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

Abstract

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 genePNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD to date. 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-driven cellular change.Significance StatementFatty liver disease affects nearly a quarter of the world’s population and has both environmental and genetic risk factors. A mutation in the genePNPLA3that converts Ile 148 to Met is the strongest known genetic risk factor for developing fatty liver disease. Using a series of techniques to track endogenous PNPLA3 and PNPLA3-I148M biogenesis and localization, we reveal new insights into how the mutation changes cellular dynamics. Although previous reports focus on its role on lipid droplets, we reveal that PNPLA3-I148M also functions at the Golgi apparatus, an organelle critical for protein transport into and out of the cell and lipid signaling. PNPLA3-I148M causes altered Golgi morphology and drives changes reminiscent of liver disease.

RAB18 Regulates Extrahepatic SiRNA-Mediated Gene Silencing Efficacy

“…Of the cell structures examined, the most noteworthy difference was colocalization of siRNA with LDs after 2 days of siRNA treatment (Figure 7B, Supplementary Figure 9), where a 20% reduction was observed in Hep3BRAB18KO compared to parental cells. To further validate this finding, label-free live-cell holotomographic 16,17 of LDs and siRNA was conducted in Hep3BCas9 and Hep3BRAB18KO cells. As shown in Figure 7C, colocalization analysis on this live-cell imaging revealed a similar reduction of siRNA-LD…”

“…The characteristic refractive index of lipid droplets clearly enables their segmentation from the remainder of the cell 16,17 . Lable-free imaging of live Hep3B cells was performed as previously described 16 .…”

“…The characteristic refractive index of lipid droplets clearly enables their segmentation from the remainder of the cell 16,17 . Lable-free imaging of live Hep3B cells was performed as previously described 16 . Briefly, Hep3BCas9 and Hep3BRAB18KO cells were seeded onto 96-well glassbottom plates (MatTek Corporation) (10,000-12,000 cells per 90 µL of phenol red-free EMEM medium (Quality Biological)) with vehicle, 1.5 µM GalNAc-HPRT siRNA (8172) or 1.5 µM AF647 conjugated GalNAc-HPRT siRNA and incubated in tissue culture incubator supplied with 5% CO2 for 1 day or 2 days.…”

“…After 1 hour of plate thermalization, refractive index and fluorescence images (CY5 channel) were acquired using the 3x3 GridScan mode (a field view of 236 x 236 µm). For lipid droplet characterization, cells and lipid droplets in refractive images were segmented and quantified using the Smart Lipid Droplet Assay module (version 2.0) in the AI/ML powered Nanolive 16,17 . For colocalization measurement between AF647 conjugated GalNAc-HPRT siRNA and lipid droplets, refractive and fluorescence images were imported into Huygens Professional software (version 22.10) (Scientific Volume Imaging B.V., Hilversum, Netherlands).…”