The Identification and Characterization of Synthetic Peptide Substrate for Protein Tyrosine Kinases

蛋白酪氨酸激酶合成肽底物的鉴定和表征

• 批准号: 9506217
• 负责人: Kit Lam
• 金额: $ 24万
• 依托单位: University of Arizona
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-15 至 1999-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9506217&HistoricalAwards=false
• 关键词: Identification; Characterization; Synthetic; Peptide; Substrate

; R o o t E n t r y F #?6 @ C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l "?6 "?6 F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT marcia steinberg marcia steinberg @ R =6 @ e = e i \ " " " " " " " L L L L L d n L C x | | | | | | | # c T T 4 " | | | | | | " " | x | | | | " | " | 6 " " " " | | 2 | 9506217 Lam The objective of this project is to apply the combinatorial synthetic peptide library method to identify peptide substrate motifs for various classes of protein tyrosine kinases (PTK). Approximately 10 million random peptides will be screened for each PTK. The two PTKs to be studied include (i) Fes, a non receptor linked cytosolic PTKs, and (ii) Src, a non receptor linked, membrane anchored PTKs. In an attempt to identify natural substrates, the GeneBank will then be searched for proteins that have sequence homology with the identified peptides. Detailed structure activity relationship (SAR) studies will be performed on synthetic analogues of these peptides. Kinetic parameters as well as specificity of these pe ptide substrates will be determined. Based on these peptide substrate motifs, pseudosubstrate based peptide inhibitors for various PTKs will be developed. The combinatorial synthetic peptide library method based on a "one bead, one peptide concept" developed by the PI is used in this proposal. Random heptapeptide libraries are screened for their ability to be phosphorylated by specific PTKs in the presence of ~32P ATP. After incubation, the library is washed and the beads immobilized on a glass plate with agarose. The 32P labeled beads are then identified by autoradiography and individual labeled beads isolated for microsequencing. This method has already been proven for both the cAMP dependent protein kinase and p60C src PTK. Efficient peptide substrates have been identified for these two protein kinases. A high throughput solid phase phosphorylation assay system, developed in the P.I. s laboratory, is used for the SAR studies. Peptide analogues are synthesized on resin beads and used as substrates for the phosphorylation reaction. No purification of peptides is required and separation of the free gamma 32P ATP from the solid phase 32P labeled peptides is easy and rapid as peptides are covalently attached to beads. Quantitative autoradiography is then used to quantitate the phosphorylation. Using this approach it is possible to synthesize and analyze hundreds of analogues within a relatively short time. %%% The objective of this research proposal is to apply a novel combinatorial peptide library method to identify peptides that can be phosphorylated (the transfer of a phosphate group to a peptide or protein) by a number of human enzymes named protein tyrosine kinases. These enzymes are especially important in regulating various cellular functions such as activation of the immune cellular function, differentiation of various cell types, cell growth and death, and even the cancerous process. The combinatorial peptide library method described in this proposal was first developed in Dr. Lam 's laboratory and involves the synthesis of millions of peptide beads. The peptide beads phosphorylated by these enzymes will be isolated and their chemical structures determined. After several peptide substrates for these enzymes are identified, the P.I. plans to synthesize many of their analogs and test their activity. Results from such studies will provide important information for the understanding of the mechanism of action of these enzymes, and also help us to understand the biochemical basis of many important cellular functions. In addition, based on these results, he may be able to design specific and potent inhibitors for these enzymes. *** @ ....()()))()() ; S u m m a r y I n f o r m a t i o n ( @ U ?6 @ v A Microsoft Word 6.0 2

;​难道你不知道我是谁吗？6 @ C o m p o b j b W o r d d o c u m e n t O b j e c t P O O l "?6”?6 F Microsoft Word 6.0文档MSWordDoc。6;Oh +' 0 $ H l D H R:\WWUSER\TEMPLATE\NORMAL。DOT马西娅·斯坦伯格马西娅·斯坦伯格@ R =6 @ e = ei \ " " " " " " " L L L L L L d n L C x | | | | | | | # C T T 4“ | | | | | | ” “ | x | | | | ” | " | 6 " " " " | | 2 | 9506217 Lam本项目的目的是应用组合合成肽库方法，以确定肽底物基序的各种类型的蛋白酪氨酸激酶（PTK）。每个PTK将筛选大约1000万个随机肽。要研究的两种PTKs包括(i) Fes，一种非受体连接的细胞质PTKs，和（ii） Src，一种非受体连接的膜锚定PTKs。为了鉴定天然底物，基因库将搜索与鉴定肽序列同源的蛋白质。将对这些肽的合成类似物进行详细的构效关系（SAR）研究。动力学参数以及这些多肽底物的特异性将被确定。基于这些肽底物基序，各种PTKs的假底物基肽抑制剂将被开发出来。本方案采用PI提出的基于“一头一肽”概念的组合合成肽库方法。筛选随机七肽库，以确定它们在~32P ATP存在下被特定PTKs磷酸化的能力。孵育后，清洗文库，用琼脂糖将珠固定在玻璃板上。然后通过放射自显影术和分离的单个标记珠进行微测序鉴定32P标记珠。该方法已被证明适用于cAMP依赖性蛋白激酶和p60C src PTK。这两种蛋白激酶的有效肽底物已被确定。高通量固相磷酸化测定系统，在P.I.的实验室开发，用于SAR研究。肽类似物在树脂珠上合成，并用作磷酸化反应的底物。不需要对多肽进行纯化，由于多肽共价附着在微球上，因此从固相32P标记的多肽中分离游离的γ 32P ATP容易而快速。定量放射自显影术用于定量磷酸化。使用这种方法可以在相对较短的时间内合成和分析数百种类似物。本研究计划的目的是应用一种新的组合肽库方法来鉴定可以被称为蛋白质酪氨酸激酶的许多人类酶磷酸化的肽（磷酸基团转移到肽或蛋白质）。这些酶在调节各种细胞功能方面尤其重要，如免疫细胞功能的激活、各种细胞类型的分化、细胞的生长和死亡，甚至是癌变过程。本提案中描述的组合肽库方法最初是在林博士的实验室开发的，涉及数百万个肽珠的合成。被这些酶磷酸化的肽珠将被分离出来并确定其化学结构。在确定了这些酶的几种肽底物之后，P.I.计划合成它们的许多类似物并测试它们的活性。这些研究结果将为了解这些酶的作用机制提供重要信息，也有助于我们了解许多重要细胞功能的生化基础。此外，基于这些结果，他可能能够为这些酶设计特异性和有效的抑制剂。*** @ ....()()))()() ;如果我是你，我是你，我是你，我是你，我是你。@ u ？Microsoft Word 6.0