ER Chaperone Availability in Cells During Homeostasis and Misfolded Protein Stres

描述（由申请人提供）：我们的目标是阐明内质网（ER）内稳态和错误折叠蛋白应激的物理和分子基础。内质网伴侣对分泌蛋白的正确折叠和质量控制对细胞和生物体的生存能力至关重要。不能正确折叠蛋白质导致蛋白质功能丧失，可激活未折叠蛋白反应（UPR），并刺激细胞凋亡。在不同的环境条件下，是否有足够的伴侣蛋白用于新生或错误折叠的蛋白质是理解伴侣蛋白功能的关键。稳定状态下的ER伴侣网络可以通过冗余和伴侣的过剩来缓冲，也可以在蛋白质折叠能力的边缘运行。内质网伴侣网络的性质对内质网如何感知和应对错误折叠的蛋白质应激具有深远的影响。一个满负荷运转的系统几乎没有犯错的余地，将有一个明确的压力阈值，并可能表现出两相极端。相反，在缓冲和冗余系统中，应力和内稳态可以作为状态梯度存在。折叠要求中的微小扰动可以被感知和响应，而不会扰乱整个折叠环境。我们假设内质网伴侣网络的复杂性和缓冲能力调节内质网应激途径的激活和内质网稳态的维持。伴侣网络的复杂性将取决于肠道ER伴侣的分布、动态、组织和占用。虽然遗传学和试管生物化学已经帮助定义了伴侣蛋白的折叠活动，但人们对ER伴侣蛋白如何在细胞中实时遇到并与其底物相互作用仍然知之甚少。我们将采用生化、药理学和定量单细胞荧光显微镜方法建立具有精细时空分辨率的内质网伴侣网络模型。我们的生物物理系统方法将定义内质网伴侣网络在稳态和错误折叠蛋白质应激期间的层次结构。正确的分泌蛋白折叠和质量控制过程对细胞活力和人体健康至关重要。我们正在研究，在细胞水平上，维持和调节分泌蛋白折叠环境的机制。

DESCRIPTION (provided by applicant): Our goal is to elucidate the physical and molecular basis of homeostasis and misfolded protein stress in the endoplasmic reticulum (ER). Correct folding and quality control of secretory proteins by ER chaperones is essential for the viability of cells and organisms. Failure to correctly fold proteins results in loss of protein function, can activate the Unfolded Protein Response (UPR), and stimulate apoptotic death. Whether chaperones are sufficiently available for nascent or misfolded proteins under different environmental conditions is critical to understanding chaperone function. The ER chaperone network at steady state could be buffered with redundancy and an excess of chaperones or the network could be operating at the edge of protein folding capacity. The nature of the ER chaperone network has deep implications for how the ER senses and copes with misfolded protein stress. A system operating at capacity leaves little room for error, will have an explicit threshold for stress, and is likely to exhibit biphasic extremes. In contrast, stress and homeostasis could exist as a gradient of states in a buffered and redundant system. Small perturbations in folding requirements could be sensed and responded to, without upsetting the global folding environment. We hypothesize that the complexity and buffering capacity of the ER chaperone network regulates activation of ER stress pathways and maintenance of ER homeostasis. Chaperone network complexity will depend on the distribution, dynamics, organization, and occupancy of lumenal ER chaperones. While genetics and test-tube biochemistry have helped define the folding activities of chaperones, it remains poorly understood how ER chaperones encounter and interact with their substrates in cells in real time. We will employ biochemical, pharmacologic, and quantitative single cell fluorescence microscopy methods to establish a model of the ER chaperone network with exquisite spatio-temporal resolution. Our biophysical systems approach will define the hierarchy of the ER chaperone network during homeostasis and misfolded protein stress. PUBLIC HEALTH RELEVANCE The processes of correct secretory protein folding and quality control are vital for cell viability and human health. We are studying, at the cellular level, the mechanisms that maintain and regulate the secretory protein folding environment.