Protein folding and stability in the stress sensing machinery of stromal interaction molecules.

所有具有离散核的动物细胞都使用钙（Ca2+）来发出信号，这是它们生命周期中不可或缺的一部分，从细胞应激反应到细胞分裂和自杀。这些真核细胞使用由蛋白质分子组成的Ca2+信号工具箱，这些蛋白质分子专门针对细胞的环境。该工具包的许多组成部分依赖于Ca2+水平的巨大差异来介导特定的细胞信号。例如，基质相互作用分子（stim）位于一个特殊的细胞室，内质网（ER），含有高水平的Ca2+，需要处理基因组中编码的许多蛋白质机制；此外，stim对内质网Ca2+的消耗做出反应，通过改变形状并移动到细胞周围附近，在那里与另一种Ca2+工具箱成分（Orai通道蛋白）发生相互作用。这种相互作用导致位于外膜上的Orai通道打开，允许Ca2+以最小的能量消耗从高外部浓度移动到低内部水平。由此产生的细胞内Ca2+的升高是触发广泛细胞反应的信号；此外，这种区室化Ca2+水平的特定系列变化被称为存储操作Ca2+进入（SOCE），因为它依赖于内质网存储的Ca2+水平。**本研究项目旨在研究蛔虫和果蝇等相对简单生物的stim如何感知内质钙+水平的变化，以及这些机制与脊椎动物等更进化的生物的比较。此外，该研究还提出要研究为什么高等动物使用两种不同的STIM分子来感知内质网Ca2+水平的变化，而低等生物只需要一种。为了以一种特定的方式回答这些问题，我们建议表达和分离高纯度的蛋白质，这些蛋白质对应于负责ER Ca2+感应的STIM机制。此外，我们计划表征各自结构特征承受化学和温度应力的能力，以及Ca2+水平如何改变耐受性。同样，我们的目标是评估内质网蛋白中经常发生的化学修饰对这些结构特征的影响，以及STIM中与Ca2+传感机制密切相关的物种特异性区域对这些特征的作用。**我们预计STIM分子内最小的Ca2+感应机制表现出由蛋白质序列的适应性变化介导的结构和相互作用差异；此外，我们认为每个STIM分子都利用最小Ca2+传感机制之外的高度可变区域以及自然化学修饰来微调细胞应激的结构反应，包括Ca2+水平、温度和活性氧的变化，以生物体和STIM亚型特异性的方式。该研究计划将提供深入了解与高等真核生物相比，低等真核生物Ca2+信号工具箱的特定感觉功能至关重要的特征，这些信息目前在广泛的Ca2+信号研究领域缺乏。此外，这项工作将为自然发生的化学修饰在介导stim结构耐久性方面的作用提供新的数据，这与其他er驻留蛋白有关。重要的是，这些数据将为开发新的研究工具提供基础，设计用于感知Ca2+，温度和活性氧的变化。最后，这项研究将通过为本科生、研究生和博士后提供多学科培训使加拿大受益，这些培训将为未来在学术界和/或工业界的职业发展提供广泛的技能。

All animal cells which have a discrete nucleus use calcium (Ca2+) to signal processes that are an integral part of their lifecycle, ranging from cell stress responses to cell division and suicide. These eukaryotic cells use a Ca2+ signaling toolkit comprised of protein molecules specifically tailored to the environment of the cell. Many components of the toolkit rely on vast differences in Ca2+ levels to mediate a specific cell signal. For example, stromal interaction molecules (STIMs) are located in a specialized cellular compartment, the endoplasmic reticulum (ER), containing high Ca2+ levels needed to process many of the protein machinery encoded in genomes; further, STIMs respond to the depletion of Ca2+ from the ER by changing shape and moving to near the periphery of the cell where interactions occur with another Ca2+ toolkit component, the Orai channel proteins. This interaction causes Orai channels located on the outer membrane to open, allowing Ca2+ to move from the high outside concentration to the low inside levels with minimal energy expenditure. The resultant elevation in intracellular Ca2+ is the signal which triggers the wide ranging cellular responses; further, this specific series of changes in compartmentalized Ca2+ levels is called store operated Ca2+ entry (SOCE) since it is dependent on ER stored Ca2+ levels. **This research program aims to study how STIMs from relatively simple organisms such as the roundworm and the fruit fly sense changes in ER Ca2+ levels and how these mechanisms compare to more evolved organisms such as vertebrates. Additionally, the work proposes to investigate why higher order animals use two different STIM molecules to sense changes in ER Ca2+ levels, while lower organisms require only one. In order to answer these questions in a specific manner, we propose to express and isolate highly pure proteins corresponding to the STIM machinery responsible for ER Ca2+ sensing. Further, we plan to characterize the ability of the respective structural features to endure chemical and temperature stresses as well as a how Ca2+ levels alter the tolerances. Similarly, we aim to assess the effects that chemical modifications often occurring in ER proteins have on these structural characteristics and the role that species-specific regions of STIM closely apposed to the Ca2+ sensing machinery have on these features.**We anticipate that the minimal Ca2+ sensing machinery within STIM molecules exhibit structural and interaction differences mediated by adaptive variations in the protein sequences; moreover, we believe that each STIM molecule employs the highly variable regions outside the minimal Ca2+ sensing machinery as well as natural chemical modifications to fine tune the structural responses to cellular stresses that include changes in Ca2+ levels, temperature and reactive oxygen species, in an organism- and STIM subtype-specific manner. This research program will provide insight into the features vital for dictating specific sensory functions of the Ca2+ signaling toolkit in lower compared to higher eukaryotes, information which is currently lacking in the broad Ca2+ signaling research field. Further, the work will provide new data on the roles that naturally occurring chemical modifications have on mediating the structural durability of STIMs, relatable to other ER-resident proteins. Importantly, this data will provide bases for the development of new research tools, engineered to sense changes in Ca2+, temperature and reactive oxygen species. Finally, the research will benefit Canada by providing multidisciplinary training for undergraduate, graduate and postdoctoral fellows that will develop a broad skill set for future careers in academia and/or industry.