The characterization of separase suppressors

分离酶抑制剂的表征

• 批准号: 8349672
• 负责人: Andy Golden
• 金额: $ 9.86万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349672
• 关键词: 26S proteasome; Alleles; Anaphase; Caenorhabditis elegans; Chromosome Segregation; Cleaved cell; Collaborations; Complex; Cysteine Protease; Cytoplasmic Granules; Defect; Development; Embryo; Excision; Exocytosis; Gene Mutation; Genes; Genetic; Goals; Homologous Gene; Meiosis; Metaphase; Methyltransferase; Mitosis; Mutation; National Institute of Diabetes and Digestive and Kidney Diseases; Orthologous Gene; Pathway interactions; Peptide Hydrolases; Phenotype; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Protein phosphatase; RNA Interference; Role; Suppressor Mutations; Temperature; Time; Yeasts; cohesin; gene function; human PTTG1 protein; inorganic phosphate; interest; mutant; separase

Given our interest in the APC/C and its downstream targets, we have been focusing our studies on an indirect target of the APC/C. Separase is the protease that cleaves the cohesin complex that holds homologs together at meiosis. Securin inhibits separase from carrying out this role until the metaphase to anaphase transition, at which time securin is ubiquitinated by the APC/C and degraded by the 26S proteasome. We have taken a genetic approach to identify regulators and substrates of separase. We have three mutant alleles of sep-1 and have recently shown, through a collaboration with Dr. Joshua Bembenek, that these mutants all have defects in cortical granule exocytosis (CGE). CGE is important for the secretion of components necessary for eggshell formation, which is indirectly required for proper polar body extrusion. In order to identify other genes that function in the separase pathway, we carried out a suppression screen with a temperature-sensitive allele of sep-1, e2406ts. We have identified three suppressors that restore viability to sep-1 mutants at the non-permissive temperature. One of these mutants is an intragenic suppressor; the other two are extragenic. We have recently determined that one of these suppressor mutations is in a phosphatase gene called pph-5. This phosphatase mutant, in an otherwise wild-type background, has no obvious phenotypes on its own. Our pph-5 allele, av101 (and a two deletion alleles of the pph-5 gene), suppresses the embryonic lethality of two of our three sep-1 alleles. RNAi of pph-5 also suppresses the embryonic lethality of two of our three sep-1 alleles. RNAi depletion or genetic mutation of pph-5 restores CGE to wildtype levels in the suppressed sep-1 backgrounds. In collaboration with Harold Smith (NIDDK), we have also carried out deep sequencing to determine the mutation in our second extragenic suppressor, av102. A mutation was found in a gene that encodes a carboxyl methylase. Orthologs of this gene in yeast are known to promote the formation of the protein phosphatase 2A complex. Thus our studies in C. elegans suggest that two distinct phosphatases may regulate separase function. Since numerous components of the separase pathway are known kinases or phosphoproteins, the removal of specific phosphate groups must play an important regulatory role in separase activity. We seek to determine the substrates of these two phosphatases and the mechanisms by which mutations suppress sep-1 lethality.

鉴于我们对APC/C及其下游目标的兴趣，我们一直将研究重点放在APC/C的间接目标上。 分离酶是裂解在减数分裂时将同源物结合在一起的粘蛋白复合物的蛋白酶。 Securin抑制分离酶执行该角色，直到中期向后期转变为止，此时Securin被APC/C泛素化并被26S蛋白酶体降解。 我们采用了一种遗传方法来鉴定分离酶的调节剂和底物。 我们有三个SEP-1的突变等位基因，最近通过与Joshua Bembenek博士的合作表明，这些突变体在皮质颗粒外胞菌病（CGE）中都有缺陷。 CGE对于蛋壳形成所需的成分很重要，这对于适当的极性体挤出是间接所需的。 为了识别在分离酶途径中起作用的其他基因，我们用SEP-1，E2406T的温度敏感等位基因进行了抑制筛选。 我们已经确定了在非耐药温度下恢复对Sep-1突变体生存能力的三个抑制剂。 这些突变体之一是基因内抑制剂。另外两个是外部的。 我们最近确定，这些抑制突变之一是在称为PPH-5的磷酸酶基因中。 在原本野生型背景下，这种磷酸酶突变体本身没有明显的表型。 我们的PPH-5等位基因AV101（和PPH-5基因的两个缺失等位基因）抑制了我们三个Sep-1等位基因中两个的胚胎致死性。 PPH-5的RNAi还抑制了我们三个Sep-1等位基因中两个的胚胎致死性。 在被抑制的SEP-1背景下，PPH-5的RNAi耗竭或PPH-5的基因突变将CGE恢复为野生型水平。 与Harold Smith（NIDDK）合作，我们还进行了深入的测序，以确定第二次抑制器AV102中的突变。 在编码羧基甲基酶的基因中发现了一个突变。 已知该基因的直系同源物已知可以促进蛋白质磷酸酶2a复合物的形成。 因此，我们在秀丽隐杆线虫中的研究表明，两种不同的磷酸酶可能调节分离酶功能。 由于分离酶途径的许多成分是已知的激酶或磷酸蛋白，因此特定磷酸基团的去除必须在分离酶活性中起重要的调节作用。 我们试图确定这两种磷酸酶的底物以及突变抑制SEP-1致死性的机制。