Engineering protein-specific proteases: targeting signaling proteins

工程蛋白特异性蛋白酶：靶向信号蛋白

• 批准号: 10431978
• 负责人: PHILIP N BRYAN
• 金额: $ 52.13万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10431978
• 关键词: Active Sites; Adopted; Biological Models; Biophysics; Cancer Model; Cancer cell line; Catalysis; Cell Culture Techniques; Cell Line; Cell model; Cells; Cellular biology; Chemicals; Complex; Crystallization; Engineering; Enzymatic Biochemistry; Enzyme Precursors; Escherichia coli; Generations; Goals; Human; Imidazole; In Vitro; KRAS2 gene; Kinetics; Knowledge; Language; Libraries; Malignant neoplasm of urinary bladder; Microscopic; Modeling; Molecular; Molecular Conformation; Multidimensional NMR Techniques; Nitrites; Oncogenic; Peptide Hydrolases; Peptides; Phage Display; Protein Engineering; Protein Isoforms; Proteins; RAS genes; Reaction; Rhabdomyosarcoma; Roentgen Rays; SWI1; Series; Signal Transduction; Signaling Protein; Site; Specificity; Structure; Subtilisins; System; Testing; Therapeutic; Variant; X-Ray Crystallography; base; cofactor; controlled release; design; experimental study; improved; insight; interdisciplinary approach; mutant; novel; pancreatic cancer cells; peptide structure; tool

Summary: Our objective is to develop general principles for engineering proteases that can control signaling in cells. This requires seven steps: 1) Choice of signaling proteins (Human RAS isoforms and oncogenic mutants); 2) Identification of dynamic regions within the signaling proteins; 3) Identification of a target sequence within dynamic regions; 4) Engineering/evolving high specificity against the target sequence; 5) Engineering zymogen activation that is catalyzed by the target protein; 6) Testing regulated RAS degradation in E. coli; 7) Testing control of signaling in human cell culture. We will complete these steps using a multi-disciplinary approach that includes computational and in vitro selection-based protein engineering, enzymology, multi- dimensional NMR, X-ray crystallography, and cell-biology. The three Specific Aims are: 1) Evolve protein-specific proteases that target active RAS; 2) Engineer zymogen activation catalyzed by active RAS; 3) Test promising proteases in cell model systems. This project will develop general principles of recognition, folding, and catalysis based on detailed understanding of structure and mechanism and use this knowledge to develop tools to control signaling in a cell. The protease and its regulators will comprise an enzymatic system that will sense the presence of active RAS and transduce a controlled release of the active RAS-specific protease, thereby modulating RAS signaling. The ability to cleave RAS in cell-based systems and thereby alter RAS signaling will represent a significant milestone towards establishing the utility of protein-specific proteases as exogenous signaling regulators. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

摘要：我们的目标是制定工程蛋白酶的一般原则 可以控制细胞中的信号传导。这需要七个步骤：1）选择信号蛋白 （人Ras同工型和致癌突变体）； 2）识别动态区域 在信号蛋白内； 3）在动态中识别目标序列 地区； 4）针对目标序列进行工程/发展高特异性； 5） 靶蛋白催化的工程酶原激活； 6）测试 大肠杆菌中的调节RAS降解； 7）测试人类细胞培养中信号传导的控制。 我们将使用包含的多学科方法来完成这些步骤 计算和体外选择的蛋白质工程，酶学，多 尺寸NMR，X射线晶体学和细胞生物学。三个具体目标是： 1）进化靶向活性RA的蛋白质特异性蛋白酶； 2）Zymogen工程师 活性RA催化激活； 3）在细胞模型系统中测试有希望的蛋白酶。 该项目将根据基于认可，折叠和催化的一般原则 对结构和机制的详细理解，并利用这些知识来发展 控制电池中信号的工具。蛋白酶及其调节器将包括 酶促系统将感觉到活性RA的存在并转导受控的 释放活性RAS特异性蛋白酶，从而调节RAS信号传导。这 能够在基于细胞的系统中裂解RA，从而改变RAS信号传导 代表建立蛋白质特异性效用的重要里程碑 蛋白酶作为外源信号调节剂。尽管这些蛋白酶靶向活跃 拉斯，基本的设计原则是基本的，将适应许多 靶蛋白。