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

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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.