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Journal of Lipid Research Oct 1990Apolipoprotein B (apoB), an apolipoprotein associated with very low density lipoproteins and the atherogenic low density lipoproteins (LDL), directs the metabolism of...
Apolipoprotein B (apoB), an apolipoprotein associated with very low density lipoproteins and the atherogenic low density lipoproteins (LDL), directs the metabolism of lipoprotein particles in plasma by interacting with the LDL receptor. Utilizing human intestinal biopsy organ cultures, we have studied the synthesis of intestinal apoB in man. Intestinal organ cultures from normal adults (n = 6) were incubated in the presence of protease inhibitors in media supplemented with [35S]methionine. Media from these cultures were evaluated by sequential NaDodSO4 polyacrylamide gel electrophoresis, radioautography, and Western blot analyses, and intestinal biopsies were studied using immunohistochemistry. The relative abundance of apoB-100 and apoB-48 mRNA was assessed using reverse transcriptase-polymerase chain reaction followed by primer extension. Although apoB-48 was the principal isoprotein that was newly synthesized by intestinal organ cultures, apoB-100 was also synthesized and secreted by human intestinal organ cultures with 16 +/- 3% of the intestinal apoB mRNA coding for apoB-100. These results establish that apoB-100 is produced by the human intestine. The synthesis of the atherogenic apoB-100 by the intestine has pathophysiologic implications for the development of diet-induced atherosclerosis.
Topics: Adult; Apolipoprotein B-100; Apolipoprotein B-48; Apolipoproteins B; Base Sequence; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Humans; Immunohistochemistry; Intestinal Mucosa; Molecular Sequence Data; Organ Culture Techniques; Protease Inhibitors; RNA, Messenger
PubMed: 2079601
DOI: No ID Found -
Journal of Lipid Research Sep 2001There is general consensus that amphipathic alpha-helices and beta sheets represent the major lipid-associating motifs of apolipoprotein (apo)B-100. In this review, we... (Review)
Review
There is general consensus that amphipathic alpha-helices and beta sheets represent the major lipid-associating motifs of apolipoprotein (apo)B-100. In this review, we examine the existing experimental and computational evidence for the pentapartite domain structure of apoB. In the pentapartite nomenclature presented in this review (NH(2)-betaalpha(1)-beta(1)-alpha(2)-beta(2)-alpha(3)-COOH), the original alpha(1) globular domain (Segrest, J. P. et al. 1994. Arterioscler. Thromb. 14: 1674;-1685) is expanded to include residues 1;-1,000 and renamed the betaalpha(1) domain. This change reflects the likelihood that the betaalpha(1) domain, like lamprey lipovitellin, is a globular composite of alpha-helical and beta-sheet secondary structures that participates in lipid accumulation in the co-translationally assembled prenascent triglyceride-rich lipoprotein particles. Evidence is presented that the hydrophobic faces of the amphipathic beta sheets of the beta(1) and beta(2) domains of apoB-100 are in direct contact with the neutral lipid core of apoB-containing lipoproteins and play a role in core lipid organization. Evidence is also presented that these beta sheets largely determine LDL particle diameter. Analysis of published data shows that with a reduction in particle size, there is an increase in the number of amphipathic helices of the alpha(2) and alpha(3) domains associated with the surface lipids of the LDL particle; these increases modulate the surface pressure decreases caused by a reduction in radius of curvature. The properties of the LDL receptor-binding region within the overall domain structure of apoB-100 are also discussed. Finally, recent three-dimensional models of LDL obtained by cryoelectron microscopy and X-ray crystallography are discussed. These models show three common features: a semidiscoidal shape, a surface knob with the dimensions of the betaC globular domain of lipovitellin, and planar multilayers in the lipid core that are approximately 35 A apart; the multilayers are thought to represent cholesteryl ester in the smectic phase. These models present a conundrum: are LDL particles circulating at 37 degrees C spheroidal in shape, as generally assumed, or are they semidiscoidal in shape, as suggested by the models? The limited evidence available supports a spheroidal shape.
Topics: Animals; Apolipoprotein B-100; Apolipoproteins B; Binding Sites; Computer Simulation; Detergents; Humans; Lipoproteins, LDL; Models, Molecular; Protein Conformation; Protein Structure, Secondary; Receptors, LDL; Solubility; Structure-Activity Relationship
PubMed: 11518754
DOI: No ID Found -
Journal of Lipid Research Sep 2022The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and...
The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cells are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine-derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The endogenous MTTP gene was ablated in human hepatoma Huh-7 cells using single guide RNA and RNA-guided clustered regularly interspaced short palindromic repeats-associated sequence 9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP, and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.
Topics: Apolipoprotein B-48; Apolipoproteins B; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line; DNA, Complementary; Humans; Lipoproteins; Liver Neoplasms; RNA, Guide, CRISPR-Cas Systems; RNA, Messenger; Ribonucleoproteins; Triglycerides
PubMed: 35931202
DOI: 10.1016/j.jlr.2022.100257 -
Trends in Endocrinology and Metabolism:... Sep 2008Because the levels of secreted apolipoprotein B (apoB) directly correlate with circulating serum cholesterol levels, there is a pressing need to define how the... (Review)
Review
Because the levels of secreted apolipoprotein B (apoB) directly correlate with circulating serum cholesterol levels, there is a pressing need to define how the biosynthesis of this protein is regulated. Most commonly, the concentration of a secreted, circulating protein corresponds to transcriptionally and/or translationally regulated events. By contrast, circulating apoB levels are controlled by degradative pathways in the cell that select the protein for disposal. This article summarizes recent findings on two apoB disposal pathways, endoplasmic reticulum (ER)-associated degradation and autophagy, and describes a role for post-ER degradation in the increased circulating lipid levels in insulin-resistant diabetics.
Topics: Animals; Apolipoproteins B; Energy Metabolism; Humans; Lipid Metabolism
PubMed: 18691900
DOI: 10.1016/j.tem.2008.07.002 -
Journal of Lipid Research Nov 2011This review integrates historical biochemical and modern genetic findings that underpin our understanding of the low-density lipoprotein (LDL) dyslipidemias that bear on... (Review)
Review
This review integrates historical biochemical and modern genetic findings that underpin our understanding of the low-density lipoprotein (LDL) dyslipidemias that bear on human disease. These range from life-threatening conditions of infancy through severe coronary heart disease of young adulthood, to indolent disorders of middle- and old-age. We particularly focus on the biological aspects of those gene mutations and variants that impact on sterol absorption and hepatobiliary excretion via specific membrane transporter systems (NPC1L1, ABCG5/8); the incorporation of dietary sterols (MTP) and of de novo synthesized lipids (HMGCR, TRIB1) into apoB-containing lipoproteins (APOB) and their release into the circulation (ANGPTL3, SARA2, SORT1); and receptor-mediated uptake of LDL and of intestinal and hepatic-derived lipoprotein remnants (LDLR, APOB, APOE, LDLRAP1, PCSK9, IDOL). The insights gained from integrating the wealth of genetic data with biological processes have important implications for the classification of clinical and presymptomatic diagnoses of traditional LDL dyslipidemias, sitosterolemia, and newly emerging phenotypes, as well as their management through both nutritional and pharmaceutical means.
Topics: Absorption; Amino Acid Sequence; Animals; Apolipoproteins B; Disease; Genetic Predisposition to Disease; Humans; Lipoproteins, LDL; Molecular Sequence Data; Sterols
PubMed: 21862702
DOI: 10.1194/jlr.R017855 -
Journal of Lipid Research Jan 2003Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are necessary for lipoprotein assembly. ApoB consists of five structural domains,... (Review)
Review
Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are necessary for lipoprotein assembly. ApoB consists of five structural domains, betaalpha(1)-beta(1)-alpha(2)-beta(2)-alpha(3). We propose that MTP contains three structural motifs (N-terminal beta-barrel, central alpha-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 betaalpha(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.
Topics: Animals; Apolipoproteins B; Carrier Proteins; Humans; Protein Binding; Protein Structure, Tertiary; Structure-Activity Relationship
PubMed: 12518019
DOI: 10.1194/jlr.r200014-jlr200 -
The Journal of Biological Chemistry Jul 1986The structural relationship between apolipoprotein B-100 (apo-B-100) and apolipoprotein B-48 (apo-B-48) has not been elucidated. A peptide fragment (MDB-18) of... (Comparative Study)
Comparative Study
The structural relationship between apolipoprotein B-100 (apo-B-100) and apolipoprotein B-48 (apo-B-48) has not been elucidated. A peptide fragment (MDB-18) of approximately 6 kDa was isolated from a tryptic digest of apo-B-100. The sequence of the first 22 amino acids of MDB-18 was determined by Edman degradation. A 15-residue peptide corresponding to this sequence was synthesized by the solid-phase method and was utilized to develop a sequence-specific polyclonal antibody. On immunoblot analysis, the antibody recognized both intact apo-B-100 and apo-B-48. In addition, preincubating the antibody with the synthetic peptide abolished the recognition of both apo-B-100 and apo-B-48. These data are interpreted as indicating that there is an amino acid sequence homology between apo-B-100 and apo-B-48. Since the MDB-18 peptide is located in the carboxyl region of the B-100 molecule, we propose apo-B-100 and apo-B-48 share a common carboxyl region sequence.
Topics: Amino Acid Sequence; Apolipoprotein B-100; Apolipoprotein B-48; Apolipoproteins B; Humans; Immunologic Techniques; Peptide Fragments; Trypsin
PubMed: 3522585
DOI: No ID Found -
The Biochemical Journal Jul 1992We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by...
We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.
Topics: Animals; Apolipoprotein B-100; Apolipoprotein B-48; Apolipoproteins B; Blotting, Western; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Endocytosis; Endoplasmic Reticulum; Golgi Apparatus; Liver; Male; Microsomes, Liver; Precipitin Tests; Rats; Rats, Inbred Strains; Subcellular Fractions
PubMed: 1637294
DOI: 10.1042/bj2850153 -
CMAJ : Canadian Medical Association... May 1992
Review
Topics: Animals; Apolipoproteins B; Blood Proteins; Complement C3a; Fatty Acids; Humans; Lipoprotein Lipase; Triglycerides
PubMed: 1596813
DOI: No ID Found -
Nature Communications Jul 2022Lipid metabolism plays an instructive role in regulating stem cell state and differentiation. However, the roles of lipid mobilization and utilization in stem...
Lipid metabolism plays an instructive role in regulating stem cell state and differentiation. However, the roles of lipid mobilization and utilization in stem cell-driven regeneration are unclear. Planarian flatworms readily restore missing tissue due to injury-induced activation of pluripotent somatic stem cells called neoblasts. Here, we identify two intestine-enriched orthologs of apolipoprotein b, apob-1 and apob-2, which mediate transport of neutral lipid stores from the intestine to target tissues including neoblasts, and are required for tissue homeostasis and regeneration. Inhibition of apob function by RNAi causes head regression and lysis in uninjured animals, and delays body axis re-establishment and regeneration of multiple organs in amputated fragments. Furthermore, apob RNAi causes expansion of the population of differentiating neoblast progeny and dysregulates expression of genes enriched in differentiating and mature cells in eight major cell type lineages. We conclude that intestine-derived lipids serve as a source of metabolites required for neoblast progeny differentiation.
Topics: Animals; Apolipoproteins; Apolipoproteins B; Intestines; Planarians; Pluripotent Stem Cells
PubMed: 35778403
DOI: 10.1038/s41467-022-31385-2