Chinese Hamster Ovary Cells Require the Coexpression of Microsomal Triglyceride Transfer Protein and Cholesterol 7α-Hydroxylase for the Assembly and Secretion of Apolipoprotein B-containing Lipoproteins

Fleming, James F.; Spitsen, Gary M.; Hui, To Y.; Olivier, Lisa M.; Du, Emma Z.; Raabe, Martin; Davis, Roger J.

doi:10.1074/jbc.274.14.9509

Cited by 23 publications

(16 citation statements)

References 48 publications

(51 reference statements)

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“…An enzyme that converts cholesterol esters to free cholesterol, cholesterol ester hydrolase (32), was identified. This result may fit in with the growing body of evidence that the pathways for hepatic cholesterol metabolism and lipoprotein secretion intersect (2,33). Another steroid metabolic enzyme, 11-␤ hydroxysteroid dehydrogenase (34), was also found.…”

Section: Fig 5-continued Identification Of Proteins That Bind Apob 2supporting

confidence: 59%

A Proteomic Approach Identifies Proteins in Hepatocytes That Bind Nascent Apolipoprotein B

Rashid¹,

Hevi

Yin³

et al. 2002

Journal of Biological Chemistry

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The biogenesis of apolipoprotein B is quite complex in view of its huge size, hydrophobicity, obligate association with lipids such as cholesterol and triglycerides prior to secretion, and intracellular degradation of a substantial proportion of newly synthesized molecules. Multiple proteins likely serve roles as molecular chaperones to assist in folding, assembly with lipids, and regulation of the secretion of apolipoprotein B. In these studies, we developed a strategy to isolate proteins associated with apolipoprotein B in rat livers. The purification consisted of two stages: first, microsomes were prepared from rat liver and treated with chemical crosslinkers, and second, the solubilized proteins were coimmunoprecipitated with antibody against apolipoprotein B. We found that several proteins were cross-linked to apolipoprotein B. The proteins were digested with trypsin, and the released peptides were sequenced by tandem mass spectrometry. The sequences precisely matched 377 peptides in 99 unique proteins. We show that at least two of the identified proteins, ferritin heavy and light chains, can directly bind apolipoprotein B. These and possibly other proteins identified by this proteomic approach are novel candidates for proteins that affect apolipoprotein B during its biogenesis. Apolipoprotein B (apoB)1 is secreted with lipids including cholesterol esters, phospholipids, cholesterol, and triglycerides, as very low density lipoproteins (1-5). The secretion of cholesterol from the liver in humans is tied to the export of apoB into plasma. Hepatic regulation of apoB secretion is chiefly posttranslational (6); secretion reflects the balance between assembly of this protein with lipids into a lipoprotein particle and intracellular degradation. Both of these competing processes appear to involve several other proteins.The biogenesis of this large (molecular mass greater than 500 kDa) (7, 8), hydrophobic protein requires the participation of several known chaperone proteins including some that are particular to the specialized physiologic role of apoB. Evidence suggests that calnexin, calreticulin, BiP, Erp72, GRP94, and protein disulfide isomerase (PDI) all interact with apoB during its translocation and further biogenesis once it has entered the endoplasmic reticulum (ER) (9 -11). These proteins also function in the proper folding and quality control of other secretory proteins. However, the assembly of apoB with lipids into a lipoprotein particle necessitates additional proteins that may be particular to apoB. The lack of solubility of apoB in an aqueous environment, such as the lumen of the ER, necessitates its co-translational association with lipids (12). This process is facilitated by microsomal triglyceride transport protein (MTP), which plays a crucial role in the initial assembly and regulation of secretion of apoB (13-15). In a second step that is sensitive to inhibition by brefeldin A, apoB is assembled with a full complement of lipids into a mature lipoprotein particle (16,17). The protein that mediat...

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Section: Fig 5-continued Identification Of Proteins That Bind Apob 2supporting

confidence: 59%

A Proteomic Approach Identifies Proteins in Hepatocytes That Bind Nascent Apolipoprotein B

Rashid¹,

Hevi

Yin³

et al. 2002

Journal of Biological Chemistry

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“…The secretion efficiency of apoB was low and apoB was secreted as HDL-size particles, indicating that other factors are needed for optimal lipoprotein assembly and secretion. In Chinese hamster ovary cells, in addition to MTP, cholesterol 7 ␣ -hydroxylase has been shown to be necessary for apoB secretion (54). Third, the importance of the lipid transfer activity in the lipidation of apoB polypeptides was reinforced by using specific inhibitors (55)(56)(57)(58)(59)(60).…”

Section: Role Of Mtp's Lipid Transfer Activity In Lipoprotein Assemblymentioning

confidence: 99%

Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly

Hussain

Shi

Dreizen

2003

Journal of Lipid Research

499

416

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Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are necessary for lipoprotein assembly. ApoB consists of five structural domains, ␤␣ 1 -␤ 1 -␣ 2 -␤ 2 -␣ 3 . We propose that MTP contains three structural motifs (N-terminal ␤ -barrel, central ␣ -helix, and C-terminal lipid cavity) and three functional domains (lipid transfer, membrane associating, and apoB binding). MTP's lipid transfer activity is required for the assembly of lipoproteins. This activity renders nascent apoB secretion-competent and may be involved in the import of triglycerides into the lumen of endoplasmic reticulum. In addition, MTP binds to apoB with high affinity involving ionic interactions. MTP interacts at multiple sites in the N-terminal ␤␣ 1 structural domain of apoB. A novel antagonist that inhibits apoB-MTP binding decreases apoB secretion. Furthermore, site-directed mutagenesis and deletion analyses that inhibit apoB-MTP binding decrease apoB secretion. Lipids modulate protein-protein interactions between apoB and MTP. Lipids associated with MTP increase apoB-MTP binding whereas lipids associated with apoB decrease this binding. Thus, specific antagonist, site-directed mutagenesis, deletion analyses, and modulation studies support the notion that apoB-MTP binding plays a role in lipoprotein biogenesis. However, specific steps in lipoprotein assembly that require apoB-MTP binding have not been identified.ApoB-MTP binding may be important for the prevention of degradation and lipidation of nascent apoB. Plasma lipoproteins are absent in abetalipoproteinemia due to mutations in the microsomal triglyceride transfer protein (MTP) gene, and plasma lipoprotein levels are low in hypobetalipoproteinemia due to mutations in the apolipoprotein B (apoB) gene (1, 2). These genetic disorders clearly underscore the importance of these two proteins in lipoprotein biogenesis, and recent findings indicate that MTP and apoB physically interact during this process. The aim of this review is to discuss specific molecular interactions between these proteins and their role in the biosynthesis of triglyceride-rich lipoproteins. A brief review of apoB and MTP is provided to aid in the understanding of protein-protein interactions between these proteins. In-depth discussion of apoB, MTP, and lipoprotein assembly can be found in several recent reviews and references therein (3-14). STRUCTURAL AND FUNCTIONAL DOMAINS IN APOBApolipoprotein B (apoB) is a non-exchangeable apolipoprotein found associated exclusively with plasma lipoproteins. In the human genome there is one apob gene of ‫ف‬ 45 kb. In the liver, it is transcribed into a single mRNA of 15 kb and is translated into a single polypeptide of 4536 amino acids called apoB-100. In the intestine, the apoB mRNA is post-transcriptionally edited, resulting in the conversion of a glutamine codon into a stop codon. The edited mRNA is translated into a single polypeptide of 2,152 amino acids called apoB48. By comparing mean hydrophobic moments per amino acid residue and the average...

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“…In HepG2 cells, the non-ERAD pathways are not sensitive to proteasome inhibitors (12)(13)(14)(15). We investigated whether the unsecreted apoB in H3 and L1 cells is a substrate for ERAD.…”

Section: The Block In Apob Secretion Induced By Ferritin H or L Is Nomentioning

confidence: 99%

“…Other factors also regulate secretion since some ubiquitin-conjugated apoB can be deubiquitinylated and secreted (11). In HepG2 cells, other non-ERAD routes for disposal exist but are not sensitive to agents that inhibit proteasomes (12)(13)(14)(15).…”

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confidence: 99%

Ferritins Can Regulate the Secretion of Apolipoprotein B

Hevi

Chuck

2003

Journal of Biological Chemistry

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Apolipoprotein B is secreted with atherogenic lipids as lipoprotein particles from hepatocytes. Regulation of the secretion of apolipoprotein B is largely post-translational and reflects the balance between processes that leads to particle assembly or to intracellular degradation. Previously, we conducted a proteomic screen to find proteins that bind apolipoprotein B in rat liver microsomes. We identified ferritin heavy and light chains in this screen among other proteins and showed that the two ferritins bind apolipoprotein B directly in vitro. In hepatocytes and other cells, ferritin heavy and light chains form cytosolic cages that store iron. We now show that ferritin heavy or light chains post-translationally inhibit the secretion of apolipoprotein B without altering the export of other hepatic proteins including albumin, factor XIII, and apolipoprotein A-I. This inhibition of apolipoprotein B secretion is not due to diminished lipid synthesis and can be partially overcome by stimulating triglyceride synthesis. The block in apolipoprotein B secretion by ferritins leads to an increase in endoplasmic reticulum-associated degradation of the apolipoprotein. Thus, despite being cytosolic proteins without known chaperone activity, ferritins can specifically regulate the secretion of apolipoprotein B post-translationally. The metabolic pathways for iron storage and intercellular cholesterol and triglyceride transport could intersect. Apolipoprotein B (apoB)1 is the large (molecular mass greater than 500 kDa), hydrophobic protein that is secreted with cholesterol esters and triglycerides in very low density lipoprotein particles (1). Regulation of the secretion of apoB from hepatocytes occurs largely post-translationally (2). Newly synthesized apoB can be assembled with lipids into lipoprotein particles or degraded intracellularly (3, 4), and it is the balance assembly and degradation that determines secretion. Since cholesterol esters and triglycerides can only be secreted into the bloodstream with apoB, factors that regulate the secretion of this protein likewise impact plasma levels of these atherogenic lipids.In HepG2 cells, unassembled apoB can be targeted for endoplasmic reticulum-associated degradation (ERAD) via the ubiquitin-proteasome pathway (5-8). ApoB is marked with ubiquitin for degradation even before it is finished being translated (7, 9, 10) while the protein is still in the translocation channel (8, 11). Furthermore, factors that slow the translocation of apoB increase the number of ubiquitin-conjugated apoB molecules marked for degradation by proteasomes (8) suggesting that translocation is a point of regulation of secretion. Other factors also regulate secretion since some ubiquitin-conjugated apoB can be deubiquitinylated and secreted (11). In HepG2 cells, other non-ERAD routes for disposal exist but are not sensitive to agents that inhibit proteasomes (12-15).The intertwined processes of lipidation, folding, translocation, intracellular degradation, quality control, and regulation of the secret...

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Chinese Hamster Ovary Cells Require the Coexpression of Microsomal Triglyceride Transfer Protein and Cholesterol 7α-Hydroxylase for the Assembly and Secretion of Apolipoprotein B-containing Lipoproteins

Cited by 23 publications

References 48 publications

A Proteomic Approach Identifies Proteins in Hepatocytes That Bind Nascent Apolipoprotein B

A Proteomic Approach Identifies Proteins in Hepatocytes That Bind Nascent Apolipoprotein B

Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly

Ferritins Can Regulate the Secretion of Apolipoprotein B

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