Physiology of Osmotic Stress in Saccharmyces cerevisiae

性状（由申请方提供）：适应渗透压的能力是所有生物体的基本特性。系统发育多样的生物体通过渗透压调节剂的积累来适应渗透胁迫，渗透压调节剂稳定细胞组分并防止细胞水分的损失。细胞对渗透压的适应对于包括人类在内的高等生物体的适当肾细胞功能也很重要。例如，已知哺乳动物肾细胞在抗利尿期间积累甘油磷酸胆碱（GPC）、甘氨酸甜菜碱、山梨糖醇、肌醇和尿素。酿酒酵母已被用作研究渗透胁迫的生理反应（主要是甘油合成和信号转导）的模型。该研究的长期目标是揭示酵母中低分子量渗透物质的功能和代谢。初步数据表明，高盐胁迫诱导磷脂酰胆碱（PC）的快速周转，产生GPC。此外，这些事件可在不存在从头蛋白质合成的情况下发生，并且依赖于Nte 1（神经病靶向酯酶）。Nte 1的结构特征表明其活性受蛋白激酶A（PKA）磷酸化的调节。 该建议的中心假设是，PC脱酰，这是耦合到GPC合成的调节磷酸化的Nte 1在响应渗透胁迫。为了验证我们的假设，我们将使用酿酒酵母作为我们的模型微生物来解决以下具体目标：（1）检查具有nte缺失突变的酵母菌株中的体内表型;（2）检查Nte 1中的氨基酸取代和其他调控突变对体内PC周转/GPC合成以及细胞提取物中的酯酶/磷脂酶活性的影响;（3）研究有机磷酸盐对细胞生长、PC周转/GPC合成和Nte活性的影响;（4）与资深科学家（乔治卡曼博士）建立合作关系;（5）增强PI实验室的研究能力，提高学生培训和研究机会。 与公共卫生的相关性：这项研究的结果适用于其他领域的研究，这取决于更好地了解PC营业额/GPC的合成，如：神经病变和麻痹的有机磷农药，NTE在胚胎，胎盘和大脑发育中的作用，血小板活化因子和花生四烯酸的合成，和GPC代谢的个体患有阿尔茨海默氏症。

DESCRIPTION (provided by applicant): The ability to adapt to osmotic stress is an essential property of all living organisms. Phylogenetically diverse organisms adapt to osmotic stress through the accumulation of osmolytes that stabilize cellular components and prevent the loss of cellular water. Cellular adaptation to osmotic stress is also important for proper renal cell function in higher organisms, including humans. For example, mammalian renal cells are known to accumulate glycerophosphocholine (GPC), glycine betaine, sorbitol, inositol and urea during anti-diuresis. The yeast Saccharomyces cerevisiae has been used as a model to study the physiological responses to osmotic stress (principally glycerol synthesis and signal transduction). The long-term goal of the proposed research is to reveal the function and metabolism of low molecular weight osmolytes in yeast. Preliminary data show that hypersaline stress induces the rapid turnover of phosphatidylcholine (PC) to generate GPC. In addition, these events can occur in the absence of de novo protein synthesis and are dependent on Nte1 (neuropathy targeted esterase). Structural features within Nte1 suggest that its activity is regulated by phosphorylation by protein kinase A (PKA). The central hypothesis of this proposal is that PC deacylation that is coupled to GPC synthesis is regulated by phosphorylation of Nte1 in response to osmotic stress. To test our hypothesis, we will use Saccharomyces cerevisiae as our model microorganism to address the following specific aims: (1) examine the in vivo phenotypes in yeast strains with nte-deletion mutations; (2) examine the effects that amino acid substitutions in Nte1 and other regulatory mutations have on PC turnover/GPC synthesis in vivo, as well as esterase/ phospholipase activity in cell extracts; (3) examine the effects of organophosphate on cell growth, PC turnover/GPC synthesis, and Nte activity; (4) develop collaborative ties with a senior scientist (Dr. George Carman); (5) augment the research capabilities of the PI's laboratory, enhance student training and research opportunities. Relevance to Public Health: The results from this study are applicable to other areas of research that depend on a greater understanding of PC turnover/GPC synthesis, such as: neuropathy and paralysis by organophosphate pesticides, the role of NTE in embryonic, placental and brain development, synthesis of platelet activating factor and arachidonic acid, and GPC metabolism in individuals suffering from Alzheimers.