The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics

Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery twenty years ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-drivencellular change. To investigate the transcriptional changes induced by PNPLA3-I148M, we established Hep3B cell lines expressing endogenous PNPLA3 (WT) or mutant PNPLA3-I148M (I148M). We then performed gene expression profiling obtained from RNA-Seq of the two cell lines with no treatment and treated with oleic acid for 0.5, 1, 2, 4, and 24 h. We compared the gene expression profiles between the cell lines under each treatment condition.

非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）近日更名为代谢功能障碍相关性脂肪性肝病（metabolic dysfunction-associated steatotic liver disease, MASLD），是一类以肝脏甘油三酯异常蓄积为起始病变的进行性代谢紊乱疾病，可进展为肝硬化与肝细胞癌。PNPLA3基因的常见变异编码PNPLA3-I148M蛋白，是目前已知的MASLD最强遗传风险因子。尽管该变异于二十年前被发现，但PNPLA3的蛋白功能以及PNPLA3-I148M的致病机制仍未明确。本研究旨在解析PNPLA3与PNPLA3-I148M的生物发生过程，并表征该致病变异内源性表达所诱导的细胞变化。与生物信息学预测及此前针对过表达蛋白的研究结论相悖，本研究证实PNPLA3与PNPLA3-I148M并非内质网驻留跨膜蛋白。为明确二者的细胞内定位关联，我们构建了一对等基因人类肝癌细胞系，可在内源性水平表达PNPLA3与PNPLA3-I148M。两种蛋白均富集于脂滴、高尔基体与内体组分中。纯化的PNPLA3与PNPLA3-I148M蛋白可与这些亚细胞分区中常见的磷酸肌醇结合。尽管与野生型蛋白具有相似的组分分离模式，PNPLA3-I148M可诱导高尔基体形态改变，包括增加脂滴-高尔基体接触位点，该现象同样在表达I148M的原代人类患者肝细胞中被观察到。除脂滴蓄积外，PNPLA3-I148M的表达还可引发显著的蛋白质组与转录组变化，该变化覆盖肝病的全病程阶段。综上，我们验证了可用于研究PNPLA3-I148M生物学特性的内源性人类细胞系统，并确定高尔基体是PNPLA3-I148M介导的细胞变化的核心枢纽。为探究PNPLA3-I148M诱导的转录变化，我们构建了可内源性表达野生型PNPLA3（WT）或突变型PNPLA3-I148M（I148M）的Hep3B细胞系。随后对两种细胞系分别进行未处理及经油酸处理0.5、1、2、4与24小时后的RNA测序（RNA-Seq）基因表达谱分析，并对比了各处理条件下两种细胞系的基因表达差异。