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.

摘要 本项目旨在阐明变构通过N-末端调节SIRT1活性的机制 SIRT1结构域，催化核心远端的构象动态区。SIRT1依赖于NAD+ 蛋白质脱乙酰酶，已被证明在许多生物途径中发挥重要作用，如胰岛素 分泌、肿瘤形成、脂代谢和神经退行性变。出于这个原因，已经确定了SIRT1 作为潜在的治疗靶点。这一进展受到了对 SIRT1活性调控的分子机制--SIRT1的C-末端和N-末端结构域 在变构影响SIRT1活性中扮演着复杂的角色。N端结构域已被显示为 增强SIRT1‘S酶的活性；该区域还含有STAC结合结构域(SBD)，这是一个结合SIRT1的位点 Sirtuin激活化合物(STAC)。然而，关于其作用的体外生物化学研究有限。 SIRT1调控中的N-末端结构域。我们的项目专注于了解变构相互作用。 在N-末端结构域和SIRT1‘S催化核心之间使用三个独立的目标，具体如下 关注白藜芦醇对SIRT1的底物依赖性调节，其他STAC对SIRT1的调节， 以及基序A对SIRT1的分子内调控，基序A位于其N-末端区域。 目的1：研究络合物稳定性和构象动力学在底物序列中的作用 白藜芦醇对SIRT1的依赖调节，白藜芦醇是一种研究得很好的STAC。我们将比较它们的结合亲和力 白藜芦醇以SIRT1为底物，在不同底物存在下，比较不同底物的稳定性 SIRT1·底物·白藜芦醇配合物，并比较不同结构的构象 SIRT1·底物·白藜芦醇复合体。 目的2：探索与白藜芦醇结构相似或不同的其他STAC是否也能调节SIRT1 底物顺序依赖方式。我们将表征底物序列依赖的其他 采用酶活性测定法和结合测定法对SIRT1活性的影响进行了研究。 目的3：阐明分子内SIRT1通过基序A的调控机制 SIRT1 N-末端的区域以及磷酸化在这一调控中的作用。我们将使用酶的活性 分析和结合分析，并辅之以分子动力学模拟，以确定 基序A上的磷酸化对SIRT1活性的调节能力。 我们的研究将使我们更详细地了解N-受体诱导的SIRT1的变构调节。 终端域。这将阐明SIRT1的活性在各种生物途径和疾病中是如何改变的 作为一种治疗方法，指导采取更有针对性的方法来调节SIRT1的活动。