An LXR protein interaction network controlling macrophage lipid transporter expression in response to inflammatory-lipid crosstalk

巨噬细胞泡沫细胞是动脉粥样硬化发生和病理的主要驱动力。暴露于 巨噬细胞对类固醇脂和炎性TLR信号的调节作用改变了控制脂质的基因调控网络 动态平衡，有利于泡沫细胞的形成。我们对这一问题的理解存在着根本性的差距 TLR-脂质信号串扰影响这些网络并调节泡沫的分子机制 细胞形成。长期目标是了解泡沫细胞形成的分子机制， 用于动脉粥样硬化的治疗干预。阐明这些机制不仅会 促进我们对疾病病理学的理解，但也提供了对功能相互作用的重要见解 这可以被改造成增强脂质转运体的表达，作为新的治疗方法的一部分 对抗动脉粥样硬化。本提案的目标是确定NCOA5、SND1和SART1如何 功能调控脂转运蛋白基因的表达、功能和泡沫细胞的形成。中心假设是 这些蛋白质是在基因调控中组装的LXR转录复合体的关键成分 脂转运蛋白ABCA1和Abcg1的元件(GRE)，以及TLR-脂质串扰促进 这些复合体的组成和活性，通过磷酸化，有利于基因抑制，胆固醇 积聚和泡沫细胞的形成。根据初步数据，这一假说将通过以下方式进行验证 三个具体目标：(1)阐明LXR相互作用蛋白在控制脂质转运蛋白中的作用 表达、功能和泡沫细胞的形成；(2)阐明LXR相互作用的磷酸化作用 控制脂转运蛋白表达、功能和泡沫细胞形成的蛋白质；以及(3)表征 在泡沫细胞形成过程中，依赖LXR的调节复合体组装在脂质转运体GRE上，以及 在体内评估NCOA5在动脉粥样硬化中的作用。在第一个目标下，生化、分子和细胞 将使用方法来解决NCOA5、SND1和SART1在调节中的信号依赖作用 脂类转运蛋白的表达与泡沫细胞的形成。在第二个目标中，创新的目标群体 光谱(MS)策略将被用来识别信号依赖的磷酸化状态的变化 NCOA5、SND1和SART1，然后将使用功能研究来确定这些因素的影响 修改。对于第三个目标，一种创新的启动子浓缩定量MS(PE-QMS)方法将 用来表征在ABCA1和Abcg1的GRE处组装的蛋白质复合体的组成 体内的泡沫细胞。此外，我们将评估NCOA5在促进泡沫细胞形成和 活体动脉粥样硬化。这项拟议的研究意义重大，因为它将提供机制来解释 泡沫细胞的形成在分子水平上受到调控，并提供了对蛋白质和相互作用的洞察 可以有针对性地重新设计基因调控网络，以防止泡沫细胞的形成并中和 动脉硬化。这有可能使患有这种疾病的患者的健康受益。

Macrophage foam cells are major drivers of the development and pathology of atherosclerosis. Exposure of macrophages to sterol lipid and inflammatory TLR signals modifies gene regulatory networks controlling lipid homeostasis, which can favor foam cell formation. There exists a fundamental gap in our understanding of the molecular mechanisms by which TLR-lipid signal crosstalk impinges upon these networks and regulates foam cell formation. The long-term goal is to understand the molecular mechanisms governing foam cell formation, for the purpose of therapeutic intervention in atherosclerosis. Elucidating these mechanisms will not only advance our understanding of disease pathology, but also provide crucial insights into functional interactions that could be engineered to enhance lipid transporter expression as part of new therapeutic approaches to counteract atherosclerosis. The objective of this proposal is to determine how NCOA5, SND1, and SART1 function to control lipid transporter gene expression, function and foam cell formation. The central hypothesis is that these proteins are critical components of LXR transcriptional complexes assembled at the gene regulatory elements (GREs) of lipid transporters Abca1 and Abcg1, and that TLR-lipid crosstalk promotes changes in the composition and activity of these complexes, through phosphorylation, that favor gene repression, cholesterol accumulation and foam cell formation. Based on preliminary data, this hypothesis will be tested by pursuing three specific aims: (1) Elucidate the function of LXR-interacting proteins in the control of lipid transporter expression, function and foam cell formation; (2) Elucidate the role of phosphorylation of LXR-interacting proteins in the control of lipid transporter expression, function and foam cell formation; and (3) Characterize LXR-dependent regulatory complexes assembled on lipid transporter GREs during foam cell formation, and assess the role of NCOA5 in atherosclerosis, in vivo. Under the first aim, biochemical, molecular, and cellular approaches will be used to address the signal-dependent role of NCOA5, SND1, and SART1 in regulating expression of lipid transporters and foam cell formation. In the second aim, an innovative targeted mass spectrometry (MS) strategy will be used to identify signal-dependent changes in the phosphorylation state of NCOA5, SND1, and SART1, which will then be pursued using functional studies to define the impact of those modifications. For the third aim, an innovative promoter enrichment quantitative MS (PE-QMS) approach will be employed to characterize the composition of protein complexes assembled at GREs of Abca1 and Abcg1 in foam cells in vivo. Moreover, we will assess the importance of NCOA5 in promoting foam cell formation and atherosclerosis in vivo. The proposed research is significant because it will provide mechanisms to explain how foam cell formation is regulated at the molecular level, and provide insights into proteins and interactions that can be targeted to re-engineer gene regulatory networks to prevent foam cell formation and counteract atherosclerosis. This has the potential to benefit the health of patients suffering from this disease.