Document Type

Senior Thesis

Publication Date

2011

Abstract

Cellular signaling is a complex system of communication that regulates cell function through a variety of molecular messengers. When cell survival is threatened by changes in environment or by malfunction of internal regulatory pathways, signals are initiated to restore homeostasis. Deficient protein processing within the endoplasmic reticulum (ER) causes an accumulation of misfolded proteins and stress signaling. The resulting stress signaling includes both adaptive signals (unfolded protein response) and in cases of severe ER stress apoptotic signals induced via the mitochondria. FKBP10 is a key ER luminal peptidy-prolyl isomerase (rotamase) that mediates protein folding. The production of chaperone proteins and foldases typically increases following the activation of ER stress signaling. However, within 6 -12 hours of ER stress induction, FKBP10 protein levels decrease dramatically. Cell fractionation studies revealed FKBP10 fragments to appear in the cytosol during ER stress. Imaging confirmed decreased ER localization following ER stress. Both calpain and the proteasome were shown to be active in our cells and required for the rapid proteolysis of FKBP10. These data lead us to a model of FKBP10 destruction in which FKBP10 or its cleavage product is retrotranslocated from the ER to the cytosol as it is destroyed. An examination of the structure of the FKBP10 protein revealed two calcium binding EFhand domains near the C-terminus. As the ER lumen is a critical site of cellular calcium storage and ER calcium stores are known to decrease with ER stress, we have chosen to examine the importance of these EF-hand domains in FKBP10 stability by mutagenizing the putative calcium binding amino acids in a recombinant FKBP10 clone. Mutant constructs have been generated and calcium binding dependence of FKBP10 has been analyzed. The destruction of FKBP10 is interesting because it may identify a unique cellular strategy for repairing/restoring ER function following ER stress.

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