MOLECULAR MECHANISMS OF MULTIVESICULAR BODY BIOGENESIS

描述(申请人提供)：内体系统从质膜内化膜和蛋白质，然后在一个不同的拓扑学过程中将膜内化到自身形成多囊泡体(MVB)。膜和相关物质内化到MVB中对于溶酶体的消化和降解是必不可少的，也被专门的细胞用来产生外切体。一组从酵母到哺乳动物保守的18种“E类”蛋白质被认为调节并可能驱动腔内小泡的形成。一些或可能所有这些蛋白质也被非裂解性病毒(如艾滋病毒)用来从受感染细胞的质膜发芽，这个过程在拓扑上相当于发芽进入内体的管腔。对酵母和最近的哺乳动物系统的优雅研究让我们深入了解了这些蛋白质是如何相互联系的，货物分子和内膜，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。由于所需的膜曲率与传统的包被囊泡驱动的内吞作用相反，很可能涉及到新的机制。我们使用快速冷冻深蚀刻电子显微镜发现，与Snf7相关的ESCRT-III蛋白(哺乳动物系统中的hSnf7/CHMP4)在质膜上组装成细丝，可以诱导形成紧密的环形阵列，并使膜偏离细胞质，在细胞表面产生芽，最终形成小管。我们假设，类似的ESCRT-III聚合物通常会产生新生内体囊泡的颈部，既限制了囊泡的内容物，又导致了膜上必要的变形。我们的计划是在体外、在模型膜上以及在正常的MVB生物发生的背景下定义ESCRT-III聚合物的结构和动力学，以确定ESCRT-III聚合物的组装和拆解如何参与MVB的生物发生。目的1探索ESCRT-III均相和杂相聚合物的结构。目标2将定义调节ESCRT-III聚合物组装的机制。目的3将研究AAA+ATPase Vps4对ESCRT-III聚合物的分解。最后，Aim 4将研究Vps4和hSnf7蛋白沉默前后受体下调过程中细胞内源性ESCRT-III蛋白的变化。 与公共健康相关：这些研究将阐明与受体下调、脂质稳态以及包括HIV在内的许多包膜病毒从细胞中释放有关的细胞机制。要研究的两种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。对这些过程的病理生理学的洞察需要对这项工作所针对的机制的理解。

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.