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MYB, a proto-oncogene, is overexpressed in prostate cancer (PCa) and promotes its growth, aggressiveness, and resistance to androgen-deprivation therapy. Here, we examined the effect of androgen signaling on MYB expression and delineated the underlying molecular mechanisms. Paralleling a dichotomous effect on growth, low-dose androgen induced MYB expression at both transcript and protein levels, whereas it was suppressed in high-dose androgen-treated PCa cells. Interestingly, treatment with both low- and high-dose androgen transcriptionally upregulated MYB by increasing the binding of androgen receptor to the MYB promoter. In a time-course assay, androgen induced MYB expression at early time points followed by a sharp decline in high-dose androgen-treated cells due to decreased stability of MYB mRNA. Additionally, profiling of MYB-targeted miRNAs demonstrated significant induction of miR-150 in high-dose androgen-treated PCa cells. We observed a differential binding of androgen receptor on miR-150 promoter with significantly greater occupancy recorded in high-dose androgen-treated cells than those treated with low-dose androgen. Functional inhibition of miR-150 relieved MYB suppression by high-dose androgen, while miR-150 mimic abolished MYB induction by low-dose androgen. Furthermore, MYB-silencing or miR-150 mimic transfection suppressed PCa cell growth induced by low-dose androgen, whereas miR-150 inhibition rescued PCa cells from growth repression by high-dose androgen. Similarly, we observed that MYB silencing suppressed the expression of androgen-responsive, cell cycle-related genes in low-dose androgen-treated cells, while miR-150 inhibition increased their expression in cells treated with high-dose androgen. Overall, these findings reveal novel androgen-mediated mechanisms of MYB regulation that support its biphasic growth control in PCa cells., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Pancreatic cancer (PC) remains a therapeutic challenge because of its intrinsic and extrinsic chemoresistance mechanisms. Here, we report that C- X -C motif chemokine receptor 4 (CXCR4) and hedgehog pathways cooperate in PC chemoresistance via bidirectional tumor-stromal crosstalk. We show that when PC cells are co-cultured with pancreatic stellate cells (PSCs) they are significantly more resistant to gemcitabine toxicity than those grown in monoculture. We also demonstrate that this co-culture-induced chemoresistance is abrogated by inhibition of the CXCR4 and hedgehog pathways. Similarly, the co-culture-induced altered expression of genes in PC cells associated with gemcitabine metabolism, antioxidant defense, and cancer stemness is also reversed upon CXCR4 and hedgehog inhibition. We have confirmed the functional impact of these genetic alterations by measuring gemcitabine metabolites, reactive oxygen species production, and sphere formation in vehicle- or gemcitabine-treated monocultures and co-cultured PC cells. Treatment of orthotopic pancreatic tumor-bearing mice with gemcitabine alone or in combination with a CXCR4 antagonist (AMD3100) or hedgehog inhibitor (GDC-0449) displays reduced tumor growth. Notably, we show that the triple combination treatment is the most effective, resulting in nearly complete suppression of tumor growth. Immunohistochemical analysis of Ki67 and cleaved caspase-3 confirm these findings from in vivo imaging and tumor measurements. Our findings provide preclinical and mechanistic evidence that a combination of gemcitabine treatment with targeted inhibition of both the CXCR4 and hedgehog pathways improves outcomes in a PC mouse model., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Khan et al.)

Extensive desmoplasia is a prominent pathological characteristic of pancreatic cancer (PC) that not only impacts tumor development, but therapeutic outcome as well. Recently, we demonstrated a novel role of MYB, an oncogenic transcription factor, in PC growth and metastasis. Here we studied its effect on pancreatic tumor histopathology and associated molecular and biological mechanisms. Tumor-xenografts derived from orthotopic-inoculation of MYB-overexpressing PC cells exhibited far-greater desmoplasia in histological analyses compared with those derived from MYB-silenced PC cells. These findings were further confirmed by immunostaining of tumor-xenograft sections with collagen-I, fibronectin (major extracellular-matrix proteins), and α-SMA (well-characterized marker of myofibroblasts or activated pancreatic stellate cells (PSCs)). Likewise, MYB-overexpressing PC cells provided significantly greater growth benefit to PSCs in a co-culture system as compared with the MYB-silenced cells. Interrogation of deep-sequencing data from MYB-overexpressing versus -silenced PC cells identified Sonic-hedgehog (SHH) and Adrenomedullin (ADM) as two differentially-expressed genes among others, which encode for secretory ligands involved in tumor-stromal cross-talk. In-silico analyses predicted putative MYB-binding sites in SHH and ADM promoters, which was later confirmed by chromatin-immunoprecipitation. A cooperative role of SHH and ADM in growth promotion of PSCs was confirmed in co-culture by using their specific-inhibitors and exogenous recombinant-proteins. Importantly, while SHH acted exclusively in a paracrine fashion on PSCs and influenced the growth of PC cells only indirectly, ADM could directly impact the growth of both PC cells and PSCs. In summary, we identified MYB as novel regulator of pancreatic tumor desmoplasia, which is suggestive of its diverse roles in PC pathobiology., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Gain-of-function mutations in the calcium channel TRPC6 lead to autosomal dominant focal segmental glomerulosclerosis and podocyte expression of TRPC6 is increased in some acquired human glomerular diseases, particularly in membranous nephropathy. These observations led to the hypothesis that TRPC6 overactivation is deleterious to podocytes through pathological calcium signaling, both in genetic and acquired diseases. Here, we show that the effects of TRPC6 on podocyte function are context-dependent. Overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure. Unexpectedly, however, overexpression of TRPC6 protected podocytes from complement-mediated injury, whereas genetic or pharmacological TRPC6 inactivation increased podocyte susceptibility to complement. Mechanistically, this effect was mediated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. Podocyte-specific TRPC6 transgenic mice showed stronger CaMKII activation, reduced podocyte foot process effacement and reduced levels of proteinuria during nephrotoxic serum nephritis, whereas TRPC6 null mice exhibited reduced CaMKII activation and higher levels of proteinuria compared with wild type littermates. Human membranous nephropathy biopsy samples showed podocyte staining for active CaMKII, which correlated with the degree of TRPC6 expression. Together, these data suggest a dual and context dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to focal segmental glomerulosclerosis.

Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation because it is abrogated by pretreatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 up-regulation by gemcitabine correlates with time-dependent accumulation of NF-κB and HIF-1α in the nucleus. Enhanced binding of NF-κB and HIF-1α to the CXCR4 promoter is observed in gemcitabine-treated PC cells, whereas their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-κB and HIF-1α, and suppression of ROS diminishes these effects. The effect of ROS on NF-κB and HIF-1α is mediated through activation of ERK1/2 and Akt, and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 up-regulation. Interestingly, our data demonstrate that nuclear accumulation of NF-κB results from phosphorylation-induced degradation of IκBα, whereas HIF-1α up-regulation is NF-κB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.

Serum amyloid A (SAA) is best known for being the main component of amyloid in the inflammation-related disease amyloid A (AA) amyloidosis. Despite the high sequence identity among different SAA isoforms, not all SAA proteins are pathogenic. In most mouse strains, the AA deposits mostly consist of SAA1.1. Conversely, the CE/J type mouse expresses a single non-pathogenic SAA2.2 protein that is 94% identical to SAA1.1. Here we show that SAA1.1 and SAA2.2 differ in their quaternary structure, fibrillation kinetics, prefibrillar oligomers, and fibril morphology. At 37 °C and inflammation-related SAA concentrations, SAA1.1 exhibits an oligomer-rich fibrillation lag phase of a few days, whereas SAA2.2 shows virtually no lag phase and forms small fibrils within a few hours. Deep UV resonance Raman, far UV-circular dichroism, atomic force microscopy, and fibrillation cross-seeding experiments suggest that SAA1.1 and SAA2.2 fibrils possess different morphology. Both the long-lived oligomers of pathogenic SAA1.1 and the fleeting prefibrillar oligomers of non-pathogenic SAA2.2, but not their respective amyloid fibrils, permeabilized synthetic bilayer membranes in vitro. This study represents the first comprehensive comparison between the biophysical properties of SAA isoforms with distinct pathogenicities, and the results suggest that structural and kinetic differences in the oligomerization-fibrillation of SAA1.1 and SAA2.2, more than their intrinsic amyloidogenicity, may contribute to their diverse pathogenicity.

Recent evidence suggests a major role of tumor-stromal interactions in pancreatic cancer pathobiology. The chemokine CXCL12 (stromal cell-derived factor 1 (SDF-1)), abundantly produced by stromal cells, promotes progression, metastasis, and chemoresistance of pancreatic cancer cells. On the other hand, pancreatic tumor cell-derived sonic hedgehog (SHH) acts predominantly on stromal cells to induce desmoplasia and, thus, has a paracrine effect on tumorigenesis and therapeutic outcome. In this study, we examined the association between these two proteins of pathological significance in pancreatic cancer. Our data demonstrate that CXCL12 leads to a dose- and time-dependent up-regulation of SHH in pancreatic cancer cells. CXCL12-induced SHH up-regulation is specifically mediated through the receptor CXCR4 and is dependent on the activation of downstream Akt and ERK signaling pathways. Both Akt and ERK cooperatively promote nuclear accumulation of NF-κB by inducing the phosphorylation and destabilization of its inhibitory protein, IκB-α. Using dominant negative IκB-α, a SHH promoter (deletion mutant) reporter, and chromatin immunoprecipitation assays, we demonstrate that CXCL12 exposure enhances direct binding of NF-κB to the SHH promoter and that suppression of NF-κB activation abrogates CXCL12-induced SHH expression. Finally, our data demonstrate a strong correlative expression of CXCR4 and SHH in human pancreatic cancer tissues, whereas their expression is not observed in the normal pancreas. Altogether, our data reveal a novel mechanism underlying aberrant SHH expression in pancreatic cancer and identify a molecular link facilitating bidirectional tumor-stromal interactions.

Anoikis is a cell death that occurs due to detachment of a cell from the extracellular matrix (ECM). Resistance to anoikis is a primary feature of a cell that undergoes metastasis. In this study for the first time, we demonstrated the potential role of Gli1 in anoikis resistance. Treatment of various ovarian cancer cells by different concentrations of diindolylmethane (DIM), an active ingredient of cruciferous vegetables, reduced the anoikis resistance in a concentration-dependent manner. Reduction in anoikis resistance was associated with a decrease in the expression of Gli1 and an increase in the cleavage of poly(ADP-ribose) polymerase (PARP). Sonic hedgehog (Shh) treatment not only increased the expression of Gli1, but also blocked anoikis induced by DIM and abrogated the change in the expression of Gli1 and cleaved PARP by DIM. To confirm the role of Gli1, hedgehog inhibitor cyclopamine, Gli1 siRNA and Gli1(-/-) mouse embryonic fibroblasts (MEFs) were used. Cyclopamine treatment alone significantly reduced anoikis resistance in A2780 and OVCAR-429 cells. Cyclopamine-mediated reduction in anoikis resistance was associated with reduced expression of Gli1 and induction of cleaved PARP. Shh treatment blocked cyclopamine-induced anoikis. Silencing Gli1 expression induced anoikis and cleavage of PARP in A2780 and OVCAR-429 cells. Furthermore, Gli1(-/-) MEFs were more sensitive to anoikis compared with Gli1(+/+) MEFs. Our in vivo studies established that DIM- or cyclopamine-treated ovarian cancer cells under suspension culture conditions drastically lost their ability of tumor formation in vivo in mice. Taken together, our results establish that Gli1 is a critical player in anoikis resistance in ovarian cancer.

The development of intratumoral hypoxia, a hallmark of rapidly progressing solid tumors, renders tumor cells resistant to chemotherapy and radiation therapy. We have recently shown that inhibition of aldose reductase (AR), an enzyme that catalyzes the reduction of lipid aldehydes and their glutathione conjugates, prevents human colon cancer cell growth in culture as well as in nude mouse xenografts by inhibiting the NF-κB-dependent activation of oxidative stress-mediated inflammatory and carcinogenic markers. However, the role of AR in mediating hypoxic stress signals is not known. We therefore investigated the molecular mechanisms by which AR inhibition prevents the hypoxia-induced human colon cancer cells growth and invasion. Our results indicate that AR inhibition by the pharmacological inhibitor fidarestat or ablation by AR-specific siRNA prevents hypoxia-induced proliferation of HT29, SW480, and Caco-2 colon cancer cells. Furthermore, hypoxia-induced increase in the level of HIF-1α in colon cancer cells was significantly decreased by AR inhibition. During hypoxic conditions, treatment of HT29 cells with the AR inhibitor fidarestat significantly decreased the expression of vascular endothelial growth factor, a down target of HIF-1α, at both mRNA and protein levels and also prevented the activation of PI3K/AKT, GSK3β, Snail, and lysyl oxidase. Furthermore, inhibition of hypoxia-induced HIF-1α protein accumulation by AR inhibition was abolished in the presence of MG132, a potent inhibitor of the 26 S proteasome. In addition, AR inhibition also prevented the hypoxia-induced inflammatory molecules such as Cox-2 and PGE2 and expression of extracellular matrix proteins such as MMP2, vimentin, uPAR, and lysyl oxidase 2. In conclusion, our results indicate that AR mediates hypoxic signals, leading to tumor progression and invasion.

Abnormal production of inflammatory cytokines and chemokines is a key feature of bacterial endotoxin, lipopolysaccharide (LPS)-induced inflammation, and cytotoxicity; however, the mechanisms regulating production of inflammatory markers remain unclear. Herein, we show that inhibition of the aldehyde-metabolizing enzyme aldose reductase (AR; AKR1B3) modulates NF-kappaB-dependent activation of inflammatory cytokines and chemokines in mouse serum, liver, heart, and spleen. Pharmacological inhibition or small interfering RNA ablation of AR prevented the biosynthesis of tumor necrosis factor-alpha, interleukin 1beta, interleukin-6, macrophage-chemoattractant protein-1, and cyclooxygenase-2 and prostaglandin E(2) in LPS-activated RAW264.7 murine macrophages. The AR inhibition or ablation significantly attenuated LPS-induced activation of protein kinase C (PKC) and phospholipase C (PLC), nuclear translocation of NF-kappaB, and phosphorylation and proteolytic degradation of IkappaBalpha in macrophages. Furthermore, treatment of macrophages with 4-hydroxy-trans-2-nonenal (HNE), and cell-permeable esters of glutathionyl-4-hydroxynonanal (GS-HNE) and glutathionyl-1,4-dihydroxynonane (GS-DHN) activated NF-kappaB and PLC/PKC. Pharmacological inhibition or antisense ablation of AR that catalyzes the reduction of GS-HNE to GS-DHN prevented PLC, PKC, IKKalpha/beta, and NF-kappaB activation caused by HNE and GS-HNE, but not by GS-DHN, suggesting that reduced GS-lipid aldehydes catalyzed by AR propagate LPS-induced production of inflammatory markers. Collectively, these data provide evidence that inhibition of AR may be a significant therapeutic approach in preventing bacterial endotoxin-induced sepsis and tissue damage.

Products of lipid peroxidation such as 4-hydroxy-trans-2-nonenal (HNE) trigger multiple signaling cascades that variably affect cell growth, differentiation, and apoptosis. Because glutathiolation is a significant metabolic fate of these aldehydes, we tested the possibility that the bioactivity of HNE depends upon its conjugation with glutathione. Addition of HNE or the cell-permeable esters of glutathionyl-4-hydroxynonenal (GS-HNE) or glutathionyl-1,4-dihydroxynonene (GS-DHN) to cultures of rat aortic smooth muscle cells stimulated protein kinase C, NF-kappaB, and AP-1, and increased cell growth. The mitogenic effects of HNE, but not GS-HNE or GS-DHN, were abolished by glutathione depletion. Pharmacological inhibition or antisense ablation of aldose reductase (which catalyzes the reduction of GS-HNE to GS-DHN) prevented protein kinase C, NF-kappaB, and AP-1 stimulation and the increase in cell growth caused by HNE and GS-HNE, but not GS-DHN. The growth stimulating effect of GS-DHN was enhanced in cells treated with antibodies directed against the glutathione conjugate transporters RLIP76 (Ral-binding protein) or the multidrug resistance protein-2. Overexpression of RLIP76 abolished the mitogenic effects of HNE and its glutathione conjugates, whereas ablation of RLIP76 using RNA interference promoted the mitogenic effects. Collectively, our findings suggest that the mitogenic effects of HNE are mediated by its glutathione conjugate, which has to be reduced by aldose reductase to stimulate cell growth. These results raise the possibility that the glutathione conjugates of lipid peroxidation products are novel mediators of cell signaling and growth.

DNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents.

We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9- and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G(2)/M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3beta. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G(2)/M arrest. HCT116 human colon cancer-derived Chk2(-/-) cells were significantly more resistant to G(2)/M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G(2)/M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.

Abnormal vascular smooth muscle cell (VSMC) proliferation is a key feature of atherosclerosis and restenosis; however, the mechanisms regulating growth remain unclear. Herein we show that inhibition of the aldehyde-metabolizing enzyme aldose reductase (AR) inhibits NF-kappa B activation during restenosis of balloon-injured rat carotid arteries as well as VSMC proliferation due to tumor necrosis factor alpha (TNF-alpha) stimulation. Inhibition of VSMC growth by AR inhibitors was not accompanied by increase in cell death or apoptosis. Inhibition of AR led to a decrease in the activity of the transcription factor NF-kappa B in culture and in the neointima of rat carotid arteries after balloon injury. Inhibition of AR in VSMC also prevented the activation of NF-kappa B by basic fibroblast growth factor (bFGF), angiotensin-II (Ang-II), and platelet-derived growth factor (PDGF-AB). The VSMC treated with AR inhibitors showed decreased nuclear translocation of NF-kappa B and diminished phosphorylation and proteolytic degradation of I kappa B-alpha. Under identical conditions, treatment with AR inhibitors also prevented the activation of protein kinase C (PKC) by TNF-alpha, bFGF, Ang-II, and PDGF-AB but not phorbol esters, indicating that AR inhibitors prevent PKC stimulation or the availability of its activator but not PKC itself. Treatment with antisense AR, which decreased the AR activity by >80%, attenuated PKC activation in TNF-alpha, bFGF, Ang-II, and PDGF-AB-stimulated VSMC and prevented TNF-alpha-induced proliferation. Collectively, these data suggest that inhibition of NF-kappa B may be a significant cause of the antimitogenic effects of AR inhibition and that this may be related to disruption of PKC-associated signaling in the AR-inhibited cells.

Lipid peroxidation products have signaling functions and at higher concentrations are toxic and may trigger cell death. The compounds are metabolized predominantly by glutathione S-transferases exemplified by mGSTA4-4, an enzyme highly efficient in glutathione conjugation of 4-hydroxyalkenals, and possessing glutathione peroxidase activity toward phospholipid hydroperoxides. mGSTA4-4 belongs to the predominant group of "canonical" glutathione S-transferases that are soluble and generally localized in the cytoplasm. The intracellular localization of mGSTA4-4 was examined in hepatocytes of normal mouse liver and in transfected HepG2 cells by fluorescence microscopy and digital deconvolution. mGSTA4-4 was found to be predominantly localized at or near the plasma membrane in transfected HepG2 cells, as well as in hepatocytes endogenously expressing the protein. In vitro, mGSTA4-4 associated with liposomes, and this interaction was potentiated when the liposomes contained negatively charged phospholipids. Mutating lysine 115 to glutamic acid resulted in a loss of the plasma membrane targeting of mGSTA4-4 as well as in a significant reduction of its binding to liposomes in vitro. These data suggest preferential targeting of mGSTA4-4 to the plasma membrane that may contain the major substrate(s) for this enzyme. Lysine 115 is critically important for the membrane association of mGSTA4-4, most likely by entering into an electrostatic interaction with negatively charged phospholipid headgroups.

Aldose reductase (AR), a member of the aldo-keto reductase superfamily, has been implicated in the etiology of secondary diabetic complications. However, the physiological functions of AR under euglycemic conditions remain unclear. We have recently demonstrated that, in intact heart, AR catalyzes the reduction of the glutathione conjugate of the lipid peroxidation product 4-hydroxy-trans-2-nonenal (Srivastava, S., Chandra, A., Wang, L., Seifert, W. E., Jr., DaGue, B. B., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998) J. Biol. Chem. 273, 10893-10900), consistent with a possible role of AR in the metabolism of glutathione conjugates of aldehydes. Herein, we present several lines of evidence suggesting that the active site of AR forms a specific glutathione-binding domain. The catalytic efficiency of AR in the reduction of the glutathione conjugates of acrolein, trans-2-hexenal, trans-2-nonenal, and trans,trans-2,4-decadienal was 4-1000-fold higher than for the corresponding free alkanal. Alterations in the structure of glutathione diminished the catalytic efficiency in the reduction of the acrolein adduct, consistent with the presence of specific interactions between the amino acid residues of glutathione and the AR active site. In addition, non-aldehydic conjugates of glutathione or glutathione analogs displayed active-site inhibition. Molecular dynamics calculations suggest that the conjugate adopts a specific low energy configuration at the active site, indicating selective binding. These observations support an important role of AR in the metabolism of glutathione conjugates of endogenous and xenobiotic aldehydes and demonstrate, for the first time, efficient binding of glutathione conjugates to an aldo-keto reductase.

The metabolism of 4-hydroxy-trans-2-nonenal (HNE), an alpha, beta-unsaturated aldehyde generated during lipid peroxidation, was studied in isolated perfused rat hearts. High performance liquid chromatography separation of radioactive metabolites recovered from [3H]HNE-treated hearts revealed four major peaks. Based on the retention times of synthesized standards, peak I, which accounted for 20% radioactivity administered to the heart, was identified to be due to glutathione conjugates of HNE. Peaks II and III, containing 2 and 37% radioactivity, were assigned to 1, 4-dihydroxy-2-nonene (DHN) and 4-hydroxy-2-nonenoic acid, respectively. Peak IV was due to unmetabolized HNE. The electrospray ionization mass spectrum of peak I revealed two prominent metabolites with m/z values corresponding to [M + H]+ of HNE and DHN conjugates with glutathione. The presence of 4-hydroxy-2-nonenoic acid in peak III was substantiated using gas chromatography-chemical ionization mass spectroscopy. When exposed to sorbinil, an inhibitor of aldose reductase, no GS-DHN was recovered in the coronary effluent, and treatment with cyanamide, an inhibitor of aldehyde dehydrogenase, attenuated 4-hydroxy-2-nonenoic acid formation. These results show that the major metabolic transformations of HNE in rat heart involve conjugation with glutathione and oxidation to 4-hydroxy-2-nonenoic acid. Further metabolism of the GS-HNE conjugate involves aldose reductase-mediated reduction, a reaction catalyzed in vitro by homogenous cardiac aldose reductase.

The kinetics of the conjugation of glutathione (GSH) with anti-7beta, 8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene (anti-BPDE) catalyzed by GSH S-transferase (GST) isoenzymes purified from the liver and forestomach of female A/J mouse has been investigated. The GST isoenzymes studied included an alpha class isoenzyme of forestomach (GST 9.5), alpha class hepatic isoenzymes mGSTA3-3 and mGSTA4-4, pi class hepatic isoenzyme mGSTP1-1, and mu class hepatic isoenzyme mGSTM1-1. When the concentration of (+)-anti-BPDE was varied (5-120 microM) at a fixed GSH concentration (2 mM), linear Lineweaver-Burk plots were observed for each isoenzyme. The kcat values for GST 9.5, mGSTA3-3, mGSTP1-1, mGSTM1-1, and mGSTA4-4 were 2.0, 0.02, 0.40, 0. 05, and 0.01 s-1, respectively, with corresponding Km values of 16, 12, 29, 27, and 49 microM. The catalytic efficiency (kcat/Km) of GST 9.5 in the conjugation of GSH with (+)-anti-BPDE, which is believed to be the ultimate carcinogenic metabolite of benzo(a)pyrene, was about 9-625-fold higher as compared with other mouse GST isoenzymes. These results indicate that GST 9.5 of forestomach is different among mammalian alpha class GSTs because (+)-anti-BPDE has been shown to be a poor substrate for alpha class rat or human GST isoenzymes. The catalytic efficiency of GST 9.5 was approximately 4.5-fold higher than that of pi class human isoenzyme (hGSTP1-1), which among human GSTs is reported to be most efficient in the detoxification of (+)-anti-BPDE. Unlike rat GST isoenzymes, linear Lineweaver-Burk plots were observed for mouse GSTs when GSH was used as a variable substrate. The catalytic efficiencies of the mouse GSTs toward (+)-anti-BPDE were about 2-20-fold higher as compared with the (-)-enantiomer of anti-BPDE. The results of the present study suggest that GST 9.5 may play an important role in the detoxification of (+)-anti-BPDE.

We have previously isolated a cDNA clone for a unique mouse lung glutathione S-transferase, mGSTA4-4 (GST 5.7) (Zimniak, P., Eckles, M. A., Saxena, M., and Awasthi, Y. C. (1992) FEBS Lett. 313, 173-176). By genomic Southern blotting and polymerase chain reaction single strand conformation polymorphism analysis we have now demonstrated the presence of at least two mGSTA4-related genes in the mouse. The heterogeneity of mGSTA4-4 was further examined by comparing the structural and kinetic properties of mGSTA4-4 isolated from mouse lung with those of recombinant rec-mGSTA4-4 expressed in Escherichia coli. Except for the isoelectric point, the physical properties of the two proteins were indistinguishable. Western blots using antibodies against rec-mGSTA4-4 have shown selective expression of the enzyme in mouse tissues. Even though the substrate specificity profiles of the tissue-isolated and recombinant enzymes, which point to a role of mGSTA4-4 in the detoxification of lipid peroxidation products, were generally similar, significant differences were observed with selected substrates. The existence of functionally distinct forms of mGSTA4-4 and the presence of more than one gene strongly suggest that the previously observed differences in properties of mGSTA4-4 isolated from various mouse tissues (Awasthi, S., Singhal, S. S., Srivastava, S. K., and Awasthi, Y. C. (1993) Arch. Biochem. Biophys. 301, 143-150) may be due to tissue-specific expression of mGSTA4-related genes.

The pH-dependence of nucleotide binding and steady-state kinetic parameters of aldehyde reduction and alcohol oxidation catalyzed by bovine lens aldose reductase were studied. The maximal velocity of aldehyde reduction with NADPH and p-chlorobenzaldehyde was pH independent at low pH but decreased at high pH with a pK of 7.6. The V/K of NADPH displayed a bell-shaped dependence on pH and decreased with a pKa of 5.3 and a pKb of 7.5. The dissociation constant of NADPH and 3-acetylpyridine adenine dinucleotide phosphate (3-APADP) increased at low pH with a pK of 5.6-5.8 and at high pH with a pK of 9.4-9.7. The pKi of NADP and NADPH decreased below a pH of 5 and 6.7 and above a pH of 8.5 and 9.7, respectively. The pK of 8.5-9.7 appears to be due to the interaction of the 2'-phosphate of the nucleotide with a protonated base, possibly a lysine residue. The maximal velocity of alcohol oxidation was pH independent at high pH but decreased at low pH with a pK of 6.5-7.0, when p-chlorobenzyl alcohol or benzyl alcohol and 3-APADP were used. The amino acid residue for alcohol binding has a pK of 7.5-8.2 and also appears in pKi profiles of sorbinil, a competitive inhibitor versus the alcohol. Large (3-3.5) isotope effects on maximal velocity obtained with benzyl alcohol and 3-APADP suggest that with these substrates the hydride transfer step is rate-limiting and a pK of 6.5-7.0 may be the true pK of the acid-base catalyst, possibly a histidine.

In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.

gamma-Glutamyl transpeptidase, present in various mammalian tissues, transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37 degrees followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration; and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The Km for gamma-glutamyl transpeptidase using the artificial substrate, gamma-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of gamma-glutamyl transpeptidase when gamma-glutamyl-p-nitroanilide was used as the substrate. Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified gamma-glutamyl transpeptidase had no phosphate-independent glutaminase activity. The ratio of gamma-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of gamma-glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney.

Glutathione peroxidase has been purified to homogeneity from human erythrocytes. The purification steps involved ammonium sulfate precipitation of hemolysate, CM-cellulose (CM-52), DEAE-cellulose (DE52), Sephadex G-200, and DEAE-Sephadex column chromatography. In the last step, i.e. DEAE-Sephadex A-25 column chromatography, the enzyme was eluted in a major peak and tailing fraction. The major peak was found to be homogeneous on polyacrylamide disc electrophoresis and disignated as glutathione peroxidase A (GSH-Px A). The tail fraction, however, separated into two protein bands on polyacrylamide disc electrophoresis. One of the bands corresponded to GSH-Px A while the other band was slower moving and was designated as GSH-Px B. GSH-Px A and GSH-Px B had specific activity of 103 and 4 enzyme units per mg of protein, respectively. Antibodies raised against the homogeneous GSH-Px A have been found to cross-react with GSH-Px B. Both, GSH-Px A and B are selenoproteins. GSH-Px A has been found to contain 3.5 g atoms of selenium per mol of protein. Selenium content of GSH-Px B, however, could not be determined accurately due to insufficient material. The molecular weight of GSH-Px A as determined by the sedimentation equilibrium method is 95,000 plus or minus 3,000. On urea-sodium dodecyl sulfate-polyacrylamide disc electrophoresis GSH-Px A and B dissociate into single subunits. The molecular weight of the subunits of GSH-Px A is 23,000 and that of GSH-Px B is 47,000. Thus, it appears that GSH-Px A is a tetramer. Our results suggest that GSH-Px B is probably an altered form of the major component, GSH-Px A, or its precursor. The properties of GSH-Px A have been studied. The isoelectric pH was found to be 4.9 and the optimum pH for enzyme activity was 8.5. The energy of activation was 8.2 kcal. The Km of the enzyme for GSH was 4.1 mM while the Km for t-butyl hydroperoxide was 52 mu-M. The effect of sulfhydryl reagents and the metal ions on the enzyme was also studied.