A suite of genome-engineered hepatic cells provide novel insights into the spatiotemporal metabolism of APOB and APOB-containing lipoprotein secretion.

ABSTRACT

AIMS: APOB-containing very-low-density lipoprotein (VLDL) production, secretion, and clearance by hepatocytes is a central determinant of hepatic and circulating lipid levels. Impairment of any of the aforementioned processes is associated with the development of multiple diseases. Despite the discovery of genes and processes that govern hepatic VLDL metabolism, our understanding of the different mechanistic steps involved is far from complete. An impediment to these studies is the lack of tractable hepatocyte-based systems to interrogate and follow APOB in cells, which the current study addresses. METHODS AND RESULTS: To facilitate the cellular study of VLDL metabolism, we generated human hepatic HepG2 and Huh-7 cell lines in which CRISPR/Cas9-based genome engineering was used to introduce the fluorescent protein mNeonGreen into the APOB gene locus. This results in the production of APOB100-mNeon that localizes predominantly to the endoplasmic reticulum (ER) and Golgi by immunofluorescence and electron microscopy imaging. The production and secretion of APOB100-mNeon can be quantitatively followed in medium over time, and results in production of lipoproteins that are taken up via the LDLR pathway. Importantly, the production and secretion of APOB-mNeon is sensitive to established pharmacological and physiological treatments, and to genetic modifiers known to influence VLDL production in humans. As a showcase, we used HepG2-APOBmNeon cells to interrogate ER-associated degradation (ERAD) of APOB. Using a dedicated sgRNA library targeting all established membrane-associated ER-resident E3 ubiquitin ligases led to identification of SYNV1 as the E3 responsible for degradation of poorly-lipidated APOB in HepG2 cells. CONCLUSIONS: In summary, the engineered cells reported here allow the study of hepatic VLDL assembly and secretion, and facilitate spatiotemporal interrogation induced by pharmacologic and genetic perturbations.