Control of vesicular trafficking in the hepatocyte.

描述(申请人提供)：内吞和自噬是许多细胞类型的健康所必需的基本过程，包括肝细胞。我们团队和其他人之前的研究已经确定了内吞作用和自噬之间的相互作用：1)它们之间共享特定的隔室；2)两者都有助于细胞蛋白分解；3)这两种途径都需要以动态方式相互作用的囊泡的受调控的运输，作为其成熟过程的一部分，经历分裂和融合。根据先前的发现，我们认为内吞或自噬转运途径的功能障碍可能对正常肝功能产生有害影响，并参与肝病的发病机制。这一提议的总体目标是在分子水平上描述内吞和自噬途径之间的功能相互作用及其对细胞内稳态的贡献。为此，我们将：1)机械地定义囊泡相关蛋白 通过分裂和融合调节内吞/自噬小泡加工所需的运动招募和活性的复合体；2)确定膜脂成分变化对内吞和自噬途径之间相互作用的影响，以及3)定量表征内吞和自噬之间的功能相互作用。我们将在培养的肝细胞系以及大鼠和小鼠的肝脏中使用化学和遗传操作，并结合生化和形态学方法来回答这些问题。Allan Wolkoff博士(细胞吞噬和肝脏病理生理学)和Ana Maria Cuervo博士(自噬和细胞生物学)两位共同PI的互补专业知识极大地增强了他们发现肝脏内吞和自噬途径之间的相互作用以及研究其中一条途径失败对另一条途径的后果的能力。拟议研究的主要意义在于，在人类疾病中观察到了内吞和自噬的变化，这些疾病包括蛋白质构象紊乱(如α1-抗胰蛋白酶缺乏)、癌症、代谢紊乱(如糖尿病、肥胖症)以及传染病和免疫性疾病。然而，对内吞和自噬细节的不完全了解限制了基于机制的治疗的发展。这些研究的成功完成将最终提供所需的工具来确定新的治疗靶点，以恢复正常的肝功能，或者减缓由内酶体/溶酶体系统功能障碍引起的常见肝病的功能衰退。

DESCRIPTION (provided by applicant): Endocytosis and autophagy represent fundamental processes that are essential to the health of many cell types, including hepatocytes. Previous studies from our groups and others have identified cross-talk between endocytosis and autophagy: 1) specific compartments are shared between them; 2) both contribute to cellular proteolysis and 3) both pathways require regulated trafficking of vesicles that interact in a dynamic manner, undergoing fission and fusion as part of their maturation process. Based on prior findings, we propose that dysfunction of the endocytic or autophagic trafficking pathways can have deleterious effects on normal liver function and contribute to the pathogenesis of hepatic disorders. The overall goal of this proposal is to characterize at the molecular level the functional interaction between the endocytic and the autophagic pathways and their contribution to cellular homeostasis. To this end we will: 1) mechanistically define vesicle- associated protein complexes that regulate motor recruitment and activity required for endocytic/autophagic vesicle processing by fission and fusion; 2) determine the consequences of changes in membrane lipid composition on the interactions between endocytic and autophagic pathways and 3) characterize quantitatively the functional interplay between endocytosis and autophagy. We will use chemical and genetic manipulations in cultured hepatocyte cell lines and in rat and mouse liver combined with biochemical and morphological approaches to answer these questions. The complementary expertise of the two co-PIs, Dr. Allan Wolkoff (endocytosis and liver pathophysiology) and Dr. Ana Maria Cuervo (autophagy and cell biology) greatly enhances their ability to discover interactions between the endocytic and the autophagic pathways in liver and to study the consequences that failure in one of these pathways has on the other. The major significance of the proposed studies resides in the fact that alterations in endocytosis and autophagy have been observed in human diseases that include protein conformational disorders (e.g. alpha1-antitrypsin deficiency), cancer, metabolic disorders (e.g. diabetes, obesity) and infectious and immune diseases. However, incomplete knowledge regarding details of endocytosis and autophagy limits development of mechanism-based therapeutics. Successful completion of these studies will ultimately provide the tools needed to identify novel therapeutic targets to restore normal liver function or perhaps slow the functional decline associated with common liver disorders resulting from dysfunction of the endosome/lysosome system.