Cell adhesion mediated by LINKIN

LINKIN 介导的细胞粘附

• 批准号: 9354504
• 负责人: Tsui-Fen Chou
• 金额: $ 37.35万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-20 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9354504
• 关键词: ATP phosphohydrolase; Adhesions; Adhesives; Alpha Cell; Amino Acid Substitution; Amino Acids; Animal Model; Area; Binding; Biological Assay; Caenorhabditis elegans; Cell Adhesion; Cell Adhesion Molecules; Cell Line; Cell-Matrix Junction; Cells; Cleaved cell; Clinical Research; Complex; Coupled; Cytoplasm; Cytoskeleton; Development; Disease; Employee Strikes; Epithelial Cells; Evolution; Extracellular Domain; Fertilization; Gene Dosage; Genes; Genetic; Genetic Screening; Genetic study; Genomics; Health; Homeostasis; Homodimerization; Human; Human Genetics; Immunomodulators; Immunoprecipitation; Integral Membrane Protein; Investigation; Knowledge; Mass Spectrum Analysis; Mediating; Membrane; Microtubules; Molecular Genetics; Mutate; Mutation; Organ; Organism; Pathway interactions; Pattern; Phenotype; Phylogenetic Analysis; Physiological; Proteins; Proteomics; RNA Interference; Reporting; Role; Signal Pathway; Stable Isotope Labeling; Structure; Systems Biology; T-Lymphocyte; Testing; Tissues; Transgenic Organisms; Vas deferens structure; Western Blotting; Work; alpha Tubulin; base; experimental study; extracellular; improved; mutant; organ growth; transcriptomics

Project Summary Cell adhesion is crucial aspect of development, fertilization, tissue homeostasis, protection from and interaction with other organisms. Many adhesion proteins have been intensively studied for decades and it was an open question whether we have sufficient knowledge of adhesion, given the large number of interacting proteins and interactions involved in adhesion. LINKIN is a conserved, transmembrane protein that we found to be involved in cell adhesion, suggesting that by studying LINKIN and its interacting proteins we can define a new cellular pathway involved in cell adhesion. The striking conservation of LINKIN sequence and gene copy number across eukaryotic evolution suggests a critical role in development. We discovered LINKIN's role in cell adhesion in the context of C. elegans development, specifically the attachment of the migrating linker cell to the vas deferens, which provides a powerful assay for its structure, function and interactions. We identified interacting proteins in human cells and provided evidence for similar molecular interactions in both organisms. We identified the RUVBL1 and RUVBL2 AAA-ATPases, and α-tubulin as cytoplasmic interactors of LINKIN, suggesting a role for LINKIN in regulating microtubule dynamics, but further investigation is required to connect LINKIN to other interactors and signaling pathways. We propose to further characterize LINKIN's role in cell adhesion in order to connect this new adhesion molecule to other known (or unknown) cellular pathways. A major limitation of human genetic, genomic and systems biology approach is our understanding of the function of many genes. By discovering a new pathway, we will increase the likelihood that genomic, clinical and genetic studies will be interpretable. We will analyze the function and interactors of both the extracellular and intracellular domains of LINKIN. Using evolutionary conservation as a guide we will mutate conserved residues in C. elegans LINKIN and test their function in transgenic worms. We will use human LINKIN proteins with cognate mutations in cell lines for immunoprecipitation and quantitative mass spectrometry proteomics to identify physiologically relevant interacting proteins. We will test the function of interacting proteins identified by the proteomics in C. elegans and cell lines. We will also use C. elegans genetics to screen prioritized candidates for a role in adhesion using the linker cell attachment assay. Candidates will be prioritized by expression in the linker cell, for which we have a deep transcriptomic profile, predictions of encoding transmembrane or secreted proteins, phylogenetic co-occurrence as well as network predictions of interactions based on orthologous proteins in other intensively-studied organisms. In this way, we will identify the portions of LINKIN that are crucial to its function in adhesion and physiologically-relevant interacting proteins both on other cells and same cell that likely mediate adhesion, and in the cytoplasm that implement the adhesive and cytoskeletal interactions. This validated set of functionally interacting proteins will define a new pathway of cellular adhesion and inform a variety of human genetic studies in normal development and disease states.

项目摘要 细胞粘附是发育，受精，组织稳态的关键方面，免受保护和相互作用 与其他生物体。数十年来，许多粘附蛋白已经深入研究了，这是一个开放的 考虑到大量相互作用的蛋白质和 广告中涉及的互动。 Linkin是一种保守的跨膜蛋白，我们发现它涉及 在细胞粘附中，表明通过研究Linkin及其相互作用蛋白，我们可以定义一个新的细胞 细胞粘附涉及的途径。 Linkin序列和基因拷贝数的惊人保守性 整个真核进化表明在发展中起关键作用。我们发现了Linkin在细胞中的作用 在秀丽隐杆线虫发育的背景下，特别是迁移接头单元的附着 VAS延迟，为其结构，功能和相互作用提供了强大的测定法。我们确定了 在人类细胞中相互作用的蛋白质，并提供了两种生物体中类似分子相互作用的证据。 我们将RUVBL1和RUVBL2 AAA-ATP酶以及α-微管蛋白确定为Linkin的细胞质相互作用者， 提出Linkin在控制微管动力学中的作用，但需要进一步研究以连接 链接到其他交互器和信号通路。我们建议进一步表征Linkin在细胞中的作用 粘附是为了将这种新的粘附分子连接到其他已知（或未知）的细胞途径。一个 人类遗传，基因组和系统生物学方法的主要局限性是我们对功能的理解 许多基因。通过发现新途径，我们将增加基因组，临床和 遗传研究将是可解释的。我们将分析细胞外和 Linkin的细胞内结构域。使用进化保护作为指南，我们将突变保守的残差 在秀丽隐杆线虫中，并测试了它们在转基因蠕虫中的功能。我们将使用人类链接蛋白与 用于免疫沉淀和定量质谱蛋白质组学的细胞系中的同源突变 确定与物理相关的相互作用蛋白。我们将测试由 秀丽隐杆线虫和细胞系中的蛋白质组学。我们还将使用秀丽隐杆线虫遗传学来筛选优先级 候选人使用链接细胞附件测定法在广告中发挥作用。候选人将优先考虑 在接头单元格中的表达，我们具有深度的转录组轮廓，编码的预测 跨膜或分泌的蛋白质，系统发育共发生以及相互作用的网络预测 基于其他深入研究生物中的直系同源蛋白。这样，我们将确定这些部分 链接蛋白在其在粘合剂和物理相关的相互作用蛋白上的功能至关重要 其他细胞和可能介导粘合剂的细胞，以及在实现粘合剂的细胞质中 细胞骨架相互作用。经过验证的一组功能相互作用的蛋白质将定义 细胞粘附并为正常发育和疾病状态中的各种人类遗传研究提供信息。