Chemical Genetic Elucidation of Histone Acetyltransferase Signaling Networks

组蛋白乙酰转移酶信号网络的化学遗传学阐明

• 批准号: 8939649
• 负责人: Hans Luecke
• 金额: $ 26.16万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939649
• 关键词: Acetyl Coenzyme A; Acetylation; Acetyltransferase; Active Sites; Acyl Coenzyme A; Affinity Chromatography; Animal Model; Binding; Cell Nucleus; Cells; Chemistry; Coenzyme A; Coenzymes; Diabetes Mellitus; Engineering; Enzymes; Event; Family; Goals; Histones; Human; Imagery; Knowledge; Language; Lead; Libraries; Link; Lysine; Malignant Neoplasms; Molecular; PCAF gene; Pathway interactions; Peptides; Phosphorylation; Post-Translational Protein Processing; Protein Acetylation; Proteins; Research; Signal Pathway; Signal Transduction; Tail; Techniques; chemical genetics; cofactor; genetic regulatory protein; histone acetyltransferase; human disease; in vivo; member; mutant; novel; programs; tissue/cell culture; tool

Regulatory proteins serve as integrators of inter-and intracellular signals. Cellular information is often communicated in a language of posttranslational protein modification. This theme has been particularly apparent in cellular signaling pathways that impinge on the transcriptional program coordinated in the nucleus of the cell. Protein acetylation has been known for many years, but our knowledge of proteins regulated by acetylation is considerably less than for protein phosphorylation. Recent studies indicate that several key transcriptional regulators are regulated by reversible acetylation, which underscores the need to better understand the relevant acetylation pathways in the cell, and to link these events to specific regulatory functions. Dissecting these circuits will hopefully lead to a better understanding and treatment of diabetes, cancer and other human diseases. We are developing a chemical genetic strategy to identify cellular substrates of GCN5 and PCAF, two human Histone Acetyltransferase (HATs). These enzymes belong to the highly conserved GNAT family of HATs. We have successfully engineered the GNAT active site to allow the acetyl coenzyme A (CoA) binding pocket to accommodate unnatural acyl CoAs. We generated a library of synthetic acyl CoAs and screened them against the engineered GNAT domain to identify unique enzyme cofactor pairs. This approach yielded a GNAT mutant that catalyzes acyl transfer from from an unnatural CoA conjugate to histone tail peptides and nucleosomal substrates. Importantly, this acyl CoA is not a cofactor for wild-type GNAT domain. Thus, the engineered HAT covalently and specifically modifies substrates with a chemically unique tag. We are now utilizing click chemistry to facilitate direct visualization and affinity purification of novel substrates for two members of the GNAT family. Enrichment of specific substrates of the GNAT enzymes will enable protein identification using mass spectrometric techniques. We are pursing this approach in mammalian tissue culture cells and a variety of eukaryotic model organisms.

调节蛋白作为细胞内和细胞间信号的整合者。细胞信息通常以翻译后蛋白质修饰的语言进行交流。这一主题在影响细胞核中协调的转录程序的细胞信号通路中尤其明显。蛋白质的乙酰化作用已经被发现很多年了，但是我们对乙酰化作用调控的蛋白质的了解远远少于对蛋白质磷酸化作用的了解。最近的研究表明，几个关键的转录调节因子的调节可逆乙酰化，这强调了需要更好地了解相关的乙酰化途径在细胞中，并将这些事件的具体调控功能。解剖这些回路将有望更好地理解和治疗糖尿病，癌症和其他人类疾病。 我们正在开发一种化学遗传策略来鉴定GCN 5和PCAF（两种人类组蛋白乙酰转移酶（HAT））的细胞底物。 这些酶属于HAT的高度保守的GNAT家族。 我们已经成功地设计了GNAT活性位点，使乙酰辅酶A（CoA）结合口袋，以适应非天然的酰基辅酶A。 我们生成了一个合成酰基辅酶A库，并针对工程化GNAT结构域对其进行筛选，以鉴定独特的酶辅因子对。 该方法产生了催化酰基从非天然CoA缀合物转移到组蛋白尾肽和核小体底物的GNAT突变体。 重要的是，这种酰基CoA不是野生型GNAT结构域的辅因子。 因此，工程化的HAT用化学独特的标签共价且特异性地修饰底物。 我们现在正在利用点击化学，以促进直接可视化和亲和纯化的新基板的两个成员的GNAT家族。 GNAT酶的特定底物的富集将使得能够使用质谱技术进行蛋白质鉴定。 我们正在哺乳动物组织培养细胞和各种真核生物模型中追求这种方法。