Structural rearrangements in disease-related STIM1 proteins

Fine-tuned regulation of Ca2+ homeostasis is essential for various cellular processes in the human body. These processes include gene transcription, programmed cell death and the activation of immune cells. Ca2+ can enter the cell via specific ion channels, constituted of two major proteins: STIM1, embedded in the endoplasmic reticulum, and Orai1 in the plasma membrane. In the present project Ca2+-dependent, structural rearrangements in disease-related STIM1 Proteins my team and I characterize previously unknown, disease-related mutations within the STIM1 protein. Orai1 and STIM1 have been associated with several forms of cancer, like testicular and abdominal cancer and brain tumors, although their role in cancer genesis is still unclear. We examine several mutations within STIM1, derived from a cancer database of human cancer studies. Together with STIM1 mutations from patients with tubular aggregate myopathy, we examine their effect onto the Ca2+ homeostasis of the cells. By the use of different experimental methods, we aim at resolving how these mutations effect the genesis of the specific disease. In cooperation with Prof. Martin Pichler, Research Unit of Non-Coding RNA and Genome Editing of the Medical University in Graz, we analyse the impact of these mutations in gene-edited cancer cells. Furthermore, we cooperate with Prof. Rüdiger Ettrich, University of Nove Hrady, Tzech Republic, in terms of molecular dynamics simulations to gather information about protein structure and folding. This project will provide fundamental insight, how these mutations effect Ca2+-dependent processes in cancer and other disease-related cells. The knowledge on Ca2+ signalling pathways in cancer is essential to understand physiological and pathophysiological processes, from which the medical treatment of cancer patients can highly benefit.

Ca2+-dependent, structural rearrangements in disease-related STIM1 proteins Mag. Dr. Irene Frischauf Fine-tuned regulation of Ca2+ homeostasis is essential for various cellular processes in the human body. These processes include gene transcription, programmed cell death and the activation of immune cells. Ca2+ can enter the cell via specific ion channels, constituted of two major proteins: STIM1, embedded in the endoplasmic reticulum, and Orai1 in the plasma membrane. Those two proteins have also been shown to be involved in human diseases including severe combined immune deficiency, tubular aggregate myopathy, Stormorken syndrome and several forms of cancer. Within the present project, I characterized previously unknown, disease-related mutations within the STIM1 protein. I also examined mutations within STIM1, derived from a cancer database of human cancer studies and analysed their effect onto the Ca2+ homeostasis of the cells. By the use of different experimental methods, I was able to resolve how these mutations may affect the genesis of specific diseases via structural alterations within the STIM1 protein. STIM1 possesses two main functions: it senses the ER-Ca2+ concentration and directly binds to the Ca2+ channel Orai1, for its activation when Ca2+ recedes. Structural rearrangements of STIM1 are essential to initiate interactions of C-terminal domains and to unleash the binding site for Orai1 channels. However, the structural basis of this luminal STIM1 aggregation has so far been elusive. During the project duration, I was able to identify a novel STIM1-Orai1 gating interface, necessary for transmission of the signal between the two proteins. In addition, we could refine the steps of STIM1 activation on a molecular level. In detail, we showed that disease-related mutations cause structural unfolding and led to downstream activation of autophagic cellular processes. Based on molecular dynamics simulation results we targeted the residues that predominantly form STIM1 interactions and engineered single point mutations. Indeed, these STIM1 mutations interfered with oligomerization of STIM1 proteins in biochemical assays and in live-cell experiments. STIM1 mutants further significantly reduced Ca2+ entry via Orai1. Hence, our experiments directly visualized a dynamically formed luminal STIM1 dimer interface as a key domain in the activation process of Orai1 Ca2+ channels. This project provides fundamental insight, how mutations in STIM1 effect Ca2+-dependent processes in disease-related cells. The knowledge on Ca2+ signalling pathways is essential to understand physiological and pathophysiological processes, from which the medical treatment of patients can highly benefit.