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Published April 30, 2024 | Published
Journal Article Open

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

Abstract

Nonalcoholic fatty liver disease, 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 20 y 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.

Copyright and License

© 2024 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).

Acknowledgement

We thank Mikhal Hardy and Mike Ollmann for initial generation of genetically modified Hep3B cells; Spiros Garbis for mass spectrometry/proteomics guidance and support; Tom Nguyen, Jiamiao Lu, Chi-Ming Li, Oliver Homann, Xuan Liu, and Xin Luo for RNA sequencing sample preparation and data analysis support; Bram Estes, Kevin Graham, and Katie Douglas for support with cloning and protein production; and Malia Potts, Simon Jackson, and Saptarsi Haldar for critical reads and discussions of manuscript drafts. The Proteome Exploration Laboratory was supported by NIH OD010788, NIH OD020013, the Betty and Gordon Moore Foundation through grant GBMF775, and the Beckman Institute at Caltech. The electron microscopy work was supported by NIH grant P50AI150464 and the Beckman Electron Microscopy Center at Caltech. Amgen Inc. paid for the research in the study, although the work does not have direct financial implications for Amgen Inc.

Contributions

D.J.S., R.V., I.C.R., and R.J.D. designed research; D.J.S., L.L., B.L., and M.S.L. performed research; J.X. and J.F. contributed new reagents/analytic tools; D.J.S., L.L., J.L.M., B.L., and M.S.L. analyzed data; J.L.M. reviewed and edited manuscript; R.V. reviewed and edited manuscript and advised on research; I.C.R. and R.J.D. reviewed and edited manuscript, advised on research, and supervised research; and D.J.S. wrote the paper.

Data Availability

Mass spectrometry-based proteomics RNA-seq data have been deposited in PRIDE (for Proteomics) (69) and GEO (for RNA-seq) (70) [PXD046335 (for PRIDE) GSE261297 (for GEO)].

Conflict of Interest

D.J.S., L.L., J.L.M., J.X., J.F., R.V., I.C.R., and R.J.D. are or were employees of Amgen Inc., although this study was conducted as postdoctoral research for D.J.S. and does not have direct financial implications for Amgen Inc. L.L., J.F., R.V., I.C.R., and R.J.D. are Amgen shareholders. The authors declare no other competing interests.

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Additional details

Created:
May 1, 2024
Modified:
June 17, 2024