Regulation of nitrogen catabolic gene expression in S. cerevisiae

描述(由申请人提供)：我们对细胞调控的理解的一个空白是真核细胞通过其检测环境中的营养物质并以综合的方式对其作出反应的详细机制。这一点对于雷帕霉素复合体靶标1(TorC1)尤其重要，它是一种整合多种环境信号并控制广泛的基本细胞过程以响应它们的全球调节因子。雷帕霉素家族药物在治疗组织排斥反应、多种癌症、早衰症和衰老相关疾病方面的临床潜力越来越大，这要求我们准确了解mTORC1调控的机制细节，进而控制其下游过程。例如，对mTORC1如何调节下游事件(增加触发凝血级联反应的组织因子的产生)的不完全理解已经引起了人们对用于治疗急性心肌梗死的雷帕霉素衍生物洗脱支架中晚期支架血栓的高度关注。本申请中提出的实验试图识别这种遗漏的或不完全理解的Tor调节机制。该计划首先利用酿酒酵母强大的遗传学和众所周知的细胞和分子生物学，通过研究TorC1依赖(雷帕霉素诱导的)和独立的调控GATA转录激活因子Gln3和Gat1的机制，更准确和深入地了解TorC1的调控。它们是非致病酵母和致病酵母中TorC1活性的最广泛使用的报告之一。所发现的机制原理将被应用于与公认的哺乳动物细胞生物学家和mTOR调节领域的专家合作，在哺乳动物细胞中研究mTORC1营养传感和对其反应的类似的中心问题。具体地说，第一个和第二个具体目标测试 假设Gln3和Gat1分别由TorC1依赖和独立的调控通路控制，具有不同的氮输入、蛋白磷酸酶(Sit4和PP2A)和激酶需求。将确定每个途径所需的磷酸酶和激酶。该研究策略使用gln3氨基酸替代突变体实现了这一目标，该突变体在基因上将假设的途径彼此隔离，从而允许在不输入或干扰第二条途径的情况下严格分析一条途径的成分和调节机制。证明这一方法成功的关键的、原则证明的突变体已经被分离出来并初步表征。第三个目标挑战和分析大多数TorC1/mTORC1研究所依赖的基本假设，并测试从描述TorC1的Vam6-GTR1/2-Ego1/3激活的令人兴奋的新模型中发出的预测。然后，这些信息被用来设计和执行在哺乳动物细胞中研究这些问题的实验。如果拟议的实验证实了我的团队最近获得的数据得出的结论，将需要对现有的TorC1和mTORC1数据进行重大重新考虑，并可能确定营养响应调节的新途径。

DESCRIPTION (provided by applicant): One of the gaps in our understanding of cellular regulation is the detailed mechanisms through which eukaryotic cells detect nutrients in their environment and respond to them in an integrated manner. This is particularly important with respect to the Target of Rapamycin Complex 1 (TorC1), a global regulator that integrates multiple environmental signals and controls a wide range of basic cellular processes in response to them. The growing clinical potential of rapamycin-family drugs for treating tissue rejection, a variety of cancers, progeria and aging-related diseases requires that we accurately understand the mechanistic details of mTorC1 regulation and in turn its control of downstream processes. For example, an incomplete understanding of how mTorC1 regulates downstream events (increases Tissue Factor production which triggers the coagulation cascade) has raised high concern about late stent thrombosis in rapamycin-derivative eluting stents used to treat acute myocardial infarction. The proposed experiments in this application seek to identify such missed or incompletely understood Tor regulatory mechanisms. The plan first utilizes the powerful genetics and well understood cellular and molecular biology of S. cerevisiae to gain a more accurate and in depth understanding of TorC1 regulation by investigating TorC1-dependent (rapamycin-inducible) and -independent mechanisms regulating the GATA transcription activators Gln3 and Gat1. They are among the most widely used reporters of TorC1 activity in non-pathogenic and pathogenic yeast. The mechanistic principles discovered will then be applied to investigate analogous central questions of mTorC1 nutrient sensing and responses to it in mammalian cells in collaboration with a recognized mammalian cell biologist and an expert in the field of mTor regulation. Specifically, the 1st and 2nd Specific Aims test the hypothesis that Gln3 and Gat1 are individually controlled by TorC1-dependent and -independent regulatory pathways with different nitrogen inputs, protein phosphatase (Sit4 and PP2A) and kinase requirements. The phosphatase and kinase requirements for each pathway will be established. The research strategy achieves this goal using gln3 amino acid substitution mutants that genetically isolate the hypothesized pathways from one another, thereby permitting one pathway's components and regulatory mechanisms to be rigorously analyzed without input or interference from the second pathway. Pivotal, proof-of-principle mutants demonstrating the success of this approach have already been isolated and preliminarily characterized. The 3rd Aim challenges and analyzes fundamental assumptions upon which most TorC1/mTorC1 investigations depend and tests predictions that emanate from the exciting new model describing Vam6-Gtr1/2-Ego1/3 activation of TorC1. This information is then used to design and perform experiments that investigate these questions in mammalian cells. If the proposed experiments substantiate the conclusions derived from recently acquired data by my group, significant reconsideration of existing TorC1 & mTorC1 data will be required, and new pathways of nutrient-responsive regulation will likely be identified.