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• W2151400756 abstract "Apolipoprotein B (ap1111190) and microsomal triglyceride transfer protein (MTP) are necessary for lipoprotein assembly. ApoB consists of five structural domains, βα1-β1-α2-β2-α3. We propose that MTP contains three structural motifs (N-terminal β-barrel, central α-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 βα1 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. Apolipoprotein B (ap1111190) and microsomal triglyceride transfer protein (MTP) are necessary for lipoprotein assembly. ApoB consists of five structural domains, βα1-β1-α2-β2-α3. We propose that MTP contains three structural motifs (N-terminal β-barrel, central α-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 βα1 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 (1Havel R.J. Kane J.P. Introduction: Structure and metabolism of plasma lipoproteins.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disorders. McGraw-Hill, Inc., New York1995: 1841-1851Google Scholar, 2Kane J.P. Havel R.J. Disorders of the biogenesis and secretion of lipoproteins containing the B apolipoproteins.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disorders. McGraw-Hill, Inc., New York1995: 1853-1885Google Scholar). 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 (3Segrest J.P. Jones M.K. De Loof H. Dashti N. Structure of apolipoprotein B-100 in low density lipoproteins.J. Lipid Res. 2001; 42: 1346-1367Google Scholar, 4Fisher E.A. Ginsberg H.N. Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins.J. Biol. Chem. 2002; 277: 17377-17380Google Scholar, 5Shelness G.S. Sellers J.A. Very-low-density lipoprotein assembly and secretion.Curr. Opin. Lipidol. 2001; 12: 151-157Google Scholar, 6Davidson N.O. Shelness G.S. Apolipoprotein B: mRNA editing, lipoprotein assembly, and presecretory degradation.Annu. Rev. Nutr. 2000; 20: 169-193Google Scholar, 7Berriot-Varoqueaux N. Aggerbeck L.P. Samson-Bouma M. Wetterau J.R. The role of the microsomal triglygeride transfer protein in abetalipoproteinemia.Annu. Rev. Nutr. 2000; 20: 663-697Google Scholar, 8Olofsson S.O. Asp L. Borén J. The assembly and secretion of apolipoprotein B-containing lipoproteins.Curr. Opin. Lipidol. 1999; 10: 341-346Google Scholar, 9Davis R.A. Cell and molecular biology of the assembly and secretion of apolipoprotein B-containing lipoproteins by the liver.Biochim. Biophys. Acta. 1999; 1440: 1-31Google Scholar, 10Gordon D.A. Jamil H. Progress towards understanding the role of microsomal triglyceride transfer protein in apolipoprotein-B lipoprotein assembly.Biochim. Biophys. Acta. 2000; 1486: 72-83Google Scholar, 11Ginsberg H.N. Role of lipid synthesis, chaperone proteins and proteasomes in the assembly and secretion of apoprotein B-containing lipoproteins from cultured liver cells.Clin. Exp. Pharmacol. Physiol. 1997; 24: A29-A32Google Scholar, 12Yao Z. Tran K. McLeod R.S. Intracellular degradation of newly synthesized apolipoprotein B.J. Lipid Res. 1997; 38: 1937-1953Google Scholar, 13Hussain M.M. Kedees M.H. Singh K. Athar H. Jamali N.Z. Signposts in the assembly of chylomicrons.Front. Biosci. 2001; 6: D320-D331Google Scholar, 14Hussain M.M. A proposed model for the assembly of chylomicrons.Atherosclerosis. 2000; 148: 1-15Google Scholar). Apolipoprotein B (apoB) is a non-exchangeable apolipoprotein found associated exclusively with plasma lipoproteins. In the human genome there is one apob gene of ∼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 hydrophobicity of the hydrophobic face of the helices, Segrest et al. have proposed a pentapartite secondary structure for apoB-100 (3Segrest J.P. Jones M.K. De Loof H. Dashti N. Structure of apolipoprotein B-100 in low density lipoproteins.J. Lipid Res. 2001; 42: 1346-1367Google Scholar, 15Segrest J.P. Jones M.K. Mishra V.K. Anantharamaiah G.M. Garber D.W. ApoB-100 has a pentapartite structure composed of three amphipathic alpha-helical domains alternating with two amphipathic beta-strand domains - Detection by the computer program LOCATE.Arterioscler. Thromb. 1994; 14: 1674-1685Google Scholar, 16Segrest J.P. Jones M.K. Mishra V.K. Pierotti V. Young S.H. Borén J. Innerarity T.L. Dashti N. Apolipoprotein B-100: conservation of lipid-associating amphipathic secondary structural motifs in nine species of vertebrates.J. Lipid Res. 1998; 39: 85-102Google Scholar, 17Segrest J.P. Jones M.K. Dashti N. N-terminal domain of apolipoprotein B has structural homology to lipovitellin and microsomal triglyceride transfer protein: a “lipid pocket” model for self-assembly of apoB-containing lipoprotein particles.J. Lipid Res. 1999; 40: 1401-1416Google Scholar). According to this model, apoB100 is comprised of three amphiphatic α-helical domains alternating with two amphiphatic β-sheet domains in an NH2-α1-β1-α2-β2-α3-COOH configuration. The α1 domain (B:58–795) is an independent globular domain. It associates with lipids but is incapable of forming a lipoprotein (18Herscovitz H. Derksen A. Walsh M.T. McKnight C.J. Gantz D.L. Hadzopoulou-cladaras M. Zannis V. Curry C. Small D.M. The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins.J. Lipid Res. 2001; 42: 51-59Google Scholar, 19DeLozier J.A. Parks J.S. Shelness G.S. Vesicle-binding properties of wild-type and cysteine mutant forms of alpha(1) domain of apolipoprotein B.J. Lipid Res. 2001; 42: 399-406Google Scholar). Nonetheless, it is required for lipoprotein assembly because its absence ablates lipoprotein assembly (20Gretch D.G. Sturley S.L. Wang L. Lipton B.A. Dunning A. Grunwald K.A.A. Wetterau J.R. Yao Z. Talmud P. Attie A.D. The amino terminus of apolipoprotein B is necessary but not sufficient for microsomal triglyceride transfer protein responsiveness.J. Biol. Chem. 1996; 271: 8682-8691Google Scholar). It contains 12 cysteine residues that form six disulfide linkages (21Yang C.Y. Kim T.W. Weng S.A. Lee B.R. Yang M.L. Gotto Jr., A.M. Isolation and characterization of sulfhydryl and disulfide peptides of human apolipoprotein B-100.Proc. Natl. Acad. Sci. USA. 1990; 87: 5523-5527Google Scholar). Proper disulfide bond formation between some cysteine residues is essential for the assembly of apoB-containing lipoproteins (22Huang X.F. Shelness G.S. Identification of cysteine pairs within the amino-terminal 5% of apolipoprotein B essential for hepatic lipoprotein assembly and secretion.J. Biol. Chem. 1997; 272: 31872-31876Google Scholar, 23Tran K. Borén J. Macri J. Wang Y.W. McLeod R. Avramoglu R.K. Adeli K. Yao Z. Functional analysis of disulfide linkages clustered within the amino terminus of human apolipoprotein B.J. Biol. Chem. 1998; 273: 7244-7251Google Scholar). Based on sequence homology with lipovitellin, this region has been predicted to consist of a β-barrel (B:1–263) and an α-helical (B:294–592) domains (24Mann C.J. Anderson T.A. Read J. Chester S.A. Harrison G.B. Köchl S. Ritchie P.J. Bradbury P. Hussain F.S. Amey J. Vanloo B. Rosseneu M. Infante R. Hancock J.M. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins.J. Mol. Biol. 1999; 285: 391-408Google Scholar) and has subsequently be called βα1 domain (3Segrest J.P. Jones M.K. De Loof H. Dashti N. Structure of apolipoprotein B-100 in low density lipoproteins.J. Lipid Res. 2001; 42: 1346-1367Google Scholar). As discussed below, this domain contains MTP binding site. The other four domains (β1-α2-β2-α3) are also comprised of several short amphiphatic β-strands and α-helices. The β-sheet domains (B:827–2001 and B:2571–4032) are essential for lipoprotein assembly and bind lipids non-reversibly. The assembly of these β-sheets into lipoproteins requires α1 domain (20Gretch D.G. Sturley S.L. Wang L. Lipton B.A. Dunning A. Grunwald K.A.A. Wetterau J.R. Yao Z. Talmud P. Attie A.D. The amino terminus of apolipoprotein B is necessary but not sufficient for microsomal triglyceride transfer protein responsiveness.J. Biol. Chem. 1996; 271: 8682-8691Google Scholar). The LDL receptor binding and heparin binding sites are in the β2 domain. ApoB-48 contains β1 domain only, whereas apoB-100 contains both the β1 and β2 domains. These two proteins are used for the assembly of two different lipoproteins, chylomicrons and VLDLs. The α2 (B:2045–2587) and α3 (B:4017–4515) domains consist of several amphiphatic helices that can reversibly associate with lipids, a characteristic property of exchangeable apolipoproteins. Their role in lipoprotein assembly is unknown. Evidence for the presence of a protein in the endoplasmic reticulum (ER) responsible for the transfer of neutral lipids between vesicles was first provided by Wetterau and Zilversmit (25Wetterau J.R. Zilversmit D.B. A triglyceride and cholesteryl ester transfer protein associated with liver microsomes.J. Biol. Chem. 1984; 259: 10863-10866Google Scholar). Subsequently, they purified MTP to homogeneity and showed that it consists of two non-covalently bound polypeptides of 97 (M subunit) and 55 (P subunit) kDa (26Wetterau J.R. Zilversmit D.B. Purification and characterization of microsomal triglyceride and cholesteryl ester transfer protein from bovine liver microsomes.Chem. Phys. Lipids. 1985; 38: 205-222Google Scholar, 27Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex.J. Biol. Chem. 1990; 265: 9800-9807Google Scholar, 28Wetterau J.R. Aggerbeck L.P. Laplaud P.M. McLean L.R. Structural properties of the microsomal triglyceride-transfer protein complex.Biochemistry. 1991; 30: 4406-4412Google Scholar). The small 55-kDa “P” subunit was the ubiquitous ER resident enzyme protein disulfide isomerase (PDI). The P subunit is inactive with respect to its isomerase activity in the MTP complex (27Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex.J. Biol. Chem. 1990; 265: 9800-9807Google Scholar, 29Wetterau J.R. Combs K.A. McLean L.R. Spinner S.N. Aggerbeck L.P. Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal tri-glyceride transfer protein.Biochemistry. 1991; 30: 9728-9735Google Scholar). Moreover, the isomerase activity is not essential for its association with the larger M subunit and for MTP activity, as PDI mutants lacking enzyme activity are fully functional in lipid transfer activity in association with a normal M subunit (30Lamberg A. Jauhiainen M. Metso J. Ehnholm C. Shoulders C. Scott J. Pihlajaniemi T. Kivirikko K.I. The role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity.Biochem. J. 1996; 315: 533-536Google Scholar). The large 97-kDa M subunit was unique and was essential for the lipid transfer activity. The kinetics of lipid transfer from membranes to lipoproteins has not yet been studied in detail. MTP enhances the rate of lipid transfer between vesicles (31Gregg R.E. Wetterau J.R. The molecular basis of abetalipoproteinemia.Curr. Opin. Lipidol. 1994; 5: 81-86Google Scholar, 32Wetterau J.R. Lin M.C.M. Jamil H. Microsomal tri-glyceride transfer protein.Biochim. Biophys. Acta. 1997; 1345: 136-150Google Scholar). Kinetic studies with model membranes suggest that MTP transfers lipids by a shuttle mechanism (33Atzel A. Wetterau J.R. Mechanism of microsomal tri-glyceride transfer protein catalyzed lipid transport.Biochemistry. 1993; 32: 10444-10450Google Scholar). In this mechanism, each MTP molecule is proposed to interact transiently with a membrane, extract lipid molecules, dissociate from the membrane, bind transiently with another membrane, deliver lipids rapidly to the second membrane, and become available for another cycle of lipid transfer. The lipid transfer activity was shown to be optimum with neutrally charged membranes and decreased in the presence of negatively charged lipids in vesicles (33Atzel A. Wetterau J.R. Mechanism of microsomal tri-glyceride transfer protein catalyzed lipid transport.Biochemistry. 1993; 32: 10444-10450Google Scholar). Kinetic studies suggest that MTP has two, one fast and one slow, lipid binding sites (34Atzel A. Wetterau J.R. Identification of two classes of lipid molecule binding sites on the microsomal triglyceride transfer protein.Biochemistry. 1994; 33: 15382-15388Google Scholar, 35Jamil H. Dickson Jr., J.K. Chu C-H. Lago M.W. Rinehart J.K. Biller S.A. Gregg R.E. Wetterau J.R. Microsomal tri-glyceride transfer protein. Specificity of lipid binding and transport.J. Biol. Chem. 1995; 270: 6549-6554Google Scholar). The fast site is implicated in lipid transfer (34Atzel A. Wetterau J.R. Identification of two classes of lipid molecule binding sites on the microsomal triglyceride transfer protein.Biochemistry. 1994; 33: 15382-15388Google Scholar). The M subunit is a single polypeptide of 894 amino acids (36Sharp D. Blinderman L. Combs K.A. Kienzle B. Ricci B. Wager-Smith K. Gil C.M. Turck C.W. Bouma M-E. Rader D.J. Aggerbeck L.P. Gregg R.E. Gordon D.A. Wetterau J.R. Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinemia.Nature. 1993; 365: 65-69Google Scholar). Based on sequence homology with lipovitellin, it is proposed (24Mann C.J. Anderson T.A. Read J. Chester S.A. Harrison G.B. Köchl S. Ritchie P.J. Bradbury P. Hussain F.S. Amey J. Vanloo B. Rosseneu M. Infante R. Hancock J.M. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins.J. Mol. Biol. 1999; 285: 391-408Google Scholar, 37Bradbury P. Mann C.J. Köchl S. Anderson T.A. Chester S.A. Hancock J.M. Ritchie P.J. Amey J. Harrison G.B. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. A common binding site on the microsomal triglyceride transfer protein for apolipoprotein B and protein disulfide isomerase.J. Biol. Chem. 1999; 274: 3159-3164Google Scholar, 38Read J. Anderson T.A. Ritchie P.J. Vanloo B. Amey J. Levitt D. Rosseneu M. Scott J. Shoulders C.C. A mechanism of membrane neutral lipid acquisition by the microsomal triglyceride transfer protein.J. Biol. Chem. 2000; 275: 30372-30377Google Scholar) that the M subunit contains three domains: N-terminal β-barrel, central α-helical domain, and C-terminal lipid-binding cavity (Fig. 1). We propose that MTP may contain at least three (lipid transfer, membrane associating, and apoB binding) functionally independent domains (Fig. 1). Kinetic studies indicate for the presence of two, one low and one high affinity, lipid-binding domains in MTP (34Atzel A. Wetterau J.R. Identification of two classes of lipid molecule binding sites on the microsomal triglyceride transfer protein.Biochemistry. 1994; 33: 15382-15388Google Scholar). The high affinity domain binds few molecules of neutral lipids and phospholipids and may represent the lipid transfer domain. The lipid transfer activity antagonists probably bind at this site and inhibit lipid transfer activity. Precise information about the lipid transfer domain in MTP is not available but based on the homology with lipovitellin (39Anderson T.A. Levitt D.G. Banaszak L.J. The structural basis of lipid interactions in lipovitellin, a soluble lipoprotein.Structure. 1998; 6: 895-909Google Scholar), Read et al. (38Read J. Anderson T.A. Ritchie P.J. Vanloo B. Amey J. Levitt D. Rosseneu M. Scott J. Shoulders C.C. A mechanism of membrane neutral lipid acquisition by the microsomal triglyceride transfer protein.J. Biol. Chem. 2000; 275: 30372-30377Google Scholar) have suggested that MTP contains a C-terminal lipid binding cavity. The walls of the lipid binding cavity in MTP are formed by the A and C β-sheets present in the M subunit (Fig. 1). The α-helical domain holds these sheets together. The back of the cavity is probably covered by the P subunit (not shown in the figure). The lipid transfer domain may be involved in the loading and unloading of lipid molecules, a step necessary for their transfer. A nonsense mutation in the A sheet, Asn780Tyr, does not affect its binding to the P subunit but abolishes MTP's lipid transfer activity (40Ohashi K. Ishibashi S. Osuga J. Tozawa R. Harada K. Yahagi N. Shionoiri F. Iizuka Y. Tamura Y. Nagai R. Illingworth D.R. Gotoda T. Yamada N. Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia.J. Lipid Res. 2000; 41: 1199-1204Google Scholar). Thus, the C-terminal 1/3rd of the M subunit and the P subunit may form a lipid transfer domain in MTP. Studying the binding of 125I-MTP to lipid vesicles and separating MTP from vesicles by density gradient ultracentrifugation, we provided evidence for the stable association of MTP with membranes (41Bakillah A. Hussain M.M. Binding of microsomal tri-glyceride transfer proten to lipids results in increased affinity for apolipoprotein B: Evidence for stable microsomal MTP-lipid complexes.J. Biol. Chem. 2001; 276: 31466-31473Google Scholar). Similarly, Read et al. (38Read J. Anderson T.A. Ritchie P.J. Vanloo B. Amey J. Levitt D. Rosseneu M. Scott J. Shoulders C.C. A mechanism of membrane neutral lipid acquisition by the microsomal triglyceride transfer protein.J. Biol. Chem. 2000; 275: 30372-30377Google Scholar) have shown association of MTP with lipid vesicles. The low affinity lipid-binding domain identified in the kinetic experiments may be involved in membrane association. At present, the structural properties of the membrane-associating domain are not known. We propose that the region between the N and the A sheets may form a membrane-association domain. It should be pointed out that the lipovitellin (LV) structure contains a lipid moiety at this site (39Anderson T.A. Levitt D.G. Banaszak L.J. The structural basis of lipid interactions in lipovitellin, a soluble lipoprotein.Structure. 1998; 6: 895-909Google Scholar). We showed that apoB-binding domain in MTP is different from the lipid transfer domain because the lipid transfer activity inhibitors do not inhibit apoB-MTP binding, and inhibitors that inhibit apoB-MTP binding have no effect on lipid transfer activity of MTP (42Bakillah A. Nayak N. Saxena U. Medford R.M. Hussain M.M. Decreased secretion of apoB follows inhibition of apoB-MTP binding by a novel antagonist.Biochemistry. 2000; 39: 4892-4899Google Scholar). Furthermore, immobilization of MTP results in partial loss of MTP's lipid transfer activity but has no effect on apoB-MTP binding (43Hussain M.M. Bakillah A. Jamil H. Apolipoprotein B binding to microsomal triglyceride transfer protein decreases with increases in length and lipidation: implications in lipoprotein biosynthesis.Biochemistry. 1997; 36: 13060-13067Google Scholar, 44Hussain M.M. Bakillah A. Nayak N. Shelness G.S. Amino acids 430-570 in apolipoprotein B are critical for its binding to microsomal triglyceride transfer protein.J. Biol. Chem. 1998; 273: 25612-25615Google Scholar). Similarly, apoB-binding and membrane associating domains in MTP appear to be dissimilar. Evidence for the independent membrane associating and apoB binding domains also comes from the modulation of apoB-MTP interactions by lipids (41Bakillah A. Hussain M.M. Binding of microsomal tri-glyceride transfer proten to lipids results in increased affinity for apolipoprotein B: Evidence for stable microsomal MTP-lipid complexes.J. Biol. Chem. 2001; 276: 31466-31473Google Scholar). If apoB and membrane binding domains were the same, then apoB-MTP binding would have decreased in the presence of lipids. Contrary to this expectation, association of MTP with lipids resulted in increased binding to apoB. Thus, apoB-binding domain in MTP appears to be different from both the lipid transfer and membrane associating domains in MTP. The M subunit requires the P subunit for its solubility, retention in the ER, and for lipid-transfer activity (27Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex.J. Biol. Chem. 1990; 265: 9800-9807Google Scholar, 29Wetterau J.R. Combs K.A. McLean L.R. Spinner S.N. Aggerbeck L.P. Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal tri-glyceride transfer protein.Biochemistry. 1991; 30: 9728-9735Google Scholar). These two subunits are held together by non-covalent interactions. Early evidence for the P subunit-binding site in the M subunit came from the identification of genetic mutations in abetalipoproteinemia. Ricci et al. (45Ricci B. Sharp D. Orourke E. Kienzle B. Blinderman L. Gordon D. Smithmonroy C. Robinson G. Gregg R.E. Rader D.J. Wetterau J.R. A 30-amino acid truncation of the microsomal triglyceride transfer protein large subunit disrupts its interaction with protein disulfide-isomerase and causes abetalipoproteinemia.J. Biol. Chem. 1995; 270: 14281-14285Google Scholar) sequenced the mttp gene, which codes for the M subunit, from an abetalipoproteinemia patient and showed that the C-terminal 30 amino acids are required for its interaction with the P subunit. Using yeast two-hybrid system, Bradbury et al. (37Bradbury P. Mann C.J. Köchl S. Anderson T.A. Chester S.A. Hancock J.M. Ritchie P.J. Amey J. Harrison G.B. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. A common binding site on the microsomal triglyceride transfer protein for apolipoprotein B and protein disulfide isomerase.J. Biol. Chem. 1999; 274: 3159-3164Google Scholar) showed that P:1–274 bind to the central α-helical region of the M (M:297–603) subunit. Within this region, M:520–598 showed maximum binding to the P subunit. The binding between the N-terminus of the P subunit and the middle region of the M subunit may constitute a nucleation site for the heterodimerization of the two subunits. Subsequent binding of other regions in the P subunit with the C-terminal region in the M subunit are probably important for the formation of a soluble and biochemically active heterodimeric MTP complex. Three independent approaches (coimmunoprecipitation, solid-liquid inter-phase binding assays, and yeast two-hybrid system) have been used to demonstrate protein-protein interactions between apoB and MTP. Wu et al. (46Wu X.J. Zhou M.Y. Huang L.S. Wetterau J. Ginsberg H.N. Demonstration of a physical interaction between microsomal triglyceride transfer protein and apolipoprotein B during the assembly of ApoB-containing lipoproteins.J. Biol. Chem. 1996; 271: 10277-10281Google Scholar) presented the first evidence for interactions between these proteins in 1996 using co-immunoprecipitation technique. They immunoprecipitated MTP from [3H]leucine-labeled HepG2 cells and found that about 5–10% of the nascent apoB was associated with MTP. Furthermore, co-immunoprecipitation of apoB was also demonstrated by Western blot analysis of proteins immunoprecipitated with anti-MTP antibodies. In the same year Patel and Grundy transfected COS cells with various C-terminally truncated apoB polypeptides with MTP, immunoprecipitated apoB with anti-apoB antibodies, and observed that MTP was precipitated with various apoB peptides (47Patel S.B. Grundy S.M. Interactions between microsomal triglyceride transfer protein and apolipoprotein B within the endoplasmic reticulum in a heterologous system.J. Biol. Chem. 1996; 271: 18686-18694Google Scholar). We published a solid-liquid inter-phase binding assay in 1997 to study apoB-MTP binding. First, we immobilized different lipoproteins to microtiter plates and incubated them with 125I-labeled heterodimeric MTP. The amounts of MTP bound to LDL and VLDL were significantly higher than those bound to HDL (43Hussain M.M. Bakillah A. Jamil H. Apolipoprotein B binding to microsomal triglyceride transfer protein decreases with increases in length and lipidation: implications in lipoprotein biosynthesis.Biochemistry. 1997; 36: 13060-13067Google Scholar). Next, the binding of MTP to different lipoproteins was compared with the binding of PDI. Lipoproteins bound to MTP but not to PDI. These studies indicated that the M subunit plays an important role in lipoprotein binding. Subsequently, we showed that immobilized heterodimeric MTP also interacted with lipoproteins present in solution. Kinetic studies demonstrated that protein-protein interactions between these proteins were of high affinity (Kd 10–30 nM). In 1999, Shoulders and associates used baculoviral expression system and yeast two-hybrid system to study protein-protein interactions (24Mann C.J. Anderson T.A. Read J. Chester S.A. Harrison G.B. Köchl S. Ritchie P.J. Bradbury P. Hussain F.S. Amey J. Vanloo B. Rosseneu M. Infante R. Hancock J.M. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins.J. Mol. Biol. 1999; 285: 391-408Google Scholar, 37Bradbury P. Mann C.J. Köchl S. Anderson T.A. Chester S.A. Hancock J.M. Ritchie P.J. Amey J. Harrison G.B. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. A common binding site on the microsomal triglyceride transfer protein for apolipoprotein B and protein disulfide isomerase.J. Biol. Chem. 1999; 274: 3159-3164Google Scholar). They expressed apoB-17 with M subunit, P subunit, or both M and P subunits in Sf 9 cells, immunoprecipitated apoB, and looked for the co-precipitation of the M and P subunits (24Mann C.J. Anderson T.A. Read J. Chester S.A. Harrison G.B. Köchl S. Ritchie P.J. Bradbury P. Hussain F.S. Amey J. Vanloo B. Rosseneu M. Infante R. Hancock J.M. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins.J. Mol. Biol. 1999; 285: 391-408Google Scholar). PDI was not precipitated with apoB. However, the M subunit was immunoprecipitated with apoB-17 when expressed with the P subunit. These studies reinforced the notion that apoB interacts with the M subunit and the P subunit is probably not required for apoB binding. Patel and Grundy made the first attempt to understand the nature of interactions between apoB and MTP (47Patel S.B. Grundy S.M. Interactions between microsomal triglycerid" @default.
• W2151400756 created "2016-06-24" @default.
• W2151400756 creator A5020617939 @default.
• W2151400756 creator A5052861667 @default.
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• W2151400756 date "2003-01-01" @default.
• W2151400756 modified "2023-10-10" @default.
• W2151400756 title "Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly" @default.
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• W2151400756 doi "https://doi.org/10.1194/jlr.r200014-jlr200" @default.
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