Regulation of autophagy by AMPK, Hsp90-Cdc37 and Ulk1 in erythroid cells

描述（由申请人提供）：该项目的长期目标是破译参与调节自噬的分子途径，以便更精确地操纵这一过程，以治疗造血疾病和恶性肿瘤。自噬是细胞内的主要循环途径，主要负责细胞器（包括线粒体）和长寿命或错误折叠的蛋白质的周转。细胞能够诱导适当水平的自噬以应对各种代谢或蛋白质毒性应激源，从而使细胞能够生存并适应环境挑战。事实上，许多肿瘤在不利条件下依靠自噬生存。最近的一项研究表明，在某些类型的癌症中，Ulk1蛋白（自噬的关键调节因子）的稳定与预后不良有关，这可能是自噬机制在应激反应中增强的能力的结果。因此，了解参与调节自噬的信号通路不仅可以深入了解自噬在维持健康细胞和疾病中的作用，还可以揭示在癌症治疗中操纵自噬的新靶点。Ulk1和Ulk2是哺乳动物中普遍表达的Atg1的同源物，Atg1是一种特性良好的丝氨酸-苏氨酸激酶，调节酵母的选择性和非选择性自噬。Ulk1是氨基酸饥饿诱导的培养细胞自噬、红细胞和肝细胞线粒体清除以及去极化损伤所必需的。Ulk2与自噬有关；然而，Ulk2敲除动物的特异性缺陷尚未报道。相反，缺乏Ulk1和Ulk2的小鼠表现出与缺乏核心非冗余基因（如mAtg5或mAtg7）的小鼠相似的围产死亡率，这表明Ulk1和Ulk2在调节自噬方面具有一些重叠的功能。鉴于核心自噬基因的缺乏对生物体是有害的，识别和表征Ulk1和Ulk2调控的差异可能会揭示选择性靶向其中一种蛋白质的独特机会，并更精确地操纵自噬以治疗癌症。最近的研究表明，虽然Ulk1和Ulk2都是能量感应激酶AMPK的底物，但只有Ulk1（而不是Ulk2）是Hsp90- cdc37伴侣复合物的客户端，其激活和稳定性对Hsp90功能的改变很敏感。尽管AMPK和Hsp90-Cdc37在激活Ulk1功能和维持细胞稳态方面都很重要，但这些参与者如何合作将自噬和线粒体周转的调节与细胞不断变化的能量需求结合起来仍不清楚。由于与体内Ulk1缺乏相关的主要表型是红细胞成熟末期线粒体清除的缺陷，我们建议通过使用细胞和体内模型，结合生化、结构和细胞生物学方法，研究在红细胞成熟背景下，AMPK和Hsp90-Cdc37调节Ulk1的分子基础和生理相关性。

DESCRIPTION (provided by applicant): The long-term goal of this project is to decipher the molecular pathways involved in regulating autophagy so that the process can be more precisely manipulated for the treatment of hematopoietic disorders and malignancies. Autophagy, the primary recycling pathway in cells, is largely responsible for the turnover of organelles, includin mitochondria, and long-lived or misfolded proteins. The ability of cells to induce appropriate levels of autophagy in response to various metabolic or proteotoxic stressors allows cells to survive and adapt to environmental challenges. Indeed, many tumors rely on autophagy for survival under adverse conditions. A recent study indicates that stabilization of Ulk1 protein (a key regulator of autophagy) in certain types of cancer is associated with a poor prognosis-perhaps as a consequence of enhanced ability to engage the autophagy machinery in response to stress. Thus, understanding the signaling pathways involved in regulating autophagy will not only provide insight into the role of autophagy in the maintenance of healthy cells and in disease, but should also reveal new targets for manipulating autophagy in the treatment of cancer. Ulk1 and Ulk2 are ubiquitously expressed mammalian homologues of Atg1, a well-characterized serine- threonine kinase that regulates both selective and non-selective autophagy in yeast. Ulk1 is required for amino acid starvation induced autophagy in cultured cells and for clearance of mitochondria in red blood cells and hepatocytes, and following depolarizing damage. Ulk2 has been implicated in autophagy; however, specific defects in Ulk2 knockout animals have not been reported. Rather, mice lacking both Ulk1 and Ulk2 show perinatal lethality similar to those lacking core non-redundant genes such as mAtg5 or mAtg7, suggesting that Ulk1 and Ulk2 share some overlapping functions in the regulation of autophagy. Given that deficiency of core autophagy genes is detrimental to organisms, identifying and characterizing differences in the regulation of Ulk1 and Ulk2 may reveal unique opportunities to selectively target one or the other protein, and more precisely manipulate autophagy in the treatment of cancer. Recent studies have demonstrated that while both Ulk1 and Ulk2 are substrates of the energy-sensing kinase AMPK, Ulk1 alone (not Ulk2) is a client of the Hsp90-Cdc37 chaperone complex whose activation and stability is sensitive to alterations in Hsp90 function. Despite the importance of both AMPK and Hsp90-Cdc37 in activating Ulk1 function and in maintaining cellular homeostasis, the way in which these players cooperate to integrate regulation of autophagy and mitochondrial turnover with the changing energy demands of the cell remains unclear. Since the primary phenotype associated with Ulk1-deficiency in vivo is a defect in the clearance of mitochondria during the terminal stages of erythroid maturation, we propose to examine the molecular basis and physiologic relevance of Ulk1 regulation by AMPK and Hsp90-Cdc37 in the context of erythroid maturation through a combination of biochemical, structural and cell biological approaches using both cell-based and in vivo models. PUBLIC HEALTH RELEVANCE: This proposal is focused on understanding the regulation of autophagy ("self-eating"), a cellular response to stress and starvation, which has been implicated in the pathogenesis of diseases, including neurodegeneration, diabetes, and cancer.