Cell adhesion mediated by LINKIN

LINKIN 介导的细胞粘附

基本信息

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
项目类别：
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

项目摘要

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.

项目概要细胞粘附是发育、受精、组织稳态、保护和相互作用的重要方面许多粘附蛋白已经被深入研究了几十年，并且它是一个开放的。考虑到大量相互作用的蛋白质，我们是否对粘附有足够的了解？ LINKIN 是一种保守的跨膜蛋白，我们发现它参与了粘附。细胞粘附，表明通过研究 LINKIN 及其相互作用蛋白，我们可以定义一种新的细胞粘附 LINKIN 序列和基因拷贝数的显着保守性。整个真核进化表明 LINKIN 在细胞发育中发挥着关键作用。秀丽隐杆线虫发育过程中的粘附，特别是迁移连接细胞的附着我们确定了输精管的结构、功能和相互作用。蛋白质在人类细胞中相互作用，并为两种生物体中类似的分子相互作用提供了证据。我们确定了 RUVBL1 和 RUVBL2 AAA-ATP 酶以及 α-微管蛋白作为 LINKIN 的细胞质相互作用子，表明 LINKIN 在调节微管动力学中发挥作用，但需要进一步研究来连接我们建议进一步表征 LINKIN 在细胞中的作用。粘附，以便将这种新的粘附分子连接到其他已知（或未知）的细胞途径。人类遗传学、基因组学和系统生物学方法的主要局限性是我们对功能的理解通过发现新的途径，我们将增加基因组、临床和基因组的可能性。遗传学研究将是可解释的。我们将分析细胞外和细胞的功能和相互作用。以进化保守为指导，我们将突变保守的残基。线虫中的 LINKIN 并测试其在转基因蠕虫中的功能我们将使用人类 LINKIN 蛋白。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鉴定生理相关的相互作用蛋白质。我们将测试鉴定的蛋白质的功能。我们还将使用线虫遗传学来筛选优先顺序。使用连接细胞附着测定对粘附作用的候选者将按优先顺序排列。连接细胞中的表达，我们对此有深入的转录组谱，编码的预测跨膜或分泌蛋白、系统发育共现以及相互作用的网络预测基于其他经过深入研究的生物体中的直系同源蛋白质，我们将识别这些部分。 LINKIN 的功能对其粘附功能和生理相关的两种蛋白质相互作用至关重要其他细胞和同一细胞可能介导粘附，并且在细胞质中实现粘附和这组经过验证的功能性蛋白质相互作用将定义一条新的途径。细胞粘附并为正常发育和疾病状态下的各种人类遗传学研究提供信息。