Bacterial N-Acetyltransferases: Resistance to Regulation

细菌 N-乙酰转移酶：对调节的抵抗

• 批准号: 7649041
• 负责人: John S Blanchard
• 金额: $ 53.67万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7649041
• 关键词: Acetylation; Affinity; Affinity Chromatography; Alkynes; Aminoglycosides; Azides; Base Pairing; Binding; Bioinformatics; Biology; Cell Extracts; Cell Wall; Cell membrane; Cells; Charge; Chemicals; Chemistry; Coenzyme A; Copper; Coupled; Crude Extracts; Escherichia coli; Eubacterium; Eukaryota; Exhibits; Funding; Glycine; Goals; Hydrolysis; In Vitro; Individual; Investigation; Label; Mammalian Cell; Mass Spectrum Analysis; Membrane; Methods; Mycobacterium tuberculosis; N-myristoyltransferase; N-terminal; Organism; Peptides; Preparation; Proteins; Proteome; Reaction; Reagent; Regulation; Resistance; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Series; Solid; Specificity; Structure; Sulfhydryl Compounds; System; Technology; Testing; Transferase; Trypsin; Yeasts; activity-based protein profiling; analog; base; cell envelope; chloroacetyl coenzyme A; enzyme mechanism; experience; fluorophore; human NAT2 protein; human disease; in vivo; inhibitor/antagonist; interest; isoniazid; member; myristoylation; novel; pathogenic bacteria; programs; public health relevance; scaffold; small molecule; success; tool; two-dimensional

DESCRIPTION (provided by applicant): The long term goals of the present application are to precisely determine the identities of all the proteins and small molecules in pathogenic bacteria that are enzymatically acetylated and to similarly define the identities of all the proteins in yeast that are myristoylated. We propose to do this using our recently developed activity-based protein profiling method. In the case of the "acetylome", we will use chloroacetyl-CoA, a reagent that we discovered and showed that it can be used as a substrate by every Gcn5-related N-acetyltransferase (GNAT's) that we have tested to date. By enzymatically transferring the chloroacetyl group to the protein, this chemically "marks" that protein as a substrate for acetylation, and that mark can be chemically reacted with thiol-containing compounds to generate stable covalent species that can be purified and identified. While our earlier studies were performed with pure or relatively pure mixtures of proteins, in order to confirm that neither specificity nor regioselectivity were compromised with the reagent, we have since demonstrated that the method can be used in crude extracts of cells with equal efficiency and selectivity. The reagent is equally useful in the preparation of bisubstrate analogues that exhibit nanomolar binding affinity to the corresponding GNAT. We will thus establish all of the substrates for all the GNATs in selected organisms. Having identified all the substrates, we will then prepare bisubstrate analogues containing these substrates coupled to solid supports and use them as "bait" to identify the cognate GNAT. Those GNATs that are essential for cellular viability will be subjected to detailed enzymatic and structural analysis. In the case of the "myristoylome", we have shown that undecynoyl-CoA is an efficient substrate for the Saccharomyces cerevisiae protein - N-myristoyl-transferase, a member of the GNAT superfamily. This substrate will cause the undecynoylation of the 1-N-terminal glycine of what we estimate is 60 protein substrates, based on amino terminal sequence in yeast. Using azide-containing fluorophores and affinity purification tags, we will identify which of these 60 proteins is, in fact, myristoylated. In both projects, we will use two- dimensional SDS-PAGE/IEF to separate the labeled protein mixtures, and the identification of the proteins will be made using trypsin hydrolysis followed by HPLC-MS/MS. We have significant experience in these methods, having recently used them to define the many potential targets for isoniazid in Mycobacterium tuberculosis crude cell extracts. PUBLIC HEALTH RELEVANCE: The GNAT superfamily is one of the largest protein superfamilies and is responsible for acetylation, succinylation and myristoylation of both small molecules and proteins. A large number of these are implicated in regulation and in human disease.

描述（由申请人提供）：当前应用的长期目标是精确确定致病细菌中所有蛋白质和小分子的身份，这些蛋白质和小分子是酶上乙酰化的，并类似地定义了酵母中所有蛋白质的蛋白质的身份。我们建议使用我们最近开发的基于活动的蛋白质分析方法来做到这一点。对于“乙酰基团”，我们将使用氯乙酰基-COA，该试剂我们发现并表明它可以由我们迄今已测试的每个与GCN5相关的N-乙酰基转移酶（GNAT）用作底物。通过将氯乙酰基酶转移到蛋白质上，该化学上的“标记”是该蛋白作为乙酰化的底物，并且该标记可以与含硫醇的化合物化学反应，从而产生稳定的共价物种，可以纯化和鉴定出来。虽然我们先前的研究是用蛋白质的纯或相对纯混合物进行的，但为了确认与试剂的特异性或区域选择性均未损害，我们此后证明，该方法可以以同样的效率和选择性为基础的细胞提取物。该试剂在制备Bisubstrate类似物的制备中同样有用，该Bisubstrate类似物表现出与相应GNAT的纳摩尔结合亲和力。因此，我们将在选定生物体中建立所有GNAT的所有底物。鉴定了所有底物后，我们将准备包含这些基板与固体支撑的这些底物的Bisubstrate类似物，并将它们用作“诱饵”以识别同源GNAT。那些对细胞活力必不可少的gn g进行了详细的酶促和结构分析。在“肉豆蔻酰斑点”的情况下，我们已经表明，decynoyl-CoA是酿酒酵母蛋白的有效底物-N-米升转移酶，GNAT超级家族的成员。该底物将基于酵母中的氨基末端序列，导致我们估计的是60个蛋白质底物的1-N末端甘氨酸的未烯醇化。使用含有叠氮化物的荧光团和亲和力纯化标签，我们将确定这60种蛋白质中的哪种实际上是肉豆蔻酰化的。在这两个项目中，我们都将使用二维SDS-PAGE/IEF分离标记的蛋白质混合物，并使用胰蛋白酶水解进行鉴定，然后使用HPLC-MS/MS进行鉴定。我们在这些方法方面具有丰富的经验，最近使用它们来定义结核分枝杆菌粗细细胞提取物中异念珠菌的许多潜在靶标。公共卫生相关性：GNAT超家族是最大的蛋白质超家族之一，负责小分子和蛋白质的乙酰化，琥珀酰化和肉豆蔻酰化。其中大量与调节和人类疾病有关。