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: 8741531
• 负责人: Hans Luecke
• 金额: $ 40.38万
• 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/8741531
• 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.

调节蛋白充当细胞间和细胞内信号的整合者。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传导途径中尤其明显。蛋白质乙酰化已为人所知很多年，但我们对乙酰化调节蛋白质的了解远远少于对蛋白质磷酸化的了解。最近的研究表明，几个关键的转录调节因子受到可逆乙酰化的调节，这强调需要更好地了解细胞中相关的乙酰化途径，并将这些事件与特定的调节功能联系起来。剖析这些回路有望有助于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识别 GCN5 和 PCAF（两种人类组蛋白乙酰转移酶 (HAT)）的细胞底物。 这些酶属于高度保守的 GNAT HAT 家族。 我们成功地设计了 GNAT 活性位点，使乙酰辅酶 A (CoA) 结合口袋能够容纳非天然酰基 CoA。 我们生成了一个合成酰基 CoA 文库，并针对工程化的 GNAT 结构域对其进行筛选，以识别独特的酶辅因子对。 这种方法产生了一种 GNAT 突变体，可催化从非天然 CoA 缀合物到组蛋白尾肽和核小体底物的酰基转移。 重要的是，该酰基 CoA 不是野生型 GNAT 结构域的辅助因子。 因此，工程化的 HAT 通过化学上独特的标签共价且特异性地修饰底物。 我们现在利用点击化学来促进 GNAT 家族两个成员的新型底物的直接可视化和亲和纯化。 富集 GNAT 酶的特定底物将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模型生物中探索这种方法。