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

总结 本项目旨在阐明SIRT 1活性通过N-末端变构调节的机制。 SIRT 1的结构域，催化核心远端的构象动态区域。SIRT 1是NAD+依赖型 蛋白质脱乙酰基酶，其已被证明在许多生物途径中起重要作用，如胰岛素 分泌、肿瘤形成、脂质代谢和神经变性。因此，SIRT 1已被鉴定为 作为潜在的治疗靶点。这一进展由于对人权的认识不足而受到阻碍。 SIRT 1活性调节的分子机制，如SIRT 1内的C-末端和N-末端结构域 在变构影响SIRT 1活性中起复杂作用。N-末端结构域已被证明 增强SIRT 1的酶活性;该区域还包含STAC结合结构域（SBD）， 去乙酰化酶激活化合物（STACs）。然而，关于细胞因子的作用的体外生物化学研究有限。 SIRT 1调节中的N-末端结构域。我们的项目重点是了解变构相互作用 在N-末端结构域和SIRT 1的催化核心之间使用三个独立的目标，如下所述， 关注白藜芦醇对SIRT 1的底物依赖性调节，其他STAC对SIRT 1的调节， 以及SIRT 1通过基序A（其N-末端区域内的结构域）的分子内调节。 目的1：研究复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SIRT 1的依赖性调节，这是一种研究充分的STAC。我们将比较 白藜芦醇对SIRT 1在不同底物存在下，比较不同底物的稳定性 SIRT 1·底物·白藜芦醇复合物，并比较不同的构象 SIRT 1·底物·白藜芦醇复合物。 目的2：探索其他与白藜芦醇结构相似或不同的STAC是否也能调节SIRT 1， 底物序列依赖方式。我们将描述其他的底物序列依赖性效应 STAC，即白藜芦醇和SRT 2104对SIRT 1活性的影响，使用酶活性测定和结合测定。 目的3：阐明SIRT 1通过基序A（一种内在无序的结构域）进行分子内调控的机制。 SIRT 1的N-末端区域以及磷酸化在该调节中的作用。我们将利用酶的活性 分析和结合分析，辅以分子动力学模拟，以确定 磷酸化对基序A调节SIRT 1活性的能力的影响。 我们的研究将提供一个更详细的了解SIRT 1的变构调节引起的N- 末端域这将阐明SIRT 1的活性如何在各种生物途径和疾病中改变。 国家，指导一个更有针对性的方法在调节SIRT 1活性作为一种治疗方法。

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.