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，其中生物学上无活性的前体蛋白通过多肽链的特异性切割而被激活。尽管其重要性，蛋白质组学分析的蛋白质水解加工的进展落后于其他翻译后修饰的类似分析。为了解决这一缺点，威尔斯实验室最近开发了一种用于检测蛋白水解加工产物的新型高通量方法。该方法利用枯草杆菌酶，一种能够将活化的肽底物连接到蛋白质的N-末端上的工程化酶。这种独特的酶使得有可能检测受调节的蛋白质水解的化学副产物，游离的N-末端氨基，选择性地超过赖氨酸侧链和许多其他生物分子中的类似反应性胺。通过使用用亲和标签如生物素标记的底物肽，可以从细胞提取物中仅分离具有游离N-末端的那些蛋白质。然后这些蛋白质可以被消化并通过质谱法鉴定。通过同位素标记，可以定量比较对照和实验浸提液、细胞或组织中推定底物的丰度。在这里，我们建议扩大这项技术的范围，包括一些医学相关的细胞外蛋白酶级联反应。该提案包含两个独立但密切相关的目标：提高枯草杆菌酶在检测不同蛋白酶底物中的性能，并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BACE 1的研究。选择这些模型系统是因为它们在医学上的重要性，也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功效。相反，BACE 1的生物学作用还没有得到很好的理解，它提供了一个很好的系统来寻找未知的蛋白酶底物。相关性：这项工作将导致更好地了解从癌症到细菌感染等许多疾病背后的基本生物学过程。此外，我们正在开发一种可能具有诊断工具价值的技术。

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.