Mallosteric Regulation of the Sterol Pathway

HMG-CoA还原酶(HMGR)是类固醇途径的关键酶。HMGR正在接受反馈- 受调节的降解是从酵母到人类的保守。我们利用这个守恒定律来理解 酵母菌Hmg2同工酶调节降解的机制和机制。降解 由HRD ER相关降解(ERAD)质量控制途径发生，也是 错误折叠的内质网蛋白的降解。Hmg2 ERAD受类固醇途径分子GGPP调控。 GGPP通过引起可逆的错误折叠来完成控制，从而触发HRD途径的降解。 GGPP对Hmg2的作用具有许多变构调控的特征，我们将这种调控命名为 “Mallostery”结合了错误折叠和变构的概念。在拟议的研究中，我们将揭示 苹果酸对Hmg2的调节，以更好地了解类固醇途径的调控，并为高潜力 Mallostery被认为是广泛适用的药物发现途径。具体来说，我们将：1)研究 允许依赖GGPP的可逆错误折叠的Hmg2的特征：GGPP对Hmg2的影响 跨膜区是高度特异的，需要广泛保守的类固醇敏感结构域。 (固态硬盘)。我们将发现GGPP调控苹果酸的Hmg2的序列特征， 研究已知Hmg2基序的作用并发现无偏遗传的序列特征 方法；2)探索GGPP依赖调节Hmg2的机制-我们将测试 假设GGPP是Hmg2的高效配体，导致可逆的错误折叠 直接相互作用分析的发展、GGPP类似物的分析和体外重组 使用上一次资助中开发的一些工具和方法来管理Hmg2的泛素化 发现INSIG蛋白在GGPP介导的Hmg2-The INSIG错误折叠中的作用 蛋白质是哺乳动物脂类平衡的关键，在酵母中是保守的(Nsg1和Nsg2)。如中所示 在哺乳动物中，酵母INSIGs通过传递固醇信号发挥作用。酵母INSIG控制GGPP依赖 Hmg2错误折叠的方式依赖于生物合成的甾醇羊毛甾醇。因此，酵母INSIGs 对GGPP的Mallosteric调控施加第二层监管。我们将使用工具，变种人， 并从前期和方法两个方面进行研究，探讨其作用机制 生理上的INSIG在Hmg2调节的稳定性中起作用。我们还将进行基因筛查来收集 INSIG的更广泛的生物学，尽管关键和高度保守，但几乎一无所知 这些蛋白质在人类脂质稳态和病理学中的作用。

HMG-CoA reductase (HMGR) is a key enzyme of the sterol pathway. HMGR undergoes feedback- regulated degradation conserved from yeast to humans. We exploit this conservation to understand the machinery and mechanisms at play in regulated degradation of the yeast Hmg2 isozyme. Degradation occurs by the HRD ER-associated degradation (ERAD) quality control pathway, also responsible for the degradation of misfolded ER proteins. Hmg2 ERAD is regulated by sterol pathway molecule GGPP. GGPP accomplishes control by causing reversible misfolding that triggers HRD pathway degradation. GGPP’s action on Hmg2 has many features of allosteric control; we have named this type of regulation “mallostery” to combine the ideas of misfolding and allostery. In the proposed studies we will unravel the mallosteric regulation of Hmg2, to better understand sterol pathway control, and for the high potential mallostery holds as broadly applicable avenue of drug discovery. Specifically, we will : 1) Study the features of Hmg2 that allow GGPP-dependent reversible misfolding: GGPP’s effect on the Hmg2 transmembrane regions is highly specific, and requires the broadly conserved sterol sensing domain (SSD). We will discover the sequence features of Hmg2 responsible for mallosteric control by GGPP, investigating the role of known Hmg2 motifs and discovering sequence features with unbiased genetic approaches; 2) Explore the mechanism of GGPP dependent regulation of Hmg2- We will test the hypothesis that GGPP is a high potency ligand for Hmg2, causing reversible misfolding through development of direct interaction assays, analysis of GGPP analogues, and through in vitro reconstitution of regulated ubiquitination of Hmg2 using a number tools and approaches developed in the last funding cycle; and 3) Discover the role of INSIG proteins in GGPP-mediated misfolding of Hmg2- The INSIG proteins are critical in mammalian lipid homeostasis, and are conserved in yeast (Nsg1 and 2). As in mammals, yeast INSIGs function by transducing sterol signals. Yeast INSIG controls GGPP-dependent Hmg2 misfolding in a manner dependent on the biosynthetic sterol lanosterol. Thus the yeast INSIGs impose a second layer of regulation on mallosteric regulation by GGPP. We will use the tools, mutants, and methods from both the previous funding period, and the above studies to explore the mechanism and physiology INSIG function in Hmg2 regulated stability. We will also execute a genetic screen to glean the broader biology of INSIGs, about which almost nothing is known despite the key and highly conserved roles of these proteins in human lipid homeostasis and pathology.