Novel mechanisms of UPR sensing and nonalcoholic fatty liver disease

 描述(由申请人提供)：该项目涉及未折叠蛋白反应(UPR)和非酒精性脂肪性肝病(NAFLD)的机制。当未折叠或错误折叠的蛋白质在内质网(ER)的管腔中积聚时，真核细胞采用一条保守的、基本重要的UPR途径来开启主要的ER伴侣和参与ER相关降解的蛋白质的表达，以缓解应激。这一关键的蛋白质质量控制机制由三个ER跨膜UPR传感器传递，包括IRE1、PERK和ATF6。普遍定期审议研究在医学上的意义是深远的；2014年授予森和志博士和彼得·沃尔特博士的阿尔伯特·拉斯克奖突出了这一点并得到了支持。然而，UPR传感器的触发机制仍然是一个激烈辩论的主题，鉴于UPR在生理和疾病中的普遍含义，解决这一问题非常重要。目前的模型包括错误折叠的蛋白质直接与ER传感器结合，以及GRP78从ER传感器的管腔结构域解离。这两个模型都不能充分解释该系统的精致敏感性和高效率，该系统允许细胞对压力做出实时反应和适应。我们已经产生了一系列令人兴奋的证据，表明CNPY2，一种以前在UPR中未知的ER蛋白，激活了所有三个UPR传感器，以响应ER压力。CNPY2在稳定状态下在肝脏中高表达，并可通过CHOP直接反式激活进一步被UPR诱导。小鼠cnpy2基因敲除(KO)沉默UPR通路，阻断CHOP表达，保护细胞免受UPR诱导的细胞死亡。值得注意的是，KO小鼠对高脂饮食(HFD)诱导的ER应激和非酒精性脂肪性肝病(NAFLD)具有高度保护作用。在内质网应激诱导下，CNPY2的过表达增加了细胞中的UPR和凋亡信号。因此，一种新的涉及CNPY2的UPR传感模型正在出现。我们假设CNPY2是肝脏中普遍的UPR的关键正向启动子，并且它是ER应激诱导的NAFLD所必需的。我们提出了两个具体的目标来解决我们的假设。目的1利用生化、生物物理和结构分析方法，确定CNPY2调控UPR启动的分子机制。目的2系统研究CNPY2在以下几个方面的作用：1)脂肪生成和脂代谢；2)调节脂蛋白分泌和低密度脂蛋白受体的生物发生；3)控制与线粒体-内质网信号传导有关的钙稳态，从而揭示CNPY2调控肝骨病的机制。总之，这一建议将对理解UPR传感的机制和NAFLD的发病机制产生重大影响。

 DESCRIPTION (provided by applicant): This project addresses the mechanism of unfolded protein response (UPR) and non-alcoholic fatty liver disease (NAFLD). Upon accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER), eukaryotic cells adopt a conserved and fundamentally important UPR pathway to turn on the expression of major ER chaperones and proteins involved in ER-associated degradation to alleviate stress. This critical protein quality control mechanism is transmitted by three ER trans-membrane UPR sensors including IRE1, PERK and ATF6. The significance of UPR research is far-reaching in medicine; it is highlighted and supported by the 2014 Albert Lasker Prize awarded to Drs. Kazutoshi Mori and Peter Walter. The mechanism whereby UPR sensors are triggered however remains a subject of intense debate, which is important to resolve given the ubiquitous implication of UPR in physiology and diseases. Present models include direct binding of ER sensors by misfolded proteins and dissociation of Grp78 from the luminal domains of ER sensors. Neither of the models sufficiently explains the exquisite sensitivity and high efficiency of the system which allows cells to react and adapt to stress in real time. We have produced an exciting body of evidence implicating CNPY2, a previously unknown ER protein in UPR, in activating all three UPR sensors in response to ER stress. CNPY2 is highly expressed in the liver in the steady state and can be further induced by UPR via direct transactivation by CHOP. Knockout (KO) of cnpy2 from mice silences UPR pathways, blocks CHOP expression, and protects cells from UPR-induced cell death. Remarkably, the KO mice are rendered highly protective against high fat diet (HFD)-induced ER stress and non-alcoholic fatty liver disease (NAFLD). In turn, CNPY2 overexpression increases the UPR and apoptotic signals in cells upon ER stress induction. Thus, a novel model of UPR sensing that involves CNPY2 is emerging. We hypothesize that CNPY2 is a critical positive initiator for general UPR in the liver, and that it i required for ER stress-induced NAFLD. We propose two specific aims to address our hypothesis. Aim 1 will determine the molecular mechanism of CNPY2- regulated UPR initiation, using biochemical, biophysical and structural approaches. Aim 2 will be focused on untangling the mechanisms of CNPY2 in regulating hepatosteatosis by systemically studying the roles of CNPY2 in: 1) lipogenesis and lipid metabolism; 2) regulating lipoprotein secretion and the biogenesis of LDL receptor; 3) controlling calcium homeostasis related to mitochondria-ER cross-talk. Collectively, this proposal shall have a significant impact in understanding the mechanism of UPR sensing, and the pathogenesis of NAFLD.