Site-directed mutagenesis clarifies the substrate position within the three-dimensional model of the active site of herpes simplex virus type-1 thymidine kinase

Site-directed mutagenesis was used to experimentally verify the 3D model of the active site of herpes simplex virus type-1 thymidine kinase (HSV 1 TK) obtained by homology modelling. For this purpose, D215 and K317 were replaced by R and G, respectively, at homologous positions in the aciclovir-insensitive bovine herpes virus type-1 thymidine kinase (BHV 1 TK). Wild-type and mutated enzymes were expressed in Escherichia coli using a gene fusion vector and purified to homogeneity. While both mutants had the same Km value for thymidine as the recombinant wild-type enzyme (0.2 microM), Vmax was decreased to 20-25% of the original wild-type value. The recombinant wild-type enzyme was inhibited by the substrate analogue aciclovir with a Ki of 146 microM. Both mutants were able to phosphorylate aciclovir to about the same extent as the wild-type enzyme. These findings suggest that neither D215 nor K317 are directly involved in substrate binding. Therefore, a rearrangement of the 3D model is suggested, concerning the assignment of the substrate-binding site and co-substrate-binding site at the right and left side of the phosphate-binding loop, respectively.     

Suicide gene therapy of chemically induced mammary tumor in rat: efficacy and distant bystander effect

We examined the efficacy of suicide gene therapy for nitrosomethylurea-induced mammary tumors in rats. Individual tumors were directly injected with a retrovirus-producing cell line that releases retroviral vectors that transduce the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene. HSV1-TK specifically converts the nucleoside analogue ganciclovir (GCV) into a toxic metabolite. Compared to control rats receiving saline, we observed a significant tumor regression of the injected tumors following GCV administration, accompanied by a stromal inflammation and an extensive lymphocyte infiltration invading the tumor epithelium. It is noteworthy that the neighboring uninjected tumors also regressed, demonstrating the occurrence of a distant bystander effect. This is the first demonstration that HSV1-TK/GCV can efficiently treat multiple solid tumors directly generated from an epithelial tissue.     

Conversion of a beta-strand to an alpha-helix induced by a single-site mutation observed in the crystal structure of Fis mutant Pro26Ala

The conversion from an alpha-helix to a beta-strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self-assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a beta-strand to an alpha-helix by a single-site mutation as observed in the crystal structure of Fis mutant Pro26Ala determined at 2.0 A resolution. Pro26 in Fis occurs at the point where a flexible extended beta-hairpin arm leaves the core structure. Thus it can be classified as a "hinge proline" located at the C-terminal end of the beta2-strand and the N-terminal cap of the A alpha-helix. The replacement of Pro26 to alanine extends the A alpha-helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted from a beta-strand to an alpha-helix. This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant's reduced ability to activate Hin-catalyzed DNA inversion. The peptide (residues 20 to 26) in the second monomer subunit presumably retains its beta-strand conformation in the crystal; therefore, this peptide shows a "chameleon-like" character since it can adopt either an alpha-helix or a beta-strand structure in different environments. The structure of Pro26Ala provides an additional example where not only the protein sequence, but also non-local interactions determine the secondary structure of proteins.     

Volume of resection in patients treated with breast conservation for ductal carcinoma in situ

The N-terminal domain (1-318 amino acids) of mouse NFkappaB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length ikappaB-alpha (MAD3, 1-317 amino acids) molecule was generated by binding the E. coli-derived ikappaB-alpha to the purified NFkappaB and purifying the complex by sequential chromatography. The stoichiometry of NFkappaB to ikappaB in the complex was determined to be 2 to 1 by light scattering and SDS-polyacrylamide gel electrophoresis. The secondary structure of the NFkappaB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFkappaB occurs upon binding of DNA. The FTIR spectrum of the NFkappaB/ikappaB complex indicates that its secondary structure is composed of 17% alpha-helix, 39% beta-strand, 18% irregular structures, and 26% beta-turns and loops. By comparing these data to the FTIR data for NFkappaB alone, it is concluded that the ikappaB (MAD3) in the complex contains 35% alpha-helix, 27% beta-strand, 22% irregular structures, and 16% beta-turns and loops. Circular dichroism (CD) analysis of a shorter form of ikappaB (pp40) indicates that it contains at least 20% alpha-helix and that the ikappaB subunit accounts for nearly all of the alpha-helix present in the NFkappaB/ikappaB complex, consistent with the FTIR results. The stabilities of NFkappaB, ikappaB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of ikappaB is enhanced upon the formation of the NFkappaB/ikappaB complex.     

Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR

Raphanus sativus Antifungal Protein 1 (Rs-AFP1) is a 51 amino acid residue plant defensin isolated from radish (Raphanus sativus L.) seeds. The three-dimensional structure in aqueous solution has been determined from two-dimensional 1H NMR data recorded at 500 MHz using the DIANA/REDAC calculation protocols. Experimental constraints consisted of 787 interproton distances extracted from NOE cross-peaks, 89 torsional constraints from 106 vicinal interproton coupling constants and 32 stereospecific assignments of prochiral protons. Further refinement by simulated annealing resulted in a set of 20 structures having pairwise root-mean-square differences of 1.35(+/- 0.35) A over the backbone heavy atoms and 2.11(+/- 0.46) A over all heavy atoms. The molecule adopts a compact globular fold comprising an alpha-helix from Asn18 till Leu28 and a triple-stranded beta-sheet (beta 1 = Lys2-Arg6, beta 2 = His33-Tyr38 and beta 3 = His43-Pro50). The central strand of this beta-sheet is connected by two disulfide bridges (Cys21-Cys45 and Cys25-Cys47) to the alpha-helix. The connection between beta-strand 2 and 3 is formed by a type VIa beta-turn. Even the loop (Pro7 to Asn17) between beta-strand 1 and the alpha-helix is relatively well defined. The structure of Raphanus sativus Antifungal Protein 1 features all the characteristics of the "cysteine stabilized alpha beta motif". A comparison of the complete structure and of the regions important for interaction with the fungal receptor according to a mutational study, is made with the structure of gamma-thionin, a plant defensin that has no antifungal activity. It is concluded that this interaction is both electrostatic and specific, and some possible scenarios for the mode of action are given.     

A detailed molecular model for human aromatase

Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P-450arom (human aromatase) based on that of P-450cam, the only cytochrome P-450 enzyme for which the crystal structure is known. The predicted structure is found to be in good agreement with current experimental data; both direct, from site-directed mutagenesis studies, and indirect, from the consideration of the structures and activities of known substrates and inhibitors.     

Raman detection of macular carotenoid pigments in intact human retina

We are developing procedures to repeatedly and noninvasively image the expression of transplanted reporter genes in living animals and in patients, using PET. We have investigated the use of the Herpes Simplex Virus type 1 thymidine kinase gene (HSV1-tk) as a reporter gene and [8-14C]-ganciclovir as a reporter probe. HSV1-tk, when expressed, leads to phosphorylation of [8-14C]-ganciclovir. As a result, specific accumulation of phosphorylated [8-14C]-ganciclovir should occur almost exclusively in tissues expressing the HSV1-tk gene. METHODS: An adenoviral vector was constructed carrying the HSV1-tk gene along with a control vector. C6 rat glioma cells were infected with either viral vector and uptake of [8-3H]-ganciclovir was determined. In addition, 12 mice were injected with varying levels of either viral vector. Adenovirus administration in mice leads primarily to liver infection. Forty-eight hours later the mice were injected with [8-14C]-ganciclovir, and 1 hr later the mice were sacrificed and biodistribution studies performed. Digital whole-body autoradiography also was performed on separate animals. HSV1-tk expression was assayed, using both normalized HSV1-tk mRNA levels and relative HSV1-TK enzyme levels, in both the cell culture and murine studies. RESULTS: Cell culture, murine tissue biodistribution and murine in vivo digital whole-body autoradiography all demonstrate the feasibility of HSV1-tk as a reporter gene and [8-14C]-ganciclovir as an imaging reporter probe. A good correlation (r2 = 0.86) between the [8-14C]-ganciclovir percent injected dose per gram tissue from HSV1-tk positive tissues and HSV1-TK enzyme levels in vivo was found. An initial study in mice with [8-18F]-fluoroganciclovir and microPET imaging supports further investigation of [8-18F]-fluoroganciclovir as a PET reporter probe for imaging HSV1-tk gene expression. CONCLUSION: These results demonstrate the feasibility of using [8-14C]-ganciclovir as a reporter probe for the HSV1-tk reporter gene, using an in vivo adenoviral mediated gene delivery system in a murine model. The results form the foundation for further investigation of [8-18F]-fluoroganciclovir for noninvasive and repeated imaging of gene expression with PET.     

Imaging herpes virus thymidine kinase gene transfer and expression by positron emission tomography

We report a series of studies that assess the feasibility and sensitivity of imaging of herpes virus type one thymidine kinase (HSV1-tk) gene transfer and expression with [124I]-5-iodo-2'-fluoro-1-beta-D-arabinofuranosyluracil ([124I]-FIAU) and positron emission tomography (PET) and the ability of [124I]-FIAU-PET imaging to discriminate different levels of HSV1-tk gene expression. Studies were performed in rats bearing multiple s.c. tumors derived from W256 rat carcinoma and RG2 rat glioma cells. In the first set, we tested the sensitivity of [124I]-FIAU-PET imaging to detect low levels of HSV1-tk gene expression after retroviral-mediated gene transfer. HSV1-tk gene transduction of one of preestablished wild-type W256 tumor in each animal was accomplished by direct intratumoral injection of retroviral vector-producer cells (W256-->W256TK* tumors). Tumors produced from W256 and W256TK+ cells served as the negative and positive control in each animal. Highly specific images of [124I]-FIAU-derived radioactivity were obtained in W256TK* tumors (that were transduced in vivo) and in W256TK+ tumors but not in nontransduced wild-type W256 tumors. The level of "specific" incorporated radioactivity in transduced portions of both W256TK* and W256TK+ tumors was relatively constant between 4 and 50 h. In the second set, we tested whether [124I]-FIAU and PET imaging can measure and discriminate between different levels of HSV1-tk gene expression. Multiple s.c. tumors were produced from wild-type RG2 cells and stably transduced RG2TK cell lines that express different levels of HSV1-tk. A highly significant relationship between the level of [124I]-FIAU accumulation [% injected dose/g and incorporation constant (Ki)] and an independent measure of HSV1-tk expression (sensitivity of the transduced tumor cells to ganciclovir, IC50) was demonstrated, and the slope of this relationship was defined as a sensitivity index. We have demonstrated for the first time that highly specific noninvasive images of HSV1-tk expression in experimental animal tumors can be obtained using radiolabeled 2'-fluoro-nucleoside [124I]-FIAU and a clinical PET system. The ability to image the location (distribution) of gene expression and the level of expression over time provides new and useful information for monitoring clinical gene therapy protocols in the future.     

Differential mechanism of cytostatic effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, and other antiherpetic drugs on tumor cells transfected by the thymidine kinase gene of herpes simplex virus type 1 or type 2

After they have been transfected with the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) thymidine kinase (TK) gene murine mammary carcinoma (FM3A) cells become highly sensitive to the growth inhibitory properties of the antiherpetic agents (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 9(-)[(2-hydroxyethoxy)methyl]guanine (acyclovir, ACV), 9(-)[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, ganciclovir), and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU). BVDU was 100-fold more potent an inhibitor of HSV TK gene-transfected tumor cell growth (50% inhibitory concentration (IC50), 0.0020-0.0047 microM) than FMAU or DHPG (IC50, 0.051-0.277 microM) and 1000-fold more potent than ACV (IC50, 0.42-4.9 microM). As a rule, the test compounds were more cytostatic to HSV-2 TK than HSV-1 TK gene-transfected FM3A cells. This may be ascribed to the higher phosphorylating capacity (Vmax/Km) of HSV-2 TK than HSV-1 TK and/or to the higher TK enzyme levels of the HSV-2 TK gene-transfected FM3A cells than the HSV-1 TK gene-transfected FM3A cells. Thymidylate synthase of the HSV TK gene-transfected FM3A cells appears to be the target enzyme for the cytostatic action of BVDU, but not FMAU, DHPG, or ACV. Instead, the cytostatic activity of DHPG seems to be correlated with its conversion to the triphosphate form and subsequent incorporation into the DNA of HSV TK gene-transfected FM3A cells.     

Recurrent acyclovir-resistant herpes simplex in an immunocompromised patient: can strain differences compensate for loss of thymidine kinase in pathogenesis?

To investigate how acyclovir-resistant (ACVr) herpes simplex virus (HSV) evades drug therapy and causes disease, HSV-1 isolates from a bone marrow transplant (BMT) patient were studied. The patient developed ACVr disease after an initial BMT and, following a second BMT, reactivated ACVr HSV despite high-dose acyclovir prophylaxis. ACVr isolates from each episode contained the same point mutation in the viral thymidine kinase (tk) gene, documenting the emergence, latency, and reactivation of this mutant. The mutants were exceedingly impaired for TK activity in sensitive enzyme, plaque autoradiography, and drug-susceptibility assays. Nevertheless, these mutants and a tk deletion mutant constructed in the same genetic background reactivated from latency in mouse trigeminal ganglia, in contrast to similar mutants from laboratory strains. It is hypothesized that alleles in the clinical isolate compensate for the loss of TK in this animal model. Such genetic variability may be important for ACVr disease in humans.     

The peptide-binding domain of the chaperone protein Hsc70 has an unusual secondary structure topology

Modern NMR methods were used to determine the secondary structure topology of the 18 kDa peptide binding domain of the chaperone protein Hsc70 in solution. This report constitutes the first experimental conformational information on this important domain of the class of Hsp70 proteins. The domain consists of two four-stranded antiparallel beta-sheets and a single alpha-helix. The topology does not resemble at all the topology observed in the human leukocyte antigen (HLA) proteins of the major histocompatibility complex. This is significant because such resemblance was predicted on the basis of limited amino acid homology, secondary structure prediction, and related function. Moreover, the exact meander-type beta-sheet topology identified in Hsc70 has to our best knowledge not been observed in any other known protein structure.     

Validity of the children and youth physical self-perception profile: a confirmatory factor analysis

MOTIVATION: The Match-Box software comprises protein sequence alignment tools based on strict statistical thresholds of similarity between protein segments. The method circumvents the gap penalty requirement: gaps being the result of the alignment and not a governing parameter of the procedure. The reliable conserved regions outlined by Match-Box are particularly relevant for homology modelling of protein structures, prediction of essential residues for site-directed mutagenesis and oligonucleotide design for cloning homologous genes by polymerase chain reaction (PCR). RESULTS: The method produces reliable results, as assessed by tests performed on protein families of known structures and of low sequence similarity. A reliability score is computed in relation to a threshold of similarity progressively raised to extend the aligned regions to their maximal length, up to the significance limit of matching segments. The score obtained at each position is printed below the sequences and allows a discriminant reading of each aligned region. AVAILABILITY: Sequences may be submitted to a Web server at http://www.fundp.ac.be/sciences/biologie/bms/+ ++matchbox_submit.html or sent by e-mail to matchbox/biq.fundp.ac.be (help available by just mailing help).     

Targeting gene therapy vectors to CNS malignancies

Multidrug resistance to anti-cancer drugs is a major medical problem. Resistance is manifested largely by the product of the human MDR1 gene, P-glycoprotein, an ABC transporter that is an integral membrane protein of 1280 amino acids arranged into two homologous halves, each comprising 6 putative transmembrane alpha-helices and an ATP binding domain. Despite the plethora of data from site-directed, scanning and domain replacement mutagenesis, epitope mapping and photoaffinity labeling, a clear structural model for P-glycoprotein remains largely elusive. In this report, we propose a new model for P-glycoprotein that is supported by the vast body of previous data. The model comprises 2 membrane-embedded 16-strand beta-barrels, attached by short loops to two 6-helix bundles beneath each barrel. Each ATP binding domain contributes 2 beta-strands and 1 alpha-helix to the structure. This model, together with an analysis of the amino acid sequence alignment of P-glycoprotein isoforms, is used to delineate drug binding and translocation sites. We show that the locations of these sites are consistent with mutational, kinetic and labeling data.     

Application of gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry for analysis of DNA and protein adducts

Olive-pollen profilin has been isolated and characterized as a significant allergen. Its molecular properties, such as a molecular mass of 15 kDa; amino-acid composition; and secondary repetitive structure percentages of 15% alpha-helix, 33% beta-strand, 20% beta-turn, and 32% random coil, have been determined. Its allergenic capability, a recognition frequency estimated at 24% of olive-hypersensitive patients, and high cross-reactivity with all the pollen used have been found. The presence of conformation epitopes in the olive profilin, as well as a high structural and immunologic similarity to other pollen sources such as birch and ash, can be established from these studies.     

Glycosaminoglycan specificity of a heparin-binding peptide

Glycosaminoglycans are complex sulfated polysaccharides with a diverse range of biological functions. Three glycosaminoglycan standards--chondroitin sulfate, dermatan sulfate and heparin--were characterized during this study. The interaction of the heparin binding site of protein C inhibitor, represented by the peptide sequence 264-283, in solution with the above glycosaminoglycan standards was studied. Circular dichroism spectroscopy was used to determine the dominant secondary structure induced in the peptide upon binding the relevant glycosaminoglycans. The various glycosaminoglycans induced different secondary structures. The level of induced secondary structure by dermatan sulfate and heparin was approximately twice that induced by chondroitin sulfate. For chondroitin sulfate and heparin, alpha-helix was the dominant ordered secondary structure, whereas for dermatan sulfate the beta-strand conformation dominated. The order of secondary structure induction of the protein C inhibitor peptide by the glycosaminoglycans paralleled the reported biological activities of these glycosaminoglycans for mediation of the biological activity in the intact protein. The strength of the interaction of dermatan sulfate and heparin with the protein C inhibitor peptide was measured by determining the concentration of salt required to inhibit 50% of the interaction. The values determined were 0.1 and 0.3 M salt for dermatan sulfate and heparin, respectively. These results show that different glycosaminoglycans can support different secondary structures in the protein C inhibitor peptide.     

SS3D-P2: a three dimensional substructure search program for protein motifs based on secondary structure elements

This paper discusses the implementation of a three-dimensional (3D) structure motif search of proteins. Each protein structure is represented by a set of secondary structure elements (SSEs) which involves alpha-helix segments and beta-strand segments. In describing it, every SSE is further reduced into a two-node graph that consists of the starting amino acid residue, the ending residue and a pseudo-bond between them. The searching algorithm is based on a graph theoretical clique-finding algorithm that has been used for 3D substructure searching in small organic molecules. The program SS3D-P2 was validated using proteins that have well-known 3D motifs, and it correctly found the Greek key motif within an eye lens protein, crystallin, that consists of four anti-parallel beta strands. The program was also successfully applied to searching for the more complex 3D motif, TIM-type beta-barrel motif, with a protein structure database from the Protein Data Bank.     

Evaluation of protein 3-D structure prediction: comparison of modelled and X-ray structure of an alkaline serine protease

We describe the modelling of the structure of the highly alkaline subtilisin protease OPTICLEAN from Bacillus alcalophilus. The model was developed through modelling by homology. We used the structure of subtilisin Carlsberg from the Brookhaven protein databank (entry 1CSE) as start structure. Amino acid changes and deletions were performed with the graphic protein design program BRAGI. Force field calculations and molecular dynamic simulations were made with AMBER 3.0 on a Multiflow TRACE 14/300. The comparison of the model and the later solved X-ray structure of OPTICLEAN shows a high similarity between the two structures, but there were also remarkable deviations between the two structures in some loop regions. The comparison shows that the deviations are due to difficulties in the prediction of correct main chain torsion angles of the prolines and the selection of correct loops in deletion or insertion regions. Strategies to avoid these mistakes are discussed.     

A phase I/II dose-escalation study of herpes simplex virus type 1 thymidine kinase "suicide" gene therapy for metastatic melanoma. Study Group on Gene Therapy of Metastatic Melanoma

We performed a dose-escalating phase I/II study of retrovirus-mediated herpes simplex virus type 1 thymidine kinase (HSV-1-TK) suicide gene therapy for metastatic melanoma. HSV-1 TK expression, which specifically sensitizes transduced and bystander cancer cells to ganciclovir (GCV) toxicity, was mediated by one (four patients, first dose step) to three (four patients, second dose step) injections of "M11" retrovirus vector-producing cells in melanoma cutaneous nodules. After a 7-day period allowed for cancer cell transduction, GCV was administered for 14 days. Safety was assessed by clinical and laboratory evaluations, and efficacy was assessed by tumor measurements and histology. M11 doses ranged from 76 to 1247 x 10(6) cells. Treatment-related adverse events were mild and transient, limited to inflammatory skin reactions at injection and fever on repeated injections. Plasma GCV was in the active range (>0.2 microg/ml); transgene was detected by polymerase chain reaction in three of six patients; treated tumor size was moderately affected under GCV as compared with untreated tumors, although 2 weeks after GCV administration important (>50%) treated-tumor necrosis was evidenced on histology in three of eight patients. All patients showed disease progression on long-term follow-up. Thus, M11-mediated HSV-1 TK gene therapy was well tolerated over a wide dose range. The limited tumor response is likely to be related to poor gene transfer efficiency. However, necrosis following GCV administration in transduced tumors indicates a potential for treatment efficacy.     

JPred: a consensus secondary structure prediction server

An interactive protein secondary structure prediction Internet server is presented. The server allows a single sequence or multiple alignment to be submitted, and returns predictions from six secondary structure prediction algorithms that exploit evolutionary information from multiple sequences. A consensus prediction is also returned which improves the average Q3 accuracy of prediction by 1% to 72.9%. The server simplifies the use of current prediction algorithms and allows conservation patterns important to structure and function to be identified. AVAILABILITY: http://barton.ebi.ac.uk/servers/jpred.h tml CONTACT: geoff@ebi.ac.uk