Solution structure of human p8MTCP1, a cysteine-rich protein encoded by the MTCP1 oncogene, reveals a new alpha-helical assembly motif

MTCP1 (for Mature-T-Cell Proliferation) is the first gene unequivocally identified in the group of uncommon leukemias with a mature phenotype. The three-dimensional solution structure of the human p8(MTCP1) protein encoded by the MTCP1 oncogene was determined by homonuclear proton two-dimensional NMR methods at 600 MHz. After sequence specific assignments, a total of 931 distance restraints and 57 dihedral restraints were collected. The location of the three previously unassigned disulfide bridges was determined from preliminary DIANA structures, using a statistical analysis of intercystinyl distances. The solution structure of p8(MTCP1) is presented as a set of 30 DIANA structures, further refined by restrained molecular dynamics using a simulated annealing protocol with the AMBER force field. The r.m.s.d. values with respect to the mean structure for the backbone and all heavy atoms for a family of 30 structures are 0.73(+/-0.28) and 1.17(+/-0.23) A, when the structured core of the protein (residues 5 to 63) is considered. The solution structure of p8(MTCP1) reveals an original scaffold consisting of three alpha helices, associated with a new cysteine motif. Two of the helices are covalently paired by two disulfide bridges, forming an alpha-hairpin which resembles an antiparallel coiled-coil. The third helix is oriented roughly parallel to the plane defined by the alpha-antiparallel motif and its axis forms an angle of approximately 60 degrees with respect to the main axis of this motif.     

Solution structure of recombinant human interleukin-6

Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivo and whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five alpha-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 A in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 A for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.     

Treatment of uveitis with recombinant human interleukin-13

AIM: To evaluate the anti-inflammatory cytokine interleukin-13 (IL-13) for the treatment of uveitis. METHODS: Uveitis was induced in monkeys by immunisation with human retinal S-antigen. Starting at the onset of disease, the animals were treated with IL-13 at 25 micrograms/kg, or vehicle control, injected subcutaneously once a day for 28 days. Intraocular inflammation was scored by indirect ophthalmoscopy for a period of 56 days. Circulating leucocyte levels were monitored. RESULTS: Uveitis started unilaterally in all but one animal. IL-13 inhibited inflammation both in the eyes in which the disease was present when the treatment was initiated (p = 0.0001), and in the contralateral initially negative eyes (p = 0.0001). After cessation of therapy, there was a progressive increase of inflammation in the IL-13 treated group. However, the beneficial effect of IL-13 extended into the 4 week follow up period. IL-13 produced an increase in circulating polymorphonuclear neutrophils and a decrease in lymphocytes. CONCLUSION: Administration of IL-13 appears to be a promising modality of treatment for severe uveitis.     

Inactivation of p27Kip1 by the viral E1A oncoprotein in TGFbeta-treated cells

The adenovirus oncoprotein E1A and the simian virus SV40 large T antigen can both reverse the strong growth-inhibitory effect of transforming growth factor(TGF)-beta on mink lung epithelial cells: exposure of TGF-beta causes these cells to arrest late in the G1 phase of the cell cycle (ref. 3). This arrest correlates with an increase in expression of the protein p15Ink4B (ref. 4), inactivation of the cyclin E/A-cdk2 complex by the inhibitory protein p27Kip1 (refs 5-7), and with the accumulation of unphosphorylated retinoblastoma protein. The rescue by E1A of cells from TGF-beta arrest is partly independent of its binding to retinoblastoma protein. Here we show that E1A directly affects the cyclin-dependent kinase inhibitor p27Kip1 in TGF-beta-treated cells by binding to it and blocking its inhibitory effect, thereby restoring the activity of the cyclin-cdk2 kinase complex. In this way, E1A can overcome the effect of TGF-beta and modulate the cell cycle. To our knowledge, E1A provides the first example of a viral oncoprotein that can disable a cellular protein whose function is to inhibit the activity of cyclin-dependent kinases.     

Solution structure of human neuropeptide Y

The three-dimensional structure of synthetic human neuropeptide Y in aqueous solution at pH 3.2 and 37 degrees C was determined from two-dimensional 1H NMR data recorded at 600 MHz. A restraint set consisting of 440 interproton distance restraints inferred from NOEs and 11 backbone and 4 side-chain dihedral angle restraints derived from spin-spin coupling constants was used as input for distance geometry calculations on DIANA and simulated annealing and restrained energy minimization in X-PLOR. The final set of 26 structures is well defined in the region of residues 11-36, with a mean pairwise rmsd of 0.51 A for the backbone heavy atoms (N, C alpha and C) and 1.34 A for all heavy atoms. Residues 13-36 form an amphipathic alpha-helix. The N-terminal 10 residues are poorly defined relative to the helical region, although some elements of local structure are apparent. At least one of the three prolines in the N-terminal region co-exists in both cis and trans conformations. An additional set of 24 distances was interpreted as intermolecular distances within a dimer. A combination of distance geometry and restrained simulated annealing yielded a model of the dimer having antiparallel packing of two helical units, whose hydrophobic faces form a well-defined core. Sedimentation equilibrium experiments confirm the observation that neuropeptide Y associates to form dimers and higher aggregates under the conditions of the NMR experiments. Our results therefore support the structural features reported for porcine neuropeptide Y [Cowley, D.J. et al. (1992) Eur. J. Biochem., 205, 1099-1106] rather than the 'aPP' fold described previously for human neuropeptide Y [Darbon, H. et al. (1992) Eur. J. Biochem., 209, 765-771].     

Solution structure of a selectively 13C-labeled intramolecular DNA triplex

The three-dimensional structure of an intramolecular triple helix whose three strands have been linked by a hexaethylene glycol chain, and selectively 13C-enriched in position C1' on the third strand was investigated by NMR spectroscopy and constrained molecular mechanics calculations. Starting from different initial conformations, we show that the NOE constraints determined by the complete relaxation matrix calculation and iterative back-calculations allowed us to reach the same final restrained triple helix, taking into account implicitly the solvent effect. We conclude that this triplex adopted a B-type conformation rather than a A-type. The sugar pucker was found predominantly in the S-type conformation, in the range of C2'-endo geometry.     

Confirmation of the localization of the human recombination activating gene 1 (RAG1) to chromosome 11p13

The human recombination activating gene 1 (RAG1) has previously been mapped to chromosomes 14q and 11p. Here we confirm the chromosome 11 assignment by two independent approaches: autoradiographic and fluorescence in situ hybridization to metaphase spreads and analysis of human-hamster somatic cell hybrid DNA by the polymerase chain reaction (PCR) and Southern blotting. Our results unequivocally localize RAG1 to 11p13.     

Solution structure of glutathione bound to human glutathione transferase P1-1: comparison of NMR measurements with the crystal structure

The conformation of the bound glutathione (GSH) in the active site of the human glutathione transferase P1-1 (EC 2.5.1.18) has been studied by transferred NOE measurements and compared with those obtained by X-ray diffraction data. Two-dimensional TRNOESY and TRROESY experiments have been performed under fast-exchange conditions. The family of GSH conformers, compatible with TRNOE distance constraints, shows a backbone structure very similar to the crystal model. Interesting differences have been found in the side chain regions. After restrained energy minimization of a representative NMR conformer in the active site, the sulfur atom is not found in hydrogen-bonding distance of the hydroxyl group of Tyr 7. This situation is similar to the one observed in an "atypical" crystal complex grown at low pH and low temperature. The NMR conformers display also a poorly defined structure of the glutamyl moiety, and the presence of an unexpected intermolecular NOE could indicate a different interaction of this substrate portion with the G-site. The NMR data seem to provide a snapshot of GSH in a precomplex where the GSH glutamyl end is bound in a different fashion. The existence of this precomplex is supported by pre-steady-state kinetic experiments [Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034] and preliminary time-resolved fluorescence data.     

Immunoexpression of mutant p53 and human papillomavirus related E6 oncoprotein in anal malignancies

Immunohistochemical expression of mutant p53 protein and human papillomavirus (HPV) 16 and 18 related E6 oncoprotein was studied in 36 biopsy proved anal cancers. Mutant p53 was detected in 61.1% cases. HPV 16 and 18 E6 protein was expressed in 22.2% cases, all of which were squamous cell carcinomas. Coexpression of both mutant p53 and E6 protein was found in only 5 cases (13.8%). In HPV 16/18 positive anal tumors, the degradation of p53 is accelerated by viral E6 oncoprotein. In HPV negative tumors, however, other mutagenic factors probably play a role in carcinogenesis.     

Solution structure of the recombinant oxidized rabbit uteroglobin using homonuclear and heteronuclear multidimensional NMR

Rabbit uteroglobin (rab-UG) is a 16-kDa homodimeric secretory protein with potent anti-inflammatory/immunomodulatory properties. Its physiological role is still unclear, although it was observed that several small hydrophobic molecules bind to the oxidized and the reduced uteroglobin. It is suggested that the formation and/or disruption of the two disulphide bridges not only regulates this binding itself, but also the affinity to the ligand. The determination of the solution structure has been started with the assignment of 1H, 15N and 13C resonances of the oxidized rabbit uteroglobin, based on several two-dimensional and three-dimensional homonuclear and heteronuclear double and triple resonance experiments. The assignment was possible with the overproduction of the wild-type as well as of uniformly 15N-labeled and 15N/13C-labeled samples of the recombinant protein. A complete assignment of 1H, 15N and 13C resonances, the secondary-structure elements and the tertiary structure in solution is presented. The tertiary solution structure was found to be in good agreement with the previously determined crystal structure of rab-UG and with the solution structure of human uteroglobin (h-UG). h-UG and rab-UG are extremely stable proteins within a wide range of pH and temperatures. Some of the binding characteristics of ligands of rab-UG and a mutant with all cysteine residues exchanged to serine residues are discussed.     

Solution structure of the Eps15 homology domain of a human POB1 (partner of RalBP1)

The solution structure of the Eps15 homology (EH) domain of a human POB1 (partner of RaIBP1) has been determined by uniform 13C/15N labeling and heteronuclear multidimensional nuclear magnetic resonance spectroscopy. The POB1 EH domain consists of two EF-hand structures, and the second one binds a calcium ion. In the calcium-bound state, the orientation of the fourth alpha-helix relative to the other helices of the POB1 EH domain is slightly different from that of calbindin, and much more different from those of calmodulin and troponin C, on the basis of their atomic coordinates.     

Assignment of cDNA encoding hyaluronic acid-binding protein 1 to human chromosome 17p12-p13

PURPOSE: To report unilateral pupil-sparing third nerve palsy after use of sildenafil citrate (Viagra). METHOD: Case report. RESULTS: A 56-year-old man with a history of tobacco abuse was treated for erectile dysfunction. Viagra, 50 mg, was taken once without adverse effect. Three weeks later, the patient took a second dose of Viagra (50 mg); 36 hours later he experienced a complete pupil-sparing third nerve palsy. Erythrocyte sedimentation rate, blood glucose level, magnetic resonance imaging, and magnetic resonance angiography were normal. CONCLUSION: In a patient with microvascular disease, use of sildenafil may be associated with pupil-sparing third nerve palsy.     

Studies of human papillomavirus infection (HPV) and expression of oncoprotein p 21 in cervix neoplasms

Frequencies of HPV type 16 and 18, evaluated by in situ hybridization technique in uterine cervix carcinomas of the IIIrd stage of clinical advancement, were 54% and 36.5% respectively. Presence of p 21 protein was detected in 85.3% cases of the cancers and showed no relation to HPV infection.     

Selection of recombinant, library-derived antibody fragments against p24 for human immunodeficiency virus type 1 diagnostics

By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VH and Vkappa regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.     

Human papillomavirus and p53 oncoprotein in verrucous carcinoma of the larynx

An in vitro screening method for selective acetylcholinesterase (AChE) inhibitors was established. Inhibitory activity of AChE and butyrylcholinesterase (BuChE) was measured and the culture broths of microorganisms that showed selective inhibition against AChE were characterized. By using this method, a strain producing the novel and selective inhibitors of AChE, arisugacins A and B, was picked out among over seven thousand microorganisms tested. Arisugacins were obtained as white powders from the culture broth together with three known compounds, territrems B and C and cyclopenin that also showed selective inhibition against AChE. Arisugacins and territrems are members of the meroterpenoid compounds. They showed potent inhibitory activities against AChE with IC50 values in range of 1.0 approximately 25.8 nM. Furthermore, they showed greater than 2,000-fold more potent inhibition against AChE than BuChE.     

Cytotoxic effects of recombinant adenovirus p53 and cell cycle regulator genes (p21 WAF1/CIP1 and p16CDKN4) in human prostate cancers

PURPOSE: Overexpressing or restoring the basal levels of tumor suppressor genes in cancer cells can suppress tumorigenicity of cancer cells. In this communication, we compared tumor suppressive activities of three well-defined tumor suppressive genes (p53, p21WAF1/CIP1, and p16CDKN2) delivered individually to prostate cancer cells with adenoviral vector (Ad). MATERIALS AND METHODS: Efficacy of growth inhibition by recombinant adenoviruses bearing p53, p21WAF1/CIP1, or p16CDKN2 (Ad5CMV-p53, Ad5CMV-p21, Ad5CMV-p16) genes were tested in vitro on androgen-dependent (LNCaP) and androgen-independent (C4-2, DU-145, and PC-3) human prostate cancer cells, ex vivo and in vivo on PC-3 tumor. RESULTS: Ad5CMV-p53 was observed to exert the greatest growth inhibitory action on all of the cell lines tested; inhibition appeared to be cytolytic. In comparison to control Ad5CMV-PA added samples, the growth inhibitory action of Ad5CMV-p21 and Ad5CMV-p16 appeared to be cytostatic. Ad5CMV-p53 is more effective than Ad5CMV-p16 and Ad5CMV-p21 in inhibiting the tumor "take" rate. A similar order of antitumor activity was observed when recombinant adenoviruses were injected intratumorily to previously established PC-3 tumors in vivo. CONCLUSION: p53 is the most effective tumor suppressor gene to target human prostate cancer.