The Role of Molecular Chaperones in Extracellular Vesicles

分子伴侣在细胞外小泡中的作用概述在细胞中，蛋白质折叠和蛋白质降解受到几个分子伴侣家族的严格调控。伴侣通过与未折叠的蛋白质分子结合来保护所有细胞，以促进它们的正确折叠或移除。如果伴侣的保护措施不足，蛋白平衡不被维持，细胞功能障碍和死亡就会随之而来。*基本的伴侣机制在所有三个王国都是保守的。Hsp70/Hsc70(70-kDa热休克蛋白和同源蛋白)伴侣家族是最丰富和高度保守的伴侣蛋白家族之一，具有广泛的细胞功能，涉及大量的靶蛋白。凭借其结合和隔离各种底物蛋白未折叠区域的能力，Hsp70能够重新折叠并使其“客户蛋白”恢复到其功能正常的状态。Hsp70在这方面并不是单独发挥作用的。J蛋白通过指定目标客户蛋白作为Hsp70的分子开关。我们小组最近的工作证明，选择的J蛋白伴侣蛋白是以细胞外小泡的形式从细胞中输出的。人们普遍认为J蛋白伴侣介导的折叠发生在细胞内，我们的观察是出乎意料的。J蛋白在细胞外小泡中的生物学功能是一个悬而未决的问题。在这个方案中，我们将进行研究，以辨别J蛋白在释放过程中的作用。*我们将从两个特定目标评估细胞外小泡J蛋白。具体目标1.J蛋白如何控制细胞外小泡的脂质含量？*在目标1中，我们将阐明哪些J蛋白影响包装到细胞外小泡中的脂类，以及这种脂质输出背后的分子事件。具体目的2.确定分泌的J蛋白是否决定了细胞外小泡的蛋白质运输量。细胞内J蛋白通过指定目标客户蛋白、增加ATP水解率和确定客户蛋白是折叠还是降解来充当Hsp70的分子开关。在目标2中，我们将确定J蛋白是否也决定‘客户蛋白是否被包装’到胞外小泡中以供出口。令人惊讶的是，人们对细胞外小泡中的J蛋白知之甚少。这项提案的核心将解决J蛋白伴侣如何输出，更具体地说，J蛋白如何调节细胞外小泡的脂质和蛋白质含量。蛋白质平衡对于所有细胞的生存都是至关重要的。在细胞外运行以确保蛋白质构象和功能的稳定性和保真度的蛋白质平衡系统在很大程度上仍然是一个黑匣子。这里提出的实验将提供一个框架，以开始阐明J蛋白在细胞-细胞信号转导中的作用。

The Role of Molecular Chaperones in Extracellular Vesicles*******Overview. In cells, protein folding and protein degradation are tightly regulated by several families of molecular chaperones. Chaperones safeguard all cells by binding to unfolded protein molecules to facilitate either their correct folding or removal. If the chaperone safeguard falls short' and proteostasis is not maintained, cell dysfunction and death ensues.*******The basic chaperone machinery is conserved throughout all three kingdoms. The Hsp70/Hsc70 (70-kDa heat shock protein and cognate protein) chaperone family constitutes one of the most abundant and highly conserved chaperone families and has a broad range of cellular roles involving a large number of target proteins. By virtue of their ability to bind and sequester unfolded regions of a variety of substrate proteins, Hsp70s are able to re-fold and return their “client proteins” to their functionally competent states. Hsp70s don't function alone in this capacity. J proteins serve as molecular switches for Hsp70s by specifying the client proteins to be targeted.*******Recent work from our group has documented that select J protein chaperones are exported from cells in extracellular vesicles. It has been widely assumed that J protein chaperone-mediated folding occurs inside cells and our observation is unexpected. The biological function of J proteins in extracellular vesicles is an open question. In this proposal, we will perform studies to discern the role of J proteins in the release process.*******We will evaluate extracellular vesicle J proteins in two specific aims.**********Specific Aim 1. How do J proteins control the lipid content of extracellular vesicles?****In aim 1, we will shed light on which J proteins influence the lipids packaged into extracellular vesicles and the molecular events underlying this lipid export.**********Specific Aim 2. To determine if secreted J proteins decide' the protein cargo of extracellular vesicles.****Intracellularly J proteins serve as molecular switches for Hsp70s by specifying the client proteins to be targeted, increasing ATP hydrolysis and determining whether client proteins are folded or degraded. In aim 2 we will establish if J proteins also decide' whether client proteins are packed' into extracellular vesicles for export.**********Significance. Surprisingly little is known about J proteins in extracellular vesicles. The core of this proposal will address how J protein chaperones are exported and more specifically how J proteins regulate lipid and protein content of extracellular vesicles. Proteostasis is essential for the survival of all cells. The proteostasis system that operates outside the cell to ensure the stability and fidelity of protein conformation and function still remains for the most part a black box'. The experiments proposed here will provide a framework to begin to elucidate the role of J proteins in cell-cell signaling.***