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

整合素是细胞黏附受体，介导多种功能，如细胞锚定、力传递和迁移。它们是α-β异源二聚体，通过其头部结构域与细胞外基质配体结合，并通过其胞外柄和跨膜结构域与细胞内细胞骨架相连。通过经历从弯曲到拉长的戏剧性构象变化，整合素被激活并发挥其功能。构象变化是头部和茎结构域围绕由阿尔法亚单位铰链域形成的枢轴的全局移动。这个铰链结构域，以及潜在的阿尔法亚单位茎中的calf2-结构域，包含一个基于半胱氨酸的硫醇开关。这解释了在生理浓度的过氧化氢氧化时整合素结合活性增强的原因。我们典型地表明，层粘连蛋白受体α7β1整合素铰链区内的硫醇开关是可逆的氧化还原修饰的，并参与整合素相关细胞功能的氧化还原调节，如迁移。因此，整合素介导的细胞功能依赖于细胞的氧化还原环境。在这个项目中，我们将通过点突变来研究氧化还原活性半胱氨酸在整合素α7亚单位的calf2结构域中的作用。此外，在SPP-合作伙伴的合作下，硫氧还蛋白家族的氧化还原酶将被确定为氧化还原调节细胞外整合素结构域，以解开氧化还原机制。我们将通过在蛋白质-化学相互作用分析、高分辨电子显微镜和原子力显微镜中使用重组整合素胞外域来确定整合素的构象变化和分子力传递如何依赖于铰链和calf2结构域中的硫醇开关(ES)。在细胞水平上，纤维肉瘤细胞和黑色素瘤细胞将在内源性整合素被CRISPR/Cas9技术敲除后，被不同的硫醇开关缺失的α7β1整合素突变体导入。其他与层粘连蛋白结合的整合素将被抑制抗体阻断。与SPP1710合作伙伴一起，这些细胞还将被表征其细胞外氧化还原修饰酶的酶系。在不同的氧化还原条件下，在氧化还原修饰酶的存在下，通过基于阻抗的黏附/迁移分析以及视频和荧光显微镜，转基因细胞株将揭示氧化还原调节的整合素功能对细胞扩散、黏附和迁移的生物学后果。粘附性形成是整合素作用的另一个结果，将用2D-DGE进行比较研究。我们将使用黑色素瘤球体作为体外肿瘤模型，通过流式细胞仪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依赖于α7β1的黑色素瘤的迁移和扩散。

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.