The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that regulates mitosis and G1 by sequentially targeting cell-cycle regulators for ubiquitination and proteasomal degradation. The mechanism of ubiquitin chain formation by APC and the resultant chain topology remains controversial. By using a single-lysine APC substrate to dissect the topology of ubiquitinated substrates, we find that APC-catalyzed ubiquitination has an intrinsic preference for the K11 linkage of ubiquitin that is essential for substrate degradation. K11 specificity is determined by an E2 enzyme, UBE2S/E2-EPF, that elongates ubiquitin chains after the substrates are pre-ubiquitinated by UbcH10 or UbcH5. UBE2S copurifies with APC; dominant-negative Ube2S slows down APC substrate degradation in functional cell-cycle extracts. We propose that Ube2S is a critical, unique component of the APC ubiquitination pathway.
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... Lys11-linked ubiquitin chains may be of particular interest as a research direction for PROTAC developers, since they have proven to be critical regulators of mitotic protein degradation through the proteasome [76]. Their formation and destruction require the recruitment of a specific set of anaphase-promoting complex (APC/C), its specific chain-elongating E2, Ube2S and deubiquitinase [71,77,78], although it was shown that homotypic Lys11 chains did not bind to pure proteasomes or proteasomeassociated ubiquitin receptors [79]. ...
... Hydrolysis of such chains can be carried out by several enzymes, among which OTULIN is specific for this type of conjugation [52,118,120,121]. Similarly, Lys11 linked chains require linkage-specific enzymes: the anaphase-promoting complex (APC/C) and its specific chain-elongating E2, Ube2S; for cleavage -the Lys11 specific Cezanne [77,[122][123][124]. The role of such ubiquitinylation increases during cell division, when these conjugates target cell cycle regulators [122,125]. ...
Ubiquitinylation of protein substrates results in various but distinct biological consequences, among which ubiquitin-mediated degradation is most well studied for its therapeutic application. Accordingly, artificially targeted ubiquitin-dependent degradation of various proteins has evolved into the therapeutically relevant PROTAC technology. This tethered ubiquitinylation of various targets coupled with a broad assortment of modifying E3 ubiquitin ligases has been made possible by rational design of bi-specific chimeric molecules that bring these proteins in proximity. However, forced ubiquitinylation inflicted by the binary warheads of a chimeric PROTAC molecule should not necessarily result in protein degradation but can be used to modulate other cellular functions. In this respect it should be noted that the ubiquitinylation of a diverse set of proteins is known to control their transport, transcriptional activity, and protein-protein interactions. This review provides examples of potential PROTAC usage based on non-degradable ubiquitinylation.
... The APC/C and its E2s can perform a vast number of different ubiquitination reactions [21][22][23][29][30][31][32][33][34]41 . Trapping each of these individual structural states has required detailed biochemical knowledge about the system to stabilize the reaction intermediates 23,27,[36][37][38]42 . ...
... UBE2C can either continue to modify substrate lysines or make short chains 21,[29][30][31] . A second E2, UBE2S, extends the K11-linked Ub chains on the substrate [32][33][34] . These reactions can all occur during a single substrate-binding event while the E2s require multiple rounds of transient binding and catalysis to modify the substrate. ...
Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and obtaining mechanistic insight has thus far required artificially trapped structures to stabilize specific steps along the enzymatic process. So far, how any ubiquitin ligase builds a proteasomal degradation signal, which is canonically regarded as four or more ubiquitins, remains unclear. Here we present time-resolved cryogenic electron microscopy studies of the 1.2 MDa E3 ubiquitin ligase, known as the anaphase-promoting complex/cyclosome (APC/C), and its E2 co-enzymes (UBE2C/UBCH10 and UBE2S) during substrate polyubiquitination. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the human APC/C during polyubiquitination, directly visualize an active E3–E2 pair modifying its substrate, and identify unexpected interactions between multiple ubiquitins with parts of the APC/C machinery, including its coactivator CDH1. Together, we demonstrate how modification of substrates with nascent ubiquitin chains helps to potentiate processive substrate polyubiquitination, allowing us to model how a ubiquitin ligase builds a proteasomal degradation signal.
... E2 enzymes play a crucial role in determining the linkage specificity of the polyubiquitin chain (19)(20)(21). For instance, the human E2s Ube2C/UbcH10 and Ube2S (15,22) and the Arabidopsis homolog UBC22 (23) are able to catalyze K11-linked ubiquitination. An in vitro ubiquitination assay showed that AtUBC10 can conjugate both K48 and K63 linkages, while AtUBC35 mostly prefers K63 sites (24). ...
Ubiquitination plays a crucial role throughout plant growth and development. The E3 ligase DA2 has been reported to activate the peptidase DA1 by ubiquitination, hereby limiting cell proliferation. However, the molecular mechanisms that regulate DA2 remain elusive. Here, we demonstrate that DA2 has a very high turnover and auto-ubiquitinates with K48-linkage polyubiquitin chains, which is counteracted by two deubiquitinating enzymes, UBIQUITIN-SPECIFIC PROTEASE 12 (UBP12) and UBP13. Unexpectedly, we found that auto-ubiquitination of DA2 does not influence its stability but determines its E3 ligase activity. We also demonstrate that impairing the protease activity of DA1 abolishes the growth-reducing effect of DA2. Last, we show that synthetic, constitutively activated DA1-ubiquitin fusion proteins overrule this complex balance of ubiquitination and deubiquitination and strongly restrict growth and promote endoreduplication. Our findings highlight a nonproteolytic function of K48-linked polyubiquitination and reveal a mechanism by which DA2 auto-ubiquitination levels, in concert with UBP12 and UBP13, precisely monitor the activity of DA1 and fine-tune plant organ size.
... K29-linked ubiquitination is essential for proteasomal degradation [46,47]. In addition, we further speculated that K6-, K27-, and K33linked ubiquitin chains may also be related to protein degradation. ...
Glioblastoma, IDH-Wild type (GBM, CNS WHO Grade 4) is a highly heterogeneous and aggressive primary malignant brain tumor with high morbidity, high mortality, and poor patient prognosis. The global burden of GBM is increasing notably due to limited treatment options, drug delivery problems, and the lack of characteristic molecular targets. OTU deubiquitinase 4 (OTUD4) is a potential predictive factor for several cancers such as breast cancer, liver cancer, and lung cancer. However, its function in GBM remains unknown. In this study, we found that high expression of OTUD4 is positively associated with poor prognosis in GBM patients. Moreover, we provided in vitro and in vivo evidence that OTUD4 promotes the proliferation and invasion of GBM cells. Mechanism studies showed that, on the one hand, OTUD4 directly interacts with cyclin-dependent kinase 1 (CDK1) and stabilizes CDK1 by removing its K11, K29, and K33-linked polyubiquitination. On the other hand, OTUD4 binds to fibroblast growth factor receptor 1 (FGFR1) and reduces FGFR1’s K6 and K27-linked polyubiquitination, thereby indirectly stabilizing CDK1, ultimately influencing the activation of the downstream MAPK signaling pathway. Collectively, our results revealed that OTUD4 promotes GBM progression via OTUD4-CDK1-MAPK axis, and may be a prospective therapeutic target for GBM treatment.
... 10 UBE2S, a K11 linkage-specific E2, can prolong K11-linked polyubiquitin chains on substrates through cooperation with Ube2c/d or anaphase-promoting complex/cyclosome (APC/C). [11][12][13] Moreover, UBE2S can regulate differentiation of Sox2-mediated embryonic stem cell and associate with Ku70 for DNA repair. 14 Recently, many studies have indicated that UBE2S is overexpressed in multiple human primary cancers and is involved in tumorigenesis. ...
Background:
Ubiquitin-conjugating enzyme E2S (UBE2S), an E2 enzyme, is associated with the development of various tumors and exerts oncogenic activities. UBE2S is overexpressed in tumors, including hepatocellular carcinoma (HCC). However, the key molecular mechanisms of UBE2S in HCC still need additional research. The aim of this study was to explore the role of UBE2S in HCC.
Methods:
The expression levels of UBE2S in HCC tissues and cells were detected by western blot analysis, quantitative real-time polymerase chain reaction analysis (qRT-PCR), and immunohistochemistry (IHC). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, wound healing assay, colony formation assay transwell assay, and animal models were used to detect the proliferation and migration ability of HCC cells. Western blot analysis, qRT-PCR, immunofluorescence, small-interfering RNA (siRNA), and plasmid transfection and coimmunoprecipitation (Co-IP) assays were performed to detect the interaction among UBE2S, von Hippel-Lindau (VHL), hypoxia-inducible factor 1-alpha (HIF-1α), Janus kinase-2 (JAK2), and signal transducer and activator of transcription 3 (STAT3).
Results:
In this study, we found that high UBE2S expression was associated with poor prognosis in HCC patients. In addition, UBE2S expression was upregulated in HCC tissues and cell lines. Knockdown of UBE2S inhibited the proliferation and migration of HCC cells in vitro and in vivo by directly interacting with VHL to downregulate the HIF-1α and JAK2/STAT3 signaling pathways. Accordingly, overexpression of UBE2S significantly enhanced the proliferation and migration of HCC cells in vitro via VHL to upregulate HIF-1α and JAK2/STAT3 signaling pathways. Furthermore, we found that downregulation of UBE2S expression enhanced the sensitivity of HCC cells to sorafenib in vivo and in vitro.
Conclusion:
UBE2S enhances malignant properties via the VHL/HIF-1α and VHL/JAK2/STAT3 signaling pathways and reduces sensitivity to sorafenib in HCC. The findings of this study may open a new approach for HCC diagnosis and provide a potential option for the treatment of HCC.
The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.
Background: At the pan-cancer level, exploring the expression and prognostic significance of a gene, such as UBE2S, will help to gain insight into the role of the gene and its feasibility for cancer screening, prognosis assessment and even gene therapy. Methods: The Cancer Genome Atlas, Human Protein Atlas, Kaplan–Meier, Tumor Immunology Estimation Resource and other databases were used to analyze the expression of UBE2S at the pan-cancer level, its prognosis and the role of the immune microenvironment. Immunohistochemistry samples of tumor tissue collected in our clinic were taken as verification. Results: UBE2S is significantly overexpressed in pan-cancer and is closely associated with malignant clinical features, poor prognosis and tumor-associated macrophages. Conclusion: UBE2S may be a potential diagnostic and prognostic marker for pan-cancer and is associated with tumor-associated macrophages.
Ubiquitin (Ub) and ubiquitin-like (Ubl) pathways are critical post-translational modifications that determine whether functional proteins are degraded or activated/inactivated. To date, >600 associated enzymes have been reported that comprise a hierarchical task network (e.g., E1–E2–E3 cascade enzymatic reaction and deubiquitination) to modulate substrates, including enormous oncoproteins and tumor-suppressive proteins. Several strategies, such as classical biochemical approaches, multiomics, and clinical sample analysis, were combined to elucidate the functional relations between these enzymes and tumors. In this regard, the fundamental advances and follow-on drug discoveries have been crucial in providing vital information concerning contemporary translational efforts to tailor individualized treatment by targeting Ub and Ubl pathways. Correspondingly, emphasizing the current progress of Ub-related pathways as therapeutic targets in cancer is deemed essential. In the present review, we summarize and discuss the functions, clinical significance, and regulatory mechanisms of Ub and Ubl pathways in tumorigenesis as well as the current progress of small-molecular drug discovery. In particular, multiomics analyses were integrated to delineate the complexity of Ub and Ubl modifications for cancer therapy. The present review will provide a focused and up-to-date overview for the researchers to pursue further studies regarding the Ub and Ubl pathways targeted anticancer strategies.
Deubiquitinylating enzymes (DUBs) regulate the deubiquitinylation process of post-translationally modified proteins and thus control protein signaling in various cellular processes. The DUB Cezanne-1 catalyzes the cleavage of the iso-peptide bond of Lys11-linked polyubiquitin chains with high selectivity. Crystal structures of Cezanne-1 in different states provide important insight regarding the complex formation and global changes during the catalytic cycle but are lacking details of dynamics and control of activation. Activity-based probes are used to isolate intermediate states upon forming covalent bonds with the DUB active site. Those, however, may lead to structures that are non-native. Conformational changes of Cezanne-1, during its process of activation and proteolytic activity, are investigated using all-atom molecular dynamics (MD) simulations of the ubiquitin-free, diubiquitin-bound, and monoubiquitin-bound Cezanne-1 DUB for a total of ∼18 μs. Our results show that ubiquitin-free Cezanne-1 dynamically shuttles between catalytically competent and incompetent states which suggests that its activation is independent of substrate binding. The catalytically competent substrate-free Cezanne-1 promotes distal ubiquitin substrate access to the catalytic center. The subsequent binding of the proximal ubiquitin shifts the equilibrium toward the catalytically competent state of the dyad, thereby promoting proteolysis of the iso-peptide bond. After cleavage of the scissile bond, sequential dissociation of first the proximal ubiquitin induces the inactivation of Cezanne-1. The subsequent release of the distal ubiquitin fully reconstitutes the inactive substrate-free state of Cezanne-1. The process of activation and catalytic turnover of DUB Cezanne-1 is a multistage cycle with several critical dynamic transitions that cannot be characterized based on protein structures alone. Activity-based probes of cysteine proteases lead to non-native protein-protein contacts, which need to be resolved in order to be able to issue statements about physiological states and substrate binding.
The anaphase-promoting complex (APC/C), a ubiquitin ligase, is the target of the spindle-assembly checkpoint (SAC), and it ubiquitylates protein substrates whose degradation regulates progress through mitosis. The identity of the ubiquitin-conjugating (E2) enzymes that work with the APC/C is unclear. In an RNA interference (RNAi) screen for factors that modify release from drug-induced SAC activation, we identified the E2 enzyme UBE2S as an APC/C auxiliary factor that promotes mitotic exit. UBE2S is dispensable in a normal mitosis, but its depletion prolongs drug-induced mitotic arrest and suppresses mitotic slippage. In vitro, UBE2S elongates ubiquitin chains initiated by the E2 enzymes UBCH10 and UBCH5, enhancing the degradation of APC/C substrates by the proteasome. Indeed, following release from SAC-induced mitotic arrest, UBE2S-depleted cells neither degrade crucial APC/C substrates, nor silence this checkpoint, whereas bypassing the SAC through BUBR1 depletion or Aurora-B inhibition negates the requirement for UBE2S. Thus, UBE2S functions with the APC/C in a two-step mechanism to control substrate ubiquitylation that is essential for mitotic exit after prolonged SAC activation, providing a new model for APC/C function in human cells.
Autoantibodies from a patient suffering from endemic pemphigus foliaceus (EPF), a blistering skin disease, were used to screen a lambda gt11 human keratinocyte cDNA library. One immunoreactive cDNA clone (lambda EPF5) containing a 900-base pair insert was isolated and subjected to further analysis. Eight of 25 EPF sera were shown to react with the EPF5 fusion protein on immunoblots. The EPF5 cDNA insert hybridized with a 1.2-kilobase epidermal RNA transcript on a Northern blot. Sequence analysis revealed that lambda EPF5 contained the complete coding sequence for a 24-kDa polypeptide exhibiting significant sequence homology with a family of enzymes known as ubiquitin carrier proteins, or E2s, which are an essential component of the ubiquitin-protein conjugation system. The homology was particularly high in the core region containing the active site cysteine. The keratinocyte ubiquitin carrier protein expressed in bacteria, and isolated either intact or as a glutathione S-transferase fusion protein, exhibited the ability to form a thiol ester linkage with ubiquitin in a ubiquitin activating enzyme (E1)-dependent manner, a characteristic property of ubiquitin carrier proteins. The E2 enzyme encoded by clone EPF5 is the first member of this protein family to be cloned from an epidermal source. Interestingly, the EPF autoantibody-reactive epitope and the ubiquitin carrier protein were shown to be encoded in two different translational reading frames. The relevance of the cloned EPF epitope in the pathogenesis of this autoimmune disorder remains to be determined.
Target protein multi-ubiquitination involving lysine 48 of ubiquitin (Ub) is known to occur during protein degradation in the ATP- and Ub-dependent proteolytic pathway (Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989) Science 243, 1576-1583). However, little is known about the enzymatic mechanism of multi-ubiquitination. We show that a purified Ub carrier protein, E2(25)K, catalyzes multi-Ub chain synthesis from purified Ub. Incubation of E2(25)K with Ub activating enzyme (E1), MgATP, and radiolabeled Ub (Mr = 8500) resulted in time dependent appearance of a "ladder" of radiolabeled Ub conjugates with molecular masses of 8.5n kDa, where n = 1, 2, 3, 4... (up to at least n = 10). The kinetics of this conjugative process were consistent with Ub2 acting as a steady-state intermediate. The putative Ub2 product of E2(25)K catalysis was purified and cleaved with a partially purified isopeptidase preparation. The sole cleavage product (Mr = 8500) had a tryptic digest identical to that of authentic Ub, confirming that the original conjugate was Ub2. Tryptic digestion of intact Ub2 gave products consistent with the existence of an isopeptide linkage between the COOH terminus of one Ub and Lys-48 of the other; this structure was confirmed by sequence analysis of the unique Ub2 tryptic fragment. Tryptic digestion of higher order Ubn adducts (n greater than or equal to 4) yielded fragments identical to those of Ub2, indicating that E2(25)K ligates successive Ub molecules primarily or exclusively via Lys-48. Although several other E2s supported synthesis of an apparent Ub2 adduct of undetermined linkage, only E2(25)K was capable of synthesizing multi-Ub chains from isolated Ub. Quantitative analysis of single turnovers showed that transfer from E2(25)K-Ub to Ub and Ub2 occurred with kappa 2 = 488 and 1170 M-1 min-1, respectively, at pH 7.3 and 37 degrees C. These results show that increasing the number of Ub molecules in a chain increases susceptibility to further ubiquitination by E2(25)K. Ub2 was a good substrate for activation by E1 and was readily transferred to E2(25)K. The labile E2(25)K-Ub2 adduct was catalytically active, and exhibited preference for Ub2 (versus Ub) as acceptor. These results suggest that E2(25)K may function as a multi-ubiquitinating enzyme in the Ub-dependent proteolytic pathway.
Energy-dependent proteolysis is lost during maturation of rabbit reticulocytes to erythrocytes (Speiser, S., and Etlinger, J.D. (1982) J. Biol Chem. 257, 14122-14127), but nothing is known about the fates of individual components in the multienzyme ATP- and ubiquitin (Ub)-dependent proteolytic pathway during this process. Rabbit reticulocytes contain five low molecular weight carrier proteins (E2s) that form labile Ub adducts in the presence of Ub-activating enzyme (E1) (Pickart, C. M. and Rose, I. A. (1985) J. Biol. Chem. 260, 1573-1581). A method to estimate levels of active E2s in erythroid cells has been developed involving: 1) stepwise anion exchange fractionation of a soluble lysate; 2) addition of purified E1, MgATP, and radioiodinated Ub to the fractions followed by gel electrophoresis of the resulting E2-Ub adducts; and 3) quantitative densitometry of autoradiographs. Levels of active E2s are much lower in (rabbit) erythrocytes than in reticulocytes. Mean -fold decreases are: E235K, 6 x; E2(25K), 11 x; E2(20K), 18 x; E2(17K), not detected in erythrocytes; E2(14K), 12 x. The large decreases in levels of E2(20K) and E2(14K) are consistent with known functions of these proteins in DNA repair and Ub-dependent proteolysis, respectively. Decreases in levels of the other E2s, whose biological roles are presently unknown, suggest diminished requirements, if any, for them in erythrocyte metabolism. The analysis revealed two previously undescribed carrier proteins, one of which has a high molecular weight. Additional catalytic properties of E2(35K) and E2(14K) are reported.
Targeting of substrates for degradation by the ATP, ubiquitin-dependent pathway requires formation of multiubiquitin chains in which the 8.6-kDa polypeptide is linked by isopeptide bonds between carboxyl termini and Lys-48 residues of successive monomers. Binding of Lys-48-linked chains by subunit 5 of the 26 S proteasome regulatory complex commits the attached target protein to degradation with concomitant release of free ubiquitin monomers following disassembly of the chains. Point mutants of ubiquitin (Lys-->Arg) were used to map the linkage specificity for ubiquitin-conjugating enzymes previously demonstrated to form novel multiubiquitin chains not attached through Lys-48. Recombinant human E2EPF catalyzed multiubiquitin chain formation exclusively through Lys-11 of ubiquitin while recombinant yeast RAD6 formed chains linked only through Lys-6. Multiubiquitin chains linked through Lys-6, Lys-11, or Lys-48 each bound to subunit 5 of partially purified human 26 S proteasome with comparable affinities. Since chains bearing different linkages are expected to pack into distinct structures, competition between Lys-11 and Lys-48 chains for binding to subunit 5 demonstrates that the latter possesses determinants for recognizing alternatively linked chains and precludes the existence of subunit 5 isoforms recognizing distinct structures. In addition, competition studies provided an estimate of Kd < or = 18 nM for the intrinsic binding of Lys-48-linked chains of linkage number n > 4. This result suggests that the principal mechanistic advantage of multiubiquitin chain formation is to enhance the affinity of the associated substrate for the 26 S complex relative to that of unconjugated target protein. Complementation studies with E1/E2-depleted rabbit reticulocyte extract demonstrated RAD6 supported isopeptide ligase-dependent degradation only through Lys-48-linked chains, while E2EPF retained the ability to target a model radiolabeled substrate through Lys-11-linked chains. Therefore, the linkage specificity exhibited by these E2 isozymes depends on their catalytic context with respect to isopeptide ligase.
A novel member of the ubiquitin carrier protein family, designated E2EPF, has been cloned by our laboratory and expressed in a bacterial system in an active form. Ubiquitin carrier proteins, or E2s, catalyze one step in a multistep process that leads to the covalent conjugation of ubiquitin to substrate proteins. In this paper, we show that recombinant E2EPF catalyzes auto/multiubiquitination, the conjugation of multiple ubiquitin molecules to itself. Multiubiquitination has been shown previously to be required for targeting of a substrate protein for rapid degradation. Using a rabbit reticulocyte lysate system, E2EPF was shown to support the degradation of a model substrate in an ATP- and ubiquitin-dependent fashion. In contrast to a previous study which showed that selective protein degradation in one system is dependent upon multiubiquitination via the lysine 48 residue of ubiquitin, multiubiquitination, and proteolytic targeting by E2EPF was shown here to be independent of the lysine 48 multiubiquitin linkage. This functional characterization of E2EPF revealed a combination of features that distinguishes this enzyme from all previously characterized members of the ubiquitin carrier protein family. These results also suggest several possible autoregulatory models for E2EPF involving auto- and multiubiquitination.
A vertebrate securin (vSecurin) was identified on the basis of its biochemical analogy to the Pds1p protein of budding yeast
and the Cut2p protein of fission yeast. The vSecurin protein bound to a vertebrate homolog of yeast separins Esp1p and Cut1p
and was degraded by proteolysis mediated by an anaphase-promoting complex in a manner dependent on a destruction motif. Furthermore,
expression of a stableXenopus securin mutant protein blocked sister-chromatid separation but did not block the embryonic cell cycle. The vSecurin proteins
share extensive sequence similarity with each other but show no sequence similarity to either of their yeast counterparts.
Human securin is identical to the product of the gene called pituitary tumor-transforming gene (PTTG), which is overexpressed in some tumors and exhibits transforming activity in NIH 3T3 cells. The oncogenic nature of increased
expression of vSecurin may result from chromosome gain or loss, produced by errors in chromatid separation.
Ubiquitination by the anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The human APC/C promotes the degradation of mitotic regulators by assembling K11-linked ubiquitin chains, the formation of which is initiated by its E2 UbcH10. Here, we identify the conserved Ube2S as a K11-specific chain elongating E2 for human and Drosophila APC/C. Ube2S depends on the cell cycle-dependent association with the APC/C activators Cdc20 and Cdh1 for its activity. While depletion of Ube2S already inhibits APC/C in cells, the loss of the complete UbcH10/Ube2S-module leads to dramatic stabilization of APC/C substrates, severe spindle defects, and a strong mitotic delay. Ube2S and UbcH10 are tightly co-regulated in the cell cycle by APC/C-dependent degradation. We conclude that UbcH10 and Ube2S constitute a physiological E2-module for APC/C, the activity of which is required for spindle assembly and cell division.
p97 is an ATP-dependent chaperone that plays an important role in endoplasmic reticulum-associated degradation but whose connections to turnover of soluble proteins remain sparse. Binding of p97 to substrates is mediated by cofactors that contain ubiquitin-binding domains. We employed "network proteomics" to show that p97 assembles with all of the 13 mammalian UBX-domain proteins. The UBX proteins that bind ubiquitin conjugates also interact with dozens of E3 ubiquitin ligases, only one of which had been previously linked to p97. In particular, UBXD7 links p97 to the ubiquitin ligase CUL2/VHL and its substrate hypoxia-inducible factor 1alpha (HIF1alpha). Depletion of p97 leads to accumulation of endogenous HIF1alpha and increased expression of a HIF1alpha target gene. The large number of ubiquitin ligases found associated with UBX proteins suggests that p97 plays a far broader role than previously anticipated in the global regulation of protein turnover.
In vitro, the anaphase-promoting complex (APC) E3 ligase functions with E2 ubiquitin-conjugating enzymes of the E2-C and Ubc4/5 families to ubiquitinate substrates. However, only the use of the E2-C family, notably UbcH10, is genetically well validated. Here, we biochemically demonstrate preferential use of UbcH10 by the APC, specified by the E2 core domain. Importantly, an additional E2-E3 interaction mediated by the N-terminal extension of UbcH10 regulates APC activity. Mutating the highly conserved N terminus increases substrate ubiquitination and the number of substrate lysines targeted, allows ubiquitination of APC substrates lacking their destruction boxes, increases resistance to the APC inhibitors Emi1 and BubR1 in vitro, and bypasses the spindle checkpoint in vivo. Fusion of the UbcH10 N terminus to UbcH5 restricts ubiquitination activity but does not direct specific interactions with the APC. Thus, UbcH10 combines a specific E2-E3 interface and regulation via its N-terminal extension to limit APC activity for substrate selection and checkpoint control.