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

钙（Ca 2+）调节肝脏中响应激素和营养信号的广泛功能。该项目的主要目标是：（1）确定营养素如何通过三磷酸肌醇受体（InsP 3R）的翻译后修饰来调节细胞核和胞质Ca 2+信号传导，InsP 3R是一种在内质网（ER）和核质网（NR）中发现的Ca 2+释放通道，（2）该途径的失调如何导致代谢性肝病。营养流导致O-连接的P-N-乙酰葡糖胺（OGlcNAc）对细胞质和核蛋白进行翻译后修饰。 这种动态和可逆的修饰正在成为细胞信号传导和代谢生理学的关键营养传感器和调节器。我们最近发现InsP 3R被0-GlcNAc修饰，并且这种修饰降低了InsP 3R单通道活性和ER的Ca 2+释放。在该PPG中产生的初步数据表明，脂肪肝诱导ER以及核被膜中的应激， NR，这可能导致受损的胞质和核Ca 2+信号传导。基于这些发现，我们假设lnsP 3R的0-GlcNAc酰化受葡萄糖和游离脂肪酸控制，这被翻译成在肝细胞的不同亚细胞区室中对lnsP 3R的调节。这些调节事件反过来又参与了非酒精性脂肪肝中ER/NR应激对Ca 2+信号的干扰。我们将通过以下具体目标来验证这一假设：（1）我们将鉴定葡萄糖和游离脂肪酸对细胞核中InsP 3R异构体的0-GlcNAc化的影响 和胞质溶胶;（2）我们将检查InsP 3R的0-GlcNAc酰化是否改变InsP 3门控通道活性和随后具有核和胞质特异性的细胞内Ca 2+信号传导;和（3）我们将确定代谢应激是否通过干扰InsP 3R 0-GlcNAc酰化和核Ca 2+信号传导促进肝脂肪变性。总的来说，这些研究将与项目1和3协同作用，以确定InsP 3R的0-GlcNAc酰化在营养传感和肝脂肪变性发展中核Ca 2+信号传导调节中的作用。

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.