The Lysine 48 and Lysine 63 Ubiquitin Conjugates Are Processed Differently by the 26 S Proteasome
ABSTRACT The role of Lys-63 ubiquitin chains in targeting proteins for proteasomal degradation is still obscure. We systematically compared proteasomal processing of Lys-63 ubiquitin chains with that of the canonical proteolytic signal, Lys-48 ubiquitin chains. Quantitative mass spectrometric analysis of ubiquitin chains in HeLa cells determines that the levels of Lys-63 ubiquitin chains are insensitive to short-time proteasome inhibition. Also, the Lys-48/Lys-63 ratio in the 26 S proteasome-bound fraction is 1.7-fold more than that in the cell lysates, likely because some cellular Lys-63 ubiquitin conjugates are sequestered by Lys-63 chain-specific binding proteins. In vitro, Lys-48 and Lys-63 ubiquitin chains bind the 26 S proteasome comparably, whereas Lys-63 chains are deubiquitinated 6-fold faster than Lys-48 chains. Also, Lys-63 tetraubiquitin-conjugated UbcH10 is rapidly deubiquitinated into the monoubiquitinated form, whereas Lys-48 tetraubiquitin targets UbcH10 for degradation. Furthermore, we found that both the ubiquitin aldehyde- and 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 S proteasome contribute to Lys-48- and Lys-63-linkage deubiquitination, albeit the inhibitory extents are different. Together, our findings suggest that compared with Lys-48 chains, cellular Lys-63 chains have less proteasomal accessibility, and proteasome-bound Lys-63 chains are more rapidly deubiquitinated, which could cause inefficient degradation of Lys-63 conjugates.
SourceAvailable from: Pádraig Bernard D'Arcy[Show abstract] [Hide abstract]
ABSTRACT: The ubiquitin proteasome system (UPS) is the main system for controlled protein degradation and a key regulator of fundamental cellular processes. The dependency of cancer cells on a functioning UPS has made this an attractive target for development of drugs that show selectivity for tumor cells. Deubiquitinases (DUBs, ubiquitin isopeptidases) are components of the UPS that catalyze the removal of ubiquitin moieties from target proteins or polyubiquitin chains, resulting in altered signaling or changes in protein stability. A number of DUBs regulate processes associated with cell proliferation and apoptosis, and as such represent candidate targets for cancer therapeutics. The majority of DUBs are cysteine proteases and are likely to be more "druggable" than E3 ligases. Cysteine residues in the active sites of DUBs are expected to be reactive to various electrophiles. Various compounds containing α,β−unsaturated ketones have indeed been demonstrated to inhibit cellular DUB activity. Inhibition of proteasomal cysteine DUB enzymes (i.e. USP14 and UCHL5) can be predicted to be particularly cytotoxic to cancer cells as it leads to blocking of proteasome function and accumulation of proteasomal substrates. We here provide an overall review of DUBs relevant to cancer and of various small molecules which have been demonstrated to inhibit DUB activity.Pharmacology [?] Therapeutics 11/2014; 147. DOI:10.1016/j.pharmthera.2014.11.002 · 7.75 Impact Factor
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ABSTRACT: Protein homeostasis is largely dependent on proteolysis by the ubiquitin-proteasome system. Diverse polyubiquitin modifications are reported to target cellular proteins to the proteasome. At the proteasome, deubiquitination is an essential pre-processing event that contributes to degradation efficiency. We characterized the specificities of two proteasome-associated deubiquitinases (DUBs), Rpn11 and Ubp6, and explored their impact on overall proteasome DUB activity. This was accomplished by constructing a panel of well-defined ubiquitin (Ub) conjugates including homogeneous linkages of varying lengths as well as a heterogeneously modified target. Rpn11 and Ubp6 processed Lys11 and Lys63 linkages with comparable efficiencies that increased with chain length. In contrast, processing of Lys48 linkages by proteasome was inversely correlated to chain length. Fluorescently labeled tetra-Ub chains revealed endo-chain preference for Ubp6 acting on Lys48, and random action for Rpn11. Proteasomes were more efficient at deconjugating identical substrates than their constituent DUBs by roughly two orders of magnitude. Incorporation into proteasomes significantly enhanced enzymatic efficiency of Rpn11, due in part to alleviation of the auto-inhibitory role of its C-terminus. The broad specificity of Rpn11 could explain how proteasomes were more effective at disassembling a heterogeneously modified conjugate compared to homogenous Lys48-linked chains. The reduced ability to disassemble homogenous Lys48-linked chains longer than four Ub units may prolong residency time on the proteasome.Journal of Biological Chemistry 11/2014; DOI:10.1074/jbc.M114.568295 · 4.60 Impact Factor
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ABSTRACT: Ubiquitin-specific protease 14 (USP14) is one of three proteasome-associated deubiquitinating enzymes that remove ubiquitin from proteasomal substrates prior to their degradation. In vitro evidence suggests that inhibiting USP14's catalytic activity alters the turnover of ubiquitinated proteins by the proteasome, although whether protein degradation is accelerated or delayed seems to be cell-type and substrate specific. For example, combined inhibition of USP14 and the proteasomal deubiquitinating enzyme UCH37 halts protein degradation and promotes apoptosis in multiple myeloma cells, whereas USP14 inhibition alone accelerates the degradation of aggregate-prone proteins in immortalized cell lines. These findings have prompted interest in USP14 as a therapeutic target both inside and outside of the nervous system. However, loss of USP14 in the spontaneously occurring ataxia mouse mutant leads to a dramatic neuromuscular phenotype and early perinatal lethality, suggesting that USP14 inhibition may have adverse consequences in the nervous system. We therefore expressed a catalytically inactive USP14 mutant in the mouse nervous system to determine whether USP14's catalytic activity is required for neuromuscular junction (NMJ) structure and function. Mice expressing catalytically inactive USP14 in the nervous system exhibited motor deficits, altered NMJ structure, and synaptic transmission deficits that were similar to what is observed in the USP14-deficient ataxia mice. Acute pharmacological inhibition of USP14 in wild type mice also reduced NMJ synaptic transmission. However, there was no evidence of altered proteasome activity when USP14 was inhibited either genetically or pharmacologically. Instead, these manipulations increased the levels of non-proteasome targeting ubiquitin conjugates. Specifically, we observed enhanced proteasome-independent ubiquitination of mixed lineage kinase 3 (MLK3). Consistent with the direct activation of MLK3 by ubiquitination, we also observed increased activation of its downstrea targets MAP kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK). In vivo inhibition of JNK improved motor function and synapse structure in the USP14 catalytic mutant mice. USP14's catalytic activity is required for nervous system structure and function and has an ongoing role in NMJ synaptic transmission. By regulating the ubiquitination status of protein kinases, USP14 can coordinate the activity of intracellular signaling pathways that control the development and activity of the NMJ.Molecular Neurodegeneration 01/2015; 10(1):3. DOI:10.1186/1750-1326-10-3 · 5.29 Impact Factor