The molecular basis for how acetyl-coenzyme A links metabolism to gene expression

乙酰辅酶 A 如何将代谢与基因表达联系起来的分子基础

• 批准号: 9125794
• 负责人: Gleb Bazilevsky
• 金额: $ 4.36万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9125794
• 关键词: ATP Citrate (pro-S)-Lyase; Acetyl Coenzyme A; Acetylation; Acetyltransferase; Acids; Adenosine Triphosphate; Affect; Automobile Driving; Binding; Biological Assay; Calorimetry; Cancerous; Cell Line; Cell physiology; Cells; Citrates; Communication; Complement; Complex; Coupled; DNA; Data; Dependence; Diet; Enzymes; Exhibits; Factor X; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Glucose; Goals; Growth; Health; Histone Acetylation; Histones; Human; In Vitro; Kinetics; Laboratories; Length; Link; Lyase; Lysine; Malate Dehydrogenase; Mammalian Cell; Maps; Measures; Metabolism; Modification; Molecular; Mutagenesis; Mutation; Nature; Phosphorylation; Production; Proliferating; Property; Proteins; Radioactive; Recombinants; Reporting; Roentgen Rays; Signal Pathway; Signal Transduction; Structure; Surface; Testing; Thermodynamics; Titrations; Transcriptional Activation; Western Blotting; X-Ray Crystallography; adipocyte differentiation; base; biophysical properties; cancer cell; cancer therapy; carcinogenesis; cofactor; differential expression; enzyme activity; in vivo; knock-down; mutant; non-histone protein; p300/CBP-Associated Factor; protein complex; research study; response; stoichiometry; transcription factor; tumor growth; ATP Citrate (pro-S)-Lyase; Acetyl Coenzyme A; Acetylation; Acetyltransferase; Acids; Adenosine Triphosphate; Affect; Automobile Driving; Binding; Biological Assay; Calorimetry; Cancerous; Cell Line; Cell physiology; Cells; Citrates; Communication; Complement; Complex; Coupled; DNA; Data; Dependence; Diet; Enzymes; Exhibits; Factor X; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Glucose; Goals; Growth; Health; Histone Acetylation; Histones; Human; In Vitro; Kinetics; Laboratories; Length; Link; Lyase; Lysine; Malate Dehydrogenase; Mammalian Cell; Maps; Measures; Metabolism; Modification; Molecular; Mutagenesis; Mutation; Nature; Phosphorylation; Production; Proliferating; Property; Proteins; Radioactive; Recombinants; Reporting; Roentgen Rays; Signal Pathway; Signal Transduction; Structure; Surface; Testing; Thermodynamics; Titrations; Transcriptional Activation; Western Blotting; X-Ray Crystallography; adipocyte differentiation; base; biophysical properties; cancer cell; cancer therapy; carcinogenesis; cofactor; differential expression; enzyme activity; in vivo; knock-down; mutant; non-histone protein; p300/CBP-Associated Factor; protein complex; research study; response; stoichiometry; transcription factor; tumor growth

DESCRIPTION (provided by applicant): The goal of this proposal is to investigate how the essential metabolite acetyl-coenzyme A (Ac-CoA) links cellular metabolism to changes in gene expression. The lysine acetyltransferases (KATs) general control nonderepressible 5 (GCN5) and p300/CBP-associated factor (PCAF) are global transcription factors that function in multisubunit complexes to acetylate histone and non-histone proteins to regulate gene transcription. As a necessary cofactor for KAT activity, Ac-CoA cellular concentration is directly related to increased rates of GCN5 and PCAF activity, protein target acetylation, and the transcriptional activation of growth-promoting genes. In metazoans, Ac-CoA is produced from glucose-derived citrate by the enzyme adenosine triphosphate (ATP)-citrate lyase (ACLY). Our collaborator, Dr. Kathryn Wellen, has reported that ACLY links cellular metabolism to histone acetylation, with ACLY-dependent production of Ac-CoA driving increased histone acetylation. In addition, many actively-proliferating cells exhibit increased levels of ACLY, and the proliferation of cancer cells in particular is sensitive to the availability of glucose and proper ACLY function. ACLY also undergoes an increase in stability in response to elevated glucose levels and activation of growth-promoting signaling pathways by KAT-dependent transcription activation. Moreover, new unpublished data from the Wellen laboratory suggest that ACLY and PCAF form a complex in cells, complementing a recent report demonstrating that PCAF acetylates ACLY to promote ACLY stability and tumor growth. Yet, the nature of the communication between ACLY and GCN5/PCAF and how this communication may regulate the activity of each of the enzymes is not known. Based on these data, this proposal seeks to test the hypothesis that ACLY forms a direct complex with the GCN5 and PCAF to facilitate both ACLY acetylation and stability and GCN5 and PCAF acetylation activity towards histone substrates, to support the activation of growth promoting genes. This hypothesis will be investigated using in vitro pull-down assays on biochemically purified ACLY and GCN5/PCAF, in order to determine if they can bind directly to one another. The biophysical properties of the interaction will then be characterized with ITC, as well as the steady state kinetic parameters of the interacting enzymes by enzyme activity assays. Lastly, X-ray crystallography structure determination and mutagenesis experiments will provide the molecular basis for the interaction of ACLY with GCN5/PCAF. Lastly, this proposal will determine the in vivo consequence of the ACLY-GCN5/PCAF interaction by disrupting this interaction and interrogating mammalian cell lines for changes in global histone acetylation, the expression of specific metabolism and growth-promoting genes, and the ability of these cells to proliferate and differentiate. Together, these studies will delineate the molecular mechanism for how the AcCoA metabolite links cellular metabolism to gene expression and will provide new avenues for targeting ACLY function for cancer therapy.

描述（由申请人提供）：该提案的目的是研究基本代谢物乙酰辅酶A（AC-COA）如何将细胞代谢与基因表达的变化联系起来。赖氨酸乙酰基转移酶（KATS）通用对照非苯甲酸5（GCN5）和P300/CBP相关因子（PCAF）是全球转录因子，它们在多亚基复合物中起作用，可用于乙酰化组蛋白和非固定蛋白，以调节基因转录。作为KAT活性的必要辅助因子，AC-COA细胞浓度与GCN5和PCAF活性，蛋白质靶乙酰化的速率以及生长促进基因的转录激活直接相关。在后生动物中，AC-COA是由葡萄糖衍生的柠檬酸盐由三磷酸腺苷（ATP） - 西氯酸酯（ACLY）产生的。我们的合作者Kathryn Wellen博士报告说，Acly将细胞代谢与组蛋白乙酰化联系起来，AC-COA驱动的ACLY依赖性产生增加了组蛋白乙酰化。此外，许多积极扩散的细胞表现出酸水水平的增加，尤其是癌细胞的增殖对葡萄糖的可用性和适当的acly功能敏感。 Acly还会通过依赖Kat依赖性转录激活的葡萄糖水平升高和激活促进生长的信号通路的稳定性增加。此外，韦尔伦实验室的新未发表的数据表明，Acly和PCAF在细胞中形成了复合物，并补充了最近的一份报告，表明PCAF乙酰酸盐Acly可以促进Acly稳定性和肿瘤生长。然而，尚不清楚ACLY和GCN5/PCAF之间的通信性质以及这种通信如何调节每种酶的活性。基于这些数据，该提案旨在检验以下假设：ACLY与GCN5和PCAF形成直接复合物，以促进乙酰化乙酰化和稳定性，以及GCN5和PCAF乙酰化活性对组蛋白底物，以支持促进生长基因的激活。该假设将使用生化纯化的Acly和GCN5/PCAF上的体外下拉测定法进行研究，以确定它们是否可以直接结合彼此结合。然后，相互作用的生物物理特性将以ITC以及通过酶活性测定的相互作用酶的稳态动力学参数来表征。最后，X射线晶体学结构的测定和诱变实验将为ACLY与GCN5/PCAF的相互作用提供分子基础。最后，该提案将通过破坏这种相互作用并询问哺乳动物细胞系的体内后果，以使哺乳动物细胞系的变化，特定的代谢和促进生长基因的表达以及这些细胞增殖和分化的能力。总之，这些研究将描述Accoa代谢产物如何将细胞代谢与基因表达联系起来的分子机制，并将为靶向癌症治疗的ACLY功能提供新的途径。