Protease Engineering

描述（由申请人提供）：设计具有量身定制的物理和催化性能的酶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特性和功能。剩下的重要的下一步是“卷起袖子”，着眼于细节来解决重要问题，这些细节可能是特定于酶的。这一建议扩展了我们的酶定向进化计划在工程蛋白酶领域的重要有利方向。我们最近对OmpT蛋白酶的研究代表了迄今为止对蛋白酶P1和P1底物特异性的最普遍的操作，同时保持了高水平的总体催化活性。除了在洗涤剂工业中的应用之外，工程蛋白酶作为蛋白质组学工具或催化疗法具有巨大的实际潜力。特别是，对含有磷酸化或O-GlcNAc修饰的底物特异性的蛋白酶将成为在高通量蛋白质组学分析中鉴定修饰蛋白的有用新工具。在Specific Aim 1下，我们将设计特异性的OmpT变体，仅用于切割含有磷酸化或O-GlcNAc丝氨酸的底物。我们还将研究我们是否可以设计出“限制性限制样”蛋白酶，它可以非常有选择性地识别包含远远超出P1和P1'残基的扩展序列。在Specific Aim 2下，我们将扩展精确的OmpT蛋白酶识别，包括P2， P3, P2‘和P3’。我们将特别针对Gln-His-Ala-Arg-Ala-Ser (QHA？RAS)， C3a过敏毒素肽c端68-73残基。C3a的c端切割会干扰其在补体活化中的生物学作用。我们认识到，通过诱变和分选在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，工程化的OmpT不太可能成为治疗性临床候选物。因此，对于Specific Aim 3，我们将设计精确的C3a裂解活性到分泌的人胰蛋白酶样蛋白酶颗粒酶A中，以期产生临床候选物。作为一项实际成果，在我们的最佳变体（章节D6）的功能分析之后，我们将首次能够验证补体抑制的蛋白水解方法，适用于各种炎症性疾病的治疗。公共卫生相关性：一般来说，目前几乎所有疾病的治疗方法都涉及以“一对一”的比例与特定疾病靶点相互作用的分子，即需要一个药物分子与每个疾病分子相互作用。我们正在提出一种称为蛋白酶的分子工程路线，它将催化破坏许多疾病目标分子（即一个药物分子破坏数百，数千甚至更多疾病分子的新范例）！特别是，我们将尝试产生一种有效的催化剂，能够“瞄准”一个目标（称为C3a过敏毒素），这将允许有效治疗广泛的炎症性疾病，包括哮喘和败血症。

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.