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 SIRT1 活性通过 N 末端变构调节的机制 SIRT1 的结构域是催化核心远端的构象动态区域，是 NAD+ 依赖性的。 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，例如胰岛素 因此，SIRT1 已被鉴定。 作为一个潜在的治疗靶点，这一进展受到了缺乏了解的阻碍。 SIRT1 活性调节的分子机制，如 SIRT1 内的 C 端和 N 端结构域 N 末端结构域在变构影响 SIRT1 活性中发挥着复杂的作用。 增强 SIRT1 的酶活性；该区域还包含 STAC 结合域 (SBD)，这是 然而，关于 sirtuin 激活化合物（STAC）的作用的体外生物化学研究有限。 我们的项目重点是了解 SIRT1 调控中的 N 端结构域。 N 端结构域和 SIRT1 催化核心之间使用三个独立的目标，如下详述： 重点关注白藜芦醇对 SIRT1 的底物依赖性调节，以及其他 STAC 对 SIRT1 的调节， SIRT1 通过基序 A（其 N 末端区域内的一个结构域）进行的分子内调节。 目标 1：检查底物序列中复合物稳定性和构象动力学的作用 白藜芦醇（一种经过充分研究的 STAC）对 SIRT1 的依赖性调节我们将比较白藜芦醇的结合亲和力。 白藜芦醇对SIRT1在不同底物存在下的稳定性比较 SIRT1•底物•白藜芦醇复合物，并比较不同的构象 SIRT1•底物•白藜芦醇复合物。 目标 2：探索与白藜芦醇具有相似或不同结构的其他 STAC 是否也可以调节 SIRT1 我们将表征其他底物序列依赖性效应。 使用酶活性测定和结合测定来研究 STAC（即白皮杉醇和 SRT2104）对 SIRT1 活性的影响。 目标 3：阐明 SIRT1 通过基序 A 进行分子内调节的机制，基序 A 是一种本质上无序的 SIRT1 N 末端区域以及磷酸化在此调节中的作用我们将使用酶活性。 测定和结合测定，辅以分子动力学模拟，以确定的影响 基序 A 的磷酸化调节 SIRT1 活性的能力。 我们的研究将更详细地了解 N- 引起的 SIRT1 变构调节 这将阐明 SIRT1 的活性在各种生物途径和疾病中的作用。 指出，指导一种更有针对性的方法来调节 SIRT1 活性作为治疗方法。