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  • The isoenzyme hGSTP is an attractive

    2021-11-25

    The isoenzyme hGSTP1-1 is an attractive drug target due to its high levels of disease association (target validation) and druggability (target tractability) properties [5,6,[17], [18], [19], [20], [21]]. hGSTP1-1 functions as a homodimer protein [18]. Each monomer has two domains, an α/β domain (N-terminal domain) that includes helices α1-α3 and a large α-helical domain (C-terminal domain). The N-terminal domain forms the GSH binding site (G-site), whereas the hydrophobic site (H-site), which binds the xenobiotic substrates, is located mainly at the C-terminal domain [18]. Several metal chelate, are effective reagents for cleavage of the polypeptide chains by reactive oxygen species (ROS) [[22], [23], [24], [25], [26]]. Such reagents that composed by a metal-chelating group and a functional group capable of reacting with a specific group VX765 on the protein have been synthesized and employed for probing structure-function relationships in proteins [26].
    Methods
    Results & discussion
    Conclusions
    Conflict of interest
    Acknowledgement This project was supported by the Deanship of Scientific Research at Prince Sattam bin Abdulaziz University under the research project 2017/03/6910.
    Introduction Glutathione S-transferases (GSTs, EC 2.5.1.18) belong to a large family of functionally different enzymes that catalyze the S-conjugation of glutathione (GSH) with a wide variety of electrophilic compounds. Through this reaction, GSTs play an important role in the protection of cells from the products of oxidative stress as well as from several environmental carcinogens (e.g., benzo(a)pyrene and other polyaromatic hydrocarbons) [1], [2]. Even though the S-conjugation reaction protects the cell from cytotoxic and carcinogenic agents, much evidence shows that some classes might be involved in anticancer drug resistance and even be considered as tumor markers [3]. In particular, elevated levels of glutathione S-transferase P1-1 (GSTP1-1) have been found in stomach, colon, bladder, testicular, prostate, breast, skin and lung tumors, as well as acute myeloid and lymphoid leukemia, when compared with corresponding normal tissues [4], [5], [6], [7]. The GSTP class is polymorphic and four VX765 have been described at the GSTP1 locus located on chromosome 11q13: GSTP1*A, GSTP1*B, GSTP1*C and GSTP1*D[8]. Two sites in the cDNA sequence are variable and are characterized by an A→G transition at nucleotide 313 (point mutation in exon 5) and a C→T transition at nucleotide 341 (point mutation in exon 6). The resulting codon variants result in the amino acids Ile105 or Val105 and Ala114 or Val114 at the electrophilic “H”-site level. The proteins encoded by the different GSTP1 alleles show different abilities to bind and possibly metabolize carcinogens and anticancer agents, and several studies have indicated an association between GSTP1 polymorphism and the risk for a variety of cancers [7], [9] as well as varying responses to cancer treatment [10] or susceptibility to some diseases such as Parkinson's disease [11], multiple sclerosis [12] and asthma [13]. Even if the results of many studies often conflict and the association between GSTP1 polymorphism and disease susceptibility/outcome has not been definitively established, it is clear that it might be an ubiquitous “disease-modifying” agent. The aim of our study was to develop a fast, accurate and automated methodology to determine the GSTP1-1 polymorphism in order to perform large-scale molecular epidemiological studies. We have applied this methodology in order to determine the genotype distribution for the GSTP1-1 “H”-site polymorphism in a cohort of free-living, apparently healthy subjects representative of an Italian population coming from the Central area (Rome).
    Materials and methods
    Results and discussion The melting peaks of exon 5 that we found in our 250 subjects were in accordance with Ko et al. [14] and are shown in Fig. 1 (panel A). The melting peaks of exon 6, obtained on our 250 subjects with our newly constructed hybridization probes and PCR primers, are shown in Fig. 1 (panel B). In particular, the distinction between the Ala/Ala (homozygous GCG), Val/Val (homozygous GTG) and the Ala/Val (heterozygous GCG/GTG) genotypes was obtained by a melting curve analysis in which the maximum of dehybridization for Ala (GCG) was 61.5 °C, and the maximum of dehybridization for Val (GTG) was 67.8 °C. Therefore, the melting curve of an Ala/Ala genotype showed a single peak at 61.5 °C, the melting curve of a Val/Val genotype showed a single peak at 67.8 °C, and the melting curve of an Ala/Val genotype showed two peaks at 61.5 and 67.8 °C.