Cellular radiosensitivity and endocytosis: The role of PINCH1 and caveolin-1

在正常细胞和癌细胞中，内吞作用在各种细胞功能如存活、增殖、代谢、迁移和信号转导中起着基本作用。小窝主要由小窝蛋白和小窝蛋白形成，介导受体非依赖性内吞作用，并且基本上参与生化信号沿着胞内和胞间信号通路的传递。这些行动从根本上参与各种细胞功能的调节，包括对不同类型压力的快速反应。我们以前的研究表明，小窝蛋白-1在恶性肿瘤中过表达，作为一个关键组成部分，适当的整合素信号转导，并作为一个有前途的癌症靶点，因为它的下调敏感人类癌细胞的放射化疗。由于小窝蛋白-1与细胞骨架蛋白和粘着斑蛋白-如衔接蛋白特别有趣的新的富含半胱氨酸组氨酸的1（PINCH 1），这本身是放射性和化学抗性的关键决定因素-聚结和共沉淀-我们在正在进行的拨款提案中研究了这种相互作用。我们的初步数据表明，（i）两个小窝相关蛋白，小窝蛋白-1和cavin 1的表达水平，是PINCH 1依赖性的和（ii）PINCH 1通过与小窝蛋白-1和cavin 1的相互作用，在X射线照射后严格调节小窝运动。由于缺乏有关辐射调节的内吞作用的信息，我们在此，在此后续的资助提案中，描述了进一步的深入研究：（i）确定X射线照射后PINCH 1-cavin 1和PINCH 1-caveolin-1的相互作用动力学，（ii）鉴定PINCH 1，cavin 1和caveolin-1上这种相互作用的物理结合位点，（iii）揭示肌动蛋白和微管蛋白网络的PINCH 1依赖性组织，以及它们在X射线照射后对cavin 1和caveolin-1的运动性有多重要，以及（iv）确定放射性应激下PINCH 1对cavin 1和caveolin-1的分子募集机制。拟议的后续项目解决了这些问题，通过小鼠胚胎PINCH 1-/-成纤维细胞与PINCH 1的不同变体重组，加上与野生型和突变形式的小窝蛋白-1和cavin 1共转染。通过可视化它们之间的相互作用动力学，并确定PINCH 1，cavin 1和caveolin-1之间的物理结合位点，该后续项目旨在深入了解小窝的形成和功能是如何调节的，这些蛋白质如何有助于抵抗过程和相关的inter- /细胞内信号传导网络，其可用于通过靶向小窝相关内吞作用的特定组分来增强放射化学敏感性。

In normal and cancer cells, endocytosis plays a fundamental role in various cell functions such as survival, proliferation, metabolism, migration and signal transduction. Caveolae, predominantly formed by caveolins and cavins, mediate receptor-independent endocytosis and are essentially involved in the transfer of biochemical cues along inter– and intracellular signaling pathways. These actions fundamentally participate in the regulation of various cell functions, including the rapid responses to different types of stress. Our previous studies show that caveolin-1 is over-expressed in malignant tumors, serves as a critical component for proper integrin signaling and acts as a promising cancer target as its downregulation sensitizes human cancer cells to radiochemotherapy. As caveolin-1 coalesces and co-precipitates with cytoskeletal proteins and focal adhesion proteins - like the adapter protein particularly interesting new cysteine-histidine-rich 1 (PINCH1), which, itself, is a key determinant of radio- and chemoresistance - we investigated this interaction in the context of endocytosis in the ongoing grant proposal. Our preliminary data show that (i) the expression levels of both caveolae associated proteins, caveolin-1 and cavin1, are PINCH1-dependent and (ii) PINCH1 critically regulates caveolae motility upon X–ray irradiation through interactions with caveolin–1 and cavin1. Given the paucity of information about radiation-modulated endocytosis, we here, in this follow-up grant proposal, describe further in-depth investigations on (i) determining the interaction dynamics of PINCH1–cavin1 and PINCH1–caveolin–1 upon X–ray irradiation, (ii) the identification of the physical binding sites for this interaction on PINCH1, cavin1 and caveolin–1, (iii) uncovering the PINCH1–dependent organization of actin and tubulin networks and how critical these are for the motility of cavin1 and caveolin–1 upon X–ray irradiation, and (iv) identifying the molecular recruitment mechanisms of cavin1 and caveolin–1 by PINCH1 under radiogenic stress. The proposed follow-up project addresses these questions by means of mouse embryonic PINCH1-/- fibroblasts reconstituted with different variants of PINCH1 plus co-transfection with wildtype and mutated forms of caveolin-1 and cavin1. Through the visualization of their interaction dynamics and the identification of the physical binding sites between PINCH1, cavin1 and caveolin–1, this follow–up project aims to provide in–depth understanding of how caveolae formation and function are regulated, how these proteins contribute to resistance processes and associated inter– / intracellular signaling networks that may be exploitable for enhancing radiochemosensitivity through targeting of particular components of caveolae associated endocytosis.