MODULATION OF APOLIPOPROTEIN B-100 DEGRADATION AND SECRETION BY HEPATIC PROTEIN QUALITY CONTROL COMPONENTS

Abstract

The central protein component of hepatic very low density lipoproteins (VLDL), apolipoprotein B-100 (apoB-100), is unique in that it is removed via endoplasmic reticulum-associated degradation (ERAD) when poorly lipidated. Protein quality control processes that govern apoB-100 production can impact the secretion rate of VLDL. The human hepatoma cell line HepG2 was used to characterize the functional role of several proteins in the apoB-100 ERAD pathway. A network of cellular protein components serves as the machinery to identify ERAD substrates, remove them from the ER and deliver them to the cytosolic proteasome. Here, the retrotranslocation of apoB-100 out of the ER was found to require p97, as the siRNA-mediated knockdown of p97 impaired the release of apoB-100 and polyubiquitinated apoB into the cytosol. Blocking p97-dependent retrotranslocation did not increase apoB-100 secretion, suggesting that apoB-100 was committed to degradation by the time it associated with p97. In contrast, knockdown of the ER-resident ubiquitin ligase gp78 decreased apoB-100 ubiquitination and increased apoB-100 secretion. Depletion of gp78 reversed the p97-dependent accumulation of apoB-100, suggesting that gp78 acted upstream of p97 during ERAD. Surprisingly, blocking gp78-dependent ubiquitination of apoB-100 allowed the assembly of more buoyant, lipid-rich VLDL. The cytosolic chaperone “holdase” Bag6 was identified by mass spectrometry and immunoblot to associate with apoB-100 following proteasome inhibition. Bag6 associated with newly synthesized, cytosolic apoB-100. Intriguingly, the depletion of Bag6 protein by siRNA reduced apoB-100 secretion efficiency by as much as 50%. Furthermore, the synthesis of apoB-100 was not affected, suggesting that the loss of Bag6 function increased the degradation of apoB-100. This degradation occured at a post-translational step of assembly by an unidentified mechanism. Taken together, these data suggest that ubiquitination is the committing step in the apoB-100 ERAD pathway and that ubiquitination may play a regulatory role in VLDL assembly. Elements of cellular quality control act as modulators of VLDL output, and may be dysregulated in several human diseases which are characterized by increased VLDL production.