C Cladaras

The complete sequence and structural analysis of human apolipoprotein B-100: relationship between apoB-100 and apoB-48 forms.

We have isolated and sequenced overlapping cDNA clones covering the entire sequence of human apolipoprotein B‐100 (apoB‐100). DNA sequence analysis and determination of the mRNA transcription initiation site by S1 nuclease mapping showed that the apoB mRNA consists of 14,112 nucleotides including the 5′ and 3′ untranslated regions which are 128 and 301 nucleotides respectively. The DNA‐derived protein sequence shows that apoB‐100 is 513,000 daltons and contains 4560 amino acids including a 24‐amino‐acid‐long signal peptide. The mol. wt of apoB‐100 implies that there is one apoB molecule per LDL particle. Computer analysis of the predicted secondary structure of the protein showed that some of the potential alpha helical and beta sheet structures are amphipathic, whereas others have non‐amphipathic neutral to apolar character. These latter regions may contribute to the formation of the lipid‐binding domains of apoB‐100. The protein contains 25 cysteines and 20 potential N‐glycosylation sites. The majority of cysteines are distributed in the amino terminal portion of the protein. Four of the potential glycosylation sites are in predicted beta turn structures and may represent true glycosylation positions. ApoB lacks the tandem repeats which are characteristic of other apolipoproteins. The mean hydrophobicity the mean value of H1 and helical hydrophobic moment the mean value of microH profiles of apoB showed the presence of several potential helical regions with strong polar character and high hydrophobic moment. The region with the highest hydrophobic moment, between amino acid residues 3352 and 3369, contains five closely spaced, positively charged residues, and has sequence homology to the LDL receptor binding site of apoE. This region is flanked by three neighbouring regions with positively charged amino acids and high hydrophobic moment that are located between residues 3174 and 3681. One or more of these closely spaced apoB sequences may be involved in the formation of the LDL receptor‐binding domain of apoB‐100. Blotting analysis of intestinal RNA and hybridization of the blots with carboxy apoB cDNA probes produced a single 15‐kb hybridization band whereas hybridization with amino terminal probes produced two hybridization bands of 15 and 8 kb. Our data indicate that both forms of apoB mRNA contain common sequences which extend from the amino terminal of apoB‐100 to the vicinity of nucleotide residue 6300. These two messages may have resulted from differential splicing of the same primary apoB mRNA transcript.

Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

Apolipoprotein and lipoprotein synthesis and modifications

Conventional and molecular biological approaches have defined the sites of apolipoprotein synthesis and have described intra- and extracellular apolipoprotein modifications. Topics selected for discussion in this review include new aspects of apolipoprotein synthesis and modifications, modification of apolipoprotein B byproducts of lipid oxidation, remodeling of lipoproteins by transfer of apolipoproteins and functional significance, in vivo and in vitro assembly of apolipoprotein B-containing lipoproteins, and intracellular versus extracellular assembly of high-density lipoprotein and lipoprotein-type particles.

Regulation of Expression of HIV-1 by Viral Factors

The expression of the human immunodeficiency virus type 1 (HIV-1) in human cells is regulated by viral factors. This regulation results in the establishment of a steady-state permitting the balanced expression of the viral components. The concerted function of two viral activator proteins tat and rev is necessary for viral expression. Tat and rev are expressed from overlapping reading frames (Figure 1). The action of tat is mediated by a cis-acting element in the R region of the LTR (TAR), while the action of rev is mediated by a cis-acting element in the env region (RRE) (Figure 1). Tat is essential for the accumulation of all viral mRNAS. On the other hand, rev is essential for the accumulation of those viral mRNAs encoding structural proteins.

Molecular Biology of Human Apolipoprotein B and Related Diseases

Apolipoprotein B is the main protein component of LDL and comprises 23.8% of the LDL particle (1,2). ApoB is the ligand for the cellular recognition and catabolism of LDL by the LDL receptor (1,3). The LDL receptor-apoB interaction and subsequent catabolism mediates the clearance of LDL from plasma and regulates cellular cholesterol biosynthesis (1,3). Early studies had shown that the human and rat apoB exist in two primary forms designated apoB-100 and apoB-48 (4–12). Apolipoprotein B seems to be important for lipoprotein assembly and secretion (14,15). Thus, absence of apoR in abetalipoproteinemia (16) or inhibition of its synthesis by cyclohexamide treatment of cells (15) abolishes secretion of chylomicrons and VLDL.