Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Waldeck-Weiermair, M.
Identification of new Ca2+-regulated proteins in endothelial cell signal transduction
[ Dissertation ] Medical University of Graz; 2008. pp.135
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- Autor*innen der Med Uni Graz:
- Waldeck-Weiermair Markus
- Betreuer*innen:
- Graier Wolfgang
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- Abstract:
- Twofold positive charged calcium ions (Ca2+) serve as ubiquitous second messengers for communication within cells. Depending on the appearance of different spatio-temporal Ca2+ signals, Ca2+ is able to activate or inhibit various Ca2+ dependent proteins. Some of these Ca2+-sensitive protein molecules work as so called scaffold proteins that act as platforms for interaction proteins. Thereby Ca2+-regultated translocations may occur resulting in an amplification, activation or inhibition of distinct signaling pathways.In the present study, it was intended to identify new, yet unknown Ca2+ dependent scaffold proteins or their interaction partners in endothelial cells by the use of two different independent approaches. In the first proteomic approach protein extracts of various cell compartments were visualized using 2D-gel electrophoresis before or after a cell stimulation with histamine. The histamine-induced cellular Ca2+ elevation resulted in the translocations of Valosin containing protein from the nucleus to the cytosol and Peroxiredoxin 1 towards cellular membranes. Further experiments are necessary to understand these still unknown Ca2+ dependent effects.In the second bioinformatic approach Gaf1, -SNAP associated factor 1, was identified to serve as a putative Ca2+ regulated scaffold protein. Using several biochemical strategies such as protein overexpression, gene silencing via siRNA or mutagenesis, the functional role of Gaf1 was tested. Thereby it was observed that Gaf1 is involved in the regulation of Ca2+ entry form the plasma membrane into the mitochondria and further into the cytosol as well as into the endoplasmic reticulum (ER). The Ca2+ release from the ER and the direct Ca2+ refilling of this Ca2+ store was not under the control of Gaf1. These results indicate that Gaf1 is involved in the selective transfer of extracellular Ca2+ into the mitochondrial matrix during cell stimulation. Therfore Gaf1 may act as a linking protein between a plasma membrane Ca2+ channel and the mitochondria. Moreover further observations confirmed this hypothesis as the inhibition of mitochondrial motility by Ca2+ in single cells during histamine stimulation was found to be Gaf1 dependent and Gaf1-Citrine was mainly observed to be localized at sites of the plasma membrane.