A monomeric, biologically active, full-length human apolipoprotein E

Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and infectious diseases. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural study of full-length apoE is hindered by apoE's oligomerization property. Using protein-engineering techniques, we generated a monomeric, biologically active, full-length apoE. Cross-linking experiments indicate that this mutant is nearly 95-100% monomeric even at 20 mg/mL. CD spectroscopy and guanidine hydrochloride denaturation demonstrate that the structure and stability of the monomeric mutant are identical to wild-type apoE. Monomeric and wild-type apoE display similar lipid-binding activities in dimyristoylphosphatidylcholine clearance assays and formation of reconstituted high-density lipoproteins. Furthermore, the monomeric and wild-type apoE proteins display an identical LDL receptor binding activity. Availability of this monomeric, biologically active, full-length apoE allows us to collect high quality NMR data for structural determination. Our initial NMR data of lipid-free apoE demonstrates that the N-terminal domain in the full-length apoE adopts a nearly identical structure as the isolated N-terminal domain, whereas the C-terminal domain appears to become more structured than the isolated C-terminal domain fragment, suggesting a weak domain-domain interaction. This interaction is confirmed by NMR examination of a segmental labeled apoE, in which the N-terminal domain is deuterated and the C-terminal domain is double-labeled. NMR titration experiments further suggest that the hinge region (residues 192-215) that connects apoE's N- and C-terminal domains may play an important role in mediating this domain-domain interaction.     

Synthesis, intracellular processing, and signal peptide of human apolipoprotein E

Northern blotting analysis has shown apo-E mRNA synthesis by human liver, HepG2 cells, and primary cultures of human monocyte macrophages but not by the macrophage-like cell line U937 and normal or transformed human fibroblasts. Cell-free translation has shown that the primary translation product of apo-E consists of one major and one minor isoprotein of apparent Mr = 28,500 and isoelectric points 6.20 and 6.02, respectively. These isoproteins differ by +1 and 0 charges from apo-E3 and have been designated preapo-E. Co-translational treatment of mRNA with dog pancreatic membranes converts both preapo-E isoproteins to a form which is undistinguishable by two-dimensional gel electrophoresis from plasma apo-E3. The isolation and nucleotide sequence analysis of a full length apo-E cDNA clone has shown that preapo-E contains an 18-amino acid NH2-terminal signal peptide compared to plasma apo-E. The signal peptide sequence is: MetLysValLeuTrpAlaAlaLeuLeuValThrPheLeuAlaGlyCysGlnAla. Comparison of co-translationally modified apo-E with intracellular, secreted, and plasma forms indicates that after the intracellular cleavage of the signal peptide, the protein is glycosylated with carbohydrate chains containing sialic acid, secreted as sialoapo-E (apo-Es), and subsequently desialated in plasma. These findings demonstrate that apo-E is synthesized as preprotein and undergoes intracellular proteolysis and glycosylation and extracellular desialation to attain the major asialoapo-E isoprotein form observed in plasma.     

Comparison of the human,canine and swine E apoproteins

A comparative study of the E apoprotein isolated from the d<1.02 lipoproteins of human, canine and swine plasma revealed that the various apo-E preparations had similar molecular weights (37,000–39,000) and had similar amino acid compositions in that glutamic acid, alanine, leucine and arginine were present in high concentrations. The various preparations showed partial immunochemical cross-reactivity, demonstrating significant sequence homology between the species. However, determination of the amino-terminal amino acid sequence by automated Edman degradation showed each apo-E was different, demonstrating that the amino-terminal portion of the E apoprotein was a variable region of this protein.     

Distribution of apolipoprotein A-I, C-II, C-III, and E mRNA in fetal human tissues. Time-dependent induction of apolipoprotein E mRNA by cultures of human monocyte-macrophages

Recently developed molecular probes for human apolipoprotein (apo) genes have been used to study the specificity of human tissue expression of the apo A-I, apo C-II, apo C-III, and apo E genes. We have found that apo E mRNA was present in all tissues examined. On the basis of total RNA concentration the relative abundance of apo E mRNA expressed as a percentage of the liver value is as follows: adrenal gland and macrophages, 74-100%; gonads and kidney, 12-15%; spleen, brain, thymus, ovaries, intestine, and pancreas, 3-9%; heart, 1.5%; stomach, striated muscle, and lung, less than 1%. The relative concentration of apo E mRNA in cultures of human peripheral blood monocyte-macrophages increases dramatically as a function of time in culture, and after 5 days, it compares to that of liver. The human tissues shown to synthesize apo E mRNA were also examined for their ability to synthesize apo A-I, apo C-II, and apo C-III mRNA. The relative abundance of apo A-I, apo C-III, and apo C-II mRNA expressed as a percentage of the liver value is as follows: apo A-I, intestine, 50%; apo A-I, pancreas and gonads, 12%; apo A-I, kidney, 4%; apo A-I, adrenal, 2.5%; apo A-I, ovaries and heart, 1%; apo A-I, stomach and thymus, less than 1%; apo C-III, intestine, 62%; apo C-III, pancreas, 7%; apo C-II, intestine, 3%; apo C-II, pancreas, less than 1%. The knowledge of tissue specificities in the synthesis of apolipoproteins is important for our understanding of the regulation of apolipoproteins and lipoprotein metabolism.     

Structural variation in human apolipoprotein E3 and E4: secondary structure, tertiary structure, and size distribution

Human apolipoprotein E (apoE) is a 299-amino-acid protein with a molecular weight of 34 kDa. The difference between the apoE3 and apoE4 isoforms is a single residue substitution involving a Cys-Arg replacement at residue 112. ApoE4 is positively associated with atherosclerosis and late-onset and sporadic Alzheimer's disease (AD). ApoE4 and its C-terminal truncated fragments have been found in the senile plaques and neurofibrillary tangles in the brain of AD patients. However, detail structural information regarding isoform and domain interaction remains poorly understood. We prepared full-length, N-, and C-terminal truncated apoE3 and apoE4 proteins and studied their structural variation. Sedimentation velocity and continuous size distribution analysis using analytical ultracentrifugation revealed apoE3(72-299) as consisting of a major species with a sedimentation coefficient of 5.9. ApoE4(72-299) showed a wider and more complicated species distribution. Both apoE3 and E4 N-terminal domain (1-191) existed with monomers as the major component together with some tetramer. The oligomerization and aggregation of apoE protein increased when the C-terminal domain (192-271) was incorporated. The structural influence of the C-terminal domain on apoE is to assist self-association with no significant isoform preference. Circular dichroism and fluorescence studies demonstrated that apoE4(72-299) possessed a more alpha-helical structure with more hydrophobic residue exposure. The structural variation of the N-terminal truncated apoE3 and apoE4 protein provides useful information that helps to explain the greater aggregation of the apoE4 isoform and thus has implication for the involvement of apoE4 in AD.     

The structure of human apolipoprotein E2, E3 and E4 in solution 1. Tertiary and quaternary structure

Three recombinant apoE isoforms fused with an amino-terminal extension of 43 amino acids were produced in a heterologous expression system in E. coli. Their state of association in aqueous phase was analyzed by size-exclusion liquid chromatography, sedimentation velocity and sedimentation equilibrium experiments. By liquid chromatography, all three isoforms consisted of three major species with Stokes radii of 4.0, 5.0 and 6.6 nm. Sedimentation velocity confirmed the presence of monomers, dimers and tetramers as major species of each isoform. The association schemes established by sedimentation equilibrium experiments corresponded to monomer-dimer-tetramer-octamer for apoE2, monomer-dimer-tetramer for apoE3 and monomer-dimer-tetramer-octamer for apoE4. Each of the three isoforms exhibits a distinct self-association pattern. The apolipoprotein multi-domain structure was mapped by limited proteolysis with trypsin, chymotrypsin, elastase, subtilisin and Staphylococcus aureus V8 protease. All five enzymes produced stable intermediates during the degradation of the three apoE isoforms, as described for plasma apoE3. The recombinant apoE isoforms, thus, consist of N- and C-terminal domains. The presence of the fusion peptide did not appear to alter the apolipoprotein tertiary organization. However, a 30 kDa amino-terminal fragment appeared during the degradation of the recombinant apoE isoforms resulting from cleavage in the 273-278 region. This region, not accessible in plasma apoE3, results from a different conformation of the C-terminal domain in the recombinant isoforms. A specific pattern for the apoE4 C-terminal domain was observed during the proteolysis. The region 230-260 in apoE4, in contrast to that of apoE3 and apoE2, was not accessible to proteases, probably due to the existence of a longer helix in this region of apoE4 stabilized by an interdomain interaction.     

Carboxyl-terminal domain of human apolipoprotein E: expression, purification, and crystallization.

Thioredoxin fusion expression vectors for two carboxyl-terminal fragments of human apolipoprotein (apo) E (residues 223-272 and 223-299) were generated from an apoE cDNA with the objective of obtaining structural information on this functionally important region of apoE by X-ray crystallography. A thrombin cleavage recognition site was positioned at the fusion junction to release the apoE fragments from the fusion protein. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates by nickel-affinity column chromatography, and cleaved with thrombin. After gel filtration and ion exchange chromatography, yields of each fragment were approximately 14 mg/L. Both fragments bind to the phospholipid dimyristoylphosphatidylcholine in a manner similar to that of the 216-299 fragment of apoE isolated from plasma, which represents the major lipid-binding region of the protein. Orthorhombic crystals of the apoE 223-272 fragment that diffracted to 1.8 A were obtained in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH 7.0-7.5), containing 30% glycerol. The space group is C222 with cell dimensions of a = 35.17 A, b = 38.95 A, and c = 133.27 A.     

Regulation of nuclear receptor activities by two human papillomavirus type 18 oncoproteins,E6 and E7

The human papillomavirus (HPV) E6 and E7 oncoproteins are two major proteins that remain expressing in HPV-associated human cancers. The high-risk HPVs synthesize E6 and E7 oncoproteins to alter the function of cellular regulatory proteins, such as p53 and retinoblastoma gene product, respectively. In this study, we demonstrated that HPV-18 E6 and E7 proteins were able to directly interact with some nuclear receptors (NRs), such as thyroid receptor, androgen receptor, and estrogen receptor (ER), whether or not appropriate hormones were present. The functional roles of these two oncoproteins in NRs depended on the cell type (including ligand), promoter context, and NR type. These two oncoproteins regulated ER functions through ER's AF-1, AF-2, or both. Hence, our results provide new insights into the mechanisms controlling the proliferation and immortalization of HPV infected cells by these two oncoproteins mediating through their regulatory functions in NR systems.     

Expression, purification and characterization of human urodilatin in E. coli

Urodilatin is a 32-amino acid peptide hormone synthesized in kidney to regulate natriuresis and diuresis. It has been shown clinically useful for the treatment of acute decompensated heart failure. A synthetic deoxyoligonucleotide encoding urodilatin was cloned into a pET32a vector immediately after the thioredoxin encoding sequence with a hexa-hisditine tag and an enterokinase recognition site incorporated in between. The fusion protein was overexpressed in Escherichia coli, which constituted 28% of the total cell proteins. More than 85% of Trx-urodilatin was soluble and purified nearly homogenous by Ni-Sepharose affinity chromatography. Urodilatin was then released from the fusion protein by the enterokinase treatment and separated from the fusion partner by the subtractive chromatography using Ni-Sepharose once again. The urodilatin sample was further purified with reverse phase HPLC. Via a biological activity assayed in vitro, it was found that urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of (2.02+/-0.36)x10(-6)mg/ml, which was similar to that of the synthetic urodilatin standard. The method described here promises to produce about 4.5mg fully active recombinant urodilatin with homogeneity over 97% from one liter shaking flask culture of E. coli.     

HPV18 E7致癌蛋白的高效表达、纯化和鉴定

目的为进一步研究人乳头状瘤病毒18（Human papillomavirus18,HPV18）E7蛋白的结构与功能。方法构建HPV18 E7的谷胱甘肽S-转移酶融合蛋白质粒pGEX-6P-1-GST-HPV18 E7,重组质粒转入大肠埃希菌BL21进行可溶性融合蛋白的高效表达。结果柱上切除法去除GST标签,表达产物经glutathione Sepharose 4B亲和层析纯化,获得了SDS-PAGE和HPLC-ESI-MS纯度的HPV18 E7均质蛋白,非变性PAGE和凝胶过滤表明HPV18 E7以稳定的单体形式存在于水溶液中。高压液相色谱-电喷雾质谱（HPLC-ESI-MS）分析得到HPV18 E7精确分子量为12865．0 Da,与其理论值吻合。纯化蛋白经HPLC-ESI-MS／MS鉴定为目的产物,鉴定出的9个匹配肽段覆盖率为HPV18 E7整个氨基酸序列的96．5％。结论本文所建立的技术可以有效地大量制备HPV18 E7,为进一步研究其结构与功能和致癌机制奠定了重要的物质基础。     

Relationships of the Col plasmids E2, E3, E4, E5, E6, and E7: Restriction mapping and colicin gene fusions

Thirteen ColE plasmids representing the E2-E7 types have been compared by restriction mapping. Over 80% of their restriction sites were found to be similarly positioned, indicating that these plasmids share a common structure. Three variants are ColE2-CA42 and ColE7-K317, both of which contain 1.8-kb DNA segments in place of a 2.5-kb segment common to the other plasmids, and ColE6-CT14, which has an additional 5.0-kb DNA segment compared to the other plasmids. The colicin (col), immunity (imm), and colicin release (hic) genes of these plasmids have been localized to regions corresponding to those known for ColE3-CA38 and ColE2-P9, with the imm and hic genes adjacent to the 3' end of the col gene. Active colicin is produced from hybrid col genes containing 5' and 3' ends from different E-type plasmids. The 3'-termini of the fused col genes specify the colicin type.     

Cyclin E in human cancers.

Regulators of the cell cycle such as cyclin E play an important part in neoplasia. The cyclin E protein forms a partnership with a specific protein kinase. This complex phosphorylates key substrates to initiate DNA synthesis. Cyclin-dependent kinase inhibitors (CKIs) are able to suppress the activity of cyclin E. Various substances (including proteins produced by oncogenic viruses) affect cyclin E directly or indirectly through an interaction with CKIs. These interactions are important in elucidating the mechanisms of neoplasia. They may also provide prognostic information in a wide range of common cancers. Cyclin E may even be a target for treatment of cancers in the future.     

Intracellular modifications of human apolipoprotein E

We have used pulse-chase techniques to study the synthesis, intracellular modification, and secretion of human apolipoprotein E by cultures of HepG2 cells and peripheral blood human monocyte macrophages. We have found that modified apoE isoproteins are detectable intracellularly after 14 min of pulse, and their relative concentration increases linearly over a 2-h pulse-chase period. At the same time, the relative concentration of unmodified apoE decreases. All the major modified apoE isoproteins appear simultaneously, and they correspond to the sialo apoE forms apooEs2, apoEs4, and apoEs6. ApoE secretion is first detected after a 30-min pulse. Secreted apoE consists of 92% of the same sialated apoE forms observed intracellularly. ApoE sialation and secretion were not affected by treatment of the HepG2 cultures with tunicamycin, whereas monensin inhibited both the intracellular sialation and secretion of this protein. These findings suggest that apoE is secreted in the form of three major isoproteins generated by intracellular modification of this protein with one or more O-linked oligosaccharide chains containing sialic acid.