Enzymology of reactive intermediate protection: kinetic analysis and temperature dependence of the mesophilic membrane protein catalyst MGST1.

Glutathione transferases (GSTs) are a class of phase II detoxifying enzymes catalysing the conjugation of glutathione (GSH) to endogenous and exogenous electrophilic molecules, with microsomal glutathione transferase 1 (MGST1) being one of its key members. MGST1 forms a homotrimer displaying third-of-the-sites-reactivity and up to 30-fold activation through modification of its Cys-49 residue. It has been shown that the steady-state behaviour of the enzyme at 5 °C can be accounted for by its pre-steady-state behaviour if the presence of a natively activated subpopulation (~ 10%) is assumed. Low temperature was used as the ligand-free enzyme is unstable at higher temperatures. Here, we overcame enzyme lability through stop-flow limited turnover analysis, whereby kinetic parameters at 30 °C were obtained. The acquired data are more physiologically relevant and enable confirmation of the previously established enzyme mechanism (at 5 °C), yielding parameters relevant for in vivo modelling. Interestingly, the kinetic parameter defining toxicant metabolism, kcat /KM , is strongly dependent on substrate reactivity (Hammett value 4.2), underscoring that glutathione transferases function as efficient and responsive interception catalysts. The temperature behaviour of the enzyme was also analysed. Both the KM and KD values decreased with increasing temperature, while the chemical step k3 displayed modest temperature dependence (Q10 : 1.1-1.2), mirrored in that of the nonenzymatic reaction (Q10 : 1.1-1.7). Unusually high Q10 values for GSH thiolate anion formation (k2 : 3.9), kcat (2.7-5.6) and kcat /KM (3.4-5.9) support that large structural transitions govern GSH binding and deprotonation, which limits steady-state catalysis.

Resin Infiltration of Non-Cavitated Proximal Caries Lesions in Primary and Permanent Teeth: A Systematic Review and Scenario Analysis of Randomized Controlled Trials.

The present study aimed to meta-analyze and evaluate the certainty of evidence for resin infiltration of proximal carious lesions in primary and permanent teeth. While resin infiltration has been shown efficacious for caries management, the certainty of evidence remains unclear. The protocol was registered with PROSPERO (CRD42018080895), and PRISMA guidelines have been followed. The databases PubMed, Embase, and Cochrane CENTRAL were systematically screened, complemented by hand searches and cross-referencing. Eleven relevant articles were identified and included, i.e., randomized controlled trials (RCTs) comparing the progression of resin infiltrated proximal caries lesions (combined with non-invasive measures) in primary or permanent teeth with non-invasive measures. Random-effects meta-analyses and trial sequential analyses (TSA) were performed for per-protocol (PP), intention-to-treat (ITT), and best/worst case (BC/WC) scenarios. Six included trials assessed lesions in permanent teeth and five trails assessed lesions in primary teeth. The trials had a high or unclear risk of bias. Risk of caries progression was significantly reduced for infiltrated lesions in the PP, ITT, and BC scenarios in both permanent teeth and primary teeth, but not in the WC scenario. According to the TSA, firm evidence was reached for all of the scenarios except the WC. In conclusion, there is firm evidence for resin infiltration arresting proximal caries lesions in permanent and primary teeth.

Cost-effectiveness and efficacy of fluoride varnish for caries prevention in South African children: A cluster-randomized controlled community trial.

OBJECTIVES: This cluster-randomized controlled community trial aimed to assess the efficacy and costs of fluoride varnish (FV) application for caries prevention in a high-risk population in South Africa. METHODS: 513 children aged 4-8 years from two schools in a township in South Africa were randomly allocated by class to the FV or Control (CO) groups. In addition to supervised toothbrushing with fluoridated toothpaste in both groups, FV was applied in 3-month intervals by trained local non-professional assistants. Intraoral examinations were conducted at baseline, 12, 21 and 24 months. Primary outcome was the increment of teeth with cavitated lesions (i.e. newly developed or progressed, formerly non-cavitated lesions), requiring restoration or extraction over the study period. Additionally, treatment and re-treatment costs were analyzed. RESULTS: 513 children (d1-4 mft 5.9 ± 4.3 (mean ± SD)) were randomly allocated to FV (n = 287) or CO (n = 226). 10.2% FV and CO teeth received or required a restoration; 3.9% FV and 4.1% CO teeth were extracted, without significant differences between groups. While FV generated high initial costs, follow-up costs were comparable in both groups, resulting in FV being significantly more expensive than CO (1667 ± 1055 ZAR vs. 950 ± 943 ZAR, p < .001). CONCLUSIONS: Regular FV application, in addition to daily supervised toothbrushing, had no significant caries-preventive effect and was not cost-effective in a primary school setting within a peri-urban, high-risk community in South Africa. Alternative interventions on community or public health level should be considered to reduce the caries burden in high-risk communities.

COVID-19 testing in a UK dental practice - results of a pilot study.

Objective In December 2019, a novel coronavirus (SARS-CoV-2) caused a disease outbreak that soon became a global pandemic. Dentists are potentially exposed to infectious microorganisms, including SARS-CoV-2, by virtue of the transmission routes and work environment. This study aims to determine the infection load in a dental healthcare setting during the onset of the pandemic in the UK, as well as to evaluate the effectiveness of recommended test regimens in order to estimate potential risks for caregivers and patients in a dynamically changing pandemic environment.Methods Twenty-four persons (dental personnel of one dental office and family contacts) were included in this pilot study, and their infection load was determined between March and May 2020 using antigen and antibody tests.Results Of the 24 subjects, three tested positive for SARS-CoV-2 and were quarantined accordingly. After six weeks, they tested negative for the virus, had built antibodies and had no remaining symptoms, enabling an efficient return to work.Conclusion This paper outlines the results of COVID-19 testing in a dental practice during the onset of the pandemic, and discusses possible strategies and protocols to gain certainty in the dental practice, assessing possible testing scenarios that can be performed in a primary healthcare setting.

Cross Talk in HEK293 Cells Between Nrf2, HIF, and NF-κB Activities upon Challenges with Redox Therapeutics Characterized with Single-Cell Resolution.

AIM: Many transcription factors with importance in health and disease are redox regulated. However, how their activities may be intertwined in responses to redox-perturbing stimuli is poorly understood. To enable in-depth characterization of this aspect, we here developed a methodology for simultaneous determination of nuclear factor E2-related factor 2 (Nrf2), hypoxia-inducible factor (HIF), and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation at single-cell resolution, using a new tool named pTRAF (plasmid for transcription factor reporter activation based upon fluorescence). The pTRAF allowed determination of Nrf2, HIF, and NF-κB activities in a high-resolution and high-throughput manner, and we here assessed how redox therapeutics affected the activities of these transcription factors in human embryonic kidney cells (HEK293). RESULTS: Cross talk was detected between the three signaling pathways upon some types of redox therapeutics, also by using inducers typically considered specific for Nrf2, such as sulforaphane or auranofin, hypoxia for HIF activation, or tumor necrosis factor alpha (TNFα) for NF-κB stimulation. Doxorubicin, at low nontoxic doses, potentiated TNFα-induced activation of NF-κB and HIF, without effects in stand-alone treatment. Stochastic activation patterns in cell cultures were also considerable upon challenges with several redox stimuli. INNOVATION: A novel strategy was here used to study simultaneous activation of Nrf2, HIF, and NF-κB in single cells. The method can also be adapted for studies of other transcription factors. CONCLUSION: The pTRAF provides new opportunities for in-depth studies of transcription factor activities. In this study, we found that upon challenges of cells with several redox-perturbing conditions, Nrf2, HIF, and NF-κB are uniquely responsive to separate stimuli, but can also display marked cross talk to each other within single cells. Antioxid. Redox Signal. 26, 229-246.

Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase.

Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.

Entinostat up-regulates the CAMP gene encoding LL-37 via activation of STAT3 and HIF-1α transcription factors.

Bacterial resistance against classical antibiotics is a growing problem and the development of new antibiotics is limited. Thus, novel alternatives to antibiotics are warranted. Antimicrobial peptides (AMPs) are effector molecules of innate immunity that can be induced by several compounds, including vitamin D and phenyl-butyrate (PBA). Utilizing a luciferase based assay, we recently discovered that the histone deacetylase inhibitor Entinostat is a potent inducer of the CAMP gene encoding the human cathelicidin LL-37. Here we investigate a mechanism for the induction and also find that Entinostat up-regulates human ß-defensin 1. Analysis of the CAMP promoter sequence revealed binding sites for the transcription factors STAT3 and HIF-1α. By using short hairpin RNA and selective inhibitors, we found that both transcription factors are involved in Entinostat-induced expression of LL-37. However, only HIF-1α was found to be recruited to the CAMP promoter, suggesting that Entinostat activates STAT3, which promotes transcription of CAMP by increasing the expression of HIF-1α. Finally, we provide in vivo relevance to our findings by showing that Entinostat-elicited LL-37 expression was impaired in macrophages from a patient with a STAT3-mutation. Combined, our findings support a role for STAT3 and HIF-1α in the regulation of LL-37 expression.

Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells.

Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS-etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS-DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS-DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS-DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance mechanisms in cells. Here we propose that GST-dependent prodrugs require a conversion rate "window" in order to selectively target GST overexpressing cells, while limiting their effects on normal cells. Prodrugs are furthermore a suitable system to specifically target GSTs and to overcome various drug resistance mechanisms that apply to the parental drug.

TrxR1 as a potent regulator of the Nrf2-Keap1 response system.

SIGNIFICANCE: All cells must maintain a balance between oxidants and reductants, while allowing for fluctuations in redox states triggered by signaling, altered metabolic flow, or extracellular stimuli. Furthermore, they must be able to rapidly sense and react to various challenges that would disrupt the redox homeostasis. RECENT ADVANCES: Many studies have identified Keap1 as a key sensor for oxidative or electrophilic stress, with modification of Keap1 by oxidation or electrophiles triggering Nrf2-mediated transcriptional induction of enzymes supporting reductive and detoxification pathways. However, additional mechanisms for Nrf2 regulation are likely to exist upstream of, or in parallel with, Keap1. CRITICAL ISSUES: Here, we propose that the mammalian selenoprotein thioredoxin reductase 1 (TrxR1) is a potent regulator of Nrf2. A high chemical reactivity of TrxR1 and its vital role for the thioredoxin (Trx) system distinguishes TrxR1 as a prime target for electrophilic challenges. Chemical modification of the selenocysteine (Sec) in TrxR1 by electrophiles leads to rapid inhibition of thioredoxin disulfide reductase activity, often combined with induction of NADPH oxidase activity of the derivatized enzyme, thereby affecting many downstream redox pathways. The notion of TrxR1 as a regulator of Nrf2 is supported by many publications on effects in human cells of selenium deficiency, oxidative stress or electrophile exposure, as well as the phenotypes of genetic mouse models. FUTURE DIRECTIONS: Investigation of the role of TrxR1 as a regulator of Nrf2 activation will facilitate further studies of redox control in diverse cells and tissues of mammals, and possibly also in animals of other classes.

Thioredoxin-related protein of 14 kDa is an efficient L-cystine reductase and S-denitrosylase.

Thioredoxin-related protein of 14 kDa (TRP14, also called TXNDC17 for thioredoxin domain containing 17, or TXNL5 for thioredoxin-like 5) is an evolutionarily well-conserved member of the thioredoxin (Trx)-fold protein family that lacks activity with classical Trx1 substrates. However, we discovered here that human TRP14 has a high enzymatic activity in reduction of l-cystine, where the catalytic efficiency (2,217 min(-1)⋅µM(-1)) coupled to Trx reductase 1 (TrxR1) using NADPH was fivefold higher compared with Trx1 (418 min(-1)⋅µM(-1)). Moreover, the l-cystine reduction with TRP14 was in contrast to that of Trx1 fully maintained in the presence of a protein disulfide substrate of Trx1 such as insulin, suggesting that TRP14 is a more dedicated l-cystine reductase compared with Trx1. We also found that TRP14 is an efficient S-denitrosylase with similar efficiency as Trx1 in catalyzing TrxR1-dependent denitrosylation of S-nitrosylated glutathione or of HEK293 cell-derived S-nitrosoproteins. Consequently, nitrosylated and thereby inactivated caspase 3 or cathepsin B could be reactivated through either Trx1- or TRP14-catalyzed denitrosylation reactions. TRP14 was also, in contrast to Trx1, completely resistant to inactivation by high concentrations of hydrogen peroxide. The oxidoreductase activities of TRP14 thereby complement those of Trx1 and must therefore be considered for the full understanding of enzymatic control of cellular thiols and nitrosothiols.

The rare TXNRD1_v3 ("v3") splice variant of human thioredoxin reductase 1 protein is targeted to membrane rafts by N-acylation and induces filopodia independently of its redox active site integrity.

The human selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the TXNRD1 gene, is a key player in redox regulation. Alternative splicing generates several TrxR1 variants, one of which is v3 that carries an atypical N-terminal glutaredoxin domain. When overexpressed, v3 associates with membranes and triggers formation of filopodia. Here we found that membrane targeting of v3 is mediated by myristoylation and palmitoylation of its N-terminal MGC motif, through which v3 specifically targets membrane rafts. This was suggested by its localization in cholera toxin subunit B-stained membrane areas and also shown using lipid fractionation experiments. Utilizing site-directed mutant variants, we also found that v3-mediated generation of filopodia is independent of the Cys residues in its redox active site, but dependent upon its membrane raft targeting. These results identify v3 as an intricately regulated protein that expands TXNRD1-derived protein functions to the membrane raft compartment.

Noble metal targeting of thioredoxin reductase--covalent complexes with thioredoxin and thioredoxin-related protein of 14 kDa triggered by cisplatin.

Palladium (Pd), platinum (Pt), and gold (Au) are noble metals, two of which have established medical use. Pt has anticancer efficacy, predominantly as cisplatin, whereas the gold compound auranofin is used against arthritis. Both compounds inhibit the selenoprotein thioredoxin reductase (TrxR), but Pd has not been studied in this regard. Using salts of Pd, Pt, and Au as well as cisplatin and auranofin we found that Pd and Au were strikingly more potent inhibitors of recombinant TrxR1 than Pt. The TrxR-related nonselenoprotein glutathione reductase in pure form (but less so in a cellular context), as well as cellular thioredoxin (Trx) activities, were inhibited by the gold salt KAuCl(4) but were little affected by auranofin or the other compounds. In an analysis of three cancer cell lines, the extent of inhibition of TrxR activity and decrease in cell viability depended upon the choice of both noble metal and ligand and also seemed independent of p53 status. During treatment of cells with cisplatin, covalent complexes of TrxR1 with either Trx1 or TRP14 (Trx-related protein of 14kDa) were formed, as verified by Western blot analyses and mass spectrometry. These results reveal that Au and Pd are strong inhibitors of TrxR, but Pt and cisplatin trigger highly specific cellular effects on the Trx system, including covalent cross-linking of TrxR1 with Trx1 and TRP14.