TCP5: ACTIVE SITE LABELING REAGENT FOR ACETYLTRANSFERASES

TCP5：乙酰转移酶活性位点标记试剂

• 批准号: 7622843
• 负责人: PHILIP A COLE
• 金额: $ 20.46万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7622843
• 关键词: Acetyltransferase; Active Sites; Affinity; Affinity Labels; Arylalkylamine N-Acetyltransferase; Biological Assay; Buffers; Cell Extracts; Chemicals; CoASH; Coenzyme A; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Condition; Consultations; DNA Sequence; EP300 gene; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Family; Funding; Gene Expression Regulation; Glass; Grant; Institution; Label; Methods; PCAF gene; Protein Microchips; Proteins; Proteome; Radiolabeled; Range; Reaction Time; Reagent; Research; Research Personnel; Resources; Scintillation Counting; Series; Slide; Source; Specificity; Technology; Testing; Two-Dimensional Gel Electrophoresis; United States National Institutes of Health; affinity labeling; analog; base; crosslink; histone acetyltransferase; human NAT2 protein; interest; novel; protein crosslink; radiotracer; research study; stoichiometry

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Technology Core Projects 5: This core technology involves the use of chemical affinity labeling reagents to discover and characterize novel histone acetyltransferase (HAT) enzymes. It is hypothesized here that families of HATs remain to be discovered, in part due to the limited approaches that have been so far employed for HAT identification. By applying active site labeling agents, it should be possible to find new HAT enzymes which can open new vistas in our understanding of gene regulation. Specific Aim 1. Synthesize a series of chemically reactive CoA analogs for affinity labeling studies. CoASH, generated in radiolabeled form containing 3''''''''''''''''''''''''''''''''-32p (or 3''''''''''''''''''''''''''''''''-33p), will be used as a precursor to synthesize a series of intrinsically reactive or photoreactive reagents. The target compounds will be varied in terms of the distance between the electrophilic/photoactive moiety from the CoA core and the degree of reactivity toward nucleophilic or non-nucleophilic enzyme groups. Specific Aim 2. Evaluate the CoA affinity reagents with known, purified HATs, and spiked HATs in mixtures and immobilized in microarrays.. The CoA affinity reagents will be tested as enzyme inhibitors individually with purified p300, PCAF, EsaI, and serotonin N-acetyltransferase to assess active site interactions. Based on these studies, crosslinking experiments with suitable ranges of compound concentration, buffer pH, and reaction times will be performed. To assess specificity, crosslinking experiments in the absence and presence of competing desulfoCoA will be carried out. Stoichiometry of labeling will be determined by scintillation counting and/or phosphorimager analysis. After optimizing conditions with purified proteins, compounds will be employed in cell extracts spiked with mixtures to determine the level of specificity that can be achieved in a more practical setting. In collaboration with Heng Zhu, they will also be examined on glass slide immobilized HATs. Specific Aim 3. Identify and characterize novel CoA-crosslinked proteins as potential HATs. A subset of compounds culled from experiments in Specific Aim 2 will be tested to identify unknown bands in extracts and with spatially separated proteomes on slides. Cell extracts will be separated by 2D-gel electrophoresis and visualized by phosphorimage analysis. Bands corresponding to labeled proteins from extracts will be isolated and identified by modern mass spec methods in collaboration with Bob Cotter. Proteins from extracts as well as from protein chips, judged to be most interesting based on their DNA sequences based on consultation with our Co-PIs Jef Boeke and Shelly Berger, will be expressed and assayed for HAT activity. Promising enzymes will be characterized more deeply in cellular stud

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 技术核心项目5： 该核心技术涉及使用化学亲和力标记试剂来发现和表征新型组蛋白乙酰转移酶（HAT）酶。在这里假设，帽子家族仍有待发现，部分原因是 到目前为止，已采用有限的方法来识别帽子。通过应用主动的位点标记剂，应该可以找到新的帽子酶，这些酶可以在我们对基因调节的理解中打开新的远景。 具体目标1。合成一系列的化学反应性COA类似物，用于亲和力标记研究。 CoASH, generated in radiolabeled form containing 3''''''''''''''''''''''''''''''''-32p (or 3''''''''''''''''''''''''''''''''-33p), will be used as a precursor to synthesize a series of intrinsically reactive or光反应性试剂。靶化合物将随着与COA核心的亲电/光活动部分之间的距离以及对亲核或非核核酶基团的反应性程度的变化。 Specific Aim 2. Evaluate the CoA affinity reagents with known, purified HATs, and spiked HATs in mixtures and immobilized in microarrays.. The CoA affinity reagents will be tested as enzyme inhibitors individually with purified p300, PCAF, EsaI, 和5-羟色胺N-乙酰转移酶，以评估活性位点相互作用。基于这些研究，将进行具有合适的复合浓度，缓冲液和反应时间的交联实验。为了评估特异性，将在不存在和存在竞争性的desulfocoa的情况下进行交联实验。标记的化学计量法将通过闪烁计数和/或磷光受益剂分析确定。 在用纯化的蛋白质优化条件后，化合物将在用混合物尖刺的细胞提取物中使用，以确定可以实现的特异性水平 更实用的环境。与Heng Zhu合作，还将在玻璃滑梯固定的帽子上进行检查。 特定目标3。识别并表征新型的CoA跨链接蛋白是潜在的帽子。将测试从特定目标2中剔除的化合物的一部分，以鉴定提取物中的未知带，并在玻片上具有空间分离的蛋白质组织。细胞提取物将通过2D凝胶电泳分离，并通过磷光图分析可视化。与鲍勃·科特（Bob Cotter）合作，将通过现代质量规格方法隔离和鉴定与提取物标记的蛋白质相对应的条带。 提取物以及蛋白质芯片的蛋白质，被认为是最有趣的 根据与我们的Co-Pis Jef Boeke和Shelly Berger的咨询，他们的DNA序列将被表达并分析以进行帽子活动。有希望的酶将在细胞螺柱中更深入地表征