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: 10595636
• 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/10595636
• 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; Communication; Complex; 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; Series; Signal Transduction; Signaling Protein; Site; Specificity; Structure; System; Testing; Therapeutic; Variant; X-Ray Crystallography; cofactor; controlled release; design; experimental study; improved; insight; interdisciplinary approach; mutant; novel; pancreatic cancer cells; 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：我们的目标是开发工程蛋白水解酶的一般原理 可以控制细胞中的信号。这需要七个步骤：1)信号蛋白的选择 (人类RAS异构体和致癌突变)；2)动态区域的鉴定 在信号蛋白内；3)在动态内鉴定靶序列 区域；4)针对目标序列的高度特异性的工程/进化；5) 由目标蛋白催化的工程酶原激活；6)检测 调节RAS在大肠杆菌中的降解；7)测试人类细胞培养中信号的控制。 我们将使用多学科方法完成这些步骤，包括 基于计算和体外选择的蛋白质工程，酶学，多- 三维核磁共振、X射线结晶学和细胞生物学。这三个具体目标是： 1)进化以活性RAS为靶点的蛋白质专一性蛋白酶；2)设计酶原 活性RAS催化的激活；3)在细胞模型系统中测试有前景的蛋白酶。 这个项目将开发识别、折叠和催化的一般原理，基于 对结构和机制有详细的了解，并利用这些知识开发 控制单元格中信令的工具。该蛋白酶及其调节器将包括一个 酶系统，将感知活性RAS的存在并转导受控的 释放活性的RAS特有的蛋白水解酶，从而调节RAS信号。这个 在基于细胞的系统中切割RAS从而改变RAS信号的能力将 代表着一个重要的里程碑，朝着建立蛋白质特异性的效用 蛋白水解酶作为外源信号调节因子。尽管这些蛋白水解酶以活性为目标 RAS，基本的设计原则是基本的，并将适用于许多 靶蛋白。