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中的一个空白是真核细胞检测其环境中的营养物质并以综合方式对其作出反应的详细机制。这对于雷帕霉素靶标复合物1 （TorC1）来说尤其重要，TorC1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剂。雷帕霉素家族药物在治疗组织排斥反应、各种癌症、早衰症和衰老相关疾病方面的临床潜力越来越大，这就要求我们准确地了解mTorC1调控的机制细节，进而了解其对下游过程的控制。例如，对mTorC1如何调节下游事件（增加触发凝血级联的组织因子产生）的不完全了解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形成的高度关注。本应用程序中提出的实验旨在确定这些被遗漏或不完全理解的调控机制。该计划首先利用酿酒酵母强大的遗传学和充分了解的细胞和分子生物学，通过研究TorC1依赖性（雷帕霉素诱导）和非依赖性调节GATA转录激活子Gln3和Gat1的机制，更准确和深入地了解TorC1调控。它们是在非致病性和致病性酵母中最广泛使用的TorC1活性报告基因。我们将与一位公认的哺乳动物细胞生物学家和一位mTor调控领域的专家合作，将发现的机制原理应用于研究哺乳动物细胞中mTorC1营养感知和对其反应的类似核心问题。具体来说，第一和第二特定目标测试

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. PUBLIC HEALTH RELEVANCE: The importance and increasing clinical use of rapamycin-family drugs to treat tissue rejection in transplant patients, multiple types of cancer and potentially progeria make it imperative that we accurately and completely understand how the target of these drugs, the mammalian Target Of Rapamycin Complex 1 (mTorC1), is regulated and in turn regulates downstream cellular processes. Despite impressive advances, one of the least well understood aspects of mTorC1 regulation are the mechanisms through which the presence of nitrogenous nutrients, such as amino acids, are detected and appropriate, integrated responses to them subsequently implemented. The proposed research will significantly contribute to elucidating these mechanisms.