Elucidation of STIM2 domains for CRAC channel activation

The aim and major goal of the presented application is to shed light on cytosolic STIM2 domains, which are essential in regulatory / modulatory processes of the CRAC channel system. Several mutants of STIM and Orai proteins - the key-players composing the CRAC channel system result in gain of function or loss of function. These mutants result in a variety of diseases and symptoms comprising susceptibility to infections, autoimmunity, the Stormorken Syndrome, Tubular aggregate myopathy (TAM) and York platelet syndrome (YPS)_ENREF_2. Most research data are available on STIM1 and Orai1, however, STIM2 has not been analyzed in equivalent detail. STIM2 is part of the CRAC channel complex and is of major importance for a cells live. In case of STIM2 malfunction, the CRAC channel system is affected. Therefore, it is essential to increase knowledge on STIM2 and its mechanistical activation steps by exploring STIM2 domains and their function(s) in detail. Specifically, human cells (HEK293) expressing the proteins of interest will be analyzed using biophysical methods like patch- clamp and confocal FRET microscopy, as well as molecular biology and biochemistry. Insights in structure function of STIM2 activation will allow to describe the wild-type system as well as pathophysiological relevant STIM2 mutants.

Elucidation of regulatory STIM2 domains for CRAC channel activation Mag. Dr. Marc Fahrner Stromal Interaction Molecule (STIM) denotes an intracellular protein family consisting of STIM1 and STIM2, whereby both STIM1 and STIM2 exist in different cell type-specific isoforms. The general structure of these proteins is identical for all isoforms. STIM proteins contain an ER luminal N-terminal part, which serves as a calcium sensor. (The endoplasmic reticulum (ER) is used as an intracellular calcium store, whereby a decrease in ER luminal calcium concentration activates the STIM protein). Following the ER luminal part, STIM contains a transmembrane domain followed by a large cytosolic C-terminal portion. This contains so-called coiled coil domains, which have been well described functionally and structurally in recent years, both in the quiescent and active STIM protein forms. Activated STIM changes its conformation and cellular localization, moving close to the cell periphery, and thus is able to couple and activate the calcium-selective channel Orai, which is located in the cell membrane. Consequently, calcium flows along the electrochemical gradient from the extracellular space into the cytosol of the cell, where it acts as a second messenger. This results in an intracellular signaling cascade leading to a cell type-specific response to the initial signal, which accounts for ER calcium store depletion. Overall, STIM and Orai are considered to be the key proteins that are primarily responsible for the store-dependent calcium influx into a cell. The system is generally described as the "Store Operated Calcium Entry" (SOCE) system. Since this SOCE system is realized in the majority of human cells, STIM and Orai mutants result in various diseases, depending on whether the genetically encoded mutation in STIM or Orai has an activating effect (gain of function) or an inactivating effect (loss of function). The diseases include Severe Combined Immunodeficiency (SCID), Stormorken Syndrome, Tubular Aggregate Myopathy, York Platelet Syndrome and more. The clinical progression can range from mild to lethal. In general, it is advantageous to understand the STIM activation/inactivation mechanism at the molecular level. This has been studied in the context of the physiological "wild type" as well as the pathophysiological system in this project. In particular, in addition to wild type STIM1, the STIM1 R304W mutant inducing Stormorken Syndrome was in focus of some of our project-relevant publications. STIM research at the molecular level increases the chance of therapeutic approaches with respect to molecular medicine. In this project, we have intensively investigated the structure-function relationship of STIM1 at the molecular level and were able to present the results in 9 scientific publications. Milestones achieved in STIM1 are to be applied to STIM2 in order to characterize this STIM variant at the molecular level as well.