The molecular mechanism and the functional role of pyruvate dehydrogenase complex regulation in macrophages

巨噬细胞丙酮酸脱氢酶复合物调控的分子机制及功能作用

• 批准号: 10324556
• 负责人: Gretchen Seim
• 金额: $ 1.9万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10324556
• 关键词: Acetyl Coenzyme A; Adopted; Adoption; Affect; Anti-Inflammatory Agents; Antigen Presentation; Arthritis; Cells; ChIP-seq; Chemicals; Citric Acid Cycle; Coenzyme A; Complex; Coupled; Data; Diabetes Mellitus; Disease; Enzymes; Epigenetic Process; Equilibrium; Gene Expression Profile; Genetic; Genetic Transcription; Health; Heart Diseases; Histone Acetylation; Human; Human Pathology; Immune; Immune response; Immunity; Infection; Inflammation; Inflammatory; Innate Immune System; Interferon Type II; Interferons; Intervention; Invaded; Ketoglutarate Dehydrogenase Complex; Lipopolysaccharides; Lysine; Macrophage Activation; Mass Spectrum Analysis; Measures; Mediating; Metabolic; Metabolism; Methods; Mitochondria; Modeling; Modification; Molecular; Natural Immunity; Nitric Oxide; Oxygen; Pathogenicity; Pathologic; Pathology; Pattern; Phagocytosis; Phase; Phenotype; Physiological; Play; Preventive therapy; Process; Production; Publishing; Pyruvate Dehydrogenase Complex; Regulation; Resolution; Rest; Role; Sepsis; Signal Transduction; Stimulus; Succinates; Sulfhydryl Compounds; Techniques; Testing; Thioctic Acid; Transacylase; Work; base; cell injury; cofactor; cost; cytokine; extracellular; immunoregulation; in vitro activity; insight; macrophage; novel; pathogen; pyruvate dehydrogenase complex E2; response; succinyl-coenzyme A; therapy development; tool

PROJECT SUMMARY: Precise control of innate immunity is critical for human health. Both insufficient or excess inflammation can have detrimental effects and both are related to a variety of common and costly human pathologies including sepsis, arthritis, heart disease, and diabetes. Macrophages are crucial players in the coordination of this balance. In response to extracellular signals, macrophages can adopt diverse phenotypes that act in both the mounting and resolution of the immune response. Therefore, detailed understanding of the mechanisms regulating macrophage function is crucial for understanding immune-mediated disease pathology. Increasing evidence has shown that metabolism is important in controlling macrophage function. When stimulated, macrophages dramatically and dynamically alter their metabolism. However, in many cases, the mechanisms controlling and functional relevance of these metabolic alterations are unknown. In response to signals associated with infection, lipopolysaccharide and interferon-γ (LPS and IFN-γ), macrophages rapidly develop a pro-inflammatory phenotype. Following this initial activation, the cells eventually transition into a more immuno-suppressive state. Coupled to the dynamic change in function is a dynamic change in metabolism. In particular, TCA cycle metabolism is substantially rewired, and this rewiring is largely driven by inhibition of pyruvate dehydrogenase complex (PDHC) activity. Altering PDHC activity affects the function of LPS and IFN-γ stimulated macrophages. However, the detailed mechanism controlling PDHC activity and the mechanisms dictating the functional importance of PDHC are unknown. Aim 1 will elucidate the molecular mechanism controlling PDHC inhibition. In response to LPS and IFN-γ stimulation, the activity of PDHC’s E2 subunit decreases. Data shows that this is due to increased covalent modification of the E2 cofactor lipoic acid, on its reactive thiol group. We will identify the modification using a targeted mass spectrometry technique and will assess its role in controlling PHDC activity using genetic or chemical perturbation of the processes required for modification. Aim 2 will test the hypothesis that PDHC inhibition, via control of its product acetyl-CoA, influences functionally relevant histone acetylation and gene expression patterns. To test this model, the impact of genetic and chemical manipulation of acetyl-CoA levels and PDHC activity on histone acetylation will be assessed. To identify the consequences of PDHC-regulated histone acetylation, ChIP-seq and qPCR analyses will assess the impact of PDHC modulation on the histone acetylation and transcriptional landscape. The proposed work will illuminate novel mechanisms directing the metabolic and epigenetic reprogramming in macrophages. It will provide a broader understanding of the control of inflammatory state in macrophages and lay the groundwork for developing metabolic interventions to modulate immunity and treat disease.

项目概要： 精确控制先天免疫对于人类健康至关重要。炎症不足或过度 可能会产生有害影响，并且两者都与各种常见且昂贵的人类病理有关，包括 败血症、关节炎、心脏病和糖尿病。巨噬细胞是协调这种平衡的关键参与者。 为了响应细胞外信号，巨噬细胞可以采取不同的表型，在安装和安装中发挥作用 和免疫反应的解决。因此，详细了解调节机制 巨噬细胞功能对于理解免疫介导的疾病病理学至关重要。 越来越多的证据表明新陈代谢对于控制巨噬细胞功能很重要。什么时候 受到刺激后，巨噬细胞会急剧地、动态地改变它们的新陈代谢。然而，在许多情况下， 这些代谢改变的控制机制和功能相关性尚不清楚。作为回应 与感染、脂多糖和干扰素-γ（LPS 和 IFN-γ）、巨噬细胞迅速相关的信号 形成促炎表型。在最初的激活之后，细胞最终转变为更 免疫抑制状态。与功能的动态变化相结合的是新陈代谢的动态变化。在 特别是，TCA 循环代谢显着重新连接，并且这种重新连接很大程度上是由抑制 丙酮酸脱氢酶复合物 (PDHC) 活性。改变 PDHC 活性会影响 LPS 和 IFN-γ 的功能 刺激巨噬细胞。然而，控制 PDHC 活性的详细机制和 决定 PDHC 功能重要性的机制尚不清楚。 目标 1 将阐明控制 PDHC 抑制的分子机制。响应 LPS 和 IFN-γ刺激后，PDHC的E2亚基活性降低。数据表明，这是由于共价键增加所致 E2 辅因子硫辛酸对其反应性硫醇基团的修饰。我们将使用 靶向质谱技术，并将使用遗传或评估其在控制 PHDC 活性中的作用 修饰所需过程的化学扰动。 目标 2 将检验以下假设：通过控制其产物乙酰辅酶 A 来抑制 PDHC， 影响功能相关的组蛋白乙酰化和基因表达模式。为了测试这个模型， 乙酰辅酶A水平和PDHC活性的遗传和化学操作对组蛋白乙酰化的影响将 进行评估。确定 PDHC 调节的组蛋白乙酰化、ChIP-seq 和 qPCR 的后果 分析将评估 PDHC 调节对组蛋白乙酰化和转录景观的影响。 拟议的工作将阐明指导代谢和表观遗传重编程的新机制 在巨噬细胞中。它将提供对巨噬细胞和炎症状态的控制的更广泛的理解。 为开发代谢干预措施以调节免疫力和治疗疾病奠定基础。