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Waltho A, Popp O, Lenz C, Pluska L, Lambert M, Dötsch V, Mertins P, Sommer T. K48- and K63-linked ubiquitin chain interactome reveals branch- and length-specific ubiquitin interactors. Life Sci Alliance 2024; 7:e202402740. [PMID: 38803224 PMCID: PMC11109483 DOI: 10.26508/lsa.202402740] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2024] [Revised: 05/08/2024] [Accepted: 05/08/2024] [Indexed: 05/29/2024] Open
Abstract
The ubiquitin (Ub) code denotes the complex Ub architectures, including Ub chains of different lengths, linkage types, and linkage combinations, which enable ubiquitination to control a wide range of protein fates. Although many linkage-specific interactors have been described, how interactors are able to decode more complex architectures is not fully understood. We conducted a Ub interactor screen, in humans and yeast, using Ub chains of varying lengths, as well as homotypic and heterotypic branched chains of the two most abundant linkage types-lysine 48-linked (K48) and lysine 63-linked (K63) Ub. We identified some of the first K48/K63-linked branch-specific Ub interactors, including histone ADP-ribosyltransferase PARP10/ARTD10, E3 ligase UBR4, and huntingtin-interacting protein HIP1. Furthermore, we revealed the importance of chain length by identifying interactors with a preference for Ub3 over Ub2 chains, including Ub-directed endoprotease DDI2, autophagy receptor CCDC50, and p97 adaptor FAF1. Crucially, we compared datasets collected using two common deubiquitinase inhibitors-chloroacetamide and N-ethylmaleimide. This revealed inhibitor-dependent interactors, highlighting the importance of inhibitor consideration during pulldown studies. This dataset is a key resource for understanding how the Ub code is read.
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Affiliation(s)
- Anita Waltho
- Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
- Institute for Biology, Humboldt-University zu Berlin, Berlin, Germany
| | - Oliver Popp
- Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
| | - Christopher Lenz
- Institute for Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt, Germany
| | - Lukas Pluska
- Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
- Institute for Biology, Humboldt-University zu Berlin, Berlin, Germany
| | - Mahil Lambert
- Institute for Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt, Germany
| | - Volker Dötsch
- Institute for Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt, Germany
| | - Philipp Mertins
- Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
| | - Thomas Sommer
- Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
- Institute for Biology, Humboldt-University zu Berlin, Berlin, Germany
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Vadolas J, Nualkaew T, Voon HPJ, Vilcassim S, Grigoriadis G. Interplay between α-thalassemia and β-hemoglobinopathies: Translating genotype-phenotype relationships into therapies. Hemasphere 2024; 8:e78. [PMID: 38752170 PMCID: PMC11094674 DOI: 10.1002/hem3.78] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2024] [Revised: 03/22/2024] [Accepted: 04/11/2024] [Indexed: 05/18/2024] Open
Abstract
α-Thalassemia represents one of the most important genetic modulators of β-hemoglobinopathies. During this last decade, the ongoing interest in characterizing genotype-phenotype relationships has yielded incredible insights into α-globin gene regulation and its impact on β-hemoglobinopathies. In this review, we provide a holistic update on α-globin gene expression stemming from DNA to RNA to protein, as well as epigenetic mechanisms that can impact gene expression and potentially influence phenotypic outcomes. Here, we highlight defined α-globin targeted strategies and rationalize the use of distinct molecular targets based on the restoration of balanced α/β-like globin chain synthesis. Considering the therapies that either increase β-globin synthesis or reactivate γ-globin gene expression, the modulation of α-globin chains as a disease modifier for β-hemoglobinopathies still remains largely uncharted in clinical studies.
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Affiliation(s)
- Jim Vadolas
- Centre for Cancer ResearchHudson Institute of Medical ResearchClaytonVictoriaAustralia
- Department of Molecular and Translational SciencesMonash UniversityClaytonVictoriaAustralia
| | - Tiwaporn Nualkaew
- Centre for Cancer ResearchHudson Institute of Medical ResearchClaytonVictoriaAustralia
- Present address:
Department of Medical Technology, School of Allied Health SciencesWalailak UniversityNakhon Si ThammaratThailand
| | - Hsiao P. J. Voon
- Department of Biochemistry and Molecular Biology, Cancer Program, Biomedicine Discovery InstituteMonash UniversityClaytonVictoriaAustralia
| | - Shahla Vilcassim
- Centre for Cancer ResearchHudson Institute of Medical ResearchClaytonVictoriaAustralia
- School of Clinical Sciences at Monash HealthMonash UniversityClaytonAustralia
| | - George Grigoriadis
- Centre for Cancer ResearchHudson Institute of Medical ResearchClaytonVictoriaAustralia
- School of Clinical Sciences at Monash HealthMonash UniversityClaytonAustralia
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Zurlo M, Zuccato C, Cosenza LC, Gamberini MR, Finotti A, Gambari R. Increased Expression of α-Hemoglobin Stabilizing Protein (AHSP) mRNA in Erythroid Precursor Cells Isolated from β-Thalassemia Patients Treated with Sirolimus (Rapamycin). J Clin Med 2024; 13:2479. [PMID: 38731008 PMCID: PMC11084795 DOI: 10.3390/jcm13092479] [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: 12/07/2023] [Revised: 04/18/2024] [Accepted: 04/20/2024] [Indexed: 05/13/2024] Open
Abstract
Background/Objectives: in β-thalassemia, important clinical complications are caused by the presence of free α-globin chains in the erythroid cells of β-thalassemia patients. These free α-globin chains are present in excess as a result of the lack of β-globin chains to bind with; they tend to aggregate and precipitate, causing deleterious effects and overall cytotoxicity, maturation arrest of the erythroid cells and, ultimately, ineffective erythropoiesis. The chaperone protein α-hemoglobin-stabilizing protein (AHSP) reversibly binds with free α-globin; the resulting AHSP-αHb complex prevents aggregation and precipitation. Sirolimus (rapamycin) has been previously demonstrated to induce expression of fetal hemoglobin and decrease the excess of free α-globin chain in the erythroid cells of β-thalassemia patients. The objective of this study was to verify whether sirolimus is also able to upregulate AHSP expression in erythroid precursor cells (ErPCs) isolated from β-thalassemia patients. Methods: the expression of AHSP genes was analyzed by measuring the AHSP mRNA content by real-time quantitative PCR (RT-qPCR) and the AHSP protein production by Western blotting. Results: AHSP gene expression was found to be higher in ErPCs of β-thalassemia patients in comparison to ErPCs isolated from healthy subjects. In addition, AHSP expression was further induced by treatment of β-thalassemia ErPCs with sirolimus. Finally, AHSP mRNA was expressed at an increased level in ErPCs of sirolimus-treated β-thalassemia patients participating in the NCT03877809 Sirthalaclin clinical trial. Conclusions: this exploratory study suggests that AHSP expression should be considered as an endpoint in clinical trials based on sirolimus.
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Affiliation(s)
- Matteo Zurlo
- Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy; (M.Z.); (C.Z.); (L.C.C.)
| | - Cristina Zuccato
- Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy; (M.Z.); (C.Z.); (L.C.C.)
- Center “Chiara Gemmo and Elio Zago” for the Research on Thalassemia, Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy;
| | - Lucia Carmela Cosenza
- Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy; (M.Z.); (C.Z.); (L.C.C.)
| | - Maria Rita Gamberini
- Center “Chiara Gemmo and Elio Zago” for the Research on Thalassemia, Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy;
| | - Alessia Finotti
- Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy; (M.Z.); (C.Z.); (L.C.C.)
- Center “Chiara Gemmo and Elio Zago” for the Research on Thalassemia, Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy;
| | - Roberto Gambari
- Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy; (M.Z.); (C.Z.); (L.C.C.)
- Center “Chiara Gemmo and Elio Zago” for the Research on Thalassemia, Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy;
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Keith J, Christakopoulos GE, Fernandez AG, Yao Y, Zhang J, Mayberry K, Telange R, Sweileh RBA, Dudley M, Westbrook C, Sheppard H, Weiss MJ, Lechauve C. Loss of miR-144/451 alleviates β-thalassemia by stimulating ULK1-mediated autophagy of free α-globin. Blood 2023; 142:918-932. [PMID: 37339583 PMCID: PMC10517214 DOI: 10.1182/blood.2022017265] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2022] [Revised: 05/08/2023] [Accepted: 05/29/2023] [Indexed: 06/22/2023] Open
Abstract
Most cells can eliminate unstable or misfolded proteins through quality control mechanisms. In the inherited red blood cell disorder β-thalassemia, mutations in the β-globin gene (HBB) lead to a reduction in the corresponding protein and the accumulation of cytotoxic free α-globin, which causes maturation arrest and apoptosis of erythroid precursors and reductions in the lifespan of circulating red blood cells. We showed previously that excess α-globin is eliminated by Unc-51-like autophagy activating kinase 1 (ULK1)-dependent autophagy and that stimulating this pathway by systemic mammalian target of rapamycin complex 1 (mTORC1) inhibition alleviates β-thalassemia pathologies. We show here that disrupting the bicistronic microRNA gene miR-144/451 alleviates β-thalassemia by reducing mTORC1 activity and stimulating ULK1-mediated autophagy of free α-globin through 2 mechanisms. Loss of miR-451 upregulated its target messenger RNA, Cab39, which encodes a cofactor for LKB1, a serine-threonine kinase that phosphorylates and activates the central metabolic sensor adenosine monophosphate-activated protein kinase (AMPK). The resultant enhancement of LKB1 activity stimulated AMPK and its downstream effects, including repression of mTORC1 and direct activation of ULK1. In addition, loss of miR-144/451 inhibited the expression of erythroblast transferrin receptor 1, causing intracellular iron restriction, which has been shown to inhibit mTORC1, reduce free α-globin precipitates, and improve hematological indices in β-thalassemia. The beneficial effects of miR-144/451 loss in β-thalassemia were inhibited by the disruption of Cab39 or Ulk1 genes. Together, our findings link the severity of β-thalassemia to a highly expressed erythroid microRNA locus and a fundamental, metabolically regulated protein quality control pathway that is amenable to therapeutic manipulation.
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Affiliation(s)
- Julia Keith
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | | | | | - Yu Yao
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Jingjing Zhang
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Kalin Mayberry
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Rahul Telange
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Razan B. A. Sweileh
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Michael Dudley
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Camilla Westbrook
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Heather Sheppard
- Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN
- Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Mitchell J. Weiss
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Christophe Lechauve
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
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Vijayasimha K, Tran MV, Leestemaker-Palmer AL, Dolan BP. Direct Conjugation of NEDD8 to the N-Terminus of a Model Protein Can Induce Degradation. Cells 2021; 10:854. [PMID: 33918652 PMCID: PMC8069691 DOI: 10.3390/cells10040854] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Revised: 04/05/2021] [Accepted: 04/08/2021] [Indexed: 12/28/2022] Open
Abstract
While the role of ubiquitin in protein degradation is well established, the role of other ubiquitin-like proteins (UBLs) in protein degradation is less clear. Neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is the UBL with the highest level of amino acids identified when compared to ubiquitin. Here we tested if the N-terminal addition of NEDD8 to a protein of interest could lead to degradation. Mutation of critical glycine residues required for normal NEDD8 processing resulted in a non-cleavable fusion protein that was rapidly degraded within the cells by both the proteasome and autophagy. Both degradation pathways were dependent on a functional ubiquitin-conjugation system as treatment with MLN7243 increased levels of non-cleavable NEDD8-GFP. The degradation of non-cleavable, N-terminal NEDD8-GFP was not due to a failure of GFP folding as different NEDD8-GFP constructs with differing abilities to fold and fluoresce were similarly degraded. Though the fusion of NEDD8 to a protein resulted in degradation, treatment of cells with MLN4924, an inhibitor of the E1 activating enzyme for NEDD8, failed to prevent degradation of other destabilized substrates. Taken together these data suggest that under certain conditions, such as the model system described here, the covalent linkage of NEDD8 to a protein substrate may result in the target proteins degradation.
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Affiliation(s)
| | | | | | - Brian P. Dolan
- Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA; (K.V.); (M.V.T.); (A.L.L.-P.)
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Gui W, Davidson GA, Zhuang Z. Chemical methods for protein site-specific ubiquitination. RSC Chem Biol 2021; 2:450-467. [PMID: 34381999 PMCID: PMC8323803 DOI: 10.1039/d0cb00215a] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Accepted: 02/02/2021] [Indexed: 12/16/2022] Open
Abstract
Ubiquitination is an important protein post-translational modification regulating many cellular processes in eukaryotes. Ubiquitination is catalyzed by a three-enzyme cascade resulting in the conjugation of the C-terminal carboxylate of ubiquitin (Ub) to the ε-amino group of a lysine residue in the acceptor protein via an isopeptide bond. In vitro enzymatic ubiquitination utilizing Ub ligases has been successfully employed to generate Ub dimers and polymers. However, limitations of the enzymatic approach exist, particularly due to the requirement of specific Ub ligase for any given target protein and the low catalytic efficiency of the Ub ligase. To achieve an in-depth understanding of the molecular mechanism of Ub signaling, new methods are needed to generate mono- and poly-ubiquitinated proteins at a specific site with defined polyubiquitin chain linkage and length. Chemical methods offer an attractive solution to the above-described challenges. In this review, we summarize the recently developed chemical methods for generating ubiquitinated proteins using synthetic and semisynthetic approaches. These new tools and approaches, as an important part of the Ub toolbox, are crucial to our understanding and exploitation of the Ub system for novel therapeutics.
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Affiliation(s)
- Weijun Gui
- Department of Chemistry and Biochemistry, University of Delaware 214A Drake Hall Newark DE 19716 USA
| | - Gregory A Davidson
- Department of Chemistry and Biochemistry, University of Delaware 214A Drake Hall Newark DE 19716 USA
| | - Zhihao Zhuang
- Department of Chemistry and Biochemistry, University of Delaware 214A Drake Hall Newark DE 19716 USA
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Tokumoto T, Hossain MF, Jyoti MMS, Ali MH, Hossain MB, Acharjee M, Rezanujjaman M, Tokumoto M. Two-Step Mechanism of Cyclin B Degradation Initiated by Proteolytic Cleavage with the 26 S Proteasome in Fish. Sci Rep 2020; 10:8924. [PMID: 32488101 PMCID: PMC7265292 DOI: 10.1038/s41598-020-65009-w] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2019] [Accepted: 04/20/2020] [Indexed: 11/25/2022] Open
Abstract
To complete meiosis II, cyclin B is degraded in a short period by the inactivation of M-phase promoting factor (MPF). Previously, we showed that the destruction of cyclin B was initiated by the ubiquitin-independent proteolytic activity of the 26 S proteasome through an initial cut in the N-terminus of cyclin (at K57 in the case of goldfish cyclin B). We hypothesized that this cut allows cyclin to be ubiquitinated for further destruction by the ubiquitin-dependent proteolytic pathway, which leads to MPF inactivation. In this study, we aimed to identify the ubiquitination site for further degradation. The destruction of cyclin B point mutants in which lysine residues in a lysine-rich stretch following the cut site of cyclin B had been mutated was analyzed. All the lysine point mutants except K57R (a point mutant in which K57 was substituted with arginine) were susceptible to proteolytic cleavage by the 26 S proteasome. However, the degradation of the K77R and K7677R mutants in Xenopus egg extracts was significantly slower than the degradation of other mutants, and a 42 kDa truncated form of cyclin B was detected during the onset of the degradation of these mutants. The truncated form of recombinant cyclin B, an N-terminal truncated cyclin BΔ57 produced as cut by the 26 S proteasome, was not further cleaved by the 26 S proteasome but rather degraded in Xenopus egg extracts. The injection of the K57R, K77R and K7677R cyclin B proteins stopped cleavage in Xenopus embryos. From the results of a series of experiments, we concluded that cyclin B degradation involves a two-step mechanism initiated by initial ubiquitin-independent cleavage by the 26 S proteasome at lysine 57 followed by its ubiquitin-dependent destruction by the 26 S proteasome following ubiquitination at lysine 77.
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Affiliation(s)
- Toshinobu Tokumoto
- Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan. .,Biological Science Course, Graduate School of Science, National University Corporation, Shizuoka University, Oya 836, Suruga-ku, Shizuoka, 422-8529, Japan.
| | - Md Forhad Hossain
- Biological Science Course, Graduate School of Science, National University Corporation, Shizuoka University, Oya 836, Suruga-ku, Shizuoka, 422-8529, Japan
| | - Md Maisum Sarwar Jyoti
- Biological Science Course, Graduate School of Science, National University Corporation, Shizuoka University, Oya 836, Suruga-ku, Shizuoka, 422-8529, Japan
| | - Md Hasan Ali
- Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan
| | - Md Babul Hossain
- Department of Bioscience, Faculty of Science, Shizuoka University, Shizuoka, 422, Japan
| | - Mrityunjoy Acharjee
- Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan
| | - Md Rezanujjaman
- Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8529, Japan
| | - Mika Tokumoto
- CREST Research Project, Japan Science and Technology Corporation, Shizuoka, Japan
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Wei F, Zhao L, Jing Y. Hemoglobin-derived peptides and mood regulation. Peptides 2020; 127:170268. [PMID: 32070683 DOI: 10.1016/j.peptides.2020.170268] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/18/2019] [Revised: 02/02/2020] [Accepted: 02/04/2020] [Indexed: 12/12/2022]
Abstract
Evidence accumulated over the past decades has revealed that red blood cells and hemoglobin (Hb) in the blood play important roles in modulating moods and emotions. The number of red blood cells affects the mood. Hb is the principal content in the red blood cells besides water. Denatured Hb is hydrolyzed to produce bioactive peptides. RVD-hemopressin α (RVD-Hpα), which is a fragment of α-chain (95-103) in Hb, functions as a negative allosteric modulator of cannabinoid receptor 1 and a positive allosteric modulator of cannabinoid receptor 2. Hemorphins, which are fragments of β-chain in Hb, exert their effects on opioid receptors. Two hemorphins, namely, LVV-hemorphin-6 and LVV-hemorphin-7, could induce anxiolytic-like effects. The use of Hb-derived bioactive peptides for the treatment of mood disorders is desirable due to cannabinoid-opioid cross modulation and the critical roles of the two systems in physiological processes, such as memory, mood and emotion.
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Affiliation(s)
- Fengmei Wei
- Department of Physiology and Psychology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu, 730000, PR China
| | - Long Zhao
- Department of Orthopaedics, Lanzhou University First Affiliated Hospital, Lanzhou, Gansu, 730000, PR China
| | - Yuhong Jing
- Institute of Anatomy and Histology & Embryology, Neuroscience, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu, 730000, PR China; Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, Lanzhou, Gansu, 730000, PR China.
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Gavrilov Y, Hagai T, Levy Y. Nonspecific yet decisive: Ubiquitination can affect the native-state dynamics of the modified protein. Protein Sci 2015; 24:1580-92. [PMID: 25970168 DOI: 10.1002/pro.2688] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2015] [Accepted: 04/05/2015] [Indexed: 11/10/2022]
Abstract
Ubiquitination is one of the most common post-translational modifications of proteins, and mediates regulated protein degradation among other cellular processes. A fundamental question regarding the mechanism of protein ubiquitination is whether and how ubiquitin affects the biophysical nature of the modified protein. For some systems, it was shown that the position of ubiquitin within the attachment site is quite flexible and ubiquitin does not specifically interact with its substrate. Nevertheless, it was revealed that polyubiquitination can decrease the thermal stability of the modified protein in a site-specific manner because of alterations of the thermodynamic properties of the folded and unfolded states. In this study, we used detailed atomistic simulations to focus on the molecular effects of ubiquitination on the native structure of the modified protein. As a model, we used Ubc7, which is an E2 enzyme whose in vivo ubiquitination process is well characterized and known to lead to degradation. We found that, despite the lack of specific direct interactions between the ubiquitin moiety and Ubc7, ubiquitination decreases the conformational flexibility of certain regions of the substrate Ubc7 protein, which reduces its entropy and thus destabilizes it. The strongest destabilizing effect was observed for systems in which Lys48-linked tetra-ubiquitin was attached to sites used for in vivo degradation. These results reveal how changes in the configurational entropy of the folded state may modulate the stability of the protein's native state. Overall, our results imply that ubiquitination can modify the biophysical properties of the attached protein in the folded state and that, in some proteins, different ubiquitination sites will lead to different biophysical outcomes. We propose that this destabilizing effect of polyubiquitin on the substrate is linked to the functions carried out by the modification, and in particular, regulatory control of protein half-life through proteasomal degradation.
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Affiliation(s)
- Yulian Gavrilov
- Department of Structural Biology, Weizmann Institute of Science, Rehovot, 76100, Israel
| | - Tzachi Hagai
- Department of Structural Biology, Weizmann Institute of Science, Rehovot, 76100, Israel
| | - Yaakov Levy
- Department of Structural Biology, Weizmann Institute of Science, Rehovot, 76100, Israel
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Snoek BC, Wilt LHAMD, Jansen G, Peters GJ. Role of E3 ubiquitin ligases in lung cancer. World J Clin Oncol 2013; 4:58-69. [PMID: 23936758 PMCID: PMC3708064 DOI: 10.5306/wjco.v4.i3.58] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/03/2012] [Revised: 01/10/2013] [Accepted: 06/06/2013] [Indexed: 02/06/2023] Open
Abstract
E3 ubiquitin ligases are a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome. Therefore, E3 ubiquitin ligases play an essential role in a variety of biological processes including cell cycle regulation, proliferation and apoptosis. E3 ubiquitin ligases are often found overexpressed in human cancers, including lung cancer, and their deregulation has been shown to contribute to cancer development. However, the lack of specific inhibitors in clinical trials is a major issue in targeting E3 ubiquitin ligases with currently only one E3 ubiquitin ligase inhibitor being tested in the clinical setting. In this review, we focus on E3 ubiquitin ligases that have been found deregulated in lung cancer. Furthermore, we discuss the processes in which they are involved and evaluate them as potential anti-cancer targets. By better understanding the mechanisms by which E3 ubiquitin ligases regulate biological processes and their exact role in carcinogenesis, we can improve the development of specific E3 ubiquitin ligase inhibitors and pave the way for novel treatment strategies for cancer patients.
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11
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Ciechanover A, Stanhill A. The complexity of recognition of ubiquitinated substrates by the 26S proteasome. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2013; 1843:86-96. [PMID: 23872423 DOI: 10.1016/j.bbamcr.2013.07.007] [Citation(s) in RCA: 112] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/30/2013] [Revised: 07/02/2013] [Accepted: 07/08/2013] [Indexed: 02/03/2023]
Abstract
The Ubiquitin Proteasome System (UPS) was discovered in two steps. Initially, APF-1 (ATP-dependent proteolytic Factor 1) later identified as ubiquitin (Ub), a hitherto known protein of unknown function, was found to covalently modify proteins. This modification led to degradation of the tagged protein by - at that time - an unknown protease. This was followed later by the identification of the 26S proteasome complex which is composed of a previously identified Multi Catalytic Protease (MCP) and an additional regulatory complex, as the protease that degrades Ub-tagged proteins. While Ub conjugation and proteasomal degradation are viewed as a continued process responsible for most of the regulated proteolysis in the cell, the two processes have also independent roles. In parallel and in the years that followed, the hallmark signal that links the substrate to the proteasome was identified as an internal Lys48-based polyUb chain. However, since these initial findings were described, our understanding of both ends of the process (i.e. Ub-conjugation to proteins, and their recognition and degradation), have advanced significantly. This enabled us to start bridging the ends of this continuous process which suffered until lately from limited structural data regarding the 26S proteasomal architecture and the structure and diversity of the Ub chains. These missing pieces are of great importance because the link between ubiquitination and proteasomal processing is subject to numerous regulatory steps and are found to function improperly in several pathologies. Recently, the molecular architecture of the 26S proteasome was resolved in great detail, enabling us to address mechanistic questions regarding the various molecular events that polyubiquitinated (polyUb) substrates undergo during binding and processing by the 26S proteasome. In addition, advancement in analytical and synthetic methods enables us to better understand the structure and diversity of the degradation signal. The review summarizes these recent findings and addresses the extrapolated meanings in light of previous reports. Finally, it addresses some of the still remaining questions to be solved in order to obtain a continuous mechanistic view of the events that a substrate undergoes from its initial ubiquitination to proteasomal degradation. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.
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Affiliation(s)
- Aaron Ciechanover
- The David and Janet Polak Cancer and Vascular Biology Research Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 31096, Israel
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Shabek N, Herman-Bachinsky Y, Buchsbaum S, Lewinson O, Haj-Yahya M, Hejjaoui M, Lashuel H, Sommer T, Brik A, Ciechanover A. The Size of the Proteasomal Substrate Determines Whether Its Degradation Will Be Mediated by Mono- or Polyubiquitylation. Mol Cell 2012; 48:87-97. [DOI: 10.1016/j.molcel.2012.07.011] [Citation(s) in RCA: 138] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2012] [Revised: 06/04/2012] [Accepted: 07/09/2012] [Indexed: 11/28/2022]
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13
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14
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Spasser L, Brik A. Chemistry and Biology of the Ubiquitin Signal. Angew Chem Int Ed Engl 2012; 51:6840-62. [DOI: 10.1002/anie.201200020] [Citation(s) in RCA: 114] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2012] [Indexed: 01/07/2023]
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15
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Bavikar SN, Spasser L, Haj-Yahya M, Karthikeyan SV, Moyal T, Ajish Kumar KS, Brik A. Chemical Synthesis of Ubiquitinated Peptides with Varying Lengths and Types of Ubiquitin Chains to Explore the Activity of Deubiquitinases. Angew Chem Int Ed Engl 2011; 51:758-63. [DOI: 10.1002/anie.201106430] [Citation(s) in RCA: 93] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2011] [Revised: 10/12/2011] [Indexed: 01/27/2023]
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16
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Bavikar SN, Spasser L, Haj-Yahya M, Karthikeyan SV, Moyal T, Ajish Kumar KS, Brik A. Chemical Synthesis of Ubiquitinated Peptides with Varying Lengths and Types of Ubiquitin Chains to Explore the Activity of Deubiquitinases. Angew Chem Int Ed Engl 2011. [DOI: 10.1002/ange.201106430] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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17
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Abstract
Erythrocytes must regulate hemoglobin synthesis to limit the toxicities of unstable free globin chain subunits. This regulation is particularly relevant in β-thalassemia, in which β-globin deficiency causes accumulation of free α-globin, which forms intracellular precipitates that destroy erythroid precursors. Experimental evidence accumulated over more than 40 years indicates that erythroid cells can neutralize moderate amounts of free α-globin through generalized protein quality control mechanisms, including molecular chaperones, the ubiquitin-proteasome system, and autophagy. In many ways, β-thalassemia resembles protein aggregation disorders of the nervous system, liver, and other tissues, which occur when levels of unstable proteins overwhelm cellular compensatory mechanisms. Information gained from studies of nonerythroid protein aggregation disorders may be exploited to further understand and perhaps treat β-thalassemia.
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18
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Calcium-SANDOZ®-induced erythrocyte exovesiculation and internalization of hemichromic material into rat brown adipocytes. ARCH BIOL SCI 2011. [DOI: 10.2298/abs1102309m] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Abstract
An ultramicroscopic study of brown adipose tissue (BAT) of rats treated with
Ca-SANDOZ? (480 mg/l) for 3 days, revealed erythrocyte exovesiculation and
migratory erythrocytic complexes from the capillaries to adipocyte cytoplasm
and mitochondria. Two types of erythrocytic material transfer were observed:
(i) numerous exocytic vesicles with electron dense material leaving the
erythrocytes; (ii) furcated complexes with microholes, embedded in amorphous
material. The content of red blood cell (RBC) complexes passed through the
capillaries and transferred to the brown adipocytes where it was detectable
in the cytoplasm and mitochondria. Light microscopy confirmed
sphenoechinocytic transformation of the RBCs in the blood smears of the
Ca-SANDOZ? treated rats.
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19
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Abstract
Protein degradation plays a central role in many cellular functions. Misfolded and damaged proteins are removed from the cell to avoid toxicity. The concentrations of regulatory proteins are adjusted by degradation at the appropriate time. Both foreign and native proteins are digested into small peptides as part of the adaptive immune response. In eukaryotic cells, an ATP-dependent protease called the proteasome is responsible for much of this proteolysis. Proteins are targeted for proteasomal degradation by a two-part degron, which consists of a proteasome binding signal and a degradation initiation site. Here we describe how both components contribute to the specificity of degradation.
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Affiliation(s)
- Erin K Schrader
- Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois, USA
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20
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Abstract
Multisubunit complexes containing molecular chaperones regulate protein production, stability, and degradation in virtually every cell type. We are beginning to recognize how generalized and tissue-specific chaperones regulate specialized aspects of erythropoiesis. For example, chaperones intersect with erythropoietin signaling pathways to protect erythroid precursors against apoptosis. Molecular chaperones also participate in hemoglobin synthesis, both directly and indirectly. Current knowledge in these areas only scratches the surface of what is to be learned. Improved understanding of how molecular chaperones regulate erythropoietic development and hemoglobin homeostasis should identify biochemical pathways amenable to pharmacologic manipulation in a variety of red blood cell disorders including thalassemia and other anemias associated with hemoglobin instability.
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21
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Yu X, Kong Y, Dore LC, Abdulmalik O, Katein AM, Zhou S, Choi JK, Gell D, Mackay JP, Gow AJ, Weiss MJ. An erythroid chaperone that facilitates folding of alpha-globin subunits for hemoglobin synthesis. J Clin Invest 2007; 117:1856-65. [PMID: 17607360 PMCID: PMC1904324 DOI: 10.1172/jci31664] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2007] [Accepted: 04/24/2007] [Indexed: 11/17/2022] Open
Abstract
Erythrocyte precursors produce abundant alpha- and beta-globin proteins, which assemble with each other to form hemoglobin A (HbA), the major blood oxygen carrier. alphaHb-stabilizing protein (AHSP) binds free alpha subunits reversibly to maintain their structure and limit their ability to generate reactive oxygen species. Accordingly, loss of AHSP aggravates the toxicity of excessive free alpha-globin caused by beta-globin gene disruption in mice. Surprisingly, we found that AHSP also has important functions when free alpha-globin is limited. Thus, compound mutants lacking both Ahsp and 1 of 4 alpha-globin genes (genotype Ahsp(-/-)alpha-globin*(alpha/alphaalpha)) exhibited more severe anemia and Hb instability than mice with either mutation alone. In vitro, recombinant AHSP promoted folding of newly translated alpha-globin, enhanced its refolding after denaturation, and facilitated its incorporation into HbA. Moreover, in erythroid precursors, newly formed free alpha-globin was destabilized by loss of AHSP. Therefore, in addition to its previously defined role in detoxification of excess alpha-globin, AHSP also acts as a molecular chaperone to stabilize nascent alpha-globin for HbA assembly. Our findings illustrate what we believe to be a novel adaptive mechanism by which a specialized cell coordinates high-level production of a multisubunit protein and protects against various synthetic imbalances.
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Affiliation(s)
- Xiang Yu
- Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
GlaxoSmithKline, King of Prussia, Pennsylvania, USA.
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Safety Assessment, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Sydney, Australia.
Department of Pharmacology, Rutgers University, Piscataway, New Jersey, USA
| | - Yi Kong
- Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
GlaxoSmithKline, King of Prussia, Pennsylvania, USA.
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Safety Assessment, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Sydney, Australia.
Department of Pharmacology, Rutgers University, Piscataway, New Jersey, USA
| | - Louis C. Dore
- Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
GlaxoSmithKline, King of Prussia, Pennsylvania, USA.
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Safety Assessment, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Sydney, Australia.
Department of Pharmacology, Rutgers University, Piscataway, New Jersey, USA
| | - Osheiza Abdulmalik
- Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
GlaxoSmithKline, King of Prussia, Pennsylvania, USA.
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Safety Assessment, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Sydney, Australia.
Department of Pharmacology, Rutgers University, Piscataway, New Jersey, USA
| | - Anne M. Katein
- Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
GlaxoSmithKline, King of Prussia, Pennsylvania, USA.
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Safety Assessment, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Sydney, Australia.
Department of Pharmacology, Rutgers University, Piscataway, New Jersey, USA
| | - Suiping Zhou
- Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
GlaxoSmithKline, King of Prussia, Pennsylvania, USA.
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Safety Assessment, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Sydney, Australia.
Department of Pharmacology, Rutgers University, Piscataway, New Jersey, USA
| | - John K. Choi
- Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
GlaxoSmithKline, King of Prussia, Pennsylvania, USA.
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Safety Assessment, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Sydney, Australia.
Department of Pharmacology, Rutgers University, Piscataway, New Jersey, USA
| | - David Gell
- Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
GlaxoSmithKline, King of Prussia, Pennsylvania, USA.
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Safety Assessment, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Sydney, Australia.
Department of Pharmacology, Rutgers University, Piscataway, New Jersey, USA
| | - Joel P. Mackay
- Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
GlaxoSmithKline, King of Prussia, Pennsylvania, USA.
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Safety Assessment, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Sydney, Australia.
Department of Pharmacology, Rutgers University, Piscataway, New Jersey, USA
| | - Andrew J. Gow
- Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
GlaxoSmithKline, King of Prussia, Pennsylvania, USA.
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Safety Assessment, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Sydney, Australia.
Department of Pharmacology, Rutgers University, Piscataway, New Jersey, USA
| | - Mitchell J. Weiss
- Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
GlaxoSmithKline, King of Prussia, Pennsylvania, USA.
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Safety Assessment, AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Sydney, Australia.
Department of Pharmacology, Rutgers University, Piscataway, New Jersey, USA
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22
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Weiss MJ, Zhou S, Feng L, Gell DA, Mackay JP, Shi Y, Gow AJ. Role of alpha-hemoglobin-stabilizing protein in normal erythropoiesis and beta-thalassemia. Ann N Y Acad Sci 2006; 1054:103-17. [PMID: 16339656 DOI: 10.1196/annals.1345.013] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Hemoglobin (Hb) synthesis is coordinated by homeostatic mechanisms to limit the accumulation of free alpha or beta subunits, which are cytotoxic. Alpha hemoglobin-stabilizing protein (AHSP) is an abundant erythroid protein that specifically binds free alphaHb, stabilizes its structure, and limits its ability to participate in chemical reactions that generate reactive oxygen species. Gene ablation studies in mice demonstrate that AHSP is required for normal erythropoiesis. AHSP-null erythrocytes are short-lived, contain Hb precipitates, and exhibit signs of oxidative damage. Loss of AHSP exacerbates beta-thalassemia in mice, indicating that altered AHSP expression or function could modify thalassemia phenotypes in humans, a topic that is beginning to be explored in clinical studies. We used biochemical, spectroscopic, and crystallographic methods to examine how AHSP stabilizes alphaHb. AHSP binds the G and H helices of alphaHb on a surface that largely overlaps with the alpha1-beta1 interface of HbA. This result explains previous findings that betaHb can competitively displace AHSP from alphaHb to form HbA tetramer. Remarkably, binding of AHSP to oxygenated alphaHb induces dramatic conformational changes and converts the heme-bound iron to an oxidized hemichrome state in which all six coordinate positions are occupied. This structure limits the reactivity of heme iron, providing a mechanism by which AHSP stabilizes alphaHb. These findings suggest a biochemical pathway through which AHSP might participate in normal Hb synthesis and modulate the severity of thalassemias. Moreover, understanding how AHSP stabilizes alphaHb provides a theoretical basis for new strategies to inhibit the damaging effects of free alphaHb that accumulates in beta-thalassemia.
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Affiliation(s)
- Mitchell J Weiss
- The Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA 19104, USA.
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23
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Kong Y, Zhou S, Kihm AJ, Katein AM, Yu X, Gell DA, Mackay JP, Adachi K, Foster-Brown L, Louden CS, Gow AJ, Weiss MJ. Loss of alpha-hemoglobin-stabilizing protein impairs erythropoiesis and exacerbates beta-thalassemia. J Clin Invest 2004; 114:1457-66. [PMID: 15545996 PMCID: PMC525742 DOI: 10.1172/jci21982] [Citation(s) in RCA: 124] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2004] [Accepted: 09/14/2004] [Indexed: 11/17/2022] Open
Abstract
Hemoglobin (Hb) A production during red blood cell development is coordinated to minimize the deleterious effects of free alpha- and beta-Hb subunits, which are unstable and cytotoxic. The alpha-Hb-stabilizing protein (AHSP) is an erythroid protein that specifically binds alpha-Hb and prevents its precipitation in vitro, which suggests that it may function to limit free alpha-Hb toxicities in vivo. We investigated this possibility through gene ablation and biochemical studies. AHSP(-/-) erythrocytes contained hemoglobin precipitates and were short-lived. In hematopoietic tissues, erythroid precursors were elevated in number but exhibited increased apoptosis. Consistent with unstable alpha-Hb, AHSP(-/-) erythrocytes contained increased ROS and evidence of oxidative damage. Moreover, purified recombinant AHSP inhibited ROS production by alpha-Hb in solution. Finally, loss of AHSP worsened the phenotype of beta-thalassemia, a common inherited anemia characterized by excess free alpha-Hb. Together, the data support a model in which AHSP binds alpha-Hb transiently to stabilize its conformation and render it biochemically inert prior to Hb A assembly. This function is essential for normal erythropoiesis and, to a greater extent, in beta-thalassemia. Our findings raise the possibility that altered AHSP expression levels could modulate the severity of beta-thalassemia in humans.
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Affiliation(s)
- Yi Kong
- Cell and Molecular Biology Graduate Program, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
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24
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Adachi K, Lakka V, Zhao Y, Surrey S. Ubiquitylation of nascent globin chains in a cell-free system. J Biol Chem 2004; 279:41767-74. [PMID: 15297454 DOI: 10.1074/jbc.m405059200] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The ubiquitin/proteasome pathway for degradation of completed and nascent globin chains was evaluated using a cell-free in vitro coupled transcription/translation assay. No decrease in radiolabeled globin chains was observed when ubiquitin, energy regenerating source (or ATP), and E1 and E2 enzymes were added 30 min after the start of translation when globin chain synthesis had plateaued. In contrast, the addition of these components prior to the start of translation resulted in no radiolabeled globin chains after 30 min. The loss of radiolabeled globin chains was dependent on ATP concentration; the higher the concentration, the less the radiolabeled globin chains formed. Prior to the initiation of transcription/translation, cell extract was preincubated with the proteasomal inhibitor MG132 in the absence of globin chain expression vector after which ubiquitin-protein isopeptidase inhibitor, Ubal, and expression vector were added in the presence of 1.5 mm ATP. Thereafter, radiolabeled monoubiquitylated and multiubiquitylated globin chains with few unmodified globin chains were formed. Our results suggest that polyubiquitylated globin chains are localized to the polysomal fractions. These results suggest that nascent globin chains are potential targets for ubiquitylation and deubiquitylation during or soon after translation and that ATP levels play a role in the balance between polypeptide synthesis and degradation.
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Affiliation(s)
- Kazuhiko Adachi
- The Children's Hospital of Philadelphia, Division of Hematology and University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104, USA.
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25
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Guterman A, Glickman MH. Complementary roles for Rpn11 and Ubp6 in deubiquitination and proteolysis by the proteasome. J Biol Chem 2003; 279:1729-38. [PMID: 14581483 DOI: 10.1074/jbc.m307050200] [Citation(s) in RCA: 113] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Substrates destined for degradation by the 26 S proteasome are labeled with polyubiquitin chains. These chains can be dismantled by deubiquitinating enzymes (DUBs). A number of reports have identified different DUBs that can hydrolyze ubiquitin from substrates bound to the proteasome. We measured deubiquitination by both isolated lid and base-core particle subcomplexes, suggesting that at least two different DUBs are intrinsic components of 26 S proteasome holoenzymes. In agreement, we find that highly purified proteasomes contain both Rpn11 and Ubp6, situated within the lid and base subcomplexes, respectively. To study their relative contributions, we purified proteasomes from a mutant in the putative metalloprotease domain of Rpn11 and from a ubp6 null. Interestingly, in both preparations we observed slower deubiquitination rates, suggesting that Rpn11 and Ubp6 serve complementary roles. In accord, the double mutant is synthetically lethal. In contrast to WT proteasomes, proteasomes lacking the lid subcomplex or those purified from the rpn11 mutant are less sensitive to metal chelators, supporting the prediction that Rpn11 may be a metalloprotein. Treatment of proteasomes with ubiquitin-aldehyde or with cysteine modifiers also inhibited deubiquitination but simultaneously promoted degradation of a monoubiquitinated substrate along with the ubiquitin tag. Degradation is unique to 26 S proteasome holoenzymes; we could not detect degradation of a ubiquitinated protein by "lidless" proteasomes, although they were competent for deubiquitination. The fascinating observation that a single ubiquitin moiety is sufficient for targeting an otherwise stable substrate to proteasomes exposes how rapid deubiquitination of poorly ubiquitinated substrates may counteract degradation.
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Affiliation(s)
- Adi Guterman
- Department of Biology and the Institute for Catalysis Science and Technology, The Technion, 32000 Haifa, Israel
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26
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Lapham CK, Romantseva T, Petricoin E, King LR, Manischewitz J, Zaitseva MB, Golding H. CXCR4 heterogeneity in primary cells: possible role of ubiquitination. J Leukoc Biol 2002. [DOI: 10.1189/jlb.72.6.1206] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Affiliation(s)
- Cheryl K. Lapham
- Divisions of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland and
| | - Tatiana Romantseva
- Divisions of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland and
| | - Emmanuel Petricoin
- Divisions of Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland
| | - Lisa R. King
- Divisions of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland and
| | - Jody Manischewitz
- Divisions of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland and
| | - Marina B. Zaitseva
- Divisions of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland and
| | - Hana Golding
- Divisions of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland and
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27
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Obin M, Lee BY, Meinke G, Bohm A, Lee RH, Gaudet R, Hopp JA, Arshavsky VY, Willardson BM, Taylor A. Ubiquitylation of the transducin betagamma subunit complex. Regulation by phosducin. J Biol Chem 2002; 277:44566-75. [PMID: 12215439 DOI: 10.1074/jbc.m205308200] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
G proteins (Galphabetagamma) are essential signaling molecules, which dissociate into Galpha and Gbetagamma upon activation by heptahelical membrane receptors. We have identified the betagamma subunit complex of the photoreceptor-specific G protein, transducin (T), as a target of the ubiquitin-proteasome pathway. Ubiquitylated species of the transducin gamma-subunit (Tgamma) but not the alpha- or beta-subunits were assembled de novo in bovine photoreceptor preparations. In addition, Tgamma was exclusively ubiquitylated when Tbetagamma was dissociated from Talpha. Ubiquitylation of Tbetagamma on Tgamma was selectively catalyzed by human ubiquitin-conjugating enzymes UbcH5 and UbcH7 and was coincident with degradation of the entire Tbetagamma subunit complex in vitro by a mechanism requiring ATP and the proteasome. We also show that Tbetagamma association with phosducin, a photoreceptor-specific protein of unknown physiological function, blocks Tbetagamma ubiquitylation and subsequent degradation. Phosphorylation of phosducin by Ca(2+)/calmodulin-dependent protein kinase II, which inhibits phosducin-Tbetagamma complex formation, completely restored Tbetagamma ubiquitylation and degradation. We conclude that Tbetagamma is a substrate of the ubiquitin-proteasome pathway and suggest that phosducin serves to protect Tbetagamma following the light-dependent dissociation of Talphabetagamma.
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Affiliation(s)
- Martin Obin
- Laboratory for Nutrition & Vision Research, JMUSDA-HNRCA at Tufts University and Tufts Center for Vision Research, Boston, Massachusetts 02111, USA.
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28
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Abstract
Ubiquitination of key cellular proteins involved in signal transduction, gene transcription and cell-cycle regulation usually condemns those proteins to proteasomal or lysosomal degradation. Additionally, cycles of reversible ubiquitination regulate the function of certain proteins in a manner analogous to phosphorylation. In this short review we describe the current methodology for measuring ubiquitin and ubiquitination, provide examples which illustrate how various techniques have been used to study protein ubiquination, alert the readers of pitfalls to avoid, and offer guidelines to investigators newly interested in this novel post-translational protein modification.
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Affiliation(s)
- E G Mimnaugh
- Tumor Cell Biology Section, Medicine Branch, National Cancer Institute, National Institutes of Health, Key West Center, Rockville, MD 20850, USA.
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29
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Verdier F, Chrétien S, Muller O, Varlet P, Yoshimura A, Gisselbrecht S, Lacombe C, Mayeux P. Proteasomes regulate erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) activation. Possible involvement of the ubiquitinated Cis protein. J Biol Chem 1998; 273:28185-90. [PMID: 9774439 DOI: 10.1074/jbc.273.43.28185] [Citation(s) in RCA: 141] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Cis is an Src homology 2 domain-containing protein, which binds to the erythropoietin receptor and decreases erythropoietin-stimulated cell proliferation. We show that Cis associates with the second tyrosine residue of the intracellular domain of the erythropoietin receptor (Tyr401). Two forms of Cis with molecular masses of 32 and 37 kDa were detected, and we demonstrate that the 37-kDa protein resulted from post-translational modifications of the 32-kDa form. Anti-ubiquitin antibodies recognized the 37-kDa form of Cis and the proteasome inhibitors N-acetyl-leucyl-leucyl-norleucinal and lactacystin inhibited its degradation, showing that the 37-kDa form of Cis is a ubiquitinated protein, which seems to be rapidly degraded by the proteasome. In erythropoietin-stimulated UT-7 cells, the activation of the erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) was transient and returned to basal levels after 30-60 min of erythropoietin stimulation. In contrast, these proteins remained strongly phosphorylated, and STAT5 remained activated for at least 120 min in the presence of proteasome inhibitors. These experiments demonstrate that the proteasomes are involved in the down-regulation of the erythropoietin receptor activation signals. Because the proteasome inhibitors induced the accumulation of both the ubiquitinated form of Cis and the Cis-erythropoietin receptor complexes, our results suggest that the ubiquitinated form of Cis could be involved in the proteasome-mediated inactivation of the erythropoietin receptor.
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Affiliation(s)
- F Verdier
- Institut Cochin de Génétique Moléculaire, INSERM U363, Université René Descartes, 27 rue du Faubourg Saint Jacques, F75014 Paris, France
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30
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Wickramasinghe SN, Lee MJ. Evidence that the ubiquitin proteolytic pathway is involved in the degradation of precipitated globin chains in thalassaemia. Br J Haematol 1998; 101:245-50. [PMID: 9609517 DOI: 10.1046/j.1365-2141.1998.00699.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Ultrastructural immunocytochemical studies were performed on sections of bone marrow from three patients with beta-thalassaemia major and two patients with haemoglobin H (HbH) disease. Some sections were reacted with either a polyclonal or a monoclonal anti-human-ubiquitin antibody and the reaction visualized using a gold-labelled secondary antibody. The inclusions of precipitated globin chains found within the erythropoietic cells of all five patients reacted much more strongly than the surrounding inclusion-free cytoplasm with both of the anti-ubiquitin antibodies, indicating that the precipitated globin chains were ubiquitinated. A non-specific reaction between the anti-ubiquitin antibodies and the inclusions was excluded by demonstrating that various other antibodies, including a polyclonal anti-human cathepsin D antibody, did not react with the inclusions. The data suggest that the ubiquitin proteolytic pathway is involved in the degradation of precipitated globin chains in alpha- and beta-thalassaemia.
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Affiliation(s)
- S N Wickramasinghe
- Department of Haematology, Imperial College School of Medicine, St Mary's Hospital, London
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31
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The 19S Regulatory Complex of the 26S Proteasome. ACTA ACUST UNITED AC 1998. [DOI: 10.1016/s1569-2558(08)60460-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
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Terrell J, Shih S, Dunn R, Hicke L. A function for monoubiquitination in the internalization of a G protein-coupled receptor. Mol Cell 1998; 1:193-202. [PMID: 9659916 DOI: 10.1016/s1097-2765(00)80020-9] [Citation(s) in RCA: 285] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Modification of an S. cerevisiae G protein-coupled receptor with ubiquitin is required for its ligand-stimulated internalization. We now demonstrate that monoubiquitination on a single lysine residue is sufficient to signal receptor internalization, a modification distinct from that required for proteasome recognition. Formation of a polyubiquitin chain is not necessary, as demonstrated by the ability of mutant ubiquitins that lack lysine residues to serve as efficient internalization signals. Fusion of ubiquitin in-frame to a receptor that lacks cytoplasmic tail lysines also promotes rapid receptor internalization, indicating that ubiquitin itself and not a specific type of linkage to the receptor acts as an internalization signal. Thus, we have defined a cellular function for monoubiquitination in alpha-factor receptor endocytosis.
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Affiliation(s)
- J Terrell
- Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500, USA
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Naturally Processed Tissue- and Differentiation Stage-Specific Autologous Peptides Bound by HLA Class I and II Molecules of Chronic Myeloid Leukemia Blasts. Blood 1997. [DOI: 10.1182/blood.v90.12.4938] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractStructural analysis of naturally processed peptides bound to the HLA class I and class II molecules of chronic myeloid leukemia (CML) blast cells was performed to characterize the antigen processing and autoantigen repertoire in this hematopoietic malignancy. Self-peptides derived from the carboxy-terminal end of the breakpoint cluster region (bcr) protein, as well as several differentiation stage- and tissue-specific self-antigens characteristic of early stages of myeloid differentiation, such as c-fes, c-pim, granulocyte-macrophage colony-stimulating factor receptor α chain, proteinase 3, and cathepsin G, were identified. A common characteristic of several of the high copy-number self-peptides identified in this study is the participation of their parent proteins in signal transduction or myeloid effector function. Because bcr-abl junctional peptides bind to a limited number of major histocompatibility complex (MHC) class I alleles, an effective peptide-based immunotherapy strategy for CML requires identification of further tumor-associated or tissue-specific peptide antigens binding to common MHC alleles such as HLA-A2. The differentiation stage- and tissue-specific MHC-bound peptides found in this study, as well as the naturally processed proteins from which they are derived, may represent autoantigens towards which T-cell responses may potentially be developed for immunotherapy of hematopoietic malignancies such as CML.
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Naturally Processed Tissue- and Differentiation Stage-Specific Autologous Peptides Bound by HLA Class I and II Molecules of Chronic Myeloid Leukemia Blasts. Blood 1997. [DOI: 10.1182/blood.v90.12.4938.4938_4938_4946] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Structural analysis of naturally processed peptides bound to the HLA class I and class II molecules of chronic myeloid leukemia (CML) blast cells was performed to characterize the antigen processing and autoantigen repertoire in this hematopoietic malignancy. Self-peptides derived from the carboxy-terminal end of the breakpoint cluster region (bcr) protein, as well as several differentiation stage- and tissue-specific self-antigens characteristic of early stages of myeloid differentiation, such as c-fes, c-pim, granulocyte-macrophage colony-stimulating factor receptor α chain, proteinase 3, and cathepsin G, were identified. A common characteristic of several of the high copy-number self-peptides identified in this study is the participation of their parent proteins in signal transduction or myeloid effector function. Because bcr-abl junctional peptides bind to a limited number of major histocompatibility complex (MHC) class I alleles, an effective peptide-based immunotherapy strategy for CML requires identification of further tumor-associated or tissue-specific peptide antigens binding to common MHC alleles such as HLA-A2. The differentiation stage- and tissue-specific MHC-bound peptides found in this study, as well as the naturally processed proteins from which they are derived, may represent autoantigens towards which T-cell responses may potentially be developed for immunotherapy of hematopoietic malignancies such as CML.
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Lam YA, DeMartino GN, Pickart CM, Cohen RE. Specificity of the ubiquitin isopeptidase in the PA700 regulatory complex of 26 S proteasomes. J Biol Chem 1997; 272:28438-46. [PMID: 9353303 DOI: 10.1074/jbc.272.45.28438] [Citation(s) in RCA: 79] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
The specificity of the ubiquitin (Ub) isopeptidase in the PA700 regulatory complex of the bovine 26 S proteasome was investigated. Disassembly of poly-Ub by this enzyme is restricted to the distal-end Ub of the substrate, i.e. the Ub farthest from the site of protein attachment in poly-Ub-protein conjugates. The determinants recognized by the isopeptidase were probed by the use of mutant ubiquitins incorporated into Lys48-linked poly-Ub substrates. PA700 could not disassemble poly-Ub chains that contained a distal Ub(L8A,I44A). This suggested either that the enzyme interacts directly with Leu8 or Ile44 or that it recognizes a higher order structure that caps the distal end of a poly-Ub substrate and is destabilized by Ub(L8A,I44A). The previously determined di-Ub crystal structure (Cook, W. J., Jeffrey, L. C., Carson, M., Chen, Z., and Pickart, C. M. (1992) J. Biol. Chem. 267, 16467-16471) offered a candidate for such a "cap." In solution, however, this structure was not observed by 1H NMR spectroscopy. This and the finding that di-Ub with a single proximal Ub(L8A,I44A) is cleaved efficiently suggest that Leu8 and Ile44 in the distal-end Ub contact the isopeptidase directly. In addition to Lys48-linked chains, PA700 also could disassemble Lys6- and Lys-11-linked poly-Ub, but, surprisingly, not alpha-linked di-Ub. Results with these and other substrates suggest that specificity determinants for the PA700 isopeptidase include Leu8, Ile44, and Lys48 on the distal Ub and, for poly-Ub, some features of the Ub-Ub linkage itself.
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Affiliation(s)
- Y A Lam
- Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA
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36
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Taylor A, Shang F, Obin M. Relationships between stress, protein damage, nutrition, and age-related eye diseases. Mol Aspects Med 1997; 18:305-414. [PMID: 9578986 DOI: 10.1016/s0098-2997(95)00049-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Affiliation(s)
- A Taylor
- Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, USA.
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Piotrowski J, Beal R, Hoffman L, Wilkinson KD, Cohen RE, Pickart CM. Inhibition of the 26 S proteasome by polyubiquitin chains synthesized to have defined lengths. J Biol Chem 1997; 272:23712-21. [PMID: 9295315 DOI: 10.1074/jbc.272.38.23712] [Citation(s) in RCA: 189] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Ubiquitin is a covalent signal that targets cellular proteins to the 26 S proteasome. Multiple ubiquitins can be ligated together through the formation of isopeptide bonds between Lys48 and Gly76 of successive ubiquitins. Such a polyubiquitin chain constitutes a highly effective proteolytic targeting signal, but its mode of interaction with the proteasome is not well understood. Experiments to address this issue have been limited by difficulties in preparing useful quantities of polyubiquitin chains of uniform length. We report a simple method for large scale synthesis of Lys48-linked polyubiquitin chains of defined length. In the first round of synthesis, two ubiquitin derivatives (K48C-ubiquitin and Asp77-ubiquitin) were used as substrates for the well characterized ubiquitin-conjugating enzyme E2-25K. Diubiquitin blocked at the nascent proximal and distal chain termini was obtained in quantitative yield. Appropriately deblocked chains were then combined to synthesize higher order chains (tetramer and octamer in the present study). Deblocking was achieved either enzymatically (proximal terminus) or by chemical alkylation (distal terminus). Chains synthesized by this method were used to obtain the first quantitative information concerning the influence of polyubiquitin chain length on binding to the 26 S proteasome; this was done through comparison of different length (unanchored) polyubiquitin chains as inhibitors of ubiquitin-conjugate degradation. K0.5 was found to decrease approximately 90-fold, from 430 to 4.8 microM, as the chain was lengthened from two to eight ubiquitins. The implications of these results for the molecular basis of chain recognition are discussed.
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Affiliation(s)
- J Piotrowski
- Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA
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Ubiquitin Aldehyde Increases Adenosine Triphosphate–Dependent Proteolysis of Hemoglobin α-Subunits in β-Thalassemic Hemolysates. Blood 1997. [DOI: 10.1182/blood.v90.3.1300] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractTwo major causes of the anemia in β-thalassemia are a deficiency in hemoglobin (Hb) β-subunit (and consequently HbA) synthesis and, due to the resulting excess of Hb α-subunits, erythroid cell hemolysis. The hemolytic component might be ameliorated by increasing the intracellular proteolysis of the excess α-subunits. Isolated 3H-labeled α-chains are known to be degraded primarily by the adenosine triphosphate (ATP)- and ubiquitin (Ub)-dependent proteolysis pathway in unfractionated β-thalassemic hemolysates. Our objective was to increase this degradation by targeted intervention. Ub aldehyde (Ubal), a synthetic inhibitor of isopeptidases (proteases that hydrolyze the bond between the Ub polypeptide and its protein adduct), was added to reaction mixtures containing a hemolysate from the blood cells of one of four β-thalassemic donors and 3H-α-chains or 3H-α-globin as a substrate. Optimum enhancement of ATP-dependent degradation occurred at 0.4 to 1.5 μmol/L Ubal and ranged from 29% to 115% for 3H-α-chains and 47% to 96% for 3H-α-globin among the four hemolysates. We suggest that Ubal stimulates 3H-α-subunit proteolysis by inhibition of an isopeptidase(s) that deubiquitinates, or “edits,” Ub-3H-α-subunit conjugates, intermediates in the degradative pathway. In control studies, similarly low Ubal concentrations did not enhance the degradation of 3H-α2β2 (HbA) tetramers or inhibit the activities of methemoglobin reductase and four selected glycolysis pathway enzymes. These and other results may be the basis for a therapeutic approach to β-thalassemia.
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Ubiquitin Aldehyde Increases Adenosine Triphosphate–Dependent Proteolysis of Hemoglobin α-Subunits in β-Thalassemic Hemolysates. Blood 1997. [DOI: 10.1182/blood.v90.3.1300.1300_1300_1308] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Two major causes of the anemia in β-thalassemia are a deficiency in hemoglobin (Hb) β-subunit (and consequently HbA) synthesis and, due to the resulting excess of Hb α-subunits, erythroid cell hemolysis. The hemolytic component might be ameliorated by increasing the intracellular proteolysis of the excess α-subunits. Isolated 3H-labeled α-chains are known to be degraded primarily by the adenosine triphosphate (ATP)- and ubiquitin (Ub)-dependent proteolysis pathway in unfractionated β-thalassemic hemolysates. Our objective was to increase this degradation by targeted intervention. Ub aldehyde (Ubal), a synthetic inhibitor of isopeptidases (proteases that hydrolyze the bond between the Ub polypeptide and its protein adduct), was added to reaction mixtures containing a hemolysate from the blood cells of one of four β-thalassemic donors and 3H-α-chains or 3H-α-globin as a substrate. Optimum enhancement of ATP-dependent degradation occurred at 0.4 to 1.5 μmol/L Ubal and ranged from 29% to 115% for 3H-α-chains and 47% to 96% for 3H-α-globin among the four hemolysates. We suggest that Ubal stimulates 3H-α-subunit proteolysis by inhibition of an isopeptidase(s) that deubiquitinates, or “edits,” Ub-3H-α-subunit conjugates, intermediates in the degradative pathway. In control studies, similarly low Ubal concentrations did not enhance the degradation of 3H-α2β2 (HbA) tetramers or inhibit the activities of methemoglobin reductase and four selected glycolysis pathway enzymes. These and other results may be the basis for a therapeutic approach to β-thalassemia.
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Haracska L, Udvardy A. Mapping the ubiquitin-binding domains in the p54 regulatory complex subunit of the Drosophila 26S protease. FEBS Lett 1997; 412:331-6. [PMID: 9256246 DOI: 10.1016/s0014-5793(97)00808-9] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Short-lived intracellular proteins, after being marked by multiubiquitination, are degraded by the 26S protease. This large ATP-dependent protease is composed of two multiprotein complexes: the regulatory complex and the 20S proteosome. The selective recognition of ubiquitinated proteins is ensured by the regulatory complex. Using an overlay assay a single 54-kDa multiubiquitin-chain-binding subunit was detected in the regulatory complex of the Drosophila 26S protease. Overlay assay with the recombinant p54 subunit confirmed its ubiquitin-binding property. The recombinant protein showed pronounced preference for higher ubiquitin multimers, in agreement with the known preference of the 26S protease for multiubiquitinated proteins as substrates. To map the ubiquitin-binding domain of the p54 subunit different segments of the recombinant protein were expressed in E. coli and tested by the overlay assay. The p54 subunit carries two independent ubiquitin-binding domains. The central domain carries two highly conserved sequence blocks: the FGVDP sequence (at position 207), which is 100% conserved from yeast till human, and the DPELALALRVSMEE sequence (at position 214), which is 100% conserved in higher eukaryotes with two amino acid changes in yeast. In the C-terminal ubiquitin-binding domain the GVDP sequence motif is repeated and 100% conserved in higher eukaryotes. This domain, however, due to the shorter size of the yeast multiubiquitin-binding subunit, is present only in higher eukaryotes.
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Affiliation(s)
- L Haracska
- Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged
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41
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Lam YA, Xu W, DeMartino GN, Cohen RE. Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature 1997; 385:737-40. [PMID: 9034192 DOI: 10.1038/385737a0] [Citation(s) in RCA: 348] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
In eukaryotes, ubiquitin (Ub)-dependent proteolysis is essential for bulk protein turnover as well as diverse processes including cell-cycle control, differentiation, antigen presentation, and the stress response. Generally, multiple ubiquitins are added onto a substrate to form an isopeptide-linked 'polyubiquitin' chain, which targets substrates for degradation by the 26S proteasome. The specificity of Ub-dependent degradation was thought to depend primarily on the selection of targets for ubiquitination, but recently we have reported evidence for a second level of specificity in which (poly)Ub-protein conjugates are partitioned among two fates: degradation of the protein substrate by the 26S proteasome; and disassembly by Ub isopeptidase(s) to regenerate the protein substrate. Potentially, an isopeptidase could influence degradation by 'editing' (poly)Ub-protein conjugates according to the extent of ubiquitination rather than the structure of the ubiquitination target itself. Here we describe a bovine isopeptidase that is well suited to such an editing function because of its unique localization and specificity. This enzyme is an intrinsic subunit of PA700, the 19S regulatory complex of the 26S proteasome. By disassembling the degradation signal from only the distal end of (poly)Ub chains, this isopeptidase can selectively rescue poorly ubiquitinated or slowly degraded Ub-protein conjugates from proteolysis.
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Affiliation(s)
- Y A Lam
- Department of Biochemistry, University of Iowa, Iowa City 52242, USA
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42
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Abstract
A growing number of cellular regulatory mechanisms are being linked to protein modification by the polypeptide ubiquitin. These include key transitions in the cell cycle, class I antigen processing, signal transduction pathways, and receptor-mediated endocytosis. In most, but not all, of these examples, ubiquitination of a protein leads to its degradation by the 26S proteasome. Following attachment of ubiquitin to a substrate and binding of the ubiquitinated protein to the proteasome, the bound substrate must be unfolded (and eventually deubiquitinated) and translocated through a narrow set of channels that leads to the proteasome interior, where the polypeptide is cleaved into short peptides. Protein ubiquitination and deubiquitination are both mediated by large enzyme families, and the proteasome itself comprises a family of related but functionally distinct particles. This diversity underlies both the high substrate specificity of the ubiquitin system and the variety of regulatory mechanisms that it serves.
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Affiliation(s)
- M Hochstrasser
- Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637, USA.
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Lee SL, Steinberg RA. Pathways for degradation of the catalytic subunit of cAMP-dependent protein kinase differ in wild-type and kinase-negative S49 mouse lymphoma cells. J Biol Chem 1996; 271:16553-8. [PMID: 8663257 DOI: 10.1074/jbc.271.28.16553] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
The catalytic subunit of cAMP-dependent protein kinase radiolabeled with [35S]methionine in wild-type S49 mouse lymphoma cells was degraded with half-lives of approximately 9.2 h in unstimulated cells and approximately 4.5 h in cells stimulated with a membrane-permeable cAMP analog. Turnover in kinase-negative mutant cells was about three times faster than in stimulated wild-type cells and appeared to involve a unique 47-kDa intermediate. Levels of catalytic subunit protein revealed by Western immunoblotting were consistent with the measured differences in turnover, but whereas the protein was mostly soluble in wild-type cell extracts, it was almost entirely insoluble in the mutant cell extracts. A substantial fraction of the catalytic subunit labeled in a 5-min pulse was soluble in kinase-negative cell extracts, but most of this material was rendered insoluble by incubating the cells for an additional 30 min before extraction. Degradation of the catalytic subunit in kinase-negative, but not in wild-type, cells was inhibited strongly by two specific peptide aldehyde inhibitors of the proteasomal chymotrypsin-like activity. An inhibitor of the proteasomal protease that prefers branched-chain amino acids had less of an effect on catalytic subunit degradation in the mutant cells.
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Affiliation(s)
- S L Lee
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA
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