of-interest, can target those proteins for degradation in response to various stimuli (temperature, chemical agents, co-expressed proteins, or light). Here we describe a new inducible degron (the photo-N-degron or PND), which when fused to the N-terminus of a protein, can induce N-end rule-mediated degradation in response to blue-light illumination and have validated its use in both yeast and Drosophila embryos. Moreover, using the Drosophila embryonic patterning protein Cactus, we show that like the PND, the previously-described B-LID domain, but not the previously-described photosensitive degron (psd), can produce detectable light-inducible phenotypes in Drosophila embryos that are consistent with the role of Cactus in dorsal-ventral patterning.
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Affiliation(s)
- Leslie M. Stevens
- Department of Molecular Biosciences and Institute for Molecular and Cellular Biology, The University of Texas at Austin, Austin, Texas, United States of America
| | - Goheun Kim
- Department of Molecular Biosciences and Institute for Molecular and Cellular Biology, The University of Texas at Austin, Austin, Texas, United States of America
| | - Theodora Koromila
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, United States of America
| | - John W. Steele
- Department of Molecular Biosciences and Institute for Molecular and Cellular Biology, The University of Texas at Austin, Austin, Texas, United States of America
| | - James McGehee
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, United States of America
| | - Angelike Stathopoulos
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, United States of America
- * E-mail: (AS); (DSS)
| | - David S. Stein
- Department of Molecular Biosciences and Institute for Molecular and Cellular Biology, The University of Texas at Austin, Austin, Texas, United States of America
- * E-mail: (AS); (DSS)
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17
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Arlow T, Kim J, Haye-Bertolozzi JE, Martínez CB, Fay C, Zorensky E, Rose MD, Gammie AE. MutSα mismatch repair protein stability is governed by subunit interaction, acetylation, and ubiquitination. G3 (BETHESDA, MD.) 2021; 11:jkaa065. [PMID: 33793773 PMCID: PMC8063085 DOI: 10.1093/g3journal/jkaa065] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/29/2020] [Accepted: 12/14/2020] [Indexed: 11/13/2022]
Abstract
In eukaryotes, DNA mismatch recognition is accomplished by the highly conserved MutSα (Msh2/Msh6) and MutSβ (Msh2/Msh3) complexes. Previously, in the yeast Saccharomyces cerevisiae, we determined that deleting MSH6 caused wild-type Msh2 levels to drop by ∼50%. In this work, we determined that Msh6 steady-state levels are coupled to increasing or decreasing levels of Msh2. Although Msh6 and Msh2 are reciprocally regulated, Msh3 and Msh2 are not. Msh2 missense variants that are able to interact with Msh6 were destabilized when Msh6 was deleted; in contrast, variants that fail to dimerize were not further destabilized in cells lacking Msh6. In the absence of Msh6, Msh2 is turned over at a faster rate and degradation is mediated by the ubiquitin-proteasome pathway. Mutagenesis of certain conserved lysines near the dimer interface restored the levels of Msh2 in the absence of Msh6, further supporting a dimer stabilization mechanism. We identified two alternative forms of regulation both with the potential to act via lysine residues, including acetylation by Gcn5 and ubiquitination by the Not4 ligase. In the absence of Gcn5, Msh2 levels were significantly decreased; in contrast, deleting Not4 stabilized Msh2 and Msh2 missense variants with partial function. The stabilizing effect on Msh2 by either the presence of Msh6 or the absence of Not4 are dependent on Gcn5. Taken together, the results suggest that the wild-type MutSα mismatch repair protein stability is governed by subunit interaction, acetylation, and ubiquitination.
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Affiliation(s)
- Tim Arlow
- Ophthalmic Associates, Johnstown, PA
| | | | | | | | | | | | - Mark D. Rose
- Georgetown University, Georgetown, Washington D.C
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18
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Wen X, Ge X, Zhou L, Zhang Y, Guo X, Yang H. PRRSV Promotes MARC-145 Cells Entry Into S Phase of the Cell Cycle to Facilitate Viral Replication via Degradation of p21 by nsp11. Front Vet Sci 2021; 8:642095. [PMID: 33869322 PMCID: PMC8044838 DOI: 10.3389/fvets.2021.642095] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Accepted: 03/03/2021] [Indexed: 12/21/2022] Open
Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most economically significant pathogens that seriously affect the global swine industry. Despite sustained efforts, the factors that affect PRRSV replication in host cells are far from being fully elucidated and thus warrants further investigation. In this study, we first demonstrated that PRRSV infection can cause downregulation of endogenous p21 protein in MARC-145 cells in a virus dose-dependent manner. Next, we analyzed the effect of p21 knockdown by RNA interference on cell cycle progression using flow cytometric analysis, and found that knockdown of p21 promotes MARC-145 cells entry into S phase of the cell cycle. Interestingly, we further discovered PRRSV infection is also able to promote MARC-145 cells entry into the S phase. Subsequently, we synchronized MARC-145 cells into G0/G1, S and G2/M phases, respectively, and then determined PRRSV replication in these cells. Results here show that the MARC-145 cells synchronized into the S phase exhibited the highest viral titer among the cells synchronized to different phases. Additionally, to reliably analyze the potential role of endogenous p21 protein in PRRSV replication, we constructed a p21 gene-knockout MARC-145 cell line (p21-/-) using CRISPR/Cas9 technology and evaluated its capability to support PRRSV replication. Our results indicate that knockout of p21 is conducive to PRRSV replication in MARC-145 cells. Furthermore, through construction of a series of eukaryotic plasmids expressing each of individual PRRSV proteins combined with cell transfection, we demonstrated that the nonstructural protein 11 (nsp11) of PRRSV mediates p21 degradation, which was further confirmed by generating a stable MARC-145 cell line constitutively expressing nsp11 using a lentivirus system. Notably, we further demonstrated that the endoribonuclease activity rather than the deubiquitinating activity of nsp11 is essential for p21 degradation via mutagenic analysis. Finally, we demonstrated that nsp11 mediates p21 degradation via a ubiquitin-independent proteasomal degradation manner. Altogether, our study not only uncovers a new pathogenesis of PRRSV, but also provides new insights into development of novel antiviral strategies.
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Affiliation(s)
- Xuexia Wen
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China.,Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China
| | - Xinna Ge
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Lei Zhou
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Yongning Zhang
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Xin Guo
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Hanchun Yang
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, China
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19
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Palicharla VR, Gupta D, Bhattacharya D, Maddika S. Ubiquitin-independent proteasomal degradation of Spindlin-1 by the E3 ligase HACE1 contributes to cell-cell adhesion. FEBS Lett 2021; 595:491-506. [PMID: 33421097 DOI: 10.1002/1873-3468.14031] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2020] [Revised: 12/10/2020] [Accepted: 12/21/2020] [Indexed: 11/06/2022]
Abstract
HECT-E3 ligases play an essential role in catalyzing the transfer of ubiquitin to protein substrates. The noncatalytic roles of HECT-E3 ligases in cells are unknown. Here, we report that a HECT-E3 ligase, HACE1, functions as an adaptor independent of its E3 ligase activity. We identified Spindlin-1, a histone reader, as a new HACE1-associated protein. Interestingly, we found that HACE1 promotes Spindlin-1 degradation via the proteasome in an ubiquitination-independent manner. Functionally, we demonstrated that the loss of HACE1 results in weak cell-cell adhesion due to Spindlin-1-mediated accumulation of GDNF, a negative regulator of cell adhesion. Together, our data suggest that HACE1 acts as a molecular adaptor and plays an important noncatalytic role in presenting selected substrates directly to the proteasome for degradation.
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Affiliation(s)
- Vivek Reddy Palicharla
- Laboratory of Cell Death & Cell Survival, Centre for DNA Fingerprinting and Diagnostics (CDFD), Uppal, India
| | - Devanshi Gupta
- Laboratory of Cell Death & Cell Survival, Centre for DNA Fingerprinting and Diagnostics (CDFD), Uppal, India.,Graduate Studies, Regional Centre for Biotechnology, Faridabad, India
| | - Debjani Bhattacharya
- Laboratory of Cell Death & Cell Survival, Centre for DNA Fingerprinting and Diagnostics (CDFD), Uppal, India
| | - Subbareddy Maddika
- Laboratory of Cell Death & Cell Survival, Centre for DNA Fingerprinting and Diagnostics (CDFD), Uppal, India
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20
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Alquezar C, Arya S, Kao AW. Tau Post-translational Modifications: Dynamic Transformers of Tau Function, Degradation, and Aggregation. Front Neurol 2021; 11:595532. [PMID: 33488497 PMCID: PMC7817643 DOI: 10.3389/fneur.2020.595532] [Citation(s) in RCA: 172] [Impact Index Per Article: 43.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2020] [Accepted: 12/07/2020] [Indexed: 12/11/2022] Open
Abstract
Post-translational modifications (PTMs) on tau have long been recognized as affecting protein function and contributing to neurodegeneration. The explosion of information on potential and observed PTMs on tau provides an opportunity to better understand these modifications in the context of tau homeostasis, which becomes perturbed with aging and disease. Prevailing views regard tau as a protein that undergoes abnormal phosphorylation prior to its accumulation into the toxic aggregates implicated in Alzheimer's disease (AD) and other tauopathies. However, the phosphorylation of tau may, in fact, represent part of the normal but interrupted function and catabolism of the protein. In addition to phosphorylation, tau undergoes another forms of post-translational modification including (but not limited to), acetylation, ubiquitination, glycation, glycosylation, SUMOylation, methylation, oxidation, and nitration. A holistic appreciation of how these PTMs regulate tau during health and are potentially hijacked in disease remains elusive. Recent studies have reinforced the idea that PTMs play a critical role in tau localization, protein-protein interactions, maintenance of levels, and modifying aggregate structure. These studies also provide tantalizing clues into the possibility that neurons actively choose how tau is post-translationally modified, in potentially competitive and combinatorial ways, to achieve broad, cellular programs commensurate with the distinctive environmental conditions found during development, aging, stress, and disease. Here, we review tau PTMs and describe what is currently known about their functional impacts. In addition, we classify these PTMs from the perspectives of protein localization, electrostatics, and stability, which all contribute to normal tau function and homeostasis. Finally, we assess the potential impact of tau PTMs on tau solubility and aggregation. Tau occupies an undoubtedly important position in the biology of neurodegenerative diseases. This review aims to provide an integrated perspective of how post-translational modifications actively, purposefully, and dynamically remodel tau function, clearance, and aggregation. In doing so, we hope to enable a more comprehensive understanding of tau PTMs that will positively impact future studies.
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Affiliation(s)
| | | | - Aimee W. Kao
- Department of Neurology, Memory and Aging Center, University of California, San Francisco, San Francisco, CA, United States
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21
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Sorensen DW, Carreon D, Williams JM, Pearce WJ. Hypoxic modulation of fetal vascular MLCK abundance, localization, and function. Am J Physiol Regul Integr Comp Physiol 2021; 320:R1-R18. [PMID: 33112654 PMCID: PMC7847055 DOI: 10.1152/ajpregu.00212.2020] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2020] [Revised: 10/01/2020] [Accepted: 10/13/2020] [Indexed: 12/26/2022]
Abstract
Changes in vascular contractility are among the most important physiological effects of acute and chronic fetal hypoxia. Given the essential role of myosin light-chain kinase (MLCK) in smooth muscle contractility and its heterogeneous distribution, this study explores the hypothesis that subcellular changes in MLCK distribution contribute to hypoxic modulation of fetal carotid artery contractility. Relative to common carotid arteries from normoxic term fetal lambs (FN), carotids from fetal lambs gestated at high altitude (3,802 m) (FH) exhibited depressed contractility without changes in MLCK mRNA or protein abundance. Patterns of confocal colocalization of MLCK with α-actin and 20-kDa regulatory myosin light chain (MLC20) enabled calculation of subcellular MLCK fractions: 1) colocalized with the contractile apparatus, 2) colocalized with α-actin distant from the contractile apparatus, and 3) not colocalized with α-actin. Chronic hypoxia did not affect MLCK abundance in the contractile fraction, despite a concurrent decrease in contractility. Organ culture for 72 h under 1% O2 decreased total MLCK abundance in FN and FH carotid arteries, but decreased the contractile MLCK abundance only in FH carotid arteries. Correspondingly, culture under 1% O2 depressed contractility more in FH than FN carotid arteries. In addition, hypoxia appeared to attenuate ubiquitin-independent proteasomal degradation of MLCK, as reported for other proteins. In aggregate, these results demonstrate that the combination of chronic hypoxia followed by hypoxic culture can induce MLCK translocation among at least three subcellular fractions with possible influences on contractility, indicating that changes in MLCK distribution are a significant component of fetal vascular responses to hypoxia.
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Affiliation(s)
- Dane W Sorensen
- Divisions of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, California
| | - Desirelys Carreon
- Divisions of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, California
| | - James M Williams
- Divisions of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, California
| | - William J Pearce
- Divisions of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, California
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22
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Gomez-Perosanz M, Ras-Carmona A, Lafuente EM, Reche PA. Identification of CD8 + T cell epitopes through proteasome cleavage site predictions. BMC Bioinformatics 2020; 21:484. [PMID: 33308150 PMCID: PMC7733697 DOI: 10.1186/s12859-020-03782-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2020] [Accepted: 09/28/2020] [Indexed: 01/08/2023] Open
Abstract
Background We previously introduced PCPS (Proteasome Cleavage Prediction Server), a web-based tool to predict proteasome cleavage sites using n-grams. Here, we evaluated the ability of PCPS immunoproteasome cleavage model to discriminate CD8+ T cell epitopes. Results We first assembled an epitope dataset consisting of 844 unique virus-specific CD8+ T cell epitopes and their source proteins. We then analyzed cleavage predictions by PCPS immunoproteasome cleavage model on this dataset and compared them with those provided by a related method implemented by NetChop web server. PCPS was clearly superior to NetChop in term of sensitivity (0.89 vs. 0.79) but somewhat inferior with regard to specificity (0.55 vs. 0.60). Judging by the Mathew’s Correlation Coefficient, PCPS predictions were overall superior to those provided by NetChop (0.46 vs. 0.39). We next analyzed the power of C-terminal cleavage predictions provided by the same PCPS model to discriminate CD8+ T cell epitopes, finding that they could be discriminated from random peptides with an accuracy of 0.74. Following these results, we tuned the PCPS web server to predict CD8+ T cell epitopes and predicted the entire SARS-CoV-2 epitope space. Conclusions We report an improved version of PCPS named iPCPS for predicting proteasome cleavage sites and peptides with CD8+ T cell epitope features. iPCPS is available for free public use at https://imed.med.ucm.es/Tools/pcps/.
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Affiliation(s)
- Marta Gomez-Perosanz
- Laboratory of Immunomedicine, Department of Immunology, Faculty of Medicine, Complutense University of Madrid, Pza Ramon y Cajal, s/n, 28040, Madrid, Spain
| | - Alvaro Ras-Carmona
- Laboratory of Immunomedicine, Department of Immunology, Faculty of Medicine, Complutense University of Madrid, Pza Ramon y Cajal, s/n, 28040, Madrid, Spain
| | - Esther M Lafuente
- Laboratory of Immunomedicine, Department of Immunology, Faculty of Medicine, Complutense University of Madrid, Pza Ramon y Cajal, s/n, 28040, Madrid, Spain
| | - Pedro A Reche
- Laboratory of Immunomedicine, Department of Immunology, Faculty of Medicine, Complutense University of Madrid, Pza Ramon y Cajal, s/n, 28040, Madrid, Spain.
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23
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Blount JR, Johnson SL, Libohova K, Todi SV, Tsou WL. Degron capability of the hydrophobic C-terminus of the polyglutamine disease protein, ataxin-3. J Neurosci Res 2020; 98:2096-2108. [PMID: 32643791 DOI: 10.1002/jnr.24684] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2019] [Revised: 05/27/2020] [Accepted: 06/08/2020] [Indexed: 11/12/2022]
Abstract
Ataxin-3 is a deubiquitinase and polyglutamine disease protein whose cellular properties and functions are not entirely understood. Mutations in ataxin-3 cause spinocerebellar ataxia type 3 (SCA3), a neurodegenerative disorder that is a member of the polyglutamine family of diseases. Two major isoforms arise from alternative splicing of ATXN3 and are differently toxic in vivo as a result of faster proteasomal degradation of one isoform compared to the other. The isoforms vary only at their C-termini, suggesting that the hydrophobic C-terminus of the more quickly degraded form of ataxin-3 (here referred to as isoform 2) functions as a degron-that is, a peptide sequence that expedites the degradation of its host protein. We explored this notion in this study and present evidence that: (a) the C-terminus of ataxin-3 isoform 2 signals its degradation in a proteasome-dependent manner, (b) this effect from the C-terminus of isoform 2 does not require the ubiquitination of ataxin-3, and (c) the isolated C-terminus of isoform 2 can enhance the degradation of an unrelated protein. According to our data, the C-terminus of ataxin-3 isoform 2 is a degron, increasing overall understanding of the cellular properties of the SCA3 protein.
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Affiliation(s)
- Jessica R Blount
- Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA
| | - Sean L Johnson
- Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA
| | - Kozeta Libohova
- Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA
| | - Sokol V Todi
- Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA.,Department of Neurology, Wayne State University School of Medicine, Detroit, MI, USA
| | - Wei-Ling Tsou
- Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA
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24
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He W, Rao HBDP, Tang S, Bhagwat N, Kulkarni DS, Ma Y, Chang MAW, Hall C, Bragg JW, Manasca HS, Baker C, Verhees GF, Ranjha L, Chen X, Hollingsworth NM, Cejka P, Hunter N. Regulated Proteolysis of MutSγ Controls Meiotic Crossing Over. Mol Cell 2020; 78:168-183.e5. [PMID: 32130890 DOI: 10.1016/j.molcel.2020.02.001] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2019] [Revised: 01/03/2020] [Accepted: 01/31/2020] [Indexed: 01/04/2023]
Abstract
Crossover recombination is essential for accurate chromosome segregation during meiosis. The MutSγ complex, Msh4-Msh5, facilitates crossing over by binding and stabilizing nascent recombination intermediates. We show that these activities are governed by regulated proteolysis. MutSγ is initially inactive for crossing over due to an N-terminal degron on Msh4 that renders it unstable by directly targeting proteasomal degradation. Activation of MutSγ requires the Dbf4-dependent kinase Cdc7 (DDK), which directly phosphorylates and thereby neutralizes the Msh4 degron. Genetic requirements for Msh4 phosphorylation indicate that DDK targets MutSγ only after it has bound to nascent joint molecules (JMs) in the context of synapsing chromosomes. Overexpression studies confirm that the steady-state level of Msh4, not phosphorylation per se, is the critical determinant for crossing over. At the DNA level, Msh4 phosphorylation enables the formation and crossover-biased resolution of double-Holliday Junction intermediates. Our study establishes regulated protein degradation as a fundamental mechanism underlying meiotic crossing over.
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Affiliation(s)
- Wei He
- Howard Hughes Medical Institute, University of California, Davis, Davis, California, USA; Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - H B D Prasada Rao
- Howard Hughes Medical Institute, University of California, Davis, Davis, California, USA; Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - Shangming Tang
- Howard Hughes Medical Institute, University of California, Davis, Davis, California, USA; Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - Nikhil Bhagwat
- Howard Hughes Medical Institute, University of California, Davis, Davis, California, USA; Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - Dhananjaya S Kulkarni
- Howard Hughes Medical Institute, University of California, Davis, Davis, California, USA; Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - Yunmei Ma
- Howard Hughes Medical Institute, University of California, Davis, Davis, California, USA; Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - Maria A W Chang
- Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - Christie Hall
- Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - Junxi Wang Bragg
- Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - Harrison S Manasca
- Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - Christa Baker
- Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - Gerrik F Verhees
- Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA
| | - Lepakshi Ranjha
- Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland
| | - Xiangyu Chen
- Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA
| | - Nancy M Hollingsworth
- Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA
| | - Petr Cejka
- Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland
| | - Neil Hunter
- Howard Hughes Medical Institute, University of California, Davis, Davis, California, USA; Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA; Department of Molecular & Cellular Biology, University of California, Davis, Davis, California, USA; Department of Cell Biology & Human Anatomy, University of California, Davis, Davis, California, USA.
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25
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Yun SI, Hong HK, Yeo SY, Kim SH, Cho YB, Kim KK. Ubiquitin-Specific Protease 21 Promotes Colorectal Cancer Metastasis by Acting as a Fra-1 Deubiquitinase. Cancers (Basel) 2020; 12:cancers12010207. [PMID: 31947604 PMCID: PMC7017141 DOI: 10.3390/cancers12010207] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Revised: 01/02/2020] [Accepted: 01/04/2020] [Indexed: 12/22/2022] Open
Abstract
Fos-related-antigen-1 (Fra-1), a member of the activator protein-1 (AP-1) transcription factor superfamily, has an essential role in cancer progress and metastasis and Fra-1 is considered a therapeutic target in metastatic cancer including metastatic colorectal cancer (mCRC). However, its regulation at protein level has not yet been clearly elucidated. We found that ubiquitin-specific protease 21 (USP21) increases Fra-1 stability by deubiquitinating Fra-1 and enhances the expression of Fra-1 target genes in colon cancer cells. We also showed that USP21 controlled Fra-1-dependent migration and invasion activities. The oncogenic property of USP21 was confirmed by a significant reduction in liver metastasis when USP21-knockdown cancer cells were injected intrasplenically into mice. Consistently, clinicopathological analysis of colorectal cancer patients revealed a correlation of USP21 expression with high-grade carcinoma and life span. These results demonstrate that USP21 enhances Fra-1 stability and AP-1 target gene expression by deubiquitinating Fra-1. Therefore, USP21 is considered an attractive therapeutic target in mCRC with high Fra-1 expression.
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Affiliation(s)
- Sun-Il Yun
- Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon 16419, Korea;
| | - Hye Kyung Hong
- Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06531, Korea;
| | - So-Young Yeo
- Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06531, Korea;
| | - Seok-Hyung Kim
- Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06531, Korea;
- Samsung Medical Center, Department of Health Science and Technology, Samsung Advanced Institute for Health Science and Technology, Sungkyunkwan University School of Medicine, Seoul 06531, Korea
- Correspondence: (S.-H.K.); (Y.B.C.); (K.K.K.); Tel.: +82-02-3410-2898 (S.-H.K.); +82-02-3410-0217 (Y.B.C.); +82-031-299-6136 (K.K.K.)
| | - Yong Beom Cho
- Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06531, Korea;
- Samsung Medical Center, Department of Health Science and Technology, Samsung Advanced Institute for Health Science and Technology, Sungkyunkwan University School of Medicine, Seoul 06531, Korea
- Correspondence: (S.-H.K.); (Y.B.C.); (K.K.K.); Tel.: +82-02-3410-2898 (S.-H.K.); +82-02-3410-0217 (Y.B.C.); +82-031-299-6136 (K.K.K.)
| | - Kyeong Kyu Kim
- Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon 16419, Korea;
- Samsung Medical Center, Department of Health Science and Technology, Samsung Advanced Institute for Health Science and Technology, Sungkyunkwan University School of Medicine, Seoul 06531, Korea
- Correspondence: (S.-H.K.); (Y.B.C.); (K.K.K.); Tel.: +82-02-3410-2898 (S.-H.K.); +82-02-3410-0217 (Y.B.C.); +82-031-299-6136 (K.K.K.)
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26
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PTPN1/2-mediated dephosphorylation of MITA/STING promotes its 20S proteasomal degradation and attenuates innate antiviral response. Proc Natl Acad Sci U S A 2019; 116:20063-20069. [PMID: 31527250 DOI: 10.1073/pnas.1906431116] [Citation(s) in RCA: 56] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Upon cytosolic viral DNA stimulation, cGMP-AMP synthase (cGAS) catalyzes synthesis of 2'3'cGMP-AMP (cGAMP), which binds to the adaptor protein MITA (mediator of IRF3 activation, also called STING, stimulator of IFN genes) and induces innate antiviral response. How the activity of MITA/STING is regulated to avoid excessive innate immune response is not fully understood. Here we identified the tyrosine-protein phosphatase nonreceptor type (PTPN) 1 and 2 as MITA/STING-associated proteins. PTPN1 and PTPN2 are associated with MITA/STING following viral infection and dephosphorylate MITA/STING at Y245. Dephosphorylation of MITA/STING leads to its degradation via the ubiquitin-independent 20S proteasomal pathway, which is dependent on the intrinsically disordered region (IDR) of MITA/STING. Deficiencies of PTPN1 and PTPN2 enhance viral DNA-induced transcription of downstream antiviral genes and innate antiviral response. Our findings reveal that PTPN1/2-mediated dephosphorylation of MITA/STING and its degradation by the 20S proteasomal pathway is an important regulatory mechanism of innate immune response to DNA virus.
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27
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Senapati D, Kushwaha R, Dutta S, Maurya JP, Biswas S, Gangappa SN, Chattopadhyay S. COP1 regulates the stability of CAM7 to promote photomorphogenic growth. PLANT DIRECT 2019; 3:e00144. [PMID: 31245782 PMCID: PMC6593147 DOI: 10.1002/pld3.144] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/07/2018] [Revised: 04/18/2019] [Accepted: 05/07/2019] [Indexed: 05/31/2023]
Abstract
The unique member of the calmodulin gene family, Calmodulin7 (CAM7), plays a crucial role as transcriptional regulator to promote Arabidopsis seedling development. CAM7 regulates the expression of HY5, which is intimately involved in the promotion of photomorphogenic growth and light-regulated gene expression. COP1 ubiquitin ligase suppresses photomorphogenesis by degrading multiple photomorphogenesis promoting factors including HY5 in darkness. Genetic interaction studies, in this report, reveal that CAM7 and COP1 co-ordinately work to promote photomorphogenic growth and light-regulated gene expression at lower intensity of light. CAM7 physically interacts with COP1 in the nucleus. Further, in vivo study suggests that CAM7 and COP1 interaction is light intensity dependent. We have also shown that functional COP1 is required for optimum accumulation of CAM7 at lower fluences of light. Taken together, this study demonstrates the coordinated function of CAM7 and COP1 in Arabidopsis seedling development.
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Affiliation(s)
| | - Ritu Kushwaha
- Department of BiotechnologyNational Institute of TechnologyDurgapurIndia
| | - Siddhartha Dutta
- Department of BiotechnologyNational Institute of TechnologyDurgapurIndia
| | - Jay Prakash Maurya
- Department of BiotechnologyNational Institute of TechnologyDurgapurIndia
| | - Srabasthi Biswas
- Department of BiotechnologyNational Institute of TechnologyDurgapurIndia
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28
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Han K, Zhao D, Liu Y, Liu Q, Huang X, Yang J, Zhang L, Li Y. The ubiquitin-proteasome system is necessary for the replication of duck Tembusu virus. Microb Pathog 2019; 132:362-368. [PMID: 31054366 PMCID: PMC7126904 DOI: 10.1016/j.micpath.2019.04.044] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2019] [Revised: 04/18/2019] [Accepted: 04/30/2019] [Indexed: 01/18/2023]
Abstract
Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. The cellular factors required for DTMUV replication have been poorly studied. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates diverse cellular processes, including endocytosis and signal transduction, which may be involved in the entry of virus. In the present study, we explored the interplay between DTMUV replication and the UPS in BHK-21 cells and found that treatment with proteasome inhibitor (MG132 and lactacystin) significantly decreased the DTMUV progency at the early infection stage. We further revealed that inhibition of the UPS mainly occurs on the level of viral protein expression and RNA transcription. In addition, using specific siRNAs targeting ubiquitin reduces the production of viral progeny. In the presence of MG132 the staining for the envelope protein of DTMUV was dramatically reduced in comparison with the untreated control cells. Overall, our observations reveal an important role of the UPS in multiple steps of the DTMUV infection cycle and identify the UPS as a potential drug target to modulate the impact of DTMUV infection.
Treatment with proteasome inhibitor significantly decreased the DTMUV progency. Inhibition of the UPS mainly occurs on the level of viral protein expression and RNA transcription. Inhibit the expression of ubiquitin reduces the production of viral progeny.
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Affiliation(s)
- Kaikai Han
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, PR China
| | - Dongmin Zhao
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, PR China
| | - Yuzhuo Liu
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, PR China
| | - Qingtao Liu
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, PR China
| | - Xinmei Huang
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, PR China
| | - Jing Yang
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, PR China
| | - Lijiao Zhang
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, PR China
| | - Yin Li
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, PR China.
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29
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Holloway RW, Thomas ML, Cohen AM, Bharadwaj AG, Rahman M, Marcato P, Marignani PA, Waisman DM. Regulation of cell surface protease receptor S100A10 by retinoic acid therapy in acute promyelocytic leukemia (APL) ☆. Cell Death Dis 2018; 9:920. [PMID: 30206209 PMCID: PMC6134137 DOI: 10.1038/s41419-018-0954-6] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2018] [Accepted: 07/31/2018] [Indexed: 01/18/2023]
Abstract
S100A10 (p11), a member of the S100 family of small dimeric EF-hand-type Ca2+-binding proteins, plays a role in a variety of both intracellular and extracellular processes. Previous studies have suggested that p11 is intrinsically unstable and requires binding to annexin A2 (p36) to prevent its rapid ubiquitylation and degradation. Our laboratory has shown that p11 levels are stimulated by the expression of the oncoprotein, PML/RARα. Furthermore, treatment of the APL cell line, NB4 with all-trans retinoic acid (ATRA) causes the rapid loss of p36 and p11 protein. However, the mechanism by which ATRA regulates p11 levels has not been established. Here, we show that the proteasomal inhibitor, lactacystin reversed the ATRA-dependent loss of p11, but did not cause an accumulation of ubiquitylated forms of p11, suggesting that ATRA promotes the proteasomal degradation of p11 in an ubiquitin-independent manner. ATRA treatment of MCF-7 breast cancer cells reduced p11 but not p36 transcript and protein levels, thus indicating that ATRA can regulate p11 levels independently of PML/RARα and p36. Overexpression of p36 upregulated p11 protein but not mRNA levels, indicating that p36 affects p11 post translationally. The forced expression of ubiquitin and p11 in 293 T cells resulted in ubiquitylation of p11 that was blocked by mutagenesis of lysine 57. This study highlights the complex regulation of p11 by retinoid signaling and challenges the hypothesis that ubiquitin-mediated proteasomal degradation of p11 represents a universal mechanism of regulation of this protein.
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Affiliation(s)
- Ryan W Holloway
- Department of Pathology, Dalhousie University, Halifax, NS, B3H 1X5, Canada
| | - Margaret L Thomas
- Department of Pathology, Dalhousie University, Halifax, NS, B3H 1X5, Canada
| | - Alejandro M Cohen
- Proteomic Core Facility, Faculty of Medicine, Dalhousie University, Halifax, NS, B3H 1X5, Canada
| | | | - Mushfiqur Rahman
- Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, B3H 1X5, Canada
| | - Paola Marcato
- Department of Pathology, Dalhousie University, Halifax, NS, B3H 1X5, Canada.,Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, B3H 1X5, Canada
| | - Paola A Marignani
- Department of Pathology, Dalhousie University, Halifax, NS, B3H 1X5, Canada.,Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, B3H 1X5, Canada
| | - David M Waisman
- Department of Pathology, Dalhousie University, Halifax, NS, B3H 1X5, Canada. .,Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, B3H 1X5, Canada.
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30
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Reporter PET Images Bortezomib Treatment-Mediated Suppression of Cancer Cell Proteasome Activity. Sci Rep 2018; 8:12290. [PMID: 30116045 PMCID: PMC6095884 DOI: 10.1038/s41598-018-29642-w] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2018] [Accepted: 07/16/2018] [Indexed: 12/31/2022] Open
Abstract
Proteasomal protein degradation is a promising target for cancer therapy. Here, we developed a positron emission tomography (PET) technique based on the sodium-iodide symporter (NIS) gene fused with the carboxyl-terminal of ornithine decarboxylase (cODC) that noninvasively images cancer cells with inhibited proteasome activity. A retroviral vector was constructed in which the murine cODC degron was fused to the human NIS gene (NIS-cODC). Transiently transduced CT26 and HT29 colon cancer cells and stably expressing CT26/NIS-cODC cells were prepared. In cancer cells transiently transduced with NIS-cODC, NIS expression and transport activity was low at baseline, but NIS protein and 125I uptake was significantly increased by inhibition of proteasome activity with bortezomib. Stable CT26/NIS-cODC cells also showed increased cytosolic and membrane NIS by bortezomib, and four different stable clones displayed bortezomib dose-dependent stimulation of 125I and 99mTc-04− uptake. Importantly, bortezomib dose-dependently suppressed survival of CT26/NIS-cODC clones in a manner that closely correlated to the magnitudes of 125I and 99mTc-04− uptake. CT26/NIS-cODC tumors of bortezomib-treated mice demonstrated greater 124I uptake on PET images and increased NIS expression on tissue staining compared to vehicle-injected animals. NIS-cODC PET imaging may allow noninvasive quantitative monitoring of proteasome activity in cancer cells treated with bortezomib.
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31
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Mittenberg AG, Kuzyk VO, Shabelnikov SV, Gorbach DP, Shatrova AN, Fedorova OA, Barlev NA. Combined treatment of human multiple myeloma cells with bortezomib and doxorubicin alters the interactome of 20S proteasomes. Cell Cycle 2018; 17:1745-1756. [PMID: 30009671 DOI: 10.1080/15384101.2018.1496742] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
The proteasome is the key player in targeted degradation of cellular proteins and serves as a therapeutic target for treating several blood malignancies. Although in general, degradation of proteins via the proteasome requires their ubiquitination, a subset of proteins can be degraded independently of their ubiquitination by direct interaction with subunits of the 20S proteasome core. Thus, investigation of the proteasome-associated proteins may help identify novel targets of proteasome degradation and provide important insights into the mechanisms of malignant cell proteostasis. Here, using biochemical purification of proteasomes from multiple myeloma (MM) cells followed by mass-spectrometry we have uncovered 77 proteins in total that specifically interacted with the 20S proteasome via its PSMA3 subunit. Our GST pull-down assays followed by western blots validated the interactions identified by mass-spectrometry. Eleven proteins were confirmed to bind PSMA3 only upon apoptotic conditions induced by a combined treatment with the proteasome inhibitor, bortezomib, and genotoxic drug, doxorubicin. Nine of these eleven proteins contained bioinformatically predicted intrinsically disordered regions thus making them susceptible to ubiquitin-independent degradation. Importantly, among those proteins five interacted with the ubiquitin binding affinity matrix suggesting that these proteins may also be ubiquitinylated and hence degraded via the ubiquitin-dependent pathway. Collectively, these PSMA3-interacting proteins represent novel potential substrates for 20S proteasomes upon apoptosis. Furthermore, these data may shed light on the molecular mechanisms of cellular response to chemotherapy. ABBREVIATIONS BD: bortezomib/doxorubicin treatment; CDK: cyclin-dependent kinases; CHCA: α-cyanohydroxycinnamic acid; IDP: intrinsically disordered proteins; IDR: intrinsically disordered regions; IPG: immobilized pI gradient; MALDI TOF/TOF: matrix-assisted laser desorption/ionization time-of-flight tandem mass-spectrometry; MM: multiple myeloma; ODC: ornithine decarboxylase; PI: proteasomal inhibitors; PSMA: alpha-type 20S proteasome subunits; PTMs: post-translational modifications; SDS-PAGE: sodium dodecylsulphate polyacrylamide gel electrophoresis; UIP: ubiquitin-independent proteasomal proteolysis.
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Affiliation(s)
- Alexey G Mittenberg
- a Institute of Cytology of the Russian Academy of Sciences , St. Petersburg , Russia
| | - Valeria O Kuzyk
- a Institute of Cytology of the Russian Academy of Sciences , St. Petersburg , Russia
| | - Sergey V Shabelnikov
- a Institute of Cytology of the Russian Academy of Sciences , St. Petersburg , Russia
| | - Daria P Gorbach
- a Institute of Cytology of the Russian Academy of Sciences , St. Petersburg , Russia
| | - Alla N Shatrova
- a Institute of Cytology of the Russian Academy of Sciences , St. Petersburg , Russia
| | - Olga A Fedorova
- a Institute of Cytology of the Russian Academy of Sciences , St. Petersburg , Russia
| | - Nickolai A Barlev
- a Institute of Cytology of the Russian Academy of Sciences , St. Petersburg , Russia.,b Moscow Institute of Physics and Technology (State University) , Moscow Region , Dolgoprudny , Russia
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Khan QA, Pediaditakis P, Malakhau Y, Esmaeilniakooshkghazi A, Ashkavand Z, Sereda V, Krupenko NI, Krupenko SA. CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase. PLoS One 2018; 13:e0199699. [PMID: 29979702 PMCID: PMC6034817 DOI: 10.1371/journal.pone.0199699] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2017] [Accepted: 06/12/2018] [Indexed: 12/27/2022] Open
Abstract
ALDH1L1 is a folate-metabolizing enzyme abundant in liver and several other tissues. In human cancers and cell lines derived from malignant tumors, the ALDH1L1 gene is commonly silenced through the promoter methylation. It was suggested that ALDH1L1 limits proliferation capacity of the cell and thus functions as putative tumor suppressor. In contrast to cancer cells, mouse cell lines NIH3T3 and AML12 do express the ALDH1L1 protein. In the present study, we show that the levels of ALDH1L1 in these cell lines fluctuate throughout the cell cycle. During S-phase, ALDH1L1 is markedly down regulated at the protein level. As the cell cultures become confluent and cells experience increased contact inhibition, ALDH1L1 accumulates in the cells. In agreement with this finding, NIH3T3 cells arrested in G1/S-phase by a thymidine block completely lose the ALDH1L1 protein. Treatment with the proteasome inhibitor MG-132 prevents such loss in proliferating NIH3T3 cells, suggesting the proteasomal degradation of the ALDH1L1 protein. The co-localization of ALDH1L1 with proteasomes, demonstrated by confocal microscopy, supports this mechanism. We further show that ALDH1L1 interacts with the chaperone-dependent E3 ligase CHIP, which plays a key role in the ALDH1L1 ubiquitination and degradation. In NIH3T3 cells, silencing of CHIP by siRNA halts, while transient expression of CHIP promotes, the ALDH1L1 loss. The downregulation of ALDH1L1 is associated with the accumulation of the ALDH1L1 substrate 10-formyltetrahydrofolate, which is required for de novo purine biosynthesis, a key pathway activated in S-phase. Overall, our data indicate that CHIP-mediated proteasomal degradation of ALDH1L1 facilitates cellular proliferation.
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Affiliation(s)
- Qasim A. Khan
- Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, North Carolina, United States of America
| | - Peter Pediaditakis
- Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, North Carolina, United States of America
| | - Yuryi Malakhau
- Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, North Carolina, United States of America
| | - Amin Esmaeilniakooshkghazi
- Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, North Carolina, United States of America
| | - Zahra Ashkavand
- Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, North Carolina, United States of America
| | - Valentin Sereda
- Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, North Carolina, United States of America
| | - Natalia I. Krupenko
- Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, North Carolina, United States of America
- Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
| | - Sergey A. Krupenko
- Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, North Carolina, United States of America
- Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
- * E-mail:
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Njomen E, Osmulski PA, Jones CL, Gaczynska M, Tepe JJ. Small Molecule Modulation of Proteasome Assembly. Biochemistry 2018; 57:4214-4224. [PMID: 29897236 DOI: 10.1021/acs.biochem.8b00579] [Citation(s) in RCA: 50] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
The 20S proteasome is the main protease that directly targets intrinsically disordered proteins (IDPs) for proteolytic degradation. Mutations, oxidative stress, or aging can induce the buildup of IDPs resulting in incorrect signaling or aggregation, associated with the pathogenesis of many cancers and neurodegenerative diseases. Drugs that facilitate 20S-mediated proteolysis therefore have many potential therapeutic applications. We report herein the modulation of proteasome assembly by the small molecule TCH-165, resulting in an increase in 20S levels. The increase in the level of free 20S corresponds to enhanced proteolysis of IDPs, including α-synuclein, tau, ornithine decarboxylase, and c-Fos, but not structured proteins. Clearance of ubiquitinated protein was largely maintained by single capped proteasome complexes (19S-20S), but accumulation occurs when all 19S capped proteasome complexes are depleted. This study illustrates the first example of a small molecule capable of targeting disordered proteins for degradation by regulating the dynamic equilibrium between different proteasome complexes.
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Affiliation(s)
- Evert Njomen
- Department of Chemistry , Michigan State University , East Lansing , Michigan 48824 , United States
| | - Pawel A Osmulski
- Institute of Biotechnology , University of Texas Health Science Center at San Antonio , 15355 Lambda Drive , San Antonio , Texas 78245 , United States
| | - Corey L Jones
- Department of Chemistry , Michigan State University , East Lansing , Michigan 48824 , United States
| | - Maria Gaczynska
- Institute of Biotechnology , University of Texas Health Science Center at San Antonio , 15355 Lambda Drive , San Antonio , Texas 78245 , United States
| | - Jetze J Tepe
- Department of Chemistry , Michigan State University , East Lansing , Michigan 48824 , United States
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34
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Xin SH, Tan L, Cao X, Yu JT, Tan L. Clearance of Amyloid Beta and Tau in Alzheimer's Disease: from Mechanisms to Therapy. Neurotox Res 2018; 34:733-748. [PMID: 29626319 DOI: 10.1007/s12640-018-9895-1] [Citation(s) in RCA: 114] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2017] [Revised: 03/19/2018] [Accepted: 03/21/2018] [Indexed: 01/02/2023]
Abstract
Alzheimer's disease (AD) is the most common neurodegenerative disease. Pathological proteins of AD mainly contain amyloid-beta (Aβ) and tau. Their deposition will lead to neuron damage by a series of pathways, and then induce memory and cognitive impairment. Thus, it is pivotal to understand the clearance pathways of Aβ and tau in order to delay or even halt AD. Aβ clearance mechanisms include ubiquitin-proteasome system, autophagy-lysosome, proteases, microglial phagocytosis, and transport from the brain to the blood via the blood-brain barrier (BBB), arachnoid villi and blood-CSF barrier, which can be named blood circulatory clearance. Recently, lymphatic clearance has been demonstrated to play a key role in transport of Aβ into cervical lymph nodes. The discovery of meningeal lymphatic vessels is another direct evidence for lymphatic clearance in the brain. Furthermore, periphery clearance also contributes to Aβ clearance. Tau clearance is almost the same as Aβ clearance. In this review, we will mainly introduce the clearance mechanisms of Aβ and tau proteins, and summarize corresponding targeted drug therapies for AD.
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Affiliation(s)
- Shu-Hui Xin
- Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, Shandong, China
| | - Lin Tan
- Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, Shandong, China
| | - Xipeng Cao
- Clinical Research Center, Qingdao Municipal Hospital, Qingdao University, Qingdao, China
| | - Jin-Tai Yu
- Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, Shandong, China. .,Clinical Research Center, Qingdao Municipal Hospital, Qingdao University, Qingdao, China.
| | - Lan Tan
- Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No.5 Donghai Middle Road, Qingdao, 266071, Shandong, China.
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The Ubiquitin-Proteasome System Is Necessary for Efficient Replication of Human Astrovirus. J Virol 2018; 92:JVI.01809-17. [PMID: 29093085 DOI: 10.1128/jvi.01809-17] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2017] [Accepted: 10/18/2017] [Indexed: 12/25/2022] Open
Abstract
Astroviruses, members of the family Astroviridae, represent an important cause of human gastroenteritis in the world. The cellular factors required for astrovirus replication have been poorly studied. In this work, we evaluated the relevance of the ubiquitin-proteasome system (UPS) in the replication of Yuc8, a human astrovirus serotype 8 strain. We found that proteasome inhibitors decrease the production of infectious viral progeny at a step in the replication cycle subsequent to virus entry. The inhibition of proteasome activity decreases viral RNA levels and viral protein synthesis; similarly, the inhibition of ubiquitination by chemical inhibitors or RNA interference (RNAi) reduces the production of viral progeny as well as viral protein synthesis. The effect on viral progeny production induced by proteasome inhibitors is not explained by a reduction in the pool of monoubiquitin or the induction of early apoptosis or autophagy. Our observations are consistent with the need of the proteolytic activity of the UPS for the efficient replication of the virus and suggest that UPS is necessary for the production of genomic and subgenomic RNA but not for antigenomic RNA.IMPORTANCE Astroviruses are a major cause of gastroenteritis in young humans and animals, and recently, it was associated with fatal encephalitis in humans. The role of the ubiquitin-proteasome system in the replication of these viruses has not been studied previously. In this work, we present evidence that supports that the proteolytic activity of the proteasome is necessary for efficient viral progeny production and that this proteolytic system is required for the accumulation of both genomic and subgenomic viral RNAs.
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Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway. Int J Mol Sci 2017; 18:ijms18122667. [PMID: 29292768 PMCID: PMC5751269 DOI: 10.3390/ijms18122667] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2017] [Revised: 11/30/2017] [Accepted: 12/04/2017] [Indexed: 11/17/2022] Open
Abstract
Spinal muscular atrophy is due to mutations affecting the SMN1 gene coding for the full-length protein (survival motor neuron; SMN) and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMNΔ7) by alternative splicing. To study SMN and SMNΔ7 degradation in the cell, we have used tagged versions at the N- (Flag) or C-terminus (V5) of both proteins. Transfection of those constructs into HeLa cells and treatment with cycloheximide showed that those protein constructs were degraded. Proteasomal degradation usually requires prior lysine ubiquitylation. Surprisingly, lysine-less variants of both proteins tagged either at N- (Flag) or C-terminus (V5) were also degraded. The degradation of the endogenous SMN protein, and the protein constructs mentioned above, was mediated by the proteasome, as it was blocked by lactacystin, a specific and irreversible proteasomal inhibitor. The results obtained allowed us to conclude that SMN and SMNΔ7 proteasomal degradation did not absolutely require internal ubiquitylation nor N-terminal ubiquitylation (prevented by N-terminal tagging). While the above conclusions are firmly supported by the experimental data presented, we discuss and justify the need of deep proteomic techniques for the study of SMN complex components (orphan and bound) turn-over to understand the physiological relevant mechanisms of degradation of SMN and SMNΔ7 in the cell.
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van IJzendoorn DGP, Forghany Z, Liebelt F, Vertegaal AC, Jochemsen AG, Bovée JVMG, Szuhai K, Baker DA. Functional analyses of a human vascular tumor FOS variant identify a novel degradation mechanism and a link to tumorigenesis. J Biol Chem 2017; 292:21282-21290. [PMID: 29150442 DOI: 10.1074/jbc.c117.815845] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2017] [Revised: 11/03/2017] [Indexed: 11/06/2022] Open
Abstract
Epithelioid hemangioma is a locally aggressive vascular neoplasm, found in bones and soft tissue, whose cause is currently unknown, but may involve oncogene activation. FOS is one of the earliest viral oncogenes to be characterized, and normal cellular FOS forms part of the activator protein 1 (AP-1) transcription factor complex, which plays a pivotal role in cell growth, differentiation, and survival as well as the DNA damage response. Despite this, a causal link between aberrant FOS function and naturally occurring tumors has not yet been established. Here, we describe a thorough molecular and biochemical analysis of a mutant FOS protein we identified in these vascular tumors. The mutant protein lacks a highly conserved helix consisting of the C-terminal four amino acids of FOS, which we show is indispensable for fast, ubiquitin-independent FOS degradation via the 20S proteasome. Our work reveals that FOS stimulates endothelial sprouting and that perturbation of normal FOS degradation could account for the abnormal vessel growth typical of epithelioid hemangioma. To the best of our knowledge, this is the first functional characterization of mutant FOS proteins found in tumors.
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Affiliation(s)
| | - Zary Forghany
- Molecular Cell Biology, Leiden University Medical Center (LUMC), 2300 RC Leiden, The Netherlands
| | - Frauke Liebelt
- Molecular Cell Biology, Leiden University Medical Center (LUMC), 2300 RC Leiden, The Netherlands
| | - Alfred C Vertegaal
- Molecular Cell Biology, Leiden University Medical Center (LUMC), 2300 RC Leiden, The Netherlands
| | - Aart G Jochemsen
- Molecular Cell Biology, Leiden University Medical Center (LUMC), 2300 RC Leiden, The Netherlands
| | | | - Karoly Szuhai
- Molecular Cell Biology, Leiden University Medical Center (LUMC), 2300 RC Leiden, The Netherlands
| | - David A Baker
- Molecular Cell Biology, Leiden University Medical Center (LUMC), 2300 RC Leiden, The Netherlands
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38
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Wei J, Zanker D, Di Carluccio AR, Smelkinson MG, Takeda K, Seedhom MO, Dersh D, Gibbs JS, Yang N, Jadhav A, Chen W, Yewdell JW. Varied Role of Ubiquitylation in Generating MHC Class I Peptide Ligands. THE JOURNAL OF IMMUNOLOGY 2017; 198:3835-3845. [PMID: 28363906 DOI: 10.4049/jimmunol.1602122] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/20/2016] [Accepted: 03/08/2017] [Indexed: 12/11/2022]
Abstract
CD8+ T cell immunosurveillance is based on recognizing oligopeptides presented by MHC class I molecules. Despite decades of study, the importance of protein ubiquitylation to peptide generation remains uncertain. In this study, we examined the ability of MLN7243, a recently described ubiquitin-activating enzyme E1 inhibitor, to block overall cytosolic peptide generation and generation of specific peptides from vaccinia- and influenza A virus-encoded proteins. We show that MLN7243 rapidly inhibits ubiquitylation in a variety of cell lines and can profoundly reduce the generation of cytosolic peptides. Kinetic analysis of specific peptide generation reveals that ubiquitylation of defective ribosomal products is rate limiting in generating class I peptide complexes. More generally, our findings demonstrate that the requirement for ubiquitylation in MHC class I-restricted Ag processing varies with class I allomorph, cell type, source protein, and peptide context. Thus, ubiquitin-dependent and -independent pathways robustly contribute to MHC class I-based immunosurveillance.
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Affiliation(s)
- Jiajie Wei
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
| | - Damien Zanker
- T Cell Laboratory, School of Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia
| | - Anthony R Di Carluccio
- T Cell Laboratory, School of Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia
| | - Margery G Smelkinson
- Biological Imaging Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
| | - Kazuyo Takeda
- Microscopy and Imaging Core Facility, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993; and
| | - Mina O Seedhom
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
| | - Devin Dersh
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
| | - James S Gibbs
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
| | - Ning Yang
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
| | - Ajit Jadhav
- Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20892
| | - Weisan Chen
- T Cell Laboratory, School of Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia
| | - Jonathan W Yewdell
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
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Crow MS, Cristea IM. Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation. Mol Cell Proteomics 2017; 16:S200-S214. [PMID: 28077445 DOI: 10.1074/mcp.m116.064741] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2016] [Revised: 01/11/2017] [Indexed: 01/05/2023] Open
Abstract
The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral functions work in part via viral transcriptional suppression and that HSV-1 has acquired mechanisms to block its functions via proteasome-dependent degradation.
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Affiliation(s)
- Marni S Crow
- From the Lewis Thomas Laboratory, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
| | - Ileana M Cristea
- From the Lewis Thomas Laboratory, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
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40
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Shi Y, Long MJ, Rosenberg MM, Li S, Kobjack A, Lessans P, Coffey RT, Hedstrom L. Boc 3Arg-Linked Ligands Induce Degradation by Localizing Target Proteins to the 20S Proteasome. ACS Chem Biol 2016; 11:3328-3337. [PMID: 27704767 DOI: 10.1021/acschembio.6b00656] [Citation(s) in RCA: 57] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Targeted protein degradation is a promising strategy for drug design and functional assessment. Several small molecule approaches have been developed that localize target proteins to ubiquitin ligases, inducing ubiquitination and subsequent degradation by the 26S proteasome. We discovered that the degradation of a target protein can also be induced by a recognition ligand linked to tert-butyl carbamate (Boc3)-protected arginine (B3A). Here, we show that this process requires the proteasome but does not involve ubiquitination of the target protein. B3A does not perturb the structure of the target protein; instead, a B3A-ligand stabilizes its target protein. B3A ligands stimulate activity of purified 20S proteasome, demonstrating that the tag binds directly to the 20S proteasome. Moreover, purified 20S proteasome is sufficient to degrade target proteins in the presence of their respective B3A-linked recognition ligands. These observations suggest a simple model for B3A-mediated degradation wherein the B3A tag localizes target proteins directly to the 20S proteasome. Thus, B3A ligands are the first example of a ubiquitin-free strategy for targeted protein degradation.
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Affiliation(s)
- Yuntao Shi
- Graduate Program in Chemistry, ‡Graduate Program
in Biochemistry and Biophysics, §Department of Biology, ∥Graduate Program
in Molecular and Cell Biology, Brandeis University, Waltham, Massachusetts, United States
- Department of Biochemistry, #Department of Chemistry, Brandeis University, Waltham, Massachusetts, United States
| | - Marcus J.C. Long
- Graduate Program in Chemistry, ‡Graduate Program
in Biochemistry and Biophysics, §Department of Biology, ∥Graduate Program
in Molecular and Cell Biology, Brandeis University, Waltham, Massachusetts, United States
- Department of Biochemistry, #Department of Chemistry, Brandeis University, Waltham, Massachusetts, United States
| | - Masha M. Rosenberg
- Graduate Program in Chemistry, ‡Graduate Program
in Biochemistry and Biophysics, §Department of Biology, ∥Graduate Program
in Molecular and Cell Biology, Brandeis University, Waltham, Massachusetts, United States
- Department of Biochemistry, #Department of Chemistry, Brandeis University, Waltham, Massachusetts, United States
| | - Shican Li
- Graduate Program in Chemistry, ‡Graduate Program
in Biochemistry and Biophysics, §Department of Biology, ∥Graduate Program
in Molecular and Cell Biology, Brandeis University, Waltham, Massachusetts, United States
- Department of Biochemistry, #Department of Chemistry, Brandeis University, Waltham, Massachusetts, United States
| | - Aimee Kobjack
- Graduate Program in Chemistry, ‡Graduate Program
in Biochemistry and Biophysics, §Department of Biology, ∥Graduate Program
in Molecular and Cell Biology, Brandeis University, Waltham, Massachusetts, United States
- Department of Biochemistry, #Department of Chemistry, Brandeis University, Waltham, Massachusetts, United States
| | - Philip Lessans
- Graduate Program in Chemistry, ‡Graduate Program
in Biochemistry and Biophysics, §Department of Biology, ∥Graduate Program
in Molecular and Cell Biology, Brandeis University, Waltham, Massachusetts, United States
- Department of Biochemistry, #Department of Chemistry, Brandeis University, Waltham, Massachusetts, United States
| | - Rory T. Coffey
- Graduate Program in Chemistry, ‡Graduate Program
in Biochemistry and Biophysics, §Department of Biology, ∥Graduate Program
in Molecular and Cell Biology, Brandeis University, Waltham, Massachusetts, United States
- Department of Biochemistry, #Department of Chemistry, Brandeis University, Waltham, Massachusetts, United States
| | - Lizbeth Hedstrom
- Graduate Program in Chemistry, ‡Graduate Program
in Biochemistry and Biophysics, §Department of Biology, ∥Graduate Program
in Molecular and Cell Biology, Brandeis University, Waltham, Massachusetts, United States
- Department of Biochemistry, #Department of Chemistry, Brandeis University, Waltham, Massachusetts, United States
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41
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Regulation of energy homeostasis by the ubiquitin-independent REGγ proteasome. Nat Commun 2016; 7:12497. [PMID: 27511885 PMCID: PMC4987533 DOI: 10.1038/ncomms12497] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2016] [Accepted: 07/07/2016] [Indexed: 12/30/2022] Open
Abstract
Maintenance of energy homeostasis is essential for cell survival. Here, we report that the ATP- and ubiquitin-independent REGγ-proteasome system plays a role in maintaining energy homeostasis and cell survival during energy starvation via repressing rDNA transcription, a major intracellular energy-consuming process. Mechanistically, REGγ-proteasome limits cellular rDNA transcription and energy consumption by targeting the rDNA transcription activator SirT7 for ubiquitin-independent degradation under normal conditions. Moreover, energy starvation induces an AMPK-directed SirT7 phosphorylation and subsequent REGγ-dependent SirT7 subcellular redistribution and degradation, thereby further reducing rDNA transcription to save energy to overcome cell death. Energy starvation is a promising strategy for cancer therapy. Our report also shows that REGγ knockdown markedly improves the anti-tumour activity of energy metabolism inhibitors in mice. Our results underscore a control mechanism for an ubiquitin-independent process in maintaining energy homeostasis and cell viability under starvation conditions, suggesting that REGγ-proteasome inhibition has a potential to provide tumour-starving benefits. In conditions of energy stress cells reduce transcription of ribosomal RNA (rRNA) to maintain cell survival. Here, the authors show that energy stress induces an AMPK-dependent phosphorylation of Sirt7, which promotes its ubiquitin-independent degradation by REGγ, resulting in the down-regulation of rRNA transcription and cell survival.
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42
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Liu YC, Lee CY, Lin CL, Chen HY, Liu GY, Hung HC. Multifaceted interactions and regulation between antizyme and its interacting proteins cyclin D1, ornithine decarboxylase and antizyme inhibitor. Oncotarget 2016; 6:23917-29. [PMID: 26172301 PMCID: PMC4695161 DOI: 10.18632/oncotarget.4469] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2015] [Accepted: 06/16/2015] [Indexed: 11/25/2022] Open
Abstract
Ornithine decarboxylase (ODC), cyclin D1 (CCND1) and antizyme inhibitor (AZI) promote cell growth. ODC and CCND1 can be degraded through antizyme (AZ)-mediated 26S proteasomal degradation. This paper describes a mechanistic study of the molecular interactions between AZ and its interacting proteins. The dissociation constant (Kd) of the binary AZ-CCND1 complex and the respective binding sites of AZ and CCND1 were determined. Our data indicate that CCND1 has a 4-fold lower binding affinity for AZ than does ODC and an approximately 40-fold lower binding affinity for AZ than does AZI. The Kd values of AZ-CCND1, AZ-ODC and AZ-AZI were 0.81, 0.21 and 0.02 μM, respectively. Furthermore, the Kd values for CCND1 binding to the AZ N-terminal peptide (AZ34–124) and AZ C-terminal peptide (AZ100–228) were 0.92 and 8.97 μM, respectively, indicating that the binding site of CCND1 may reside at the N-terminus of AZ, rather than the C-terminus. Our data also show that the ODC-AZ-CCND1 ternary complex may exist in equilibrium. The Kd values of the [AZ-CCND1]-ODC and [AZ-ODC]-CCND1 complexes were 1.26 and 4.93 μM, respectively. This is the first paper to report the reciprocal regulation of CCND1 and ODC through AZ-dependent 26S proteasomal degradation.
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Affiliation(s)
- Yen-Chin Liu
- Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan
| | - Chien-Yun Lee
- Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan.,Graduate Institute of Biotechnology, National Chung-Hsing University (NCHU), Taichung, Taiwan.,Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan
| | - Chi-Li Lin
- Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan
| | - Hui-Yi Chen
- Biotechnology Center, National Chung-Hsing University (NCHU), Taichung, Taiwan.,Agricultural Biotechnology Center (ABC), National Chung-Hsing University (NCHU), Taichung, Taiwan
| | - Guang-Yaw Liu
- Institute of Microbiology & Immunology, Chung Shan Medical University, Taichung, Taiwan.,Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan
| | - Hui-Chih Hung
- Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan.,Agricultural Biotechnology Center (ABC), National Chung-Hsing University (NCHU), Taichung, Taiwan.,Institute of Genomics and Bioinformatics, National Chung Hsing University (NCHU), Taichung, Taiwan
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43
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Curcumin inhibits HIV-1 by promoting Tat protein degradation. Sci Rep 2016; 6:27539. [PMID: 27283735 PMCID: PMC4901322 DOI: 10.1038/srep27539] [Citation(s) in RCA: 76] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2016] [Accepted: 05/09/2016] [Indexed: 02/07/2023] Open
Abstract
HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity.
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44
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Paci A, Liu PXH, Zhang L, Zhao R. The Proteasome Subunit Rpn8 Interacts with the Small Nucleolar RNA Protein (snoRNP) Assembly Protein Pih1 and Mediates Its Ubiquitin-independent Degradation in Saccharomyces cerevisiae. J Biol Chem 2016; 291:11761-75. [PMID: 27053109 DOI: 10.1074/jbc.m115.702043] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2015] [Indexed: 11/06/2022] Open
Abstract
Pih1 is a scaffold protein of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) protein complex, which is conserved in fungi and animals. The chaperone-like activity of the R2TP complex has been implicated in the assembly of multiple protein complexes, such as the small nucleolar RNA protein complex. However, the mechanism of the R2TP complex activity in vivo and the assembly of the complex itself are still largely unknown. Pih1 is an unstable protein and tends to aggregate when expressed alone. The C-terminal fragment of Pih1 contains multiple destabilization factors and acts as a degron when fused to other proteins. In this study, we investigated Pih1 interactors and identified a specific interaction between Pih1 and the proteasome subunit Rpn8 in yeast Saccharomyces cerevisiae when HSP90 co-chaperone Tah1 is depleted. By analyzing truncation mutants, we identified that the C-terminal 30 amino acids of Rpn8 are sufficient for the binding to Pih1 C terminus. With in vitro and in vivo degradation assays, we showed that the Pih1 C-terminal fragment Pih1(282-344) is able to induce a ubiquitin-independent degradation of GFP. Additionally, we demonstrated that truncation of the Rpn8 C-terminal disordered region does not affect proteasome assembly but specifically inhibits the degradation of the GFP-Pih1(282-344) fusion protein in vivo and Pih1 in vitro We propose that Pih1 is a ubiquitin-independent proteasome substrate, and the direct interaction with Rpn8 C terminus mediates its proteasomal degradation.
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Affiliation(s)
- Alexandr Paci
- From the Department of Biological Sciences, University of Toronto, Toronto, Ontario M1C 1A4, Canada
| | - Peter X H Liu
- From the Department of Biological Sciences, University of Toronto, Toronto, Ontario M1C 1A4, Canada
| | - Lingjie Zhang
- From the Department of Biological Sciences, University of Toronto, Toronto, Ontario M1C 1A4, Canada
| | - Rongmin Zhao
- From the Department of Biological Sciences, University of Toronto, Toronto, Ontario M1C 1A4, Canada
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45
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Guharoy M, Bhowmick P, Tompa P. Design Principles Involving Protein Disorder Facilitate Specific Substrate Selection and Degradation by the Ubiquitin-Proteasome System. J Biol Chem 2016; 291:6723-31. [PMID: 26851277 DOI: 10.1074/jbc.r115.692665] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
The ubiquitin-proteasome system (UPS) regulates diverse cellular pathways by the timely removal (or processing) of proteins. Here we review the role of structural disorder and conformational flexibility in the different aspects of degradation. First, we discuss post-translational modifications within disordered regions that regulate E3 ligase localization, conformation, and enzymatic activity, and also the role of flexible linkers in mediating ubiquitin transfer and reaction processivity. Next we review well studied substrates and discuss that substrate elements (degrons) recognized by E3 ligases are highly disordered: short linear motifs recognized by many E3s constitute an important class of degrons, and these are almost always present in disordered regions. Substrate lysines targeted for ubiquitination are also often located in neighboring regions of the E3 docking motifs and are therefore part of the disordered segment. Finally, biochemical experiments and predictions show that initiation of degradation at the 26S proteasome requires a partially unfolded region to facilitate substrate entry into the proteasomal core.
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Affiliation(s)
- Mainak Guharoy
- From the VIB Structural Biology Research Center (SBRC), Vlaams Instituut voor Biotechnologie, 1050 Brussel, Belgium, the Structural Biology Brussels (SBB), Vrije Universiteit Brussel, 1050 Brussels, Belgium, and
| | - Pallab Bhowmick
- From the VIB Structural Biology Research Center (SBRC), Vlaams Instituut voor Biotechnologie, 1050 Brussel, Belgium, the Structural Biology Brussels (SBB), Vrije Universiteit Brussel, 1050 Brussels, Belgium, and
| | - Peter Tompa
- From the VIB Structural Biology Research Center (SBRC), Vlaams Instituut voor Biotechnologie, 1050 Brussel, Belgium, the Structural Biology Brussels (SBB), Vrije Universiteit Brussel, 1050 Brussels, Belgium, and the Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, 1117 Budapest, Hungary
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46
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Control of Pim2 kinase stability and expression in transformed human haematopoietic cells. Biosci Rep 2015; 35:BSR20150217. [PMID: 26500282 PMCID: PMC4672348 DOI: 10.1042/bsr20150217] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2015] [Accepted: 10/05/2015] [Indexed: 01/02/2023] Open
Abstract
The oncogenic Pim2 kinase is overexpressed in several haematological malignancies, such as multiple myeloma and acute myeloid leukaemia (AML), and constitutes a strong therapeutic target candidate. Like other Pim kinases, Pim2 is constitutively active and is believed to be essentially regulated through its accumulation. We show that in leukaemic cells, the three Pim2 isoforms have dramatically short half-lives although the longer isoform is significantly more stable than the shorter isoforms. All isoforms present a cytoplasmic localization and their degradation was neither modified by broad-spectrum kinase or phosphatase inhibitors such as staurosporine or okadaic acid nor by specific inhibition of several intracellular signalling pathways including Erk, Akt and mTORC1. Pim2 degradation was inhibited by proteasome inhibitors but Pim2 ubiquitination was not detected even by blocking both proteasome activity and protein de-ubiquitinases (DUBs). Moreover, Pyr41, an ubiquitin-activating enzyme (E1) inhibitor, did not stabilize Pim2, strongly suggesting that Pim2 was degraded by the proteasome without ubiquitination. In agreement, we observed that purified 20S proteasome particles could degrade Pim2 molecule in vitro. Pim2 mRNA accumulation in UT7 cells was controlled by erythropoietin (Epo) through STAT5 transcription factors. In contrast, the translation of Pim2 mRNA was not regulated by mTORC1. Overall, our results suggest that Pim2 is only controlled by its mRNA accumulation level. Catalytically active Pim2 accumulated in proteasome inhibitor-treated myeloma cells. We show that Pim2 inhibitors and proteasome inhibitors, such as bortezomib, have additive effects to inhibit the growth of myeloma cells, suggesting that Pim2 could be an interesting target for the treatment of multiple myeloma.
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Tsvetkov P, Mendillo ML, Zhao J, Carette JE, Merrill PH, Cikes D, Varadarajan M, van Diemen FR, Penninger JM, Goldberg AL, Brummelkamp TR, Santagata S, Lindquist S. Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome. eLife 2015; 4. [PMID: 26327695 PMCID: PMC4551903 DOI: 10.7554/elife.08467] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2015] [Accepted: 07/29/2015] [Indexed: 12/11/2022] Open
Abstract
Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans.
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Affiliation(s)
- Peter Tsvetkov
- Whitehead Institute for Biomedical Research, Cambridge, United States
| | - Marc L Mendillo
- Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States
| | - Jinghui Zhao
- Department of Cell Biology, Harvard Medical School, Boston, United States
| | - Jan E Carette
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, United States
| | - Parker H Merrill
- Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, United States
| | - Domagoj Cikes
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria
| | | | - Ferdy R van Diemen
- Department of Biochemistry, Netherlands Cancer Institute, Amsterdam, Netherlands
| | - Josef M Penninger
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria
| | - Alfred L Goldberg
- Department of Cell Biology, Harvard Medical School, Boston, United States
| | - Thijn R Brummelkamp
- Department of Biochemistry, Netherlands Cancer Institute, Amsterdam, Netherlands
| | - Sandro Santagata
- Whitehead Institute for Biomedical Research, Cambridge, United States
| | - Susan Lindquist
- Whitehead Institute for Biomedical Research, Cambridge, United States
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48
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Fortmann KT, Lewis RD, Ngo KA, Fagerlund R, Hoffmann A. A Regulated, Ubiquitin-Independent Degron in IκBα. J Mol Biol 2015; 427:2748-56. [PMID: 26191773 DOI: 10.1016/j.jmb.2015.07.008] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2014] [Revised: 06/12/2015] [Accepted: 07/13/2015] [Indexed: 12/30/2022]
Abstract
Whereas ubiquitin-dependent degrons have been characterized in some detail, how proteins may be targeted to ubiquitin-independent proteasomal degradation remains unclear. Here we show that IκBα contains an ubiquitin-independent degron whose activity is portable to heterologous proteins such as the globular protein GFP (green fluorescent protein) via a proteasome-dependent, ubiquitin-independent, non-lysosomal pathway. The ubiquitin-independent degradation signal resides in an 11-amino-acid sequence, which is not only sufficient but also required for IκBα's short half-life. Finally, we show that this degron's activity is regulated by the interaction with NFκB, which controls its solvent exposure, and we demonstrate that this regulation of the degron's activity is critical for IκBα's signaling functions.
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Affiliation(s)
- Karen T Fortmann
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA
| | - Russell D Lewis
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA
| | - Kim A Ngo
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA; Institute for Quantitative and Computational Biosciences, Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Riku Fagerlund
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA
| | - Alexander Hoffmann
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA; Institute for Quantitative and Computational Biosciences, Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
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49
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Pereira-Neves A, Gonzaga L, Menna-Barreto RFS, Benchimol M. Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form. PLoS One 2015; 10:e0129165. [PMID: 26047503 PMCID: PMC4457923 DOI: 10.1371/journal.pone.0129165] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2015] [Accepted: 05/05/2015] [Indexed: 11/30/2022] Open
Abstract
Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in T. foetus.
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MESH Headings
- Acetylcysteine/analogs & derivatives
- Acetylcysteine/pharmacology
- Amino Acid Sequence
- Blotting, Western
- Cell Cycle
- Cysteine Proteinase Inhibitors/pharmacology
- Endoplasmic Reticulum/drug effects
- Endoplasmic Reticulum/metabolism
- Endoplasmic Reticulum/ultrastructure
- Flagella/metabolism
- Flagella/ultrastructure
- Life Cycle Stages/drug effects
- Microscopy, Electron, Scanning
- Microscopy, Electron, Transmission
- Microscopy, Fluorescence
- Molecular Sequence Data
- Phylogeny
- Proteasome Endopeptidase Complex/classification
- Proteasome Endopeptidase Complex/genetics
- Proteasome Endopeptidase Complex/metabolism
- Protein Subunits/antagonists & inhibitors
- Protein Subunits/genetics
- Protein Subunits/metabolism
- Protozoan Proteins/genetics
- Protozoan Proteins/metabolism
- Protozoan Proteins/ultrastructure
- Sequence Homology, Amino Acid
- Spores, Protozoan/drug effects
- Spores, Protozoan/metabolism
- Spores, Protozoan/ultrastructure
- Tritrichomonas foetus/genetics
- Tritrichomonas foetus/growth & development
- Tritrichomonas foetus/metabolism
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Affiliation(s)
- Antonio Pereira-Neves
- Programa de Pós-graduação em Ciências Morfológicas, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
- Fiocruz, Centro de Pesquisa Aggeu Magalhães, Departamento de Microbiologia, Laboratório de Microbiologia e Biologia Celular, Recife, PE, Brazil
| | - Luiz Gonzaga
- Laboratório Nacional de Computação Cientifica (LNCC/MCT), Petrópolis, RJ, Brazil
| | | | - Marlene Benchimol
- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
- UNIGRANRIO- Universidade do Grande Rio, Duque de Caxias, RJ, Brazil
- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
- * E-mail:
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50
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Goitea VE, Hallak ME. Calreticulin and Arginylated Calreticulin Have Different Susceptibilities to Proteasomal Degradation. J Biol Chem 2015; 290:16403-14. [PMID: 25969538 DOI: 10.1074/jbc.m114.626127] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2014] [Indexed: 12/31/2022] Open
Abstract
Post-translational arginylation has been suggested to target proteins for proteasomal degradation. The degradation mechanism for arginylated calreticulin (R-CRT) localized in the cytoplasm is unknown. To evaluate the effect of arginylation on CRT stability, we examined the metabolic fates and degradation mechanisms of cytoplasmic CRT and R-CRT in NIH 3T3 and CHO cells. Both CRT isoforms were found to be proteasomal substrates, but the half-life of R-CRT (2 h) was longer than that of cytoplasmic CRT (0.7 h). Arginylation was not required for proteasomal degradation of CRT, although R-CRT displays ubiquitin modification. A CRT mutant incapable of dimerization showed reduced metabolic stability of R-CRT, indicating that R-CRT dimerization may protect it from proteasomal degradation. Our findings, taken together, demonstrate a novel function of arginylation: increasing the half-life of CRT in cytoplasm.
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Affiliation(s)
- Victor E Goitea
- From the Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), Consejo Nacional de Investigaciones Científicas y Técnicas, and Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba X5000HUA, Argentina
| | - Marta E Hallak
- From the Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), Consejo Nacional de Investigaciones Científicas y Técnicas, and Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba X5000HUA, Argentina
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