Intracellular Assembly and Degradation of Apolipoprotein B-100-containing Lipoproteins in Digitonin-permeabilized HEP G2 Cells

Abstract

Permeabilized Hep G2 cells have been used to investigate the turnover of apolipoprotein B-100 (apoB-100). When such cells were chased in the presence of buffer, there was no biosynthesis of apoB-100, nor was the protein secreted from the cells. Thus the turnover of apoB-100 in these cells reflected the posttranslational degradation of the protein. Pulse-chase studies indicated that apoB-100 was degraded both when associated with the membrane and when present as lipoproteins in the secretory pathway. Neither albumin nor ␣ 1 -antitrypsin showed any significant posttranslational intracellular degradation under the same condition. The kinetics for the turnover of apoB-100 in the luminal content differed from that of apoB-100 that was associated with the microsomal membrane. Moreover, while the degradation of the luminal apoB-100 was inhibited by N-acetylleucyl-leucyl-norleucinal (ALLN), this was not the case for the membrane-associated protein. Together these results suggest the existence of different pathways for the degradation of luminal apoB-100 and membrane-associated apoB-100. This was further supported by results from pulse-chase studies in intact cells, showing that ALLN increased the amount of radioactive apoB-100 that associated with the microsomal membrane during the pulse-labeling of the cells. However, ALLN did not influence the rate of turnover of the membrane-associated apoB-100.The presence of an ATP-generating system during the chase of the permeabilized cells prevented the disappearance of pulse-labeled apoB-100 from the luminal lipoprotein-associated pool. The ATP-generating system combined with cytosol protected the total apoB-100 in the system from being degraded. The cells cultured in the presence of oleic acid and chased after permeabilization in the presence of cytosol and the ATP-generating system showed an increase in the amount of apoB-100 present on dense ("high density lipoprotein-like") particles. This increase was linear during the time investigated (i.e. from 0 to 2 h chase) and independent of protein biosynthesis. Our results indicate that the dense particle was generated by a redistribution of apoB-100 within the secretory pathway and that it most likely was assembled from the membrane-associated form of apoB-100. These results indicate that the release of apoB-100 from this membrane-associated form to the microsomal lumen is dependent on cytosolic factors and a source of metabolic energy.

Effects of Atorvastatin on the Intracellular Stability and Secretion of Apolipoprotein B in HepG2 Cells

“…Recent data from our laboratory confirm that both membrane-bound apoB and lipoprotein-associated apoB are subject to intracellular degradation, possibly involving distinct degradative mechanisms. 36 Yeung and coworkers 37 have recently demonstrated the involvement of the ubiquitindependent proteasome system in degradation of apoB. Recent data from two other laboratories provide further evidence for the role of the proteasome in apoB degradation.…”

“…33,34,36,41,42 Briefly, permeabilized cells incubated for 0 to 2 hours were washed once with 250 mmol/L sucrose and 3 mmol/L imidazole, pH 7.4, and once with 50 mmol/L sucrose and 3 mmol/L imidazole, pH 7.4. The cells were then scraped in 0.5 mL of 50-mmol/L sucrose solution supplemented with a cocktail of protease inhibitors (0.1 mmol/L leupeptin, 1 mmol/L PMSF, 100 KIU/mL Trasylol (aprotinin), 1 mol/L pepstatin A, and 5 mol/L ALLN) and homogenized with a glass Dounce homogenizer as described.…”

“…It is important to note that the necessary control experiments showing the integrity of microsomal membranes in digitonin-permeabilized HepG2 cells previously have been published. 8,23,36 Intact HepG2 cells were treated with atorvastatin, pulsed with […”

“…The cells were then scraped in 0.5 mL of 50-mmol/L sucrose solution supplemented with a cocktail of protease inhibitors (0.1 mmol/L leupeptin, 1 mmol/L PMSF, 100 KIU/mL Trasylol (aprotinin), 1 mol/L pepstatin A, and 5 mol/L ALLN) and homogenized with a glass Dounce homogenizer as described. 36,41,42 The homogenate was centrifuged for 10 minutes at 2200g, and the supernatant containing the crude microsomes was subjected to carbonate extraction followed by fractionation of membrane and luminal fractions by ultracentrifugation, as described. 32 In some experiments, luminal contents isolated from microsomes were supplemented with protease inhibitors (0.1 mmol/L leupeptin, 1 mmol/L PMSF, 100 KIU/mL Trasylol, 1 mol/L pepstatin A, and 5 mol/L ALLN) and then subjected to ultracentrifugation on a step sucrose gradient (1.5 mL 49%/3.0 mL 25%/2.0 mL 20%/3.5 mL sample/1.9 mL 5%/0.9 mL 0% sucrose) at 35 000 rpm in a SW41 rotor for 65 hours at 12°C.…”

Hepatitis C Virus Nonstructural Proteins Inhibit Apolipoprotein B100 Secretion

“…In the presence of sufficient lipids, mainly triglycerides, apoB is efficiently assembled into secretion-competent VLDL particles (23). If, however, apoB is poorly lipidated, the majority of newly synthesized apoB is degraded by the proteasome in the cytosol (24) or by ER-resident proteases (25,26). MTP stabilizes apoB by lipidation, and the subsequently lipidated apoB fuses with triglyceride-rich particles, leading to the formation of VLDLs (27,28).…”