Protease Engineering

蛋白酶工程

• 批准号: 7581398
• 负责人: BRENT L IVERSON
• 金额: $ 32.16万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7581398
• 关键词: Active Sites; Address; Affinity; Amino Acids; Anaphylatoxins; Antibodies; Area; Asthma; Back; Bacterial Proteins; Basic Science; Binding; Biological; Biological Assay; Biotechnology; Birds; C-terminal; Cardiovascular Diseases; Characteristics; Charge; Chicago; Cleaved cell; Clinical; Complement; Complement 3a; Complement Activation; Detergents; Disease; Drug Industry; Engineering; Enzymes; Exhibits; Experimental Designs; Eye; Face; Family; Flow Cytometry; Frequencies; Generations; Goals; Granzyme; Hand; Human; Human Engineering; In Vitro; Individual; Industry; Inflammatory; Laboratories; Lead; Libraries; Ligands; Light; Location; Malignant Neoplasms; Marketing; Mediating; Medical; Methodology; Methods; Modification; Mutagenesis; Nature; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Phosphorylation; Process; Property; Protein Engineering; Proteins; Proteomics; Randomized; Reporting; Research; Route; Science; Screening procedure; Sepsis; Serine; Signal Transduction; Site; Sorting - Cell Movement; Specificity; Staging; Structure; Substrate Specificity; Techniques; Technology; Testing; Therapeutic; Time; Translating; Trypsin; Universities; Variant; Work; alanylglutamine; angiogenesis; base; catalyst; design; direct application; directed evolution; effective therapy; experience; granzyme A; high throughput screening; immunogenicity; insight; interest; member; novel; pre-clinical; programs; protein aminoacid sequence; public health relevance; research study; success; therapeutic protein; tool; tyrosine O-sulfate; ward

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.

描述（由申请人提供）：设计具有定制物理和催化特性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沿着与合理的设计原则，现已成功地应用于提高蛋白质的性质和功能。剩下的是重要的下一步，即“卷起袖子”，着眼于细节，解决重要问题，这可能是酶特异性的。这一建议在工程蛋白酶领域的重要使能方向上扩展了我们的酶定向进化计划。我们最近的工作与OmpT蛋白酶代表了迄今为止最普遍的操纵P1和P1'底物特异性的蛋白酶，同时保持高水平的整体催化活性。除了在洗涤剂工业中的应用外，工程蛋白酶作为蛋白质组学工具或催化治疗剂具有巨大的实用潜力。特别地，对含有修饰（例如磷酸化或O-GlcNAc）的底物具有特异性的蛋白酶将代表用于在高通量蛋白质组学测定中鉴定修饰的蛋白质的有用的新工具。在特定目标1下，我们将工程化OmpT变体，其仅特异性切割含有磷酸化或O-GlcNAc丝氨酸的底物。我们还将研究我们是否可以工程化“限制样”蛋白酶，其可以非常选择性地识别包含远远超过P1和P1 '的残基的延伸序列。在特定目标2下，我们将扩展精确OmpT蛋白酶识别以包括P2、P3、P2 '和P3'。特别是，我们将针对Gln-His-Ala-Arg-Ala-Ser（QHA？RAS），C3 a过敏毒素肽C-末端的残基68-73。C3 a的C-末端裂解干扰其在补体激活中的生物学效应。我们认识到，通过诱变和分选在OmpT中产生高度特异性的亚位点是一个令人兴奋但又有风险的目标，并且由于其细菌来源，工程化OmpT不太可能是治疗性临床候选物。因此，对于特异性目标3，我们将精确的C3 a切割活性工程化到分泌的人胰蛋白酶样蛋白酶颗粒酶A中，希望产生临床候选物。作为一个实际的可交付成果，在对我们最好的变体进行功能测定后（第D 6节），我们将首次能够验证蛋白水解方法对补体抑制的作用，适用于各种炎症性疾病治疗。公共卫生相关性：一般而言，目前几乎每种疾病的治疗都涉及以“一对一”比例与特定疾病靶标相互作用的分子，即需要一种药物分子与每种疾病分子相互作用。我们正在提出一种分子工程的途径，称为蛋白酶，它将催化破坏许多疾病靶分子（即一种新的范例，其中一种药物分子破坏数百，数千甚至更多的疾病分子）！特别是，我们将试图产生一种能够“归零”靶点（称为C3 a过敏毒素）的有效催化剂，从而有效治疗包括哮喘和败血症在内的各种炎症性疾病。