Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells

BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS) and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity

Microbe-host interplay in atopic dermatitis and psoriasis

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.Peer reviewe

Exploring thioredoxin reductase as an anticancer drug target

Mammalian thioredoxin reductase (TrxR; EC 1.8.1.9) is a homodimeric NADPH-dependent selenium-containing flavoenzyme with disulfide oxidoreductase activity. The mammalian thioredoxin (Trx) system exerts an impressive spectrum of functions either directly through reactions catalyzed by TrxR or by its prime substrate Trx. It is involved in redox regulation, antioxidant defense, cell growth, replication and regulation of transcription factors. TrxR was found to be overexpressed in a variety of tumor cells and may be involved in several, if not all, steps of carcinogenesis. Therefore, TrxR has been proposed as a potential target for anticancer therapy. In fact, inhibition of TrxR has been shown by a number of chemotherapeutic drugs as part of their mechanism of action. The rather unique biochemistry of TrxR indeed supports efficient targeting by electrophiles. TrxR has an N-terminal active site with two cysteines (Cys) and a Cterminal active site with a Cys and a selenocysteine (Sec). Both active sites are necessary for native TrxR function. Sec is a rare, naturally occurring amino acid encoded by a UGA codon. As this codon normally signals a termination of translation, a recoding signal, the Sec insertion sequence is present in the 3´ untranslated region of the mRNA. A specific translation machinery then guides the insertion of the Sec into the polypeptide chain. Sec is a highly reactive Cys analog with a selenium atom in place of the sulfur, resulting in a significantly lower pKa value. Therefore, Sec is mainly deprotonated at physiological pH and thus highly reactive towards alkylation by electrophilic agents. This results in abrogation of redox activity of the enzyme´s C-terminal active site that is essential for reduction of Trx and most other substrates. However, specific targeting of the Sec does not render a completely inactive protein. With an intact N-terminal active site, inhibited TrxR may still induce a rapid cell death in cancer cells presumably by redox cycling with an endogenous substrate inducing oxidative stress as shown in this thesis. We named this cytotoxic form of TrxR SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins). Introducing SecTRAPs directly into A549 (lung carcinoma) or HeLa (cervical adenocarcinoma) cells using the protein delivery reagent BioPORTER® we observed DNA condensation, phosphatidylserine exposure prior to loss of membrane integrity and production of reactive oxygen species. Cell death was caspase-dependent but independent of novel protein synthesis. Cisplatin was shown to inhibit TrxR and form SecTRAPs in vitro. We investigated the elemental differences in TrxR inhibition of simple Pt, Pd and Au salts, showing Pd and Au to be superior in this aspect. All salts seemingly target the C-terminal active site of TrxR in a specific manner, potentially forming SecTRAPs. Inhibition of cellular TrxR was shown to correlate with cytotoxicity of nitroaromatic and quinone compounds involving caspase activation. However, the extent of cell death was in some cases, but not all, dependent on TrxR1 levels. Using siRNA constructs to specifically knock down TrxR1 by 90% in A549 cells resulted in increased susceptibility towards DNCB and menadione, and decreased sensitivity to cisplatin. This may indicate that different TrxR inhibition mechanisms can lead to different treatment outcomes and that cisplatin may indeed form toxic SecTRAPs within cells. This occurs possibly at a greater extent in tumors where high expression of TrxR is prevalent. The potential of TrxR as an anticancer drug target is discussed in this thesis with special focus on drugs likely to form SecTRAPs and possibly to induce selective tumor killing

Fecal transfers from children on the ketogenic diet mimic the anti-seizure effect in mice

The ketogenic diet (KD) mediates its anti-seizure effect through the gut microbiota in epilepsy mouse models.1 Lum et al.2 demonstrated that fecal microbiota from children with epilepsy treated with the KD decreases seizure susceptibility in mice after transfer

Both SecTRAPs and TrxR1 are efficient in reducing juglone and thereby produce superoxide.

<p>In (<i>A</i>) the formation of FAD-reduced disulfide charge transfer complex by NADPH (80 µM) in a SecTRAP preparation (truncated TrxR1, 16 µM subunit) and full-length TrxR1 (12 µM subunit) was analyzed with stopped-flow spectroscopy at 540 nm, showing similar kinetics for both enzymes. In (<i>B</i>) Michaelis-Menten kinetics for both full-length (filled symbols) and truncated (open symbols) TrxR1 using juglone as a substrate is demonstrated. In (C) it is shown that Trx1 and juglone compete for the reduction by full-length TrxR1 (filled bars) but that truncated TrxR1 (open bars) can only use juglone and not Trx1 as a substrate. This is illustrated from the initial NADPH consumption rate (0–200s) followed at 340 nm with or without Trx1 and insulin (upper panels). After 30 min of reaction, the number of exposed free thiols was determined (lower panels) in order to estimate to which extent the electrons from the NADPH oxidation were passed on to Trx1 and subsequently to insulin. The juglone concentration is indicated at the x-axes and each bar represents the mean±S.D. of three measurements. In (D), the reduction of juglone (5 µM) catalyzed by 10 nM SecTRAP (truncated TrxR1, left panel) or full-length TrxR1 (right panel) is shown following the consumption of NADPH (initial concentration 250 µM) by the decrease in absorbance at 340 nm (open symbols). Concomitantly, superoxide formation was detected at 480 nm with the adrenochrome method using 2 mM epinephrine (filled symbols). The formation of adrenochrome was completely inhibited by addition of 5 U SOD (circles), which also reduced the elevated NADPH consumption seen upon addition of only epinephrine (squares).</p

Model for the formation and function of SecTRAPs.

<p>We propose that during most conditions of normal cell growth the Sec residue in TrxR1 is intact and the role of this enzyme is thus to sustain the many cellular functions of the thioredoxin system. This activity is dependent upon the redox active C-terminal Sec-containing active site in TrxR1. SecTRAPs may however be formed from TrxR1 if its Sec residue becomes compromised, either by removal or by derivatization with electrophilic compounds, while the FAD and N-terminal redox active CVNVGC motif of the enzyme are kept intact. SecTRAPs lack the Trx reducing activity of native TrxR1 but become potent inducers of cell death by a gain of function. This cell death is, as shown in the present study, rapid, does not require induction of protein synthesis, involves production of reactive oxygen species and is prevented by caspase inhibitors. If, on the other hand, both the CVNVGC motif and the C-terminal selenolthiol motif become inactivated, e.g. by certain types of TrxR1 inhibitors or if the overall expression of TrxR1 is diminished in cells, impaired cell function or cell death may still occur. In such cases the cellular consequences would however not be due to a gain of function in derivatives of TrxR1, but rather to hampered functions of the complete cellular thioredoxin system.</p

Mucosal and Plasma Metabolomes in New-onset Paediatric Inflammatory Bowel Disease : Correlations with Disease Characteristics and Plasma Inflammation Protein Markers

Background and Aims To advance the understanding of inflammatory bowel disease [IBD] pathophysiology, we compared the mucosal and plasma metabolomes between new-onset paediatric IBD patients and symptomatic non-IBD controls, and correlated plasma inflammation markers and disease characteristics with the altered metabolites. Methods Paired colonic and ileal biopsies and plasma from 67 treatment-naïve children with incident Crohn’s disease [CD; n = 47], ulcerative colitis [UC; n = 9], and non-IBD controls [n = 11] were analysed using ultra-performance liquid chromatography-mass spectrometry [UPLC-MS/MS]. Inflammatory plasma proteins [n = 92] were assessed. Results The metabolomes in inflamed mucosal biopsies differed between IBD patients and controls. In CD, mucosal levels of several lysophospholipids [lysophosphatidylcholines, lysophosphatidyletanolamines, lysophosphatidylinositols, and lysophosphatidylserines] were decreased, correlating with various plasma metabolites including amino acid analogues and N-acetylated compounds. In both CD and UC, mucosal sphingolipids, including ceramide [d18:2/24:1, d18:1/24:2], lactosyl-N-palmitoyl-sphingosine [d18:1/16:0], behenoyl sphingomyelin [d18:1/22:0], lignoceroyl sphingomyelin [d18:1/24:0], and/or sphingomyelin [d18:1/24:1, d18:2/24:0] were increased, correlating with sphingolipids, bile acids, and/or N-acetylated metabolites in plasma. Among proteins associated with CD, interleukin-24 correlated with plasma metabolites, including lactosyl-N-palmitoyl sphingosine [d18:1/16:0] and phosphatidyletanolamine [18:1/18:1], haemoglobin, and faecal calprotectin. In UC, interleukin-24, interleukin-17A, and C-C motif chemokine 11 correlated with several plasma metabolites, including N-acetyltryptophan, tryptophan, glycerate, and threonate, and with the Paediatric Ulcerative Colitis Activity Index, C-reactive protein, and faecal calprotectin. Conclusions Mucosal perturbations of lysophospholipids and sphingolipids characterised the metabolome in new-onset paediatric IBD and correlated with plasma metabolites. By integrating plasma metabolomics data with inflammatory proteins and clinical data, we identified clinical and inflammatory markers associated with metabolomic signatures for IBD

C59S/C64S mutant SecTRAPs cannot induce cell death in A549 cells.

<p>A549 cells were treated with 100 ng of the C59S/C64S mutant rat TrxR or native rat TrxR1 preparations, with or without cisplatin (CDDP) derivatization of the Sec residue, using <i>BioPORTER,</i> as indicated in the figure and described further in the text. Cell death was significantly increased after treatment with truncated TrxR or TrxR1 derivatized with cisplatin compared to control using only <i>BioPORTER,</i> whereas the other proteins gave no significant difference in cell death compared to the control (n.s., p>0.05; *, p<0.05; **, p<0.01).</p

Either α-tocopherol or ascorbic acid can prevent cell killing by SecTRAPs but not the combinatory treatment.

<p>A549 cells were preincubated for 1 h with α-tocopherol (100 µM), ascorbic acid (100 µM) or a combination of the two compounds, as indicated in the figure. SecTRAP/<i>BioPORTER</i>-complex was subsequently added to the cells, which were then incubated for additional 4 h before analysis of cell death as described in the text. A significant increase in cell death compared to non-treated cells (***, p<0.001) or cells treated with only <i>BioPORTER</i> (##, p<0.01, ###, p<0.001) was seen in cells treated with SecTRAPs either in absence of the antioxidant compounds or together with the combination of both α-tocopherol and ascorbic acid. All other treatments lacked a significant difference in cell death compared to either of the two controls (p>0.05).</p