Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n ‫؍‬ 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n ‫؍‬ 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the ␣-helical domain within the N terminus of apoB. Thus, proper folding of the ␣-helical domain of apoB-100 is essential for efficient secretion. Apolipoprotein (apo)6 B, a large amphipathic glycoprotein, plays a central role in human lipoprotein metabolism (1, 2). The human apoB gene (APOB) is located on chromosome 2 and produces, via a unique mRNA editing process (3), two forms of apoB, namely apoB-48 (2152 amino acids) and apoB-100 (4536 amino acids) (4, 5). ApoB-48 is the truncated form of apoB-100 consisting of the N-terminal 48% of full-length apoB-100. ApoB-48 is synthesized in the intestine and is essential for the formation and secretion of chylomicrons. ApoB-100 is synthesized in the liver and is an essential structural component of very low density lipoprotein (VLDL) and its metabolic products, intermediate density lipoprotein (IDL) and low density lipoprotein (LDL), and is also a ligand for the LDL receptor. Unlike humans, the rat liver produces both apoB-100 and apoB-48, and both forms can assemble VLDL (6).A pentapartite model for human apoB-100 has been proposed, which depicts a five-domain structure composed of alternating amphipathic ␣-helices and amphipathic ␤-strands, namely ␤␣1-␤1-␣2-␤2-␣3 (7). The ␤␣1 domain is a mixture of 13 amphipathic ␤-strands and 17 amphipathic ␣-helices, whose amino acids share extensive sequence homologies to the yolk protein lipovitellin (7-9). The apoB ␤␣1 domain has been modeled on the structure of silver lamprey lipovitellin, in which the 13 ␤-strands (amino acids 21-263) form a ␤-barrel, whereas the 17 ␣-helices (amino acids 440 -592) form a two-layered helical bundle (10). There is an interface between the ␣-helical bundle * This work was supported by the Heart and Stroke Foundation of Ontario . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article is available online at http://www.jlr.org Apolipoprotein (apo) B-100 is the major protein component of VLDLs. Assembly of VLDL in the liver begins at the endoplasmic reticulum (ER) with the formation of a primordial lipoprotein. As apoB-100 enters the ER lumen cotranslationally, it must associate with suffi cient lipids for VLDL assembly to proceed. The microsomal triglyceride transfer protein (MTP) facilitates transfer of lipids onto nascent apoB-100 ( 1 ). ApoB-100 is somewhat unique in that its secretion can be regulated by degradation ( 2 ), whereas control of expression of most proteins is at the level of mRNA transcription or translation. During conditions that limit lipid supply, such as low MTP activity ( 3 ) or reduced lipid availability ( 4, 5 ), apoB-100 is delivered to and degraded by the cytosolic proteasome in a process termed ER-associated degradation (ERAD). ApoB-100 contains large hydrophobic regions that require lipidation during apoB-100 synthesis or the nascent protein is targeted to ERAD ( 6 ). In a process that remains poorly defi ned, apoB-100 can be secreted only if lipidation/assembly satisfi es the quality control surveillance system in the secretory pathway.The ERAD pathway removes malfolded proteins from the ER lumen or membrane [reviewed in ( 7 )]. ERAD helps reduce the burden on ER-resident chaperones and allows the cell to maintain ER homeostasis. The typical ERAD pathway for a protein in the secretory pathway consists of at least the following steps: substrate recognition, retrotranslocation from the ER into the cytosol and ubiquitination, followed by degradation in the proteasome.Abstract Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase 1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumulation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retr...

The ATPase associated with various cellular activities (AAA-ATPase) p97 (p97) has been implicated in the retrotranslocation of target proteins for delivery to the cytosolic proteasome during endoplasmic reticulum-associated degradation (ERAD). Apolipoprotein B-100 (apoB-100) is an ERAD substrate in liver cells, including the human hepatoma, HepG2. We studied the potential role of p97 in the ERAD of apoB-100 in HepG2 cells using cell permeabilization, coimmunoprecipitation, and gene silencing. Degradation was abolished when HepG2 cytosol was removed by digitonin permeabilization, and treatment of intact cells with the proteasome inhibitor MG132 caused accumulation of ubiquitinated apoB protein in the cytosol. Cross-linking of intact cells with the thiol-cleavable agent dithiobis(succinimidylpropionate) (DSP), as well as nondenaturing immunoprecipitation, demonstrated an interaction between p97 and intracellular apoB. Small interfering ribonucleic acid (siRNA)-mediated reduction of p97 protein increased the intracellular levels of newly synthesized apoB-100, predominantly because of a decrease in the turnover of newly synthesized apoB-100 protein. However, although the posttranslational degradation of newly synthesized apoB-100 was delayed by p97 knockdown, secretion of apoB-100 was not affected. Knockdown of p97 also impaired the release of apoB-100 and polyubiquitinated apoB into the cytosol. In summary, our results suggest that retrotranslocation and proteasomal degradation of apoB-100 can be dissociated in HepG2 cells, and that the AAA-ATPase p97 is involved in the removal of full-length apoB from the biosynthetic pathway to the cytosolic proteasome.-Fisher, E. A., L. R. Lapierre, R. D. Junkins, and R. S. McLeod. The AAA-ATPase p97 facilitates degradation of apolipoprotein B by the ubiquitin-proteasome pathway .

Apolipoprotein B (apoB) is the main protein component of very low density lipoprotein (VLDL) and is necessary for the assembly and secretion of these triglyceride (TG)-rich particles. Following release from the liver, VLDL is converted to low density lipoprotein (LDL) in the plasma and increased production of VLDL can therefore play a detrimental role in cardiovascular disease. Increasing evidence has helped to establish VLDL assembly as a target for the treatment of dyslipidemias. Multiple factors are involved in the folding of the apoB protein and the formation of a secretion-competent VLDL particle. Failed VLDL assembly can initiate quality control mechanisms in the hepatocyte that target apoB for degradation. ApoB is a substrate for endoplasmic reticulum associated degradation (ERAD) by the ubiquitin proteasome system and for autophagy. Efficient targeting and disposal of apoB is a regulated process that modulates VLDL secretion and partitioning of TG. Emerging evidence suggests that significant overlap exists between these degradative pathways. For example, the insulin-mediated targeting of apoB to autophagy and postprandial activation of the unfolded protein response (UPR) may employ the same cellular machinery and regulatory cues. Changes in the quality control mechanisms for apoB impact hepatic physiology and pathology states, including insulin resistance and fatty liver. Insulin signaling, lipid metabolism and the hepatic UPR may impact VLDL production, particularly during the postprandial state. In this review we summarize our current understanding of VLDL assembly, apoB degradation, quality control mechanisms and the role of these processes in liver physiology and in pathologic states.

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