Regulation of nitrogen catabolic gene expression in S. cerevisiae

描述（由申请人提供）：我们对细胞调节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。这对于雷帕霉素复合物1（TorC 1）的靶标尤其重要，TorC 1是一种整合多种环境信号并控制多种基本细胞过程以响应它们的全局调节剂。雷帕霉素家族药物用于治疗组织排斥反应、各种癌症、早衰症和衰老相关疾病的临床潜力不断增长，这要求我们准确了解mTorC 1调控的机制细节，进而了解其对下游过程的控制。例如，对mTorC 1如何调节下游事件（增加组织因子产生，从而触发凝血级联反应）的不完全理解引起了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。本申请中提出的实验试图鉴定这种遗漏或不完全理解的Tor调节机制。该计划首先利用强大的遗传学和充分理解的细胞和分子生物学的S。通过研究TorC 1依赖性（雷帕霉素诱导）和非依赖性机制调节加塔转录激活因子Gln 3和Gat 1，从而更准确和深入地了解TorC 1的调节。它们是在非致病性和致病性酵母中最广泛使用的TorC 1活性报告基因之一。然后，将发现的机械原理应用于与公认的哺乳动物细胞生物学家和mTor调控领域的专家合作，研究mTorC 1营养感测和哺乳动物细胞对其反应的类似中心问题。具体而言，第一和第二个具体目标测试 假设Gln 3和Gat 1分别由TorC 1依赖性和非依赖性调节途径控制，具有不同的氮输入、蛋白磷酸酶（Sit 4和PP 2A）和激酶需求。将确定每种途径的磷酸酶和激酶要求。该研究策略使用gln 3氨基酸取代突变体实现了这一目标，这些突变体在遗传上将假设的途径彼此隔离，从而允许一种途径的组分和调节机制在没有第二种途径的输入或干扰的情况下进行严格分析。证明这种方法成功的原理性突变体已经被分离并初步表征。第三目标挑战和分析大多数TorC 1/mTorC 1研究所依赖的基本假设，并测试来自描述TorC 1的Vam 6-Gtr 1/2-Ego 1/3激活的令人兴奋的新模型的预测。然后，这些信息用于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小组从最近获得的数据中得出的结论，则需要对现有的TorC 1和mTorC 1数据进行重大重新考虑，并且可能会发现营养响应调节的新途径。 公共卫生相关性：雷帕霉素家族药物治疗移植患者组织排斥反应、多种类型癌症和潜在早衰症的重要性和日益增加的临床用途使得我们必须准确和完整地了解这些药物的靶点，即哺乳动物雷帕霉素复合物靶点1（mTorC 1）是如何调节的，进而调节下游细胞过程。尽管取得了令人印象深刻的进展，mTorC 1调节的一个最不为人所知的方面是通过其存在的含氮营养物质，如氨基酸，检测和适当的，综合的反应，随后实施的机制。拟议的研究将大大有助于阐明这些机制。

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.