Functional Ocular Chemoproteomics for Retinal Biology Insight and in vivo Enzyme Activity

项目摘要 基于组学的方法的最新进展几乎允许任何科学家执行强大的 对正常组织、病变组织、 甚至是单个细胞。这些分析提供的信息提供了重要的见解 由于发育、疾病和风险因素而发生的细胞变化 与疾病相关，并确定疾病干预的潜在靶点。然而， 太常见了，绝大多数的组学研究只能推断出 观察对蛋白质功能的影响。例如，许多研究人员通常推断， 转录物或蛋白质水平上丰度的增加与相应的 增加特定蛋白质功能;但情况并非如此。现实中 蛋白质的功能不仅仅由其mRNA或蛋白质丰度决定，而是由 细胞适当折叠蛋白质的能力达到顶峰， 细胞/细胞外区域，以及抑制剂、激活剂和/或辅因子的存在。到 克服了传统组学方法的这些固有局限性，一种独特的化学- 一种称为基于活性的蛋白质谱分析（ABPP）的生物学方法已被用于提供 功能蛋白活性数据。为此，在这个探索/发展项目中，我们 将评估i）ABPP的效用，以确定年龄相关的视网膜改变是否最终 影响蛋白质在生理环境中的功能，以及ii）我们是否可以利用新设计的， 酶特异性ABPP探针以产生体内实时酶活性。成功完成 该项目将积极影响我们对眼部酶的认识，并将提供更多的 用于探究与年龄相关的遗传性眼睛的复杂生物学基础的工具 疾病

PROJECT SUMMARY Recent advances in ‘omics-based approaches allow virtually any scientist to perform powerful genome-wide, transcriptome-wide and proteome-wide analyses of normal tissue, diseasedtissue, or even single cells. The information provided by these analyses has afforded important insight into cellular changes that occur as a consequence of development, disease and risk factors associated with disease, and the identification of potential targets for disease intervention. Yet, all too commonly, the vast majority of ‘omics studies enable only inferences regarding the ultimate effect of the observation on protein function. For example, many researchers routinely infer that an increase in abundance either at the transcript or protein level correlates with a corresponding increase in that particular protein’s function; but this is not necessarily the case. In reality, the function of a protein is not simply determined by its mRNA or protein abundance, but rather by the culmination of the cell’s ability to fold the protein appropriately, traffic it to the proper cellular/extracellular locale, and the presence of inhibitors, activators, and/or cofactors. To overcome these inherent limitations of conventional ‘omics approaches, a unique chemical- biology approach called activity-based protein profiling (ABPP) has been used to provide functional protein activity data. Towards this end, in this exploratory/developmental project, we will assess i) the utility of ABPP to determine whether age-related retinal alterations ultimately affect protein function in a physiologic context and ii) whether we can leverage newly designed, enzyme-specific ABPP probes to yield real-time enzyme activity in vivo. Successful completion of this project will positively impact our knowledge of ocular enzymes and will provide additional tools for interrogating the intricate biology underlying incurable age-related and inherited eye diseases.