Allosteric regulation of SIRT1 by a PACS-2 and DBC1 regulatory hub

项目总结 “营养感应”酶SIRT1位于一系列复杂的分子相互作用的十字路口， 影响各种疾病的易感性，如肥胖、糖尿病、神经变性和癌症。一 治疗肥胖相关疾病的有希望的治疗策略包括小分子STAC， 它们结合SIRT1的N-末端区域以变构增加SIRT1的活性，从而提高 PGC-1分解代谢/PPAR靶基因的表达有助于预防饮食诱导的肥胖。STAC 将SIRT1结合在位于SIRT1 N-末端区域远端的3-螺旋束中。这个3螺旋束是 被SIRT1 N-末端区域的上游元件保护，保护酶不受不明原因的 细胞调节器。因此，通过相互作用控制酶活性的细胞蛋白的鉴定 3-螺旋束是理解SIRT1调控的关键。我们公布的和初步的数据表明 PACS-2就是这样一种SIRT1调节器，而PACS-2、DBC1和SIRT1形成了一种新的三方集线器，其 控制SIRT1脱乙酰酶活性。DBC1与SIRT1的N-末端区域结合，在那里它促进PACS-2 招募和约束SIRT1。Pacs-2破坏关键的3-螺旋束的稳定性，从而抑制SIRT1的活性 从而抑制依赖sirt1的pGC-1/pPAR靶基因的激活。我们的长期目标 是为了了解SIRT1是如何被调控来控制人类的能量稳态的。这样做的目的是 具体的应用是确定PACS-2和DBC1如何协同作用以变构调制SIRT1 酶的活性以及这个调节中枢如何在体内控制对禁食或营养过剩的反应。我们的 中心假说是SIRT1/PACS-2或SIRT1/DBC1相互作用的中断将阻止高脂- 饮食诱导抑制SIRT1，因此，保护小鼠免受肝脏脂肪变性和胰岛素抵抗的影响。 在强大的初步数据的指导下，我们将通过追求三个具体目标来验证我们的假设：1)确定 PACS-2/SIRT1相互作用调控酶活性的构象和机制步骤； 确定PACS-2和DBC1的协同作用如何抑制SIRT1；以及3)确定生理 PACS-2、DBC1和SIRT1之间相互作用对肝脏代谢的重要性。方法是 创新是因为它将表征从原子结构到整个有机体的功能，以前的 在禁食和营养过剩期间控制能量代谢的未被认可的调节中枢。这项研究是 意义重大，因为它将揭示SIRT1调节器如何控制酶活性以及我们如何影响 他们的功能是改善健康。

PROJECT SUMMARY The “nutrient-sensing” enzyme SIRT1 lays at the crossroads of a complex array of molecular interactions that impact susceptibility to diseases as diverse as obesity, diabetes, neurodegeneration, and cancer. One promising therapeutic strategy for the treatment of obesity-related morbidity involves small-molecule STACs, which bind the SIRT1 N-terminal region to allosterically increase SIRT1 activity, thereby elevating the expression of catabolic PGC-1/PPAR target genes that help to protect against diet-induced obesity. STACs bind SIRT1 in a 3-helix bundle located at the distal part of the SIRT1 N-terminal region. This 3-helix bundle is shielded by an upstream element of the SIRT1 N-terminal region, protecting the enzyme from an unidentified cellular regulator. Thus, identification of cellular proteins that control enzyme activity through interaction with the 3-helix bundle is key to understanding SIRT1 regulation. Our published and preliminary data suggest PACS-2 is one such SIRT1 regulator and that PACS-2, DBC1 and SIRT1 form a novel tripartite hub that controls SIRT1 deacetylase activity. DBC1 binds to the N-terminal region of SIRT1, where it promotes PACS-2 recruitment and binding to SIRT1. PACS-2 destabilizes the critical 3-helix bundle, which inhibits SIRT1 activity and consequently represses SIRT1-dependent activation of PGC-1/PPAR target genes. Our long-term goal is to understand how SIRT1 is regulated to control energy homeostasis in humans. The objective of this particular application is to determine how PACS-2 and DBC1 synergize to allosterically modulate SIRT1 enzyme activity and how this regulatory hub controls the response to fasting or overnutrition in vivo. Our central hypothesis is that disruption of the SIRT1/PACS-2 or SIRT1/DBC1 interactions will prevent high-fat- diet-induced repression of SIRT1 and, therefore, protect mice from hepatic steatosis and insulin resistance. Guided by strong preliminary data, we will test our hypothesis by pursuing three specific aims: 1) Determine the conformational and mechanistic steps by which the PACS-2/SIRT1 interaction regulates enzyme activity; 2) Determine how synergistic actions by PACS-2 and DBC1 inhibit SIRT1; and 3) Determine the physiologic importance of interactions between PACS-2, DBC1, and SIRT1 on hepatic metabolism. The approach is innovative because it will characterize, from the atomic structure to whole-organism function, a previously unrecognized regulatory hub controlling energy metabolism during fasting and overnutrition. This research is significant because it will uncover how SIRT1 regulators control enzyme activity and how we can influence their functions to improve health.