The Inhibition of Microsomal Triglyceride Transfer Protein Activity in Rat Hepatoma Cells Promotes Proteasomal and Nonproteasomal Degradation of Apoprotein B100

Abstract

In the human hepatic cell line, HepG2, apolipoprotein B100 (apoB100) degradation is increased by inhibiting lipid transfer mediated by the microsomal triglyceride transfer protein (MTP) and is predominantly accomplished by the ubiquitin-proteasome pathway. In the current study, we determined whether this degradative pathway was restricted to HepG2 cells or was of more general importance in hepatic apoB100 metabolism. Rat hepatoma McArdle RH7777 cells (McA), compared to HepG2 cells, secrete a large fraction of apoB100 associated with VLDL particles, as does the normal mammalian liver. In McA cells studied under basal conditions, the proteasome inhibitor lactacystin (LAC) increased apoB100 recovery, indicating that the role of the proteasome in apoB100 metabolism is not restricted to HepG2 cells. When apoB100 lipidation was blocked by an inhibitor of MTP (MTPI), recovery of cellular apoB100 was markedly reduced, but LAC was only partially ( approximately 50%) effective in reversing the induced degradation. This partial effectiveness of LAC may have represented either (1) incomplete inhibition by LAC of its preferred target, the chymotrypsin-like activity of the proteasome, (2) the presence of an apoB100 proteolytic activity of the proteasome resistant to LAC, or (3) a nonproteasomal proteolytic pathway of apoB100 degradation. By studying immunoisolated proteasomes and McA cells treated with LAC and/or MTPI and a variety of protease inhibitors, we determined that the proteasomal component of apoB100 degradation was entirely attributable to the chymotrypsin-like catalytic activity, but only accounted for part of apoB100 degradation induced by MTPI. The nonproteasomal apoB100 degradative pathway was nonlysosomal and resistant to E64d, DTT, and peptide aldehydes such as MG132 or ALLN but was partially sensitive to the serine protease inhibitor APMSF. Furthermore, when the protein trafficking inhibitor, brefeldin A, was used to block endoplasmic reticulum (ER) to Golgi transport in MTPI-treated McA cells, degradative activity resistant to LAC was increased, suggesting that the nonproteasomal pathway is associated with the ER.

Lipid aldehyde-mediated cross-linking of apolipoprotein B-100 inhibits secretion from HepG2 cells

“…In contrast, 4-ONE treatment was observed to strongly inhibit ApoB secretion in a concentration-dependent manner (Figure 4B). Brefeldin A, an agent that disrupts ER to Golgi transport [45], was used as a positive control for inhibition of VLDL secretion (Figure 4B). Thus, 4-HNE and 4-ONE had differing effects on ApoB secretion from HepG2 cells, suggesting that temporary microtubule disruption mediated by lipid aldehydes was not primarily responsible for alterations in ApoB secretion.…”

Recent discoveries on absorption of dietary fat: Presence, synthesis, and metabolism of cytoplasmic lipid droplets within enterocytes

“…MTP physically interacts with APOB and stabilizes its structure [101]. When MTP is inhibited or missing, APOB is misfolded and undergoes ubiquitination and degradation through the proteasomal pathway [102]. In addition, MTP is involved in the fusion of APOB-containing primordial CMs and APOB-free ER LLDs for expansion of CMs [74, 103, 104].…”

Future Challenges for Microsomal Transport Protein Inhibitors

“…Different compounds act at the different sites involved in these actions of MTP [75] and there is also the potential to increase degradation of apoB by the proteolytic activity of proteasomes when MTP activity is inhibited [76]. Since we still only have limited knowledge of the structure of MTP, efforts to synthesise inhibitors have relied upon the results of techniques such as high-throughput screening [77].…”