Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of similar to 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the similar to 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. (c) 2006 Elsevier B.V. All rights reserved.

Involvement of novel autophosphorylation sites in ATM activation

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs

ROS as signalling molecules in T cells - Evidence for abnormal redox signalling in the autoimmune disease, rheumatoid arthritis

Reactive oxygen species are recognised as important signalling molecules within cells of the immune system. This is, at least in part, due to the reversible activation of kinases, phosphatases and transcription factors by modification of critical thiol residues. However, in the chronic inflammatory disease rheumatoid arthritis, cells of the immune system are exposed to increased levels of oxidative stress and the T cell becomes refractory to growth and death stimuli. This contributes to the perpetuation of the immune response. As many of the effective therapies used in the treatment of rheumatoid arthritis modulate intracellular redox state, this raises the question of whether increased oxidative stress is causative of T-cell hyporesponsiveness. To address this hypothesis, this review considers the putative sources of ROS involved in normal intracellular signalling in T cells and the evidence in support of abnormal ROS fluxes contributing to T-cell hyporesponsiveness. © W. S. Maney & Son Ltd.

Redox signalling and detection of protein oxidation by mass spectrometry

It is now recognised that redox control of proteins plays an important role in many signalling pathways both in health and disease. Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while the reversible modifications are thought to be most important in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM (chlorination, nitration, etc.), and the susceptibilities of residues vary depending on their structural location. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to the disulfide, glutathionylated or sulfenic acid forms, but there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. Well understood examples of oxidative regulation include protein tyrosine phosphatases, e.g. PTP1B/C, and members of the MAPK pathways such as MEKK1 and ASK1. Transcription factors such as NFkB and Nrf-2 are also regulated by redox-active cysteines. Improved methods for analysing specific oxPTMs in biological samples are critical for understanding the physiological and pathological roles of these changes, and tandem or MS3 mass spectrometry techniques interfaced with nano-LC separation are being now used. MS3 fragmentation markers for a variety of oxidized residues including tyrosine, tryptophan and proline have been identified, and a precursor ion scanning method that allows the selective identification of these oxPTMs in complex samples has been developed. Such advances in technology offer potential for biomarker development, disease diagnosis and understanding pathology.

Thioredoxin and Jab1 control estrogen- and antiestrogen-mediated progression of the cell cycle through p27 interactions

A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.

Understanding the Effect of Protein Tyrosine Phosphatase Receptor Type 0 on Mammalian Sensory Development: Behavioral Studies on PTPRO Knockout Mice

Nerve development, which includes axon outgrowth and guidance, is regulated by many protein families, including receptor protein tyrosine phosphatases (RPTP's).Protein tyrosine phosphatase receptor type 0 (PTPRO) is a type III RPTP that is important for axon growth and guidance, as observed in chicks and flies. In order to examine the effects ofPTPRO on mammalian development, standard behavioral tests were used to compare mice lacking the gene for PTPRO (ROKO mice) to wild-type (WT) mice. The ROKO mice showed a significant delay in reacting to a thermal noxious stimulus, hotplate analgesia, when compared to the WT mice suggesting deficient nociceptive function. In a rotarod test for proprioceptive function the ROKO mice exhibited a significant decrease in the amount of time spent on the rotating rod than did the WT mice. Additional proprioception tests were performed including the climb, step reflex, beam, and mesh walk tests. In the climb and step (place) test, the ROKO group had a significantly lower accuracy in performing the tests than did the WT mice. Thus, mice lacking the PTPRO gene showed behavioral deficiencies that reflect impairment in sensory function, specifically for nociception and proprioception.

An investigation of the role of TRIB2 in steady state and stressed haematopoiesis

TRIB2 is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1-3). Here, we studied murine haematopoiesis after Trib2 ablation under steady state and proliferative stress conditions, including genotoxic and oncogenic stress. At the steady state, we found that TRIB2 loss did not adversely affect peripheral blood cell counts and populations. No detectable significant differences were found in the populations of haematopoietic stem and progenitor cells. However, Trib2-/- mice had significantly higher thymic cellularity due to the increased proliferation of Trib2-/- developing thymocytes which give rise to increased number of mature thymic subsets. During stressed haematopoiesis, Trib2-/- developing thymocytes demonstrate hypersensitivity to 5-fluorouracil-induced cell death. Nevertheless, Trib2-/- mice exhibit accelerated thymopoietic recovery post 5-fluorouracil treatment due to increased cell division kinetics of developing thymocytes. In an experimental murine T-cell acute lymphoblastic leukaemia (T-ALL) model, Trib2-/- mice had reduced latency in vivo which associated with aggressive T-ALL phenotypes and impaired activation of mitogen-activated protein kinase. Gene set enrichment analysis showed that TRIB2 expression is elevated in immature subtype of human T-ALL enriched with mitogen-activated protein kinase signalling. However, TRIB2 expression is suppressed in mature subtype of human T-ALL. Thus, TRIB2 emerges as a novel regulator of thymocyte cellular proliferation, important for the thymopoietic response to genotoxic and oncogenic stress, and possessing tumour suppressor function. In Drosophila, Tribbles promotes degradation of String which is an orthologue of mammalian CDC25 phosphatases in order to arrest cell cycle during embryonic development. Here, we showed that the role of Tribbles-induced degradation of String is evolutionarily conserved in TRIB2. We found that TRIB2 interacts with CDC25B/C but not CDC25A isoform. Overexpression of TRIB2 promotes polyubiquitination and degradation of CDC25C. Hence, future works are warranted to examine TRIB2-CDC25C interaction in the context of developing thymocytes and in T-cell acute lymphoblastic leukaemia, the malignant counterpart.

Targeting protein tyrosine phosphatase SHP2 for therapeutic intervention

Protein tyrosine phosphatases have been the focus of considerable research efforts aimed at developing novel therapeutics; however, these targets are often characterized as being ‘undruggable’ due to the challenge of achieving selectivity, potency and cell permeability. More recently, there has been renewed interest in developing inhibitors of the tyrosine phosphatase SHP2 (PTPN11) in the light of its broad role in cancer, specifically juvenile myelomonocytic leukemia, and recent studies that implicate SHP2 as a key factor in breast cancer progression. Recent significant advances in the field of SHP2 inhibitor development raise the question: are we on the verge of a new era of protein tyrosine phosphatase-directed therapeutics? This article critically appraises recent developments, assesses ongoing challenges and presents a perspective on possible future directions.

Rôle de la protéine DUSP5 dans l’autophagie des cardiomyocytes

Résumé: L’autophagie est un processus essentiel au maintien de l’homéostasie cellulaire. Elle permet de dégrader et recycler aussi bien des organelles entières que des composants cytoplasmiques non fonctionnels. De plus, l’augmentation d’autophagie en condition de stress constitue une réponse adaptative favorisant la survie cellulaire. Chez les cardiomyocytes, l’autophagie en condition basale est indispensable au renouvellement, entre autres, des mitochondries et des protéines formant les sarcomères. De plus, les stress tels l’ischémie cardiaque ou la carence en nutriments induisent une augmentation de l’autophagie protectrice. Dans certaines conditions extrêmes, il a été suggéré qu’un surcroît d’autophagie puisse toutefois exacerber la pathologie cardiaque en provoquant la mort des cardiomyocytes. Considérant l’importance de ce processus dans la physiopathologie cardiaque, l’identification des mécanismes signalétiques régulant l’autophagie chez les cardiomyocytes a été le sujet de recherches intenses. À cet effet, l’activation des Mitogen-Activated Protein Kinase (MAPK) a été démontrée pour réguler, avec d’autres voies signalétiques, l’autophagie et l’apoptose des cardiomyocytes. Il est donc probable que les Dual-Specificity Phosphatase (DUSP), enzymes clés contrôlant l’activité des MAPK, participent aussi à la régulation de l’autophagie. Afin de vérifier cette hypothèse, nous avons induit l’autophagie chez des cardiomyocytes isolés de rats nouveau-nés en culture. L’analyse de marqueurs d’autophagie par immunobuvardage démontre que l’activation des MAPK ERK1/2 et p38 corrèle avec l’activité autophagique chez les cardiomyocytes. Dans ces conditions, la diminution d’expression de la majorité des ARNm encodant les différentes DUSP retrouvées chez les cardiomyocytes contraste de façon marquée avec l’augmentation d’expression de l’ARNm Dusp5. De plus, nous avons démontré par une étude de gain de fonction que l’activation soutenue de p38 par surexpression d’un mutant MKK6 constitutivement actif stimule l’autophagie chez les cardiomyocytes. De façon surprenante, la perte de fonction de p38 obtenue par surexpression d’un mutant p38 dominant négatif n’altère en rien la réponse autophagique initiatrice dans notre modèle in vitro. Nos résultats suggèrent que les DUSP puissent réguler, via leurs actions sur les MAPK, d’importantes étapes du processus autophagique chez les cardiomyocytes.

Studies on the synthesis of novel PP2A inhibitors and a GPCR detergent

Chapter 1 While targeting kinases in oncology research has been explored extensively, targeting protein phosphatases is currently in its infancy. However, a number of pharmaceutical companies are currently looking to expand their research efforts in this area. PP2A has been shown to down-regulate ERK5, a mitogen-activated protein kinase (MAPK) that has been shown to be important in driving the invasive phenotype of prostate cancer. Fostriecin and its related structural analogues PD 113,270 and 113,271 have been shown to inhibit a mitotic entry checkpoint in cell growth through the potent and selective inhibition of protein phosphatases PP1, PP2A, and PP4 (IC50 of 45 μM, 1.5 nM, and 3 nM respectively). Fostriecin is one of the most selective protein phosphatase inhibitors disclosed to date with a 104 fold selectivity for PP2A/PP4 versus PP1. Unfortunately, fostriecin and its analogues are very unstable, and this instability has effectively prevented them from being used as effective therapeutic leads. The microcystins and nodularins on the other hand, exhibit significant inhibitory activity against PP1 and PP2A (IC50 = 26 pM and 1.8 nM respectively), but their high toxicity has prevented any therapeutic application. Truncation of the ADDA chain from these polypeptides completely attenuates PP inhibitory activity. Simpler analogues incorporating the N-acylated ADDA chain and D-Ala retain moderate activity against PP1 and PP2A (IC50 = 1.0 μM and 0.17 μM respectively). The generation of a new series of fostriecin analogues to further expand its structure-activity relationship is envisaged with a view to creating new more stable PP2A inhibitors. It was hoped that by incorporating some of the more stable structural features of ADDA into fostriecin that stability and activity could be reconciled. With that in mind a series of PP2A inhibitors were synthesised and biologically evaluated. Chapter 2 GPCRs are an important area of research and are the targets of a quarter of the drugs on the market (2005). As a result, GPCRs continue to be at the forefront of research in both small and large drug companies. However one of the difficulties in studying this diverse class of membrane proteins is their tendency to denature in aqueous solution. As a result there is a pressing need to develop new detergents to solubilise, stabilise and crystallise GPCRs in their native form for further study. Cholesterol analogues have been shown to be important for stabilising membrane proteins and preventing their thermal inactivation. In addition the β2-adrenergic receptor, a GPCR membrane protein, has been crystallised in the active state with two cholesterol molecules bound between the I, II, III and IV helices of the protein. This appears to represent a distinct cholesterol binding pocket on the membrane protein that is speculated to be conserved across up to 44% of the rhodopsin class of GPCRs. CHOBIMALT is a cholesterol-based detergent that has been shown to exhibit promising GPCR-stabilising properties. When benchmarked against other cholesterol based detergents it was found to be superior to all others tested except for cholesteryl hemisuccinate.1 CHOBIMALT has an aggregation number of roughly 200 and forms 210 ± 30 kDa micelles, which are significantly larger than those of most detergents used for biological systems which is likely due to the packing constraints associated with CHOBMALT’s large polar headgroup.2 As a result, CHOBIMALT is used mostly as an additive to other commercially available detergents in order to decrease micelle size. A branched dimaltoside motif is common in recently synthesised detergents by Chae and co-workers. These detergents have shown promising detergent properties, for example the maltose neopentyl glycol (MNG) detergent synthesised by Chae. This branched dimaltoside detergent was shown to be able to solubilise and stabilise the very labile light harvesting complex I (LHI) from Rhodopsin capsulatus in its active form for 20 days with little loss of protein conformation.3 A cholesterol-based detergent was envisaged that combines the cholesterol framework of CHOBIMALT but replaces its linear tetrasaccharide with a branched dimaltoside. This detergent would then be investigated to assess its ability to solubilise, stabilise and crystallise GPCR proteins. This cholesterol-based detergent (shown below) was eventually synthesised in 9 linear steps from cholesterol.

Regulatory crosstalk by protein kinases on CFTR trafficking and activity

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

Transcription of TIR1-Controlled Genes Can be Regulated within 10 Min by an Auxin-Induced Process. Can TIR1 be the Receptor?

ABP1 and TIR1/AFBs are known as auxin receptors. ABP1 is linked to auxin responses several of which are faster than 10 min. TIR1 regulates auxin-induced transcription of early auxin genes also within minutes. We use transcription of such TIR1-dependent genes as indicator of TIR1 activity to show the rapid regulation of TIR1 by exogenous auxin. To this end, we used quantification of transcription of a set of fifteen early auxin-induced reporter genes at t = 10 and t = 30 min to measure this as a TIR1-dependent auxin response. We conducted this study in 22 mutants of auxin transporters (pin5, abcb1, abcb19, and aux1/lax3), protein kinases and phosphatases (ibr5, npr1, cpk3, CPK3-OX, d6pk1, d6pkl1-1, d6pkl3-2, d6pkl1-1/d6pkl2-2, and d6pkl1-1/d6pkl3-2), of fatty acid metabolism (fad2-1, fad6-1, ssi2, lacs4, lacs9, and lacs4/lacs9) and receptors (tir1, tir1/afb2, and tir1/afb3) and compared them to the wild type. After 10 min auxin application, in 18 out of 22 mutants mis-regulated expression of at least one reporter was found, and in 15 mutants transcription of two-to-three out of five selected auxin reporter genes was mis-regulated. After 30 min of auxin application to mutant plants, mis-regulation of reporter genes ranged from one to 13 out of 15 tested reporter genes. Those genes chosen as mutants were themselves not regulated in their expression by auxin for at least 1 h, excluding an influence of TIR1/AFBs on their transcription. The expression of TIR1/AFB genes was also not modulated by auxin for up to 3 h. Together, this excludes a feedback or feedforward of these mutant genes/proteins on TIR1/AFBs output of transcription in this auxin-induced response. However, an auxin-induced response needed an as yet unknown auxin receptor. We suggest that the auxin receptor necessary for the fast auxin-induced transcription modulation could be, instead, ABP1. The alternative hypothesis would be that auxin-induced expression of a protein, initiated by TIR1/AFBs receptors, could initiate these responses and that this unknown protein regulated TIR1/AFB activities within 10 min.

Synteza i właściwości oksiranowych pochodnych estrów kwasów fosfonowych

Wydział Chemii

L’ischémie des membres inférieurs chez les diabétiques : le rôle du récepteur AT2

Résumé : Les patients diabétiques ont plus de risques d’être amputés d’une jambe en raison d’une plus faible néovascularisation suite à une ischémie. Nous avons montré une association entre une plus faible réponse angiogénique du VEGF chez les souris diabétiques (DM) et une augmentation de l’expression de SHP-1, pouvant être activée par les récepteurs (AT[indice inférieur 1]/AT[indice inférieur 2]). La délétion du récepteur AT[indice inférieur 2] chez des souris favorise l’angiogenèse dans le muscle ischémique, mais son rôle en condition diabétique demeure inconnu. Notre objectif est de vérifier si la délétion du récepteur AT[indice inférieur 2] chez des souris DM favorise l’angiogenèse suivant l’induction d’une ischémie. Des souris DM de type 1 déficientes (KO) ou non pour le récepteur AT[indice inférieur 2] ont été utilisées. L’ischémie a été induite par la ligature de l'artère fémorale. La perfusion sanguine a été mesurée pendant 2 ou 4 semaines avant la récolte des tissus. Les effets de l’ischémie sur l’expression des récepteurs AT[indice inférieur 1] et AT[indice inférieur 2], des phosphatases SHP-1, SHP-2 et PTP1B, ainsi que l’état de la voie de signalisation du VEGF ont été mesurés. Un essai phosphatase a aussi été effectué suite à l’immunoprécipitation de SHP-1 chez des BAECs stimulés au CGP42112A. Quatre semaines après la chirurgie, le flot sanguin dans le muscle ischémique des souris DM AT[indice inférieur 2]KO s’est rétabli plus rapidement (80%) comparativement à une récupération de 47% chez les souris DM contrôles. L’expression des facteurs pro-angiogéniques (HIF-1α et VEGF) était similaire dans tous les groupes après 2 semaines d’ischémie, mais diminuée chez les DM et retournait à un niveau basal chez les DM-AT[indice inférieur 2]KO après 4 semaines, suggérant un reperfusion plus rapide chez ces souris. La phosphorylation de Akt était aussi plus faible chez les souris DM contrôles mais était rétablie chez les souris AT[indice inférieur 2]KO après 4 semaines d’ischémie. L'expression de SHP-1 était doublée dans le muscle ischémique des souris DM, en comparaison aux souris non DM, un effet absent chez les souris DM AT[indice inférieur 2]KO. L’expression de SHP-2 et PTP1B ne variait pas chez les souris DM sauvages et AT[indice inférieur 2]KO. De plus, l’expression des récepteurs AT[indice inférieur 1] et AT[indice inférieur 2] est augmentée chez les souris DM sauvages en comparaison aux souris NDM. La stimulation du récepteur AT[indice inférieur 2] chez les BAECs a permis d’augmenter l’activité phosphatase de SHP-1. Nos résultats suggèrent que l’expression élevée d’AT[indice inférieur 2] chez les souris DM mène à la surexpression et/ou l’activation de SHP-1, inhibant le signal angiogénique issu du VEGF et empêchant la reperfusion sanguine suite à l’ischémie.