Highly active anti-retroviral therapies, which incorporate HIV protease inhibitors, resolve many AIDS-defining illnesses. However, patients receiving protease inhibitors develop a marked lipodystrophy and hyperlipidemia. Using cultured human and rat hepatoma cells and primary hepatocytes from transgenic mice, we demonstrate that protease inhibitor treatment inhibits proteasomal degradation of nascent apolipoprotein B, the principal protein component of triglyceride and cholesterol-rich plasma lipoproteins. Unexpectedly, protease inhibitors also inhibited the secretion of apolipoprotein B. This was associated with inhibition of cholesteryl-ester synthesis and microsomal triglyceride transfer-protein activity. However, in the presence of oleic acid, which stimulates neutral-lipid biosynthesis, protease-inhibitor treatment increased secretion of apolipoprotein B-lipoproteins above controls. These findings suggest a molecular basis for protease-inhibitor-associated hyperlipidemia, a serious adverse effect of an otherwise efficacious treatment for HIV infection.

Apolipoprotein B100 (apoB) is a large (520-kDa) complex secretory protein; its secretion is regulated posttranscriptionally by several degradation pathways. The best described of these degradative processes is cotranslational ubiquitinylation and proteasomal degradation of nascent apoB, involving the 70-and 90-kDa heat shock proteins and the multiple components of the proteasomal pathway. Ubiquitinylation involves several proteins, including ligases called E3s, that coordinate the covalent binding of ubiquitin to target proteins. The recent discovery that tumor autocrine motility factor receptor, also known as gp78, is an endoplasmic reticulum (ER)-associated E3, raised the possibility that this E3 might be involved in the ER-associated degradation of nascent apoB. In a series of experiments in HepG2 cells, we demonstrated that overexpression of gp78 was sufficient for increased ubiquitinylation and proteasomal degradation of apoB, with reduced secretion of apoB-lipoproteins. This action of gp78 was specific: overexpression of the protein did not affect secretion of either albumin or apolipoprotein AI. Furthermore, overexpression of a cytosolic E3, Itch, had no effect on apoB secretion. Finally, using an in vitro translation system, we demonstrated that gp78 led to increased ubiquitinylation and proteasomal degradation of apoB48. Together, these results indicate that an ER-associated protein, gp78, is a bona fide E3 ligase in the apoB ERassociated degradation pathway.Apolipoprotein B100 (apoB) 1 is the essential protein component of atherogenic very low density and low density lipoproteins (1), and overproduction of these lipoproteins is a common feature of human dyslipidemia (2). Extensive studies of cultured primary hepatocytes and hepatoma cells have established that significant control over apoB secretion can be achieved at the post-transcriptional level by degradation. One form of presecretory degradation, namely the ER-associated degradation (ERAD) of apoB, is regulated by the availability of newly synthesized core lipids (triglyceride, cholesterol ester) and microsomal triglyceride transfer protein activity (1, 3, 4). When lipid availability or microsomal triglyceride transfer protein activity is limited, translocation of apoB is incomplete, and apoB is ubiquitinylated co-translationally and degraded by the cytosolic ubiquitin-proteasome pathway (5-9).Cytosolic and nuclear proteins are targeted for proteasomal degradation by the addition of multi-ubiquitin chains. The specificity of this process is largely conferred by ubiquitin (Ub) protein ligases (E3s) (10 -12). E3s interact directly or indirectly with substrate and mediate the transfer of Ub from Ub-conjugating enzymes to target proteins. Two major E3 classes have been identified: HECT domain E3s and RING finger E3s (10 -12). HECT domain E3s are homologous to E6-AP C-terminal domain E3s, which accept Ub from Ub-conjugating enzymes, thereby forming thiol-ester intermediates with Ub. The Ub is then transferred to the target protein. In contrast, RING ...

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Apolipoprotein (apo) B100 is an atypical secretory protein in that its translocation across the endoplasmic reticulum membrane is inefficient, resulting in the partial translocation and exposure of apoB100 on the cytoplasmic surface of the endoplasmic reticulum. Cytosolic exposure leads to the association of nascent apoB with heat shock protein 70 and to its predisposition to ubiquitination and proteasomal degradation. The basis for the inefficient translocation of apoB100 remains unclear and controversial. To test the hypothesis that ␤ sheet domains present in apoB100 contribute to its inefficient translocation, we created human apoB chimeric constructs apoB13,16 and apoB13,13,16, which contain amino-terminal ␣ globular domains but no ␤ sheet domains, and apoB13,16,␤, which has an amphipathic ␤ sheet domain of apoB100 inserted into apoB13,16. These constructs, along with carboxyl-terminal truncations of apoB100, apoB34 and apoB42, were used to transfect HepG2 and Chinese hamster ovary cells. In contrast to the lack of effect of proteinase K on apoB13,16 and apoB13,13,16, the levels of apoB34, apoB42, and apoB13,16,␤ were decreased by 70 -85% after proteinase K-induced proteolysis in both HepG2 and Chinese hamster ovary cells. Either oleic acid or proteasomal inhibitors (N-acetyl-leucinyl-leucinyl-norleucinal and lactacystin) significantly increased the cell levels of apoB13,16,␤, apoB34, apoB42, and full-length apoB100 but had no effect on the cell levels of apoB13,16 and apoB13,13,16. When HepG2 cells were incubated with a microsomal triglyceride transfer protein inhibitor, the cellular levels of apoB13,16,␤, apoB34, and apoB42 were decreased by 70 -80%, whereas the levels of apoB13,16 and apoB13,13,16 were unaffected. The effects of microsomal triglyceride transfer protein inhibition were reversed by lactacystin. Our results clearly demonstrate that the translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apoB were determined by the presence of a lipid binding ␤ sheet domain. It is possible that ␤ sheet domains may at least transiently facilitate the interaction of apoB with the lipid bilayer surrounding the translocation channel. Apolipoprotein (apo)1 B100 is the major protein of atherogenic very low density and low density lipoproteins (1). There is a wide range of apoB100 secretion from the liver in humans, and overproduction of very low density lipoprotein and low density lipoprotein is a common feature of human dyslipidemia (2). ApoB100 is a constitutively synthesized protein (3), and the secretion of apoB is regulated at the posttranslational level, where the availability of newly synthesized core lipids seems to facilitate the translocation of the nascent polypeptide and targets it for secretion rather than degradation via the proteasomal pathway (3-8). The basis for the posttranslational regulation of apoB100 seems to derive from the fact that it is an atypical secretory protein whose translocation across the endoplasmic reticulum (ER) membrane is inefficien...

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