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1.
Recruitment of Mad1–Mad2 complexes to unattached kinetochores is a central event in spindle checkpoint signaling. Despite its importance, the mechanism that recruits Mad1–Mad2 to kinetochores is unclear. In this paper, we show that MAD-1 interacts with BUB-1 in Caenorhabditis elegans. Mutagenesis identified specific residues in a segment of the MAD-1 coiled coil that mediate the BUB-1 interaction. In addition to unattached kinetochores, MAD-1 localized between separating meiotic chromosomes and to the nuclear periphery. Mutations in the MAD-1 coiled coil that selectively disrupt interaction with BUB-1 eliminated MAD-1 localization to unattached kinetochores and between meiotic chromosomes, both of which require BUB-1, and abrogated checkpoint signaling. The identified MAD-1 coiled-coil segment interacted with a C-terminal region of BUB-1 that contains its kinase domain, and mutations in this region prevented MAD-1 kinetochore targeting independently of kinase activity. These results delineate an interaction between BUB-1 and MAD-1 that targets MAD-1–MAD-2 complexes to kinetochores and is essential for spindle checkpoint signaling.  相似文献   

2.
目的:研究Bub1基因在肝癌中的表达以及对肝癌细胞系MHCC97-H增殖、周期和凋亡的影响。方法:利用RNA干扰技术下调肝癌细胞系MHCC97-H中Bub1的表达;qRT-PCR和Western Blot分别检测Bub1在mRNA和蛋白水平表达的变化;CCK-8实验检测肿瘤细胞增殖能力的改变;流式细胞术检测细胞周期和凋亡的变化。结果:qRT-PCR和Western Blot结果显示si-Bub1能够成功下调Bub1的表达;下调Bub1后肝癌MHCC97-H细胞的增殖能力下降(P0.05),细胞的凋亡比例升高(P0.05),细胞发生S期阻滞。结论:Bub1基因在肝癌中高表达,下调Bub1的表达后能够降低肝癌细胞的增殖能力,促进细胞凋亡,诱导细胞发生S期阻滞。  相似文献   

3.
Bub1 is an evolutionarily conserved mitotic checkpoint control protein that is present in diverse organisms including yeast and humans. Bub1 is a serine/threonine protein kinase and is required for recruitment of Mad1, Mad2, Bub3, and CENP-E to kinetochores (Sharp-Baker and Chen in J Cell Biol 153:1239–1250, 2001). The evolutionarily conserved amino acid region in the N-terminus has been called as the CD1 domain. To clarify the action mechanism of Bub1 in controlling check point signals, we initiated an NMR structure determination of the Bub1 CD1 domain. Here, we report the sequence-specific backbone resonance assignments of CD1 domain of human Bub1 (hBub1CD1).  相似文献   

4.
The bioinformatic search identified 14 plant homologs of the mitotic spindle assembly checkpoint protein kinase Bub1 of animals, yeasts, and myxomycetes. It was demonstrated that the closest plant homologs of the protein kinase Bub1 were proteins with previously unknown features: XP_002274770.1 (CBI21878.1) from Vitis vinifera, EEC82122.1 from Oryza sativa Indica, EEE67244.1 from O. sativa Japonica, EEF44403.1 from Ricinus communis, and CAL57156.1 from Ostreococcus tauri. The simulation and analysis of spatial structures of the catalytic domains of the EEC82122.1, EEE67244.1, and XP_002274770.1 (CBI21878.1) proteins confirmed their conformity with the spindle assembly checkpoint protein kinases Bub1.  相似文献   

5.
3′–nucleases/nucleotidases of the S1–P1 family (EC 3.1.30.1) are single–strand–specific or non-specific zinc–dependent phosphoesterases present in plants, fungi, protozoan parasites, and in some bacteria. They participate in a wide variety of biological processes and their current biotechnological applications rely on their single–strand preference, nucleotide non-specificity, a broad range of catalytic conditions and high stability. We summarize the present and potential utilization of these enzymes in biotechnology and medicine in the context of their biochemical and structure–function properties. Explanation of unanswered questions for bacterial and trypanosomatid representatives could facilitate development of emerging applications in medicine.  相似文献   

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Antimicrobial peptides (AMPs) are important components of the innate immune system of animals, plants, fungi and bacteria and are recently under discussion as promising alternatives to conventional antibiotics. We have investigated two cecropin-like synthetic peptides, Gm1, which corresponds to the natural overall uncharged Galleria mellonella native peptide and ΔGm1, a modified overall positively charged Gm1 variant. We have analysed these peptides for their potential to inhibit the endotoxin-induced secretion of tumour necrosis factor-α (TNF-α) from human mononuclear cells. Furthermore, in a conventional microbiological assay, the ability of these peptides to inhibit the growth of the rough mutant bacteria Salmonella enterica Minnesota R60 and the polymyxin B-resistant Proteus mirabilis R45 was investigated and atomic force microscopy (AFM) measurements were performed to characterize the morphology of the bacteria treated by the two peptides. We have also studied their cytotoxic properties in a haemolysis assay to clarify potential toxic effects.Our data revealed for both peptides minor anti-inflammatory (anti-endotoxin) activity, but demonstrated antimicrobial activity with differences depending on the endotoxin composition of the respective bacteria. In accordance with the antimicrobial assay, AFM data revealed a stronger morphology change of the R45 bacteria than for the R60. Furthermore, Gm1 had a stronger effect on the bacteria than ΔGm1, leading to a different morphology regarding indentations and coalescing of bacterial structures. The findings verify the biophysical measurements with the peptides on model systems. Both peptides lack any haemolytic activity up to an amount of 100 μg/ml, making them suitable as new anti-infective agents.  相似文献   

8.
Rap1GAP is a GTPase-activating protein (GAP) that specifically stimulates the GTP hydrolysis of Rap1 GTPase. Although Rap1GAP is recognized as a tumor suppressor gene and downregulated in various cancers, little is known regarding the regulation of Rap1GAP ubiquitination and degradation under physiological conditions. Here, we demonstrated that Rap1GAP is ubiquitinated and degraded through proteasome pathway in mitosis. Proteolysis of Rap1GAP requires the PLK1 kinase and β-TrCP ubiquitin ligase complex. We revealed that PLK1 interacts with Rap1GAP in vivo through recognition of an SSP motif within Rap1GAP. PLK1 phosphorylates Ser525 in conserved 524DSGHVS529 degron of Rap1GAP and promotes its interaction with β-TrCP. We also showed that Rap1GAP was a cell cycle regulator and that tight regulation of the Rap1GAP degradation in mitosis is required for cell proliferation.  相似文献   

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IL-1α and β are key players in the innate immune system. The secretion of these cytokines by dendritic cells (DC) is integral to the development of proinflammatory responses. These cytokines are not secreted via the classical secretory pathway. Instead, 2 independent processes are required; an initial signal to induce up-regulation of the precursor pro-IL-1α and -β, and a second signal to drive cleavage and consequent secretion. Pro-IL-1α and -β are both cytosolic and thus, are potentially subject to post-translational modifications. These modifications may, in turn, have a functional outcome in the context of IL-1α and -β secretion and hence inflammation. We report here that IL-1α and -β were degraded intracellularly in murine bone marrow-derived DC and that this degradation was dependent on active cellular processes. In addition, we demonstrate that degradation was ablated when the proteasome was inhibited, whereas autophagy did not appear to play a major role. Furthermore, inhibition of the proteasome led to an accumulation of polyubiquitinated IL-1α and -β, indicating that IL-1α and -β were polyubiquitinated prior to proteasomal degradation. Finally, our investigations suggest that polyubiquitination and proteasomal degradation are not continuous processes but instead are up-regulated following DC activation. Overall, these data highlight that IL-1α and -β polyubiquitination and proteasomal degradation are central mechanisms in the regulation of intracellular IL-1 levels in DC.  相似文献   

13.
Sphingosine-1-phosphate-induced α1B-adrenergic receptor desensitization and phosphorylation were studied in rat-1 fibroblasts stably expressing enhanced green fluorescent protein-tagged adrenoceptors. Sphingosine-1-phosphate induced adrenoceptor desensitization and phosphorylation through a signaling cascade that involved phosphoinositide 3-kinase and protein kinase C activities. The autocrine/paracrine role of sphingosine-1-phosphate was also studied. It was observed that activation of receptor tyrosine kinases, such as insulin growth factor-1 (IGF-I) and epidermal growth factor (EGF) receptors increased sphingosine kinase activity. Such activation and consequent production of sphingosine-1-phosphate appear to be functionally relevant in IGF-I- and EGF-induced α1B-adrenoceptor phosphorylation and desensitization as evidenced by the following facts: a) expression of a catalytically inactive (dominant-negative) mutant of sphingosine kinase 1 or b) S1P1 receptor knockdown markedly reduced this growth factor action. This action of sphingosine-1-phosphate involves EGF receptor transactivation. In addition, taking advantage of the presence of the eGFP tag in the receptor construction, we showed that S1P was capable of inducing α1B-adrenergic receptor internalization and that its autocrine/paracrine generation was relevant for internalization induced by IGF-I. Four distinct hormone receptors and two autocrine/paracrine mediators participate in IGF-I receptor-α1B-adrenergic receptor crosstalk.  相似文献   

14.
Gene–environment interactions have been extensively studied in lung cancer. It is likely that several genetic polymorphisms cooperate in increasing the individual risk. Therefore, the study of gene–gene interactions might be important to identify high-susceptibility subgroups. GSEC is an initiative aimed at collecting available data sets on metabolic polymorphisms and the risks of cancer at several sites and performing pooled analyses of the original data. Authors of published papers have provided original data sets. The present paper refers to gene–gene interactions in lung cancer and considers three polymorphisms in three metabolic genes: CYP1A1, GSTM1 and GSTT1. The present analyses compare the gene–gene interactions of the CYP1A1*2A, GSTM1 and GSTT1 polymorphisms from studies on lung cancer conducted in Europe and the USA between 1991 and 2000. Only Caucasians have been included. The data set includes 1466 cases and 1488 controls. The only clear-cut association was found with CYP1A1*2A. This association remained unchanged after stratification by polymorphisms in other genes (with an odds ratio [OR] of approximately 2.5), except when interaction with GSTM1 was considered. When the OR for CYP1A1*2A was stratified according to the GSTM1 genotype, the OR was increased only among the subjects who had the null (homozygous deletion) GSTM1 genotype (OR=2.8, 95% CI=0.9–8.4). The odds ratio for the interactive term (CYP1A1*2A by GSTM1) in logistic regression was 2.7 (95% CI=0.5–15.3). An association between lung cancer and the homozygous CYP1A1*2A genotype is confirmed. An apparent and biologically plausible interaction is suggested between this genotype and GSTM1.  相似文献   

15.
Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme.  相似文献   

16.
Niemann–Pick C1-Like 1 (NPC1L1) is a polytopic transmembrane protein responsible for dietary cholesterol and biliary cholesterol absorption. Consistent with its functions, NPC1L1 distributes on the brush border membrane of enterocytes and the canalicular membrane of hepatocytes in humans. As the molecular target of ezetimibe, a hypocholesterolemic drug, its physiological and pathological significance has been recognized and intensively studied for years. Recently, plenty of new findings reveal the molecular mechanism of NPC1L1's role in cholesterol uptake, which may provide new insights on our understanding of cholesterol absorption. In this review, we summarized recent progress in these studies and proposed a working model, hoping to provide new perspectives on the regulation of cholesterol transport and metabolism.  相似文献   

17.
《Mutation Research Letters》1994,323(1-2):69-74
Ascorbingen, which occurs naturally in the human diet, and a synthetic analogue (1′-methylascorbigen), were assayed for cytotoxic and clastogenic activities in a SV40-transformed Indian Muntjac cell line (SVM), and for mutagenic activity in the Ames test using Salmonella typhimurium strains TA98 and TA100. Ascorbigen had no effect upon the clonal survival of SVM at concentrations below 0.21 mg/ml and did not induce either chromosome aberrations or sister-chromatid exchanges (SCEs) at any concentration tested up to the maximum compatible with the assay conditions; nor did it induce mutations in either Salmonella strain. In contrast, 1′-methylascorbigen was an order of magnitude more cytotoxic, demonstrating a Dq of 0.03 mg/ml, and whilst it too was not found to induce chromosome aberrations it did induce SCEs in SVM (although only at higly cytotoxic doses) and mutations in the Ames test.  相似文献   

18.
It has been reported that feeding mice resveratrol activates AMPK and SIRT1 in skeletal muscle leading to deacetylation and activation of PGC-1α, increased mitochondrial biogenesis, and improved running endurance. This study was done to further evaluate the effects of resveratrol, SIRT1, and PGC-1α deacetylation on mitochondrial biogenesis in muscle. Feeding rats or mice a diet containing 4 g resveratrol/kg diet had no effect on mitochondrial protein levels in muscle. High concentrations of resveratrol lowered ATP concentration and activated AMPK in C2C12 myotubes, resulting in an increase in mitochondrial proteins. Knockdown of SIRT1, or suppression of SIRT1 activity with a dominant-negative (DN) SIRT1 construct, increased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C2C12 cells. Expression of a DN SIRT1 in rat triceps muscle also induced an increase in mitochondrial proteins. Overexpression of SIRT1 decreased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C2C12 myotubes. Overexpression of SIRT1 also resulted in a decrease in mitochondrial proteins in rat triceps muscle. We conclude that, contrary to some previous reports, the mechanism by which SIRT1 regulates mitochondrial biogenesis is by inhibiting PGC-1α coactivator activity, resulting in a decrease in mitochondria. We also conclude that feeding rodents resveratrol has no effect on mitochondrial biogenesis in muscle.  相似文献   

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The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1β was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.  相似文献   

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