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）中稳态和错误折叠蛋白应激的物理和分子基础。ER分子伴侣对分泌蛋白的正确折叠和质量控制对于细胞和生物体的生存力是必不可少的。不能正确折叠蛋白质导致蛋白质功能丧失，可以激活未折叠蛋白质反应（UPR），并刺激凋亡性死亡。在不同的环境条件下，是否有足够的分子伴侣可用于新生或错误折叠的蛋白质是理解分子伴侣功能的关键。稳态下的ER分子伴侣网络可以用冗余和过量的分子伴侣缓冲，或者网络可以在蛋白质折叠能力的边缘操作。内质网分子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刻的意义。一个以最大能力运行的系统几乎没有犯错的余地，它会有一个明确的压力阈值，并且很可能表现出双相极端。相反，压力和内稳态可以作为缓冲和冗余系统中的状态梯度存在。折叠需求中的小扰动可以被感知并做出响应，而不会扰乱全局折叠环境。我们推测，ER分子伴侣网络的复杂性和缓冲能力调节ER应激途径的激活和ER稳态的维持。伴侣网络的复杂性将取决于内腔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分子伴侣的折叠活动，但对ER分子伴侣如何在真实的时间内与细胞中的底物相遇并相互作用仍然知之甚少。我们将采用生物化学，药理学和定量单细胞荧光显微镜方法建立一个模型的ER伴侣网络与精致的时空分辨率。我们的生物物理系统的方法将定义在稳态和错误折叠的蛋白质压力的ER分子伴侣网络的层次结构。正确的分泌蛋白折叠和质量控制过程对细胞活力和人类健康至关重要。我们正在细胞水平上研究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.