Elucidation of Tubby and Tulp 1 Mechanisms by Comparitive Functional Proteomics

项目描述（由申请人提供）：本项目的目标是开发一种新技术，在蛋白质水平上系统地描述基因突变的疾病机制。对大量疾病相关突变的识别已经将定义潜在疾病机制的研究挑战从基因组学转移到功能蛋白质组学。其中一个例子是管状蛋白1 (Tulp1)，它有23个已知的与视网膜变性相关的突变。尽管各种功能蛋白质组学技术已被用于鉴定一些tulp1结合蛋白，但仍面临三个挑战：(a)如何敏感地鉴定较少的蛋白-蛋白相互作用（PPIs）；(b)如何量化整个PPI数据集的结合活性；(c)如何系统地区分与疾病相关的ppi。因此，Tulp1 PPIs的疾病相关性尚未确定，其病理机制仍然难以捉摸。我们开发了开放阅读框噬菌体展示（OPD）作为功能蛋白质组学的一项新技术，以补充现有技术。由于OPD能够通过多轮选择和扩增来丰富显示结合蛋白的克隆，因此它应该比其他缺乏蛋白质扩增能力的技术更敏感地识别出丰度较低的PPIs。OPD的其他优势包括其独特的能力，通过下一代测序（NGS）绘制突变特异性PPI的全球图谱，并定量比较整个PPI数据集与野生型蛋白及其致病突变体的结合活性，从而可靠地描绘突变特异性PPI。假设OPD是一种新技术，可用于Tulp1的功能相关和疾病相关结合蛋白的全局定位。具体目的是：(1)验证OPD系统识别突变特异性ppi的工作假设；(2)验证OPD-NGS混合技术在全球范围内绘制总ppi和突变特异性ppi的工作假设。(3)验证OPD在生化途径中可靠地描述突变特异性和生物学相关PPIs的工作假设。该项目将有助于描述Tulp1疾病的视网膜变性机制。鉴于其广泛的适用性，OPD-NGS作为突变特异性PPIs全局定位的唯一技术，将促进我们对许多其他蛋白质及其基因突变的疾病机制的理解。

DESCRIPTION (provided by applicant): The goal of this project is to develop a new technology to systematically delineate disease mechanisms of genetic mutations at the protein level. Identification of the large number of disease-associated mutations has shifted the research challenge of defining underlying disease mechanisms from genomics to functional proteomics. One of the examples is tubby-like protein 1 (Tulp1) that has 23 known mutations associated with retinal degeneration. Although various technologies of functional proteomics have been used to identify a few Tulp1-binding proteins, three challenges are: (a) how to sensitively identify less abundant protein-protein interactions (PPIs); (b) how to quantify binding activity of the entire PPI dataset; and (c) how to systematically distinguish disease-relevant PPIs. Consequently, disease relevance of Tulp1 PPIs is yet to be defined, and its pathological mechanisms remain elusive. We developed open reading frame phage display (OPD) as a new technology of functional proteomics to complement the existing technologies. Owing to its capacity to enrich clones displaying binding proteins through multi-round selection and amplification, OPD should be more sensitive to identify less abundant PPIs than other technologies that lack protein amplification capacity. Other advantages of OPD include its unique capacity to globally map mutation-specific PPIs by next generation sequencing (NGS) and to quantitatively compare the binding activity of entire PPI datasets to wild-type proteins and their pathogenic mutants for reliable delineation of mutation-specific PPIs. The hypothesis is that OPD is a new technology that can be used for global mapping of functionally-relevant and disease-relevant binding proteins of Tulp1. The specific aims are: (1) To test the working hypothesis that OPD systematically identifies mutation-specific PPIs; (2) to test the working hypothesis that OPD-NGS hybrid technology globally maps total PPIs and mutation-specific PPIs. (3) To test the working hypothesis that OPD reliably delineates mutation- specific and biologically-relevant PPIs in a biochemical pathway. This project will facilitate delineation of Tulp1 disease mechanisms of retinal degeneration. Given its broad applicability, OPD-NGS as the only technology for global mapping of mutation-specific PPIs will advance our understanding of disease mechanisms for many other proteins and their genetic mutations.