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，然后以一种拓扑结构不同的过程将膜内化到自身形成多泡体（MVBs）。膜和相关货物内化到MVB中对于溶酶体的消化和降解是必不可少的，也被特殊细胞用来产生外泌体。从酵母到哺乳动物，一组18个“E类”蛋白质被认为调节并可能驱动腔内囊泡的形成。非溶性病毒（如HIV）也会利用这些蛋白质中的一些或可能全部从被感染细胞的质膜中萌发，这一过程在拓扑结构上相当于出芽进入核内体的管腔。对酵母和最近哺乳动物系统的细致研究，让我们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。由于所需的膜曲率与传统的包被囊泡驱动的内吞作用相反，因此可能涉及新的机制。我们使用速冻深刻痕电子显微镜发现，与Snf7（哺乳动物系统中的hSnf7/CHMP4）相关的ESCRT-III蛋白在质膜上组装成细丝，可以诱导形成紧密的圆形阵列，并使膜弯曲远离细胞质，形成芽，最终在细胞表面形成小管。我们假设含有聚合物的类似ESCRT-III通常会形成新生内体囊泡的颈部，既限制囊泡的内容物，又在膜中诱导必要的变形。我们的计划是确定ESCRT-III聚合物在体外、模型膜上和正常MVB生物发生背景下的结构和动力学，以确定ESCRT-III聚合物的组装和拆卸如何参与MVB生物发生。目的1将探讨ESCRT-III的同质和异质聚合物的结构。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+ atp酶VPS4对ESCRT-III聚合物的降解作用。最后，Aim 4将研究沉默VPS4和hSnf7蛋白表达前后受体下调过程中细胞内的内源性ESCRT-III蛋白。

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.