A suite of genome-engineered hepatic cells provides novel insights into the spatiotemporal metabolism of apolipoprotein B and apolipoprotein B-containing lipoprotein secretion.

Autor:	Meurs A; Department of Medical Biochemistry, Amsterdam UMC, Amsterdam Gastroenterology Endocrinology Metabolism and Amsterdam Cardiovascular Sciences, University of Amsterdam, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands., Ndoj K; Department of Medical Biochemistry, Amsterdam UMC, Amsterdam Gastroenterology Endocrinology Metabolism and Amsterdam Cardiovascular Sciences, University of Amsterdam, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands., van den Berg M; Department of Medical Biochemistry, Amsterdam UMC, Amsterdam Gastroenterology Endocrinology Metabolism and Amsterdam Cardiovascular Sciences, University of Amsterdam, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands., Marinković G; Department of Medical Biochemistry, Amsterdam UMC, Amsterdam Gastroenterology Endocrinology Metabolism and Amsterdam Cardiovascular Sciences, University of Amsterdam, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands., Tantucci M; Center for Molecular Medicine-Cell Biology, University Medical Center Utrecht, University of Utrecht, Heidelberglaan 100, 3584CX Utrecht, The Netherlands., Veenendaal T; Center for Molecular Medicine-Cell Biology, University Medical Center Utrecht, University of Utrecht, Heidelberglaan 100, 3584CX Utrecht, The Netherlands., Kuivenhoven JA; Department of Pediatrics, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands., Klumperman J; Center for Molecular Medicine-Cell Biology, University Medical Center Utrecht, University of Utrecht, Heidelberglaan 100, 3584CX Utrecht, The Netherlands., Zelcer N; Department of Medical Biochemistry, Amsterdam UMC, Amsterdam Gastroenterology Endocrinology Metabolism and Amsterdam Cardiovascular Sciences, University of Amsterdam, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands.
Jazyk:	angličtina
Zdroj:	Cardiovascular research [Cardiovasc Res] 2024 Sep 21; Vol. 120 (11), pp. 1253-1264.
DOI:	10.1093/cvr/cvae121
Abstrakt:	Aims: Apolipoprotein B (APOB)-containing very LDL (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 the production of lipoproteins that are taken up via the LDL receptor 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 of APOB. The use of a dedicated sgRNA library targeting all established membrane-associated ER-resident E3 ubiquitin ligases led to the identification of SYNV1 as the E3 responsible for the 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. Competing Interests: Conflict of interest: none declared. (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)
Databáze:	MEDLINE
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