Engineering An Enzyme for Rapid Profiling of Proteolytic Processing

描述（由申请人提供）：蛋白水解加工是一种常见的翻译后修饰，在多种生物过程中至关重要，其中生物无活性前体蛋白通过多肽链的特定切割被激活。尽管其重要性，蛋白质水解过程的蛋白质组学分析的进展落后于其他翻译后修饰的类似分析。为了解决这个缺点，Wells实验室最近开发了一种新的、高通量的检测蛋白水解产物的方法。这种方法利用枯草酶，一种工程酶，能够将激活的肽底物连接到蛋白质的n端。这种独特的酶可以检测调节蛋白水解的化学副产物，一个自由的n端氨基，选择性地超过赖氨酸侧链中的类似活性胺和许多其他生物分子。通过使用带有生物素等亲和标签的底物肽，可以从细胞提取物中仅分离出具有游离n末端的蛋白质。这些蛋白质可以通过质谱法消化和鉴定。通过标记同位素标签，可以定量比较对照和实验提取物、细胞或组织中假定底物的丰度。在这里，我们建议扩大这项技术的范围，以涵盖一些医学上相关的细胞外蛋白酶级联。本提案包含两个独立但密切相关的目标：提高枯草化酶检测多种蛋白酶底物的性能，以及将原始和改进的酶应用于补体级联和阿尔茨海默病β -分泌酶BACE1的研究。选择这些模型系统既是因为它们的医学重要性，也是因为它们是优化该方法的良好测试系统。补体是一个经过深入研究和充分理解的过程，可用于校准方法的定量能力。相比之下，BACE1的生物学作用尚不清楚，它为寻找未知蛋白酶底物提供了一个很好的系统。相关性：这项工作将使我们更好地理解从癌症到细菌感染等许多情况下的基本生物学过程。此外，我们正在开发一种可能作为诊断工具有价值的技术。

DESCRIPTION (provided by applicant): Proteolytic processing, where biologically inactive precursor proteins are activated by specific cleavage of the polypeptide chain, is a common postranslational modification that is critical in diverse biological processes. Despite its importance, progress in proteomic analysis of proteolytic processing has lagged behind similar analyses of other posttranslational modifications. To address this shortcoming, a novel, high- throughput method for detecting products of proteolytic processing has recently been developed by the Wells lab. This method makes use of subtiligase, an engineered enzyme capable of ligating activated peptide substrates onto the N-termini of proteins. This unique enzyme makes it possible to detect the chemical byproduct of regulated proteolysis, a free N-terminal amino group, selectively over similarly reactive amines in lysine side chains and numerous other biomolecules. By using substrate peptides labeled with an affinity tag like biotin, it is possible to isolate only those proteins with free N-termini from a cell extract. These proteins can then be digested and identified by mass spectrometry. By also labeling with an isotope tag, it is possible to quantitatively compare the abundances of putative substrates in control and experimental extracts, cells, or tissues. Here we propose to expand the scope of this technique to encompass a number of medically relevant extracellular protease cascades. This proposal contains two independent but closely related goals: to improve the performance of the subtiligase enzyme in detecting diverse protease substrates, and to apply the original and improved enzymes to investigations of the complement cascade and the Alzheimer's disease beta-secretase BACE1. These model systems were chosen both for their medical importance and because they are good test systems to optimize this method. Complement is an intensely-studied and well- understood process that can be used to calibrate the quantitative power of the method. The biological role of BACE1, in contrast, is not well understood, and it provides an excellent system in which to hunt for unknown protease substrates. Relevance: This work will lead to a better understanding of a basic biological process underlying many conditions, from cancer to bacterial infection. In addition, we are developing a technique that may have value as a diagnostic tool.