Disassembly of Lys11 and Mixed Linkage Polyubiquitin Conjugates Provides Insights into Function of Proteasomal Deubiquitinases Rpn11 and Ubp6

Mansour, Wissam; Nakasone, Mark A.; Delbrück, Maximilian von; Yu, Zanlin; Krutauz, Daria; Reis, Noa; Kleifeld, Oded; Sommer, Thomas; Fushman, David; Glickman, Michael S.

doi:10.1074/jbc.m114.568295

Cited by 41 publications

(60 citation statements)

References 101 publications

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“…5C). These results indicated that USP14 has a broad DUB specificity toward various polyUb chain linkages as previously reported [13, 37], not having a preference for a specific type of chain linkages.…”

Section: Resultssupporting

confidence: 69%

“…These DUB enzymes residing on the proteasome belong to distinct classes: USP14 to the USP family, UCHL5 to UCH, and RPN11 to the JAMM family. It is possible that these DUB enzymes have different substrate preferences depending on their specificity for the type of linkage in the polyUb chain; however, polyUb chains composed of different Ub linkages showed only comparable affinity for the 26S proteasome and were similarly disassembled by proteasomal DUBs [12, 13]. …”

Section: Introductionmentioning

confidence: 99%

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Inactivation of USP14 Perturbs Ubiquitin Homeostasis and Delays the Cell Cycle in Mouse Embryonic Fibroblasts and in Fruit Fly Drosophila

Lee

Park

Yun

et al. 2018

Cell Physiol Biochem

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Background/Aims: The 26S proteasome is the key proteolytic complex for recognition and degradation of polyubiquitinated target substrates in eukaryotes. Among numerous proteasome-associated proteins, a deubiquitinating enzyme (DUB) USP14 has been identified as an endogenous inhibitor of the proteasome. Here, we explored the complex regulatory functions of USP14 that involve ubiquitin (Ub) homeostasis and substrate degradation in flies and mammals. Methods: USP14-null primary and immortalized mouse embryonic fibroblasts (MEFs) and USP14 knocked-down Drosophila were analyzed in this study. We measured proteasome and DUB activities using fluorogenic reporter substrates and adduct-forming probes. To examine the levels of ubiquitin, we performed immunoblotting and immunohistochemistry. Mass spectrometry (MS) was used to examine polyUb chain linkages and USP14-interacing proteins. Cell cycle was analyzed by flow cytometry, BrdU labeling, and phospho-histone H3 staining. Results: The homeostasis of Ub in USP14^–/–MEFs was markedly perturbed because of facilitated clearance of Ub. This phenomenon was recapitulated in muscles of USP14-deficient Drosophila with old ages. Absolute quantitation using MS also revealed that USP14^–/– MEFs contained significantly increased amounts of Ub, compared with wild-type. The key phenotype of USP14^–/– MEFs was their delayed proliferation originated from prolonged interphase possibly through aberrant degradation of cyclins A and B1. We found that knocking down USP14 in Drosophila resulted in delayed eye development associated with reduced mitotic activity. Conclusion: Our study identifies novel cellular functions of USP14 not only in cellular Ub hometostasis but also in cell cycle progression. USP14 was also essential for proper Drosophila eye development. These results strongly suggest that the USP14-mediated proteasome activity regulation may be directly related to various human diseases including cancer.

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Section: Resultssupporting

confidence: 69%

Section: Introductionmentioning

confidence: 99%

Inactivation of USP14 Perturbs Ubiquitin Homeostasis and Delays the Cell Cycle in Mouse Embryonic Fibroblasts and in Fruit Fly Drosophila

Lee

Park

Yun

et al. 2018

Cell Physiol Biochem

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“…Although Ub is a canonical degradation signal, both Ub-48 Ub and Ub-63 Ub are hydrolyzed by purified proteasomes in vitro (16,17), and some studies have observed Ub-63 Ub specificity in the polyUb amputation activity of Poh1 (18,19). Whether Ub-63 Ub has a role at the proteasome remains unclear; its presence at this location would seem incongruous with a model in which only Ub-48 Ublabeled proteins are genuine substrates (20,21).…”

mentioning

confidence: 75%

“…Ub must be removed from a substrate before translocation into the proteolytic proteasome core particle (15). Poh1 is a degradationcoupled DUB (25,29), with Ub-63 Ub specificity in vitro (18,19) and in cells (30). Electron microscopy experiments position Poh1's active site directly above the pore formed by the heterohexameric ATPase ring of the 19S base, through which a substrate must pass before entering the core particle (31)(32)(33).…”

Section: Discussionmentioning

confidence: 99%

Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21

Fletcher

Mallery

Watkinson

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

110

160

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Tripartite motif (TRIM) 21 is a cytosolic antibody receptor that neutralizes antibody-coated viruses that penetrate the cell and simultaneously activates innate immunity. Here we show that the conjugation of TRIM21 with K63-linked ubiquitin (Ub-63 Ub) catalyzed by the sequential activity of nonredundant E2 Ub enzymes is required for its dual antiviral functions. TRIM21 is first labeled with monoubiquitin (monoUb) by the E2 Ube2W. The monoUb is a substrate for the heterodimeric E2 Ube2N/Ube2V2, resulting in TRIM21-anchored Ub-63 Ub. Depletion of either E2 abolishes Ub-63 Ub and Ub-48 Ub conjugation of TRIM21, NF-κB signaling, and virus neutralization. The formation of TRIM21-Ub-63 Ub precedes proteasome recruitment, and we identify an essential role for the 19S-resident and degradation-coupled deubiquitinase Poh1 in TRIM21 neutralization, signaling, and cytokine induction. This study elucidates a complex mechanism of step-wise ubiquitination and deubiquitination activities that allows contemporaneous innate immune signaling and neutralization by TRIM21.

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“…Recent work revealed that K11-linked chains of six or more Ub units signal substrates for rapid degradation by the proteasome (Meyer and Rape, 2014) and that proteasomal DUBs rapidly disassemble K11 linkages (Mansour et al, 2015). Using K11-Ub 6+ as a substrate, in the absence of ubiB the proteasome produced numerous cleavage intermediates after just 30 minutes, and virtually all of the starting material was depleted at the end of the 20 hr time course (Fig 6C).…”

Section: Resultsmentioning

confidence: 99%

Structural Basis for the Inhibitory Effects of Ubistatins in the Ubiquitin-Proteasome Pathway

et al. 2017

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Summary The discovery of ubistatins, small molecules that impair proteasomal degradation of proteins by directly binding to polyubiquitin, makes ubiquitin itself a potential therapeutic target. Although ubistatins have the potential for drug development and clinical applications, the lack of structural details of ubiquitin-ubistatin interactions has impeded their development. Here, we characterized a panel of new ubistatin-derivatives using functional and binding assays. The structures of ubiquitin complexes with ubistatin-B and hemi-ubistatin revealed direct interactions with ubiquitin's hydrophobic surface-patch and the basic/polar residues surrounding it. Ubistatin-B binds ubiquitin and diubiquitin tighter than a high-affinity ubiquitin-receptor and shows strong preference for K48-linkages over K11 and K63. Furthermore, ubistatin-B shields ubiquitin conjugates from disassembly by a range of deubiquitinases and by the 26S-proteasome. Finally, ubistatin-B penetrates cancer cells and alters the cellular ubiquitin landscape. These findings highlight versatile properties of ubistatins and have implications for their future development and use in targeting ubiquitin-signaling pathways.

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Disassembly of Lys11 and Mixed Linkage Polyubiquitin Conjugates Provides Insights into Function of Proteasomal Deubiquitinases Rpn11 and Ubp6

Cited by 41 publications

References 101 publications

Inactivation of USP14 Perturbs Ubiquitin Homeostasis and Delays the Cell Cycle in Mouse Embryonic Fibroblasts and in Fruit Fly Drosophila

Inactivation of USP14 Perturbs Ubiquitin Homeostasis and Delays the Cell Cycle in Mouse Embryonic Fibroblasts and in Fruit Fly Drosophila

Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21

Structural Basis for the Inhibitory Effects of Ubistatins in the Ubiquitin-Proteasome Pathway

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