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

整合素是细胞粘附受体，介导多种功能，如细胞锚定、力传递和迁移。它们是α-β异二聚体，其通过其头部结构域与细胞外基质配体结合，并通过其细胞外柄和跨膜结构域将它们连接到细胞内细胞骨架。通过经历从弯曲到伸长形式的巨大构象变化，整合素被激活并发挥其功能。构象变化是头部和柄部结构域围绕由α亚基铰链结构域形成的枢轴的全局运动。该铰链结构域，以及潜在的α亚基的茎内的calf 2结构域，含有基于半胱氨酸的巯基开关。这解释了在生理浓度下用过氧化氢氧化后整合素结合活性增强。我们原型表明，α 7 β 1整联蛋白（一种层粘连蛋白受体）铰链区内的巯基开关被可逆地氧化还原修饰，并参与整联蛋白相关细胞功能（如迁移）的氧化还原调节。因此，整合素介导的细胞功能依赖于细胞的氧化还原环境。在本项目中，我们将通过点突变来研究整合素α 7亚基calf 2结构域中氧化还原活性半胱氨酸的作用。此外，与SPP合作伙伴，氧化还原调节细胞外整合素结构域的硫氧还蛋白家族的氧化还原酶，将被确定为解开氧化还原机制。我们将确定整合素的构象变化和分子力传递如何依赖于铰链和calf 2结构域内的巯基开关（ES）通过使用重组整合素胞外域蛋白质化学相互作用测定，高分辨率电子显微镜和原子力显微镜。在细胞水平上，纤维肉瘤细胞，以及在一种新的方法中，黑色素瘤细胞将在内源性整联蛋白被CRISPR/Cas9技术敲除后，用不同的巯基开关缺失的α 7 β 1整联蛋白突变体转染。其他层粘连蛋白结合整联蛋白将被抑制抗体阻断。与SPP 1710配偶体一起，还将表征细胞的细胞外氧化还原修饰酶的库。在不同的氧化还原条件下，并在氧化还原修饰酶的存在下，转染的细胞系将揭示氧化还原调节的整合素功能的生物学后果的细胞扩散，粘附，和迁移在阻抗为基础的粘附/迁移试验，以及通过视频和荧光显微镜。粘附体的形成，整合素功能的另一个后果，将比较研究的2D-DiGE。在翻译方面，我们将采用黑色素瘤球体作为体外肿瘤模型，通过流式细胞术和生命荧光显微镜检查肿瘤核心内缺氧影响的氧化还原环境是否以及如何影响α 7 β 1依赖性黑色素瘤的迁移和传播。

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.