TCP5: ACTIVE SITE LABELING REAGENT FOR ACETYLTRANSFERASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 技术核心项目5： 这项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)酶。这里假设帽子家族仍有待发现，部分原因是 迄今用于HAT鉴定的方法有限。通过应用活性位点标记剂，应该有可能找到新的HAT酶，从而为我们理解基因调控开辟新的前景。 具体目的1.合成一系列具有化学活性的辅酶A类似物，用于亲和标记研究。以含有3‘-32P(或3’-33P的放射性标记形式生成的Coash将被用作合成一系列本征反应性或光反应性试剂的前体。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程度而有所不同。 具体目的2.评价已知的、纯化的HAT和混合物中添加HAT的CoA亲和试剂，并将其固定在微阵列中。CoA亲和试剂将作为酶抑制剂分别与纯化的p300、PCAF、ESAI、 和5-羟色胺N-乙酰转移酶来评估活性部位的相互作用。在这些研究的基础上，将进行具有适当范围的化合物浓度、缓冲液pH和反应时间的交联性实验。为了评估特异性，将在没有和存在竞争脱硫剂CoA的情况下进行交联实验。标记的化学计量将通过闪烁计数和/或磷成像仪分析来确定。在优化了纯化蛋白质的条件后，化合物将被用于掺入混合物的细胞提取液中，以确定在 一个更实际的环境。在与恒珠的合作下，他们还将在玻璃片固定帽子上接受检查。 特定目的3.鉴定和表征新的CoA-交联蛋白作为潜在的HATS。将测试从特定目标2的实验中挑选出来的化合物的子集，以识别提取液中的未知条带和载玻片上空间分离的蛋白质组。细胞提取液将通过2D-Gel电泳法分离，并通过磷图像分析进行可视化。与提取物中标记的蛋白质相对应的条带将通过与鲍勃·科特合作的现代质谱学方法进行分离和鉴定。从提取物和蛋白质芯片中提取的蛋白质，被认为是最有趣的基础 在与我们的联合PIS Jef Boeke和Shelly Berger协商的基础上，他们的DNA序列将被表达并检测HAT活性。有希望的酶将在细胞学研究中得到更深入的表征

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