Pi Variants Research Articles

PINIR: a comprehensive information resource for Pin-II type protease inhibitors

BackgroundSerine protease inhibitors belonging to the Potato type-II Inhibitor family Protease Inhibitors (Pin-II type PIs) are essential plant defense molecules. They are characterized by multiple inhibitory repeat domains, conserved disulfide bond pattern, and a tripeptide reactive center loop. These features of Pin-II type PIs make them potential molecules for protein engineering and designing inhibitors for agricultural and therapeutic applications. However, the diversity in these PIs remains unexplored due to the lack of annotated protein sequences and their functional attributes in the available databases.ResultsWe have developed a database, PINIR (Pin-II type PIs Information Resource), by systematic collection and manual annotation of 415 Pin-II type PI protein sequences. For each PI, the number and position for signature sequences are specified: 695 domains, 75 linkers, 63 reactive center loops, and 10 disulfide bond patterns are identified and mapped. Database analysis revealed novel subcategories of PIs, species-correlated occurrence of inhibitory domains, reactive center loops, and disulfide bond patterns. By analyzing linker regions, we predict that alternative processing at linker regions could generate PI variants in the Solanaceae family.ConclusionPINIR (https://pinir.ncl.res.in) provides a web interface for browsing and analyzing the protein sequences of Pin-II type PIs. Information about signature sequences, spatio-temporal expression, biochemical properties, gene sequences, and literature references are provided. Analysis of PINIR depicts conserved species-specific features of Pin-II type PI protein sequences. Diversity in the sequence of inhibitory domains and reactive loops directs potential applications to engineer Pin-II type PIs. The PINIR database will serve as a comprehensive information resource for further research into Pin-II type PIs.

Presence, persistence and effects of pre-treatment HIV-1 drug resistance variants detected using next generation sequencing: A Retrospective longitudinal study from rural coastal Kenya.

BackgroundThe epidemiology of HIV-1 drug resistance (HIVDR) determined by Sanger capillary sequencing, has been widely studied. However, much less is known about HIVDR detected using next generation sequencing (NGS) methods. We aimed to determine the presence, persistence and effect of pre-treatment HIVDR variants detected using NGS in HIV-1 infected antiretroviral treatment (ART) naïve participants from rural Coastal Kenya.MethodsIn a retrospective longitudinal study, samples from HIV-1 infected participants collected prior [n = 2 time-points] and after [n = 1 time-point] ART initiation were considered. An ultra-deep amplicon-based NGS assay, calling for nucleotide variants at >2.0% frequency of viral population, was used. Suspected virologic failure (sVF) was defined as a one-off HIV-1 viral load of >1000 copies/ml whilst on ART.ResultsOf the 50 eligible participants, 12 (24.0% [95% CI: 13.1–38.2]) had at least one detectable pre-treatment HIVDR variant against Protease Inhibitors (PIs, n = 6 [12%]), Nucleoside Reverse Transcriptase Inhibitors (NRTIs, n = 4 [8.0%]) and Non-NRTIs (n = 3 [6.0%]). Overall, 15 pre-treatment resistance variants were detected (frequency, range: 2.3–92.0%). A positive correlation was observed between mutation frequency and absolute load for NRTI and/or NNRTI variants (r = 0.761 [p = 0.028]), but not for PI variants (r = -0.117 [p = 0.803]). Participants with pre-treatment NRTI and/or NNRTI resistance had increased odds of sVF (OR = 6.0; 95% CI = 1.0–36.9; p = 0.054).ConclusionsUsing NGS, pre-treatment resistance variants were common, though observed PI variants were unlikely transmitted, but rather probably generated de novo. Even when detected from a low frequency, pre-treatment NRTI and/or NNRTI resistance variants may adversely affect treatment outcomes.

High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface.

Capillary isoelectric focusing directly coupled to high resolution mass spectrometry (cIEF-MS) provides information on amphoteric molecules, including isoelectric point and accurate mass, which enables structural interrogation of biopolymer pI variants. The coupling of cIEF with MS was facilitated by a flow-through microvial interface, made by stainless steel with high chemical resistance and mechanical robustness. Two on-column electrolyte configurations of cIEF-MS were demonstrated using peptide and protein pI markers. The pI resolution was 0.02 pH unit in the pH range of 5.5 to 7.0, with no anticonvective reagent (glycerol) added. High resolution Orbitrap detector provides mass spectra for midsized proteins (<30 kDa), enabling deconvolution with high accuracy for IEF-focused low abundance species. Charge heterogeneity of therapeutic monoclonal antibodies (mAb) is one of the most important attributes in the biopharmaceutical industry, and it is routinely monitored by IEF and fractionation-based methods. As a proof of concept, the commercial formulation of infliximab was directly analyzed using cIEF-MS for separation and online identification of mAb charge variants. The main intact antibody species along with two basic and one acidic variants were observed, and their accurate molecular weights ( Mw) recorded by MS detector readily revealed the structural differences of these variants. Variants with 0.1 unit in pI difference and 1 Da difference in molecular weight were readily resolved. The deconvoluted intact Mw values showed ppm level accuracy compared to theoretical predictions.

A bifunctional α-amylase/trypsin inhibitor from pigeonpea seeds: Purification, biochemical characterization and its bio-efficacy against Helicoverpa armigera

This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing (N-LP-IEF), affinity chromatography and preparative electrophoresis. Its in-vivo and in-vitro interaction with midgut amylases of H. armigera was studied along with growth inhibitory activity. One and two dimensional (2D) zymographic analyses revealed that the purified inhibitor is dimeric glycoprotein (60.2kDa and 56kDa) exist in a multi-isomeric form with five pI variants (pI 5.5 to 6.3). It was found to be heat labile with complete inactivation up to 80°C and stable over a wide range of pH (4–11). The slow binding and competitive type of α-amylase inhibition was observed with 0.08μM of dissociation constant (Ki) for the enzyme–inhibitor complex (EI). The internal protein sequence of two subunits obtained by mass spectrometry matched with cereal-type α-AI, a conserved domain from AAI_LTSS superfamily and sialyltransferase-like protein respectively. In-vivo studies indicated up-regulation of total midgut α-amylase activity with negative effect on growth rate of H. armigera suggesting its suitability for pest control.

The Enterococcus faecalis Exoproteome: Identification and Temporal Regulation by Fsr

Analysis of the culture supernatant exoproteins produced by two PFGE clusters of high-level gentamicin and ciprofloxacin-resistant clinical isolates of Enterococcus faecalis from the UK and Ireland revealed two distinct protein profiles. This grouping distinguished OG1RF and GelE metalloprotease-expressing isolates from JH2-2 and other GelE-negative isolates. The integrity of the fsrABDC operon was found to determine the exoproteome composition, since an fsrB mutant of strain OG1RF appeared very similar to that of strain JH2-2, and complementation of the latter with the fsrABDC operon produced an OG1RF-like exoproteome. The proteins present in the supernatant fraction of OG1RF were separated using 2D gels and identified by mass spectrometry and comprised many mass and pI variants of the GelE and SprE proteases. In addition cell wall synthesis and cell division proteins were identified. An OG1RF fsrB mutant had a distinct exoprotein fraction with an absence of the Fsr-regulated proteases and was characterised by general stress and glycolytic proteins. The exoproteome of the OG1RF fsrB mutant resembles that of a divIVA mutant of E. faecalis, suggestive of a stress phenotype.

Variants of glutathione s-transferase pi 1 exhibit differential enzymatic activity and inhibition by heavy metals

Nonsynonymous single nucleotide polymorphisms in glutathione s-transferase pi 1 (GSTP1; Ile/Val 105, Ala/Val 114) have been associated with altered toxicant metabolism in epidemiological cohorts. We explored the impact of GSTP1 genotype on enzyme kinetics and heavy metal inhibition in vitro. Four GSTP1 allozymes (105/114: Ile/Ala, Val/Ala, Ile/Val, Val/Val) were expressed in and purified from Escherichia coli. Enzyme activity assays quantifying the rate of glutathione conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) revealed significant differences in kinetic parameters depending on genotype (p<0.01). Allozymes with Ile105 had better catalytic efficiency and greater affinity for CDNB (mean±SEM: Ile105 Ala114 Km=0.33±0.07mM vs. Val105 Ala114 Km=1.15±0.07mM). Inhibition of GSTP1 activity by heavy metals was assessed following treatment with mercury (inorganic-HgCl2, methylmercury-MeHg), selenium, cadmium, lead, arsenic, and manganese. All allozymes were inhibited by HgCl2 (IC50 range: 24.1–172μM), MeHg (93.9–480μM), and selenium (43.7–62.8μM). Genotype significantly influenced the potency of mercury with GSTP1 Ile105 Val114 the least sensitive and Val105 Ala114 the most sensitive to inhibition by HgCl2 and MeHg. Overall, genotype of two nonsynonymous polymorphisms in GSTP1 influenced enzyme kinetics pertaining to an electrophilic substrate and inhibition by two mercury species.

Mass spectrometric characterization of theCampylobacter jejuniadherence factor CadF reveals post-translational processing that removes immunogenicity while retaining fibronectin binding

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn-binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2-DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF(24)) and 22 kDa (CadF(22)) mass variants. CadF variants were fully characterized by MALDI-TOF MS and MALDI-MS/MS. These data confirmed that CadF forms re-folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post-translationally. CadF(22) and CadF(24), however, were characterized as N-terminal, membrane-associated polypeptides resulting from cleavage between serine(195) and leucine(196), and glycine(201) and phenylalanine(202), respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full-length CadF (34 kDa), CadF(24), and the deleted C-terminal OmpA domain (14 kDa; CadF(14)) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full-length CadF retained reactivity. Binding assays showed that CadF(24) retained Fn-binding capability, while CadF(14) did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn-binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.

Analysis of association of polymorphic variants of genes GSTP1 and GSTM1 with survival rate sick of liver cirrhosis of a virus and alcoholic etiology

With the purpose of studying of association of polymorphic variants of genes glutathione S-transferases pi and mu (GSTP1 and GSTM1) with survival rate sick of liver cirrhosis (LC), it is lead uniinstantly research of 189 sick of LC of a virus (HBV, HCV, H V+H V) and an alcoholic etiology. Have been В С compared a frequencies of allocation of polymorphic variants of genes GSTP1 and GSTM1 between the died and persisted patients for the season 4 years. With greater survival rate of sick of LC associated: presence of genotype АА of gene GSTP1 (χ 2 Pearson, р = 0,008) and «null» genotype GSTM1 (χ 2 Pearson, р = 0,027). Chances of development of a lethal outcome at sick of LC with presence of genotypes GG and AG on gene GSTP1 in 2,5 times above, than at patients with genotype АА. Presence of active gene replica GSTM1 enlarges chances of development of a lethal outcome in 2 times. The received results testify to necessity of the count of genetical factors for the forecast of LC.

Analysis and Quantification of Diagnostic Serum Markers and Protein Signatures for Gaucher Disease

Novel approaches for the qualitative and quantitative proteomics analysis by nanoscale LC-MS applied to the study of protein expression response in depleted and undepleted serum of Gaucher patients undergoing enzyme replacement therapy are presented. Particular emphasis is given to the method reproducibility of these LC-MS experiments without the use of isotopic labels. The level of chitotriosidase, an established Gaucher biomarker, was assessed by means of an absolute concentration determination technique for alternate scanning LC-MS generated data. Disease associated proteins, including fibrinogens, complement cascade proteins, and members of the high density lipoprotein serum content, were recognized by various clustering methods and sorting and intensity profile grouping of identified peptides. Condition-unique LC-MS protein signatures could be generated utilizing the measured serum protein concentrations and are presented for all investigated conditions. The clustering results of the study were also used as input for gene ontology searches to determine the correlation between the molecular functions of the identified peptides and proteins.

Glutathione S-transferase mu, omega, pi, and theta class variants and smoking in Parkinson's disease

GSTs are a family of inducible phase II enzymes that may play a neuroprotective role in Parkinson's disease (PD). GSTs may also modify PD risk by metabolizing compounds in cigarettes, as cigarette smoking is generally found to be associated with a decrease in PD risk. Using a population-based case–control study design, we examined polymorphisms of the mu, omega, pi, and theta classes of GST to elucidate the main effects and smoking–GST interactions on PD risk. From three rural California counties, we recruited 289 incident idiopathic PD cases, clinically confirmed by our study neurologist, and 270 population controls, marginally matched by age, gender, and race. We assessed main gene polymorphism associations and evaluated interactions between smoking and GST polymorphisms as departures from a multiplicative scale adjusting for age, gender, and race. We also restricted analyses to Caucasian subjects to address the potential for population stratification ( n = 235 cases, 220 controls). Among Caucasians, we observed a risk reduction in subjects carrying at least one variant allele for GSTO1 (OR = 0.68, 95% CI: 0.47–0.98) and also GSTO2 (OR = 0.64, 95% CI: 0.44–0.93); both genes were in strong linkage disequilibrium. No main gene effects were observed for the remaining polymorphisms. We noted a multiplicative interaction between ever having smoked regularly and GSTO1 (OR interaction = 0.55, 95% CI: 0.33–0.92) and GSTO2 (OR interaction = 0.54, 95% CI: 0.32–0.90). Results were similar when combining all races. These findings and the paucity of similar studies suggest a need for further inquiry into the association between GSTs, smoking, and PD risk.

Ischemia-specific phosphorylation and myofilament translocation of heat shock protein 27 precedes alpha B-crystallin and occurs independently of reactive oxygen species in rabbit myocardium

Heat shock protein 27 (Hsp27) and alpha B-crystallin (alphaBC) are small heat shock proteins that stabilize the myofilament during stress. We utilized two-dimensional gel electrophoresis (2-DE), phospho-fluorescence staining, titanium dioxide (TiO(2)) phosphopeptide purification and mass spectrometry (MS) to fully characterize isoelectric point (pI) variants of Hsp27 and alphaBC in rabbit myocardium subjected to brief ischemia/reperfusion (I/R) injury. Four variants of Hsp27 were detected, two of which were phosphorylated: HSP1 (at three sites, Ser15, Ser78 and Ser82) and HSP2 (at Ser15 and Ser82, but not Ser78). Three variants of alphaBC were detected: alphaBC1 was phosphorylated (at Ser59 alone) and alphaBC2 was deamidated (at Asn146). No modifications were found in the remaining variants. Both phospho-Hsp27 variants increased in abundance in tissue subjected to brief I/R injury (15 min I/60 min R) and ischemia without subsequent reflow (15I/0R), and these increases were not affected by addition of the potent antioxidant, N-(2-mercaptopropionyl) glycine (MPG; 15I/60R + MPG and 15I/0R + MPG). Abundance of native and phosphorylated (but not deamidated) alphaBC was elevated following 15I/60R; however, these increases were ameliorated by the presence of MPG, and did not occur in tissue subjected to 15I/0R. Both phospho-Hsp27 variants and phospho-alphaBC translocated to the myofilament following 15I/60R. Increased myofilament association of phospho-Hsp27 was not influenced by MPG, and there was a greater proportion of HSP2 than HSP1 in this fraction. MPG inhibited phospho-alphaBC translocation and increased alphaBC association with the myofilament did not occur during 15I/0R. Increased phosphorylation of Hsp27 is ischemia-specific and not influenced by reactive oxygen species (ROS), while increased expression and phosphorylation of alphaBC are ROS-dependant.

Gluthatione-S-transferase P1 polymorphism I105V in familial and sporadic prostate cancer

Several reports suggest that the glutathione-S-transferase (GST) family of enzymes is involved in a variety of cancers, due to their carcinogen-detoxification properties. A polymorphism in codon 105 of the pi variant (GSTP1 I105V), which affects the enzymatic activity of the enzyme, has been linked to the incidence of cancers from different organs. However, the published data in prostate cancer (PCa) is controversial. Some studies report an association with the GSTP1 I105V polymorphism and sporadic PCa, whereas other studies report no association. Recently, one study showed a positive correlation between the GSTP1 I105V polymorphism and familial PCa in a Japanese population. In the present study, we assessed the correlation of the GSTP1 I105V polymorphism with familial and sporadic PCa in an American population. We analyzed DNA samples from 438 patients with familial PCa, 499 patients with sporadic PCa, and 510 controls. We found no significant association between the GSTP1 I105V polymorphism and familial or sporadic PCa when compared to the control group [odds ratio (OR) =1.0 (0.74–1.37); P = 0.58]. Moreover, no association was found after stratification for age of diagnosis, Gleason grade, or lymph node involvement [OR =0.84 (0.65–1.09), P = 0.37]. These data indicate that there is no associated risk for sporadic or familial PCa in American families containing the GSTP1 I105V polymorphism.

MSAP, the meichroacidin homolog of carp (Cyprinus carpio), differs from the rodent counterpart in germline expression and involves flagellar differentiation.

To gain access to the molecular mechanisms of spermatogenesis, the genes from a subtractive screen of the carp testis cDNA library were investigated. In this study, a male-specific homolog of the meichroacidin gene, called MSAP (MORN motif-containing sperm-specific axonemal protein), was isolated and further characterized. Database search and zoo-Western blot analyses revealed that MSAP homologs might be widespread in a variety of phyla but divergent in their C-terminal length and sequences. Carp MSAP is exclusively transcribed in testis, while mouse meichroacidin message is present in gonads of both sexes, although especially enriched in testis. In mouse, meichroacidin is expressed in male germ cells of meiotic stages, while carp MSAP is expressed during late spermiogenesis and accumulated in mature spermatozoa, in which MSAP is localized to the basal body and flagellum. Contrary to mouse meichroacidin revealed previously, existence of multiple pI variants of MSAP in two-dimensional electrophoresis suggested regulatory differences of the homologous molecules between mammal and teleost. These results indicate that MSAP homologs may play different roles in male germline development between vertebrates. Proteomic analysis and immunolocalization disclosed that MSAP is associated with septin7, a conserved GTPase that may participate in cellular morphogenesis, in the basal body of carp sperm. These findings suggest the involvement of carp MSAP in flagellar differentiation during spermiogenesis.

Transmission/disequilibrium tests of androgen receptor and glutathione S-transferase pi variants in prostate cancer families.

Population-based case-control studies have found relationships between risk of prostate cancer and genetic polymorphisms in the CAG repeat and GGC repeat of the X-linked androgen receptor gene (AR) as well as the autosomal gene coding for glutathione S-transferase pi (GSTP1). This family-based study utilized the transmission disequilibrium test to examine whether there was evidence that these polymorphisms could account for familial aggregation of prostate cancer. Seventy-nine North American pedigrees were studied. Most of these families had 3 or more affected first-degree relatives. Genotype information was obtained on 578 individuals. The reconstruction combined transmission disequilibrium test (RC-TDT) was used to test for linkage. There was no evidence of linkage to the CAG and GGC repeat sequences in the AR gene or the pentanucleotide (ATAAA) repeat in the GSTP1 gene when each allele was analyzed separately or when alleles were grouped by repeat length. Our findings do not support the hypothesis that familial clustering of prostate cancer in high-risk families is attributable to these genetic variants.

Identification in Saccharomyces cerevisiae of a New Stable Variant of Alkyl Hydroperoxide Reductase 1 (Ahp1) Induced by Oxidative Stress

Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern of Saccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point. This form was also present in wild type strains after treatment with tert-butyl hydroperoxide. In vitro enzyme assays showed that Ahp1p had lower specific activity in strains lacking Cu,Zn-SOD. We studied three mutants presenting a reduced production of the low pI variant under oxidative stress conditions. Two of the mutants (C62S and S59D) were totally inactive, thus suggesting that the acidic form of Ahp1p may only appear when the enzyme is functional. The other mutant (S59A) was active in vitro and was more resistant to inactivation by tert-butyl hydroperoxide than the wild type enzyme. Furthermore, the inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress (alkyl hydroperoxide treatment or lack of Cu,Zn-SOD activity but not hydrogen peroxide treatment), the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid (a natural occurring intermediate of the enzymatic reaction) and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive.

Location of the epoxide function determines specificity of the allelic variants of human glutathione transferase Pi toward benzo[ c]chrysene diol epoxide isomers

Carcinogenic activity of many polycyclic aromatic hydrocarbons (PAHs) is mainly attributed to their respective diol epoxides, which can be classified as either bay or fjord region depending upon the location of the epoxide function. The Pi class human glutathione (GSH) transferase (hGSTP1-1), which is polymorphic in humans with respect to amino acid residues in positions 104 (isoleucine or valine) and/or 113 (alanine or valine), plays an important role in the detoxification of PAH-diol epoxides. Here, we report that the location of the epoxide function determines specificity of allelic variants of hGSTP1-1 toward racemic anti-diol epoxide isomers of benzo[ c]chrysene (B[ c]C). The catalytic efficiency ( k cat/ K m) of V104,A113 (VA) and V104,V113 (VV) variants of hGSTP1-1 was approximately 2.3- and 1.7-fold higher, respectively, than that of the I104,A113 (IA) isoform toward bay region isomer (±)- anti-B[ c]C-1,2-diol-3,4-epoxide. On the other hand, the IA variant was approximately 1.6- and 3.5-fold more efficient than VA and VV isoforms, respectively, in catalyzing the GSH conjugation of fjord region isomer (±)- anti-B[ c]C-9,10-diol-11,12-epoxide. The results of the present study clearly indicate that the location of the epoxide function determines specificity of the allelic variants of hGSTP1-1 in the GSH conjugation of activated diol epoxide isomers of B[ c]C.

The charge-heterogeneity of human fibrinogen as investigated by 2D electrophoresis

The charge-heterogeneity of human plasma fibrinogen subunit chains was characterized by two-dimensional electrophoresis (2DE). Western blotting with antibodies specific for the γ-chain demonstrated that the γ-chains focus at varying isoelectric points (pI). This microheterogeneity was also observed in fibrinogen secreted from hepatocytic cells and in recombinant fibrinogen expressed in Chinese hamster ovary (CHO) cells. Further, covalent γγ-dimerization by FXIIIa was not influenced by the charge-heterogeneity, and removal of the carbohydrate did not reduce the number of γ-chain pI variants. These observations suggest that the microheterogeneity of the γ-chain is a multifactorial phenomenon that is not due to physiologic modification of the glycoprotein in circulation.

Catalytic efficiencies of allelic variants of human glutathione S-transferase Pi in the glutathione conjugation of α,β-unsaturated aldehydes

The catalytic efficiencies of the allelic variants of human glutathione (GSH) S-transferase Pi (hGSTP1-1), which differ in their primary structures by the amino acids in positions 104 (isoleucine or valine) and/or 113 (alanine or valine), in the GSH conjugation (detoxification) of acrolein and crotonaldehyde have been determined. The k cat/ K m values for hGSTP1-1 isoforms I104,A113 (IA), I104,V113 (IV), V104,A113 (VA) and V104,V113 (VV) toward acrolein were 129±3, 116±3, 128±4 and 92±3 mM −1 s −1, respectively. The catalytic efficiencies of the hGSTP1-1 variants IA, IV, and VA in the GSH conjugation of acrolein were statistically significantly higher (at P=0.05) compared with the VV isoform. On the other hand, the catalytic efficiencies of the hGSTP1-1 isoforms IA, IV, VA and VV toward crotonaldehyde (16±2, 12±1, 17±2, and 12±2 mM −1s −1, respectively) were not statistically significantly different from each other. Our results suggest that hGSTP1-1 polymorphism may be an important factor in differential susceptibility of individuals to the toxic effects of acrolein, which is a widely spread environmental pollutant and generated endogenously during metabolic activation of anticancer drug cyclophosphamide.

Differential Catalytic Efficiency of Allelic Variants of Human GlutathioneS-Transferase Pi in Catalyzing the Glutathione Conjugation of Thiotepa

Alkylating agents are extensively used in the treatment of cancer. The clinical usefulness of this class of anticancer drugs, however, is often limited by the emergence of drug-resistant tumor cells. Increased glutathione (GSH) conjugation through catalysis by GSHS-transferases (GSTs) is believed to be an important mechanism in tumor cell resistance to alkylating agents. In the present study, we report that the allelic variants of human Pi class GST (hGSTP1-1), which differ in their primary structures at amino acids in positions 104 and/or 113, exhibit significant differences in their activity in the GSH conjugation of alkylating anticancer drug thiotepa. Mass spectrometry revealed that the major product of the reaction between thiotepa and GSH was the monoglutathionyl–thiotepa conjugate. While nonenzymatic formation of monoglutathionyl–thiotepa was negligible, the formation of this conjugate was increased significantly in the presence of hGSTP1-1 protein. The hGSTP1-1-catalyzed GSH conjugation of thiotepa was time and protein dependent and followed Michaelis–Menten kinetics. The catalytic efficiency of hGSTP1-1(I104,A113) variant was approximately 1.9- and 2.6-fold higher compared with hGSTP1-1(V104,A113) and hGSTP1-1(V104,V113) isoforms, respectively. The results of the present study indicate that the hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to thiotepa, and that subjects homozygous for thehGSTP1-1(I104,A113)allele, which is most frequent in human populations, are likely to be at a greater risk for developing GST-mediated resistance to thiotepa than heterozygotes or homozygotes with valine 104 background.

Regulatory implications of protein assemblies at the gamma origin of plasmid R6K - a review.

Recognition of the replication origin (ori) by initiator protein is a recurring theme for the regulated initiation of DNA replication in diverse biological systems. The objective of the work reviewed here is to understand the initiation process focusing specifically on the gamma-ori of the antibiotic-resistance plasmid R6K. The control of gamma-ori copy number is determined by both plasmid-encoded and host-encoded factors. The two central regulatory elements of the plasmid are a multifunctional initiator protein pi, and sequence-related DNA target sites, the inverted half-repeats (IRs) and the direct repeats (DRs). The replication activator and inhibitor activities of pi seem to be at least partially distributed between two naturally occurring pi polypeptides (designated by their molecular weights pi35.0 and pi30.5). Regulatory variants of pi with altered states of oligomerization in nucleoprotein complexes with DRs and IRs have been isolated. The properties of these mutants laid the foundation for our model of pi protein activity which proposes that different protein surfaces are required for the formation of functionally distinct complexes of pi with DRs and IRs. These mutants also suggest that pi polypeptides have a modular structure; the C-terminus contains the DNA-binding domain while the N-terminus controls protein oligomerization. Additionally, pi35.0 binds to a novel DNA sequence in the A+T-rich segment of gamma-ori. This binding site is at or near the site from which synthesis of the leading strand begins.