TCP5: ACTIVE SITE LABELING REAGENT FOR ACETYLTRANSFERASES

TCP5：乙酰转移酶活性位点标记试剂

• 批准号: 7380814
• 负责人: PHILIP A COLE
• 金额: $ 19.4万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7380814
• 关键词: TCP5; ACTIVE; SITE; LABELING; REAGENT

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 stu

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。技术核心项目5：该核心技术涉及使用化学亲和标记试剂来发现和表征新型组蛋白乙酰转移酶（HAT）。这里假设HAT家族仍有待发现，部分原因是迄今为止用于HAT鉴定的方法有限。通过应用活性位点标记剂，应该有可能发现新的HAT酶，这可以为我们对基因调控的理解开辟新的前景。 具体目标1。合成一系列化学反应性CoA类似物用于亲和标记研究。CoASH以放射性标记的形式产生，含有3 ''目标化合物将根据亲电/光活性部分与CoA核心之间的距离以及对亲核或非亲核酶基团的反应性程度而变化。 具体目标2。用已知的纯化HAT和混合物中的加标HAT以及固定在微阵列中的HAT评价CoA亲和试剂。CoA亲和试剂将作为酶抑制剂单独与纯化的p300、PCAF、EsaI和血清素N-乙酰转移酶进行检测，以评估活性位点相互作用。基于这些研究，将进行具有适当范围的化合物浓度、缓冲液pH值和反应时间的交联实验。为了评估专属性，将在不存在和存在竞争性辅酶A的情况下进行交联实验。将通过闪烁计数和/或磷光成像仪分析确定标记的化学计量。在用纯化的蛋白质优化条件后，化合物将用于掺有混合物的细胞提取物中，以确定在更实际的环境中可以实现的特异性水平。与Heng Zhu合作，他们还将在固定HAT的载玻片上进行检查。 具体目标3。鉴定和表征作为潜在HAT的新型CoA交联蛋白。将检测从特定目标2中的实验中挑选的化合物子集，以识别提取物中的未知条带和载玻片上空间分离的蛋白质组。将通过2D凝胶电泳分离细胞提取物，并通过磷光图像分析进行可视化。将与Bob Cotter合作，通过现代质谱方法分离和鉴定与提取物中标记蛋白质对应的条带。将表达来自提取物以及来自蛋白芯片的蛋白质，并测定其HAT活性，所述蛋白质基于与我们的共同PI Jef Boeke和Shelly Berger协商的DNA序列被判定为最感兴趣的。有前途的酶将在细胞研究中得到更深入的表征。