MOLECULAR MECHANISMS OF MULTIVESICULAR BODY BIOGENESIS

描述（由申请人提供）：内体系统内化来自质膜的膜和蛋白质，然后在拓扑学上不同的过程中将膜内化到自身中以形成多泡体（MVB）。膜和相关货物内化到MVB中对于溶酶体中的消化和降解是必需的，并且也被特化细胞用于产生外泌体。从酵母到哺乳动物保守的一组18个“E类”蛋白被认为调节并可能驱动腔内囊泡的形成。这些蛋白质中的一些或可能全部也被非裂解性病毒（例如HIV）用于从感染细胞的质膜出芽，其过程在拓扑学上等同于出芽进入内体的内腔。在酵母和最近的哺乳动物系统中的优雅研究已经深入了解了这些蛋白质如何相互关联，货物分子和内体膜，但基本上对这些或其他蛋白质如何驱动膜内陷和囊泡释放一无所知。由于所需的膜曲率是相反的，传统的包被囊泡驱动的内吞作用，它很可能是新的机制参与。我们发现，使用速冻深蚀刻电子显微镜，ESCRT-III蛋白相关的Snf 7（hSnf 7/CHMP 4在哺乳动物系统）组装成细丝的质膜上，可以诱导形成紧密的圆形阵列和弯曲膜远离细胞质，创造芽，并最终在细胞表面的小管。我们假设，类似的ESCRT-III含有聚合物通常会产生新生内体囊泡的颈部，既限制囊泡的内容物，又诱导膜中必要的变形。我们的计划是定义ESCRT-III聚合物的结构和动力学在体外，模型膜，并在正常的MVB生物发生的背景下，以确定如何组装和拆卸ESCRT-III聚合物参与MVB生物发生。目的1将探索ESCRT-Ⅲ均聚物和杂聚物的结构。目的2将定义调节ESCRT-III聚合物组装的机制。目的3将研究AAA+ ATP酶VPS 4对ESCRT-III聚合物的分解。最后，目的4将研究在VPS 4和hSnf 7蛋白表达沉默之前和之后，在受体下调过程中细胞中内源性ESCRT-III蛋白。 公共卫生相关性：这些研究将阐明参与受体下调、脂质稳态和包括HIV在内的许多包膜病毒从细胞中释放的细胞机制。两种待研究蛋白质的突变直接导致家族性额颞叶痴呆和早发性白内障。深入了解这些过程的病理生理学需要机械的理解，这项工作的目的。

DESCRIPTION (provided by applicant): The endosomal system internalizes membrane and protein from the plasma membrane, and then in a topologically distinct process internalizes membrane into itself to form multivesicular bodies (MVBs). Internalization of membrane and associated cargo into the MVB is essential for digestion and degradation in the lysosome, and is also used by specialized cells to generate exosomes. A set of eighteen "class E" proteins conserved from yeast to mammals is thought to regulate and probably drive the formation of intralumenal vesicles. Some or possibly all of these proteins are also used by nonlytic viruses such as HIV to bud from the plasma membrane of infected cells in a process that is topologically equivalent to budding into the lumen of the endosome. Elegant studies in yeast and more recently mammalian systems have lent insight into how these proteins associate with each other, cargo molecules, and the endosomal membrane, but essentially nothing is known about how these or other proteins drive membrane invagination and vesicle release. Because the required membrane curvature is opposite to that of traditional coated vesicle-driven endocytosis, it is likely that novel mechanisms are involved. We found using quick-freeze deep-etch electron microscopy that ESCRT-III proteins related to Snf7 (hSnf7/CHMP4 in mammalian systems) assemble into filaments on the plasma membrane that can be induced to form tight circular arrays and bend the membrane away from the cytoplasm, creating buds and eventually tubules on the cell surface. We hypothesize that similar ESCRT-III containing polymers normally create the neck of nascent endosomal vesicles, both confining the vesicle's contents and inducing the requisite deformation in the membrane. Our plan is to define the structure and dynamics of ESCRT-III polymers in vitro, on model membranes, and in the context of normal MVB biogenesis in order to determine how assembly and disassembly of ESCRT-III polymers participate in MVB biogenesis. Aim 1 will explore the structure of ESCRT-III homo- and hetero- polymers. Aim 2 will define mechanisms that regulate ESCRT-III polymer assembly. Aim 3 will study disassembly of ESCRT-III polymers by the AAA+ ATPase VPS4. Finally, Aim 4 will study endogenous ESCRT-III proteins in cells during the process of receptor downregulation before and after silencing expression of VPS4 and hSnf7 proteins. PUBLIC HEALTH RELEVANCE: These studies will illuminate cellular mechanisms involved in receptor downregulation, lipid homeostasis, and the release of many enveloped viruses including HIV from the cell. Mutations in two of the proteins to be studied are directly responsible for familial forms of frontotemporal dementia and early-onset cataracts. Insight into the pathophysiology of these processes requires the mechanistic understanding at which this work is aimed.