Thiol-based switches in integrins: molecular analysis and redox-regulatory implications in cell-matrix interaction and migration.

点击翻译按钮获取中文摘要

Integrins are cell adhesion receptors and mediate various functions, such as cell anchorage, force transmission, and migration. They are alpha-beta heterodimers, which bind to extracellular matrix ligands via their head domains and connect them via their extracellular stalks and transmembrane domains to the intracellular cytoskeleton. By undergoing dramatic conformational changes from a bent to an elongated form, integrins are activated and exert their functions. The conformational change is a global movement of the head and stalk domains around a pivot formed by the alpha subunit hinge domain. This hinge domain, and potentially also the calf2-domain within the stalk of the alpha subunit, contains a cysteine-based thiol switch. This explains the enhanced integrin binding activity upon oxidation with hydrogen peroxide at physiological concentrations. We prototypically showed that the thiol switch within the hinge region of alpha7 beta1 integrin, a laminin receptor, is reversibly redox-modified and involved in redox-regulation of integrin-related cell functions, such as migration. Therefore, integrin-mediated cellular functions depend on the redox environment of cells.In this project, we will examine the role of redox-active cysteines within the calf2-domain of the integrin alpha7 subunit by point mutations. Moreover, in cooperation with SPP-partners, redox enzymes of the thioredoxin family, which redox-regulate the extracellular integrin domains, will be identified to unravel the redox mechanism. We will determine how conformational changes and molecular force transmission of the integrin depend on the thiol switch(es) within the hinge and calf2-domain by using recombinant integrin ectodomains in protein-chemical interaction assays, high resolution electron microscopy and atomic force microscopy. At the cellular level, fibrosarcoma cells and, in a novel approach, melanoma cells will be transfected with different thiol switch-deleted alpha7 beta1 integrin mutants, after the endogenous integrin has been knocked-out by CRISPR/Cas9-technology. Other laminin-binding integrins will be blocked by inhibiting antibodies. Together with SPP1710 partners, the cells will also be characterized for their repertoire of extracellular redox-modifying enzymes. Under different redox conditions and in the presence of redox-modifying enzymes, the transfected cell lines will reveal the biological consequences of the redox-regulated integrin function on cell spreading, adhesion, and migration in impedance-based adhesion/migration assays as well as by video and fluorescence microscopy. Adhesome formation, another consequence of integrin function, will comparatively be studied by 2D-DiGE. Translationally, we will employ melanoma spheroids as in vitro-tumor models to examine by flow cytometry and life fluorescence microscopy whether and how the hypoxia-affected redox milieu within a tumor core influences alpha7 beta1-dependent melanoma migration and dissemination.