Anaphase-promoting Complex Research Articles

Glutamine synthetase licenses APC/C-mediated mitotic progression to drive cell growth.

Tumors can reprogram the functions of metabolic enzymes to fuel malignant growth; however, beyond their conventional functions, key metabolic enzymes have not been found to directly govern cell mitosis. Here, we report that glutamine synthetase (GS) promotes cell proliferation by licensing mitotic progression independently of its metabolic function. GS depletion, but not impairment of its enzymatic activity, results in mitotic arrest and multinucleation across multiple lung and liver cancer cell lines, patient-derived organoids and xenografted tumors. Mechanistically, GS directly interacts with the nuclear pore protein NUP88 to prevent its binding to CDC20. Such interaction licenses activation of the CDC20-mediated anaphase-promoting complex or cyclosome to ensure proper metaphase-to-anaphase transition. In addition, GS is overexpressed in human non-small cell lung cancer and its depletion reduces tumor growth in mice and increases the efficacy of microtubule-targeted chemotherapy. Our findings highlight a moonlighting function of GS in governing mitosis and illustrate how an essential metabolic enzyme promotes cell proliferation and tumor development, beyond its main metabolic function.

SAPCD2 promotes neuroblastoma progression by altering the subcellular distribution of E2F7

Recent studies uncovered the emerging roles of SAPCD2 (suppressor anaphase-promoting complex domain containing 2) in several types of human cancer. However, the functions and underlying mechanisms of SAPCD2 in the progression of neuroblastoma (NB) remain elusive. Herein, through integrative analysis of public datasets and regulatory network of GSK-J4, a small-molecule drug with anti-NB activity, we identified SAPCD2 as an appealing target with a high connection to poor prognosis in NB. SAPCD2 promoted NB progression in vitro and in vivo. Mechanistically, SAPCD2 could directly bind to cytoplasmic E2F7 but not E2F1, alter the subcellular distribution of E2F7 and regulate E2F activity. Among the E2F family members, the roles of E2F7 in NB are poorly understood. We found that an increasing level of nuclear E2F7 was induced by SAPCD2 knockdown, thereby affecting the expression of genes involved in the cell cycle and chromosome instability. In addition, Selinexor (KTP-330), a clinically available inhibitor of exportin 1 (XPO1), could induce nuclear accumulation of E2F7 and suppress the growth of NB. Overall, our studies suggested a previously unrecognized role of SAPCD2 in the E2F signaling pathway and a potential therapeutic approach for NB, as well as clues for understanding the differences in subcellular distribution of E2F1 and E2F7 during their nucleocytoplasmic shuttling.

Ubiquitin-proteasome pathway plays an essential regulatory role during spermatangium formation in Neopyropia yezoensis

Gerproductive cell formation is one of the most important physiological processes of organisms. The Rhodophyta, Neopyropia yezoensis, is a typical Archaeplastida and the spermatangia is usually formed along the edge of thalli and visible to the naked eye. It is therefore meaningful to invest the mechanisms of cell development. Here, the cell clumps at four typical differentiation stages of spermatangium in N. yezoensis were collected and transcriptome analysis was performed. A total of 12,188 differentially expressed genes (DEGs) were identified and the number of DEGs increased compared to the control as spermatangium formation progressed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that carbon fixation, glycolysis/gluconeogenesis, photosynthesis-antenna proteins and some primary metabolic processes were down-regulated. In contrast, sphingolipid metabolism, inositol phosphate metabolism, ubiquitin mediated proteolysis and DNA replication were up-regulated. The development of spermatangia is believed to be induced by the sex signaling molecules due to the up-regulation of sphingolipid metabolism, the biosynthesis of phytosterol and the expression of a steroid bound receptor of heme-binding b5-domain containing protein at the initial stage of spermatangia formation. During the course of development, a total of 163 DEGs related to the ubiquitin-proteasome (Ub-proteasome) system were identified and half of these were up-regulated. Additionally, a series of ubiquitin-protein ligases (E3s) related to development, cell cycle, and chromosome replication were analyzed by qRT-PCR. The continuously upregulated of histone H2A/H2B implied that the shift of global gene expression profile modulated by the Ub-proteasome system was necessary during the differentiation. The up-regulation of the anaphase-promoting complex (APC) and its related protein factors meant that ubiquitin-mediated proteolysis enhanced the cell-cycle and chromosome replication. All the results suggest that the development of male germ cells is a highly modulated process and the Ub-proteasome system might play an essential regulatory role.

MultiFLEX-LF: A Computational Approach to Quantify the Modification Stoichiometries in Label-Free Proteomics Data Sets.

In liquid-chromatography-tandem-mass-spectrometry-based proteomics, information about the presence and stoichiometry of protein modifications is not readily available. To overcome this problem, we developed multiFLEX-LF, a computational tool that builds upon FLEXIQuant, which detects modified peptide precursors and quantifies their modification extent by monitoring the differences between observed and expected intensities of the unmodified precursors. multiFLEX-LF relies on robust linear regression to calculate the modification extent of a given precursor relative to a within-study reference. multiFLEX-LF can analyze entire label-free discovery proteomics data sets in a precursor-centric manner without preselecting a protein of interest. To analyze modification dynamics and coregulated modifications, we hierarchically clustered the precursors of all proteins based on their computed relative modification scores. We applied multiFLEX-LF to a data-independent-acquisition-based data set acquired using the anaphase-promoting complex/cyclosome (APC/C) isolated at various time points during mitosis. The clustering of the precursors allows for identifying varying modification dynamics and ordering the modification events. Overall, multiFLEX-LF enables the fast identification of potentially differentially modified peptide precursors and the quantification of their differential modification extent in large data sets using a personal computer. Additionally, multiFLEX-LF can drive the large-scale investigation of the modification dynamics of peptide precursors in time-series and case-control studies. multiFLEX-LF is available at https://gitlab.com/SteenOmicsLab/multiflex-lf.

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.

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

ABSTRACTThe 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 an APC/C substrate. Previous studies have shown that Cep152 forms a complex with Cep57 and Cep63. The APC/C-mediated ubiquitylation 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.

TORC1 inactivation promotes APC/C-dependent mitotic slippage in yeast and human cells

SummaryUnsatisfied kinetochore-microtubule attachment activates the spindle assembly checkpoint to inhibit the metaphase-anaphase transition. However, some cells eventually override mitotic arrest by mitotic slippage. Here, we show that inactivation of TORC1 kinase elicits mitotic slippage in budding yeast and human cells. Yeast mitotic slippage was accompanied with aberrant aspects, such as degradation of the nucleolar protein Net1, release of phosphatase Cdc14, and anaphase-promoting complex/cyclosome (APC/C)-Cdh1-dependent degradation of securin and cyclin B in metaphase. This mitotic slippage caused chromosome instability. In human cells, mammalian TORC1 (mTORC1) inactivation also invoked mitotic slippage, indicating that TORC1 inactivation-induced mitotic slippage is conserved from yeast to mammalian cells. However, the invoked mitotic slippage in human cells was not dependent on APC/C-Cdh1. This study revealed an unexpected involvement of TORC1 in mitosis and provides information on undesirable side effects of the use of TORC1 inhibitors as immunosuppressants and anti-tumor drugs.

APC7 mediates ubiquitin signaling in constitutive heterochromatin in the developing mammalian brain

Neurodevelopmental cognitive disorders provide insights into mechanisms of human brain development. Here, we report an intellectual disability syndrome caused by the loss of APC7, a core component of the E3 ubiquitin ligase anaphase promoting complex (APC). In mechanistic studies, we uncover a critical role for APC7 during the recruitment and ubiquitination of APC substrates. In proteomics analyses of the brain from mice harboring the patient-specific APC7 mutation, we identify the chromatin-associated protein Ki-67 as an APC7-dependent substrate of the APC in neurons. Conditional knockout of the APC coactivator protein Cdh1, but not Cdc20, leads to the accumulation of Ki-67 protein in neurons invivo, suggesting that APC7 is required for the function of Cdh1-APC in the brain. Deregulated neuronal Ki-67 upon APC7 loss localizes predominantly to constitutive heterochromatin. Our findings define an essential function for APC7 and Cdh1-APC in neuronal heterochromatin regulation, with implications for understanding human brain development and disease.

GhAPC8 regulates leaf blade angle by modulating multiple hormones in cotton (Gossypium hirsutum L.)

Leaf angle, including leaf petiole angle (LPA) and leaf blade angle (LBA), is an important trait affecting plant architecture. Anaphase-promoting complex/cyclosome (APC/C) genes play a vital role in plant growth and development, including regulation of leaf angle. Here, we identified and characterized the APC genes in Upland cotton (G. hirsutum L.) with a focus on GhAPC8, a homolog of soybean GmILPA1 involved in regulation of LPA. We showed that independently silencing the At or Dt sub-genome homoeolog of GhAPC8 using virus-induced gene silencing reduced plant height and LBA, and that reduction of LBA could be caused by uneven growth of cortex parenchyma cells on the adaxial and abaxial sides of the junction between leaf blade and leaf petiole. The junction between leaf blade and leaf petiole of the GhAPC8-silenced plants had an elevated level of brassinosteroid (BR) and a decreased levels of auxin and gibberellin. Consistently, comparative transcriptome analysis found that silencing GhAPC8 activated genes of the BR biosynthesis and signaling pathways as well as genes related to ubiquitin-mediated proteolysis. Weighted gene co-expression network analysis (WGCNA) identified gene modules significantly associated with plant height and LBA, and candidate genes bridging GhAPC8, the pathways of BR biosynthesis and signaling and ubiquitin-mediated proteolysis. These results demonstrated a role of GhAPC8 in regulating LBA, likely achieved by modulating the accumulation and signaling of multiple phytohormones.

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.

Abstract PO-060: N-terminal RHAMM cooperates with dysfunctional p53 to accelerate the progression of pancreatic cancer

Abstract Pancreatic cancer has the lowest survival rate in all types of cancer. Pancreatic cancer patients are often diagnosed at advanced stages. A better therapeutic development for this devastating disease is urgently needed. Receptor for hyaluronan-mediated motility (RHAMM), not expressed in adult normal pancreas, has been suggested as a prognostic factor and a potential therapeutic target for pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumor (PNET). In this study, we initially sought to determine whether genetic deletion of RHAMM would slow down pancreatic cancer progression using Rhamm−/− mice. However, we found that Rhamm−/− mice expressed a N-terminal RHAMM protein. While N-terminal RHAMM did not enable malignant progression of pancreatic intraepithelial neoplasia in p48-Cre; LSL-KRASG12D mice, it accelerated the formation of invasive PDAC and shortened the survival of p48-Cre; LSL-KRASG12D mice with heterozygous p53 knockout. KrasG12D PDAC mice with homozygous p53 knockout mice died around 10 weeks, and the effect of N-terminal RHAMM was not apparent in these mice with short life span. In addition, N-terminal RHAMM shortened the survival of PNET-bearing RIP-Tag mice, which had inactivated p53. In our analysis of TCGA dataset, pancreatic cancer patients with mutant TP53 or loss of one copy of TP53 had higher RHAMM expression, which, combined, predicted worse outcomes. Taken together, by collaborating with dysfunctional p53, N-terminal RHAMM that lacks the centrosome targeting domain and degrons for interaction with the Anaphase-Promoting Complex (APC) accelerated pancreatic cancer progression. Citation Format: Anthony Lin, Jennifer Feng, Xiang Chen, Dunrui Wang, Megan Wong, George Zhang, Joseph Na, Tiantian Zhang, Zhengming Chen, Yao-Tseng Chen, Yi-Chieh Nancy Du. N-terminal RHAMM cooperates with dysfunctional p53 to accelerate the progression of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-060.

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.

De novo FZR1 loss-of-function variants cause developmental and epileptic encephalopathies.

FZR1, which encodes the Cdh1 subunit of the anaphase-promoting complex, plays an important role in neurodevelopment by regulating the cell cycle and by its multiple post-mitotic functions in neurons. In this study, evaluation of 250 unrelated patients with developmental and epileptic encephalopathies and a connection on GeneMatcher led to the identification of three de novo missense variants in FZR1. Whole-exome sequencing in 39 patient-parent trios and subsequent targeted sequencing in an additional cohort of 211 patients was performed to identify novel genes involved in developmental and epileptic encephalopathy. Functional studies in Drosophila were performed using three different mutant alleles of the Drosophila homologue of FZR1 fzr. All three individuals carrying de novo variants in FZR1 had childhood-onset generalized epilepsy, intellectual disability, mild ataxia and normal head circumference. Two individuals were diagnosed with the developmental and epileptic encephalopathy subtype myoclonic atonic epilepsy. We provide genetic-association testing using two independent statistical tests to support FZR1 association with developmental and epileptic encephalopathies. Further, we provide functional evidence that the missense variants are loss-of-function alleles using Drosophila neurodevelopment assays. Using three fly mutant alleles of the Drosophila homologue fzr and overexpression studies, we show that patient variants can affect proper neurodevelopment. With the recent report of a patient with neonatal-onset with microcephaly who also carries a de novo FZR1 missense variant, our study consolidates the relationship between FZR1 and developmental and epileptic encephalopathy and expands the associated phenotype. We conclude that heterozygous loss-of-function of FZR1 leads to developmental and epileptic encephalopathies associated with a spectrum of neonatal to childhood-onset seizure types, developmental delay and mild ataxia. Microcephaly can be present but is not an essential feature of FZR1-encephalopathy. In summary, our approach of targeted sequencing using novel gene candidates and functional testing in Drosophila will help solve undiagnosed myoclonic atonic epilepsy or developmental and epileptic encephalopathy cases.

CCT4 suppression inhibits tumor growth in hepatocellular carcinoma by interacting with Cdc20.

Background:The chaperonin containing t-complex (CCT) proteins play an important role in cell cycle-related protein degradation in yeast and mammals. The role of the chaperonin containing t-complex 4 (CCT4), one subtype of CCT proteins, in the progress of hepatocellular carcinoma (HCC) was not fully elucidated. Here, we aimed to explore the mechanisms of CCT4 in HCC.Methods:In this study, we used the UALCAN platform to analyze the relationship between CCT4 and HCC, and the association of CCT4 with the overall survival (OS) of HCC patients was also analyzed. CCT4 expression in HCC tumor tissues and normal tissues was also determined by western blot (WB) assay. Lentivirus vector was used to knock down the CCT4 expression, and quantitative polymerase chain reaction and WB were used to determine the level of CCT4 in HCC cell lines. Cell counting kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assays were used to detect the cell proliferation, and flow cytometry (FCM) was performed to evaluate the effect of CCT4 on the apoptosis of HCC cells. Co-immunoprecipitation (co-IP) assay and WB were used to explore the mechanisms of CCT4 regulating the growth of HCC. Data were calculated from at least three replicate experiments and expressed as mean ± standard deviation. Student's t test, paired t test, and Kaplan–Meier analysis were used to compare across different groups.Results:We found CCT4 was upregulated in HCC tissues compared with normal tissues, and its high expression was associated with poor prognosis (P < 0.001). CCT4 was significantly increased in HCC tumor tissues compared with normal tissues (0.98 ± 0.12 vs. 0.23 ± 0.05, t = 7.73, P < 0.001). After being transfected with CCT4 short-hairpin RNA (shRNA), CCT4 was decreased in mRNA level and protein level in both Huh7 (mRNA level: 0.41 ± 0.07 vs. 1.01 ± 0.11, t = 8.09, P = 0.001; protein level: 0.61 ± 0.03 vs. 0.93 ± 0.07, t = 7.19, P = 0.002) and Hep3b cells (mRNA level: 0.55 ± 0.11 vs. 1.04 ± 0.15, t = 4.51, P = 0.011; protein level: 0.64 ± 0.10 vs. 0.95 ± 0.08, t = 4.32, P = 0.012). CCK8 assay indicated that CCT4 knockdown inhibited cell proliferation in both Huh7 (OD value of 3 days: 0.60 ± 0.14 vs. 0.97 ± 0.16, t = 3.13, P = 0.036; OD value of 4 days: 1.03 ± 0.07 vs. 1.50 ± 0.12, t = 5.97, P = 0.004) and Hep3b (OD value of 3 days: 0.69 ± 0.14 vs. 1.10 ± 0.11, t = 3.91, P = 0.017; OD value of 4 days: 1.12 ± 0.12 vs. 1.48 ± 0.13, t = 3.55, P = 0.024) cells. EdU assay showed that CCT4 knockdown inhibited the cell proliferation in both Huh7 (EdU positive rate: [31.25 ± 3.41]% vs. [58.72 ± 3.78]%, t = 9.34, P = 0.001) and Hep3b cells (EdU positive rate: [44.13 ± 7.02]% vs. [61.79 ± 3.96]%, t = 3.79, P = 0.019). FCM assay suggested that CCT4 knockdown induced apoptosis in HCC cells (apoptosis rate of Huh7: [9.10 ± 0.80]% vs. [3.66 ± 0.64]%, t = −9.18, P = 0.001; apoptosis rate of Hep3b: [6.69 ± 0.72]% vs. [4.20 ± 0.86]%, t = −3.84, P = 0.018). We also found that CCT4 could regulate anaphase-promoting complex (APC)Cdc20 activity via interacting with Cdc20. Furthermore, CCT4 knockdown induced securin (0.65 ± 0.06 vs. 0.44 ± 0.05, t = −4.69, P = 0.009) and B-cell lymphoma-2 (Bcl-2) interacting mediator of cell death (Bim; 0.96 ± 0.06 vs. 0.61 ± 0.09, t = −5.65, P = 0.005) accumulation. The upregulation of securin inhibited cell growth by downregulating cyclin D1 (0.65 ± 0.05 vs. 1.04 ± 0.07, t = 8.12, P = 0.001), and the accumulation of Bim inhibited Bcl-2 (0.77 ± 0.04 vs. 0.87 ± 0.04, t = 3.00, P = 0.040) and activated caspase 9 (caspase 9: 0.77 ± 0.04 vs. 0.84 ± 0.05, t = 1.81, P = 0.145; cleaved caspase 9: 0.64 ± 0.06 vs. 0.16 ± 0.07, t = 1.81, P = 0.001), which led to elevated apoptosis.Conclusions:Overall, these results showed that CCT4 played an important role in HCC pathogenesis through, at least partly, interacting with Cdc20.

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.

NF2 Gene Participates in Regulation of the Cell Cycle of Meningiomas by Restoring Spindle Assembly Checkpoint Function and Inhibiting the Binding of Cdc20 Protein to Anaphase Promoting Complex/Cyclosome

The neurofibromatosis type 2 (NF2) gene mutation is the leading genetic event in meningiomas, usually accompanied by malignant features. Dysfunction of the spindle assembly checkpoint (SAC) induces tumorigenesis. However, the crosstalk between NF2 and SAC in meningiomas remains unclear. Cell proliferation, invasion, apoptosis, and cell cycle of meningiomas were determined by cell counting kit-8 assay, transwell assay, and flow cytometry, respectively. The expression of SAC in meningioma cells was detected by quantitative real-time polymerase chain reaction and Western blot. The interaction between anaphase promoting complex/cyclosome (APC/C) and cell division cycle 20 (Cdc20) protein in meningioma cells was further explored by co-immunoprecipitation. We found that the expression of NF2/merlin was low or absent in malignant meningiomas. Overexpression of NF2 suppressed the proliferation and invasion of meningioma cells, prolonged the G2/M phase, and elevated the expression of SAC proteins at posttranscription. Furthermore, the interaction between APC/C and Cdc20 was inhibited by NF2. Our findings suggested that NF2 might restore SAC function by impairing the binding of APC/C and Cdc20, thereby limiting the mitotic rate and inhibiting proliferation of meningiomas.

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

BackgroundMoscatilin 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. ResultsMoscatilin 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. ConclusionsThe 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▪

The APC/CFZY-1/Cdc20 Complex Coordinates With OMA-1 to Regulate the Oocyte-to-Embryo Transition in Caenorhabditis elegans.

During oocyte maturation and the oocyte-to-embryo transition, key developmental regulators such as RNA-binding proteins coordinate translation of particular messenger RNA (mRNAs) and related developmental processes by binding to their cognate maternal mRNAs. In the nematode Caenorhabditis elegans, these processes are regulated by a set of CCCH zinc finger proteins. Oocyte maturation defective-1 (OMA-1) and OMA-2 are two functionally redundant CCCH zinc finger proteins that turnover rapidly during the first embryonic cell division. These turnovers are required for proper transition from oogenesis to embryogenesis. A gain-of-function mutant of OMA-1, oma-1(zu405), stabilizes and delays degradation of OMA-1, resulting in delayed turnover and mis-segregation of other cell fate determinants, which eventually causes embryonic lethality. We performed a large-scale forward genetic screen to identify suppressors of the oma-1(zu405) mutant. We show here that multiple alleles affecting functions of various anaphase promoting complex/cyclosome (APC/C) subunits, including MAT-1, MAT-2, MAT-3, EMB-30, and FZY-1, suppress the gain-of-function mutant of OMA-1. Transcriptome analysis suggested that overall transcription in early embryos occurred after introducing mutations in APC/C genes into the oma-1(zu405) mutant. Mutations in APC/C genes prevent OMA-1 enrichment in P granules and correct delayed degradation of downstream cell fate determinants including pharynx and intestine in excess-1 (PIE-1), posterior segregation-1 (POS-1), muscle excess-3 (MEX-3), and maternal effect germ-cell defective-1 (MEG-1). We demonstrated that only the activator FZY-1, but not FZR-1, is incorporated in the APC/C complex to regulate the oocyte-to-embryo transition. Our findings suggested a genetic relationship linking the APC/C complex and OMA-1, and support a model in which the APC/C complex promotes P granule accumulation and modifies RNA binding of OMA-1 to regulate the oocyte-to-embryo transition process.

A homozygous loss-of-function mutation in FBXO43 causes human non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) represents one of the most serious forms of male infertility caused by spermatogenic failure. Despite multiple genes found to be associated with human NOA, the genetic basis of this idiopathic disease remains largely unknown. FBXO43 is a direct inhibitor of the anaphase-promoting complex/cyclosome (APC/C) E3 ligase and crucially important in mouse spermatogenesis. In this study, for the first time, we identified a homozygous nonsense mutation in FBXO43 c.1747C > T:p.Gln583X in two NOA brothers from a Chinese consanguineous family via whole-exome sequencing. FBXO43 was absent from testicular tissue of the proband, and FBXO43-immunostaining signals were invisible in the affected seminiferous tubules. Furthermore, in humans, FBXO43 defects cause meiotic arrest within early diplotene of prophase I. The results here demonstrate the pathogenicity of this loss-of-function mutation and confirmed that spermatocytes were unable to complete meiotic divisions without FBXO43 in humans. In mouse testicular protein extracts, three subunits of the APC/C, including ANAPC2, ANAPC8 and ANAPC10, were validated to interact directly with FBXO43, whereas no interactions were detected for FBXO43 and SKP1. This study furthers our understanding of the genetic basis of human NOA and provides insights into FBXO43 and male infertility.

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 down-regulation by the spindle checkpoint and increased resistance to checkpoint-activating drugs. These results suggest that down-regulation of Ubc1 in response to spindle checkpoint signaling is necessary for a robust cell cycle arrest.