Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ubiquitin-mediated proteolysis has emerged as a key mechanism of regulation in eukaryotic cells. During cell division, a multi-subunit ubiquitin ligase termed the anaphase promoting complex (APC) targets critical regulatory proteins such as securin and mitotic cyclins, and thereby triggers chromosome separation and exit from mitosis. Previous studies in the yeast Saccharomyces cerevisiae identified the conserved WD40 proteins Cdc20 and Hct1 (Cdh1) as substrate-specific activators of the APC, but their precise mechanism of action has remained unclear. This study provides evidence that Hct1 functions as a substrate receptor that recognizes target proteins and recruits them to the APC for ubiquitylation and subsequent proteolysis. By co-immunoprecipitation, we found that Hct1 interacted with the mitotic cyclins Clb2 and Clb3 and the polo-related kinase Cdc5, whereas Cdc20 interacted with the securin Pds1. Failure to interact with Hct1 resulted in stabilization of Clb2. Analysis of Hct1 derivatives identified the C-box, a motif required for APC association of Hct1 and conserved among Cdc20-related proteins. We propose that proteins of the Cdc20 family are substrate recognition subunits of the ubiquitin ligase APC.
 ...
PMID:Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC. 1156 80

Ubiquitin-mediated proteolysis of securin and mitotic cyclins is essential for exit from mitosis. The final step in ubiquitination of these and other proteins is catalysed by the anaphase-promoting complex (APC), a multi-subunit ubiquitin-protein ligase (E3). Little is known about the molecular reaction resulting in APC-dependent substrate ubiquitination or the role of individual APC subunits in the reaction. Using a well-defined in vitro system, we show that highly purified APC from Saccharomyces cerevisiae ubiquitinates a model cyclin substrate in a processive manner. Analysis of mutant APC lacking the Doc1/Apc10 subunit (APC(doc1 Delta)) indicates that Doc1 is required for processivity. The specific molecular defect in APC(doc1 Delta) is identified by a large increase in apparent K(M) for the cyclin substrate relative to the wild-type enzyme. This suggests that Doc1 stimulates processivity by limiting substrate dissociation. Addition of recombinant Doc1 to APC(doc1 Delta) fully restores enzyme function. Doc1-related domains are found in mechanistically distinct ubiquitin-ligase enzymes and may generally stimulate ubiquitination by contributing to substrate-enzyme affinity.
 ...
PMID:The Doc1 subunit is a processivity factor for the anaphase-promoting complex. 1240 45

Ubiquitin-mediated proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for sister chromatid separation and the mitotic exit. Like ubiquitylation, protein modification with the small ubiquitin-related modifier SUMO appears to be important during mitosis, because yeast cells impaired in the SUMO-conjugating enzyme Ubc9 were found to be blocked in mitosis and defective in cyclin degradation. Here, we analysed the role of SUMOylation in the metaphase/anaphase transition and in APC/C-mediated proteolysis in Saccharomyces cerevisiae. We show that cells depleted of Ubc9 or Smt3, the yeast SUMO protein, mostly arrested with undivided nuclei and with high levels of securin Pds1. This metaphase block was partially relieved by a deletion of PDS1. The absence of Ubc9 or Smt3 also resulted in defects in chromosome segregation. Temperature-sensitive ubc9-2 mutants were delayed in proteolysis of Pds1 and of cyclin Clb2 during mitosis. The requirement of SUMOylation for APC/C-mediated degradation was tested more directly in G1-arrested cells. Both ubc9-2 and smt3-331 mutants were defective in efficient degradation of Pds1 and mitotic cyclins, whereas proteolysis of unstable proteins that are not APC/C substrates was unaffected. We conclude that SUMOylation is needed for efficient proteolysis mediated by APC/C in budding yeast.
 ...
PMID:Smt3/SUMO and Ubc9 are required for efficient APC/C-mediated proteolysis in budding yeast. 1498 31

The anaphase-promoting complex (APC) is an E3 ubiquitin ligase that mediates the ubiquitination and degradation of the securin protein and mitotic cyclins, resulting in the regulation of the onset of sister-chromatid separation and mitotic exit. In an effort to identify novel therapeutic compounds that modulate cell proliferation and, therefore, have potential applications in oncology, a plate-based in vitro ubiquitination assay that uses recombinant purified E1, E2 (UbcH5c), E3 (APC11/APC2), and Flag-ubiquitin has been established and used to screen for small molecule inhibitors of APC E3 ligase activity. In this assay, APC2/APC11 is immobilized on the plate, and its E3 ligase activity (i.e., the incorporation of Flag-tagged polyubiquitin chain onto APC2/APC11 as a result of auto-ubiquitination) is detected with anti-Flag-horseradish peroxidase-conjugated antibody by monitoring the luminescence signal from the plate. Here we describe in detail the protocol for high-throughput screening of APC, including expression and purification of the individual proteins, assay development, and optimization. This assay has been validated in a 96-well plate format and successfully implemented to identify novel small molecule compounds that potently inhibit APC2/APC11 ligase activity.
 ...
PMID:High-throughput screening for inhibitors of the e3 ubiquitin ligase APC. 1633 93

Several models have been suggested above, describing possible modes of spindle checkpoint action: 1. Cdc20 sequestration (by Mad2-Cdc20 and/or MCC). 2. Stable MCC-APC/C association. 3. Cdc20 turnover (in budding yeast). 4. Cdc20-APC/C modification (by Mps1, Bub1, MAPK, Aurora B or BubR1 kinases). Several of these mechanisms could affect APC/C activity by modifying, competing for, and/or blocking the binding site(s) for its substrates. Alternatively, they could reduce the processivity of ubiquitination of substrates, or prevent the release of substrates and thereby reduce substrate turnover. Indeed, the processivity of ubiquitination can determine the order of destruction of APC/C substrates (Rape et al., 2006). Most substrates require multiple APC/C binding events in order to build polyubiquitin chains, and only polyubiquitinated substrates are recognised by the 26S proteasome for destruction. Thus, if the processivity of ubiquitination or the turnover of APC/C substrates were impaired in mitosis, the degradation of securin and cyclin would no longer take place, which would result in mitotic arrest. Our results have highlighted the importance of Mad3 as an anaphase inhibitor, and suggest that it usually acts in concert with Mad2 to efficiently inhibit Cdc20-APC/C. Further experiments are necessary to fully understand their mechanism of action, and this will require a wide range of approaches including dynamic studies of the 'flux' of Mad2 and BubR1 through signalling scaffolds, further structural insights, the identification of important phosphorylation sites on both the checkpoint proteins and Cdc20-APC/C, and an in vitro reconstitution of MCC inhibition of the APC/C. We look forward to seeing the complex regulation of mitotic progression being described over the coming years.
 ...
PMID:The spindle checkpoint: how do cells delay anaphase onset? 1836 27

Completion of the first meiotic division, manifested by extrusion of the first polar body (PBI), depends on proteasomal degradation of cyclin B1 and securin and the subsequent respective CDK1 inactivation and chromosome segregation. We aimed at identifying the polyubiquitin signal that mediates proteasomal action and at a better characterization of the role of CDK1 inactivation at this stage of meiosis. Microinjections of mutated ubiquitin proteins into mouse oocytes revealed that interference with lysine-11 polyubiquitin chains abrogated chromosome segregation and reduced PBI extrusion by 63% as compared to WT ubiquitin-injected controls. Inactivation of CDK1 in oocytes arrested at first metaphase by a proteasome inhibitor fully rescued PBI extrusion. However, removal of CDK1 inhibition failed to allow progression to the second metaphase, rather, inducing PBI reengulfment in 62% of the oocytes. Inhibition of either PLK1 or MEK1/2 during the first anaphase changed spindle dimensions. The PLK1 inhibitor also blocked PBI emission and prevented RhoA translocation. Our results identified lysine-11 rather than the canonic lysine-48 ubiquitin chains as the degradation signal in oocytes resuming meiosis, further disclosing that CDK1 inactivation is necessary and sufficient for PBI emission. This information significantly contributes to our understanding of faulty chromosome segregation that may lead to aneuploidy.
 ...
PMID:From ubiquitin-proteasomal degradation to CDK1 inactivation: requirements for the first polar body extrusion in mouse oocytes. 2285 67

Ubiquitin specific peptidase 44 (USP44) has been identified as an important component of spindle assemble checkpoint (SAC) to prevent the formation of aneuploidy. However, recent study raised a controversy about the effect of USP44 in tumor. Here, we first confirmed the intranuclear localization of USP44 by testing several specific antibodies to recognize endogenous USP44. Then, data from IHC and qRT-PCR assay indicated that the high expression of USP44 existed in high-grade glioma tissues and signified a poor prognosis. Knockdown of USP44 inhibited proliferation, migration and invasion, induced apoptosis, and arrested cell cycle in G2/M phase in the established glioma cell lines. Down-regulation of oncoprotein securin was detected in USP44 deficient cells, and the interaction of endogenous USP44 and securin was confirmed by immunoprecipitation in U251MG cells, which indicated that securin was a substrate of USP44, and might be stabilized by USP44. In vivo, knockdown of USP44 inhibited the tumorigenicity of U87MG cells significantly. Consequently, our findings suggested that overexpression of USP44 could enhance the malignancy of glioma via securin. USP44 might serve as a predictive biomarker, and the USP44-securin pathway might provide a new therapeutic strategy for the treatment of glioma.
 ...
PMID:Overexpression of ubiquitin specific proteases 44 promotes the malignancy of glioma by stabilizing tumor-promoter securin. 2893 51

Correct segregation of the mitotic chromosomes into daughter cells is a highly regulated process critical to safeguard genome stability. During M phase the spindle assembly checkpoint (SAC) ensures that all kinetochores are correctly attached before its inactivation allows progression into anaphase. Upon SAC inactivation, the anaphase promoting complex/cyclosome (APC/C) E3 ligase ubiquitinates and targets cyclin B and securin for proteasomal degradation. Here, we describe the identification of Ribonucleic Acid Export protein 1 (RAE1), a protein previously shown to be involved in SAC regulation and bipolar spindle formation, as a novel substrate of the deubiquitinating enzyme (DUB) Ubiquitin Specific Protease 11 (USP11). Lentiviral knock-down of USP11 or RAE1 in U2OS cells drastically reduces cell proliferation and increases multipolar spindle formation. We show that USP11 is associated with the mitotic spindle, does not regulate SAC inactivation, but controls ubiquitination of RAE1 at the mitotic spindle, hereby functionally modulating its interaction with Nuclear Mitotic Apparatus protein (NuMA).
 ...
PMID:USP11 deubiquitinates RAE1 and plays a key role in bipolar spindle formation. 2929 52

Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase-promoting complex/cyclosome and its coactivator CDC20 (APC/CCDC20) form the main ubiquitin E3 ligase for these two proteins. APC/CCDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylation. Here, we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilizes cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1 phosphorylation-defective CDC206A mutants. These CDC206A cells show a normal spindle checkpoint response and rapidly destroy cyclin B once all chromosomes have aligned and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase transition by promoting APC/CCDC20-dependent destruction of cyclin B in human cells.
 ...
PMID:PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20. 3275 77