A CRAF/glutathione-S-transferase P1 complex sustains autocrine growth of cancers with KRAS and BRAF mutations.

The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutant KRAS (mKRAS) and BRAF (mBRAF). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of mKRAS and mBRAF cancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of mKRAS and mBRAF cells in vitro and suppressed tumorigenesis of the xenografted mKRAS tumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of mKRAS colon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers.

Drug- and Drug Abuse-Associated Hyperbilirubinemia: Experience With Atazanavir.

Hyperbilirubinemia is a common finding in individuals with a history of substance abuse. Although this may indicate a serious disorder of liver function, this is not always the case. An understanding of bilirubin formation, metabolism, and transport can provide a helpful approach to dealing with these patients. This is typified by studies of patients treated with the antiretroviral drug atazanavir. Atazanavir has been associated with hyperbilirubinemia in as many as one-third of individuals for whom it has been prescribed, evoking concerns of hepatotoxicity. The studies in this report were designed to determine mechanisms by which this occurs. The data show that this drug inhibits the enzyme UDP-glucuronosyl transferase-1A1, responsible for conjugating bilirubin with glucuronic acid. This conjugation step is required for bilirubin excretion into bile, and when it is inhibited, bilirubin refluxes from the liver into the circulation, causing unconjugated hyperbilirubinemia. Other parameters of bilirubin formation, binding to albumin in the circulation, uptake into hepatocytes, and intracellular protein binding in hepatocytes were unaffected by atazanavir. The effect of atazanavir on serum bilirubin levels is reversible, consistent with lack of structural damage to the liver.

Reaction mechanisms of allicin and allyl-mixed disulfides with proteins and small thiol molecules.

Allylsulfides from garlic are chemopreventive agents. Entering cells they are expected to initially interact with glutathione. Accordingly, reaction mechanisms of the product, S-allylthio-glutathione, with model proteins and thiols were analyzed in cell free systems. With glutathionyl, cysteinyl or captopril representing S-allyl aliphatic adducts, the reaction with sulfhydryl groups resulted in mixed disulfide mixtures, formed by both, S-allyl and aliphatic moieties. To improve conventional prodrug treatment of blood pressure, cancer and intestinal inflammation S-allylthio prodrugs, such as S-allylthio-6-mercaptopurine and S-allylthio-captopril were synthesized. Synergistic activities of the 2 constituents, as well as increased cell permeability allow for efficient in vivo activity. Upon reaction of these derivatives with glutathione, S-allylthio-glutathione is formed, while 6-mercaptopurine is the leaving group. Excess cellular glutathione enables several cycles of sulfhydryl-disulfide exchange reactions to occur, extending the hybrid drug's pharmacodynamics.

The proximal promoter governs germ cell-specific expression of the mouse glutathione transferase mGstm5 gene.

To explain the tissue-selective expression patterns of a distinct subclass of glutathione S-transferase (GST), transgenic mice expressing EGFP under control of a 2 kb promoter sequence in the 5'-flanking region of the mGstm5 gene were produced. The intent of the study was to establish whether the promoter itself or whether posttranscriptional mechanisms, particularly at the levels of mRNA translation and stability or protein targeting, based on unique properties of mGSTM5, determine the restricted expression pattern. Indeed, the transgene expression was limited to testis as the reporter was not detected in somatic tissues such as brain, kidney or liver, indicating that the mGstm5 proximal promoter is sufficient to target testis-specific expression of the gene. EGFP expression was also more restricted vis-a-vis the natural mGstm5 gene and exclusively found in germ but not in somatic cells. Real-time quantitative PCR (qPCR) data were consistent with alternate transcription start sites in which the promoter region of the natural mGstm5 gene in somatic cells is part of exon 1 of the germ cell transcript. Thus, the primary transcription start site for mGstm5 is upstream of a TATA box in testis and downstream of this motif in somatic cells. The 5' flanking sequence of the mGstm5 gene imparts germ cell-specific transcription.

Transition state model and mechanism of nucleophilic aromatic substitution reactions catalyzed by human glutathione S-transferase M1a-1a.

An active site His107 residue distinguishes human glutathione S-transferase hGSTM1-1 from other mammalian Mu-class GSTs. The crystal structure of hGSTM1a-1a with bound glutathione (GSH) was solved to 1.9 A resolution, and site-directed mutagenesis supports the conclusion that a proton transfer occurs in which bound water at the catalytic site acts as a primary proton acceptor from the GSH thiol group to transfer the proton to His107. The structure of the second substrate-binding site (H-site) was determined from hGSTM1a-1a complexed with 1-glutathionyl-2,4-dinitrobenzene (GS-DNB) formed by a reaction in the crystal between GSH and 1-chloro-2,4-dinitrobenzene (CDNB). In that structure, the GSH-binding site (G-site) is occupied by the GSH moiety of the product in the same configuration as that of the enzyme-GSH complex, and the dinitrobenzene ring is anchored between the side chains of Tyr6, Leu12, His107, Met108, and Tyr115. This orientation suggested a distinct transition state that was substantiated from the structure of hGSTM1a-1a complexed with transition state analogue 1-S-(glutathionyl)-2,4,6-trinitrocyclohexadienate (Meisenheimer complex). Kinetic data for GSTM1a-1a indicate that kcat(CDNB) for the reaction is more than 3 times greater than kcat(FDNB), even though the nonenzymatic second-order rate constant is more than 50-fold greater for 1-fluoro-2,4-dinitrobenzene (FDNB), and the product is the same for both substrates. In addition, Km(FDNB) is about 20 times less than Km(CDNB). The results are consistent with a mechanism in which the formation of the transition state is rate-limiting in the nucleophilic aromatic substitution reactions. Data obtained with active-site mutants support transition states in which Tyr115, Tyr6, and His107 side chains are involved in the stabilization of the Meisenheimer complex via interactions with the ortho nitro group of CDNB or FDNB and provide insight into the means by which GSTs adapt to accommodate different substrates.

Diverse expression profiles of glutathione-S-transferase subunits in mammalian urinary bladders.

The hGSTM1 null genotype has been associated with increased susceptibility to urinary bladder cancer. However, the extent to which the GSTM1 subunit actually contributes to GST activities in mammalian urinary bladders is not clear. For adult mice, urinary bladders exhibited GST activity which was among the highest observed in the tissues tested. The mouse bladder GST activity with the 1-chloro 2,4-dinitrobenzene substrate was also more than 10-fold greater than that of rat and human bladders. A large increase in mouse bladder GST activity occurs during early development with the sharpest increase between 7 and 17 days of age. Subunit compositions of GSTs in adult mouse, human, and rat bladders are also markedly different. The mGSTM1 subunit is by far the predominant GST in mouse bladder, with increases in mGSTM1 between 7 and 17 days accounting for the sharp rise in GST activity during maturation. By contrast, Pi class GSTs predominate in both human and rat bladders. Investigators seeking to establish direct connections between susceptibility to bladder cancer and the hGSTM1 gene deletion should take into account the fact that the hGSTM1 subunit, even when present, represents a very minor fraction of the GST protein in human bladder.

Nomenclature for mammalian soluble glutathione transferases.

The nomenclature for human soluble glutathione transferases (GSTs) is extended to include new members of the GST superfamily that have been discovered, sequenced, and shown to be expressed. The GST nomenclature is based on primary structure similarities and the division of GSTs into classes of more closely related sequences. The classes are designated by the names of the Greek letters: Alpha, Mu, Pi, etc., abbreviated in Roman capitals: A, M, P, and so on. (The Greek characters should not be used.) Class members are distinguished by Arabic numerals and the native dimeric protein structures are named according to their subunit composition (e.g., GST A1-2 is the enzyme composed of subunits 1 and 2 in the Alpha class). Soluble GSTs from other mammalian species can be classified in the same manner as the human enzymes, and this chapter presents the application of the nomenclature to the rat and mouse GSTs.

A subclass of mu glutathione S-transferases selectively expressed in testis and brain.

A subclass of glutathione S-transferases (GSTs), exemplified by the human hGSTM3 and rodent GSTM5 subunits, has properties that distinguish it from other Mu class GSTs. Thus, they originate from single copy genes that are in an inverted order and, apart from the coding regions, share little sequence homology relative to the others in the Mu cluster. The genes for this M3/M5 subgroup encode for proteins that are in many ways unique, including their extended lengths with key amino acid substitutions. The M3/M5 subclass is selectively expressed in testis and brain and could function differently from the other GSTs.

Glutathione S-transferase hGSTM3 and ageing-associated neurodegeneration: relationship to Alzheimer's disease.

Glutathione S-transferases (GSTs) are detoxification enzymes that can counter ageing-associated oxidative and chemical stresses. The transcript of a distinct subclass of human GSTs (hGSTM3) was shown by RNA blot analysis to be widely distributed in different regions of adult brain. HPLC profiles indicated that the hGSTM3 subunit was the second most abundant GST subunit in brain. Immunocytochemistry performed with hGSTM3-specific antisera, showed prominent staining of neuritic plaques, neurofibrillary tangles and microglia in sections of hippocampus obtained from patients with Alzheimer's disease. The staining pattern was distinct from that obtained with normal brains. Because hGSTM3 is rich in cysteine residues and readily undergoes S-glutathiolation reactions, deposition of this protein could originate from cross-links produced by oxidative stress.

Selective expression of glutathione S-transferase genes in the murine gastrointestinal tract in response to dietary organosulfur compounds.

A short-term feeding regimen was designed to analyze the effects of compounds such as diallyl disulfide (DADS), diallylthiosulfinate (allicin) from garlic and butylated hydroxyanisole (BHA) on glutathione S-transferase (GST) expression in the gastrointestinal tract and liver of male mice. After animals were force-fed these compounds, tissue GSTs were purified and individual subunits resolved by HPLC and identified on the basis of mass spectrometry (ESI MS) and immunoreactivity data. The effects of DADS and allicin on GST expression were especially prominent in stomach and small intestine, where there were major coordinate changes in GST subunit profiles. In particular, the transcripts of the mGSTM1 and mGSTM4 genes, which share large segments of common 5'-flanking sequences, and their corresponding subunits were selectively induced. Levels of alpha class subunits also increased, whereas mGSTM3 and mGSTP1 were not affected. The inducible mGSTA5 and non-responsive mGSTM3 subunits had not been identified previously. Liver and colon GSTs were also affected to a lesser extent, but this short-term feeding regimen had no effect on GST subunit patterns from other organs, including heart, brain and testis. Real-time PCR (TaqMan) methods were used for quantitative estimations of relative amounts of the mRNAs encoding the GSTs. Effects on the transcripts generally paralleled changes at the protein level, for the most part, however, the greatest relative increases were observed for those mRNAs that were expressed at low abundance constituitively. Mechanisms by which the organosulfur compounds operate to affect GST transcription could involve reversible modification of certain protein sulfhydryl groups, shifts in reduced glutathione/oxidized glutathione ratios and resultant changes in cellular redox status.