Phospholipase-Mediated Stress Response in Fission Yeast

描述（由申请人提供）： 磷脂酶B（PL B）酶包括在生物学功能方面可能是最不了解的磷脂酶类别。虽然在遗传上易处理的酵母中已经鉴定出几种PLB同工酶，但它们的生理功能在很大程度上是未知的。我们已经阐明了一个新发现的真菌PLB家族成员，Plb 1，作为一个重要的调解人的渗透胁迫反应的裂殖酵母，裂殖酵母pombe，plb 1 δ突变体表现出有丝分裂和胞质分裂缺陷的条件下，渗透胁迫的缺陷承担相似的缺陷，观察到的蛋白酶体缺陷突变体的裂殖酵母。粟球。它们还能够在丰富的培养基上交配，这是cAMP途径功能缺陷的突变体的表型特征。这些表型不同于先前表征的S. pombe，表明Plb 1调节一种新的途径。与由Plb 1功能丧失引起的表型一致，筛选plb 1突变细胞的多拷贝敏感性生长缺陷抑制因子导致分离编码腺苷酸环化酶激活剂的基因、26S蛋白酶体亚基（pra7）和编码新蛋白的几个基因，其中几个在哺乳动物中是保守的。我们已经确定，cAMP途径，如PIb 1，是必不可少的生存S。pombe细胞在高渗介质中的作用，cAMP途径功能的丧失被plb 1 δ的几个多拷贝抑制因子（包括pra7）拯救。我们的研究结果使我们假设，PIb 1的功能，部分，通过一种新的cAMP和26 S蛋白酶介导的途径，以调节细胞周期的进展渗透压条件下。我们将利用酵母遗传学的力量，结合分子和生物化学方法，进一步表征S.粟球。我们的具体目标是：（1）确定Plb 1的酶活性和亚细胞定位，并确定它们是否在响应渗透胁迫时发生改变，（2）建立Plb 1与裂殖酵母中先前表征的渗透胁迫响应途径之间的功能关系，（3）研究cAMP途径和26 S蛋白酶体在介导Plb 1依赖性胁迫响应功能中的作用，以及（4）确定PLB 1的其它多拷贝抑制器是否是PLB 1的多拷贝抑制器？编码PIb 1依赖性应激反应途径的组分。我们预计，我们提出的研究将提供深入了解新的机制，压力诱导的信号转导和细胞周期调控，可能是保守的高等生物。拟议的项目与几种人类疾病有关，包括心血管疾病、炎症性疾病、神经退行性疾病和肿瘤性疾病。

DESCRIPTION (provided by applicant): Phospholipase B (PLB) enzymes comprise what is perhaps the most poorly understood class of phospholipases in terms of biological functions. Although several PLB isozymes have been identified in genetically tractable yeasts, their physiological functions are largely unknown. We have elucidated a role for a newly identified member of the fungal PLB family, Plbl, as an essential mediator of osmotic stress response in the fission yeast, Schizosaccharomyces pombe, plb 1delta mutants exhibit mitotic and cytokinesis defects under conditions of osmotic stress that bear resemblance to defects observed in proteasome-defective mutants of S. pombe. They are also able to mate on rich media, a phenotype characteristic of mutants defective in function for the cAMP pathway. These phenotypes are distinct from those caused by loss of function of previously characterized stress response pathways in S. pombe, suggesting that Plb1 regulates a novel pathway. Consistent with the phenotypes caused by loss of Plb1 function, a screen for multi-copy suppressors of the osmosensitive growth defect of plb1 mutant cells resulted in the isolation of genes encoding activators of adenylate cyclase, a 26S proteasome subunit (pra7), and several genes encoding novel proteins, several of which are conserved in mammals. We have determined that the cAMP pathway, like PIb1, is essential for survival of S. pombe cells in hyperosmotic media and that loss of function of the cAMP pathway is rescued by several multi-copy suppressors of plb1delta, including pra7. Our findings lead us to hypothesize that PIb1 functions, in part, to regulate cell cycle progression under conditions of osmotic stress through a novel cAMP and 26S proteasomemediated pathway. We will use the power of yeast genetics, in combination with molecular and biochemical approaches, to further characterize the Plb1-dependent stress response pathway(s) in S. pombe. Our Specific Aims are to: (1) define the enzymatic activities and subcellular localization of Plb1 and determine whether they are altered in response to osmotic stress, (2) establish functional relationships between Plb1 and previously characterized osmotic stress response pathways in fission yeast, (3) investigate roles for the cAMP pathway and 26S proteasome in mediating Plb1-dependent stress response functions, and (4) determine whether other multicopy suppressors of plb1? encode components of the PIb1-dependent stress response pathway. We anticipate that our proposed studies will provide insights into novel mechanisms of stress-induced signal transduction and cell cycle regulation that are likely to be conserved in higher organisms. The proposed project is relevant to several human diseases, including cardiovascular, inflammatory, neurodesenerative, and neoplastic diseases.