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The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3–dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.The cell cycle regulatory E3 ligase APC/C cooperates with UBE2C to prime substrates with ubiquitin and UBE2S to extend the ubiquitin chains. Careful analysis reveals that binding of the UBE2S to APC/C accelerates the rate-limiting step of APC/C–UBE2C.

The Anaphase Promoting Complex/Cyclosome (APC/C) is a ubiquitin E3 ligase that functions as the gatekeeper to mitotic exit. APC/C activity is controlled by an interplay of multiple pathways during mitosis, including the spindle assembly checkpoint (SAC), that are not yet fully understood. Here, we show that sumoylation of the APC4 subunit of the APC/C peaks during mitosis and is critical for timely APC/C activation and anaphase onset. We have also identified a functionally important SUMO interacting motif in the cullin-homology domain of APC2 located near the APC4 sumoylation sites and APC/C catalytic core. Our findings provide evidence of an important regulatory role for SUMO modification and binding in affecting APC/C activation and mitotic exit.

Signal transduction by small ubiquitin-like modifier (SUMO) regulates a myriad of nuclear processes. Here we report on the role of SUMO in mitosis in human cell lines. Knocking down the SUMO conjugation machinery results in a delay in mitosis and defects in mitotic chromosome separation. Searching for relevant SUMOylated proteins in mitosis, we identify the anaphase-promoting complex/cyclosome (APC/C), a master regulator of metaphase to anaphase transition. The APC4 subunit is the major SUMO target in the complex, containing SUMO acceptor lysines at positions 772 and 798. SUMOylation is crucial for accurate progression of cells through mitosis and increases APC/C ubiquitylation activity toward a subset of its targets, including the newly identified target KIF18B. Combined, our findings demonstrate the importance of SUMO signal transduction for genome integrity during mitotic progression and reveal how SUMO and ubiquitin cooperate to drive mitosis. Signal transduction by small ubiquitin-like modifier (SUMO) is important for cell cycle progression. Here the authors show that SUMOylation regulates the APC/C complex, a master orchestrator of metaphase to anaphase transition, with consequences for mitotic progression.

MicroRNA-452 (miRNA-452) was overexpressed in docetaxel-resistant human breast cancer MCF-7 cells (MCF-7/DOC). However, its role in modulating the sensitivity of breast cancer cells to docetaxel (DOC) remains unclear. The aim of this study is to investigate the role of miRNA-452 in the sensitivity of breast cancer cells to DOC. Real-time quantitative PCR (RT-qPCR) were used to identify the differential expression of miRNA-452 between MCF-7/DOC and MCF-7 cells. MiRNA-452 mimic was transfected into MCF-7 cells and miRNA-452 inhibitor was transfected into MCF-7/DOC cells. The role of miRNA-452 in these transfected cells was evaluated using RT-qPCR, MTT assay, and flow cytometry assay. The relationship of miRNA-452 and its predictive target gene “anaphase-promoting complex 4” ( APC4 ) was analyzed by RT-qPCR and Western blot. MiRNA-452 showed significantly higher expression (78.9-folds) in MCF-7/DOC cells compared to parental MCF-7 cells. The expression of miRNA-452 in the mimic transfected MCF-7 cells was upregulated 212.2-folds ( P  < 0.05) compared to its negative control (NC), and the half maximal inhibitory concentration (IC50) value of DOC (1.98 ± 0.15 μM) was significantly higher than that in its NC (0.85 ± 0.08 μM, P  < 0.05) or blank control (1.01 ± 0.19 μM, P  < 0.05). Furthermore, its apoptotic rate (6.3 ± 1.3 %) was distinctly decreased compared with that in its NC (23.8 ± 6.6 %, P  < 0.05) or blank control (18.6 ± 4.7 %, P  < 0.05). In contrast, the expression of miRNA-452 in the inhibitor-transfected MCF-7/DOC cells was downregulated 0.58-fold ( P  < 0.05) compared to its NC, the IC50 value of DOC (44.5 ± 3.2 μM) was significantly lower than that in its NC (107.3 ± 6.63 μM, P  < 0.05) or blank control (102.22 ± 11.34 μM, P  < 0.05), and the apoptotic rate (45.5 ± 10.8 %) was distinctly increased compared with its NC (9.9 ± 2.2 %, P  < 0.05) and blank control (9.4 ± 2.5 %, P  < 0.05). Further, there was an inverse association between miRNA-452 and APC4 expression in breast cancer cells in vitro. Dysregulation of miRNA-452 involved in the DOC resistance formation of breast cancer cells may be, in part, via targeting APC4 .

Human mutations in PQBP1 , a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1 -knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo . Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1 -cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4 , a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1 -cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1 -cKO mice by intraperitoneal injection of the PQBP1 -AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1 -cKO mice, opening a new strategy for treating this intractable developmental disorder.

Controls of stem cell maintenance and early differentiation are known in several systems. However, the progression from stem cell self-renewal to overt signs of early differentiation is a poorly understood but important problem in stem cell biology. The Caenorhabditis elegans germ line provides a genetically defined model for studying that progression. In this system, a single-celled mesenchymal niche, the distal tip cell (DTC), employs GLP-1/Notch signaling and an RNA regulatory network to balance self-renewal and early differentiation within the \"mitotic region,\" which continuously self-renews while generating new gametes. Here, we investigate germ cells in the mitotic region for their capacity to differentiate and their state of maturation. Two distinct pools emerge. The \"distal pool\" is maintained by the DTC in an essentially uniform and immature or \"stem cell-like\" state; the \"proximal pool,\" by contrast, contains cells that are maturing toward early differentiation and are likely transit-amplifying cells. A rough estimate of pool sizes is 30-70 germ cells in the distal immature pool and [almost equal to]150 in the proximal transit-amplifying pool. We present a simple model for how the network underlying the switch between self-renewal and early differentiation may be acting in these two pools. According to our model, the self-renewal mode of the network maintains the distal pool in an immature state, whereas the transition between self-renewal and early differentiation modes of the network underlies the graded maturation of germ cells in the proximal pool. We discuss implications of this model for controls of stem cells more broadly.

The anaphase-promoting complex (APC) is an E3 ubiquitin ligase which controls ubiquitination and degradation of multiple cell cycle regulatory proteins. During infection, human cytomegalovirus (HCMV), a widespread pathogen, not only phosphorylates the APC coactivator Cdh1 via the multifunctional viral kinase pUL97, it also promotes degradation of APC subunits via an unknown mechanism. Using a proteomics approach, we found that a recently identified HCMV protein, pUL21a, interacted with the APC. Importantly, we determined that expression of pUL21a was necessary and sufficient for proteasome-dependent degradation of APC subunits APC4 and APC5. This resulted in APC disruption and required pUL21a binding to the APC. We have identified the proline-arginine amino acid pair at residues 109-110 in pUL21a to be critical for its ability to bind and regulate the APC. A point mutant virus in which proline-arginine were mutated to alanines (PR-AA) grew at wild-type levels. However, a double mutant virus in which the viral ability to regulate the APC was abrogated by both PR-AA point mutation and UL97 deletion was markedly more attenuated compared to the UL97 deletion virus alone. This suggests that these mutations are synthetically lethal, and that HCMV exploits two viral factors to ensure successful disruption of the APC to overcome its restriction on virus infection. This study reveals the HCMV protein pUL21a as a novel APC regulator and uncovers a unique viral mechanism to subvert APC activity.

The anaphase-promoting complex is composed of eight protein subunits, including BimE (APC1), CDC27 (APC3), CDC16 (APC6), and CDC23 (APC8). The remaining four human APC subunits, APC2, APC4, APC5, and APC7, as well as human CDC23, were cloned. APC7 contains multiple copies of the tetratrico peptide repeat, similar to CDC16, CDC23, and CDC27. Whereas APC4 and APC5 share no similarity to proteins of known function, APC2 contains a region that is similar to a sequence in cullins, a family of proteins implicated in the ubiquitination of G$_1$ phase cyclins and cyclin-dependent kinase inhibitors. The APC2 gene is essential in Saccharomyces cerevisiae, and apc2 mutants arrest at metaphase and are defective in the degradation of Pds1p. APC2 and cullins may be distantly related members of a ubiquitin ligase family that targets cell cycle regulators for degradation.