Date of Award

1999

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physics

Keywords

Chemistry, Biochemistry.

Rights

CC BY-NC-ND 4.0

Abstract

Acute modulation of apolipoprotein B (apoB) biogenesis is essential for the proper assembly and secretion of the hepatic apoB-containing lipoproteins. There is substantial evidence suggesting that translocation of apoB is a major regulatory factor influencing the rate of apoB and apoB-containing lipoprotein secretion and intracellular stability. Studies were conducted to examine the relationship between the length of apoB and its intracellular translocation and stability using McA-RH7777 cells expressing recombinant human apoB variants, ranging in size from B 15 to B 100. Translocational status of apoB was assessed based on trypsin sensitivity of apoB using isolated permeabilized McARH7777 cells. Shorter apoB variants (B15 to B72) were relatively resistant to exogenous trypsin (percent trypsin-resistant apoB ranged from 69--79%) in contrast to recombinant human B100 which was only 42% trypsin resistant. Thus, an inverse correlation between the length of apoB and its sensitivity to exogenous trypsin was established. An inverse relationship was also observed between the size of apoB and its co-translational (during the pulse) and post-translational (over a 2 h chase) resistance to proteasomal degradation. As the size of the nascent apoB polypeptide increases there appears to be a higher sensitivity to proteasomal degradation. It has been well established that the biogenesis of apoB is mediated by the cytosolic proteasome. Here, the roles of both the cytosolic proteasome as well as nonproteasome mediated degradation systems were investigated. Although post-translational apoB degradation in intact HepG2 cells was sensitive to the proteasome inhibitor lactacystin, in permeabilized cells there was no post-translational protection of apoB by lactacystin, nor clasto -lactacystin (3-lactone. Further investigations of proteasomal activity in HepG2 cells revealed that, following permeabilization, there was a dramatic loss of the 20S proteasomal subunits, and consequently the cells exhibited no detectable lactacystin-inhibitable activity. Interestingly, apoB fragmentation and the generation of the 70 kDa apoB degradation fragment characteristic to permeabilized cells, continued to occur in these cells despite the absence of functional cytosolic proteasome. These data thus suggest that although the cytosolic proteasome appears to be involved in the post-translational turnover of apoB in intact cells, the specific post-translational fragmentation of apoB generating the 70 kDa fragment observed in permeabilized cells occurs independent of the cytosolic proteasome. Secretion of apoB was restored in permeabilized HepG2 cells supplemented with cytosol and an ATP generating system, and interestingly, apoB secretion was also sensitive to MG 132, thus suggesting a role for the proteasome in the post-translational regulation of the 70 kDa apoB fragment. Studies were also conducted to explore the potential role of retrograde translocation of apoB from the secretory pathway, as a targeting mechanism for proteasomal degradation. Subcellular fractionation of HepG2 cells revealed apoB in both the cytosolic and microsomal fractions of these cells. The accumulation of cytosolic apoB in comparison to microsomal apoB appeared to increase significantly post-trranslationally, and was also sensitive to MG132, thus suggesting a role for the cytosolic proteasome in the post-translational degradation of apoB. Further characterization of cytosolic apoB revealed that this pool of apoB was ubiquitinated and glycosylated. Ubiquitination indicated that the cytosolic apoB was targeted for proteasomal degradation. Glycosylation implied that cytosolic apoB had been previously exposed to the ER lumen. Inhibition of glycosylation increased the early accumulation of apoB in the cytosol. In the presence of Brefeldin A the rate of apoB accumulation in the cytosol was reduced and the degradation of apoB in the microsomes appeared to be enhanced. Thus, it is postulated that apoB which accumulates in the cytosol of HepG2 cells may have been retrograde translocated from the secretory pathway (endoplasmic reticulum) to the cytosol. A model for the potential role of retrograde translocation was also developed in an attempt to elucidate a general mechanism to explain how luminal apoB may be targeted to the cytosol for proteasomal degradation.Dept. of Physical Sciences. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1999 .C38. Source: Dissertation Abstracts International, Volume: 61-09, Section: B, page: 4695. Thesis (Ph.D.)--University of Windsor (Canada), 1999.

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