Protease Engineering

描述(申请人提供)：具有量身定制的物理和催化特性的酶的设计是现代蛋白质科学的基本推动力之一，具有深远的技术和医学影响的潜力。实验室指导的进化方法以及合理的设计原则现在已经成功地应用于增强蛋白质的性质和功能。剩下的是重要的下一步，“卷起袖子”，着眼于细节来解决重要的问题，这些问题很可能是特定于酶的。这项提议扩展了我们的酶导向进化计划，在工程蛋白酶领域的重要使能方向上。我们最近对OmpT蛋白水解酶的研究代表了迄今为止对蛋白水解酶的P1和P1‘底物特异性的最普遍的操作，同时保持了较高的催化活性水平。除了在洗涤剂工业中的应用外，工程酶作为蛋白质组学工具或催化疗法具有巨大的实用潜力。特别是，含有磷酸化或O-GlcNAc修饰的底物特有的蛋白酶将是在高通量蛋白质组学分析中识别修饰蛋白质的有用的新工具。在具体目标1下，我们将设计专门用于切割含有磷酸化或O-GlcNAc丝氨酸的底物的OmpT变体。我们还将调查是否可以设计出“限制类”的蛋白酶，这种酶可以非常选择性地识别包含远超过P1和P1‘的残基的延伸序列。在特定目标2下，我们将扩展对OmpT蛋白酶的精确识别，以包括P2、P3、P2‘和P3’。特别是，我们将针对C3a过敏毒素多肽C末端的68-73个残基的Gln-His-Ala-Arg-Ala-Ser(QHA？RAS)。C3a的C端裂解干扰了其在补体激活中的生物学作用。我们认识到，通过突变和分选在OmpT中创建高度特异性的亚位点是一个令人兴奋但有风险的目标，而且由于其细菌来源，工程OmpT不太可能成为临床治疗的候选者。因此，对于特定的目标3，我们将精确的C3a切割活性工程到分泌的人胰酶样蛋白酶颗粒酶A中，希望产生一个临床候选基因。作为一个实际的交付，在我们最好的变体的功能分析之后(D6节)，我们将第一次能够验证蛋白质分解方法来抑制补体，适用于各种炎症性疾病的治疗。公共卫生相关性：总的来说，目前几乎每种疾病的治疗方法都涉及与特定疾病目标以“一对一”比率相互作用的分子，即需要一个药物分子与每个疾病分子相互作用。我们正在提出一种被称为蛋白酶的分子工程路线，它将催化摧毁许多疾病靶标分子(即一个药物分子摧毁数百、数千甚至更多疾病分子的新范式)！特别是，我们将试图产生一种有效的催化剂，能够“瞄准”目标(称为C3a过敏毒素)，从而能够有效治疗包括哮喘和败血症在内的一系列炎症性疾病。

DESCRIPTION (provided by applicant): The design of enzymes with tailored physical and catalytic properties is one of the fundamental thrusts of modern protein science, with the potential for profound technological and medical impact. Laboratory directed evolutionary approaches along with rational design principles have now been successfully applied to enhance protein properties and function. What remains is the important next step of "rolling up our sleeves" and attacking important problems with an eye toward details, which are likely to be enzyme specific. This proposal extends our enzyme directed evolution program in important enabling directions in the area of engineered proteases. Our most recent work with the OmpT protease represents by far the most general manipulation of P1 and P1' substrate specificity of a protease while retaining high overall levels of catalytic activity. Beyond their use in the detergent industry, engineered proteases have tremendous practical potential as either proteomic tools or catalytic therapeutics. In particular, proteases specific for substrates containing modifications such as phosphorylation or O-GlcNAc would represent useful new tools for identifying modified proteins in high throughput proteomics assays. Under Specific Aim 1, we will engineer OmpT variants specific for cleaving only substrates containing phosphorylated or O-GlcNAc serine. We will also investigate whether we can engineer "restriction-like" proteases that can very selectively recognize an extended sequence comprising residues well beyond P1 and P1'. Under Specific Aim 2, we will extend precise OmpT protease recognition to include P2, P3, P2', and P3'. In particular, we will target Gln-His-Ala-Arg-Ala-Ser (QHA?RAS), residues 68-73 of the C-terminus of the C3a anaphylatoxin peptide. C-terminal cleavage of C3a interferes with its biological effects in complement activation. We recognize that creating highly specific subsites in OmpT by mutagenesis and sorting is an exciting yet risky goal and that an engineered OmpT is an unlikely therapeutic clinical candidate because of its bacterial origin. Therefore, for Specific Aim 3, we will engineer precise C3a cleavage activity into the secreted human trypsin-like protease granzyme A in hopes of producing a clinical candidate. As a practical deliverable, following the functional assays of our best variants (Section D6) we will, for the first time, be able to validate the proteolytic approach to complement inhibition, applicable to a wide variety of inflammatory disease therapies. PUBLIC HEALTH RELEVANCE: In general terms, current therapies for almost every disease involve molecules that interact with specific disease targets in a "one-for-one" ratio, i.e. one drug molecule is required to interact with each disease molecule. We are proposing a route to the engineering of molecules, called proteases, that will catalytically destroy many disease target molecules (i.e. a new paradigm in which one drug molecule destroys hundreds, thousands or even more disease molecules)! In particular, we will be attempting to generate a potent catalyst capable of "zeroing in" on a target (called the C3a anaphylatoxin) that would allow effective treatment of a wide range of inflammatory diseases including asthma and sepsis.