The characterization of separase suppressors

分离酶抑制剂的表征

• 批准号: 7733995
• 负责人: Andy Golden
• 金额: $ 24.89万
• 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/7733995
• 关键词: 26S proteasome; Alleles; Anaphase; Caenorhabditis elegans; Chromosome Segregation; Cleaved cell; Complex; Cysteine Protease; Data; Defect; Development; Embryo; Endopeptidases; Genes; Genetic; Goals; Heat-Shock Proteins 90; Homologous Gene; Mass Spectrum Analysis; Meiosis; Metaphase; Mitosis; Modeling; Mutation; Orthologous Gene; Pathway interactions; Peptide Hydrolases; Phenotype; Phosphoric Monoester Hydrolases; Play; Proteins; Proteomics; RNA Interference; Role; Sterility; Suppressor Mutations; Temperature; Thinking; Time; Transgenic Animals; Work; cohesin; gain of function; human PTTG1 protein; inhibitor/antagonist; 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 carried out a suppression screen with one temperature-sensitive alleles of sep-1. 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. RNAi of this pph-5 suppresses the embryonic lethality of sep-1 alleles as does a deletion allele of this gene. To further understand how PPH-5 works in the separase pathway, we have undertaken a proteomics approach and have generated transgenic animals that express a TAP-tagged version of PPH-5. Purification of the tagged phosphatase and its associated proteins, followed by mass spectrometry, has revealed two interacting proteins. One of these interacting proteins is DAF-21, a C. elegans HSP90 ortholog. RNAi of daf-21 has previously been shown to cause sterility in C. elegans, a phenotype similar to that of RNAi of wee-1.3, an inhibitor of CDK-1. Our current model is that PPH-5 influences the activity of DAF-21, which in turn regulates WEE-1.3 and CDK-1. Since CDK-1 is a known regulator of SEP-1 activity, it may be the indirect mechanism by which pph-5 mutations suppress the embryonic lethality of sep-1 mutants. Alternatively, PPH-5 is also thought to function in the RAS/RAF pathway and we have some genetic data to suggest that our pph-5 mutant can enhance a ras gain-of-function allele. We are pursuing this analysis to fully understand the mechanism of suppression by pph-5.

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，并进行了一个具有一个温度敏感等位基因Sep-1的抑制筛网。 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。 该PPH-5的RNAi抑制了Sep-1等位基因的胚胎致死性，该基因的缺失等位基因也是如此。 为了进一步了解PPH-5在分离酶途径中的工作原理，我们采用了一种蛋白质组学方法，并产生了表达pph-5的Tags Tagg-5版本的转基因动物。 标记的磷酸酶及其相关蛋白的纯化，其次是质谱法，揭示了两种相互作用的蛋白质。 这些相互作用的蛋白质之一是DAF-21，秀丽隐杆线虫HSP90直系同源物。 DAF-21的RNAi先前已显示在秀丽隐杆线虫中引起无菌性，这是一种与CDK-1的抑制剂Wee-1.3相似的表型。 我们当前的模型是PPH-5影响DAF-21的活性，而DAF-21的活性反过来调节WEE-1.3和CDK-1。 由于CDK-1是已知的SEP-1活性调节剂，因此可能是PPH-5突变抑制Sep-1突变体的胚胎致死性的间接机制。 另外，PPH-5也被认为在RAS/RAF途径中起作用，我们有一些遗传数据表明我们的PPH-5突变体可以增强RAS功能获得等位基因。 我们正在进行此分析，以充分了解PPH-5抑制的机理。