Attachment of the N-terminal domain of Salmonella typhimurium AhpF to Escherichia coli thioredoxin reductase confers AhpC reductase activity but does not affect thioredoxin reductase activity

AhpF of Salmonella typhimurium, the flavoprotein reductase required for catalytic turnover of AhpC with hydroperoxide substrates in the alkyl hydroperoxide reductase system, is a 57 kDa protein with homology to thioredoxin reductase (TrR) from Escherichia coli. Like TrR, AhpF employs tightly bound FAD and redox-active disulfide center(s) in catalyzing electron transfer from reduced pyridine nucleotides to the disulfide bond of its protein substrate. Homology of AhpF to the smaller (35 kDa) TrR protein occurs in the C-terminal part of AhpF; a stretch of about 200 amino acids at the N-terminus of AhpF contains an additional redox-active disulfide center and is required for catalysis of AhpC reduction. We have demonstrated that fusion of the N-terminal 207 amino acids of AhpF to full-length TrR results in a chimeric protein (Nt-TrR) with essentially the same catalytic efficiency (k(cat)/K(m)) as AhpF in AhpC reductase assays; both k(cat) and the K(m) for AhpC are decreased about 3-4-fold for Nt-TrR compared with AhpF. In addition, Nt-TrR retains essentially full TrR activity. Based on results from two mutants of Nt-TrR (C129, 132S and C342,345S), AhpC reductase activity requires both centers while TrR activity requires only the C-terminal-most disulfide center in Nt-TrR. The high catalytic efficiency with which Nt-TrR can reduce thioredoxin implies that the attached N-terminal domain does not block access of thioredoxin to the TrR-derived Cys342-Cys345 center of Nt-TrR nor does it impede the putative conformational changes that this part of Nt-TrR is proposed to undergo during catalysis. These studies indicate that the C-terminal part of AhpF and bacterial TrR have very similar mechanistic properties. These findings also confirm that the N-terminal domain of AhpF plays a direct role in AhpC reduction.     

Cloning of a novel human caspase-9 splice variant containing only the CARD domain

Caspase-9 plays a key role in the intrinsic apoptotic pathway and currently two splice variants (caspase-9-alpha and -beta) have been identified. The present study cloned and characterized a novel caspase-9 splice variant, hereby designated Casp9-gamma. Casp9-gamma is generated from an additional alternative 3' splice site in the fourth exon of caspase-9, resulting in a 58-nucleotide fragment insertion compared with the full-length caspase-9-alpha. The fragment introduces an in-frame stop codon, and the resulting open reading frame (ORF) is preterminated. The Casp9-gamma comprises the deduced 154 amino acid residues containing only the caspase recruitment domain (CARD) and does not contain the large and small subunits. The Casp9-gamma does not promote apoptosis when overexpressed in mammalian cells. Moreover, it inhibits the cleavage of procaspase-3 mediated by proapoptotic member Bax or apoptosis inductor staurosporine. Therefore, Casp9-gamma may function as an endogenous apoptotic inhibitor by interfering with the CARD-CARD interaction between Apaf-1 (apoptotic protease activating factor-1) and procaspase-9. In addition, Casp9-gamma does not enhance NF-kappaB activation in transfected 293T cells, conflicting with previous evidence that the isolated CARD of caspase-9 activates NF-kappaB in ND7 cells. This suggests that the procaspase-9-mediated NF-kappaB activation in response to cellular stresses is cell type-specific through an unidentified mechanism.     

Induction of thioredoxin reductase gene expression by peroxynitrite in human umbilical vein endothelial cells

Thioredoxin reductase (TR), a flavoprotein, catalyzes the reduction of oxidized thioredoxin in a NADPH-dependent manner, and contains a selenocysteine residue near the C-terminus. TR plays an important role in protecting against oxidative stress and in regulating cell growth and cell death. Constitutive TR expression has been observed in several cell types of the mammalian body, including endothelial cells. The latter are continually exposed to both exogenous and endogenous sources of nitric oxide (NO) and NO-derived species. Reactive nitrogen species (RNS) are associated with pathological events, contributing to the cell and tissue damage accompanying inflammation, atherogenesis and autoimmune diseases. In this study, we report on the effect of peroxynitrite on TR in human umbilical vein endothelial cells (HUVECs). Exposure to the peroxynitrite donor SIN-1 for 1 h resulted in a decrease in TR activity. Interestingly, the activity was completely restored within 24 h. To further examine this mechanism, the expression of TR at the mRNA and protein level was examined. TR mRNA levels were markedly increased by treatment of SIN-1 within 6 h, and TR protein level was also increased after the treatment in HUVECs. These results suggest that the inactivation of TR by peroxynitrite might be involved in the upregulation of the TR gene in HUVECs. Therefore, HUVECs have a unique protective mechanism that allows the maintenance of balance in intracellular redox status via TR induction as an adaptive response to nitrooxidative stress.     

Molecular cloning of an N-terminal splice variant of the capsaicin receptor. Loss of N-terminal domain suggests functional divergence among capsaicin receptor subtypes

Recently a cDNA clone, vanilloid receptor subtype-1 (VR1), was isolated and found to encode an ion channel that is activated by both capsaicin, the pain producing compound in chili peppers, and by noxious thermal stimuli. Subsequently, two related cDNAs have been isolated, a stretch inactivating channel with mechanosensitive properties and a vanilloid receptor-like protein that is responsive to high temperatures (52-53 degrees C). Here, we report the isolation of a vanilloid receptor 5'-splice variant (VR.5'sv) which differs from VR1 by elimination of the majority of the intracellular N-terminal domain and ankyrin repeat elements. Both VR.5'sv and VR1 mRNA were shown to be expressed in tissues reportedly responsive to capsaicin including dorsal root ganglion, brain, and peripheral blood mononuclear cells. Functional expression of VR.5'sv in Xenopus oocytes and mammalian cells showed no sensitivity to capsaicin, the potent vanilloid resiniferatoxin, hydrogen ions (pH 6.2), or noxious thermal stimuli (50 degrees C). Since VR.5'sv is otherwise identical to VR1 throughout its transmembrane spanning domains and C-terminal region, these results support the hypothesis that the N-terminal intracellular domain is essential for the formation of functional receptors activated by vanilloid compounds and noxious thermal stimuli.     

Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death

Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 and thioredoxin reductase 1 (TrxR1) to be significantly decreased. The human neuroblastoma cell line SH-SY5Y and the nematode Caenorhabditis elegans were used as model systems to explore the potential protective effects of the redox proteins against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. 6-OHDA is highly prone to oxidation, resulting in the formation of the quinone of 6-OHDA, a highly reactive species and powerful neurotoxin. Treatment of human cells with 6-OHDA resulted in an increased expression of Trx1, TrxR1, Grx1, and Grx2, and small interfering RNA for these genes significantly increased the cytotoxic effects exerted by the 6-OHDA neurotoxin. Evaluation of the dopaminergic neurons in C. elegans revealed that nematodes lacking trxr-1 were significantly more sensitive to 6-OHDA, with significantly increased neuronal degradation. Importantly, both the Trx and the Grx systems were also found to directly mediate reduction of the 6-OHDA-quinone in vitro and thus render its cytotoxic effects. In conclusion, our results suggest that the two redox systems are important for neuronal survival in dopamine-induced cell death.     

Oxidation of structural cysteine residues in thioredoxin 1 by aromatic arsenicals enhances cancer cell cytotoxicity caused by the inhibition of thioredoxin reductase 1

Thioredoxin systems, composed of thioredoxin reductase (TrxR), thioredoxin (Trx) and NADPH, play important roles in maintaining cellular redox homeostasis and redox signaling. Recently the cytosolic Trx1 system has been shown to be a cellular target of arsenic containing compounds. To elucidate the relationship of the structure of arsenic compounds with their ability of inhibiting TrxR1 and Trx1, and cytotoxicity, we have investigated the reaction of Trx1 system with seven arsenic trithiolates: As(Cys)₃, As(GS)₃, As(Penicillamine)₃, As(Mercaptoethanesulfonate)₃, As(Mercaptopurine)₃, As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃. The cytotoxicity of these arsenicals was consistent with their ability to inhibit TrxR1 in vitro and in cells. Unlike other arsenicals, As(Mercaptopurine)₃ which did not show inhibitory effects on TrxR1 had very weak cytotoxicity, indicating that TrxR1 is a reliable drug target for arsenicals. Moreover, the two aromatic compounds As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃ showed stronger cytotoxicity than the others. As(2-mercaptopyridine)₃ which selectively oxidized two structural cysteines (Cys62 and Cys69) in Trx1 showed mild improvement in cytotoxicity. As(2-mercaptopyridine N-oxide)₃ oxidized all the Cys residues in Trx1, exhibiting the strongest cytotoxicity. Oxidation of Trx1 by As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃ affected electron transfer from NADPH and TrxR1 to peroxiredoxin 1 (Prx1), which could result in the reactive oxygen species elevation and trigger cell death process. These results suggest that oxidation of structural cysteine residues in Trx1 by aromatic group in TrxR1-targeting drugs may sensitize tumor cells to cell death, providing a novel approach to regulate cellular redox signaling and also a basis for rational design of new anticancer agents.     

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.     

Characterization of an N-terminal secreted domain of the type-1 human metabotropic glutamate receptor produced by a mammalian cell line

A Chinese hamster ovary cell line has been established which secretes the N-terminal domain of human mGlu1 receptor. The secreted protein has been modified to contain a C-terminal hexa-histidine tag and can be purified by metal-chelate chromatography to yield a protein with an apparent molecular weight of 130 kDa. Following treatment with dithiothreitol the apparent molecular weight is reduced to 75 kDa showing that the protein is a disulphide-bonded dimer. N-terminal protein sequencing of both the reduced and unreduced forms of the protein yielded identical sequences, confirming that they were derived from the same protein, and identifying the site of signal-peptide cleavage of the receptor as residue 32 in the predicted amino acid sequence. Endoglycosidase treatment of the secreted and intracellular forms of the protein showed that the latter was present as an endoglycosidase H-sensitive dimer, indicating that dimerization is taking place in the endoplasmic reticulum. Characterization of the binding of [3H]quisqualic acid showed that the protein was secreted at levels of up to 2.4 pmol/mL and the secreted protein has a Kd of 5.6 +/- 1.8 nm compared with 10 +/- 1 nm for baby hamster kidney (BHK)-mGlu1alpha receptor-expressing cell membranes. The secreted protein maintained a pharmacological profile similar to that of the native receptor and the binding of glutamate and quisqualate were unaffected by changes in Ca2+ concentration.     

EGF-like domain calcium affinity modulated by N-terminal domain linkage in human fibrillin-1

Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins.We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor beta-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+ affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.     

Identification and characterisation of a novel splice variant of the human CB1 receptor

Cannabinoid ligands are implicated in many physiological processes and to date two receptors have been identified. However, a growing body of evidence exists that suggests the presence of additional receptors. Whilst cloning the previously described hCB1a, we have identified a novel variant that we call hCB1b. Characterising these two splice variants demonstrates that they have a unique pharmacological profile and that their RNA's are expressed at low levels in a variety of tissues.     

Molecular analysis of a new splice variant of the human melanocortin-1 receptor.

The primary hormonal regulator of pigmentation is melanocyte stimulating hormone derived from proopiomelanocortin by proteolytic processing. The melanocortin-1 receptor serves a key role in the regulation of pigmentation. We describe the identification of the first intron within a melanocortin receptor. A new melanocortin-1 receptor isoform, generated by alternative mRNA splicing, encodes an additional 65 amino acids at the predicted intracellular, C-terminal tail of the melanocortin-1 receptor. When expressed in heterologous cells, the new spliced form of the melanocortin-1 receptor (melanocortin-1 receptor B) appears pharmacologically similar to the non-spliced melanocortin-1 receptor. Melanocortin-1 receptor B is expressed in testis, fetal heart and melanomas.     

Induction of thioredoxin by ultraviolet-A radiation prevents oxidative-mediated cell death in human skin fibroblasts

The present study analyzes the expression of the thioredoxin/thioredoxin reductase (Trx/TR) system in UVA-irradiated human skin fibroblasts. Irradiation increases the intracellular level of Trx and a time-dependent increase of Trx mRNA is observed. Our data indicate that Trx translocates from the cytoplasm to the nucleus. In addition, UV exposure results in an increase in TR synthesis. In order to evaluate the function of Trx/TR system, we investigated the antioxidant role of Trx in transient transfected cells. The ROS accumulation in UVA irradiated cells was assessed using flow cytometry. A 3-fold decrease in ROS production was observed in transiently transfected fibroblasts. These results indicate that Trx acts as an antioxidant protein in UVA irradiated fibroblasts. As ROS are inducers of cell death, this raises the question as to whether Trx is able to protect cells from apoptosis and/or necrosis induced by UVA. Six hours after UVA-irradiation, 29.92% of cells were annexin-V positive. This population was significantly reduced in Trx-transfected cells (8.58%). Moreover, this work demonstrates that Trx prevents the loss of the membrane potential of the mitochondria, the depletion of cellular ATP content, and the loss of cell viability induced by irradiation.     

Analysis of the inhibition of mammalian thioredoxin, thioredoxin reductase, and glutaredoxin by cis-diamminedichloroplatinum (II) and its major metabolite, the glutathione-platinum complex.

Several studies have demonstrated a correlation between cellular toxicity of cis-diamminedichloroplatinum (II) (cisplatin, CDDP) and inhibited intracellular activity of the thioredoxin system, i.e., thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. Conversely, increased cellular activity of the Trx system confers resistance to CDDP. In this study, we have analyzed the interaction of CDDP with Trx and TrxR in order to clarify the mechanism. The inhibition with time-dependent kinetics by CDDP of NADPH-reduced (but not oxidized) TrxR was irreversible, strongly suggesting covalent modification of the reduced selenocysteine-containing active site. Assuming second order kinetics, the rate constant of TrxR inhibition by CDDP was 21 +/- 3 M(-1) x s(-1). Transplatin was found to be an even more efficient inhibitor, with a second order rate constant of 84 +/- 22 M(-1) x s(-1), whereas carboplatin (up to 1 mM) gave no inhibition of the enzyme under the same conditions. Escherichia coli Trx or human or bacterial glutaredoxin (Grx) activities were in comparison only slightly or not at all inhibited by either CDDP, transplatin, or carboplatin. However, glutaredoxins were found to be inhibited by the purified glutathione adduct of cisplatin, bis-(glutathionato)platinum(II) (GS-Platinum complex, GS-Pt), with an IC50 = 350 microM in the standard beta-hydroxyethyl disulfide-coupled assay for human Grx. Also the mammalian Trx system was inhibited by GS-Pt with similar efficiency (IC(50) = 325 microM), whereas neither the E. coli Trx system nor glutathione reductase were inhibited. Formation of GS-Pt is a major route for cellular elimination of CDDP. The fact that GS-Pt inhibits the mammalian Trx as well as Grx systems shows that CDDP may exert effects at several stages of its metabolism, including after conjugation with GSH, which are intimately linked with the cellular disulfide/dithiol redox regulatory systems.