Elucidation of Tubby and Tulp 1 Mechanisms by Comparitive Functional Proteomics

描述(申请人提供)：该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。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Tubby-like Protein 1(Tulp1)，它有23个已知突变与视网膜退化有关。尽管功能蛋白质组学的各种技术已经被用来识别一些Tulp1结合蛋白，但三个挑战是：(A)如何敏感地识别不那么丰富的蛋白质-蛋白质相互作用(PPI)；(B)如何量化整个PPI数据集的结合活性；以及(C)如何系统地区分与疾病相关的PPI。因此，Tulp1PPI与疾病的相关性尚不明确，其病理机制仍不清楚。我们开发了开放阅读框噬菌体展示(OPD)作为一种新的功能蛋白质组学技术，以补充现有的技术。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，因此与其他缺乏蛋白质扩增能力的技术相比，OPD应该更敏感地识别丰度较低的PPI。OPD的其他优势包括其独特的能力，通过下一代测序(NGS)全球绘制突变特异性PPI，并定量比较整个PPI数据集与野生型蛋白质及其致病突变的结合活性，以可靠地描绘突变特异性PPI。假设OPD是一种新技术，可以用于Tulp1功能相关和疾病相关结合蛋白的全球定位。具体目的是：(1)检验OPD系统地识别突变特定的PPI的工作假设；(2)检验OPD-NGS混合技术全局映射总的PPI和突变特定的PPI的工作假设。(3)检验OPD在生物化学途径中可靠地描绘突变特异性和生物相关的PPI的工作假设。该项目将有助于描述Tulp1视网膜变性的发病机制。鉴于其广泛的适用性，OPD-NGS作为全球唯一的突变特异性PPI定位技术，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。 公共卫生相关性：该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。这项新技术将被开发来识别和表征与疾病相关的结合蛋白和眼睛蛋白的病理机制，这种蛋白有23个已知的致盲突变。这项技术也适用于其他蛋白质及其致病突变。因此，这项研究对我们理解与基因突变相关的疾病机制具有广泛的影响。

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. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a new technology to systematically delineate disease mechanisms of genetic mutations at the protein level. The new technology will be developed to identify and characterize disease-relevant binding proteins and pathological mechanisms for an eye protein that has 23 known blindness-causing mutations. This technology is applicable to other proteins and their pathogenic mutations. Thus, this study has broad impact on our understanding of disease mechanisms associated with genetic mutations.