Molecular mechanisms that regulate lysosomal protein transport

 描述(由申请人提供)：这项提案的长期目标是确定调节完整的膜蛋白运输到溶酶体进行降解的分子机制。ESCRT机制是一组保守的内体蛋白复合体，被认为直接与泛素化的膜蛋白结合，并调控它们进入专门的多囊泡内小体(MVEs)的囊泡。这一过程对于激素受体的下调和防止可能导致发育异常和疾病的结构性信号尤为重要。ESCRT机制的后期作用组件如何协调微血管内小泡的形成将在本提案中讨论。线虫生殖系和早期胚胎是研究完整、发育中的动物的膜动力学的强大模式系统。利用RNA干扰可以有效地去除卵母细胞中的特定蛋白质。此外，卵母细胞的成熟和受精可重复地触发多种跨膜蛋白的内化和ESCRT介导的降解，为研究溶酶体蛋白运输提供了一个理想的、生理上相关的系统。线虫对基因操作具有很高的适应性，可以很容易地进行工程改造，以稳定表达转基因，以实现基因替换策略。此外，我们还建立了在胚胎发育的特定时间点对动物进行高压冷冻的方法，以便能够使用基于电子显微镜(EM)的方法逐步表征从头开始的MVE生物发生。鉴于早期胚胎发育的刻板印象，我们可以使用我们在该系统中首创的活细胞成像分析将这些EM数据与我们的发现直接关联起来。利用这种独特的属性组合，第一次更新应用的具体目标是：)定义指定微血管上ILV形成位置的调控机制，2)确定促进ESCRT-III细丝成核的机制，以及3)定义控制ESCRT-III聚合物动力学的调控机制。在赠款支持的第一阶段进行的遗传和生化研究确定了研究ESCRT-III聚合物组装的新方法和工具，提出了关于这一过程如何控制的有趣假设。利用电子分子模拟、体外重建实验和体内高分辨率显微镜分析相结合的方法，我们将定义在MVE形成过程中调节ESCRT-III复合体组装的新机制。这些研究将为未来研究哺乳动物细胞中高度相关的通路提供一个关键框架。

 DESCRIPTION (provided by applicant): The long-term goal of this proposal is to define molecular mechanisms that regulate the trafficking of integral membrane proteins to the lysosome for degradation. The ESCRT machinery, a set of conserved endosomal protein complexes, is proposed to bind directly to ubiquitinylated membrane proteins and govern their entry into vesicles that bud into the lumen of specialized multivesicular endosomes (MVEs). This process is particularly important for the downregulation of hormone receptors and to prevent constitutive signaling, which can lead to developmental abnormalities and disease. How the late-acting components of the ESCRT machinery coordinate the formation of intraluminal vesicles at MVEs will be addressed in this proposal. The C. elegans germline and early embryo are powerful model systems to study membrane dynamics in an intact, developing animal. Specific proteins can be efficiently depleted from oocytes using RNA interference. Additionally, oocyte maturation and fertilization reproducibly trigger the internalization and ESCRT-mediated degradation of multiple transmembrane proteins, providing an ideal, physiologically relevant system for studying lysosomal protein transport. C. elegans is highly amenable to genetic manipulation and can be engineered easily to stably express transgenes for gene replacement strategies. Additionally, we have established methods to high pressure freeze animals at specific time points during embryo development to enable the stepwise characterization of de novo MVE biogenesis using electron microscopy (EM)-based approaches. Given the stereotypic nature of early embryo development, we can correlate these EM data directly with our findings using live cell imaging assays, which we have pioneered in this system. Taking advantage of this unique combination of attributes, the specific aims of this first renewal application are to: ) define regulatory mechanisms that specify the site of ILV formation on MVEs, 2) determine mechanisms that promote the nucleation of ESCRT-III filaments, and 3) define regulatory mechanisms that control ESCRT-III polymer dynamics. The genetic and biochemical studies conducted during the first period of grant support defined new methods and tools to study ESCRT-III polymer assembly, raising intriguing hypotheses regarding how this process is controlled. Using a combination of in silico molecular modeling, in vitro reconstitution experiments, and in vivo high resolution microscopy-based assays, we will define new mechanisms that regulate ESCRT-III complex assembly during MVE formation. These studies will provide a key framework for future investigation into highly related pathways in mammalian cells.