Elucidating the Mechanism for Allosteric Regulation of SIRT1 through the N-terminal Region

阐明 SIRT1 通过 N 末端区域变构调节的机制

• 批准号: 10627735
• 负责人: Ningkun Wang
• 金额: $ 14.28万
• 依托单位: SAN JOSE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-05 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10627735
• 关键词: Affect; Affinity; Allosteric Regulation; Alzheimer' s Disease; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Assay; C-terminal; Catalytic Domain; Cell Culture Techniques; Cells; Complement; Complex; Disease; Distal; Enzymes; Event; Goals; Health; Human; In Vitro; Knowledge; Light; Methods; Molecular; Molecular Conformation; N-terminal; Nature; Nerve Degeneration; Non-Insulin-Dependent Diabetes Mellitus; Pathway interactions; Phosphorylation; Phosphorylation Site; Piceatannol; Play; Post-Translational Protein Processing; Property; Proteins; Regulation; Research; Rest; Resveratrol; Role; SIRT1 gene; Sir2-like Deacetylases; Sirtuins; Structure; Therapeutic; Work; design; enzyme activity; inhibitor; insulin secretion; lipid metabolism; molecular dynamics; small molecule; targeted treatment; therapeutic target; tumor

Summary This project aims to elucidate the mechanism for allosteric regulation of SIRT1 activity via the N-terminal domain of SIRT1, a conformationally dynamic region distal to the catalytic core. SIRT1 is an NAD+-dependent protein deacetylase which has been shown to play a significant role in many biological pathways, such as insulin secretion, tumor formation, lipid metabolism and neurodegeneration. For this reason, SIRT1 has been identified as a potential therapeutic target. This progress has been hampered by insufficient understanding of the molecular mechanism of the regulation of SIRT1 activity, as the C-terminal and N-terminal domains within SIRT1 play complicated roles in allosterically affecting SIRT1 activity. The N-terminal domain has been shown to potentiate SIRT1’s enzyme activity; this region also contains the STAC binding domain (SBD), a binding site for sirtuin activating compounds (STACs). However, there is limited in vitro biochemistry study regarding the role of the N-terminal domain in SIRT1 regulation. Our project is focused on understanding the allosteric interactions between the N-terminal domain and SIRT1’s catalytic core using three independent aims as detailed below that focus on the substrate-dependent regulation of SIRT1 by resveratrol, the regulation of SIRT1 by other STACs, and the intramolecular regulation of SIRT1 by motif A, a domain within its N-terminal region. Aim 1: Examine the role of complex stability and conformational dynamics in the substrate-sequence dependent regulation of SIRT1 by resveratrol, a well-studied STAC. We will compare the binding affinity of resveratrol to SIRT1 in the presence of different substrates, compare the stability of different SIRT1•substrate•resveratrol complexes, and compare the conformations of different SIRT1•substrate•resveratrol complexes. Aim 2: Explore if other STACs with similar or different structures as resveratrol can also regulate SIRT1 in a substrate-sequence dependent manner. We will characterize the substrate-sequence dependent effect of other STACs, namely piceatannol and SRT2104 on SIRT1 activity using enzyme activity assays and binding assays. Aim 3: Elucidate the mechanism of intramolecular SIRT1 regulation via motif A, an intrinsically disordered region in the N-terminus of SIRT1 and the role of phosphorylation in this regulation. We will use enzyme activity assays and binding assays, complemented by molecular dynamics simulations, to determine the effects of phosphorylation on motif A’s ability to regulate SIRT1 activity. Our studies will afford a more detailed understanding of the allosteric regulation of SIRT1 elicited by the N- terminal domain. This would clarify how the activity of SIRT1 is altered in various biological pathways and disease states, guiding a more targeted approach in modulating SIRT1 activity as a therapeutic method.

概括 该项目旨在阐明通过N末端对SIRT1活性变构调节的机制 SIRT1的结构域，催化核心远端的构象动态区域。 SIRT1是NAD+依赖性的 蛋白质脱乙酰基酶在许多生物学途径中都起着重要作用，例如胰岛素 分泌，肿瘤形成，脂质代谢和神经退行性。因此，已经确定了SIRT1 作为潜在的治疗靶标。由于不足的了解 SIRT1活性调节的分子机制，作为SIRT1中的C末端和N末端结构域 在变构影响SIRT1活动中扮演复杂的角色。 N末端结构域已显示为 潜在的SIRT1的酶活性；该区域还包含STAC结合域（SBD），这是一个结合位点 Sirtuin激活化合物（Stacs）。但是，关于体外生物化学研究的作用有限 SIRT1调节中的N末端结构域。我们的项目专注于理解变构互动 在N末端域和Sirt1的催化核心之间使用三个独立目标，如下所述 专注于白藜芦醇对SIRT1的底物依赖性调节，其他Stacs对SIRT1的调节， 以及通过其N末端区域内的一个结构域对SIRT1的分子内调节。 目标1：检查复杂稳定性和构象动力学在基材序列中的作用 白藜芦醇对SIRT1的依赖调节，这是一个经过良好研究的STAC。我们将比较 白藜芦醇在存在不同底物的情况下与SIRT1，比较不同的稳定性 SIRT1•基板•白藜芦醇复合物，并比较不同的构象 SIRT1•基材•白藜芦醇复合物。 AIM 2：探索其他与白藜芦醇相似或不同结构的Stacs是否也可以调节SIRT1 基材 - 序列依赖性方式。我们将表征其他其他依赖性效应 STACS，即使用酶活性测定和结合测定的SIRT1活性上的Piceatannol和Srt2104。 AIM 3：阐明分子内SIRT1调节的机理，通过本质上无序 SIRT1 N末端的区域以及该调节中磷酸化的作用。我们将使用酶活性 通过分子动力学模拟完成的测定和结合测定，以确定 图案A调节SIRT1活性的能力上的磷酸化。 我们的研究将对N-引起的SIRT1的变构调节有更详细的了解 终端域。这将阐明SIRT1的活性如何在各种生物学途径和疾病中改变 状态，指导一种更具针对性的方法来调节SIRT1活性作为治疗方法。