Richard J. Heads

Protein Sulfenation as a Redox Sensor Proteomics Studies Using a Novel Biotinylated Dimedone Analogue

Protein sulfenic acids are reactive intermediates in the catalytic cycles of many enzymes as well as the in formation of other redox states. Sulfenic acid formation is a reversible post-translational modification with potential for protein regulation. Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is commonly used in vitro to study sulfenation of purified proteins, selectively “tagging” them, allowing monitoring by mass spectrometry. However dimedone is of little use in complex protein mixtures because selective monitoring of labeling is not possible. To address this issue, we synthesized a novel biotinylated derivative of dimedone, keeping the dione cassette required for sulfenate reactivity but adding the functionality of a biotin tag. Biotin-amido(5-methyl-5-carboxamidocyclohexane 1,3-dione) tetragol (biotin dimedone) was prepared in six steps, combining 3,5-dimethoxybenzoic acid (Birch reduction, ultimately leading to the dimedone unit with a carboxylate functionality), 1-amino-11-azido-3,6,9-trioxaundecane (a differentially substituted tetragol spacer), and biotin. We loaded biotin dimedone (0.1 mm, 30 min) into rat ventricular myocytes, treated them with H2O2 (0.1–10,000 μm, 5 min), and monitored derivatization on Western blots using streptavidin-horseradish peroxidase. There was a dose-dependent increase in labeling of multiple proteins that was maximal at 0.1 or 1 mm H2O2 and declined sharply below basal with 10 mm treatment. Cell-wide labeling was observed in fixed cells probed with avidin-FITC using a confocal fluorescence microscope. Similar H2O2-induced labeling was observed in isolated rat hearts. Hearts loaded and subjected to hypoxia showed a striking loss of labeling, which returned when oxygen was resupplied, highlighting the protein sulfenates as oxygen sensors. Cardiac proteins that were sulfenated during oxidative stress were purified with avidin-agarose and identified by separation of tryptic digests by liquid chromatography with on-line analysis by mass spectrometry.

The role of differential activation of p38-mitogen-activated protein kinase in preconditioned ventricular myocytes

Activation of protein kinase C (PKC) and more recently mitogen‐activated protein kinases (MAPKs) have been associated with the cardioprotective effect of ischemic preconditioning. We examined the interplay between these kinases in a characterized model of ischemic preconditioning in cultured rat neonatal ventricular cardiocytes where ectopic expression of active PKC‐δ results in protection. Two members of the MAPK family, p38 and p42/44, were activated transiently during preconditioning by brief simulated ischemia/reoxygenation. Overexpression of active PKC‐δ, rather than augmenting, completely abolished this activation. We therefore determined whether a similar process occurred during lethal prolonged simulated ischemia. In contrast to ischemia, brief, lethal‐simulated ischemia activated only p38 (2.8±0.45 vs. basal, P<0.01), which was attenuated by expression of active PKC‐δ or by preconditioning (0.48 ±0.1 vs. ischemia, P<0.01). To determine whether reduced p38 activation was the cause or an effect of protection, we used SB203580, a p38 inhibitor. SB203580 reduced ischemic injury (CK release 38.0± 3.1%, LDH release 77.3±4.0%, and MTT bioreduction 127.1 ±4.8% of control, n=20, P<0.05). To determine whether p38 activation was isoform selective, myocytes were infected with adenoviruses encoding wild‐type p38a or p38β. Transfected p38a and β show differential activation (P<0.001) during sustained simulated ischemia, with p38a remaining activated (1.48±0.36 vs. basal) but p38β deactivated (0.36± 0.1 vs. basal, P<0.01). Prior preconditioning prevented the activation of p38a (0.65±0.11 vs. ischemia, P<0.05). Moreover, cells expressing a dominant negative p38α, which prevented ischemic p38 activation, were resistant to lethal simulated ischemia (CK release 82.9±3.9% and MTT bioreduction 130.2±6.5% of control, n=8, P<0.05). Thus, inhibition of p38α activation during ischemia reduces injury and may contribute to preconditioning‐induced cardioprotection in this model.—Saurin, A. T., Martin, J. L., Heads, R. J., Foley, C., Mockridge, J. W., Wright, M. J., Wang, Y., Marber, M. S. The role of differential activation of p38‐mitogen‐activated protein kinase in preconditioned ventricular myocytes. FASEB J. 14, 2237–2246 (2000)

Cyclophilins and their possible role in the stress response

Cyclophilins are proteins which are remarkably conserved through evolution; moreover they have been found in every possible existing organism, which indicates their fundamental importance. Due to their enzymatic properties, multiplicity, cellular localization and role in protein folding they belong to the group of proteins termed molecular chaperones. All the proteins of the cyclophilin family possess enzymatic peptidyl‐prolyl isomerase activity (PPI‐ase), which is essential to protein folding in vivo. Recently PPI‐ase activity was suggested as playing a role in regulation of transcription and differentiation. However, not all cyclophilin functions are explained by PPI‐ase activity. For instance, one of the cyclophilins plays a regulatory role in the heat shock response and the mitochondrial cyclophilin (Cyclophilin D) is an integral part of the mitochondrial permeability transition complex, which is regarded as having a crucial role in mechanisms of cell death. In support of a role in the stress response, the expression of certain cyclophilins has recently been shown to be up‐regulated under various stressful conditions. Current evidence of functional involvement of cyclophilins in various intracellular pathways is reviewed along with the indications that cyclophilin D (Cyp D) represents a crucial part of the mitochondrial permeability transition pore, which is detrimental in apoptotic and necrotic cell death. This review does not attempt to cover all the existing information related to cyclophilin family of proteins, but focus on the existing evidence of the involvement of these proteins in the intracellular stress response.

Diverse Mechanisms of Myocardial p38 Mitogen-Activated Protein Kinase Activation Evidence for MKK-Independent Activation by a TAB1-Associated Mechanism Contributing to Injury During Myocardial Ischemia

Abstract— The ischemic activation of p38&agr; mitogen-activated protein kinase (p38&agr;-MAPK) is thought to contribute to myocardial injury. Under other circumstances, activation is through dual phosphorylation by MAPK kinase 3 (MKK3). Therefore, the mkk3−/− murine heart should be protected during ischemia. In retrogradely perfused mkk3−/− and mkk3+/+ mouse hearts subjected to 30 minutes of global ischemia and 120 minutes of reperfusion, infarction/risk volume was similar (50±5 versus 51±4, P =0.93, respectively), as was intraischemic p38-MAPK phosphorylation (10 minutes ischemia as percent basal, 608±224 versus 384±104, P =0.43, respectively). This occurred despite undetectable activation of MKK3/6 in mkk3−/− hearts. However, tumor necrosis factor (TNF)-induced p38-MAPK phosphorylation was markedly diminished in mkk3−/− vs mkk3+/+ hearts (percent basal, 127±23 versus 540±267, respectively, P =0.04), suggesting an MKK-independent activation mechanism by ischemia. Hence, we examined p38-MAPK activation by TAB1-associated autophosphorylation. In wild-type mice and mkk3−/− mice, the p38-MAPK catalytic site inhibitor SB203580 (1 &mgr;mol/L) diminished phosphorylation during ischemia versus control (10 minutes ischemia as percent basal, 143±2 versus 436±96, P =0.003, and 122±25 versus 623±176, P =0.05, respectively) and reduced infarction volume (infarction/risk volume, 57±5 versus 36±3, P <0.001, and 50±5 versus 29±3, P =0.003, respectively) but did not alter TNF-induced activation, although in homogenates of ischemic hearts but not TNF-exposed hearts, p38-MAPK was associated with TAB1. Furthermore, adenovirally expressed wild-type and drug-resistant p38&agr;-MAPK, lacking the SB203580 binding site, was phosphorylated when H9c2 myoblasts were subjected to simulated ischemia. However, SB203580 (1 &mgr;mol/L) did not prevent the phosphorylation of resistant p38&agr;-MAPK. These findings suggest the ischemic activation of p38-MAPK contributing to myocardial injury is by TAB1-associated autophosphorylation.

Targeting p38-MAPK in the ischaemic heart: kill or cure?

The p38-MAPK pathway plays an important role in myocardial ischaemia/reperfusion injury and has been implicated in the regulation of cardiac gene expression, myocyte hypertrophy, inflammation, energetic metabolism, contractility, proliferation and apoptosis. The activation of p38-MAPK by dual phosphorylation during myocardial ischaemia aggravates lethal injury. However, under other circumstances activation can protect the heart, and recent evidence suggests that the mechanism of p38-MAPK activation may differ by circumstance. Determining the precise mechanism of activation during myocardial ischaemia is of considerable importance, since it may allow prevention of the detrimental, but not the beneficial, activation of p38-MAPK and lead to the identification of the relevant signalling molecules to be targeted for pharmaceutical intervention.

p42/p44-MAPK and PI3K are sufficient for IL-6 family cytokines/gp130 to signal to hypertrophy and survival in cardiomyocytes in the absence of JAK/STAT activation.

The effect of differential signalling by IL-6 and leukaemia inhibitory factor (LIF) which signal by gp130 homodimerisation or LIFRβ/gp130 heterodimerisation on survival and hypertrophy was studied in neonatal rat cardiomyocytes. Both LIF and IL-6 [in the absence of soluble IL-6 receptor (sIL-6Rα)] activated Erk1/2, JNK1/2, p38-MAPK and PI3K signalling peaking at 20 min and induced cytoprotection against simulated ischemia-reperfusion injury which was blocked by the MEK1/2 inhibitor PD98059 but not the p38-MAPK inhibitor SB203580. In the absence of sIL-6R, IL-6 did not induce STAT1/3 phosphorylation, whereas IL-6/sIL-6R and LIF induced STAT1 and STAT3 phosphorylation. Furthermore, IL-6/sIL-6R induced phosphorylation of STAT1 Tyr701 and STAT3 Tyr705 were enhanced by SB203580. IL-6 and pheneylephrine (PE), but not LIF, induced cardiomyocyte iNOS expression and nitric oxide (NO) production. IL-6, LIF and PE induced cardiomyocyte hypertrophy, but with phenotypic differences in ANF and SERCA2 expression and myofilament organisation with IL-6 more resembling PE than LIF. Transfection of cardiomyocytes with full length or truncated chimaeric gp130 cytoplasmic domain/Erythropoietin receptor (EpoR) extracellular domain fusion constructs showed that the membrane proximal Box 1 and Box 2 containing region of gp130 was necessary and sufficient for MAPK and PI3K activation; hypertrophy; SERCA2 expression and iNOS/NO induction in the absence of JAK/STAT activation. In conclusion, IL-6 can signal in cardiomyocytes independent of sIL-6R and STAT1/3 and furthermore, that Erk1/2 and PI3K activation by IL-6 are both necessary and sufficient for induced cardioprotection. In addition, p38-MAPK may act as a negative feedback regulator of JAK/STAT activation in cardiomyocytes.

TRPA1 is essential for the vascular response to environmental cold exposure

The cold-induced vascular response, consisting of vasoconstriction followed by vasodilatation, is critical for protecting the cutaneous tissues against cold injury. Whilst this physiological reflex response is historic knowledge, the mechanisms involved are unclear. Here by using a murine model of local environmental cold exposure, we show that TRPA1 acts as a primary vascular cold sensor, as determined through TRPA1 pharmacological antagonism or gene deletion. The initial cold-induced vasoconstriction is mediated via TRPA1-dependent superoxide production that stimulates α2C-adrenoceptors and Rho-kinase-mediated MLC phosphorylation, downstream of TRPA1 activation. The subsequent restorative blood flow component is also dependent on TRPA1 activation being mediated by sensory nerve-derived dilator neuropeptides CGRP and substance P, and also nNOS-derived NO. The results allow a new understanding of the importance of TRPA1 in cold exposure and provide impetus for further research into developing therapeutic agents aimed at the local protection of the skin in disease and adverse climates.

Superoxide generation and leukocyte accumulation: key elements in the mediation of leukotriene B4-induced itch by transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1

The underlying mechanisms of itch are poorly understood. We have investigated a model involving the chemoattractant leukotriene B4 (LTB4) that is up‐regulated in common skin diseases. Intradermal injection of LTB4 (0.1 nmol/site) into female CD1 mice induced significant scratching movements (used as an itch index) compared with vehicle‐injected (0.1% bovine serum albumin‐saline) mice. Intraperitoneal transient receptor potential (TRP) channel antagonist treatment significantly inhibited itch as follows: TRP vanilloid 1 (TRPV1) antagonist SB366791 (0.5 mg/kg, by 97%) and the TRP ankyrin 1 (TRPA1) antagonists TCS 5861528 (10 mg/kg; 82%) and HC‐030031 (100 mg/kg; 76%). Leukotriene B4 receptor 2 antagonism by LY255283 (5 mg/kg i.p.; 62%) reduced itch. Neither TRPV1‐knockout (TRPV1‐KO) nor TRPA1‐knockout (TRPA1‐KO mice exhibited LTB4‐induced itch compared with their wild‐type counterparts. The reactive oxygen species scavengers N‐acetylcysteine (NAC; 204 mg/kg i.p.; 86%) or superoxide dismutase (SOD; 10 mg/kg i.p.; 83%) also inhibited itch. LTB4‐induced superoxide release was attenuated by TCS 5861528 (56%) and HC‐030031 (66%), NAC (58%), SOD (50%), and LY255283 (59%) but not by the leukotriene B4 receptor 1 antagonist U‐75302 (9 nmol/site) or SB366791. Itch, superoxide, and myeloperoxidase generation were inhibited by the leukocyte migration inhibitor fucoidan (10 mg/kg i.v.) by 80, 61, and 34%, respectively. Myeloperoxidase activity was also reduced by SB366791 (35%) and SOD (28%). TRPV1‐KO mice showed impaired myeloperoxidase release, whereas TRPA1‐KO mice exhibited diminished production of superoxide. This result provides novel evidence that TRPA1 and TRPV1 contribute to itch via distinct mechanisms.—Fernandes, E. S., Vong, C. T., Quek, S., Cheong, J., Awal, S., Gentry, C., Aubdool, A. A., Liang, L., Bodkin, J.V., Bevan, S., Heads, R., Brain, S.D. Superoxide generation and leukocyte accumulation: key elements in the mediation of leukotriene B4‐induced itch by transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1. FASEB J. 27, 1664–1673 (2013). www.fasebj.org

Expression of the Brn-3b transcription factor correlates with expression of HSP-27 in breast cancer biopsies and is required for maximal activation of the HSP-27 promoter

In breast cancer, overexpression of the small heat shock protein, HSP-27, is associated with increased anchorage-independent growth, increased invasiveness, and resistance to chemotherapeutic drugs and is associated with poor prognosis and reduced disease-free survival. Therefore, factors that increase the expression of HSP-27 in breast cancer are likely to affect the prognosis and outcome of treatment. In this study, we show a strong correlation between elevated levels of the Brn-3b POU transcription factor and high levels of HSP-27 protein in manipulated MCF-7 breast cancer cells as well as in human breast biopsies. Conversely, HSP-27 is decreased on loss of Brn-3b. In cotransfection assays, Brn-3b can strongly transactivate the HSP-27 promoter, supporting a role for direct regulation of HSP-27 expression. Brn-3b also cooperates with the estrogen receptor (ER) to facilitate maximal stimulation of the HSP-27 promoter, with significantly enhanced activity of this promoter observed on coexpression of Brn-3b and ER compared with either alone. RNA interference and site-directed mutagenesis support the requirement for the Brn-3b binding site on the HSP-27 promoter, which facilitates maximal transactivation either alone or on interaction with the ER. Chromatin immunoprecipitation provides evidence for association of Brn-3b with the HSP-27 promoter in the intact cell. Thus, Brn-3b can, directly and indirectly (via interaction with the ER), activate HSP-27 expression, and this may represent one mechanism by which Brn-3b mediates its effects in breast cancer cells.

Regulation of Hsp27 expression and cell survival by the POU transcription factor Brn3a

The POU family transcription factor Brn3a is expressed in specific regions of the developing and adult nervous system. Both in vivo and in vitro studies demonstrate a critical role for Brn3a in the survival and specification of sensory neurons. In the sensory neuron-derived cell line ND7 Brn3a is associated with neurite outgrowth, cell cycle arrest, differentiation and protection against apoptosis. cAMP or serum withdrawal (SW) causes the upregulation of Brn3a expression in ND7 cells and correlates with the transactivation of target promoters associated with survival or differentiation. Hsp27 is a member of the small stress protein (sHsp) family and can confer resistance to a number of cytotoxic stresses, including heat, ischemia, anticancer drugs, oxidative stress and excitotoxicity. Hsp27 is highly expressed in neuronal cells and is shown to protect against neuronal cell apoptosis. Mehlen et al. reported an increase in Hsp27 expression and Hsp27-dependent protection against apoptosis during dopamine-induced, cAMP-dependent differentiation of rat olfactory neuroblasts. Since there are direct parallels between survival and differentiation in these two experimental systems, we hypothesised that Hsp27 may represent a downstream target of the Brn3a transcription factor during neuronal cell survival and differentiation. Investigation of the DNA sequences of the mouse and human Hsp27 promoters revealed an A–T-rich sequence TTGCCATTAATAG, which corresponded to a consensus Brn3a binding site and consisting of a core ATTAAT element intersecting two half oestrogen response elements (EREs) at 87 to 91 and 71 to 73. We subcloned the Hsp27 promoter region into the pGL2-luciferase reporter plasmid and co-transfected this into ND7 cells along with plasmids expressing full-length Brn3a(l), which contains the N-terminal transactivation and POU DNA binding domains, Brn3a(s) that lacks the N-terminal transactivation domain and the Brn3a POU domain in isolation. Co-transfection with the oestrogen receptor (ER) was also used as a positive control. As shown in Figure 1a, the wild-type promoter was activated 29-fold by full-length Brn3a(l), compared to a seven-fold activation by co-expression of the ER, but not by Brn3a(s) or the POU domain alone. This suggests that the N-terminal activation domain of Brn3a is required. The long (l) and short (s) isoforms of Brn3a result from alternate promoter usage. The 46 kDa Brn3a(l) contains an N-terminal activation domain not present in the 35 kDa Brn3a(s). Interestingly, while some promoters are activated by both Brn3a isoforms (i.e. SNAP25; synaptophysin) others require the N-terminal activation domain, particularly those involved in cell survival, such as Bcl-2 and Bcl-XL. 13 Site-directed mutagenesis of three nucleotides within the CATTAAT sequence to CGCCAAT within the context of the intact promoter abolished the ability of Brn3a(l) to transactivate the promoter (Figure 1b), but did not affect basal promoter activity. An enhanced activation of the promoter by ER in this case is consistent with the observation that Brn3a is able to interact with ER and represses its effect on an ERE-containing promoter. Furthermore, activation of the promoter by ER in the presence of the mutated site indicates that promoter activity is otherwise intact and that the CATTAAT site is not simply functioning as a TATA box. We investigated whether this effect of Brn3a was mediated by binding to the CATTAAT sequence within the proximal Hsp27 promoter by performing EMSAs using labelled oligonucleotides corresponding to the putative Brn3a binding site (50-TTG CCA TTA ATA GAG-30) and in vitro-translated (IVT) Brn3a. Incubation of labelled probe with IVT Brn3a resulted in two major bands that were specifically competed upon addition of unlabelled (‘cold’) oligonucleotide, but not by a nonspecific oligonucleotide or an oligonucleotide in which the Brn3a site was mutated (Figure 1c). Moreover, Brn3a failed to bind the labelled mutant probe. Functional analysis of whether Brn3a modified endogenous Hsp27 levels in ND7 cells showed that either SW alone or overexpression of Brn3a(l) clearly induced expression of Hsp27 (Figure 1e and f). This induction was further potentiated by overexpression of Brn3a followed by SW. Brn3a(l) also induced expression of the Brn3a(s) isoform as previously described. The effect of increased Brn3a and Hsp27 expression on survival and apoptosis following SW in ND7 cells was assessed by staining of the cells with annexin V and propidium iodide (PI) and subsequent FACS analysis. Annexin V staining (Figure 1e and f) showed that SW caused a substantial increase in the number of cells undergoing apoptosis (total annexin V positive cells mean7S.E.M: pLTR 12.974.0% versus pLTRþSW 65.872.8%, Pr0.01). Overexpression of Brn3a(l) significantly reduced the number of cells that were undergoing apoptosis, as indicated by a substantial reduction in both annexin V positive subpopulations (3a 21.473.1%, Pr0.01). This observation was supported by PI staining and cell cycle analysis. SW in (control) pLTR transfected cells resulted Cell Death and Differentiation (2004) 11, 1242–1244 & 2004 Nature Publishing Group All rights reserved 1350-9047/04