Ferbas, J. | Search bioRxiv

bioRxiv2024

PNPLA3-I148M is a Neomorph that Interferes with Two Primary Hepatic Triglyceride Clearance Pathways

A common variant of PNPLA3, encoding PNPLA3-I148M, is the most significant genetic determinant of fatty liver disease worldwide. However, it is unclear precisely how PNPLA3-I148M drives disease risk. Here, we show that endogenous human PNPLA3-I148M impairs secretion of Apolipoprotein B (ApoB), the scaffolding protein of very low-density lipoproteins (VLDLs), from hepatocytes. This is not due to a generalized secretory pathway defect, nor is it equivalent to loss of function of PNPLA3. The VLDL secretory defect is conserved in mice expressing human I148M. Untargeted lipidomics reveal that I148M human cells are enriched in polyunsaturated fatty acid (PUFA)-containing triglycerides at the expense of PUFA-containing phosphatidylcholine, causing reduced membrane dynamics, concomitantly hindering biogenesis of secreted VLDLs. ApoB secretion is substantially rescued in I148M cells that overexpress ABHD5/CGI-58, an I148M binding partner that activates lipolysis by ATGL/PNPLA2 when not bound to I148M. Conversely, knocking down CGI-58 or PNPLA2 mimics I148M. We propose that neomorphic PNPLA3-I148M exacerbates fatty liver risk by simultaneously impeding two major CGI-58-dependent pathways for liver triglyceride clearance: lipolysis and secretion.

cell biology↗

bioRxiv2023

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 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 worlds population and has both environmental and genetic risk factors. A mutation in the gene PNPLA3 that 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.

cell biology↗