PLEKHA5 regulates mitotic progression by promoting APC/C localization to microtubules

The anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination of effector proteins. Despite the long-appreciated spatial organization of key events in mitosis mediated largely by cytoskeletal networks, the spatial regulation of APC/C, the major mitotic E3 ligase, is poorly understood. Here, we describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5-APC/C interaction. A microtubule-localized proximity biotinylation tool revealed that depletion of PLEKHA5 decreased the extent of APC/C association with microtubules. This decreased APC/C microtubule-localization in turn prevented efficient loading of APC/C with its co-activator CDC20, leading to defects in E3 ligase catalytic activity. We propose that PLEKHA5 functions as an adaptor of APC/C that promotes its subcellular localization to microtubules and facilitates its activation by CDC20, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis.

The APC/C targets the Cep152-Cep63 complex at the centrosome to regulate mitotic spindle assembly

The control of protein abundance is a fundamental regulatory mechanism during mitosis. The anaphase promoting complex/cyclosome (APC/C) is the main protein ubiquitin ligase responsible for the temporal regulation of mitotic progression. It has been proposed that the APC/C might fulfil other functions including assembly of the mitotic spindle. Here, we show that the APC/C localizes to centrosomes, the organizers of the eukaryotic microtubule cytoskeleton, specifically during mitosis. Recruitment of the APC/C to spindle poles requires the centrosomal protein Cep152, and we identified Cep152 as both an APC/C interaction partner and as an APC/C substrate. Previous studies showed that Cep152 forms a complex with Cep57 and Cep63. The APC/C-mediated ubiquitination of Cep152 at the centrosome releases Cep57 from this inhibitory complex and enables its interaction with pericentrin, a critical step in promoting microtubule nucleation. Thus, our study extends the function of the APC/C from being a regulator of mitosis to also acting as a positive governor of spindle assembly. The APC/C thereby integrates control of these two important processes in a temporal manner.

The Anaphase Promoting Complex/Cyclosome Subunit 11 and Its Role in Organ Size and Plant Development

The anaphase promoting complex/cyclosome (APC/C), a member of the E3 ubiquitin ligase family, plays an important role in recognizing the substrates to be ubiquitylated. Progression of anaphase, and therefore, of the cell cycle, is coordinated through cyclin degradation cycles dependent on proteolysis triggered by APC/C. The APC/C activity depends on the formation of a pocket comprising the catalytic subunits, APC2, APC11, and APC10. Among these, the role of APC11 outside the cell division cycle is poorly understood. Therefore, the goal of this work was to analyze the function of APC11 during plant development by characterizing apc11 knock-down mutant lines. Accordingly, we observed decreased apc11 expression in the mutant lines, followed by a reduction in meristem root size based on the cortical cell length, and an overall size diminishment throughout the development. Additionally, crosses of apc11-1 and amiR-apc11 with plants carrying a WUSCHEL-RELATED HOMEOBOX5 (WOX5) fluorescent marker showed a weakening of the green fluorescent protein-positive cells in the Quiescent Center. Moreover, plants with apc11-1 show a decreased leaf area, together with a decrease in the cell area when the shoot development was observed by kinematics analysis. Finally, we observed a decreased APC/C activity in the root and shoot meristems in crosses of pCYCB1;1:D-box-GUS with apc11-1 plants. Our results indicate that APC11 is important in the early stages of development, mediating meristematic architecture through APC/C activity affecting the overall plant growth.

Mitotic phosphorylation of tumor suppressor DAB2IP maintains spindle assembly checkpoint and chromosomal stability through activating PLK1-Mps1 signal pathway and stabilizing mitotic checkpoint complex

Chromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP’s interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.

The Anaphase Promoting Complex/ cyclosome co-activator, Cdh1, is a novel target of human Papillomavirus 16 E7 oncoprotein in cervical oncogenesis

The transforming properties of the high risk human papillomavirus E7 oncoprotein are indispensable for driving the virus life cycle and pathogenesis. Besides inactivation of retinoblastoma (Rb) family of tumor suppressors as part of its oncogenic endeavors, E7-mediated perturbations of eminent cell cycle regulators, checkpoint proteins and proto-oncogenes are considered to be the tricks of its transformative traits. However, many such critical interactions are still unknown. In the present study, we have identified the anaphase promoting complex/ cyclosome (APC/C) co-activator, Cdh1, as a novel interacting partner and a degradation target of E7. We found that HPV16 E7-induced inactivation of Cdh1 promoted abnormal accumulation of multiple Cdh1 substrates. Such a mode of deregulation possibly contributes to HPV-mediated cervical oncogenesis. Our mapping studies recognized the carboxyl-terminal zinc finger motif of E7 to associate with Cdh1 and interfere with the timely degradation of FoxM1, a bona fide Cdh1 substrate and a potent oncogene. Importantly, the E7 mutant with impaired interaction with Cdh1 exhibited defects in its ability for overriding typical cell cycle transition and oncogenic transformation, thereby validating the functional and pathological significance of the E7-Cdh1 axis during cervical carcinoma progression. Altogether, the findings from our study discover a unique nexus between E7 and APC/C-Cdh1, thereby adding to our understanding of the mechanism of E7-induced carcinogenesis and provide a promising target for the management of cervical carcinoma.

Bifidobacterium infantis Maintains Genome Stability in Ulcerative Colitis via Regulating Anaphase-Promoting Complex Subunit 7

Probiotics represents a promising intestinal microbiota-targeted therapeutic method for the treatment of ulcerative colitis (UC). Several lines of evidence implicate that Bifidobacterium infantis serves as a probiotic strain with proven efficacy in maintaining the remission of UC. However, the exact mechanisms underlying the beneficial effects of B. infantis on UC progression have yet to be elucidated. Herein, we provide evidence that B. infantis acts as a key predisposing factor for the maintenance of host genome stability. First, we showed that the fecal microbiota transplantation (FMT) of UC-derived feces contributes to more severely DNA damage in dextran sodium sulfate (DSS)-induced mice likely due to mucosa-associated microbiota alterations, as reflected by the rapid appearance of DNA double strand breaks (DSBs), a typical marker of genome instability. Genomic DNA damage analysis of colon tissues derived from healthy controls, patients with UC or dysplasia, and colitis associated cancer (CAC) patients, revealed an enhanced level of DSBs with aggravation in the degree of the intestinal mucosal lesions. To evaluate whether B. infantis modulates the host genome stability, we employed the DSS-induced colitis model and a TNFα-induced intestinal epithelial cell model. Following the administration of C57BL/6 mice with B. infantis via oral gavage, we found that the development of DSS-induced colitis in mice was significantly alleviated, in contrast to the colitis model group. Notably, B. infantis administration decreased DSB levels in both DSS-induced colitis and TNF-treated colonial cell model. Accordingly, our bioinformatic and functional studies demonstrated that B. infantis altered signal pathways involved in ubiquitin-mediated proteolysis, transcriptional misregulation in cancer, and the bacterial invasion of epithelial cells. Mechanistically, B. infantis upregulated anaphase-promoting complex subunit 7 (APC7), which was significantly suppressed in colitis condition, to activate the DNA repair pathway and alter the genome stability, while downregulation of APC7 abolished the efficiency of B. infantis treatment to induce a decrease in the level of DSBs in TNFα-induced colonial cells. Collectively, our results support that B. infantis orchestrates a molecular network involving in APC7 and genome stability, to control UC development at the clinical, biological, and mechanistic levels. Supplying B. infantis and targeting its associated pathway will yield valuable insight into the clinical management of UC patients.

Docking and ADMET studies for investigating the anticancer potency of Moscatilin on APC10/DOC1 and PKM2 against five clinical drugs

Background Moscatilin is a bibenzyl derivative (stilbenoid), mainly found in Dendrobium species. This plant-derived chemical is a potential cytotoxic anticancer drug that acts against different cancer types. The present study compared the structural interactions of Moscatilin along with five clinically relevant drugs against two target proteins, viz., Anaphase-Promoting Complex subunit 10/Death of Cyclase 1 and Pyruvate Kinase Muscle isozyme M2 in silico. Out of five clinical ligands, four were plant-derived compounds, viz., Resveratrol, Paclitaxel, Shikonin, and Colchicine. The synthetic chemotherapeutic agent, Mitomycin-C, was used as a ligand to compare the mechanistic insights. The objective of the study was to determine the anticancer potency of Moscatilin in silico. Results Moscatilin was found to have an advantage over other drugs of interest due to its structural simplicity and folding bridge connecting the bibenzyl structures. Moscatilin exhibited dual function by exclusively affecting the cancer cells, creating instabilities in biochemical and molecular cascades. Conclusions The study demonstrates that Moscatilin is has a multi-antimetastatic function. Moscatilin interaction with APC10/DOC1 indicated that the drug is involved with post-replicative inhibition, and with PKM2 showed glycolytic pathway inhibition in cancer cells. Moscatilin can function as an effective cell cycle inhibitor. Graphical abstract

Ubc1 turnover contributes to the spindle assembly checkpoint in Saccharomyces cerevisiae

The spindle assembly checkpoint protects the integrity of the genome by ensuring that chromosomes are properly attached to the mitotic spindle before they are segregated during anaphase. Activation of the spindle checkpoint results in inhibition of the Anaphase Promoting Complex (APC), an E3 ubiquitin ligase that triggers the metaphase-anaphase transition. Here we show that levels of Ubc1, an E2 enzyme that functions in complex with the APC, modulate the response to spindle checkpoint activation in Saccharomyces cerevisiae. Overexpression of Ubc1 increased resistance to microtubule poisons, whereas Ubc1 shut-off sensitized cells. We also found that Ubc1 levels are regulated by the spindle checkpoint. Checkpoint activation or direct APC inhibition led to a decrease in Ubc1 levels, charging and half-life. Additionally, stabilization of Ubc1 prevented its downregulation by the spindle checkpoint and increased resistance to checkpoint-activating drugs. These results suggest that downregulation of Ubc1 in response to spindle checkpoint signaling is necessary for a robust cell cycle arrest.

Single-molecule analysis of specificity and multivalency in binding of short linear substrate motifs to the APC/C

Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.

Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer

SUMMARYCDC20 is a co-activator of the anaphase promoting complex/cyclosome (APC/C) and is essential for mitotic progression. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC), which prevents premature separation of sister chromatids and aneuploidy in daughter cells. Although overexpression of CDC20 is common in many cancers, oncogenic mutations have never been identified in humans. Using whole exome sequencing, we identified heterozygous missense CDC20 variants (L151R and N331K) that segregate with cancer in two families. Characterization of these mutants showed they retain APC/C activation activity but show reduced binding to BUBR1, a component of the SAC. Expression of L151R and N331K promoted mitotic slippage in HeLa cells and primary skin fibroblasts derived from carriers. CRISPR/Cas9 was used to generate mice carrying N331K. Homozygous mice carrying N331K were non-viable, however, heterozygotes displayed accelerated oncogenicity in Myc-driven cancers. These findings highlight an unappreciated role for CDC20 variants as tumor promoting genes in humans.