Interaction between protein dissulfide isomerase-A1 (PDIA1) and RhoGDI (guanine nucleotide dissociation inhibitor)-a: new frontier in RhoGTPases redox regulation and cellular remodeling

Abstract

A fundamental problem in cellular physiology is understanding the control of cell and tissue remodeling, which involves fine adjustments and homeostasis equilibrium, together accounting for cell form and function in contexts of adhesion, migration and mechanoadaptation. Central pathways involved in the upstream control of these processes are unknown and may contribute to vascular disease. In this project, we propose a novel regulatory mechanism of these processes related to cell signaling involving redox chaperones from the protein disulfide isomerase family (specifically PDIA1), well studied by our group. Our hypothesis is that PDIA1, through its interaction with RhoGDIa (guanine nucleotide dissociation inhibitor), may act as an upstream regulator of cellular remodeling and mechanical homeostasis through the regulation of RhoGTPases. RhoGTPases (such as Rac1 and RhoA) are small G-proteins transducing signals at the interface of membrane/cytoskeleton/cell adhesion, able to essentially regulate cytoskeletal organization and cell migration, form and size. While there are indirect evidences supporting the PDIA1/RhoGDIa interaction, the molecular mechanisms and variables affecting such interaction are unknown. Our central aim is to investigate the molecular basis and intervening variables of a new model of RhoGDIa regulation by PDIA1 and the effects of such interaction in RhoGTPase dynamic activation and cell responses. The specific aims are: 1) to build an in silico model of the interaction between PDIA1 and RhoGDIa and test it in conditions including RhoA or Rac1 binding, as well as PDIA1 or RhoGDIa post-translational modifications; 2) to validate in vitro the interaction between recombinant PDIA1 and RhoGDIa using PDIA1 individual domain constructs, under different conditions, and to identify RhoGDIa mutants able to inhibit such interaction; 3) to investigate in vitro the effect of PDIA1 in the dissociation of RhoGTPases from RhoGDIa induced by ERM (ezrin/radixin/moesin) proteins; 4) to assess in vascular smooth muscle cells with or without PDIA1 overexpression the effect of transfecting wild-type or mutant (according to aims 1 and 2) RhoGDIa in the activity of RhoGTPases and cell force distribution. Elucidating such molecular pathways central to RhoGTPase function has the potential to expand the understanding of cell remodeling and pathophysiological mechanisms of vascular diseases. (AU)