Biomedical subjects

Y Langelier

Publications and source records attributed to Y Langelier.

At least 19 recordsLinked to original sources

PubMed1992

Structure-function studies of peptides inhibiting the ribonucleotide reductase activity of herpes simplex virus type I.

Ac-Tyr298-Ala299-Gly300-Thr301-Val302-I le303-Asn304-Asp305-Leu306-OH (Ac-VZV R2-(298-306)) represents the acetylated form of the C-terminus of varicella-zoster virus (VZV) ribonucleotide reductase subunit 2 (R2). This peptide possesses a high degree of homology with the C-terminus nonapeptide of the herpes simplex virus (HSV) type I and II ribonucleotide reductase R2 protein and is 15 times more potent than the latter in its in vitro inhibition of HSV-1 reductase activity. Accordingly, a new series of analogues based on this structure was studied in vitro. The replacement of Asp305 by Asn, Glu, Gln, Ser, or Cys; of Asn304 by Gln or Ser; of Ile303 and Val302 by D-Val; and of Tyr298 by Cha induced an important loss of inhibitory potency. The substitution of Asn304 by Asp; of Thr301 by Cys, Ser, or Val; of Gly300 by Ala or Val; of Ala299 by Val; or of Tyr298 by homoPhe, 4'-fluoro-Phe, 4'-chloro-Phe, 3'-iodo-Tyr, Me-Tyr, or For-Trp led to a moderate decrease of the Ac-VZV R2-(298-306) potency. The replacement of Val302 by Ile; Ala299 by Cys, Ser, or Thr; or the insertion of a six- or eight-carbon chain between Tyr298 and the NH2 terminus either preserved or slightly increased the inhibitory potency of Ac-VZV R2-(298-306). Finally, the substitution of Tyr298 by Trp or the addition of 4'-nitro-Phe at the amino terminus resulted in a 3-fold increase of potency. Altogether, these results stress the importance of the structural integrity of the minimum active core 302-306 in preserving the inhibitory potency and suggest that further studies on monosubstitutions could be directed at the portion 298-301 of the peptide.

Amino Acid Sequence↗

PubMed1991

Affinity purification of active subunit 1 of herpes simplex virus type 1 ribonucleotide reductase exhibiting a protein kinase activity.

Herpes simplex virus (HSV) ribonucleotide reductase is formed by the association of two distinct dimeric subunits, R1 and R2. Attempts to purify either the HSV holoenzyme or its R1 subunit in their active form have been unsuccessful until now. The C terminus of the R2 protein being involved in the association with R1, the synthetic nonapeptide corresponding to this terminus, impedes the formation of the holoenzyme by competing with R2 for a critical site on R1. Based upon these observations, we developed an affinity chromatographic procedure to purify the R1 protein from HSV-1-infected baby hamster kidney cells. Specific binding of R1 to an affinity column made by linking the peptide HSV R2-(326-337) to Affi-Gel 10, followed by specific elution with an excess of an analogous peptide exhibiting a higher affinity for R1 yielded, in a single step, highly purified R1 protein. The purified R1 preparations contained approximately 95% of intact R1, the remaining 5% consisting of two R1 copurifying proteolytic breakdown products. The purified R1 protein exhibited a high reductase specific activity when mixed with an excess of the R2 subunit. Moreover, in vitro kinase assays revealed that the purified R1 protein of HSV-1 possesses an autophosphorylating activity also able to phosphorylate alpha-casein and histone II-S. The intrinsic protein kinase activity of HSV R1 is associated with its unique N-terminal domain which is absent from all other reductase subunits 1 and contains consensus motifs found in Ser/Thr protein kinases. A preliminary characterization of the kinase activity of the R1 protein of HSV-1 ribonucleotide reductase is presented.

Animals↗

PubMed1991

Coordination of copper(II) ions to catechoyl-dipeptides, the inhibitors of leucine aminopeptidase and ribonucleotide reductase.

Potentiometric and spectroscopic study of the coordination ability of 2,3- and 3,4-dihydroxybenzoyl-dipeptides has shown that catechoyl-dipeptides are very strongly-binding ligands. The coordination begins at the catechol site at pH below 4. In the case of 2,3-dihydroxy derivatives the peptide unit may also be involved in the copper(II) ion coordination via its peptide nitrogens. The biological tests have indicated that the studies ligands may be effective inhibitors of ribonucleotide reductase in some cases, with a reasonable level of selectivity.

Animals↗

PubMed1991

Studies on in vitro proteolytic sensitivity of peptides inhibiting herpes simplex virus ribonucleotide reductases lead to discovery of a stable and potent inhibitor.

The nonapeptide, HSV R2-(329-337), corresponding to the subunit 2 (R2) carboxyl terminus of herpes simplex virus (HSV) ribonucleotide reductases, specifically inhibits this enzyme activity. We report here that under standard reductase assay conditions, this peptide was rapidly degraded by proteases present in the partially purified enzyme extract. The main process of proteolysis involves the successive removal of Tyr329 and Ala330, which corresponds to an aminopeptidase activity. Determination of the proteolytic susceptibility of HSV R2-(329-337) analogs showed that natural modifications which are present in the homologous varicella zoster virus (VZV) nonapeptide decreased its susceptibility to protease action 1.5-fold. Nx-acetylation, a modification known to protect peptides against aminopeptidase attacks, greatly improved the proteolytic resistance of HSV and VZV nonapeptides. Moreover, Ac-VZV R2-(298-306) exhibited a 15-fold higher potency on reductase inhibition than HSV R2-(329-337). The degradation process of HSV R2-(329-337) was partially inhibited by amastatin, bestatin, and leupeptin whereas it was completely abolished by bacitracin, suggesting a combined action of more than one aminopeptidase activity. Moreover, bacitracin protected most of these nonapeptide analogs from proteolysis, although it was less effective in preventing HSV R2-(332-337) degradation. Our results indicate that it is possible to determine, in the presence of bacitracin, the relative inhibitory potencies of HSV R2-(329-337) analogs with minimal error due to proteolytic susceptibility. Moreover, HSV R2-(329-337) modifications that were found to protect the peptide against degradation might be useful to increase its efficacy in vivo.

Amino Acid Sequence↗

PubMed1990

Synthesis and inhibitory potency of peptides corresponding to the subunit 2 C-terminal region of herpes virus ribonucleotide reductases.

H-Tyr329-Ala330-Gly331-Ala332-Val333-Va l334-Asn335-Asp336-Leu337-OH, the C-terminal end of herpes simplex virus ribonucleotide reductase subunit 2 (HSV R2), specifically inhibits viral enzyme activity by interacting with subunit 1 (HSV R1). In a previous structure-activity study, we identified four sites on the nonapeptide where the inhibitory potency could be modulated: a minimum active core 333-337, a spacer segment 330-332, and the N- and C-termini. To further explore the structural features of HSV R2-(329-337) that are required to obtain a potent inhibition, a series of analogues comprising modifications in these four regions were synthesized by solid-phase methodology. Changes in the segment 333-337 of the molecule decreased the inhibitory potency by more than 2-fold, except for the Ile334 substitution, which resulted in a 1.5-fold increase in potency. Replacement of Tyr329 by other aromatic or aliphatic amino acids diminished the nonapeptide activity from 1.4-fold to 5.9-fold. The spacer segment contributed to enhance potency. Modification with amino acids that could induce conformational changes, such as Pro or D-Ala, generated compounds with a similar or lower activity, respectively. Amidation or amino acyl addition at the carboxylic end was detrimental while acylation of the N-terminus was generally beneficial for the inhibitory potency. Disubstitution in position 332 and 334 by Thr and Ile, which are present in the C-terminal portion of varicella-zoster virus ribonucleotide reductase subunit 2, resulted in a peptide that is 4.0 times more potent than HSV R2-(329-337), while each monosubstitution alone generated peptides with 150% of the activity of HSV R2-(329-337) nonapeptide. These results indicate a synergistic effect of the disubstitution which confers to this analogue physicochemical properties enhancing its ability to interact with its R1 binding site.

Amino Acid Sequence↗

PubMed1990

High level expression in 293 cells of the herpes simplex virus type 2 ribonucleotide reductase subunit 2 using an adenovirus vector.

The herpes simplex viruses (HSV-1 and HSV-2) encode a ribonucleotide reductase consisting of two non-identical subunits (RR1 and RR2) which associate to form the active holoenzyme. To facilitate the purification and subsequent biochemical characterization of this enzyme, we have cloned the small subunit 2 of the HSV-2 ribonucleotide reductase (RR2HSV-2) in a helper-independent adenovirus type 5 vector under the control of the adenovirus type 2 major late promoter. After infection of 293 cells with the recombinant virus, the amount of RR2HSV-2 protein produced was eightfold higher than in HSV-2-infected cells. The specific activities of the RR2HSV-2 recombinant subunit and the RR2HSV-2 protein in HSV-2-infected cells were determined by their mixing with saturating amounts of isolated RR1HSV-1 subunit. By comparison of the relative amount of each RR2HSV-2 subunit with its specific activity, we calculated that the recombinant protein intrinsic activity was similar to that of the protein produced in HSV-2-infected cells. These results demonstrated that the adenovirus expression vector is a good system to produce an active RR2HSV-2 subunit in fairly high amounts.

Adenoviridae↗

PubMed1989

Transformation of NIH 3T3 cells by herpes simplex type 2 BglII n fragment and sub-fragments is independent from induction of mutation at the HPRT locus.

The effect of transfection of the herpes simplex virus type 2 transforming fragment BglII n and of its three Xhol subfragments on mutagenesis and morphological transformation was assayed in NIH 3T3 cells. While BglII n and the right hand portion of this fragment increased the number of transformed foci, no significant effects on the mutation frequency at the hprt locus were observed. Our results indicate that transformation by BglII n is independent from the induction of somatic mutations and suggest that other mechanisms must be considered to explain transformation by this sequence.

Animals↗

PubMed1989

Retention and expression of the left end subfragment of the herpes simplex virus type 2 BglII N DNA fragment do not correlate with tumorigenic conversion of NIH 3T3 cells.

Cotransfection experiments have been carried out using recombinant plasmids pAG60, conferring resistance to antibiotic G418, and pXho3 which contains the left end subfragment (map coordinates 0.583 to 0.596) of the transforming herpes simplex virus type 2 BglII N DNA fragment and encodes the 36K polypeptide associated with the viral ribonucleotide reductase activity. Several NIH 3T3 cell clones resistant to G418 and having morphological changes commonly observed for transformed NIH 3T3 cells were isolated and examined for the presence and stable retention of the viral sequences. Seven of the clones that retained the transfected viral sequences were analysed for the expression of the 36K polypeptide and the tumorigenic phenotype. The results gathered from these studies show that neither the retention of the viral DNA nor the expression of the 36K polypeptide correlated with tumorigenic conversion of these cells.

Animals↗

PubMed1988

Mechanism of inhibition of herpes simplex virus (HSV) ribonucleotide reductase by a nonapeptide corresponding to the carboxyl terminus of its subunit 2. Specific binding of a photoaffinity analog, [4'- azido-Phe6] HSV H2-6(6-15), to subunit 1.

Herpes simplex virus (HSV) ribonucleotide reductase activity is specifically inhibited by a synthetic peptide, Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu (HSV H2-(7-15], corresponding to the carboxyl terminus of its subunit 2 (H2). In order to elucidate the mechanism of action of the nonapeptide a photoreactive analog, [4'-azido-Phe6]HSV H2-(6-15), was synthesized. The photoaffinity probe inhibits HSV ribonucleotide reductase activity, and when radioiodinated, it specifically labeled three viral proteins of 144, 95, and 85 kDa. We demonstrated by immunoprecipitation of the 144- and 95-kDa photolabeled proteins with antibodies specific to subunit 1 (H1) of HSV ribonucleotide reductase that the nonapeptide interacts with H1 and probably with its degradation products. Moreover, we obtained evidence that this specific binding is directly responsible for the ribonucleotide reductase inhibition.

Affinity Labels↗

PubMed1987

Structure-activity studies on synthetic peptides inhibiting herpes simplex virus ribonucleotide reductase.

Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) ribonucleotide reductase is formed by the association of two nonidentical subunits. A peptide corresponding to the COOH terminus of the subunit 2, Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu (H2-(7-15)), has been shown to completely inhibit the reductase activity (IC50 = 36 microM) without affecting the host isoenzyme. In order to study the relationship between chemical requirements and inhibitory potencies, a series of peptides, including fragments and analogs of H2-(7-15), were synthesized. The minimum active core can be assigned to the Val-Val-Asn-Asp-Leu sequence (IC50 = 760 microM). N alpha-Extended peptides, such as Ser-Thr-Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu (H2-(4-15)) and Glu-Cys-Arg-Ser-Thr-Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu (H2-(1-15) ), respectively, have inhibitory potencies 2.1- and 1.4-fold greater than the nonapeptide H2-(7-15). N alpha-Deamination or acetylation of H2-(7-15) increases its potency by 1.8- and 3.0-fold, respectively, whereas amidation of the alpha-carboxylic function diminishes its activity by 3.2-fold. These results indicate that the alpha-amino group is not essential for maximum potency but suggest that a free carboxylic function is required. Substitution of Tyr7 or Ala8 by their respective D-isomer leads to a decrease of potency, suggesting that a specific conformation of the NH2-terminal portion is required to have a maximum activity. Monosubstitution in positions 11, 13, 14, and 15, by L-alanine completely abolishes activity stressing the importance of each amino acid residue contained in the minimum active core. Finally, nonapeptides corresponding to the COOH-terminal portion of the subunit 2 of Epstein-Barr and varicella-zoster virus ribonucleotide reductases also inhibit the HSV-1 reductase activity. The varicella-zoster virus nonapeptide is 4.0 times more potent than H2-(7-15), whereas the Epstein-Barr virus nonapeptide is 3.1 times less potent. These results should help us to design a new generation of potent inhibitors of herpes virus ribonucleotide reductases.

Amino Acid Sequence↗

PubMed1987

Identification of viral polypeptides involved in pseudorabies virus ribonucleotide reductase activity.

We studied pseudorabies virus-induced ribonucleotide reductase and found that it exhibited biochemical properties very similar to those of herpes simplex virus reductase. A polyclonal rabbit antiserum (P9) directed against the carboxy terminus of subunit H2 polypeptide (38,000 daltons) of herpes simplex virus reductase neutralized the pseudorabies virus reductase, as well as the herpes simplex virus isozyme. This serum recognized two pseudorabies virus-specified polypeptides of 34,000 and 110,000 daltons, which may represent the two subunits of the enzyme. Furthermore, as already shown for herpes simplex virus reductase (E. A. Cohen, P. Gaudreau, P. Brazeau, and Y. Langelier, Nature [London] 321:441-443, 1986), we show that the nonapeptide itself specifically inhibited pseudorabies reductase activity.

Herpesvirus 1, Suid↗

PubMed1987

Activities of both ribonucleotide reductase subunits, M1 and M2, decrease upon serum starvation of baby hamster kidney 21/C13 cells.

Ribonucleotide reductase from mammalian cells is composed of two nonidentical subunits M1 and M2 which are both required to form the catalytic site. The level of ribonucleotide reductase activity is cell cycle controlled and several reports suggest that this control is achieved mainly by the regulation of M2 subunit synthesis. In the present study, we have found that the activities of both subunits decreased markedly upon serum starvation in the Syrian baby hamster kidney 21/C13 cell line. These decreases did not seem to be correlated with the appearance of an inhibitory factor in serum-starved cells. Quantification of the amount of the M1 subunit protein (89,000 molecular weight) by [32P]dTTP photoaffinity labelling revealed that the decrease in M1 activity was not due to variation in M1 protein level. Therefore, a posttranslational mechanism probably exists which inactivates M1 subunit when cells stay in the quiescent (G0) state and this mechanism could play an important role in the control of ribonucleotide reductase activity.

Animals↗

PubMed1986

5-Fluorouracil-metronidazole combination therapy in metastatic colorectal cancer. Clinical, pharmacokinetic and in vitro cytotoxicity studies.

We have investigated the role of metronidazole (MND) combined with 5-fluorouracil (5-FU) in the treatment of metastatic colorectal cancer. MND (750 mg/m2) was administered i.v. 1 h before 5-FU (600 mg/m2) i.v., daily for 5 consecutive days. Treatment was repeated every 4 weeks until disease progression or prohibitive toxicity occurred. Of the 27 patients entered in the study, 4 (15%) had an objective complete or partial response lasting an average of 7 months. 5-FU toxicity was greatly enhanced by the administration of MND, however, 74% of patients having granulocytopenia (less than 1500/microliter). We investigated the possible mechanisms underlying this enhanced 5-FU toxicity by examining whether MND modified 5-FU pharmacokinetics or whether the two drugs had a synergistic effect in vitro against the HCT-8 colon cancer cell line. While the in vitro studies failed to reveal any synergism between 5-FU and MND, pharmacokinetic evaluation revealed that 5-FU clearance was significantly reduced (26.9%, P less than 0.001) by prior MND administration. MND reduces 5-FU's therapeutic index in the treatment of colorectal cancer by impairing its clearance, which leads to increased toxicity without enhanced therapeutic efficacy.

Adult↗

PubMed1986

Neutralization of herpes simplex virus ribonucleotide reductase activity by an oligopeptide-induced antiserum directed against subunit H2.

Herpes simplex virus type 1 ribonucleotide reductase is associated with two polypeptides of apparent molecular weights 136,000 and 38,000. The two polypeptides form a tight complex and, therefore, are often coprecipitated by monoclonal antibodies. We report here that immunoglobulins G purified from polyclonal rabbit antisera (P9) raised against a nonapeptide corresponding to the carboxy terminus of the 38,000-dalton polypeptide specifically neutralize the herpes simplex virus ribonucleotide reductase activity. We suggest that the P9 immunoglobulin G neutralizes the reductase activity by impairing the association of the two subunits (H1 and H2) of the enzyme.

Antigen-Antibody Reactions↗

PubMed1986

Herpes simplex virus type 2 mutagenesis: characterization of mutants induced at the hprt locus of nonpermissive XC cells.

In a previous report, herpes simplex virus type 2 (HSV-2) was shown to increase the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus of nonpermissive rat XC cells (L. Pilon, A. Royal, and Y. Langelier, J. Gen. Virol. 66:259-265, 1985). A series of 17 independent mutants were isolated after viral infection together with 12 spontaneous noninfected mutants to characterize the nature of the mutations induced by the virus at the molecular level. The DNA of the mutants isolated after viral infection was probed with cloned HSV-2 fragments representing the entire genome. In these mutants, no authentic HSV-2 hybridization could be detected. This was indicative of a mechanism of mutagenesis which did not require the permanent integration of viral sequences in the host genome. The structure of the hprt gene was determined by the method of Southern (J. Mol. Biol. 98:503-517, 1975), and the level of hprt mRNA was analyzed by Northern blots. Except for the identification of one deletion mutant in each of the two groups, the HPRT- clones showed no evidence of alteration in their hprt gene. A total of 7 of 12 spontaneous mutants and 11 of 15 mutants isolated from the infected population transcribed an hprt mRNA of the same size and abundance as did the wild-type cells. Thus, the majority of the mutants seemed to have a point mutation in their hprt structural gene. Interestingly, the proportion of the different types of mutations was similar in the two groups of mutants. This analysis revealed that HSV-2 infection did not increase the frequency of rearrangements but rather that it probably induced a general increase of the level of mutations in the cells. This type of response is thought to be compatible with the biology of the virus, and the possible mechanisms by which HSV-2 induces somatic mutations in mammalian cells are discussed.

Animals↗

PubMed1985

Increased mutation frequency after herpes simplex virus type 2 infection in non-permissive XC cells.

The effect of herpes simplex virus type 2 (HSV-2) infection on the frequency of mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was studied in the non-permissive XC cell line. When the cells were infected with 20 to 800 p.f.u./cell, there was initially a lag in cell growth and cell death, but after 4 days there was no difference in growth rate between infected and control cultures. However, the mutation frequency, as determined by the number of 6-thioguanine-resistant colonies, was increased in infected cultures by factors ranging from 2.5 to 10.3. This effect was found to be dependent on the multiplicity of infection. The maximum effect was obtained between 20 and 100 p.f.u./cell while further increase in the amount of virus resulted in a drop in the yield of mutants. The optimum multiplicity of infection was a reproducible characteristic but was variable between viral stocks. When a number of mutant clones were examined they were found to have HGPRT activities ranging from undetectable to 6.9% of wild-type, indicating that the mutations were in the HGPRT gene. These results show that, in a non-productive infection, HSV-2 particles can increase the mutation frequency. The possible mechanisms by which this effect is brought about in the host genome are discussed.

Animals↗

PubMed1985

Herpes simplex virus ribonucleotide reductase induced in infected BHK-21/C13 cells: biochemical evidence for the existence of two non-identical subunits, H1 and H2.

In nearly all systems studied, ribonucleotide reductase consists of two non-identical subunits. We present here the results of our study on herpes simplex virus (HSV) ribonucleotide reductase in favour of the existence of two subunits, H1 and H2, different from the mammalian subunits, M1 and M2. First, although the viral subunits could not be separated by Blue Sepharose chromatography (unlike mammalian subunits), they seemed to dissociate at very low protein concentration as suggested by the non-linear relationship between activity and low protein concentration. Second, pyridoxal phosphate (Pyr.P)-NaBH4 treatment and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ) treatment of partially purified extract of mammalian ribonucleotide reductase which inactivated M1 and M2 respectively also inhibited the HSV ribonucleotide reductase. This activity could be restored by mixing Pyr.P-NaBH4-treated extracts with MAIQ-treated extracts of viral ribonucleotide reductase, suggesting that each treated extract contains one active subunit. Moreover, the addition of exogenous M1 or M2 subunits to one or the other of these two treated extracts did not produce any detectable reductase activity. Our interpretation of these results is that the two subunits H1 and H2 which could dissociate upon treatment did not form enzymically active hybrids with the mammalian subunits. Also, the higher degree of resistance to heat inactivation and to hydroxyurea of the viral reductase as compared to the mammalian enzyme suggests that H1 differs from M1 and H2 from M2.

Animals↗

PubMed1978

Aanlysis of dCMP deaminase and CDP reductase levels in hamster cells infected by herpes simplex virus.

Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of gene transfer, are induced by herpes simplex virus (HSV). The utility of additional markers prompted us to reanalyze the effects of HSV infection on the activities of two other enzymes for which direct selective methods can be devised: dCMP deaminase and CDP reductase. For this purpose, mutant Chinese hamster (lA1) cells devoid of dCMP deaminase activity or Syrian hamster (BHK-21/C13) cells were infected by HSV type 1 or 2, and the activities of thymidine kinase, dCMP deaminase, and CDP reductase were measured in the cell extracts. The reported induction of thymidine kinase and CDP reductase by HSV was confirmed, whereas the stimulation of dCMP deaminase activity could not be observed. For both cell lines, the HSV-induced CDP reductase differed from the host enzyme by sensitivity to inhibition by both dTTP and dATP. This property should be helpful in developing a selection system for this activity.

Animals↗