Wnt Signaling and Endosomal Trafficking as Regulators of Cellular Protein Degradation

项目摘要/摘要 我们对监管的基本机制的理解存在着根本性的差距 胞浆蛋白降解。一部分胞质蛋白通过一个过程被内溶酶吞噬。 然而，被称为微自噬的微自噬在多大程度上可以被调控，以及哪些特定的 通过这种机制靶向的蛋白质目前还不清楚。这一差距是一个重要的问题 因为胞浆蛋白周转的失调是神经退行性变和 代表了癌症研究中的一个新兴主题。这个项目的长期目标是确定 蛋白质分解代谢如何受到Wnt信号的调节，这是一条在发育中必不可少的途径，也是 在疾病中管理不善。我的初步数据支持概念上的新假设，即微型自噬是 由Canonical WNT协调。此外，初步数据表明，胞浆底物被 翻译后关键的精氨酸甲基化修饰Wnt信号过程中的微自噬 修饰最近涉及到细胞质信号转导。基于这些发现，中心假说 Wnt信号刺激精氨酸甲基化促进胞浆蛋白 通过微自噬降解内溶体(图1)。我提出两个具体目标：(1)澄清 精氨酸甲基化在细胞生长和胚胎发育过程中对Wnt信号的作用；以及(2)决定如何 WNT信号和微自噬调节内溶酶体内胞浆蛋白的降解。目标1将 检测精氨酸甲基化和精氨酸甲基转移酶1(PRMT1)在促进WNT中的作用 人类细胞和非洲爪哇细胞生长和胚胎发育过程中的信号传递。目标1在概念上是创新的 因为它研究了精氨酸甲基化的新作用，一种最近才在细胞质中发现的修饰 信号，在Wnt途径和内溶酶体中。此外，通过使用化学品进行技术创新-- 基因蛋白标记策略将提供最高水平的敏感性来检查甲基化在 促进活细胞中GSK3的磷酸化。目标2将定义WNT信令通过的机制 利用遗传生化方法调节微自噬以促进胞浆蛋白分解 培养的人类细胞中的内溶酶体蛋白分解。我将应用一种技术创新的体内生物素-蛋白质 检测微自噬以特定胞浆蛋白为靶点这一新概念的标记策略 WNT信令。这项拟议的研究具有重要意义，因为它阐明了胞浆蛋白水解物的调节 途径可有助于开发针对WNT的创新治疗干预措施 在癌症中传递信号，并可能揭示疾病中蛋白质降解机制的关键细节。总而言之， 这一建议为研究蛋白质的病理机制提供了一种新的方法。 在发育和组织形态发生过程中受细胞外信号因子调节的降解。

Project Summary/Abstract There is a fundamental gap in our understanding of the basic mechanisms underlying the regulation of cytosolic protein degradation. A portion of cytosolic proteins are engulfed by endolysosomes through a process known as microautophagy, however, the extent to which microautophagy can be regulated and which specific proteins are targeted through this mechanism are unknown. This gap represents an important problem because the misregulation of cytosolic protein turnover is a critical component of neurodegeneration and represents an emerging theme in cancer research. The long-term objective of this project is to determine the how protein catabolism is regulated by Wnt signaling, a pathway that is essential in development and is misregulated in disease. My preliminary data support the conceptually novel hypothesis that microautophagy is coordinated by canonical Wnt. Further, preliminary data suggests that cytosolic substrates targeted by microautophagy during Wnt signaling are modified by arginine methylation, a critical post-translation modification recently implicated in cytoplasmic signaling. Based on these findings, the central hypothesis of this proposal is that Wnt signaling stimulates arginine methylation to promote cytosolic protein degradation in endolysosomes through microautophagy (Fig. 1). I propose two specific aims: (1) elucidate the role of arginine methylation in Wnt signaling during cell growth and embryogenesis; and (2) determine how Wnt signaling and microautophagy regulate cytosolic protein degradation in endolysosomes. Aim 1 will examine the roles arginine methylation and Protein Arginine Methyl-Transferase 1 (PRMT1) in promoting Wnt signaling during cell growth and embryogenesis in human cells and Xenopus. Aim 1 is conceptually innovative as it examines a novel role for arginine methylation, a modification only recently discovered in cytoplasmic signaling, in the Wnt pathway and endolysosomes. Further, technical innovation through the use of chemical- genetic protein labeling strategies will offer the highest levels of sensitivity to examine the role of methylation in promoting GSK3 phosphorylation in live cells. Aim 2 will define the mechanism through which Wnt signaling regulates microautophagy to promote cytosolic proteolysis using genetic biochemical approaches to assess endolysosomal proteolysis in cultured human cells. I will apply a technically innovative in vivo biotin-protein labeling strategy to examine the novel concept that microautophagy targets specific cytosolic proteins during Wnt signaling. The proposed research is significant because elucidating the regulation of cytosolic proteolytic pathways could contribute to the development of innovative therapeutic interventions for targeting Wnt signaling in cancer and may reveal key details into the mechanisms of protein degradation in disease. In sum, this proposal offers a novel approach to investigate an integrated view of the pathologic mechanisms of protein degradation regulated by extracellular signaling factors during development and tissue morphogenesis.