The Identification and Characterization of Synthetic Peptide Substrates and Inhibitors for Protein Kinases

LAM 9728399 1.这项研究的重点是蛋白酪氨酸激酶(PTK)，它在一系列细胞活动的信号转导中发挥重要作用，包括T细胞和B细胞的激活、对细胞外刺激的反应、有丝分裂、分化和肿瘤发生。目的是加深我们对这些酶的范围和底物特异性的了解，特别是p60c-src PTK的多肽底物和抑制剂。结构-活性关系，动力学参数，物理化学相互作用，以及对v-src转基因细胞或c-src活性结构性升高的细胞的生物和生化效应是我们研究的基本问题。实验包括：用p60c-src PTK评价“一珠一化合物”组合多肽文库，以鉴定额外的底物基序；设计有效的基于假底物的p60c-src PTK多肽抑制剂；对已鉴定的底物和抑制剂进行详细的动力学研究；探索多肽抑制剂对细胞蛋白磷酸化的生化作用；检测多肽抑制剂对转染v-src 3T3和其他细胞系的生物和生化效应；计算机模拟(DOCK程序)已知晶体结构的多肽底物或抑制剂之间的相互作用；以及确定共晶体的X射线晶体结构(与哈佛大学的斯蒂芬·哈里森教授和迈克尔·埃克合作)。2.非技术性蛋白酪氨酸激酶是一类在细胞分裂、细胞死亡和癌症形成等多种细胞活动中发挥极其重要作用的酶。尽管它们非常重要，但人们对这些酶的作用机制知之甚少。这项研究的目的是建立在这个实验室(美国国家科学基金会授予MCB 9506217)在p60c-src蛋白酪氨酸激酶项目上已经发现的东西的基础上。已确定的多肽底物和抑制剂将用于进一步研究该酶的作用机制。还将对这些多肽进行动力学研究。此外，林博士还与哈佛大学的埃克和哈里森博士建立了合作关系，以确定多肽-酶复合体的X射线结构。还计划使用计算机模拟技术来了解这些肽如何与酶相互作用。最后，将采用不同的方法将这些肽抑制剂输送到细胞中，并研究它们对p60c-src酪氨酸激酶蛋白水平升高的细胞系的基本生物学效应。

Lam 9728399 1. Technical This study focuses on protein tyrosine kinases (PTKs), which play an important role in signal transduction for a broad spectrum of cellular activities, including T-cell and B-cell activation, responses to extracellular stimuli, mitogenesis, differentiation, and oncogenesis. The objective is to further our understanding of the range and substrate specificity of these enzymes, particularly peptide substrates and inhibitors for p60c-src PTK. The structure-activity relationship, kinetic parameters, physico-chemical interaction, and biological and biochemical effects on v-src transfected cells or cells that have constitutively elevated c-src activity are basic issues that are studied. Experiments include: evaluation "one-bead one-compound" combinatorial peptide libraries with p60c-src PTK for identification of additional substrate motifs; design of potent pseudosubstrate-based peptide inhibitors for p60c-src PTK; detailed kinetic studies on the identified substrates and inhibitors; probing the biochemical effects of the peptide inhibitors on the phosphorylation of cellular proteins; examining the biological and biochemical effects of the peptide inhibitors on v-src transfected 3T3 and other cell lines; computer modelling ( DOCK program) the interaction between the peptide substrates or inhibitors with the known crystal structure of p60c-src PTK; and determining the X-ray crystal structure of the co-crystal (in collaboration with Professor Stephen Harrison and Michael Eck of Harvard University). 2. Non-technical Protein tyrosine kinases represent a class of enzymes that play extremely important roles in various cellular activities such as cell division, cell death, and cancer formation. Despite their great importance, very little is known about the mechanism of action of these enzymes. The objective of this study is to build on what has already been discovered in this laboratory in the last two years (NSF Grant MCB 9506217) on the p60c-src protein tyrosine ki nase project. The peptide substrates and inhibitors that have already been identified will be used to further study the mechanism of action of this enzyme. Kinetic studies will also be performed with these peptides. Additionally, Dr. Lam has established a collaboration with Drs. Eck and Harrison of Harvard University to determine the X-ray structure of the peptide-enzyme complex. Also planned is the use of computer modelling techniques to understand how these peptides interact with the enzyme. Finally, different approaches will be followed to deliver the peptide inhibitors into cells and study their fundamental biological effect on cell lines which have an elevated level of protein for p60c-src tyrosine kinase.