Peptide name : Glutathione S-transferase P

Source/Organism : Human

Linear/Cyclic : Not found

Chirality : Not found

Peptide length: 210

C-terminal modification: Not found

N-terminal modification : Not found

Non-natural peptide information: None

Assay type : Antibody-based assay

Assay time : 48h

Activity : Not found

Cell line : U937

Cancer type : Not specified

Other activity : Anti-apoptotic activity

Amino acid composition bar chart :

Molecular mass : 23355.5457 Dalton

Aliphatic index : 0.989

Instability index : 32.5952

Hydrophobicity (GRAVY) : -0.121

Isoelectric point : 5.4334

Charge (pH 7) : -3.3463

Aromaticity : 0.1

Molar extinction coefficient (cysteine, cystine): (28880, 29130)

Hydrophobic/hydrophilic ratio : 1.16494845

hydrophobic moment : 0.1058

Missing amino acid : None

Most occurring amino acid : L

Most occurring amino acid frequency : 32

Least occurring amino acid : W

Least occurring amino acid frequency : 2

Secondary structure fraction (Helix, Turn, Sheet): (0.3, 0.2, 0.4)

SMILES Notation: CC[C@H](C)[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(=O)O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CS)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(=N)N)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@@H](NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)[C@H](CCCNC(=N)N)NC(=O)CNC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](NC(=O)[C@@H]1CCCN1C(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H]1CCCN1C(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCSC)[C@@H](C)O)C(C)C)C(C)C)C(C)C)C(C)C)C(C)C)[C@@H](C)O)C(C)C)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)O)C(C)C)C(C)C)C(C)C)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)C(C)C)[C@@H](C)O)[C@@H](C)O)[C@@H](C)CC)C(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)O)[C@@H](C)CC)C(C)C)C(C)C)C(C)C)[C@@H](C)CC

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Paper title : Primary and secondary structural analyses of glutathione S-transferase pi from human placenta.

Doi : https://doi.org/10.1016/0003-9861(90)90277-6

Abstract : The primary structure of glutathione S-transferase (GST) pi from a single human placenta was determined. The structure was established by chemical characterization of tryptic and cyanogen bromide peptides as well as automated sequence analysis of the intact enzyme. The structural analysis indicated that the protein is comprised of 209 amino acid residues and gave no evidence of post-translational modifications. The amino acid sequence differed from that of the deduced amino acid sequence determined by nucleotide sequence analysis of a cDNA clone (Kano, T., Sakai, M., and Muramatsu, M., 1987, Cancer Res. 47, 5626-5630) at position 104 which contained both valine and isoleucine whereas the deduced sequence from nucleotide sequence analysis identified only isoleucine at this position. These results demonstrated that in the one individual placenta studied at least two GST pi genes are coexpressed, probably as a result of allelomorphism. Computer assisted consensus sequence evaluation identified a hydrophobic region in GST pi (residues 155-181) that was predicted to be either a buried transmembrane helical region or a signal sequence region. The significance of this hydrophobic region was interpreted in relation to the mode of action of the enzyme especially in regard to the potential involvement of a histidine in the active site mechanism. A comparison of the chemical similarity of five known human GST complete enzyme structures, one of pi, one of mu, two of alpha, and one microsomal, gave evidence that all five enzymes have evolved by a divergent evolutionary process after gene duplication, with the microsomal enzyme representing the most divergent form.

Paper title : Structure and expression of a human class pi glutathione S-transferase messenger RNA.

Doi : https://doi.org/Not available

Abstract : We have used a rat glutathione S-transferase P (GST-P) complementary DNA as a probe to screen a human placenta complementary DNA library constructed in the lambda gt11 vector. One of the positive clones contained the complete coding region (630 base pair) and the entire 3'-noncoding region (78 base pair) of the putative human glutathione S-transferase pi (GST-pi) subunit mRNA. From the nucleotide sequence we deduced the complete amino acid sequence of the GST-pi subunit. It contained 209 amino acids with the relative molecular mass of Mr 23,224. Comparison of the amino acid sequences between GST-pi and GST-P subunits suggests that they are the corresponding enzymes in these species. GST-pi and GST-P both consist of 209 amino acids and differ in only 30 amino acids (85.6% homology). The difference in amino acid composition can explain the large difference in isoelectric point between GST-pi subunit (pI 5.5) and GST-P subunit (pI 6.9). The expression of GST-pi mRNA in some normal and cancerous tissues, including some hepatoma cell lines, hepatoma, and colon carcinoma specimens was determined using complementary DNA as a probe. The results indicate that the mode of the expression of GST-pi in humans is different from that of GST-P in rats.

Paper title : Expression of anionic glutathione-S-transferase and P-glycoprotein genes in human tissues and tumors.

Doi : https://doi.org/Not available

Abstract : The development of multidrug resistance in MCF-7 human breast cancer cells and the acquisition of broad resistance to xenobiotics in rat hyperplastic nodules are both associated with increased P-glycoprotein (mdr) gene expression as well as changes in activities of intracellular detoxication enzymes; among these changes is a significant increase in the activity of the anionic isozyme of glutathione-S-transferase (GST). We have isolated a cDNA encoding the human anionic glutathione-S-transferase, GST pi-1, from a cDNA library constructed from multidrug-resistant MCF-7 cells. The deduced amino acid sequence of GST pi-1 shows that while the human anionic GST displays 85% nucleotide and amino acid sequence homology to the rat anionic isozyme, it is markedly less related to human basic GST isozymes. We have examined the expression of GST pi and P-glycoprotein in 170 specimens of human tissues and tumors. P-Glycoprotein RNA expression was positive in eight of 23 lymphomas and two of 12 colon tumors; however, many other normal and malignant tissues, including lung, bladder, and breast tumors, had low or undetectable levels of P-glycoprotein RNA expression. In contrast, GST pi was readily detected in a wide variety of normal and malignant tissues. The level of GST pi mRNA expression in normal tissues was heterogeneous, with lowest levels found in liver and the highest levels found in lung, esophagus, and placenta. GST pi was also variably expressed in human tumors, with the lowest relative levels occurring in lymphoma and breast cancer and the highest levels found in lung cancer and head and neck tumors. In addition, comparison of paired specimens from the same patient indicated that GST pi expression was increased in many tumors relative to matched normal tissue.

Paper title : A two-dimensional gel database of human colon carcinoma proteins.

Doi : https://doi.org/10.1002/elps.1150180344

Abstract : The master two-dimensional gel database of human colon carcinoma cells currently lists cellular proteins from normal crypts and the colorectal cancer cell lines LIM 1863, LIM 1215 and LIM 1899 (Ward et al., Electrophoresis 1990, 11, 883-891; Ji et al., Electrophoresis 1994, 15, 391-405). Updated two-dimensional electrophoretic (2-DE) maps of cellular proteins from LIM 1215 cells, acquired under both nonreducing and reducing conditions, are presented. Fifteen cellular proteins are identified in the reducing 2-DE gel map, and seven in the nonreducing gel map, along with a tabular listing of their M(r)/pI loci and mode of identification. We also include our mass spectrometric based procedures for identifying 2-DE resolved proteins. This procedure relies on a combination of capillary column (0.10-0.32 mm internal diameter) reversed-phase HPLC peptide mapping of in-gel digested proteins, peptide mass fingerprinting, sequence analysis by either collision-induced dissociation or post-source-decay fragmentation, and protein identification using available database search algorithms. These data, and descriptions of the micro-techniques employed in this laboratory for identifying 2-DE resolved proteins can be accessed via the internet URL: http:(/)/www.ludwig.edu.au.

Paper title : Structure and function of residue 104 and water molecules in the xenobiotic substrate-binding site in human glutathione S-transferase P1-1.

Doi : https://doi.org/10.1021/bi990668u

Abstract : Two variants of human class pi glutathione (GSH) S-transferase 1-1 with either isoleucine or valine in position 104 (hGSTP1-1[I104] and hGSTP1-1[V104]) have distinct activity toward (+)-anti-7, 8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]. To elucidate their structure-function relationship, we determined the crystal structures of the two variants in complex with GSBpd, the GSH conjugate of (+)-anti-BPDE, at 2.1 and 2.0 A resolution, respectively. The crystal structures reveal that residue 104 in the xenobiotic substrate-binding site (H-site) dictates the binding modes of the product molecule GSBpd with the following three consequences. First, the distance between the hydroxyl group of Y7 and the sulfur atom of GSBpd is 5.9 A in the hGSTP1-1[I104].GSBpd complex versus 3.2 A in the V104 variant. Second, one of the hydroxyl groups of GSBpd forms a direct hydrogen bond with R13 in hGSTP1-1[V104].GSBpd; in contrast, this hydrogen bond is not observed in the I104 complex. Third, in the hydrophilic portion of the H-site of the I104 complex, five H-site water molecules [Ji, X., et al. (1997) Biochemistry 36, 9690-9702] are observed, whereas in the V104 complex, two of the five have been displaced by the Bpd moiety of GSBpd. Although there is no direct hydrogen bond between Y108 (OH) and the hydroxyl groups of GSBpd, indirect hydrogen bonds mediated by water molecules are observed in both complexes, supporting the previously suggested role of the hydroxyl group of Y108 as an electrophilic participant in the addition of GSH to epoxides.

Paper title : Stereoselective conjugation of prostaglandin A2 and prostaglandin J2 with glutathione, catalyzed by the human glutathione S-transferases A1-1, A2-2, M1a-1a, and P1-1.

Doi : https://doi.org/10.1021/tx9601770

Abstract : Prostaglandins containing an alpha,beta-unsaturated keto group, such as prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2), inhibit cell proliferation. These cyclopentenone prostaglandins may be conjugated with GSH chemically or enzymatically via glutathione S-transferases, and this has been suggested to result in inhibition of the antiproliferative mode of action. In the present study, the role of the major human GSTs in the conjugation of PGA2 and PGJ2 with GSH was investigated with purified enzymes, i.e., the Alpha-class enzymes GST A1-1 and GST A2-2, the Mu-class enzyme GST M1a-1a, and the Pi-class enzyme GST P1-1. The GSH conjugates were separated from the parent compound by HPLC and identified by fast atom bombardment mass spectrometry and 1H-NMR. Two GSH conjugates were found for both PGA2 and PGJ2, the R- and S-GSH conjugates of both prostaglandins. Incubation experiments with PGA2 and PGJ2 (70-600 microM) clearly showed the role of individual GSTs in the conjugation of PGA2 and PGJ2. Compared to the chemical reaction, enzyme activities towards PGA2 were up to 5.4 times as high (GSTA1-1) at the lowest concentration (70 microM), while at the highest concentration (600 microM) enzyme activities were up to 3.0 times as high (GST P1-1). For PGJ2, enzyme activities were up to 4.3 (GSTM1a-1a, 70 microM) and up to 3.1 (GSTM1a-1a, 600 microM) times as high. As expected, similar amounts of the R- and S-conjugates of both prostaglandins were found in the chemical reaction. Striking stereoselectivities in conjugating activities were observed for GST A1-1 and GST P1-1. GST A1-1 favors the formation of the R-GSH conjugates of both prostaglandins. GST P1-1 showed a clear selectivity with regard to the formation of the S-GSH conjugate of PGA2. However, this selectivity was not found for the formation of the S-GSH conjugate of PGJ2. GSTM1a-1a showed no stereoselectivity with regard to the GSH conjugation of both PGA2 and PGJ2. GSTA2-2 only showed some minor formation of the R-GSH conjugate of PGJ2. The possible implications of the observed stereoselectivity on the effects of PGA2 and PGJ2 are discussed.

Paper title : Purification and characterization of unique glutathione S-transferases from human muscle.

Doi : https://doi.org/10.1016/0003-9861(88)90564-4

Abstract : Results of studies designed to investigate the origin of the diversity of glutathione S-transferase (GST) isozymes in human tissues indicated that human muscle has at least three forms of GST with pI values of 5.0, 5.1, and 5.2 that are distinct from GST isozymes characterized so far. The major muscle isozyme which was expressed in all the six samples analyzed in this study was a unique GST of pI 5.2 that was designated as GST zeta. It had a blocked N-terminal and did not correspond to any of the known three classes (alpha, mu, or pi) of human GST as evidenced by its immunological properties and substrate specificities. The N-terminal regions of human muscle GST 5.1 and 5.0 had identical amino acid sequences except at residue 5, but demonstrated significant differences in amino acid composition and substrate specificities. These two isozymes showed homology with the mu class of human GST in their N-terminal region and were also immunologically related to the mu class of human GST although their subunit molecular weight values (Mr 23,000) were lower than that reported for GST psi. The substrate specificities of these isozymes were also significantly different from those of other human GST isozymes characterized so far. Significantly, muscle tissue did not express the alpha class of GST isozymes; however, two other isozymes were identified, GST 4.8 and GST 4.5, which had identical N-terminal amino acid sequences that were similar to that reported for the pi class of human GST. GST 4.8 was present in all six samples analyzed in this study whereas GST 4.5 was present in only two of these samples, indicating a possibility of polymorphism at the GST3 locus. This study indicated the occurrence of at least three distinct isozymes in muscle tissue, providing further evidence for tissue specific expression of GST isozymes in humans.

Paper title : Structure of the human genomic glutathione S-transferase-pi gene.

Doi : https://doi.org/10.1016/0378-1119(89)90377-6

Abstract : The complete human genomic glutathione-S-transferase-pi gene (GST-pi) was isolated from a lambda Charon 4A bacteriophage library which was screened by hybridization to a human GST-pi cDNA. We have sequenced 4261 bp which include the entire GST-pi gene as well as over 1200 bp of the 5' and 200 bp of the 3' flanking regions. The GST-pi gene has 7 exons and 6 introns contained within approximately 2.8 kilobases. Primer extension experiments identified four possible transcription start points closely spaced between 29 and 33 nucleotides (nt) 5' to the start of translation. Analysis of the GST-pi promoter region revealed 4 putative transcription regulatory motifs; these sequences include a 'TATA' box 29 bp upstream from the major transcription start point (nt position -29), 2 Sp1 recognition sequences (GGGCGG, nt positions -46 to -41 and -56 to -51), and an AP-1 recognition sequence (TGACTCA, nt positions -69 to -63). The first 200 nt 5' to the start point of transcription contain a G + C-rich region (79%). Additionally, an intriguing A + T-rich region was found between nt positions -505 and -413 which contained 17 AAAAT tandem repeats. Comparison of the GST-pi gene with the homologous rate gene, GST-P, disclosed extensive conservation of genomic organization between the two species.

Paper title : Tyrosine-7 in human class Pi glutathione S-transferase is important for lowering the pKa of the thiol group of glutathione in the enzyme-glutathione complex.

Doi : https://doi.org/10.1016/0006-291x(92)91177-r

Abstract : Previously, we reported the importance of Tyr7 for the catalytic activity of human class Pi glutathione S-transferase [Kong et al. (1992) Biochem. Biophys. Res. Comm., 182, 1122]. As an extension of this study, we investigated the pH dependence of kinetic parameters of the wild-type enzyme and the Y7F mutant. The replacement of Tyr7 with phenylalanine was found to alter the pH dependence of Vmax and Vmax/KmCDNB of the enzyme for conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB). The pKa of the thiol of GSH in the wild-type enzyme-GSH complex was estimated to be about 2.4 pK units lower than that in the Y7F-GSH complex. Tyr7 is thus considered to be important for catalytic activity in lowering the pKa of the thiol of GSH in the enzyme-GSH complex.

Paper title : The structure of the human glutathione S-transferase pi gene.

Doi : https://doi.org/10.1042/bj2550079

Abstract : The human glutathione S-transferase pi gene has been isolated from a cosmid library. The gene spans approximately 3 kb, is interrupted by six introns and the region around its 5' end has the high G + C and CpG content typical of an HTF (HpaII tiny fragment) island. In addition to a TATA box at position -28 relative to the transcription initiation site and two G + C boxes (GGGCGG), the promoter contains a sequence motif matching the phorbol ester- and ras-responsive element from the polyoma virus enhancer.

Paper title : Structures of class pi glutathione S-transferase from human placenta in complex with substrate, transition-state analogue and inhibitor.

Doi : https://doi.org/10.1016/s0969-2126(97)00281-5

Abstract : BACKGROUND: Glutathione S-transferases (GSTs) are detoxification enzymes, found in all aerobic organisms, which catalyse the conjugation of glutathione with a wide range of hydrophobic electrophilic substrates, thereby protecting the cell from serious damage caused by electrophilic compounds. GSTs are classified into five distinct classes (alpha, mu, pi, sigma and theta) by their substrate specificity and primary structure. Human GSTs are of interest because tumour cells show increased levels of expression of single classes of GSTs, which leads to drug resistance. Structural differences between classes of GST can therefore be utilised to develop new anti-cancer drugs. Many mutational and structural studies have been carried out on the mu and alpha classes of GST to elucidate the reaction mechanism, whereas knowledge about the pi class is still limited. RESULTS: We have solved the structures of the pi class GST hP1-1 in complex with its substrate, glutathione, a transition-state complex, the Meisenheimer complex, and an inhibitor, S-(rho-bromobenzyl)-glutathione, and refined them to resolutions of 1.8 A, 2.0 A and 1.9 A, respectively. All ligand molecules are well-defined in the electron density. In all three structures, an additionally bound N-morpholino-ethansulfonic acid molecule from the buffer solution was found. CONCLUSIONS: In the structure of the GST-glutathione complex, two conserved water molecules are observed, one of which hydrogen bonds directly to the sulphur atom of glutathione and the other forms hydrogen bonds with residues around the glutathione-binding site. These water molecules are absent from the structure of the Meisenheimer complex bound to GST, implicating that deprotonation of the cysteine occurs during formation of the ternary complex which involves expulsion of the inner bound water molecule. The comparison of our structures with known mu class GST structures show differences in the location of the electrophile-binding site (H-site), explaining the different substrate specificities of the two classes. Fluorescence measurements are in agreement with the position of the N-morpholino-ethansulfonic acid, close to Trp28, identifying a possible ligandin-substrate binding site.

Paper title : Comparative large scale characterization of plant versus mammal proteins reveals similar and idiosyncratic N-α-acetylation features.

Doi : https://doi.org/10.1074/mcp.M111.015131

Abstract : N-terminal modifications play a major role in the fate of proteins in terms of activity, stability, or subcellular compartmentalization. Such modifications remain poorly described and badly characterized in proteomic studies, and only a few comparison studies among organisms have been made available so far. Recent advances in the field now allow the enrichment and selection of N-terminal peptides in the course of proteome-wide mass spectrometry analyses. These targeted approaches unravel as a result the extent and nature of the protein N-terminal modifications. Here, we aimed at studying such modifications in the model plant Arabidopsis thaliana to compare these results with those obtained from a human sample analyzed in parallel. We applied large scale analysis to compile robust conclusions on both data sets. Our data show strong convergence of the characterized modifications especially for protein N-terminal methionine excision, co-translational N-α-acetylation, or N-myristoylation between animal and plant kingdoms. Because of the convergence of both the substrates and the N-α-acetylation machinery, it was possible to identify the N-acetyltransferases involved in such modifications for a small number of model plants. Finally, a high proportion of nuclear-encoded chloroplast proteins feature post-translational N-α-acetylation of the mature protein after removal of the transit peptide. Unlike animals, plants feature in a dedicated pathway for post-translational acetylation of organelle-targeted proteins. The corresponding machinery is yet to be discovered.

Paper title : Biosynthesis of 14,15-hepoxilins in human l1236 Hodgkin lymphoma cells and eosinophils.

Doi : https://doi.org/10.1007/s11745-010-3485-1

Abstract : Hepoxilins are epoxy alcohols synthesized through the 12-lipoxygenase (12-LO) pathway in animal cells. The epidermis is the principal source of hepoxilins in humans. Here we report on the formation of novel hepoxilin regioisomers formed by the 15-LO pathway in human cells. The Hodgkin lymphoma cell line L1236 possesses high 15-lipoxygenase-1 (15-LO-1) activity and incubation of L1236 cells with arachidonic acid led to the formation of 11(S)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),12(E) eicosatrienoic acid (14,15-HxA(3) 11(S)) and 13(R)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),11(Z) eicosatrienoic acid (14,15-HxB(3) 13(R)). In addition, two hitherto unidentified products were detected and these products were collected and analyzed by positive ion electrospray tandem mass spectrometry. These metabolites were identified as 11(S),15(S)-dihydroxy-14(R)-glutathionyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA(3)-C) and 11(S),15(S)-dihydroxy-14(R)-cysteinyl-glycyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA(3)-D). Incubation of L1236 cells with synthetic 14,15-HxA(3) 11(S) also led to the formation of 14,15-HxA(3)-C and 14,15-HxA(3)-D. Several soluble glutathione transferases, in particular GST M1-1 and GST P1-1, were found to catalyze the conversion of 14,15-HxA(3) to 14,15-HxA(3)-C. L1236 cells produced approximately twice as much eoxins as cysteinyl-containing hepoxilins upon stimulation with arachidonic acid. Human eosinophils, nasal polyps and dendritic cells selectively formed 14,15-HxA(3) 11(S) and 14,15-HxB(3) 13(R) stereoisomers, but not cysteinyl-containing hepoxilins, after stimulation with arachidonic acid. Furthermore, purified recombinant 15-LO-1 alone catalyzed the conversion of arachidonic acid to 14,15-HxA(3) 11(S) and 14,15-HxB(3) 13(R), showing that human 15-LO-1 possesses intrinsic 14,15-hepoxilin synthase activity.

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

Doi : https://doi.org/10.1021/bi971902o

Abstract : 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.

Paper title : Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

Doi : https://doi.org/10.1038/nbt810

Abstract : Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

Paper title : Structural evidence for three different types of glutathione transferase in human tissues.

Doi : https://doi.org/10.1016/0014-5793(85)80324-0

Abstract : Cytosolic glutathione transferase was purified from human placenta and human liver. Three different forms of the enzyme were obtained, the acidic (pi), the near-neutral (mu), and the basic (alpha-epsilon) forms; two had free alpha-amino groups (pi, mu) and one had a blocked alpha-amino group (alpha-epsilon). N-terminal sequence analyses and total compositions gave clearly different results for each form, although transferases pi and mu showed 35% sequence homology in the N-terminal regions, with a 1-residue shift in starting position. Consequently, the proteins are concluded to be products of three discrete but related genes.

Paper title : Increased expression of androgen receptor sensitizes prostate cancer cells to low levels of androgens.

Doi : https://doi.org/10.1158/0008-5472.CAN-09-0919

Abstract : Androgen receptor (AR) is known to be overexpressed in castration-resistant prostate cancer. To interrogate the functional significance of the AR level, we established two LNCaP cell sublines expressing in a stable fashion two to four times (LNCaP-ARmo) and four to six times (LNCaP-ARhi) higher level of AR than the parental cell line expressing the empty vector (LNCaP-pcDNA3.1). LNCaP-ARhi cell line grew faster than the control line in low concentrations, especially in 1 nmol/L 5alpha-dihydrotestosterone (DHT). Microarray-based transcript profiling and subsequent unsupervised hierarchical clustering showed that LNCaP-ARhi cells clustered together with VCaP cells, containing endogenous AR gene amplification and overexpression, indicating the central role of AR in the overall regulation of gene expression in prostate cancer cells. Two hundred forty genes showed >2-fold changes on DHT treatment in LNCaP-ARhi at 4 h time point, whereas only 164 and 52 showed changes in LNCaP-ARmo and LNCaP-pcDNA3.1, respectively. Many androgen-regulated genes were upregulated in LNCaP-ARhi at 10-fold lower concentration of DHT than in control cells. DHT (1 nmol/L) increased expression of several cell cycle-associated genes in LNCaP-ARhi cells. ChIP-on-chip assay revealed the presence of chromatin binding sites for AR within +/-200 kb of most of these genes. The growth of LNCaP-ARhi cells was also highly sensitive to cyclin-dependent kinase inhibitor, roscovitine, at 1nmol/L DHT. In conclusion, our results show that overexpression of AR sensitizes castration-resistant prostate cancer cells to the low levels of androgens. The activity of AR signaling pathway is regulated by the levels of both ligand and the receptor.

Paper title : N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB.

Doi : https://doi.org/10.1073/pnas.1210303109

Abstract : Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

Paper title : Rational design of an organometallic glutathione transferase inhibitor.

Doi : https://doi.org/10.1002/anie.200900185

Abstract : Double trouble: A hybrid organic-inorganic (organometallic) inhibitor was designed to target glutathione transferases. The metal center is used to direct protein binding, while the organic moiety acts as the active-site inhibitor (see picture). The mechanism of inhibition was studied using a range of biophysical and biochemical methods.

Paper title : N-terminome analysis of the human mitochondrial proteome.

Doi : https://doi.org/10.1002/pmic.201400617

Abstract : The high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and proteogenomics fields. The present study describes the free N-terminome analysis of human mitochondria-enriched samples using trimethoxyphenyl phosphonium (TMPP) labelling approaches. Owing to the extent of protein import and cleavage for mitochondrial proteins, determining the new N-termini generated after translocation/processing events for mitochondrial proteins is crucial to understand the transformation of precursors to mature proteins. The doublet N-terminal oriented proteomics (dN-TOP) strategy based on a double light/heavy TMPP labelling has been optimized in order to improve and automate the workflow for efficient, fast and reliable high throughput N-terminome analysis. A total of 2714 proteins were identified and 897 N-terminal peptides were characterized (424 N-α-acetylated and 473 TMPP-labelled peptides). These results allowed the precise identification of the N-terminus of 693 unique proteins corresponding to 26% of all identified proteins. Overall, 120 already annotated processing cleavage sites were confirmed while 302 new cleavage sites were characterized. The accumulation of experimental evidence of mature N-termini should allow increasing the knowledge of processing mechanisms and consequently also enhance cleavage sites prediction algorithms. Complete datasets have been deposited to the ProteomeXchange Consortium with identifiers PXD001521, PXD001522 and PXD001523 (http://proteomecentral.proteomexchange.org/dataset/PXD001521, http://proteomecentral.proteomexchange.org/dataset/PXD0001522 and http://proteomecentral.proteomexchange.org/dataset/PXD001523, respectively).

Paper title : Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 A resolution.

Doi : https://doi.org/10.1016/0022-2836(92)90692-d

Abstract : The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitor's glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.

Paper title : Structure and function of the xenobiotic substrate-binding site and location of a potential non-substrate-binding site in a class pi glutathione S-transferase.

Doi : https://doi.org/10.1021/bi970805s

Abstract : Complex structures of a naturally occurring variant of human class pi glutathione S-transferase 1-1 (hGSTP1-1) with either S-hexylglutathione or (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9, 10-dihydrophenanthrene [(9R,10R)-GSPhen] have been determined at resolutions of 1.8 and 1.9 A, respectively. The crystal structures reveal that the xenobiotic substrate-binding site (H-site) is located at a position similar to that observed in class mu GST 1-1 from rat liver (rGSTM1-1). In rGSTM1-1, the H-site is a hydrophobic cavity defined by the side chains of Y6, W7, V9, L12, I111, Y115, F208, and S209. In hGSTP1-1, the cavity is approximately half hydrophobic and half hydrophilic and is defined by the side chains of Y7, F8, V10, R13, V104, Y108, N204, and G205 and five water molecules. A hydrogen bond network connects the five water molecules and the side chains of R13 and N204. V104 is positioned such that the introduction of a methyl group (the result of the V104I mutation) disturbs the H-site water structure and alters the substrate-binding properties of the isozyme. The hydroxyl group of Y7 forms a hydrogen bond (3.2 A) with the sulfur atom of the product. There is a short hydrogen bond (2.5 A) between Y108 (OH) and (9R, 10R)-GSPhen (O5), indicating the hydroxyl group of Y108 as an electrophilic participant in the addition of glutathione to epoxides. An N-(2-hydroxethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) molecule is found in the cavity between beta2 and alphaI. The location and properties of this HEPES-binding site fit a possible non-substrate-binding site that is involved in noncompetitive inhibition of the enzyme.

Paper title : The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

Doi : https://doi.org/10.1101/gr.2596504

Abstract : The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

Paper title : The three-dimensional structure of the human Pi class glutathione transferase P1-1 in complex with the inhibitor ethacrynic acid and its glutathione conjugate.

Doi : https://doi.org/10.1021/bi962316i

Abstract : The potent diuretic drug ethacrynic acid has been tested in clinical trials as an adjuvant in chemotherapy. Its target is the detoxifying enzyme glutathione transferase which is often found overexpressed in cancer tissues. We have solved the crystal structures of human pi class glutathione transferase P1-1 in complex with the inhibitor ethacrynic acid and its glutathione conjugate. Ethacrynic acid is found to bind in a nonproductive mode to one of the ligand binding sites of the enzyme (the H site) while the glutathione binding site (G site) is occupied by solvent molecules. There are no structural rearrangements of the G site in the absence of ligand. The structure indicates that bound glutathione is required for ethacrynic acid to dock into the H site in a productive binding mode. The binding of the ethacrynic acid-glutathione conjugate shows that the contacts of the glutathione moiety with the protein are identical to those observed in crystal structures of the enzyme with other glutathione-based substrates and inhibitors. The ethacrynic acid moiety of the conjugate binds in the H site in a fashion that has not been observed in crystal structures of other glutathione-based inhibitor complexes. The crystal structures implicate Tyr 108 as an electrophilic participant in the Michael addition of glutathione to ethacrynic acid.

Paper title : Initial characterization of the human central proteome.

Doi : https://doi.org/10.1186/1752-0509-5-17

Abstract : BACKGROUND: On the basis of large proteomics datasets measured from seven human cell lines we consider their intersection as an approximation of the human central proteome, which is the set of proteins ubiquitously expressed in all human cells. Composition and properties of the central proteome are investigated through bioinformatics analyses. RESULTS: We experimentally identify a central proteome comprising 1,124 proteins that are ubiquitously and abundantly expressed in human cells using state of the art mass spectrometry and protein identification bioinformatics. The main represented functions are proteostasis, primary metabolism and proliferation. We further characterize the central proteome considering gene structures, conservation, interaction networks, pathways, drug targets, and coordination of biological processes. Among other new findings, we show that the central proteome is encoded by exon-rich genes, indicating an increased regulatory flexibility through alternative splicing to adapt to multiple environments, and that the protein interaction network linking the central proteome is very efficient for synchronizing translation with other biological processes. Surprisingly, at least 10% of the central proteome has no or very limited functional annotation. CONCLUSIONS: Our data and analysis provide a new and deeper description of the human central proteome compared to previous results thereby extending and complementing our knowledge of commonly expressed human proteins. All the data are made publicly available to help other researchers who, for instance, need to compare or link focused datasets to a common background.

Paper title : Tyrosine phosphorylation of the human glutathione S-transferase P1 by epidermal growth factor receptor.

Doi : https://doi.org/10.1074/jbc.M808153200

Abstract : Epidermal growth factor receptor (EGFR) gene amplification, mutations, and/or aberrant activation are frequent abnormalities in malignant gliomas and other human cancers and have been associated with an aggressive clinical course and a poor therapeutic outcome. Elevated glutathione S-transferase P1 (GSTP1), a major drug-metabolizing and stress response signaling protein, is also associated with drug resistance and poor clinical outcome in gliomas and other cancers. Here, we provide evidence that GSTP1 is a downstream EGFR target and that EGFR binds to and phosphorylates tyrosine residues in the GSTP1 protein in vitro and in vivo. Mass spectrometry and mutagenesis analyses in a cell-free system and in gliomas cells identified Tyr-7 and Tyr-198 as major EGFR-specific phospho-acceptor residues in the GSTP1 protein. The phosphorylation increased GSTP1 enzymatic activity significantly, and computer-based modeling showed a corresponding increase in electronegativity of the GSTP1 active site. In human glioma and breast cancer cells, epidermal growth factor stimulation rapidly increased GSTP1 tyrosine phosphorylation and decreased cisplatin sensitivity. Lapatinib, a clinically active EGFR inhibitor, significantly reversed the epidermal growth factor-induced cisplatin resistance. These data define phosphorylation and activation of GSTP1 by EGFR as a novel, heretofore unrecognized component of the EGFR signaling network and a novel mechanism of tumor drug resistance, particularly in tumors with elevated GSTP1 and/or activated EGFR.

Paper title : Site-directed mutagenesis study on the roles of evolutionally conserved aspartic acid residues in human glutathione S-transferase P1-1.

Doi : https://doi.org/10.1093/protein/6.1.93

Abstract : The evolutionally conserved aspartyl residues (Asp57, Asp98 and Asp152) in human glutathione S-transferase P1-1 were replaced with alanine by site-directed mutagenesis to obtain the mutants (D57A, D98A and D152A). The replacement of Asp98 with alanine resulted in a decrease of the affinity for S-hexyl-GSH-agarose, a 5.5-fold increase of the KmGSH and a 2.9-fold increase of the I50 of S-hexyl-GSH for GSH-CDNB conjugation. Asp98 seems to participate in the binding of GSH through hydrogen bonding with the alpha-carboxylate of the gamma-glutamyl residue of GSH. The kcat of D98A was 2.6-fold smaller than that of the wild-type, and the pKa of the thiol group of GSH bound in D98A was approximately 0.8 pK units higher than those in the wild-type. Asp98 also seems to contribute to the activation of GSH to some extent. On the other hand, most of the kinetic parameters of D57A and D152A were similar to those of the wild-type. However, the thermostabilities of D57A and D152A were significantly lower than that of the wild-type. Asp57 and Asp152 seem to be important for maintaining the proper conformation of the enzyme.

Paper title : Glutathione S-transferase pi localizes in mitochondria and protects against oxidative stress.

Doi : https://doi.org/10.1016/j.freeradbiomed.2009.02.025

Abstract : Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the protection of cellular components against anti-cancer drugs or peroxidative stress. Previously we found that GST pi, an isoform of the GSTs, is transported into the nucleus. In the present study, we found that GST pi is present in mitochondria as well as in the cytosol and nucleus in mammalian cell lines. A construct comprising the 84 amino acid residues in the amino-terminal region of GST pi and green fluorescent protein was detected in the mitochondria. The mutation of arginine to alanine at positions 12, 14, 19, 71, and 75 in full-length GST pi completely abrogated the ability to distribute in the mitochondria, suggesting that arginine, a positively charged residue, is required for the mitochondrial transport of GST pi. Chemicals generating reactive oxygen species, such as rotenone and antimycin A, decreased cell viability and reduced mitochondrial membrane potential. The overexpression of GST pi diminished these changes. GST pi-targeting siRNA abolished the protective effect of GST pi on the mitochondria under oxidative stress. The findings indicate that the peptide signal is conducive to the mitochondrial localization of GST pi under steady-state conditions without alternative splicing or posttranslational modifications such as proteolysis, suggesting that GST pi protects mitochondria against oxidative stress.

Paper title : The structures of human glutathione transferase P1-1 in complex with glutathione and various inhibitors at high resolution.

Doi : https://doi.org/10.1006/jmbi.1997.1364

Abstract : The human pi-class glutathione S-transferase (hGST P1-1) is a target for structure-based inhibitor design with the aim of developing drugs that could be used as adjuvants in chemotherapeutic treatment. Here we present seven crystal structures of the enzyme in complex with substrate (glutathione) and two inhibitors (S-hexyl glutathione and gamma-glutamyl- (S-benzyl)cysteinyl-D-phenylglycine). The binding of the modified glutathione inhibitor, gamma-glutamyl-(S-benzyl)cysteinyl-D-phenylglycine, has been characterized with the phenyl group stacking against the benzyl moiety of the inhibitor and making interactions with the active-site residues Phe8 and Trp38. The structure provides an explanation as to why this compound inhibits the pi-class GST much better than the other GST classes. The structure of the enzyme in complex with glutathione has been determined to high resolution (1.9 to 2.2 A) in three different crystal forms and at two different temperatures (100 and 288 K). In one crystal form, the direct hydrogen-bonding interaction between the hydroxyl group of Tyr7, a residue involved in catalysis, and the thiol group of the substrate, glutathione, is broken and replaced by a water molecule that mediates the interaction. The hydrogen-bonding partner of the hydroxyl group of Tyr108, another residue implicated in the catalysis, is space-group dependent. A high-resolution (2.0 A) structure of the enzyme in complex with S-hexyl glutathione in a new crystal form is presented. The enzyme-inhibitor complexes show that the binding of ligand into the electrophilic binding site does not lead to any conformational changes of the protein.

Paper title : Tyrosine-7 is an essential residue for the catalytic activity of human class PI glutathione S-transferase: chemical modification and site-directed mutagenesis studies.

Doi : https://doi.org/10.1016/0006-291x(92)91848-k

Abstract : The glutathione (GSH)-conjugating activity of human class Pi glutathione S-transferase (GST pi) toward 1-chloro-2,4-dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an O-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GST pi, which is conserved in all cytosolic GSTs, with phenylalanine by site-directed mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The Km values of the mutant for both GSH and CDNB were almost equivalent to those of the wild type, while the Vmax of the former was about 55-fold smaller than that of the latter. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GST pi.

Paper title : Lysine acetylation targets protein complexes and co-regulates major cellular functions.

Doi : https://doi.org/10.1126/science.1175371

Abstract : Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.

Paper title : Toward a comprehensive characterization of a human cancer cell phosphoproteome.

Doi : https://doi.org/10.1021/pr300630k

Abstract : Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level of phosphorylation in a cell is placed at well above 100,000 sites, there is still much room for improvement. Here, we attempt to extend the depth of phosphoproteome coverage while maintaining realistic aspirations in terms of available material, robustness, and instrument running time. We developed three strategies, where each provided a different balance between these three key parameters. The first strategy simply used enrichment by Ti(4+)-IMAC followed by reversed chromatography LC-MS (termed 1D). The second strategy incorporated an additional fractionation step through the use of HILIC (2D). Finally, a third strategy was designed employing first an SCX fractionation, followed by Ti(4+)-IMAC enrichment and additional fractionation by HILIC (3D). A preliminary evaluation was performed on the HeLa cell line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy was found to be the most sensitive but limited in comprehensivity, mainly due to issues with complexity and dynamic range. Overall, the best balance was achieved using the 2D based strategy, identifying close to 17,000 phosphopeptides with less than 1 mg of material in about 48 h. Subsequently, we confirmed the findings with the K562 cell sample. When sufficient material was available, the 3D strategy increased phosphoproteome allowing over 22,000 unique phosphopeptides to be identified. Unfortunately, the 3D strategy required more time and over 1 mg of material before it started to outperform 2D. Ultimately, combining all strategies, we were able to identify over 16,000 and nearly 24,000 unique phosphorylation sites from the cancer cell lines HeLa and K562, respectively. In summary, we demonstrate the need to carry out extensive fractionation for deep mining of the phosphoproteome and provide a guide for appropriate strategies depending on sample amount and/or analysis time.

Paper title : Molecular cloning, characterization, and expression in Escherichia coli of full-length cDNAs of three human glutathione S-transferase Pi gene variants. Evidence for differential catalytic activity of the encoded proteins.

Doi : https://doi.org/10.1074/jbc.272.15.10004

Abstract : We report the isolation of three full-length cDNAs corresponding to the mRNAs of closely related glutathione S-transferase (GST) Pi genes, designated hGSTP1*A, hGSTP1*B, and hGSTP1*C, expressed in normal cells and malignant gliomas. The variant cDNAs result from A --> G and C --> T transitions at nucleotides +313 and +341, respectively. The transitions changed codon 104 from ATC (Ile) in hGSTP1*A to GTC (Val) in hGSTP1*B and hGSTP1*C and changed codon 113 from GCG (Ala) to GTG (Val) in hGSTP1*C. Both amino changes are in the electrophile-binding active site of the GST Pi peptide. Computer modeling of the deduced crystal structures of the encoded peptides showed significant deviations in the interatomic distances of critical electrophile-binding active site amino acids as a consequence of the amino acid changes. The encoded proteins expressed in Escherichia coli and purified by GSH affinity chromatography showed a 3-fold lower Km (CDNB) and a 3-4-fold higher Kcat/Km for the hGSTP1*A encoded protein than the proteins encoded by hGSTP1*B and hGSTP1*C. Analysis of 75 cases showed the relative frequency of hGSTP1*C to be 4-fold higher in malignant gliomas than in normal tissues. These data provide conclusive molecular evidence of allelopolymorphism of the human GST Pi gene locus, resulting in active, functionally different GST Pi proteins, and should facilitate studies of the role of this gene in xenobiotic metabolism, cancer, and other human diseases.

Paper title : Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties.

Doi : https://doi.org/10.1073/pnas.82.21.7202

Abstract : The major isoenzymes of cytosolic glutathione transferase (EC 2.5.1.18) from rat, mouse, and man are shown to share structural and catalytic properties that can be used for species-independent classification. Rat, mouse, and human isoenzymes were grouped with respect to amino-terminal amino acid sequences, after correlation of seven structures analyzed in the present investigation with structures determined earlier. The isoenzymes were also characterized by substrate specificities and sensitivities to inhibitors, and the data were subjected to pattern recognition analysis. In addition, the various isoenzymes were tested for cross-reactivity by immunoprecipitation with antibodies raised against rat and human transferases. The different types of data were clearly correlated and afforded an unambiguous division of the isoenzymes into three classes named alpha, mu, and pi. Each of the three mammalian species studied contains at least one isoenzyme of each class. It is suggested that the similarities of the isoenzymes in a class reflect evolutionary relationships and that the classification applies generally.

Paper title : Glutathione-S-transferase P1 is a critical regulator of Cdk5 kinase activity.

Doi : https://doi.org/10.1111/j.1471-4159.2011.07343.x

Abstract : Cyclin dependent kinase-5 (Cdk5) activity is deregulated in Alzheimer's disease (AD) and contributes to all three hallmarks: neurotoxic β-amyloid formation, neurofibrillary tangles, and neuronal death. However, the mechanism leading to Cdk5 deregulation remains controversial. Cdk5 deregulation in AD is usually linked to the formation of p25, a proteolysis product of Cdk5 activator p35, which leads to Cdk5 mislocalization and hyperactivation. A few studies have indeed shown increased p25 levels in AD brains; however, others have refuted this observation. These contradictory findings suggest that additional factors contribute to Cdk5 deregulation. This study identified glutathione-S-transferase pi 1 (GSTP1) as a novel Cdk5 regulatory protein. We demonstrate that it is a critical determinant of Cdk5 activity in human AD brains and various cancer and neuronal cells. Increased GSTP1 levels were consistently associated with reduced Cdk5 activity. GSTP1 directly inhibits Cdk5 by dislodging p25/p35, and indirectly by eliminating oxidative stress. Cdk5 promotes and is activated by oxidative stress, thereby engaging a feedback loop which ultimately leads to cell death. Not surprisingly, GSTP1 transduction conferred a high degree of neuroprotection under neurotoxic conditions. Given the critical role of oxidative stress in AD pathogenesis, an increase in GSTP1 level may be an alternative way to modulate Cdk5 signaling, eliminate oxidative stress, and prevent neurodegeneration.

Paper title : An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome.

Doi : https://doi.org/10.1016/j.jprot.2013.11.014

Abstract : UNLABELLED: Protein phosphorylation is one of the most common post-translational modifications. It plays key roles in regulating diverse biological processes of liver tissues. To better understand the role of protein phosphorylation in liver functions, it is essential to perform in-depth phosphoproteome analysis of human liver. Here, an enzyme assisted reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach with both RPLC separations operated with optimized acidic mobile phase was developed. High orthogonal separation was achieved by trypsin digestion of the Glu-C generated peptides in the fractions collected from the first RPLC separation. The phosphoproteome coverage was further improved by using two types of instruments, i.e. TripleTOF 5600 and LTQ Orbitrap Velos. A total of 22,446 phosphorylation sites, corresponding to 6526 nonredundant phosphoproteins were finally identified from normal human liver tissues. Of these sites, 15,229 sites were confidently localized with Ascore≥13. This dataset was the largest phosphoproteome dataset of human liver. It can be a public resource for the liver research community and holds promise for further biology studies. BIOLOGICAL SIGNIFICANCE: The enzyme assisted approach enabled the two RPLC separations operated both with optimized acidic mobile phases. The identifications from TripleTOF 5600 and Orbitrap Velos are highly complementary. The largest phosphoproteome dataset of human liver was generated.