The Role of O-Glycosylation on the InsP3R in Liver Metabolism

钙(Ca~(2+))调节肝脏的一系列功能，以响应激素和营养信号。这个项目的主要目标是(1)确定营养物质如何通过三磷酸肌醇受体(LnsP3R)的翻译后修饰来调节细胞核和细胞质中的钙信号，lnsP3R是内质网(ER)和核质网(NR)中发现的一种钙释放通道，以及(2)这一途径的失调如何导致代谢性肝病。营养通量导致O-连接的P-N-乙酰氨基葡萄糖(OGlcNAc)对细胞质和核蛋白的翻译后修饰。 这种动态和可逆的修饰正在成为细胞信号和代谢生理的关键营养传感器和调节因子。我们最近发现，lnsP3R被0-GlcNAc修饰，这种修饰降低了lnsP3R的单通道活性和内质网钙释放。这项PPG产生的初步数据表明，脂肪肝在内质网和核膜中诱导应激，并 NR，这可能导致胞浆和核内钙信号转导受损。基于这些发现，我们假设lnsP3R的0-GlcN酰化受葡萄糖和游离脂肪酸的控制，这转化为对肝细胞不同亚细胞亚室中lnsP3R的调节。在非酒精性脂肪性肝病中，这些调节事件反过来又参与ER/NR应激对钙信号的干扰。我们将通过以下具体目标来检验这一假说：(1)我们将确定葡萄糖和游离脂肪酸对细胞核中lnsP3R异构体0-GlcN酰化的影响 (2)我们将检测lnsP3R的0-GlcN酰化是否改变了lnsP3门控通道的活性和随后的具有核浆特异性的细胞内钙信号；以及(3)我们将确定代谢应激是否通过干扰lnsP3R0-GlcN酰化和核钙信号来促进肝脏脂肪变性。总的来说，这些研究将与项目1和项目3协同工作，以确定0-GlcN酰化lnsP3R在营养感知中的作用以及核钙信号在肝脏脂肪变性发展中的调节作用。

Calcium (Ca2+) regulates a wide range of functions in the liver in response to hormonal and nutritional signals. The broad goals of this project are to (1) define how nutrients regulate nuclear and cytosolic Ca2+ signaling through post-translational modifications of the inositol 1,4,5 trisphosphate receptor (lnsP3R), a Ca2+ release channel found in both the endoplasmic reticulum (ER) and nucleoplasmic reticulum (NR) and (2) how the dysregulation of this pathway contributes to metabolic liver disease. Nutrient flux leads to posttranslational modifications of cytoplasmic and nuclear proteins by O-linked P-N-acetylglucosamine (OGlcNAc). This dynamic and reversible modification is emerging as a key nutrient sensor and regulator of cell signaling and metabolic physiology. We recently discovered that the lnsP3R is modified by 0-GlcNAc and that this modification decreases lnsP3R single channel activity and Ca2+ release from ER. Preliminary data generated in this PPG suggest that fatty liver induces stress in the ER as well as the nuclear envelope and NR, which may result in impaired cytosolic and nuclear Ca2+ signaling. Based on these findings, we hypothesize that 0-GlcNAcylation of the lnsP3R is controlled by glucose and free fatty acids, which is translated into regulation of the lnsP3R in distinct subcellular compartments of hepatocytes. These regulatory events are, in turn, involved in the perturbation of Ca2+ signaling by ER/NR stress in nonalcoholic fatty liver disease. We will test this hypothesis through the following specific aims: (1) We will identify the effects of glucose and free fatty acids on 0-GlcNAcylation of the lnsP3R isofomns in the nucleus and cytosol; (2) we will examine whether 0-GlcNAcylation of the lnsP3R alters lnsP3-gated channel activity and subsequent intracellular Ca2+ signaling with nuclear and cytoplasmic specificity; and (3) we will detemnine whether metabolic stress promotes hepatic steatosis by perturbing lnsP3R 0-GlcNAcylation and nuclear Ca2+ signaling. Collectively, these studies will synergize with Projects 1 and 3 to define the role of 0-GlcNAcylation of the lnsP3R in nutrient sensing and the regulation of nuclear Ca2+ signaling in the development of hepatic steatosis.