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

所有具有离散细胞核的动物细胞都使用钙(Ca~(2+))来传递信号，这些过程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。这些真核细胞使用钙信号工具包，该工具包由专为细胞环境量身定做的蛋白质分子组成。该工具包的许多组件依赖于钙水平的巨大差异来调节特定的细胞信号。例如，基质相互作用分子(STIMs)位于一个特殊的细胞室，内质网(ER)，含有处理基因组中编码的许多蛋白质机制所需的高钙水平；此外，STIMs通过改变形状并移动到靠近细胞外围的位置来响应内质网中钙的耗尽，在那里发生与另一个钙离子工具包组件Orai通道蛋白的相互作用。这种相互作用导致位于外膜上的Orai通道开放，允许钙离子以最小的能量消耗从高的外部浓度移动到低的内部水平。由此产生的细胞内钙的升高是触发广泛的细胞反应的信号；此外，这种特定的分区化钙水平的变化被称为存储操作的钙进入(SOCE)，因为它依赖于内质网存储的钙水平。**这项研究计划旨在研究相对简单的生物(如蛔虫和果蝇)的刺激物如何感知ER钙水平的变化，以及这些机制与进化程度较高的生物(如脊椎动物)相比如何。此外，这项工作还建议调查为什么高等动物使用两种不同的STIM分子来感知ER钙水平的变化，而低等生物只需要一个。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与负责ER钙传感的STIM机制相对应的高纯度蛋白质。此外，我们计划表征各自结构特征承受化学和温度压力的能力，以及钙水平如何改变耐受性。同样，我们的目标是评估内质网蛋白质中经常发生的化学修饰对这些结构特征的影响，以及与钙离子感应机制密切相对的STIM物种特异性区域对这些特征的作用。**我们预计，STIM分子中最小的钙离子感应机制表现出由蛋白质序列中的适应性变异介导的结构和相互作用的差异；此外，我们相信每个STIM分子都利用最小的钙离子感应机制外的高度可变区域以及自然的化学修饰来微调对细胞应激的结构反应，包括钙水平、温度和活性氧物种的变化，以一种生物体和STIM亚型特有的方式。这项研究计划将深入了解与高等真核生物相比，低等真核生物中钙信号工具包的特定感觉功能至关重要的特征，这是目前广泛的钙信号研究领域所缺乏的信息。此外，这项工作将提供关于自然发生的化学修饰在调节STIM结构持久性方面所起作用的新数据，这与其他内质网驻留蛋白有关。重要的是，这些数据将为开发新的研究工具提供基础，这些工具旨在感知钙离子、温度和活性氧物种的变化。最后，这项研究将通过为本科生、研究生和博士后研究员提供多学科培训而使加拿大受益，这些培训将为未来在学术界和/或行业的职业发展广泛的技能集。

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.