Arrest of Cell Cycle by Avian Reovirus p17 through Its Interaction with Bub3

Avian reoviruses (ARV) are a group of poultry pathogens that cause runting and stunting syndrome (RSS), a condition otherwise known as “frozen chicken”, which are characterized by dramatically delayed growth in broilers. It has been known that p17, a nonstructural protein encoded by ARV, prohibits cellular proliferation by halting the cell cycle at the G2/M phase, the result of which is directly associated with the typical clinical sign of RSS. Nevertheless, the mechanism by which p17 modulates cell-cycle progression remains largely unknown. Here, we screened the interactome of ectopically expressed p17 through a yeast two-hybrid assay and identified Bub3, a cellular mitotic checkpoint protein, as a binding partner of p17. The infection of the Vero cells by ARV downregulated the Bub3 expression, while the knockdown of Bub3 alleviated the p17-modulated cell-cycle arrest during ARV infection. Remarkably, the suppression of Bub3 by RNAi in the Vero cells significantly reduced the viral mRNA and protein abundance, which eventually led to diminished virus replication. Altogether, our findings reveal that ARV p17 impedes host cell proliferation through a Bub3-dependent cell-cycle arrest, which eventually contributes to efficient virus replication. These results also unveil a hitherto unknown therapeutic target for RSS.     

Disassembly of mitotic checkpoint complexes by the joint action of the AAA-ATPase TRIP13 and p31comet

The mitotic (or spindle assembly) checkpoint system delays anaphase until all chromosomes are correctly attached to the mitotic spindle. When the checkpoint is active, a Mitotic Checkpoint Complex (MCC) assembles and inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C). MCC is composed of the checkpoint proteins Mad2, BubR1, and Bub3 associated with the APC/C activator Cdc20. When the checkpoint signal is turned off, MCC is disassembled and the checkpoint is inactivated. The mechanisms of the disassembly of MCC are not sufficiently understood. We have previously observed that ATP hydrolysis is required for the action of the Mad2-binding protein p31^comet to disassemble MCC. We now show that HeLa cell extracts contain a factor that promotes ATP- and p31^comet-dependent disassembly of a Cdc20–Mad2 subcomplex and identify it as Thyroid Receptor Interacting Protein 13 (TRIP13), an AAA-ATPase known to interact with p31^comet. The joint action of TRIP13 and p31^comet also promotes the release of Mad2 from MCC, participates in the complete disassembly of MCC and abrogates checkpoint inhibition of APC/C. We propose that TRIP13 plays centrally important roles in the sequence of events leading to MCC disassembly and checkpoint inactivation.The mitotic (or spindle assembly) checkpoint is a surveillance system that prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. It monitors the existence of chromatids that are not attached yet correctly to the mitotic spindle through their kinetochores and delays anaphase until correct bipolar attachment is achieved. It acts by inhibiting the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that targets for degradation cyclin B and securin, an inhibitor of anaphase initiation. APC/C is inhibited by the Mitotic Checkpoint Complex (MCC), which consists of the checkpoint proteins Mad2, BubR1, and Bub3 associated with the APC/C activator Cdc20 (reviewed in refs. 1–4). The molecular mechanisms of the assembly of MCC when the checkpoint is turned on, and of its disassembly when the checkpoint is turned off, are not sufficiently understood. A key event in MCC formation appears to be a marked conformational transition in Mad2 promoted by the checkpoint signal. Mad2 exists in two conformations: an open inactive form (O-Mad2) and a closed active form (C-Mad2). C-Mad2 binds to Cdc20 in a structure in which the C-terminal tail of Mad2 is tightly wrapped around its partner (5, 6). It is thought that the active mitotic checkpoint stimulates the formation of the C-Mad2–Cdc20 subcomplex, which then combines with the constitutively present BubR1–Bub3 subcomplex to form the MCC (2, 3). The structure of MCC shows extensive binding interfaces between BubR1 and Cdc20, as well as between Cdc20 and C-Mad2 (7). In addition, C-Mad2 also binds to BubR1 (7, 8), thus stabilizing the structure of MCC.We have been studying the mechanisms of the disassembly of MCC that are necessary for the inactivation of the mitotic checkpoint. We observed that ATP cleavage at the β−γ position was required for the disassembly of MCC and for the release of APC/C from checkpoint inhibition (9). It was furthermore found that ATP is also required for the action of p31^comet to stimulate the disassembly of MCC (10). p31^comet is a Mad2-binding protein involved in exit from mitosis (11). It preferentially associates with C-Mad2, to the same interface that also binds O-Mad2 or BubR1 (7, 12, 13). p31^comet inactivates the mitotic checkpoint in vivo and in vitro in a manner that requires its interaction with Mad2 (12, 14). We found that p31^comet stimulates the phosphorylation of Cdc20 in MCC by cyclin-dependent kinase (Cdk), a process that promotes MCC disassembly (15). However, this explained only a part of ATP requirement for p31^comet action: We noted the existence of an additional pathway of MCC disassembly that required p31^comet and ATP, but not phosphorylation (15). By the use of a purified in vitro system, we found that the phosphorylation-dependent MCC disassembly pathway promoted the dissociation of Cdc20 from BubR1, resulting in the release of a subcomplex in which Cdc20 was still bound to Mad2 (10).The present study was initially undertaken to investigate the mechanism of the disassembly of the Cdc20–Mad2 subcomplex. An ATP-dependent disassembly factor was isolated and identified as Thyroid Receptor Interacting Protein 13 (TRIP13), an AAA-ATPase that had been found to interact with p31^comet (16). Furthermore, we then found that the joint action of TRIP13 and p31^comet also promotes MCC disassembly, releases APC/C from checkpoint inhibition, and inactivates the mitotic checkpoint.     

Up-regulation of the mitotic checkpoint component Mad1 causes chromosomal instability and resistance to microtubule poisons

The mitotic checkpoint is the major cell cycle checkpoint acting during mitosis to prevent aneuploidy and chromosomal instability, which are hallmarks of tumor cells. Reduced expression of the mitotic checkpoint component Mad1 causes aneuploidy and promotes tumors in mice [Iwanaga Y, et al. (2007) Cancer Res 67:160-166]. However, the prevalence and consequences of Mad1 overexpression are currently unclear. Here we show that Mad1 is frequently overexpressed in human cancers and that Mad1 up-regulation is a marker of poor prognosis. Overexpression of Mad1 causes aneuploidy and chromosomal instability through weakening mitotic checkpoint signaling caused by mislocalization of the Mad1 binding partner Mad2. Cells overexpressing Mad1 are resistant to microtubule poisons, including currently used chemotherapeutic agents. These results suggest that levels of Mad1 must be tightly regulated to prevent aneuploidy and transformation and that Mad1 up-regulation may promote tumors and cause resistance to current therapies.     

Expression and clinical significance of TAp73α, p53, PCNA and apoptosis in hepatocellular carcinoma

AIM: To study the prognostic role of TAp73α, p53,proliferating cell nuclear antigen (PCNA) and apoptosis in patients with hepatocellular carcinoma (HCC) after surgical tumor ablation.METHODS: Forty-seven human resected HCC tissues and 42 adjacent non-cancerous tissues were studied with 10 normal liver tissues as control group. TAp73α, p53, and PCNA were detected with Elivision immunohistochemistry.Terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick-end labeling (TUNEL) method was used to detect the apoptosis cells. All clinical and pathological materials were analyzed by SPSS10.0statistical package.RESULTS: TAp73α overexpressed in HCC tissues (36.2%)when compared with adjacent non-cancerous tissues(2.38%, P<0.005) and normal liver tissues (0, P<0.01).Mutant type p53 (rot-p53) overexpressed in HCC tissues(38.3%) when contracted with adjacent non-cancerous tissues (16.7%, P<0.05) and normal liver tissues (0,P<0.01). Proliferation index (PI) level in HCC tissues was significantly higher than that in adjacent non-cancerous tissues (30.34%±4.46% vs27.88%±5.89%, t, P= 0.028).Apoptosis index (AI) level in HCC tissues was higher than that in adjacent non-cancerous tissues (8.62%±2.28%vs7.38%±2.61%, t, P = 0.019). Expression of TAp73α was associated with lymph node metastasis and rot-p53,with r = 0.407 and 0.265, respectively. Expression of rot-p53 was associated with Edmondson‘s stage and AFP,with r = 0.295 and -0.357, respectively. In Kaplan-Meier univariant analysis, TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and HBsAg correlated with prognosis (log rank, P= 0.039, 0.012, 0.002, 0.000,0.014, 0.007, respectively). Multivariant Cox regression analysis showed that TAp73α, AFP, TNM stage, portalve in invasion, liver membrane invasion and age were independent factors of prognosis.CONCLUSION: These results suggest that TAp73α can be used as a prognostic indicator of patients with HCC undergoing surgical tumor ablation. AFP, TNM, portal vein invasion, liver membrane invasion and age also have a potency of predicting the prognosis of HCC.     

MDC1 regulates intra-S-phase checkpoint by targeting NBS1 to DNA double-strand breaks

The product of the Nijmegen breakage syndrome gene (NBS1) plays crucial roles in DNA damage response through its association with many proteins, including MRE11 and RAD50. However, it remains to be determined exactly how NBS1 accumulates at or near DNA double-strand breaks. Here we report that MDC1 directly binds to NBS1 and targets NBS1 to the sites of DNA damage. The MDC1-NBS1 interaction occurs through a specific region (residues 200-420) of MDC1, which contains multiple consensus casein kinase 2 (CK2) phosphorylation sites. In addition, this interaction requires both the forkhead-associated (FHA) and tandem BRCA1 C-terminal (BRCT) domains of NBS1. Disruption of the MDC1-NBS1 interaction results in failure of NBS1 accumulation at DNA double-strand breaks and impairment of intra-S checkpoint activation. These studies provide important mechanistic insights as to how MDC1 regulates NBS1 and the intra-S-phase checkpoint in response to DNA damage.     

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C–MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.Faithful distribution of the duplicated genome into two daughter cells during mitosis depends on proper kinetochore–microtubule (MT) attachments. Defects in kinetochore–MT attachments result in chromosome missegregation, causing aneuploidy, a hallmark of cancer (1, 2). To ensure accurate chromosome segregation, cells use the spindle assembly checkpoint (SAC) to monitor kinetochore biorientation and to control the metaphase-to-anaphase transition. Cells enter anaphase only after the SAC is satisfied, requiring that all kinetochores be attached to MTs and be properly bioriented (3, 4). The core components of SAC signaling include mitotic arrest deficient-like 1 (Mad1), Mad2, Mad3/BubR1 (budding uninhibited by benzimidazole-related 1), Bub1, Bub3, monopolar spindle 1 (Mps1), and aurora B. The full SAC function requires the correct centromere/kinetochore localization of all SAC proteins (5).Among the SAC components, Mps1 was identified originally in budding yeast as a gene required for duplication of the spindle pole body (6). Subsequently, Mps1 orthologs were found in various species, from fungi to mammals. The stringent requirement of Mps1 for SAC activity is conserved in evolution (6–13). Human Mps1 kinase (also known as “TTK”) is expressed in a cell-cycle–dependent manner and has highest expression levels and activity during mitosis. Its localization is also dynamic (8, 14). Although the molecular mechanism remains unclear, Mps1 is required to recruit Mad1 and Mad2 to unattached kinetochores, supporting its essential role in SAC activity (15–18). It also is clear that aurora B kinase activity and the outer-layer kinetochore protein nuclear division cycle 80 (Ndc80)/Hec1 are required for Mps1 localization to kinetochores, as evidenced by recent work, including ours (17, 19–24). How Mps1 activates the SAC is now becoming clear. Mps1 recruits Bub1/Bub3 and BubR1/Bub3 to kinetochores through phosphorylation of KNL1, the kinetochore receptor protein of Bub1 and BubR1 (25–30).Despite much progress in understanding Mps1 functions, it remains unclear how Mps1 is involved in regulating chromosome alignment. In budding yeast mitosis, Mps1 regulates mitotic chromosome alignment by promoting kinetochore biorientation independently of Ipl1 (aurora B in humans) (31), but in budding yeast meiosis Mps1 must collaborate with Ipl1 to mediate meiotic kinetochore biorientation (32). In humans, Mps1 regulates chromosomal alignment by modulating aurora B kinase activity (33), but recent chemical biology studies show that Mps1 kinase activity is important for proper chromosome alignment and segregation, independently of aurora B (22, 34–36). Therefore whether Mps1 regulates chromosome alignment through modulation of aurora B kinase activity is still under debate (37).In this study, we reexamined the function of human Mps1 in chromosome alignment. We found that chromosomal alignment is largely achieved in Mps1 knockdown cells, provided that cells are arrested in metaphase in the presence of MG132, a proteasome inhibitor. However, disrupting Mps1 activity via small molecule inhibitors perturbs chromosomal alignment, even in the presence of MG132. This chromosome misalignment is caused by the abnormal accumulation of inactive Mps1 in the kinetochore and the subsequent failure of correct kinetochore–MT attachments. Further, we demonstrate that inactive Mps1 does not depend on the previously reported tetratricopeptide repeat (TPR) domain for localizing to kinetochores, and we identify a previously unidentified region adjacent to the C terminus of the TPR domain that is responsible for localizing inactive Mps1 to kinetochores. Thus, our work highlights that Mps1 kinase activity is necessary in regulating chromosome alignment and that it must be tightly regulated in space and time to ensure proper localization of Mps1 at kinetochores.     

Long non-coding RNA TP73-AS1 promotes pancreatic cancer growth and metastasis through miRNA-128-3p/GOLM1 axis

BACKGROUNDPrevious studies have suggested that long non-coding RNAs (lncRNA) TP73-AS1 is significantly upregulated in several cancers. However, the biological role and clinical significance of TP73-AS1 in pancreatic cancer (PC) remain unclear. AIMTo investigate the role of TP73-AS1 in the growth and metastasis of PC.METHODSThe expression of lncRNA TP73-AS1, miR-128-3p, and GOLM1 in PC tissues and cells was detected by quantitative real-time polymerase chain reaction. The bioinformatics prediction software ENCORI was used to predict the putative binding sites of miR-128-3p. The regulatory roles of TP73-AS1 and miR-128-3p in cell proliferation, migration, and invasion abilities were verified by Cell Counting Kit-8, wound-healing, and transwell assays, as well as flow cytometry and Western blot analysis. The interactions among TP73-AS1, miR-128-3p, and GOLM1 were explored by bioinformatics prediction, luciferase assay, and Western blot. RESULTSThe expression of TP73-AS1 and miRNA-128-3p was dysregulated in PC tissues and cells. High TP73-AS1 expression was correlated with a poor prognosis. TP73-AS1 silencing inhibited PC cell proliferation, migration, and invasion in vitro as well as suppressed tumor growth in vivo. Mechanistically, TP73-AS1 was validated to promote PC progression through GOLM1 upregulation by competitively binding to miR-128-3p. CONCLUSIONOur results demonstrated that TP73-AS1 promotes PC progression by regulating the miR-128-3p/GOLM1 axis, which might provide a potential treatment strategy for patients with PC.     

BRCA2 regulates DMC1-mediated recombination through the BRC repeats

In somatic cells, BRCA2 is needed for RAD51-mediated homologous recombination. The meiosis-specific DNA strand exchange protein, DMC1, promotes the formation of DNA strand invasion products (joint molecules) between homologous molecules in a fashion similar to RAD51. BRCA2 interacts directly with both human RAD51 and DMC1; in the case of RAD51, this interaction results in stimulation of RAD51-promoted DNA strand exchange. However, for DMC1, little is known regarding the basis and functional consequences of its interaction with BRCA2. Here we report that human DMC1 interacts directly with each of the BRC repeats of BRCA2, albeit most tightly with repeats 1–3 and 6–8. However, BRC1–3 bind with higher affinity to RAD51 than to DMC1, whereas BRC6–8 bind with higher affinity to DMC1, providing potential spatial organization to nascent filament formation. With the exception of BRC4, each BRC repeat stimulates joint molecule formation by DMC1. The basis for this stimulation is an enhancement of DMC1–ssDNA complex formation by the stimulatory BRC repeats. Lastly, we demonstrate that full-length BRCA2 protein stimulates DMC1-mediated DNA strand exchange between RPA–ssDNA complexes and duplex DNA, thus identifying BRCA2 as a mediator of DMC1 recombination function. Collectively, our results suggest unique and specialized functions for the BRC motifs of BRCA2 in promoting homologous recombination in meiotic and mitotic cells.The breast cancer susceptibility protein 2, BRCA2, regulates RAD51-mediated homologous recombination (HR) (1–3). Both RAD51, a DNA strand exchange protein, and its meiotic counterpart, DMC1 (disrupted meiotic cDNA 1 or DNA meiotic recombinase 1), promote HR through the formation of a nucleoprotein filament on ssDNA (4). This filament finds and invades a homologous template, resulting in a DNA strand invasion product called a joint molecule or a displacement-loop (D-loop). The joint molecule provides a primer template for the new DNA synthesis required to repair the DNA double strand break (DSB).The first evidence implicating BRCA2 in meiosis came from studies in Ustilago maydis, where strains lacking the BRCA2 ortholog, Brh2, resulted in absence of meiotic products (5). Shortly thereafter, mouse BRCA2 was inferred to coordinate the activities of RAD51 and DMC1 (6). The first direct interaction between BRCA2 and DMC1 was observed in plants (7) and later in humans (8). In the plant, Arabidopsis thaliana, the interaction between Brca2 and Dmc1 was mapped to the BRC repeats (9), a highly conserved motif comprising a sequence of ∼35 amino acids that is present at least once in all BRCA2-like proteins (10). In humans, BRCA2 contains eight BRC repeats that bind with different affinities to RAD51, and they segregate into two functional classes (11). Within a BRC repeat, two motifs that bind RAD51 have been identified: one comprising the consensus sequence FxxA that mimics the oligomerization interface (12) and contacts the catalytic domain of RAD51; the other binding module comprises the alpha-helical region of the BRC repeat, contains the consensus sequence LFDE, and binds to RAD51 through a different hydrophobic pocket (13).Importantly, loss of Brca2 in plants causes chromosomal aberrations during meiosis (14). In humans, GST-pull down assays using peptide fragments of BRCA2 mapped a unique DMC1 interacting site to residues 2386–2411 (8). However, in mouse, mutation of a key residue (Phe-2406) within this site, which had been shown to disrupt the interaction of BRCA2 with DMC1 by peptide array analysis, had no effect in meiosis (15), suggesting that another site or sites in BRCA2 provide the functions needed during meiosis in this organism (6). A direct physical interaction was indeed established for purified full-length human BRCA2 and DMC1 (2), but the functional relevance of this interaction was not elaborated.We have previously shown that the BRC repeats of BRCA2 modulate the DNA binding selectivity of RAD51 to stimulate the assembly on ssDNA by inhibiting its ATP hydrolysis and preventing its association with dsDNA (2, 11, 16); as a result, BRCA2 catalyzes the recombination activity of RAD51 (17).A comprehensive analysis of aligned sequences of RAD51 orthologs and human RAD51 paralogues suggested that most eukaryotic RAD51 proteins, including DMC1, could interact with the BRC repeats, at least in principle (10). Here we investigate whether and how BRCA2 modulates DMC1-mediated recombination.     

Methyltransferase Set7/9 regulates p53 activity by interacting with Sirtuin 1 (SIRT1)

Numerous studies indicate that Sirtuin 1 (SIRT1), a mammalian nicotinamide adenine dinucleotide (NAD(+))-dependent histone deacetylase (HDAC), plays a crucial role in p53-mediated stress responses by deacetylating p53. Nevertheless, the acetylation levels of p53 are dramatically increased upon DNA damage, and it is not well understood how the SIRT1-p53 interaction is regulated during the stress responses. Here, we identified Set7/9 as a unique regulator of SIRT1. SIRT1 interacts with Set7/9 both in vitro and in vivo. In response to DNA damage in human cells, the interaction between Set7/9 and SIRT1 is significantly enhanced and coincident with an increase in p53 acetylation levels. Importantly, the interaction of SIRT1 and p53 is strongly suppressed in the presence of Set7/9. Consequently, SIRT1-mediated deacetylation of p53 is abrogated by Set7/9, and p53-mediated transactivation is increased during the DNA damage response. Of note, whereas SIRT1 can be methylated at multiple sites within its N terminus by Set7/9, a methylation-defective mutant of SIRT1 still retains its ability to inhibit p53 activity. Taken together, our results reveal that Set7/9 is a critical regulator of the SIRT1-p53 interaction and suggest that Set7/9 can modulate p53 function indirectly in addition to acting through a methylation-dependent mechanism.     

From the Cover: PHS1 regulates meiotic recombination and homologous chromosome pairing by controlling the transport of RAD50 to the nucleus

Recombination and pairing of homologous chromosomes are critical for bivalent formation in meiotic prophase. In many organisms, including yeast, mammals, and plants, pairing and recombination are intimately interconnected. The POOR HOMOLOGOUS SYNAPSIS1 (PHS1) gene acts in coordination of chromosome pairing and early recombination steps in plants, ensuring pairing fidelity and proper repair of meiotic DNA double-strand-breaks. In phs1 mutants, chromosomes exhibit early recombination defects and frequently associate with non-homologous partners, instead of pairing with their proper homologs. Here, we show that the product of the PHS1 gene is a cytoplasmic protein that functions by controlling transport of RAD50 from cytoplasm to the nucleus. RAD50 is a component of the MRN protein complex that processes meiotic double-strand-breaks to produce single-stranded DNA ends, which act in the homology search and recombination. We demonstrate that PHS1 plays the same role in homologous pairing in both Arabidopsis and maize, whose genomes differ dramatically in size and repetitive element content. This suggests that PHS1 affects pairing of the gene-rich fraction of the genome rather than preventing pairing between repetitive DNA elements. We propose that PHS1 is part of a system that regulates the progression of meiotic prophase by controlling entry of meiotic proteins into the nucleus. We also document that in phs1 mutants in Arabidopsis, centromeres interact before pairing commences along chromosome arms. Centromere coupling was previously observed in yeast and polyploid wheat while our data suggest that it may be a more common feature of meiosis.     

GLP-1通过调节巨噬细胞泡沫化抑制ApoE-/-小鼠动脉粥样硬化的作用机制研究

目的 研究胰高血糖素样肽1(GLP-1)是否能抑制高脂饮食喂养的载脂蛋白E基因敲除(ApoE-/-)小鼠动脉粥样硬化(AS)进程,从巨噬细胞泡沫化角度探讨相关作用机制.方法 选择6周龄雄性ApoE-/-小鼠40只,随机分为对照组(普通饮食)、模型组(高脂饮食)、GLP-1组(高脂饮食+GLP-1)和GLP-1阻断组(高...