Plasma concentrations of thioredoxin, thioredoxin reductase and peroxiredoxin-4 can identify high risk patients and predict outcome in patients with acute coronary syndrome: A clinical observation.

BACKGROUND: Oxidative stress is a pathological feature of acute coronary syndrome (ACS), a complex disease with varying clinical outcomes. Surrogate biomarkers of oxidative stress including, peroxiredoxin-2 (PRDX2), PRDX4, thioredoxin (TRX) and thioredoxin reductase (TRXR) were measured in ACS patients at presentation and follow-up, to assess their clinical utility in diagnosis and risk stratification. METHODS: Plasma from 145 participants (80 ACS and 65 healthy) at diagnosis, 1-3 month (first) and 6-month follow-up (second) was analysed by ELISA. ACS patients were monitored for 12-months. RESULTS: ACS patients at diagnosis had significantly higher concentrations of TRX (p < 0.05), TRXR (p < 0.01) and PRDX4 (p < 0.01), compared to healthy donors. This was increase was driven by non-ST elevated myocardial infarction for TRX (p < 0.01) and PRDX4 (p < 0.05). For TRXR, ACS females were significantly higher than males (p < 0.05). TRX was also higher in older females (>55 years) at diagnosis (p < 0.05). At first follow-up, TRX had lowered, whereas PRDX4 remained significantly high (p < 0.05). Stratification of ACS patients according to percutaneous coronary intervention (PCI) revealed that TRXR was significantly higher in patients receiving PCI to the right coronary artery (p < 0.05). Whereas both TRXR (p < 0.01) and PRDX4 (p < 0.01) were significantly higher in patients receiving PCI to the left anterior descending (LAD) artery. ACS patients who had plasma TRX >13.40 ng/ml at second follow-up were at high risk of readmission (p < 0.05), as were patients with TRXR of <1000 pg/ml at diagnosis having PCI to the LAD (p < 0.05). CONCLUSION: This study indicates that TRX, TRXR and PRDX4 may have clinical utility for ACS stratification.

The BCAT1 CXXC Motif Provides Protection against ROS in Acute Myeloid Leukaemia Cells.

The cytosolic branched-chain aminotransferase (BCAT1) has received attention for its role in myeloid leukaemia development, where studies indicate metabolic adaptations due to BCAT1 up-regulation. BCAT1, like the mitochondria isoform (BCAT2), shares a conserved CXXC motif ~10 Å from the active site. This CXXC motif has been shown to act as a 'redox-switch' in the enzymatic regulation of the BCAT proteins, however the response to reactive oxygen species (ROS) differs between BCAT isoforms. Studies indicate that the BCAT1 CXXC motif is several orders of magnitude less sensitive to the effects of ROS compared with BCAT2. Moreover, estimation of the reduction mid-point potential of BCAT1, indicates that BCAT1 is more reductive in nature and may possess antioxidant properties. Therefore, the aim of this study was to further characterise the BCAT1 CXXC motif and evaluate its role in acute myeloid leukaemia. Our biochemical analyses show that purified wild-type (WT) BCAT1 protein could metabolise H2O2 in vitro, whereas CXXC motif mutant or WT BCAT2 could not, demonstrating for the first time a novel antioxidant role for the BCAT1 CXXC motif. Transformed U937 AML cells over-expressing WT BCAT1, showed lower levels of intracellular ROS compared with cells over-expressing the CXXC motif mutant (CXXS) or Vector Controls, indicating that the BCAT1 CXXC motif may buffer intracellular ROS, impacting on cell proliferation. U937 AML cells over-expressing WT BCAT1 displayed less cellular differentiation, as observed by a reduction of the myeloid markers; CD11b, CD14, CD68, and CD36. This finding suggests a role for the BCAT1 CXXC motif in cell development, which is an important pathological feature of myeloid leukaemia, a disease characterised by a block in myeloid differentiation. Furthermore, WT BCAT1 cells were more resistant to apoptosis compared with CXXS BCAT1 cells, an important observation given the role of ROS in apoptotic signalling and myeloid leukaemia development. Since CD36 has been shown to be Nrf2 regulated, we investigated the expression of the Nrf2 regulated gene, TrxRD1. Our data show that the expression of TrxRD1 was downregulated in transformed U937 AML cells overexpressing WT BCAT1, which taken with the reduction in CD36 implicates less Nrf2 activation. Therefore, this finding may implicate the BCAT1 CXXC motif in wider cellular redox-mediated processes. Altogether, this study provides the first evidence to suggest that the BCAT1 CXXC motif may contribute to the buffering of ROS levels inside AML cells, which may impact ROS-mediated processes in the development of myeloid leukaemia.

Use of an anti-CD200-blocking antibody improves immune responses to AML in vitro and in vivo.

Treatment of relapsed/resistant acute myeloid leukaemia (AML) remains a significant area of unmet patient need, the outlook for most patients remaining extremely poor. A promising approach is to augment the anti-tumour immune response in these patients; most cancers do not activate immune effector cells because they express immunosuppressive ligands. We have previously shown that CD200 (an immunosuppressive ligand) is overexpressed in AML and confers an inferior overall survival compared to CD200low/neg patients. Here we show that a fully human anti-CD200 antibody (TTI-CD200) can block the interaction of CD200 with its receptor and restore AML immune responses in vitro and in vivo.

High intensity interval exercise increases the frequency of peripheral PD-1+ CD8+ central memory T-cells and soluble PD-L1 in humans.

Exercise can exert anti-inflammatory effects in an intensity-dependent manner; however, the mechanisms mediating these effects are continually being established. Programme Death Receptor-1 (PD-1) is a membrane bound receptor that maintains immune tolerance by dampening immune cell interactions, such as those mediated by cytotoxic T-cell lymphocytes (CD8+). The aim of this study was to characterise sub-populations of CD8+ T-cells with regards to their expression of PD-1 before and immediately after exercise. Interleukin (IL)-6, soluble PD-1 (sPD-1) and its ligand (sPD-L1) were also quantified in plasma. Eight individuals (mean â€‹± â€‹SD: age 29 â€‹± â€‹5 years; BMI 24.2 â€‹± â€‹3.4 â€‹kg â€‹m2; V ˙ O2max 44.5 â€‹± â€‹6.4 â€‹ml â€‹kg-1·min-1) undertook two time and energy-matched cycling bouts in a counterbalanced study design: one of moderate intensity (MOD) and a bout of high intensity interval exercise (HIIE). Both MOD and HIIE increased the number, but not the proportion of circulating CD8+ PD-1+ cells, with no differences between trials. Within the CD8+ PD-1+ pool, the expression of PD-1 increased on central memory cells following HIIE only (fold change: MOD 1.0 vs HIIE +1.4), as well the concentration of CD8+PD-1+ memory cells within the circulation (cells/uL: MOD -0.4 vs HIIE +5.8). This response composed a very small part of the exercise-induced CD8+ lymphocytosis (Pre-Ex: 0.38% to Post-Ex: 0.69%; p â€‹> â€‹0.05). sPD-L1 and IL-6 concentration increased in tandem following MOD and HIIE (r â€‹= â€‹0.57; P â€‹= â€‹0.021), with a reciprocal decline in sPD-1 observed. The current data demonstrate that PD-1+ CD8+ lymphocytes were mobilised following both MOD and HIIE. Both the number of central memory CD8+ T-cells expressing PD-1 and the expression level on these cells were increased following HIIE only. This intensity-dependent phenotypic response, in conjunction with increased circulatory sPD-L1 may represent an aspect of the anti-inflammatory response to exercise and warrants further investigation.

In Vitro Evaluation of the Inhibitory Effect of Topical Ophthalmic Agents on Acanthamoeba Viability.

PURPOSE: To compare the antimicrobial effect of topical anesthetics, antivirals, antibiotics, and biocides on the viability of Acanthamoeba cysts and trophozoites in vitro. METHODS: Amoebicidal and cysticidal assays were performed against both trophozoites and cysts of Acanthamoeba castellanii (ATCC 50370) and Acanthamoeba polyphaga (ATCC 30461). Test agents included topical ophthalmic preparations of common anesthetics, antivirals, antibiotics, and biocides. Organisms were exposed to serial two-fold dilutions of the test compounds in the wells of a microtiter plate to examine the effect on Acanthamoeba spp. In addition, the toxicity of each of the test compounds was determined against a mammalian cell line. RESULTS: Proxymetacaine, oxybuprocaine, and especially tetracaine were all toxic to the trophozoites and cysts of Acanthamoeba spp., but lidocaine was well tolerated. The presence of the benzalkonium chloride (BAC) preservative in levofloxacin caused a high level of toxicity to trophozoites and cysts. With the diamidines, the presence of BAC in the propamidine drops was responsible for the activity against Acanthamoeba spp. Hexamidine drops without BAC showed good activity against trophozoites, and the biguanides polyhexamethylene biguanide, chlorhexidine, alexidine, and octenidine all showed excellent activity against trophozoites and cysts of both species. CONCLUSIONS: The antiamoebic effects of BAC, povidone iodine, and tetracaine are superior to the current diamidines and slightly inferior to the biguanides used in the treatment for Acanthamoeba keratitis. TRANSLATIONAL RELEVANCE: Ophthalmologists should be aware that certain topical anesthetics and ophthalmic preparations containing BAC prior to specimen sampling may affect the viability of Acanthamoeba spp. in vivo, resulting in false-negative results in diagnostic tests.

Characterization of extracellular redox enzyme concentrations in response to exercise in humans.

Redox enzymes modulate intracellular redox balance and are secreted in response to cellular oxidative stress, potentially modulating systemic inflammation. Both aerobic and resistance exercise are known to cause acute systemic oxidative stress and inflammation; however, how redox enzyme concentrations alter in extracellular fluids following bouts of either type of exercise is unknown. Recreationally active men (n = 26, mean ± SD: age 28 ± 8 yr) took part in either: 1) two separate energy-matched cycling bouts: one of moderate intensity (MOD) and a bout of high intensity interval exercise (HIIE) or 2) an eccentric-based resistance exercise protocol (RES). Alterations in plasma (study 1) and serum (study 2) peroxiredoxin (PRDX)-2, PRDX-4, superoxide dismutase-3 (SOD3), thioredoxin (TRX-1), TRX-reductase and interleukin (IL)-6 were assessed before and at various timepoints after exercise. There was a significant increase in SOD3 (+1.5 ng/mL) and PRDX-4 (+5.9 ng/mL) concentration following HIIE only, peaking at 30- and 60-min post-exercise respectively. TRX-R decreased immediately and 60 min following HIIE (-7.3 ng/mL) and MOD (-8.6 ng/mL), respectively. In non-resistance trained men, no significant changes in redox enzyme concentrations were observed up to 48 h following RES, despite significant muscle damage. IL-6 concentration increased in response to all trials, however there was no significant relationship between absolute or exercise-induced changes in redox enzyme concentrations. These results collectively suggest that HIIE, but not MOD or RES increase the extracellular concentration of PRDX-4 and SOD3. Exercise-induced changes in redox enzyme concentrations do not appear to directly relate to systemic changes in IL-6 concentration.NEW & NOTEWORTHY Two studies were conducted to characterize changes in redox enzyme concentrations after single bouts of exercise to investigate the emerging association between extracellular redox enzymes and inflammation. We provide evidence that SOD3 and PRDX-4 concentration increased following high-intensity aerobic but not eccentric-based resistance exercise. Changes were not associated with IL-6. The results provide a platform to investigate the utility of SOD3 and PRDX-4 as biomarkers of oxidative stress following exercise.

Using Flow Cytometry to Detect and Measure Intracellular Thiol Redox Status in Viable T Cells from Heterogeneous Populations.

Increased production of reactive oxygen species (ROS) and deficiencies in cellular antioxidant defenses are the principal causes of cellular oxidative stress. ROS can react with a variety intracellular molecules, including redox active cysteine thiols (-SH) within proteins. Cysteine thiols can occupy several redox states and conversion between them is highly dynamic during, for example, cell growth, resulting in modification and subsequent loss of the "reduced thiol" form (-SH or -S-). The challenge lies with detecting and measuring thiol redox status inside viable heterogeneous cell populations (e.g., peripheral blood mononuclear cells (PBMCs)). Here we describe a flow cytometric approach for the evaluation of intracellular thiol redox status in human CD3+ T cells within a viable PBMC preparation. Using the thiol reactive probe, fluorescein-5 maleimide (F5M), we demonstrate that loss of reduced intracellular thiol correlates with a decrease in F5M fluorescence. We also detected a loss of F5M fluorescence in Jurkat cell cultures exposed to exogenous H2O2 generated by glucose oxidase. Since F5M binds irreversibly to reduced cysteine thiols, cells may be sorted based on F5M fluorescence intensity and redox active proteins can subsequently be extracted and separated using SDS-PAGE. This final step facilitates identification of redox active proteins from individual cell populations in live heterogeneous cell mixes using proteomic analysis.

Detecting intracellular thiol redox state in leukaemia and heterogeneous immune cell populations: An optimised protocol for digital flow cytometers.

Flow cytometric methods for detecting and quantifying reduced intracellular thiol content using fluorescein-5-maleimide (F5M) in viable eukaryotic cells date back to 1983 (Durand and Olive [1]). There has been little development in these methodologies since that time, a period that has witnessed huge technological advances, particularly with the emergence of digital multi-parameter flow cytometric systems. Concurrent advancement in our understanding of redox regulation within eukaryotic cellular systems has also followed, whereby it is now accepted that cysteine thiols partake in redox reactions, which regulate protein activity and function (Groitl and Jakob (2014), Won et al. (2012)). Moreover, we are at the dawn of a new era in redox biology whereby the importance of 'reductive stress' in eukaryotic cellular systems is gathering momentum (Wadley et al. (2018) [4]). It is therefore critical that methods be continually advanced to better understand these concepts in more detail at the cellular level. Flow cytometry is a powerful technique that may be used for this purpose. Henceforth we have rejuvenated these methods to address modern scientific questions. In this paper, essential detail is provided on: •The adaption of a protocol initially described by Durand and Olive [1] for use with modern digital flow cytometer configurations. Here we provide optimal conditions for labelling intracellular thiols with F5M for detection using digital flow cytometers. Our modifications avoid the use of methanol fixation thus preserving cell viability in single cell suspension cultures.•Demonstration that flow cytometry can detect the gain and loss of reduced intracellular thiols in cells exposed to physiological doses of hydrogen peroxide mediated by glucose oxidase (Hole et al. (2013) [5]).•Validation of F5M protein labelling by coupling method to confocal microscopy and downstream proteomics, thus permitting a powerful experimental platform for potential use with next generation flow cytometry e.g. CyTOF (Lin and Maecker (2018) [6]).

Preliminary evidence of reductive stress in human cytotoxic T cells following exercise.

This study investigated immunophenotypic differences in intracellular thiol redox state of peripheral blood mononuclear cells (PBMCs) isolated from trained [ n = 9, means ± SD: age 28 ± 5 yr; (body mass index) BMI 23.2 ± 2.6 kg/m2; V̇o2max (maximal oxygen intake)56.9 ± 6.1 ml·kg-1·min-1] and recreationally active (RA, n = 11, means ± SD: age 27 ± 6 yr; BMI 24.2 ± 3.7 kg/m2; V̇o2max 45.1 ± 6.4 ml·kg-1·min-1) participants before and after a maximal aerobic exercise tolerance test. Blood samples were taken before (Pre), during (sample acquired at 70% maximum heart rate), immediately after (Post + 0), and 15 min postexercise (Post + 15). PBMCs were isolated, and reduced thiol analysis [fluorescein-5 maleimide (F5M)] by immunophenotype [cluster of differentiation (CD)3+, CD4+, and CD8+] was performed using flow cytometry. A significant increase in cellular F5M fluorescence was observed in CD3+ T cells at Post + 0, with changes driven to a greater extent by CD8+ T cells (fold change in both groups CD4: +2.3, CD8: +2.8; P < 0.05). Further analysis revealed a population of highly reduced CD8+ T cells (CD8+T-reduced+) that significantly increased from Pre to Post + 0 in RA participants only (RA: +272 cell/µl, P < 0.05). To understand these results further, CD8+T-reduced+ and CD8+T-reduced- cells were analyzed for immunophenotype in response to the same exercise protocol ( n = 6, means ± SD: age 24 ± 5 yr; BMI 25.7 ± 4.1 kg·m-2; V̇o2max 41.33 ± 7.63 ml·kg-1·min-1). CD8+T-reduced+ had significantly less lymphoid homing potential (chemokine receptor type 7) Post + 0 compared with Pre. This study is the first, to our knowledge, to demonstrate that lymphocyte populations become more reductive in response to acute exercise. NEW & NOTEWORTHY The study presented provides the first evidence to suggest that cytotoxic T cells become transiently reductive in healthy individuals following a single bout of cycling. Detection of these cells was enabled via the use of a flow cytometric assay that incorporates the thiol reactive probe fluorescein-5 maleimide. Using this method, transient reductive stress in viable T cells is permissible and provides the basis for further research in the area of exercise immunology.

Questioning diagnoses in clinical practice: a thematic analysis of clinical psychologists' accounts of working beyond diagnosis in the United Kingdom.

BACKGROUND: The British Psychological Society proposes that clinical psychologists are well placed to move beyond psychiatric diagnoses and develop alternative practices. AIMS: This study sought to explore what the application of these guiding principles looks like in clinical practice, the challenges faced and possible routes forward. METHODS: A purpose-designed survey was completed by 305 respondents and a thematic analysis completed. RESULTS: Thematic analysis was used to identify five superordinate themes relating to individuals, relational, others, structures and society, comprising of a total of 21 group themes. The presented group themes highlight an array of approaches to practicing beyond diagnosis and factors that help and hinder such action; from scaffolding change, becoming leaders, relating to the multi-disciplinary team, restructuring services and the processes of change. A key concept was "playing the diagnostic game". CONCLUSIONS: "Playing the diagnostic game" enables psychologists to manage an array of tensions and anxieties: conflicts between belief and practice, relationships with colleagues, and dilemmas of position and power. It also potentially limits a concerted questioning of diagnosis and consideration of alternatives. An alternative conceptual framework for non-diagnostic practice is needed to aid the collective efforts of clinical psychologists developing their practice beyond diagnosis, some of which have been highlighted in this study. Until then, ways of mitigating the perceived threats to questioning diagnosis need further exploration, theorising and backing.

An unexplored role for Peroxiredoxin in exercise-induced redox signalling?

Peroxiredoxin (PRDX) is a ubiquitous oxidoreductase protein with a conserved ionised thiol that permits catalysis of hydrogen peroxide (H2O2) up to a million times faster than any thiol-containing signalling protein. The increased production of H2O2 within active tissues during exercise is thought to oxidise conserved cysteine thiols, which may in turn facilitate a wide variety of physiological adaptations. The precise mechanisms linking H2O2 with the oxidation of signalling thiol proteins (phosphates, kinases and transcription factors) are unclear due to these proteins' low reactivity with H2O2 relative to abundant thiol peroxidases such as PRDX. Recent work has shown that following exposure to H2O2 in vitro, the sulfenic acid of the PRDX cysteine can form mixed disulphides with transcription factors associated with cell survival. This implicates PRDX as an 'active' redox relay in transmitting the oxidising equivalent of H2O2 to downstream proteins. Furthermore, under oxidative stress, PRDX can form stable oxidised dimers that can be secreted into the extracellular space, potentially acting as an extracellular 'stress' signal. There is extensive literature assessing non-specific markers of oxidative stress in response to exercise, however the PRDX catalytic cycle may offer a more robust approach for measuring changes in redox balance following exercise. This review discusses studies assessing PRDX-mediated cellular signalling and integrates the recent advances in redox biology with investigations that have examined the role of PRDX during exercise in humans and animals. Future studies should explore the role of PRDX as a key regulator of peroxide mediated-signal transduction during exercise in humans.

Differential redox potential between the human cytosolic and mitochondrial branched-chain aminotransferase.

The human branched-chain aminotransferase (hBCAT) isoenzymes are CXXC motif redox sensitive homodimers central to glutamate metabolism in the central nervous system. These proteins respond differently to oxidation by H(2)O(2), NO, and S-glutathionylation, suggesting that the redox potential is distinct between isoenzymes. Using various reduced to oxidized glutathione ratios (GSH:GSSG) to alter the redox environment, we demonstrate that hBCATc (cytosolic) has an overall redox potential that is 30 mV lower than hBCATm (mitochondrial). Furthermore, the CXXC motif of hBCATc was estimated to be 80 mV lower, suggesting that hBCATm is more oxidizing in nature. Western blot analysis revealed close correlations between hBCAT S-glutathionylation and the redox status of the assay environment, offering the hBCAT isoenzymes as novel biomarkers for cytosolic and mitochondrial oxidative stress.

The topoisomerase II inhibitor voreloxin causes cell cycle arrest and apoptosis in myeloid leukemia cells and acts in synergy with cytarabine.

BACKGROUND: Topoisomerase II is essential for the maintenance of DNA integrity and the survival of proliferating cells. Topoisomerase II poisons, including etoposide and doxorubicin, inhibit enzyme-mediated DNA ligation causing the accumulation of double-stranded breaks and have been front-line drugs for the treatment of leukemia for many years. Voreloxin is a first-in-class anti-cancer quinolone derivative that intercalates DNA and inhibits topoisomerase II. The efficacy and mechanisms of action of voreloxin in acute myeloid leukaemia were addressed in this study. DESIGN AND METHODS: Primary acute myeloid leukemia blasts (n = 88) and myeloid cell lines were used in vitro to study voreloxin through viability assays to assess cell killing and synergy with other drugs. Apoptosis and cell cycling were assessed by flow cytometry. DNA relaxation assays were utilized to determine that voreloxin was active on topoisomerase II. RESULTS: The mean lethal dose 50% (LD(50)) (± standard deviation) of voreloxin for primary acute myeloid leukemia blasts was 2.30 µM (± 1.87). Synergy experiments between voreloxin and cytarabine identified synergism in 22 of 25 primary acute myeloid leukemia samples tested, with a mean combination index of 0.79. Apoptosis was shown to increase in a dose-dependent manner. Furthermore, voreloxin was active in the p53-null K562 cell line suggesting that the action of voreloxin is not affected by p53 status. The action of voreloxin on topoisomerase II was confirmed using a DNA relaxation assay. CONCLUSIONS: Voreloxin may provide an interesting addition to the cache of drugs available for the treatment of acute myeloid leukemia, a disease with a poor long-term survival. In addition to its potent action as a single agent in dividing cells, the synergy we demonstrated between voreloxin and cytarabine recommends further investigation of this topoisomerase II inhibitor.

S-Nitrosoglutathione inactivation of the mitochondrial and cytosolic BCAT proteins: S-nitrosation and S-thiolation.

Specific proteins with reactive thiol(ate) groups are susceptible to nitric oxide (NO) modification, which can result in S-nitrosation, S-thiolation, or disulfide bond formation. In the present study the effect of NO modification on the functionality of human mitochondrial and cytosolic branched-chain aminotransferases (hBCATm and hBCATc, respectively) was investigated. Here, the NO reactive agents, S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-dl-penacillamine, and sodium nitroprusside, inactivated both isoforms in a dose-dependent manner. Furthermore, low concentrations of GSNO caused a time-dependent loss in BCAT activity (50 +/- 3% and 77 +/- 2% for hBCATc and hBCATm, respectively) correlating with the loss of four and one to two thiol groups, respectively, confirming the thiols as targets for NO modification. Analysis of GSNO-modified hBCATc by quadrupole time-of-flight mass spectrometry identified a major peak containing three NO adducts and a minor peak equivalent to two NO adducts and one glutathione (GSH) molecule, the latter confirmed by Western blot analysis. Moreover, prolonged exposure or increased levels of GSNO caused increased S-glutathionylation and partial dimerization of hBCATc, suggesting a possible shift from regulation by NO to one of adaptation during nitrosated stress. Although GSNO inactivated hBCATm, neither S-nitrosation, S-glutathionylation, nor dimerization could be detected, suggesting differential mechanisms of regulation through NO between isoforms in the mitochondria and cytosol. Reversal of GSNO-modified hBCAT using GSH alone was only partial, and complete reactivation was only possible using the glutaredoxin/GSH system (97 +/- 4% and 91 +/- 3% for hBCATc and hBCATm, respectively), implicating the importance of a full physiological redox system for activation/inactivation. To conclude, these results clearly demonstrate distinct functional/mechanistic responses to GSNO modification between BCAT isoforms and offer intriguing comparisons between the BCAT proteins and the respective cytosolic and mitochondrial hTrx and hGrx proteins.

Regulatory control of human cytosolic branched-chain aminotransferase by oxidation and S-glutathionylation and its interactions with redox sensitive neuronal proteins.

Redox regulation of proteins through oxidation and S-thiolation are important regulatory processes, acting in both a protective and adaptive role in the cell. In the current study, we investigated the sensitivity of the neuronal human cytosolic branched-chain aminotransferase (hBCATc) protein to oxidation and S-thiolation, with particular attention focused on functionality and modulation of its CXXC motif. Thiol specific reagents showed significant redox cycling between the reactive thiols and the TNB anion, and using NEM, four of the six reactive thiols are critical to the functionality of hBCATc. Site-directed mutagenesis studies supported these findings where a reduced kcat (ranging from 50-70% of hBCATc) for C335S, C338S, C335/8S, and C221S, respectively, followed by a modest effect on C242S was observed. However, only the thiols of the CXXC motif (C335 and C338) were directly involved in the reversible redox regulation of hBCATc through oxidation (with a loss of 40-45% BCAT activity on air oxidation alone). Concurrent with these findings, under air oxidation, the X-ray crystallography structure of hBCATc showed a disulphide bond between C335 and C338. Further oxidation of the other four thiols was not evident until levels of hydrogen peroxide were elevated. S-thiolation experiments of hBCATc exposed to GSH provided evidence for significant recycling between GSH and the thiols of hBCATc, which implied that under reducing conditions GSH was operating as a thiol donor with minimal S-glutathionylation. Western blot analysis of WT hBCATc and mutant proteins showed that as the ratio of GSH:GSSG decreased significant S-glutathionylation occurred (with a further loss of 20% BCAT activity), preferentially at the thiols of the CXXC motif, suggesting a shift in function toward a more protective role for GSH. Furthermore, the extent of S-glutathionylation increased in response to oxidative stress induced by hydrogen peroxide potentially through a C335 sulfenic acid intermediate. Deglutathionylation of hBCATc-SSG using the GSH/glutaredoxin system provides evidence that this protein may play an important role in cellular redox regulation. Moreover, redox associations between hBCATc and several neuronal proteins were identified using targeted proteomics. Thus, our data provides strong evidence that the reactive thiol groups, in particular the thiols of the CXXC motif, play an integral role in redox regulation and that hBCATc has redox mediated associations with several neuronal proteins involved in G-protein cell signaling, indicating a novel role for hBCATc in cellular redox control.

Redox regulation and trapping sulfenic acid in the peroxide-sensitive human mitochondrial branched chain aminotransferase.

The human branched chain aminotransferase enzymes are key regulators of glutamate metabolism in the brain and are among a growing number of redox-sensitive proteins. Studies that use thiol-specific reagents and electrospray ionization mass spectrometry demonstrate that the mitochondrial BCAT enzyme has a redox-active CXXC center, which on oxidation forms a disulfide bond (RSSR), via a cysteine sulfenic acid intermediate. Mechanistic details of this redox regulation were revealed by the use of mass spectrometry and dimedone modification. We discovered that the thiol group at position C315 of the CXXC motif acts a redox sensor, whereas the thiol group at position C318 permits reversible regulation by forming an intrasubunit disulphide bond. Because of their roles in redox regulation and catalysis, there is a growing interest in cysteine sulphenic acids. Therefore, development of chemical tags/methods to trap these transient intermediates is of immense importance.