Molecular Basis of ILK/PINCH Function in Cell Adhesion

描述(申请人提供)：细胞与细胞外基质(ECM)的附着对各种生理和病理过程至关重要。这种相互作用(细胞黏附)主要由整合素介导，整合素是一组异源二聚体跨膜受体，通过它们的胞外区与ECM蛋白结合。当细胞外基质结合时，整合素聚集并将信号传递到细胞内，导致形成被称为焦点粘连(FA)的大型蛋白质复合体，将整合素细胞质尾巴(CT)连接到肌动蛋白细胞骨架。后一步，即FAs的形成及其与肌动蛋白的连接，促进了细胞的牢固黏附。此外，它还允许调节动态黏附过程，如细胞扩散和迁移。我们的长期目标是获得对FAs的详细分子理解，并阐明它们如何与肌动蛋白连接并在不同的黏附过程中进行调节。为此，我们一直专注于FAs的一个主要组成部分-整合素连接激酶(ILK)。ILK最初被发现是一种与整合素2结合的整合素连接蛋白，现已被确定为一种多功能蛋白，在整合素和肌动蛋白之间传递不同的机械和生化信号。ILK功能的一个关键初始步骤是它与Pinch的紧密绑定--一个含有LIM的适配器。这种相互作用不仅促进了ILK在整合素黏附部位的定位，而且还创造了一个稳定的平台，其中含有许多蛋白质来调节动态的FA组装和不同的信号通路。在过去的几年里，我们在建立ILK/Pinch网络的分子图谱方面取得了重大进展，并展示了它如何在各种细胞过程中以时空方式发挥作用。在与临床科学家的合作中，我们还表明，在衰竭的人类心脏中，ILK/Pinch复合体异常升高，表明它直接参与了心功能障碍。巧合的是，最近在小鼠身上的一项研究表明，G-肌动蛋白隔离肽胸腺素β-4(Tb4)可能通过调节ILK/Pinch介导的细胞迁移和存活来修复心脏损伤。虽然这导致了广泛的后续研究，并启动了一项基于TB4的治疗心脏损伤患者的阶段1临床试验，但其潜在的分子机制仍不清楚。在初步研究中，我们发现了一种新的ILK/PINCH介导的整合素-肌动蛋白连接，它可能对细胞迁移和生存至关重要。这种连锁似乎受Tb4的动态调节。在我们下一阶段的研究中，我们将使用多学科的结构/功能方法来积极研究这种联系及其由TB4调控。这些研究将为理解ILK/Pinch介导的细胞黏附提供一个新的范式。它们还将影响基于TB4的心脏疾病和可能其他疾病的治疗。公共卫生相关性：整合素连接激酶(ILK)和LIM-Only接头Pinch之间的异源复合体在细胞外基质和肌动蛋白细胞骨架之间的信息传递中发挥着核心作用。最近，这种复合体的调节失调与心脏病发作有关，它的调节由一种自然产生的人类多肽-胸腺素β-4来调节，已被证明在小鼠模型中发挥了治疗作用。我们的建议将阐明ILK/Pinch介导的ECM/肌动蛋白连接的分子基础以及TB4是如何调节它的，这可能有助于从根本上理解ILK/Pinch的功能，并对基于TB4的心脏病治疗产生影响。

DESCRIPTION (provided by applicant): The attachment of cells to extracellular matrix (ECM) is crucial for a variety of physiological and pathological processes. This interaction (cell adhesion) is mediated primarily by integrins, a group of heterodimeric transmembrane receptors that bind to ECM proteins via their extracellular domains. Upon ECM engagement, integrins cluster and transduce signals into intracellular compartment leading to the formation of large protein complexes called focal adhesions (FAs) that connect integrin cytoplasmic tails (CTs) to the actin cytoskeleton. This latter step, i.e., the formation of FAs and their linkage to actin, promotes firm cell adhesion. Furthermore, it allows regulation of dynamic adhesive processes such as cell spreading and migration. Our long term goal is to obtain a detailed molecular understanding of FAs and to elucidate how they are connected to actin and modulated during various adhesive processes. To this end, we have been focusing on a major component of FAs - integrin-linked kinase (ILK). Originally discovered as an integrin linking protein that binds to integrin 2 CTs, ILK has been established as a multifunctional protein that transmits diverse mechanical and biochemical signals between integrins and actin. A key initial step for ILK function is its tight binding to PINCH - a LIM- containing adaptor. This interaction not only promotes the localization of ILK to integrin adhesion sites but also creates a stable platform that harbors many proteins to regulate dynamic FA assembly and diverse signaling pathways. Over the past several years, we have made a major progress towards building a molecular landscape of the ILK/PINCH network and showed how it functions in a spatiotemporal manner in various cellular processes. In collaboration with clinical scientists, we have also shown that the ILK/PINCH complex is abnormally elevated in failing human hearts, suggesting its direct involvement in cardiac dysfunction. Coincidently, a recent study in mice has shown that a G-actin sequestering peptide, thymosin beta-4 (tb4), may repair cardiac damage by modulating the ILK/PINCH-mediated cell migration and survival. While this has led to widespread follow-up investigations and the launching of a tb4-based phase1A clinical trial on treating heart injury patients, the underlying molecular mechanism remains obscure. In preliminary investigation, we have discovered a novel ILK/PINCH-mediated integrin-actin linkage that may be crucial for cell migration and survival. This linkage appears to be dynamically regulated by tb4. In the next phase of our study, we will use multidisciplinary structural/functional approach to vigorously investigate this linkage and its regulation by tb4. The studies will lead to a new paradigm for understanding the ILK/PINCH-mediated cell adhesion. They will also impact on the tb4-based therapy of cardiac disorder and possibly other diseases. PUBLIC HEALTH RELEVANCE: The heterocomplex between integrin-linked kinase (ILK) and LIM-only adaptor PINCH plays a central role in transmitting information between extracellular matrix and actin cytoskeleton. Dysregulation of this complex has been recently linked to heart attack and its regulation by a naturally occurring human peptide, thymosin beta-4, has been shown to exert therapeutic effect in mouse models. Our proposal will elucidate the molecular basis of the ILK/PINCH-mediated ECM/actin linkage and how it is regulated by tb4, which may lead to fundamental understanding of the ILK/PINCH function and also impact on tb4-based therapy of heart disease.