Specification of binding modes between a transmembrane peptide mimic of ATP6V0C and polytopic E5 of human papillomavirus-16

Interaction of E5 of papillomavirus-16 based on its three transmembrane domains (TMDs) with a peptide mimicking the fourth TMD (TMD-A) of the 16 kDa c subunit of the human vacuolar H⁺-ATPase, ATP6V0C, and one of its mutant is investigated. Docking reveals binding of the peptide between the second and third TMD of E5. A series of hydrophobic residues are responsible for the contact. Estimated weak binding energies based on potential of mean force calculations reveal marginal differences of the estimated binding energies between wild type (WT) and mutant peptide. Also differences in estimated binding energies of dimers of the individual TMDs of E5 with the WT peptide are marginal. Correlation of rotational data derived from coarse-grained molecular dynamics simulations of the peptides and the protein as well as from the principal component analysis reveal that the binding of TMD-A with TMD3 is enthalpy driven and binding with TMD2 is guided by entropic conditions.     

The HPV16 E6/E7 oncogene sensitizes human ovarian surface epithelial cells to low-dose but not high-dose 5-FU and 5-FUdR

To evaluate the effect of HPV16 E6/E7 on drug sensitivity, primary human OSE cells were infected with HPV16 E6/E7 expressing retrovirus and then exposed to chemotherapeutic agents. Apoptosis induced by mitomycin C was dose-dependent in both primary OSE and E6E7/OSE cells. E6E7/OSE cells were more sensitive to mitomycin C than parental OSE cells. HPV16 E6/E7 also sensitized OSE cells to 5-FU and its derivative 5-FUdR, but only at low doses. This phenomenon was also observed in cervical cancer cells and was independent of thymidylate synthase, a target of thymine and thymidine analogues. We conclude that HPV16 E6/E7 specifically modulates the activity of 5-FU and 5-FUdR, and confers OSE cells hypersensitivity to low-dose but not high-dose 5-FU and 5-FUdR. Molecular analysis indicates that induction of p53 and p21, and suppression of pRB are associated with apoptosis induced by 5-FUdR and may partly explain the hypersensitivity of E6E7/OSE cells to low-dose 5-FUdR.     

Defining the minimal interacting regions of the tight junction protein MAGI-1 and HPV16 E6 oncoprotein for solution structure studies

The oncoprotein E6 produced by tumorigenic high-risk genital human papillomaviruses targets a number of cellular proteins containing PDZ domains for proteasome-mediated degradation. In particular, E6 targets the tight junction protein MAGI-1 by binding to its PDZ1 domain. Using light scattering and NMR, we explored different fragments of both the HPV16 E6 and the MAGI-1 PDZ1 domain to define the best-behaving complex for solution structure studies. We showed that the 70-residue HPV16 E6 C-terminal domain (E6C) can be efficiently substituted by a peptide spanning the 11 C-terminal residues of E6. The construct of MAGI-1 PDZ1 best suited for solution structure analysis presents a 14-residue N-terminal extension and a 26-residue C-terminal extension as compared to the construct used for the recently solved X-ray structure of a MAGI-1 PDZ1/HPV18 E6 complex. These data suggest a stabilizing role for the interdomain linker regions which separate the PDZ1 domain from its neighboring domains.     

人乳头瘤病毒16型E5与IL-12联合基因疫苗的免疫活性

为了研制人乳头瘤病毒16型(HPV16)防治性疫苗,分析了HPV16 E5与IL-12联合基因疫苗的免疫活性。将构建的pcDNA3.1(+)/E5与pcDNA3.1(+)/IL-12联合免疫BALB/c小鼠,以ELISA测定小鼠血清中抗HPV16 E5 IgG水平、小鼠脾细胞培养上清中IFN-γ和IL-4含量;MTT法检测脾淋巴细胞增殖反应。结果显示末次免疫后,联合基因疫苗组和单基因疫苗组血清IgG A450值分别明显高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组(P<0.01);且联合基因疫苗组显著高于单基因疫苗组(P<0.01)。联合基因疫苗组和单基因疫苗组的IFN-γ和IL-4含量分别均明显高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组IFN-γ和IL-4含量(P<0.01),且联合基因疫苗组含量显著高于单基因疫苗组(P<0.01)。联合基因疫苗组和单基因疫苗组脾淋巴细胞刺激指数(SI)分别显著高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组(P<0.01);联合基因疫苗组与单基因疫苗组比较,SI差异无统计学意义(P>0.05)。结果表明HPV16 E5单基因疫苗以及与IL-12联合基因疫苗均能刺激机体产生较强的免疫应答,且联合基因疫苗优于单基因疫苗。     

宫颈癌组织中人乳头瘤病毒16型E5基因的变异

探讨了HPV16 E5基因突变与宫颈癌发病的关系.应用银染聚合酶链反应-单链构象多态性(PCR-SSCP)分析对50份人宫颈癌活检标本进行基因突变的筛查.PCR检测显示,50例宫颈癌组织中HPV16 E5的检出率为30%(15/50),其中6例宫颈癌HPV16 E5扩增片段在行SSCP分析时发现有泳动变位,突变率为40%(6/15).可见HPV16 E5基因的突变在宫颈癌的发生过程中具有重要作用.     

Anti-tumor DNA vaccines based on the expression of human papillomavirus-16 E6/E7 oncoproteins genetically fused with the glycoprotein D from herpes simplex virus-1

DNA vaccines encoding the human papillomavirus type-16 (HPV-16) E6 and E7 oncoproteins genetically fused to the human herpes simplex virus type 1 (HSV-1) gD protein were tested in mice for induction of T cell-mediated immunity and protection against tumor cell challenge. Hybrid genes, generated after insertion of E6 or E7-encoding sequences into internal sites of the gD-encoding gene, were transcribed in vitro and the chimeric proteins were expressed at the surface of in vitro-transfected mammalian cells. Female C57BL/6 mice immunized with 4 intramuscular doses (100 microg of DNA/dose) of the DNA vaccines encoding E7 efficiently generated E7-specific CD8(+) T cells. Vaccination of mice with the DNA vaccines encoding the E7, or both E6 and E7, conferred complete protection to challenges from TC-1 tumor cells and partial therapeutic effect (40%) in mice inoculated with TC-1 cells on the same day or 5 days prior to the first vaccine dose.     

Effects of single nucleotide changes on the binding and activity of RNA aptamers to human papillomavirus 16 E7 oncoprotein

A virally-encoded oncoprotein (E7 from human papillomavirus 16, involved in the initiation of cell transformation) was the target for RNA aptamer development by the process of systematic evolution of ligands by exponential enrichment (SELEX). A number of aptamers were identified, one of which was shown to inhibit the interaction between E7 and its major binding partner, pRb. Aptamers with very similar sequences (more than 92% similarity in the random regions) did not share this activity. This study demonstrates the potential of aptamers to be highly specific, with small differences in aptamer sequence having profound effects on function.     

<Superscript>1</Superscript>H and <Superscript>15</Superscript>N resonance assignment,secondary structure and dynamic behaviour of the C-terminal domain of human papillomavirus oncoprotein E6

E6 is a viral oncoprotein implicated in cervical cancers, produced by human papillomaviruses (HPVs). E6 contains two putative zinc-binding domains of about 75 residues each. The difficulty in producing recombinant E6 has long hindered the obtention of structural data. Recently, we described the expression and purification of E6-C 4C/4S, a stable, folded mutant of the C-terminal domain of HPV16 E6. Here, we have produced ¹⁵N-labelled samples of E6-C 4C/4S for structural studies by NMR. We have assigned most ¹H and ¹⁵N resonances and identified the elements of secondary structure of the domain. The domain displays an original / topology with roughly equal proportions of -helix and -sheet. The PDZ-binding region of E6, located at the extreme C-terminus of the domain, is in a random conformation. Mass spectrometry demonstrated the presence of one zinc ion per protein molecule. Kinetics of replacement of zinc by cadmium followed by ¹H,¹⁵N-HSQC experiments revealed specific frequency changes for the zinc-binding cysteines and their immediate neighbours. NMR spectra were affected by severe line-broadening effects which seriously hindered the assignment work. Investigation of these effects by ¹⁵N relaxation experiments showed that they are due to heterogeneous dynamic behaviour with s–ms time scale motions occurring in localised regions of the monomeric domain.     

人乳头瘤病毒16型E6和E7基因及其突变体转化活性的研究

为筛选出可用于研制HPV治疗性疫苗的HPV16型E6和E7基因突变体,故将HPV16型原型株(德国株)E6和E7基因及其各种突变体分别转染Balb/c3T3细胞,观察转染后的细胞在软琼脂培养中的集落形成能力和在裸鼠体内的成瘤能力.结果表明,单独转染和共转染HPV16野生型E6和E7基因的Balb/c3T3细胞系,在软琼脂中呈集落样生长,并在裸鼠体内成瘤;而转染E6基因突变体mE6(50G)、E7基因的两种突变体mE7-1(24G26G)和mE7-3(24G26G67R)以及共转染mE6和mE7-1的Balb/c3T3细胞,在软琼脂培养中极少形成集落,也不能在裸鼠体内成瘤.提示经结构改造后的HPV16 E6和E7基因已失去了对Balb/c3T3细胞的转化活性,而保留了免疫原性,可用于HPV16相关肿瘤治疗性疫苗的构建.     

Sulphoconjugation of steroids in porcine liver: Partial purification of the cytosolic sulphotransferases for pregnenolone and 5α-androst-16-en-3β-ol

Steroid sulphotransferase activities for 5α-androst-16-en-3β-ol and pregnenolone in porcine liver cytosol have been assayed using 3′-phosphoadenosine-5′-phospho[³⁵S]sulphate as sulphate donor. 5α-Androst-16-en-3β-ol sulphotransferase activity was obtained from porcine liver cytosol by gel filtration chromatography; activity was linear with time up to about 5 min., the optimum pH was near 8.0 and optimum temperature 37°C. Pregnenolone sulphotransferase activity was partially purified from porcine liver cytosol using DEAE-cellulose chromatography with an ionic gradient of KCl. This enzyme activity was linear with time up to 10 min and had optimum pH and temperature of 8.0 and 37°C, respectively.     

PI 3-kinase p110beta: a new target for antithrombotic therapy

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.     

A non-chromatographic radioimmunoassay of estrone and estradiol-17beta serum

A sensitive and efficient non-chromatographic procedure employing the Girard reagent and solvent-partitioning has been developed for the accurate radioimmunoassay (RIA) of estrone (E1) and estradiol-17β(E2) in a single 1.0 ml specimen of male or female serum. Using standard curves which permitted the discrimination of zero from 0.75–1.5 pg (p=0.05), the following mean procedural blanks (pg ± S.D.) were determined (1.0 ml water, n= 24): estrone, 2. 1 ± 1.1 (range 0–4.1); estradiol 1.0± 1.1 (range 0–3.6).A comparison of RIA of estrogens (1) in serum after separation by the Girard procedure and by TLC yielded correlation coefficients of 0.99 and 0.98 for E1 and E2 respectively. The following results (pg/ml ± S.D.) were obtained on RIA of E1 and E2 in 12 different 1.0 ml specimens of male and female serum using the Girard procedure: male. E1 (32.0 ± 9.2), E2 (24.1 ± 10.9); female, E1 (108.5 ± 60.8), E2 (126.4 ± 63.2).The intra-assay variability (c.v.) was found to be 12.6% for E1 and 9.4% for E2. The interassay variability was 14.2% for both estrogens.Twenty-four assays of E1 and E2 can be completed by one person in 2 working days.