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

项目概要： 先天免疫的精确控制对人类健康至关重要。炎症不足或过度 可能具有有害影响，并且两者都与各种常见且昂贵的人类病理学有关， 脓毒症关节炎心脏病和糖尿病宏观经济是协调这种平衡的关键角色。 为了响应细胞外信号，巨噬细胞可以采取不同的表型，这些表型在安装 和免疫反应的消退。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是至关重要的。 越来越多的证据表明，代谢在控制巨噬细胞功能中是重要的。当 受刺激的巨噬细胞显著且动态地改变它们的代谢。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。响应于 与感染相关的信号，脂多糖和干扰素-γ（LPS和IFN-γ），巨噬细胞迅速 产生促炎表型。在这种初始激活之后，细胞最终过渡到一个更复杂的状态。 免疫抑制状态。与功能的动态变化相耦合的是新陈代谢的动态变化。在 特别地，TCA循环代谢实质上被重新布线，并且这种重新布线主要由抑制 丙酮酸脱氢酶复合物（PDHC）活性。PDHC活性改变影响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。 目的1阐明PDHC抑制的分子机制。为了应对LPS和 IFN-γ刺激后，PDHC的E2亚基活性降低。数据显示，这是由于共价键增加， E2辅因子硫辛酸在其反应性巯基上的修饰。我们将使用 靶向质谱技术，并将评估其在控制PHDC活动中的作用，使用遗传或 化学扰动的过程中所需的修改。 目的2将检验PDHC抑制，通过控制其产物乙酰辅酶A， 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活性遗传和化学操作对组蛋白乙酰化的影响将 进行评估。为了确定PDHC调节的组蛋白乙酰化的结果，ChIP-seq和qPCR 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中炎症状态控制的更广泛理解， 为开发代谢干预措施以调节免疫力和治疗疾病奠定基础。

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.