The E3 ubiquitin ligase HECTD1 contributes to cell cycle progression through an effect on mitosis

Mechanistic studies of how protein ubiquitylation regulates the cell cycle, in particular during mitosis, has provided unique insights which have contributed to the emergence of the Ubiquitin code. In contrast to RING E3 ubiquitin ligases such as the APC/c ligase complex, the contribution of other E3 ligase families during cell cycle progression remains less well understood. Similarly, the contribution of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains assembled by APC/c during mitosis, also remains to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate its function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell proliferation and we established that this is mediated through loss of its ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase). We confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. To explore the mechanisms involved, we used proteomics to explore the endogenous HECTD1 interactome in mitosis and validated the Mitosis Checkpoint Complex protein BUB3 as a novel HECTD1 interactor. In line with this, we found that HECTD1 depletion reduces the activity of the Spindle Assembly Checkpoint. Overall, our data suggests a novel role for HECTD1 ubiquitin ligase activity in mitosis.

Mitotic and DNA Damage Response Proteins: Maintaining the Genome Stability and Working for the Common Good

Cellular function is highly dependent on genomic stability, which is mainly ensured by two cellular mechanisms: the DNA damage response (DDR) and the Spindle Assembly Checkpoint (SAC). The former provides the repair of damaged DNA, and the latter ensures correct chromosome segregation. This review focuses on recently emerging data indicating that the SAC and the DDR proteins function together throughout the cell cycle, suggesting crosstalk between both checkpoints to maintain genome stability.

Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores

In metazoans, a ≍1 megadalton (MDa) super-complex comprising the Dynein-Dynactin adaptor Spindly and the ROD-Zwilch-ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high-resolution cryo-EM structure that captures the essential features of the RZZ complex, including a farnesyl binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ-Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation-dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long-sought-for molecular basis of corona assembly on metazoan kinetochores.

Inhibitors of the Bub1 spindle assembly checkpoint kinase: synthesis of BAY-320 and comparison with 2OH-BNPP1

Bub1 is a serine/threonine kinase proposed to function centrally in mitotic chromosome alignment and the spindle assembly checkpoint (SAC); however, its role remains controversial. Although it is well documented that Bub1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors are invaluable tools for investigating kinase function, we evaluated two potential Bub1 inhibitors: 2OH-BNPPI and BAY-320. After confirming that both inhibit Bub1 in vitro , we developed a cell-based assay for Bub1 inhibition. We overexpressed a fusion of Histone 2B and Bub1 kinase region, tethering it in proximity to H2A to generate a strong ectopic H2ApT120 signal along chromosome arms. Ectopic signal was effectively inhibited by BAY-320, but not 2OH-BNPP1 at concentrations tested. In addition, only BAY-320 was able to inhibit endogenous Bub1-mediated Sgo1 localization. Preliminary experiments using BAY-320 suggest a minor role for Bub1 kinase activity in chromosome alignment and the SAC; however, BAY-320 may exhibit off-target effects at the concentration required. Thus, 2OH-BNPP1 may not be an effective Bub1 inhibitor in cellulo , and while BAY-320 can inhibit Bub1 in cells, off-target effects highlight the need for improved Bub1 inhibitors.

TTK inhibition increases cisplatin sensitivity in high-grade serous ovarian carcinoma through the mTOR/autophagy pathway

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. However, the molecular mechanisms underlying HGSOC development, progression, chemotherapy insensitivity and resistance remain unclear. Two independent GEO datasets, including the gene expression profile of primary ovarian carcinoma and normal controls, were analyzed to identify genes related to HGSOC development and progression. A KEGG pathway analysis of the differentially expressed genes (DEGs) revealed that the cell cycle pathway was the most enriched pathway, among which TTK protein kinase (TTK) was the only gene with a clinical-grade inhibitor that has been investigated in a clinical trial but had not been studied in HGSOC. TTK was also upregulated in cisplatin-resistant ovarian cancer cells from two other datasets. TTK is a regulator of spindle assembly checkpoint signaling, playing an important role in cell cycle control and tumorigenesis in various cancers. However, the function and regulatory mechanism of TTK in HGSOC remain to be determined. In this study, we observed TTK upregulation in patients with HGSOC. High TTK expression was related to a poor prognosis. Genetic and pharmacological inhibition of TTK impeded the proliferation of ovarian cancer cells by disturbing cell cycle progression and increasing apoptosis. TTK silencing increased cisplatin sensitivity by activating the mammalian target of rapamycin (mTOR) complex to further suppress cisplatin-induced autophagy in vitro. In addition, the enhanced sensitivity was partially diminished by rapamycin-mediated inhibition of mTOR in TTK knockdown cells. Furthermore, TTK knockdown increased the toxicity of cisplatin in vivo by decreasing autophagy. These findings suggest that the administration of TTK inhibitors in combination with cisplatin may lead to improved response rates to cisplatin in patients with HGSOC presenting high TTK expression. In summary, our study may provide a theoretical foundation for using the combination therapy of cisplatin and TTK inhibitors as a treatment for HGSOC in the future.

BP-M345, a New Diarylpentanoid with Promising Antimitotic Activity

Previously, we reported the in vitro growth inhibitory effect of diarylpentanoid BP-M345 on human cancer cells. Nevertheless, at that time, the cellular mechanism through which BP-M345 exerts its growth inhibitory effect remained to be explored. In the present work, we report its mechanism of action on cancer cells. The compound exhibits a potent tumor growth inhibitory activity with high selectivity index. Mechanistically, it induces perturbation of the spindles through microtubule instability. As a consequence, treated cells exhibit irreversible defects in chromosome congression during mitosis, which induce a prolonged spindle assembly checkpoint-dependent mitotic arrest, followed by massive apoptosis, as revealed by live cell imaging. Collectively, the results indicate that the diarylpentanoid BP-M345 exerts its antiproliferative activity by inhibiting mitosis through microtubule perturbation and causing cancer cell death, thereby highlighting its potential as antitumor agent.

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.