The regulation of autophagy pathways in eukaryotic cells

描述(由申请人提供)：我们有兴趣更好地了解当条件不利于继续生长时真核细胞进入的不分裂休眠状态的生物学。我们的主要目标之一是确定在这些静止期诱导的过程是如何调节的，以及它们如何有助于一般细胞的生存。这一提议集中在一系列相关的途径上，统称为自噬，这是这种生存所必需的。自噬途径负责细胞质物质的周转，包括大量蛋白质和受损或多余的细胞器。这种自噬介导的降解与多种与人类健康相关的过程有关，包括肿瘤抑制、先天免疫和神经疾病，如亨廷顿病。在许多这样的情况下，自噬途径被认为是治疗干预的一个主要点。因此，我们必须对真核细胞中自噬的正常控制机制有一个透彻的了解。这项提案将研究酵母中自噬调节的两个关键方面，酿酒酵母。对这种生物的研究极大地促进了我们对包括人类在内的所有真核生物中自噬的基本理解。首先，我们将结合多种方法来研究cAMP依赖的蛋白激酶(PKA)信号通路失活所引起的自噬过程。我们的初步工作表明，这种由PKA调控的过程可能类似于最近在哺乳动物中发现的另一种形式的巨型自噬。这里的实验将探索这种可能性，并定义控制这一PKA调节途径的分子机制。这些研究还将表征磷脂酰肌醇PtdIns(3，5)P2在PKA调节的大自噬中的潜在作用。这项建议的第二个主要目标是鉴定Atg1蛋白激酶的底物。ATG1是自噬机制中的一个关键调控靶点，识别该酶的靶标是当今自噬领域的主要目标之一。总之，我们认为这项工作的完成将为真核细胞中自噬的控制方式提供重要的见解，这些见解应该有助于以临床有益的方式操纵这一途径。本研究的具体目的是：(1)研究PKA信号失活诱导的自噬途径；(2)研究PKA信号、肌醇磷脂代谢与自噬调控之间的联系；(3)鉴定和表征ATG1蛋白激酶底物。 与公共健康相关：这项提议旨在加深我们对自噬过程的理解，该过程与许多严重的人类疾病有关，包括乳腺癌和卵巢癌、克罗恩病以及亨廷顿病等神经疾病。有趣的是，针对自噬的药物正在被开发为治疗其中许多疾病的潜在疗法。通过增加我们对自噬过程正常控制的理解，这里的工作将为这一药物发现过程提供潜在的新途径。

DESCRIPTION (provided by applicant): We are interested in developing a better understanding of the biology of the non-dividing resting states that eukaryotic cells enter when conditions are not conducive to continued growth. One of our primary goals is to define how the processes that are induced during these periods of quiescence are regulated and how they contribute to general cell survival. This proposal is focused upon a set of related pathways, known collectively as autophagy, that are required for this survival. Autophagy pathways are responsible for the turnover of cytoplasmic material, including bulk protein and damaged or superfluous organelles. This autophagy-mediated degradation has been linked to a variety of processes relevant to human health, including tumor suppression, innate immunity and neurological disorders, such as Huntington's disease. In many of these conditions, the autophagy pathway is being considered as a major point of therapeutic intervention. It is therefore critical that we develop a thorough understanding of the mechanisms normally controlling autophagy in eukaryotic cells. This proposal will examine two key aspects of the regulation of autophagy in the yeast, Saccharomyces cerevisiae. Studies with this organism have contributed tremendously to our basic understanding of autophagy in all eukaryotes, including humans. First, a combination of approaches will be used to characterize the autophagy process induced by the inactivation of the cAMP-dependent protein kinase (PKA) signaling pathway. Our preliminary work indicates that this PKA-regulated process may be similar to an alternative form of macroautophagy recently identified in mammals. The experiments here will explore this possibility and define the molecular machineries governing this PKA-regulated pathway. These studies will also characterize a potential role for the phosphoinositide, PtdIns (3,5)P2, in this PKA-regulated macroautophagy. The second major goal of this proposal is the identification of the substrates of the Atg1 protein kinase. Atg1 is a key regulatory target within the autophagy machinery and identifying the targets of this enzyme represents one of the major goals in the autophagy field today. In all, we feel that the completion of this work will provide important insights into the manner in which autophagy is controlled in eukaryotic cells, insights that should facilitate efforts to manipulate this pathway in clinically beneficial ways. The specific aims in this proposal are: (1) to characterize the autophagy pathway induced upon inactivation of PKA signaling; (2) to characterize the link between PKA signaling, phosphoinositide metabolism and the regulation of autophagy; and (3) to identify and characterize substrates of the Atg1 protein kinase. PUBLIC HEALTH RELEVANCE: This proposal aims to further our understanding of a process known as autophagy that has been linked to a number of serious human ailments, including breast and ovarian cancer, Crohn's disease and neurological disorders, such as Huntington's disease. Interestingly, drugs that target autophagy are being developed as potential therapeutics for many of these conditions. By increasing our understanding of the normal control of the autophagy process, the work here would provide potentially novel avenues for this drug discovery process.