Reactive oxygen species (ROS) are products of normal cellular metabolism and derive from various sources in different cellular compartments. Oxidative stress resultant from imbalance between ROS generation and antioxidant defense mechanisms is important in pathogenesis of cardiovascular diseases, such as hypertension, heart failure, atherosclerosis, diabetes, and cardiac hypertrophy. In this review we focus on hypertension and address sources of cellular ROS generation, mechanisms involved in regulation of radical homeostasis, superoxide dismutase isoforms in pathophysiology of hypertension; as well as radical intracellular signaling and phosphorylation processes in proteins of the affected cardiovascular tissues. Finally, we discuss the transcriptional factors involved in redox-sensitive gene transcription and antioxidant response, as well as their roles in hypertension. This significantly shapes cellular metabolism and energy production. The associated oxygen intake results in the formation of toxic reactive oxygen species (ROS) in the mitochondrial respiratory chain. These radicals are electron donors and can damage DNA, RNA, proteins, and lipids. They can also propagate deleterious reactions throughout cells and tissues resulting in cell death and apoptosis [Living organisms can reduce oxygen to water poptosis . In addipoptosis .2O2) and RNS. These findings suggest that in many signaling events where ROS and RNS take part, enzymatic catalysis is more likely to be involved than a non-enzymatic reaction [Studies of the role of ROS and reactive nitrogen species (RNS) in signal transduction have grown in number during the past decade. The main interest in the field is to recognize the specific targets and chemical reactions involved in the signaling pathways triggered by ROS and RNS as second messenger molecules. It was observed that cysteine residues in the thiolate (ionized) form are present in several classes of signaling proteins, and these can be the specific targets for reactions with hydrogen peroxide (Hreaction . As suchreaction -7. In threaction -12. We s4), leading to uncoupling of the endothelial NO synthase (eNOS) and to formation of large amounts of “bonfire” radicals [2-•) and H2O2 in the vessel wall. NADPH oxidase-derived ROS then act as “kindling” and activate secondary , and tertiary  sources of ROS, which contribute to the “bonfire” of radicals and oxidative stress seen at later stages of diseases [Several mechanisms or pathways are responsible for the production of free radicals in the cell ,14. A paradicals . As a rediseases  , endothelin-1 (ET-1) and norepinephrine (NE). The action of Ang II through angiotensin type 1 (AT1) receptors plays an important role in vasoconstriction. Activation of AT1 receptors results in induction of a number of ROS-producing events in the cell. Infusion of Ang II to normotensive rats stimulates the production of O2-• by NADPH oxidase in vessels and induces pressor responses [A and ETB. Activation of ETA receptors causes contraction of blood vessels via activation of the NADPH oxidase and generation of ROS; activation of ETB receptors leads to relaxation of vessels [via α1-adrenergic receptors (α1-AR). α1-AR, AT1, and ET-1 receptors are known to activate NADPH oxidase through the G-protein, resulting in the production of ROS [ROS are derived from many sources in different cellular compartments. In the vascular smooth muscle cells and endothelial cells, NADPH oxidase acts as the primary source and is particularly important in pathophysiology of hypertension Figure . In the esponses . NADPH oesponses . ET-1, t vessels . In cardn of ROS . Signifin of ROS ,21-23.4, produces NO. When BH4 levels are reduced, stability of NOS dimer formation is altered such that the oxidase domain yields molecular uncoupling and the catalytic activity becomes functionally 'uncoupled’ [2-• rather than protective NO [Under physiological conditions, NOS, in the presence of substrate L-arginine and co-factor BHcoupled’ . Enzymatctive NO . This eNctive NO ,26, inclctive NO . Detailective NO  for deta2-• and H2O2 formation [2-• and further exacerbating in ROS production. Finally, cytosolic ROS, via opening of the redox-dependent mitochondrial ATP-sensitive potassium channels [Mitochondrial electron transport chain is another important source of ROS generation. Any damage at enzyme complexes of the mitochondrial respiratory chain leads to dysfunction of the mitochondrial respiration with subsequent transfer of electrons to molecular oxygen increasing the mitochondrial Oormation . In addiormation , which rchannels  and chanchannels , stimulachannels .via the increase in mitochondrial ROS [1R blocker, losartan. Glutamate-supported respiration, the rate of ATP production, and concentrations of coenzyme Q9 (CoQ9), coenzyme Q10 (CoQ10), and alpha-tocopherol have all been reported to improve mitochondrial dysfunctions and promote antihypertension in SHR after the therapy [Mitochondria are integral component in energy metabolism, ROS generation, and cell signaling, thus play an important role in oxidative stress leading to the development of many cardiovascular conditions, including hypertension. The functional significance of mitochondria-derived ROS, particularly in vascular cells, has recently received a great attention. For example, hyperinsulinemia induces migration and proliferation of vascular smooth muscle cells rial ROS . Interes therapy .2-• released from mitochondrial Complex III of smooth muscle cells diffuses into the cytosol and triggers increases in intracellular calcium and causes acute hypoxic pulmonary vasoconstriction [During acute hypoxia, Otriction ,37. Moretriction . This crtriction .The term “redox signaling” describes a process in which physiological levels of ROS induce modifications to proteins that are discrete, site-specific, and reversible . Evidenc2-•), and these, in turn, activate cell phosphorylation pathways: the mitogen-activated protein kinases (MAPKs), tyrosine kinases, and phosphoinositol-3-kinase/Akt kinase (PI3K/Akt). The activated phosphorylation pathways activate transcription factors such as activated protein-1 (AP-1), p53, nuclear factor kappa B (NFκB), and nuclear E2-related factor 2 (Nrf2), which stimulate transcription of genes after moving into the nucleus. Proteins encoded by these target genes in turn mediate cellular consequences leading to changes in the phenotypes, such as hypertrophy, inflammation, necrosis and apoptosis of cells and, on the other hand, stimulate the production of antioxidants involved in antioxidant defense [Until recently, ROS were considered only as harmful molecules. However, recent studies suggest that ROS may serve as an important physiological regulator of intracellular phosphorylation signaling pathways. The specific effects of ROS are largely modulated by covalent modification of specific cysteine residues, which were discovered in redox-sensitive proteins. Oxidation of these reactive cysteine residues can lead to reversible modification of enzyme activity. There is evidence that the ROS regulate various physiological parameters in response to stimulation with growth factors to generate inflammatory response. Disruption of normal ROS signaling may lead to several diseases in humans, including hypertension . In hype defense ,20,41  in the brain ,21,42-44The MAPK family includes serine-threonine protein kinases that play an important role in the transmission of extracellular signals from cell membrane to the nucleus. Activation of MAPKs and signal transduction depends on series of phosphorylating events that allow interaction of multiple signaling pathways. MAPK phosphorylation cascades can be negatively regulated by MAP phosphatases that dephosphorylate/deactivate individual MAPKs . The MAPvia ROS. Its activation is necessary for activation of the ERKs and for the growth of vascular smooth muscle cells [Activation of tyrosine kinases by ROS plays an important role in the remodeling of the cardiovascular system associated with hypertension. In vascular smooth muscle cells and other cells, the ROS activate non-receptor tyrosine kinases such as Janus kinase 2 (JAK2), proline-rich tyrosine kinase 2/cell adhesion kinase (PYK2/CAK) and Src tyrosine kinase, or the receptor tyrosine kinases, such as the receptor for epidermal growth factor (EGF) -57. Partle cells . The JAKle cells .The Akt kinase (or protein kinase B) is a serine/threonine kinase that plays an important role in many cellular processes and its activation in the PI3K/Akt pathway depends on the upstream PI3K. The activated Akt kinase phosphorylates various downstream effector molecules, such as glycogen synthase kinase-3, procaspase-9, transcription factors AP-1 and E2F , and is via oxidation and phosphorylation of the phosphatase and tensin homolog deleted on chromosome 10, which is a negative regulator of PI3K signaling [The PI3K/Akt kinase signaling pathway is also involved in the metabolic control and regulation of glucose transport and glycogen synthesis. The Akt kinase can be activated by a variety of growth stimuli: the platelet-derived growth factor, EGF, insulin, thrombin, and the nerve growth factor (NGF) . It is aignaling . This reignaling . In humaignaling .The Rho-associated protein kinase (ROCK) is a serine/threonine kinase. For its activity, ROCK requires the binding of the small G-protein RhoA. The RhoA/ROCK signaling pathway is stimulated by many agonists such as Ang II, thrombin, ET-1, and NE. Increased activity of this pathway was observed in atherosclerosis and pulmonary hypertension, and this signaling has been implicated in many major cardiovascular diseases such as hypertension, heart failure, and myocardial infarction. A role of this pathway in the regulation of several cellular functions such as gene expression, contraction, migration, and proliferation has been reported . Moreovecis-element of the methallothionein human genes encoding proteins that are involved in cell proliferation and apoptosis in response to various stimuli, including the oxidative stress. Redox-sensitive upregulation of AP-1, c-jun or c-fos have been observed in the RVLM of the hypertensive animals [A receptor-mediated and oxidative stress-associated overexpression of p53 [There are several transcription factors, including AP-1, p53, NF-κB, Nrf2, whose activity is sensitive to oxidative stress. AP-1 was first identified as a transcription factor that binds to the  animals . In deox animals . In thisn of p53 . In addin of p53 .via the ARE is mediated primarily through the Nrf2 transcription factor. Under normal conditions, the activity of Nrf2 is negatively regulated by protein Kelch-like ECH associated protein 1 (Keap1) that binds to Nrf2 and keeps it in the cytoplasm. Exposure to xenobiotics, NO and/or ROS induce the transcription of Keap1/Nrf2/ARE‒dependent genes whose functional products protect against a wide array of subsequent challenges [AP-1 binds to the antioxidant response element (ARE) of target genes, where Fos and Jun proteins may heterodimerize Nrf2 in the presence of electrophiles and oxidants [allenges . Free Nrallenges ,79. TherIn addition to enzymatic degradation, detoxification of ROS and electrophiles is also important in protecting cells from oxidative damage. Genes regulated through the ARE are involved in the production of antioxidants that directly inactivate ROS , detoxify xenobiotics H quinone oxidoreductase) and affect the production of glutathione and phase-II detoxification enzymes . All these genes are highly sensitive to increases in oxidative stress . Moreovevia the ARE genes [Emerging evidence indicates that activation of Nrf2/ARE signaling differs during acute versus chronic stress conditions. During acute oxidative stress, oxidized molecules modify multiple cysteine residues in Keap1, causing Nrf2 to be released from connection with Keap1. Nrf2 is translocated into the nucleus and induces transcription RE genes ,82. DuriRE genes . Fyn phoRE genes . This meRE genes . CatalasRE genes . On the RE genes .2-• to H2O2[For the cell to maintain ROS homeostasis there is a delicate balance existing between the production and degradation of the radicals ,89. The • to H2O2. The SOD• to H2O2,91. Appl• to H2O2. Applica• to H2O2.in vivo gene delivery of adenoviral vectors encoding SOD1. They found that both superoxide production from Ang II infusion and the induced hypertension were prevented by overexpression of CuZnSOD. A causal relationship between biochemical correlates of oxidative stress and neurological hypertension was established after a gene transfer by microinjection of adenovirus encoding SOD1, SOD2 or catalase into the RVLM, which promotes a long-term reduction of blood pressure in SHR [Zimmerman et al.  were thee in SHR . In conse in SHR .SOD2 is present mainly in the mitochondria of endothelial cells and less in mitochondria of vascular smooth muscle cells . MitochoSOD3 is located in the extracellular space and binds to the cell membrane through a heparin-binding domain. It is the second most abundant SOD isoform in blood vessels and is produced mainly by smooth muscle cells. SOD3 is very important in endothelium-mediated vasodilation because of its location in the interstitium between endothelial cells and vascular smooth muscle cells. It enables safe diffusion of NO from endothelial cells into smooth muscle cells . EcSOD-d4, modulation of the signaling and phosphorylation pathways, such as MAPK, Akt kinase, and RhoA/ROCK signaling pathways. All these possibilities and strategies have been reported to alleviate ROS accumulation and to protect against oxidative stress to a different degree in the animal models of hypertension. In addition, modulation of the redox-sensitive transcription factors, as well as stimulation of antioxidant response through the Nrf2-ARE pathway may also prove to be a useful strategy in future. All these possibilities and strategies should be further studied in the search for new therapeutical approaches in management of hypertension.Radical signaling offers several possibilities and strategies of modulating ROS homeostasis in the management of hypertension. Individual SOD isoforms can be influenced using gene transduction and SOD mimetics. Other strategies include inhibition of NADPH oxidase activity, preservation of mitochondrial electron transport chain capacity, increases in BH1-adrenergic receptors; BH4: Tetrahydrobiopterin; CoQ: Coenzyme Q; Cu/Zn SOD: Cupper/zinc superoxide dismutase; ecSOD: extracellular superoxide dismutase; EGF: Epidermal growth factor; eNOS: endothelial nitric oxidase synthase; ERK: Extracellular signal-regulated kinase; ET-1: Endothelin-1; GSK-3β: Glycogen synthase kinase-3β; H2O2: Hydrogen peroxide; HO-1: Heme oxygenase-1; JAK2: Janus kinase 2; JNK: c-Jun N-terminal kinase; Keap1: Kelch-like ECH associated protein 1; NADPH oxidase: Nicotinamide adenine dinucleotide phosphate oxidase; MAPK: Mitogen activated protein kinase; MnSOD: Manganese superoxide dismutase; NE: Norepinephrine; NF-κB: Nuclear factor kappa B; NGF: Nerve growth factor; NO: Nitric oxide; NOS: Nitric oxide synthase; Nrf2: Nuclear transcription factor E2-related factor 2; O2•–: Superoxide; PI3K/Akt: Phosphoinositol-3-kinase/Akt kinase; PYK2/CAK: Proline - rich tyrosine kinase 2/cell adhesion kinase; RAS: Renin-angiotensin system; RhoA: Small G-protein RhoA; RNS: Reactive nitrogen species; ROCK: Rho-associated protein kinase (serine/threonine kinase); ROS: Reactive oxygen species; RVLM: Rostral ventrolateral medulla; SHR: Spontaneously hypertensive rats.Akt kinase: Serine/threonine protein kinase; Ang II: Angiotensin II; AP-1: Activator protein 1; ARE: Antioxidant response element; AT1R: Angiotensin type 1 receptor; α1-AR: alphaThe authors declare that they have no competing interests.MM, ID, MB, and JYHC collected information and prepared manuscript. MM and JYHC prepared the figures, ID, MB and JYHC finalized the manuscript. All authors read and approved the final manuscript.


Dictyostelium transcription factor STATc is tyrosine phosphorylated and accumulates in the nucleus when cells are exposed either to hyper-osmotic stress or to the prestalk-inducing polyketide DIF-1. In the case of stress STAT activation is mediated by regulated dephosphorylation; whereby two serine residues on PTP3, the tyrosine phosphatase that de-activates STATc, become phosphorylated after exposure to stress so inhibiting enzymatic activity. We now show that the more highly regulated of the two PTP3 serine residues, S747, is also phosphorylated in response to DIF-1, suggesting a common activation mechanism. Hyper-osmotic stress causes a re-distribution of F-actin to the cortex, cell rounding and shrinkage and we show that DIF-1 induces a similar but transient F-actin re-distribution and rounding response. We also find that two mechanistically distinct inhibitors of actin polymerization, latrunculin A and cytochalasin A induce phosphorylation at S747 of PTP3 and activate STATc. We suggest that PTP3 phosphorylation, and consequent STATc activation, are regulated by changes in F-actin polymerization status during stress and DIF-induced cytoskeletal remodelling.The  Metazoans STATs are activated as transcription factors and accumulate in the nucleus when a cytokine binds to its receptor  cells were grown axenically in HL-5 medium  at a concentration of 1 × 107 cells/ml and shaken for 4 h at 200 rpm. Sorbitol was added to a final 200 mM, DIF-1 to 100 nM, latrunculin A to 2 μM and cytochalasin A to 20 μM.5 medium . For indPolyclonal, phospho-specific PTP3 and monoclonal STATc (CP22) antibodies were used in Western transfer as described previously . In the 7 cells/ml) were lysed by adding 2× Triton lysis buffer . The cell lysate was incubated on ice for 10 min and then at 21 °C for 10 min. The lysate was spun at 11,000 × g for 4 min and the pellet was washed once in 1× Triton lysis buffer. This pellet was deemed the Triton X-100 insoluble fraction and was analysed by SDS-PAGE followed by Colloidal Blue staining. Quantification of actin was performed by gel scanning and analysis using ImageJ software.Determination of F-actin levels was performed using a slightly modified method from that originally described . DIF-1 t


In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFκB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO).-/- mice the interleukin (IL)-1β induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFκB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKα2-/- mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IκB and p65, indicating a link between AMPK and the IκB kinase (IKK). Indeed, IKK  was found to be a direct substrate of AMPKα2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKα2+/+ versus AMPKα2-/- mice.Overexpression of a dominant negative AMPKα2 mutant in tumor necrosis factor-α-stimulated human endothelial cells resulted in increased NFκB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKα2These results demonstrate that the IKK is a direct substrate of AMPKα2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFκB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK. The AMP-activated protein kinase (AMPK) is a member of the Snf1/AMPK family of serine/threonine protein kinases and is an evolutionarily conserved sensor of the cellular energy status. Although the AMPK pathway is traditionally thought of as an intracellular fuel gauge and regulator of metabolism, recent evidence indicates that it may also be important for the maintenance of endothelial function and to redress the disturbed redox balance associated with vascular disease. Certainly, the AMPK can influence a number of signaling cascades that would be expected to result in anti-atherosclerotic effects, such as attenuated free radical generation and the activation of angiogenic factors  which has a tn Thr172 . The pho minutes . The effTo determine the involvement of the AMPK in the prevention of NFκB activation, we assessed the effects of constitutively active and dominant negative AMPKα2 mutants on the TNF-α-induced activation of NFκB. While TNF-α enhanced the activity of NFκB in cells infected with a control virus, this response was blunted in cells overexpressing the constitutively active AMPK  and pote-/- mice or their wild-type littermates (AMPKα2+/+).Theoretically a dominant negative AMPKα2 mutant could affect the activity of the AMPKα1 by, for example, sequestering AMPKβ and γ subunits from the endogenous α1 subunit. To ensure that the effects observed could indeed be attributed to the AMPKα2, we performed additional experiments in endothelial cells from AMPKα2+/+ and α2-/- mice as well as AMPKα2 expression in α1+/+ and α1-/- mice. We found that the deletion of the AMPKα2 did not affect α1 expression while the α2 subunit was upregulated in tissue from AMPKα1-/- mice  or to LPS  in the absence and presence of DETA-NO and the surface expression of E-selectin was assessed. The basal expression of E-selectin was slightly (but not significantly) enhanced in cells isolated from the AMPKα2-/- compared to the AMPKα2+/+ animals. While the basal expression of the adhesion molecule was attenuated by NO, levels remained slightly elevated in the AMPKα2-/- cells. Cell stimulation with IL-1β  in the cells from AMPKα2-/- mice than in cells from their wild-type littermates.To determine the effect of AMPK deletion on the response to inflammatory mediators, murine microvascular endothelial cells from AMPKα2th IL-1β  or LPS  resulted in a significant increase in mononuclear cell attachment .The activation of NFκB mainly occurs via the phosphorylation and degradation of inhibitory molecules, including IκB. Interestingly, the activation of the AMPK was associated with the increased expression of IκB protein, a phenomenon that was evident on comparing pulmonary endothelial cells from AMPKα2-/- mice  or COS-7-/- mice . Both thCOS-7 cells expressing constitutively active or dominant negative AMPKα2 were used to determine the consequences of AMPK activation and inhibition on the IκB kinase (IKK)-mediated phosphorylation of IκB independent of potential interference by endogenously generated NO. Neither of the AMPKα2 mutants studied affected the basal IκB phosphorylation. However, following stimulation with TNF-α (10 ng/mL), IκB phosphorylation was enhanced in cells expressing the dominant negative mutant . The lacThe TNF-α-activated IKK complex also phosphorylates p65 on Ser536, a step thought to be required for enhanced p65 transactivation potential and the optimal induction of NFκB target genes in vitro kinase assays using the purified AMPKα2 (together with β1 and γ1) we identified IKKβ as an AMPK substrate  in IKKβ phosphorylated by AMPK we replaced its major phosphorylation sites i.e. Ser177 and Ser181 with alanine by site-directed mutagenesis, and overexpressed these mutants in COS-7 cells. The wild-type and mutant IKKβ were then immunoprecipitated and incubated with AMPKα2. We found that the mutation of IKKβ on Ser177 and Ser181 resulted in a lower level of phosphorylation than the wild-type enzyme. Moreover, phosphorylation was barely detectable in the S177A/S181A double mutant. When IKKβ was replaced with GFP, no phosphorylation was observed in the presence of AMPKα2 . Also in-/- mice .To assess IKKβ activity we next determined the phosphorylation of GST-IκBα in the presence of wild-type IKKβ and either AMPKα1 or α2. We found that the phosphorylation of IκBα was reduced in the presence of AMPKα2 but not AMPKα1 .-/- mice. Thus, it appears that the NO-mediated inhibition of NFκB activity is, at least partially, dependent on the activation of the AMPKα2 subunit.The results of the present investigation indicate that the AMPKα2 subunit plays an important role in regulating inflammatory responses, adhesion molecule expression (E-selectin and VCAM-1) and monocyte adherence to endothelial cells. These effects could be related, at least partly to the AMPK-mediated phosphorylation of IKK and subsequent inhibition of IκB and p65 phosphorylation as well as DNA binding see . Moreove+/+ and AMPKα2-/- mice to determine which of the catalytic subunits was most likely to affect transcription factor activity. The results obtained using murine lung endothelial cells indicate that the AMPKα2 plays a prominent role in modulating NFκB phosphorylation in endothelial cells.The possibility that the AMPK can regulate the DNA binding activity of NFκB was initially indicated by the fact that overexpression of a dominant negative AMPK increased, while a constitutively active AMPK decreased the TNF-α-induced expression of E-selectin -/- versus AMPKα2+/+ endothelial cells. As the expression of IκB is known to be regulated by the activity of NFκB Which point in the NFκB activation cascade could be affected by AMPK? NFκB is a dimer consisting of the transcription factors p65 (RelA) or p50, and under resting conditions it is associated with its inhibitory protein IκB which retains the complex in the cytosol. The activation of NFκB mainly occurs via the phosphorylation of IκB, which results in its degradation - leaving the NFκB dimer free to translocate into the nucleus and stimulate transcription The serine phosphorylation of IκB is mediated by a large multi-unit complex containing two catalytic subunits (IKKα and IKKβ) as well as the regulatory subunit IKKγ or NEMO, which has no kinase domain. The p65 subunit is also a IKK substrate and p65 phosphorylation on Ser536 is thought to be required for enhanced p65 transactivation potential and the optimal induction of NFκB target genes in vitro assays we identified IKKβ as an AMPK substrate and were able to detect the elevated phosphorylation of IKK in intact AMPK-overexpressing cells. The phosphorylation of IKKβ on Ser 177 and/or 181 causes the rapid phosphorylation of a C-terminal serine cluster which in turn elicits the auto-inhibition of kinase activity and is thought to be an effective means of limiting the duration of IKK activation +/+ versus AMPKα2-/- mouse endothelial cells.To date there has only been circumstantial evidence to suggest a role of the AMPK in the modulation of IKK activity. For example, AICAR has been reported to be without effect on IKK in rat skeletal muscle cells in vitro, and thus one further mechanism by which the AMPK could affect NFκB would be by modulating basal NO output , at least Taken together, the results of the present investigation indicate that the activation of the AMPK in endothelial cells can limit inflammatory responses via the phosphorylation and inhibition of IKK activity. Moreover, as the AMPK could be activated by NO, the AMPK dependent inhibition of IKK activity may contribute to the anti-inflammatory and anti-atherosclerotic actions of the endothelium-derived autacoid.Antibodies for Western blotting directed against phospho-Thr172 AMPK and total AMPK, phospho-Ser536 NFκB, phospho-Ser32/36 IκB and phospho Ser 177/181 IκB kinase (IKK) were purchased from New England Biolabs . Antibodies recognizing IκB, NFκB or IKK from Santa Cruz Biotechnology . Horseradish peroxidase conjugated secondary antibodies were from Calbiochem  and the fluorescent antibodies recognizing E-selectin and VCAM-1 used for FACS analyses were from BD Biosciences . TNF-α and interleukin (IL)-1β, were from PeproTech , and DETA-NONOate (DETA-NO), DEA-NONOate and DPTA-NONOate were from Alexis . The antibody against β-actin and other chemicals were obtained from Sigma .-/- or AMPKα2-/- mice or the respective littermate wild-type animals Human umbilical vein endothelial cells were isolated and cultured as described st passage) were infected with adenoviruses  to over-express constitutively active AMPKα2 Subconfluent endothelial cells -labeled double-stranded oligonucleotides containing the consensus sequence of the binding site for transcription factor NFκB  was assessed as described Nuclear and cytosolic proteins were isolated, and binding to γ[Following stimulation endothelial cells were harvested using accutase  and washed twice with PBS. After blocking  cells were incubated with the specific conjugated antibodies , washed twice with PBS and fixed in 1% paraformaldehyde. Antibody binding was analyzed using a flow cytometer  by counting 20,000 cells per sample. The values are presented following subtraction of the isotype-matched, control IgG.Peripheral blood mononuclear cells were freshly isolated from blood obtained from healthy volunteers using a Biocoll® (1.077 g/ml) gradient  according to the manufacturer's protocol. Confluent cultures of human endothelial cells were stimulated with TNF-α for 6 hours, and were washed with culture medium before freshly isolated mononuclear cells  were added. After 10 minutes of incubation the non-adherent cells were removed the number of firmly adherent cells was quantified.The phosphorylation of IKKβ by AMPKα2 and the activity of IKK β were assessed in in vitro kinase assays. Ser177 and Ser181 in a flag-tagged IKK plasmid t test for unpaired data and one-way ANOVA or ANOVA for repeated measures followed by followed by a Bonferroni t test where appropriate. Values of P<0.05 were considered statistically significant.Values are expressed as the mean ± SEM and statistical evaluation was performed using Student's Figure S1Effect of NO donors on the phosphorylation of AMPK. (A) Human endothelial cells (passage 2) were treated with either solvent (Sol) or DEA-NO  for up to 30 minutes. Identical results were obtained in two additional experiments. (B) Human endothelial cells were treated with different concentrations of the NO donor DPTA NONOate (t½ 5 hours) and the phosphorylation of the AMPK was detected by Western blotting. The graph summarizes the data obtained in 3 independent experiments; **P<0.01 versus the appropriate Sol-treated group.(TIF)Click here for additional data file.Figure S2Effect of AMPKα subunit deletion on the expression of the second isoform in aortic lysates. While deletion of the AMPKα2 subunit had no effect on the expression of the AMPKα1 isoform, the deletion of AMPKα1 induced a compensatory increase in AMPKα2 expression. Each lane represents tissue from a different animal and identical results were obtained in tissue from 4 additional animals.(TIF)Click here for additional data file.Figure S3Role of AMPKα2 in regulating the expression and phosphorylation of p65. The phosphorylation of p65 NF-kB was assessed in COS-7 cells expressing either constitutively active (CA) or dominant negative (DN) AMPKα2 and stimulated with solvent (Sol) or TNF-α (10 ng/mL). Data are expressed relative to values obtained in control virus-infected cells. The bar graph summarizes the results of 4 to 5 independent experiments; *P<0.05 versus CA.(TIF)Click here for additional data file.Figure S4Effect of the AMPKa1 deletion on the IL1β (30 ng/mL)-mediated phosphorylation of IKKα/β and IκB in mouse lung endothelial cells. The bar graph summarizes the results of 4–5 independent experiments.(TIF)Click here for additional data file.


S. pombe. However, whether it contains a robust ATR-activation domain (AAD) required for checkpoint signaling like its orthologs TopBP1 in humans and Dpb11 in budding yeast has been incompletely clear. To identify the putative AAD in Rad4, we carried out an extensive genetic screen looking for novel mutants with an enhanced sensitivity to replication stress or DNA damage in which the function of the AAD can be eliminated by the mutations. Two new mutations near the N-terminus were identified that caused significantly higher sensitivities to DNA damage or chronic replication stress than all previously reported mutants, suggesting that most of the checkpoint function of the protein is eliminated. However, these mutations did not affect the activation of Rad3 (ATR in humans) yet eliminated the scaffolding function of the protein required for the activation of Chk1. Several mutations were also identified in or near the recently reported AAD in the C-terminus of Rad4. However, all mutations in the C-terminus only slightly sensitized the cells to DNA damage. Interestingly, a mutant lacking the whole C-terminus was found resistant to DNA damage and replication stress almost like the wild type cells. Consistent with the resistance, all known Rad3 dependent phosphorylations of checkpoint proteins remained intact in the C-terminal deletion mutant, indicating that unlike that in Dpb11, the C-terminus of Rad4 does not contain a robust AAD. These results, together with those from the biochemical studies, show that Rad4 mainly functions as a scaffold protein in the Chk1, not the Cds1(CHK2 in humans), checkpoint pathway. It plays a minor role or is functionally redundant with an unknown factor in Rad3 activation.Rad4/Cut5 is a scaffold protein in the Chk1-mediated DNA damage checkpoint in  Faithful duplication of the genome in eukaryotes depends upon the precise operation of the DNA replication machinery and upon the checkpoint pathways that deal with various perturbations of DNA replication  S. pombeAt the apex of the two checkpoint pathways, Rad3-Rad26, Rad9-Rad1-Hus1 (9-1-1 clamp), and Rad17 (9-1-1 clamp loader) function as checkpoint sensors  mutants S. pombein vitro and in vivo via its ATR activation domain (AAD) located between the sixth and the seventh BRCT repeats in vitroinet al. 2012). However, mutations of the AAD in Rad4 only slightly sensitize the cells to DNA damage.The rad4 mutants showed a high sensitivity. In addition, all previously reported rad4 mutations appear to affect the scaffolding but not the activation of Rad3. To provide a clear answer, we carried out an extensive genetic screen looking for new rad4 mutants in which most, if not all, of the checkpoint function is eliminated. Two new mutants C13Y and K56R were identified near the N-terminus that are significantly more sensitive to chronic HU exposure and the DNA damage induced by methyl methanesulfonate (MMS) than all previously reported mutants, suggesting that most of the checkpoint activity is eliminated. However, these mutations did not affect much of the Cds1 activation following HU treatment yet eliminated the scaffolding function of Rad4 required for Chk1, but not Rad3, activation. We also identified several mutations around the recently reported AAD in the C-terminus of Rad4 in vitro biochemical analyses, show that unlike that in Dpb11, the C-terminus of Rad4 does not contain a robust AAD. We believe that in fission yeast, Rad4 plays a minor role or is functionally redundant with an unknown factor in Rad3 activation.To investigate the potential function of Rad4 AAD in the Cds1-mediated replication checkpoint, we tested several previously reported mutants S. pombe strains . All mutations and sequences were confirmed by DNA sequencing . Mutational PCRs were performed following the described protocol using the thermal stable Taq DNA polymerase S. pombe cells by electroporation. To test the drug sensitivity by spot assay, 2×107 cells/ml of logarithmically growing S. pombe were diluted in fivefold steps and spotted onto YE6S plates containing HU or MMS at the indicated concentrations. The plates were usually incubated at 30°C for 3 days and then photographed. Acute drug treatment was performed in liquid cultures containing 2×106 cells/ml. After the drugs were added at the indicated concentrations, small aliquots of the cultures were diluted 1000 fold and then spread on YE6S plates for the cells to recover. After incubation at 30°C for three days, colonies were counted and presented as percentages relative to the untreated cells.Growth of  strains  and medi11, Mrc1-Thr645 and Rad9-Thr412 were prepared by Bethyl Laboratories, Inc  E. coli as the antigen. The antibodies, purified from the anti-serum using GST-Rad4(Δ498–648aa) as the antigen, were used for the Western blotting analyses.The affinity-purified phospho-specific antibodies against phosphorylated Cds1-Thr3VO4, 20 mM NaP2O7, 100 mM NaF, 120 mM glycerol phosphate) containing protease inhibitors. After clarification by centrifugation at 4°C, 13,000 rpm  for 5 min, the supernatants were incubated with anti-HA antibody beads by rotating at 4°C for 2 h. After extensive washes with Tris-buffered saline containing 0.5% Tween-20 (TBS-T), the protein was used for in vitro binding reactions or separated by SDS-PAGE for Western blotting analyses. The loading of Cds1, Rad9 and Crb2 was monitored first by Western blotting using anti-HA antibody. The same blots were stripped and reprobed with phospho-specific antibodies for detection of the site-specific phosphorylation of the proteins. Phosphorylated Chk1 was assessed by mobility shift assay using anti-HA antibody The phosphorylation of hemagglutinin (HA) epitope tagged Cds1, Rad9 and Crb2 was assessed by Western blottings using phospho-specific antibodies in proteins IPed with anti-HA antibody beads . Cell lysates were made by a mini-bead beater with acid-washed glass beads in 2× IP buffer  cells by incubation at 30°C for 4 h after isopropyl 1-thio-D-galactopyranoside was added to 0.4 mM. The E. coli cells were harvested and lysed in a cell homogenizer  in 20 mM phosphate buffer (pH 7.5) containing 500 mM NaCl, 1 mM DTT, 0.2% Tween-20, 0.2% NP-40, and protease inhibitors. Cell lysate was clarified by centrifugation at 4°C, 13,000 rpm  for 30 min. The supernatant was loaded onto a GSTrap column . After extensive washes with the lysis buffer, the proteins were eluted in the lysis buffer containing 10 mM glutathione. The eluted proteins were dialyzed overnight against the binding buffer . Full-length Rad4 was fused with GST at the N-terminus and overexpressed in S. pombe under the control of +nmt1 promoter. The cell lysate was made by grinding the frozen cells in a coffee-mill with dry ice. The clarified supernatant was loaded onto the GSTrap column, washed, and eluted as described above. While dialyzing overnight, the GST tag was cleaved off with C3 protease. The cleaved full-length Rad4 was directly used without further purification.GST tag was fused with Rad4 fragments at the N-terminal ends for expression and purification in rad3Δrad26 S. pombe under the +nmt1 (Rad3) and +nmt41 (Rad26-Flag) promoters. The yeast cells were homogenized in 2× IP buffer by the cell homogenizer. The clarified cell extract was incubated with anti-Flag M2 antibody beads (Sigma) by rotating at 4°C for 2 h. The beads were washed five times in ice-cold TBS-T buffer and the proteins eluted in TBS-T buffer containing 0.1 mg/ml Flag peptide for 60 min at 4°C. The kinase-inactive Rad3(D2249E)-Rad26-Flag complex The Rad3-Rad26-Flag complex was overexpressed in ΔEach purified Rad4 fragment was incubated with HA-Rad9 or HA-Crb2 bound to anti-HA antibody beads at a final concentration of 0.5 μM by rotating at 4°C for 2 h in 100 μl binding buffer . After washing several times with the binding buffer, the bound proteins were eluted with SDS gel loading buffer followed by heating at 98°C for 10 min. The samples were analyzed by SDS-PAGE or Western blotting.E. coli. was used as the substrate 2, 2 mM DTT, 0.1 mM EDTA, 0.01% NP-40, 200 μM ATP, pH 7.5) containing 500 ng of the Cds1(D312E) substrate. The reactions were assembled on ice and started by incubating at 30°C. To stop the reactions, the samples were chilled on ice and immediately mixed with the SDS gel-loading buffer. All samples were examined by Western blotting analyses. Loading of the substrate Cds1(D312E) was shown by Ponceau S staining. Phosphorylation of Cds1-Thr11 was examined by the phospho-specific antibody on the same membrane. Anti-HA and anti-Flag antibodies were used to detect Rad3 and Rad26, respectively.To assess the kinase activity of Rad3-Rad26, the kinase-inactive Cds1(D312E) mutant protein purified from rad4 mutants showed a high sensitivity to the replication stress induced by HU (see lower parts in ts) mutants, which may cause an unnoticeable defect in DNA replication or other functions. In order to provide a clear answer, we decided to screen new rad4 mutants with an enhanced sensitivity to replication stress. To facilitate the screening, we made a shut-off strain in which the promoter of +rad4 at the genomic locus was replaced with a thiamine repressive nmt81 promoter so that expression of Rad4 can be switched off by adding thiamine to the medium. The essential replication function is supported by wild type or mutant Rad4 expressed on a vector. As shown in In addition to its roles in DNA replication and the DNA damage checkpoint as a scaffold protein, previous studies have suggested that Rad4 may also function in the replication checkpoint mediated by Cds1 rad4 by first two rounds of mutational PCRs +leu colonies grown on plates without thiamine were replicated onto plates containing thiamine alone or thiamine plus HU or MMS. The thiamine alone plates were used to remove mutants with defects in DNA replication. The checkpoint mutants were isolated by comparing the colonies on plates containing thiamine alone with those on the plates containing thiamine plus the drugs. After extensive screenings and removal of the replication and the by-stander mutations, seven new checkpoint mutations were identified that are distributed across the molecule .Once the shut-off strain was made available, random mutations were generated in molecule . For com+rad4 genomic locus by a two-step marker switching method , indicating that Rad4 functions below Rad3 in the checkpoint pathways. Interestingly, the ΔC mutant with the deletion of the whole C-terminus between amino acid 498 and 648 was resistant to HU and MMS almost like the wild type cells .Since the new N-terminal mutants are highly sensitive to HU and MMS , we examecreased , indicatecreased  and actiy intact . These ry intact . To ruley intact . Similar1-Thr645 3 and Radrad3 or Δcds1 cells, the C13Y, K56R and E368K mutants remained resistant or slightly sensitive to HU like the Δchk1 mutant even after 6 h of treatment. However, under similar conditions, all three rad4 mutants were sensitive to MMS like the Δchk1 mutant. These results suggest that Rad4 mainly works in the Chk1-mediated DNA damage checkpoint pathway. Since all tested rad4 mutants were less sensitive than Δrad3 mutant, Rad4 functions below Rad3.To understand why Cds1 activation is only minimally affected in the C13Y and K56R mutants, we examined their sensitivities to acute HU treatments . We founrad4 mutants are sensitive to HU only in chronic exposure, we crossed the mutations into Δcds1 and Δchk1 strains and examined the possible synthetic effects  was unaffected by the C13Y and K56R mutations, it is unlikely that these mutations affected the activation of Rad3. Previous studies mainly by yeast two hybrid and Co-IP methods showed that Rad4 interacts with Crb2 and Rad9 via its N- and C-terminal pairs of BRCT repeats, respectively Since the Rad3 dependent phosphorylation of Mrc1-Thrin vivo, we performed the Co-IP experiments. Consistent with the in vitro results, the E368K mutation abolished the binding to Rad9 as previously reported 412 by Rad3  purified from E. coli was used as the substrate 11, the authentic Rad3 phosphorylation site in vivoin vitroin vitroin vitro biochemical studies showed that purified Rad4 had no or a minimal direct effect on Rad3 activation. This result is consistent with all in vivo data described above and suggests an evolutionary pressure for the convergence of functions in other eukaryotic organisms. However, the in vitro reactions lacking other known checkpoint components may be too simple to explain the physiological conditions inside the cells. Further in vitro biochemical studies that include the rest of the genetically defined components should provide a better explanation of how Rad3 is activated under various stress conditions in S. pombe.To investigate this possibility, we purified full-length Rad4 from S. pombe  and asseS. pombe. Earlier reports have also suggested that Rad4 may function in the replication checkpoint when DNA replication is perturbed. Here, we show that unlike that in budding yeast Dpb11, the C-terminus of Rad4 does not contain a robust AAD required for Rad3 activation. Our genetic screen and the in vitro studies also suggested that as previously reported cds1 but not with Δchk1 mutant when DNA damage occurs, indicating that Rad4 mainly function in the Chk1 pathway. Consistent with this, the relative sensitivities of all rad4 mutants to HU and MMS were quite similar  has only a minimal effect on checkpoint signaling rad9 null mutant under replication stress The exact mechanism of how Rad3, Mec1 and ATR are activated remains unknown because the crystal structures of these kinases are not yet available. The recently solved structures of a truncated mTOR, one of the Rad3 related PIKK kinases, may provide some implications Figure S1Examination of temperature sensitivity of the newly identified rad4 mutants with severe drug sensitivities. Wild type +rad4 or rad4 with the newly identified mutations C13Y, K56R, and A60P in the first BRCT repeat were integrated at the +rad4 genomic locus as described in (PDF)Click here for additional data file.Figure S2The N-terminal C13Y and K56R mutations may have maximally eliminated the checkpoint function of Rad4. (A) In order to search for an ideal rad4 mutation that can completely separate the checkpoint function from the DNA replication function of Rad4, C13Y, K56R and the previously described E368K (Taricani and Wang 2006) mutations were combined to see whether the combined mutations can further increase the drug sensitivity. Combination of K56R with E368K is lethal, suggesting that the replication function is affected. The C13Y-K56R and C13Y-E368K mutants grow normally, however, no significant enhancement of drug sensitivity was observed. (B) Combination of F305S with E368K is lethal, consistent with the essential function of the third BRCT repeat in DNA replication. Together, these results showed that combinations of the point mutations cannot further eliminate the checkpoint function of Rad4. Since the C13Y and K56R mutants are even more sensitive to MMS than the Δchk1 mutant Click here for additional data file.Figure S3645 by Rad3 was not affected by the Rad4 mutations.Phosphorylation of Mrc1-Thr Wild type cells or cells with the integrated rad4 mutations were incubated with (+) or without (-) HU for three hours at 30°C. The cells were fixed in 15% TCA at 4°C for more than three hours. Whole cell lysates were made from the TCA-fixed cells by the mini-bead beater method. The samples were separated by SDS PAGE and transferred to a nitrocellulose membrane. The membrane was strained with Ponceau S. A section of the stained membrane is shown for the loading (lower panel). The top section of the membrane containing Mrc1 was cut out, destained, and blotted with phosphor-specific antibody against phosphorylated Mrc1-Thr645 (top panel). Asterisk indicates the cross-reactive material. The same membrane was stripped and reprobed with anti-Mrc1 antibodies to detect Mrc1 (middle panel). Under the replication stress induced by HU, phosphorylation of Mrc1-Thr645 by Rad3 remains intact in all tested rad4 mutants.(PDF)Click here for additional data file.Figure S411 by Rad3 in Time course study of the phosphorylation of Chk1 and Cds1-Thrrad4 mutants. (A) Whole cell lysates made from MMS-treated cells under neutral conditions were separated by SDS PAGE and transferred to nitrocellulose membrane for Western blotting analysis. Phosphorylation of Chk1 was assessed by mobility shift assay using anti-HA antibody. A section of the Ponceau S stained membrane was shown below as the loading control. (B) Cds1 was IPed from the HU-treated cell and separated on an 8% SDS PAGE gel for Western blotting. Phosphorylated Cds1-Thr11 was detected by phospho-specific antibody (top). The same blot was stripped and reprobed with anti-HA antibody to detect Cds1 (bottom). IgG indicates the heavy chain of the anti-HA antibody used for the IP. This result ruled out the possibility of indirect cell cycle effect of the rad4 mutations on the phosphorylation of Chk1 and Cds1.(PDF)Click here for additional data file.Figure S5412 by Rad3 recruits Rad4.Phosphorylated Rad9-Thr (A) Binding of Rad4 to Rad9 is dependent on the phosphorylation of Rad9-Thr412 by Rad3. Rad4 was Co-IPed with Rad9 from HU-treated cells using anti-HA antibody beads. The beads with the IPed proteins were then treated with λ phosphatase in the presence or absence of the phosphatase inhibitor. Samples in lanes 3 and 5 were similarly treated except the beads in lane 5 were washed once before the SDS PAGE analysis. The phosphatase treatment removed the phosphate group on Rad9-Thr412 (compare lanes 3 with lane 4), which eliminated the binding of Rad4 to the Rad9 (compare lane 3 with lane 5). (B) Mutations of the Rad9 phosphorylation sites have a stronger effect than the Rad4(E368K) mutation on Rad3 dependent phosphorylation of Cds1-Thr11. Cds1 was IPed from the Rad9 phosphorylation site T412A or T412A-S423A mutants or the Rad4(E368K) mutants or the double mutants containing both Rad9 and Rad4 mutations. Phosphorylation of Cds1-Thr11 was detected by Western blotting using the phospho-specific antibody and quantitated. The levels of Cds1-Thr11 phosphorylation are shown as percentages with that in HU-treated wild type cells being set as 100%. Loading of Cds1 was shown in the lower panel by Western blotting using anti-HA antibody. (C) Phosphorylation of Rad9 functions in the upstream of Rad4 recruitment in the Chk1-mediated DNA damage response. The phosphorylation site mutants of Rad9 T412A and T412A-S423A were crossed with the Rad4(E368K) mutant. The single and double mutants were tested for their sensitivities to MMS by spot assay. Since Rad4(E368K) mutation affects the interaction of Rad4 with phosphorylated Rad9 and the Rad9 phosphorylation site mutations have a dominant effect over the Rad4(E368K) mutation, phosphorylated Rad9 recruits Rad4 to the DNA damage sites for efficient activation of Chk1. Together, these results suggest that phosphorylation of Rad9 by Rad3 (or activation of Rad3) does not absolutely require the recruitment of Rad4.(PDF)Click here for additional data file.Figure S6Minimal drug sensitivity caused by the C-terminal mutations and the dominant effect of the N-terminal mutations in the DNA damage response. (A) Diagram of the Rad4 C-terminus with relative locations of the mutations. The enlarged portion contains the recently reported Y599R (open circle) in the AAD domain (underlined)  and the three point mutations isolated by this study (solid circles). ΔC is the mutant in which the whole C-terminus from amino acids 498 to 648 is deleted. (B) Minimal drug sensitivities of the C-terminal mutants. The C-terminal mutations, including the previously described Y599R  mutation, were all integrated at +rad4 genomic locus as described in inet al. 2012) (strains SJ3 and SJ5) included in this experiment for comparison. (C) Dominant effects of the N-terminal mutation in the DNA damage checkpoint pathway. The N-terminal mutations C13Y and K56R (bold face) were combined with the C-terminal Y599R or D601N-K608E-L615S mutations and expressed on the vector under the control of +rad4 promoter in the shut-off strain. The drug sensitivities of the indicated mutants were tested on plates containing 0.01% MMS or HU at 2.5 mM or 5 mM concentrations. When cells are treated with MMS or 5 mM HU, the N-terminal mutations have the dominant effect over the C-terminal mutations. However, under the moderate replication stress generated by HU at the low level of 2.5 mM, which may cause minimal DNA damage, the C-terminal mutations are dominant over the N-terminal mutations in promoting the cell survival.(PDF)Click here for additional data file.Figure S7Minimal drug sensitivities caused by all mutations in the C-terminus of Rad4. (A) Diagram of Rad4 with the relative locations of the four BRCT repeats  and the three point mutations D601N, K608E, and L615S newly identified by this study. A series of deletion mutations were also made that are indicated by the lines of various lengths. (B) The three new C-terminal mutations were integrated at the +rad4 genomic locus. Various combinations of the three mutations were also integrated by the same method. Sensitivities of the cells with the integrated mutations to HU, MMS and UV were assessed by spot assay. Wild type cells and the Δrad3 mutant were used as the controls. The results showed that mutations in or near the recently identified AAD domain in the C-terminus only had a minor defect in checkpoint responses to DNA damage and the replication stress. (C) Wild type Rad4 or Rad4 with the indicated deletion mutations were expressed on a vector in the shut-off strain and tested for drug sensitivity. The three point mutations D601N, K698E, and L615S were included for comparison. Small deletions from the C-terminal ends did not affect much of the checkpoint functions except the Δ498–648 mutation, which generated a minimal sensitivity to MMS but not to HU similar to that in the three point mutations (top panel). The ΔC mutant, in which the whole C-terminus from amino acid 498 to 648 is deleted, behaved like the wild type cells. A larger deletion (Δ474–648) slightly sensitized the cells to HU but not MMS indicating a minor defect in DNA replication (lower panel). Further deletions into the third and fourth BRCT repeat (Δ376–648) and (Δ301–648) are lethal indicating that these repeats are required for DNA replication. (D) Mutations of the two tryptophan residues W485 and W580 in the C-terminus as well as the tryptophan W253 located between the third and the fourth BRCT repeats did not affect the checkpoint function of Rad4. (E) The protein levels of wild type Rad4 and Rad4 with various C-terminal mutations expressed on the vector in the shut-off strain were examined by Western blotting using anti-Rad4 antibodies. Asterisk indicates the cross-reactive material. All mutations in Rad4 C-terminus did not affect the level of the protein.(PDF)Click here for additional data file.Table S1The S. pombe strains used in this study.(DOCX)Click here for additional data file.


In the central nervous system, levels of extraneuronal dopamine are controlled primarily by the action of the dopamine transporter (DAT). Multiple signaling pathways regulate transport activity, substrate efflux, and other DAT functions through currently unknown mechanisms but presumably by oligomerization, protein-protein interactions and post-translational modification, such as phosphorylation. DAT is phosphorylated by protein kinase C within a serine cluster at the distal end of the cytoplasmic N-terminus, while recent work in model cells revealed proline-directed phosphorylation of rat DAT at membrane proximal residue Thr53. However, specific phosphorylation sites in native DAT under basal condition with associated functional properties have not been ascertained so far.in vivo phosphorylation sites (ii) generated a phospho-specific antibody (pT53Ab) for the confirmation of the identified phosphorylation site and for the determination of a stoichiometry of phosphorylation, involvement of PKC and phosphatase. Functional implications of this identified phosphorylation site have been tested by dopamine uptake and amphetamine-stimulated substrate efflux.We (i) applied mass spectrometry in rodent striatal tissue and heterologous cell systems to identify max and ablated amphetamine-induced substrate efflux.Phosphorylation of Thr53 (pThr53), occurred with a stoichiometry of ~50% under basal condition in rat striatal tissue, was unambiguously identified by mass spectrometry and immunoassay with phospho-specific antibody. pThr53 was strongly increased by phorbol esters and protein phosphatase inhibitors. Mutations of Thr53 to alanine to mimic dephosphorylation reduced dopamine transport VDAT is constitutively phosphorylated at Thr53 and its phosphorylation/dephosphorylation status plays a role in the transport mechanism, particularly in dopamine uptake and amphetamine-stimulated substrate efflux.


Daily i.p. injection of CB3 to ZDF rats inhibited the phosphorylation of JNK and p38MAPK, and prevented the expression of thioredoxin-interacting-protein (TXNIP/TBP-2) in ZDF rat brain. Although plasma glucose/insulin remained high, CB3 also increased the phosphorylation of AMP-ribose activating kinase (AMPK) and inhibited p70S6K kinase in the brain. Both CB3 and CB4 reversed apoptosis induced by inhibiting thioredoxin reductase as monitored by decreasing caspase 3 cleavage and PARP dissociation in SH-SY5Y cells. The decrease in JNK and p38MAPK activity in the absence of a change in plasma glucose implies a decrease in oxidative or neuroinflammatory stress in the ZDF rat brain. CB3 not only attenuated MAPK phosphorylation and activated AMPK in the brain, but it also diminished apoptotic markers, most likely acting via the MAPK–AMPK–mTOR pathway. These results were correlated with CB3 and CB4 inhibiting inflammation progression and protection from oxidative stress induced apoptosis in human neuronal cells. We suggest that by attenuating neuro-inflammatory processes in the brain Trx1 mimetic peptides could become beneficial for preventing neurological disorders associated with diabetes.Diabetes is a high risk factor for dementia. High glucose may be a risk factor for dementia even among persons without diabetes, and in transgenic animals it has been shown to cause a potentiation of indices that are pre-symptomatic of Alzheimer's disease. To further elucidate the underlying mechanisms linking inflammatory events elicited in the brain during oxidative stress and diabetes, we monitored the activation of mitogen-activated kinsase (MAPKs), c-jun NH  •Thioredoxin mimeitics peptides (TXM) lower apoptosis in the brain of ZDF rat.•TxM peptides prevent TXNIP/TBP-2 expression in the brain of ZDF rat.•TxM peptides could become beneficial for preventing diabetes associated neurological disorders. MAPK pathways 2+. It was shown to regulate the transcription factor c-jun in cerebellar granule neurons ob/ob) obesity models, there was a significant increase in total JNK activity, highlighting JNK as a crucial mediator of obesity and insulin resistance, and a potential target for therapeutics MAPK activation and prevention of NF-kB nuclear translocation MAPK phosphorylation in fibroblasts S6K pathway. Hence, CB3 could have a potential benefit for decreasing detrimentaleffects elicited in the brain during chronic hyperglycemia.Aging patients with Type 2 diabetes (T2D) are at a high risk of developing cognitive and memory impairments including some of Alzheimer disease's (AD) most significant symptoms d-thiopyranosato-S) gold(I); thioredoxin mimetic (TXM) peptides TXM-CB3 and -CB4 were custom synthesized by Novetide, Ltd. Haifa; Thinkpeptides, Oxford, UK, and GL Biochem., Shanghai, China; tissue culture serum and medium were from Biological Industries, Kibbutz Beit-Haemek, Israel.All materials were purchased from Sigma, Jerusalem, if not otherwise stated; Auranofin , triethylphosphine . The cells were cultured in DMEM/F12 HAM 1:1 medium supplemented with 10% fetal bovine serum (FBS) and penicillin–streptomycin, incubated at 37 °C with 5% COSH-SY5Y cells were seeded in 96-well plates and treated with 5 µM AuF for 30 min, or high glucose, washed and cultured with or without increasing concentrations of CB3, or CB4, as indicated. Twenty-four hours later, the cells were fixed with glutaraldehyde in a final concentration of 0.5% for 10 min. Cells were washed 3 times with DDW, dried over night, and washed once with borate buffer . The fixed cells were stained with 200 µl of 1% methylene blue in borate buffer for 1 h. After extensive washing and drying, the color was extracted with 200 µl of 0.1 M HCl for 1 h at 37 °C. Later cell viability was measured using spectrophotometer at 630 nm.In this study we used the obese diabetic Zucker rat, a widely used animal model of obesity and type 2 diabetes. These animals display insulin resistance, dyslipidemia, hyperinsulinemia Six-weeks old male ZDF rats were injected with either CB3 (1 mg/kg and 10 mg/kg) or with Rosiglidasone (Rosi) for 28 days. Blood glucose was measured every week (glucometer). At day 26 an OGTT assay was obtained and measured. At day 28, the animals were sacrificed and different biochemical blood markers  were meaTwenty to thirty micrograms of protein samples were loaded on 10–12% SDS-PAGE gels. The proteins were then transferred electrophoretically to nitrocellulose . The blots were blocked by incubation for 1 h at RT in TBS-T  with 4% Difco skim milk , and incubated over-night at 4 °C with the primary antibody: pERK1/2 (Thr 202/Tyr204), mouse mAb; ERK2  rabbit Ab; p-SAPK/JNK (Thr183/Tyr185), rabbit mAb; SAPK/JNK, mouse mAb; p-p38MAP kinase (Thr180/Tyr182), rabbit mAb; p38, rabbit Ab; cleaved caspase 3, rabbit mAb; PARP (Poly (ADP-ribose) polymerase), rabbit Ab; GAPDH , rabbit mAb; TXNIP/TBP-2 mouse mAb. Antibodies were from Cell Signaling Tech. USA, if not otherwise stated, used at 1:1000. Purified b Catenin, mouse mAb,  diluted in 5% BSA, 0.04% azide in TBS-T. Proteins were detected with anti-mouse or anti-rabbit IgG-HRP linked antibody . For data analysis, the amounts of each band were quantified by using the EZ-Quant software (version 2.2) and plotted with a linear regression program.We used ZDF rats characterized by a progressive β-cell dysfunction and a leptin receptor defect, which result in hyperglycemia. The ZDF rats were divided into 4 groups. Animals were i.p. injected with vehicle , 1 mg/kg CB3, 10 mg/kg CB3, or p.o. with 10 mg/kg rosiglitazone (Rosi), an antidiabetic agent, which activates peroxisome proliferator-activated receptor gamma (PPAR-γ agonist). Blood glucose levels and plasma insulin were tested as indicated . Rats tr2-terminal kinase (JNK), p38 MAP kinase (p38MAPK) and the extracellular-signal-regulated kinases 1 and 2 (ERK1/2). Rats injected with 1 mg/kg of CB3 showed no significant change in p38MAPK, JNK, or ERK1/2 phosphorylation compared to the untreated group. In contrast, the phosphorylation level of both p38MAPK and JNK was significantly lowered in animals treated with 10 mg/kg CB3 or with 10 mg/kg Rosi , a potent TrxR inhibitor. By keeping Trx1 in the oxidized-state, AuF leads to the dissociation of oxidized Trx1 from ASK1, activating the ASK1–MAPK cascade MAPK, JNK, and ERK1/2, and the corresponding non-phosphorylated MAPKs , which was previously shown to be effective in reversing amyloid beta-induced protein oxidation, loss of mitochondrial function and DNA fragmentation in primary neuronal cells ependent A and B. rylation A and B crylation C. This rrylation C. This sFurther evidences for the anti inflammatory effects of the TxM peptides were achieved by examining TNFα, a ROS-independent inflammatory reagent known as a JNK activator Next we explored the expression and the impact of CB3 on the expression of TXNIP/TBP-2 in the ZDF rat. As shown in Unlike the high glucose up-regulation of TXNIP/TBP-2 in beta cells The anti-diabetic drugs, Rosi and metformin are known as activators of the AMPK pathway, which reduce intracellular ATP by inhibiting complex I of the mitochondrial electron transport chain The phosphorylation level of AMPK, which leads to inhibition of the mammalian target of rapamycin (m-TOR) pathway, was further evaluated in the ZDF brain. AMPK mediates m-TOR inhibition through binding of Raptor and phosphorylation of p70S6 kinase, a protein involved in numerous cell-signaling pathways. We observed that in both CB3 and Rosi treated animals phosphorylation of p70S6 kinase in the ZDF brain was reduced B. These The effects of AuF on cell viability and the protection offered by CB3 and CB4 were visualized and quantified in SH-SY5Y cells. The cells were treated with AuF (5 µM) for 30 min, washed, and visualized 24 h later. Phase contrast microscopy demonstrated a considerable change in cell morphology and cell number A. In conNext we tested the effect of CB3 on caspase 3-cleavage in SH-SY5Y cells. The cells were incubated with 100 µM CB3 for 24 h in serum-free medium. A reduction in caspase 3-cleavage was observed in CB3 treated cells in a concentration dependent manner, seen already at 50 µM A. We theIn this study we analyzed the protection of ZDF rat brain and human SH-5Y5Y neuroblastoma cells from oxidative induced inflammation damages and from inflammatory consequences accompanying diabetes or through disruption of the TrxR–Trx redox system. For this purpose we used the thioredoxin mimetic peptides, CB3 and CB4. These peptides derived from the canonical CxxC motif of the Trx1 active site and a modified CxC motif, which are responsible for the redox activity of Trx1.MAPK phosphorylations and preventing NF-kB nuclear translocation in vitro and in vivo The TxM thiol peptides alleviate oxidative stress by inhibiting JNK and p38MAPK in the brain might indicate that CB3 crosses the blood brain barrier (BBB) in order to protect against inflammatory neurodegenerative consequences in the ZDF rats, more direct studies are required to establish BBB penetration of TxM peptides.Here we tested inhibition by CB3 of inflammatory pathways that are activated by MAP-Kinases, JNK and p38, in the ZDF rat brain. Although no changes in blood glucose were observed, the CB3 treated mice displayed a decrease in the phosphorylation/activation of the MAPK inflammatory-stress pathway with its ensuing apoptotic effects. Although the decrease in phosphorylated JNK and 38Interestingly, in previous studies N-acetyl cysteine (NAC), which is a much weaker reducing reagent compared to CB3 In studies carried out using insulinoma cells, CB3 appeared to prevent apoptosis through inhibiting the Trx1–ASK1–MAPK pathway Human neuronal SH-5Y5Y cells are often considered a model for Alzheimer's disease. These cells, when treated with CB3 or CB4, displayed protection from oxidative stress induced by blocking the [TrxR–Trx] redox system. The increase in cell viability, which was accompanied by a decrease in caspase-3 cleavage, prevention of PARP dissociation, as well as the ability to reverse TNF-alpha induced JNK phosphorylation in SH-SH5Y cells, further supports the anti-inflammatory properties of these peptides. TxM putative activity pathway is shown schematically in TXNIP/TBP-2 is a key stress-responsive inhibitory switch of Trx1 activity playing an important role in the preservation of cellular viability We observed a significant decrease in TXNIP/TBP-2 levels in CB3 treated ZDF rats. The mechanism by which CB3 lowers TXNIP/TBP-2 currently remains unknown. It is possible that by lowering ROS, CB3 prevents TXNIP/TBP-2 up regulation through inhibiting transcription. This possibility is consistent with a recent study demonstrating that TXNIP/TBP-2 expression in the brain was induced by oxidative stress without glucose In the Rosi-treated animals, in which glucose and triglycerides levels were low, TXNIP/TBP-2 level was not decreased. In contrast, in CB3-treated animals in which glucose and triglycerides levels were high, altering of the Trx/TXNIP redox balance, CB3 appeared to regulate TXNIP/TBP-2 in a glucose independent mechanism.Unlike a strong induction of TXNIP/TBP-2 by high glucose in insulinoma cells The involvement of AMPK in diabetes was reported in previous studies Here we show that in the ZDF brain phosphorylated AMPK was significantly elevated in both CB3- and Rosi-treated rats. To our knowledge, this is the first study demonstrating AMPK activation by a Trx1 mimetic peptide. The increase in AMPK phosphorylation in the brain of CB3 treated ZDF-rats, was in good correlation with a decrease in TXNIP/TBP-2 expression and subsequent blocking of the phosphorylation of p70S6K through the mTOR–p70S6K pathway .In the Rosi-treated rats AMPK phosphorylation was significantly increased as expected; however, no apparent reduction in TXNIP/TBP-2 was observed. Further studies are needed to explore why repression of TXNIP/TBP-2 transcription, shown by metformin MAPK and JNK activity, diminished TXNIP/TBP-2 over expression, and activated AMPK in the brain of ZDF rats. The anti inflammatory effects of CB3 in the brain of the ZDF rat were mediated through the MAPK-AMPK-mTOR-p70SP6 signaling pathway. CB3 displayed these protective effects without reducing glucose triglyceride levels or insulin indexes, as opposed to Rosi-treated ZDF rats, in which the decrease in glucose triglyceride levels or insulin indexes account for the decrease in the neuro-inflammatory signaling. The significant decrease in TXNIP/TBP-2 expression in the brain was observed only in the CB3- and not in the Rosi-treated rats. This is the first study that demonstrates significant protective effects by a Trx1 mimetic peptide in the brain of diabetic animals. We suggest that the reduction in the activation of the stress signaling in the brain could lower the risk factor for an accelerated rate of cognitive decline and memory impairments associated with diabetes..In summary, in the present 28 days-term experiment the thioredoxin mimetic peptide CB3, attenuated p38M.C.-K. researched data, contributed discussion, reviewed/edited manuscript; L.K. researched data, reviewed manuscript; M.T. researched data, contributed discussion, reviewed manuscript; H.B. researched data; J.M.L. research data reviewed manuscript T.M. and Y.L. researched data reviewed manuscript; D.A. wrote manuscript and is the guarantor responsible for the study design, access to data, and the decision to submit and publish the manuscript.


We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.Recent advancements in isolation techniques for cytochrome  Moreover, we must further our knowledge of cell survival signaling that prevent post-ischemic release of Cytc to improve our development of therapies.Release of Cytc did not preserve the in vivo phosphorylation state during protein purification. Recent studies by our group have demonstrated that mammalian Cytc can be post-translationally modified by tyrosine phosphorylation at two distinct residues in a tissue-specific manner c is targeted by cell signaling by yet unidentified mitochondrial tyrosine kinases. Phosphorylation of Cytc occurs at Tyr48 in liver and Tyr97 in heart tissue, which causes partial inhibition in the reaction with isolated cytochrome c oxidase c from human skeletal muscle in a high throughput mass spectrometry study c function has not been studied. Notably, in all the tissues investigated using conditions that preserve phosphorylation state, Cytc has been shown to be phosphorylated. Based on these findings we proposed a new model in which Cytc phosphorylation maintains controlled electron transfer rates, thereby preventing hyperpolarization of the mitochondrial membrane potential and limiting ROS production c and other OxPhos components become dephosphorylated leading to maximal flux in the ETC resulting in the mitochondrial membrane potentials exceeding 140 mV, a condition known to promote excessive mitochondrial ROS generation Traditional studies of isolated Cytc in mitochondrial type II apoptosis may also be modulated by Cytc phosphorylation. Interestingly, phosphomimetic substitution of Cytc Tyr48 with Glu completely abolished the ability of this protein to induce apoptosis in vitro, suggesting that cell signaling can regulate the execution of apoptosis at the level of Cytc. Additionally, this same alteration reduces the binding affinity of Cytc for the inner mitochondrial membrane lipid, cardiolipin c from cardiolipin is a prerequisite for its release into the cytosol c phosphorylation on respiration and apoptosis, along with the anti-apoptotic effects of insulin administration, led us to investigate the role of Cytc phosphorylation in the neuroprotection conferred by insulin following an ischemic insult to the brain.The role of CytGuide for the Care and Use of Laboratory Animals, 8th Edition and stated in the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. Animal numbers were kept to a minimum consistent with statistical significance. Global brain ischemia of 8 minutes duration was induced using bilateral carotid occlusion and hypotension as described by Smith et al. Chemicals were purchased from Sigma unless otherwise stated. Animal experiments in this study were approved by the Wayne State University Animal Investigation Care and Use Committee and conform to the guidelines on the ethical treatment of animals presented in the National Research Council's 2, 1 mM EGTA, 1 mM EDTA) supplemented with protease inhibitors (0.2 mM phenylmethylsulfonylfluoride (PMSF), 1 µg/mL pepstatin, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 12.5 µg/mL calpain inhibitor I), and then centrifuged at 750 x g for 10 min. The resulting supernatant was centrifuged at 14,000 x g for 10 min and the pellet taken as the crude mitochondrial fraction. The remaining supernatant was centrifuged at 100,000 x g for 60 min and the supernatant was taken as the cytosolic fraction. Purity of the preparation was assessed by immunoblotting each fraction with antibodies against the mitochondrial marker CcOIV  and the cytosolic marker β-actin , and all data were normalized to the appropriate marker.The hippocampal tissue was weighed, homogenized using a Dounce homogenizer in 1∶5 (wt∶vol) isotonic HEPES isolation media , and the supernatants containing Cytc were adjusted to pH 7.5 and at the same time supplemented with unspecific phosphatase inhibitors KF (10 mM) and activated vanadate (1 mM) to prevent dephosphorylation c-containing supernatants were subjected to DE52 anion exchange column and CM52 cation exchange column chromatography  as described c was concentrated under vacuum to 2 mL, desalted via Sephadex G50 gel filtration, and subjected to HPLC purification  using a Macrosphere™ GPC column c was concentrated, desalted, and stored at −80°C.Pig brains were obtained as discarded tissue from a slaughterhouse  with consent for use and immediately frozen on dry ice. Pooled tissue (1.8 kg) was ground using a commercial meat grinder and split into two equal fractions. The first (control) fraction was supplemented with 3 L of extraction buffer  and immediately homogenized using a commercial blender to extract Cytc isolation 8 brains  were combined for each condition. To improve yield, the ratio of extraction buffer volume (800 mL) to tissue was increased compared to the large scale pig brain isolation procedure. The subsequent steps were performed as above except for the DE52 anion exchange column and CM52 cation exchange column chromatography steps, which were each performed only once.For small scale rat brain Cytc , β-actin , and CcOIV .For Western blot of subcellular fractions, equal amounts of protein  were separated using SDS-PAGE, and then transferred to nitrocellulose. Membranes were incubated in primary antibody  followed by secondary antibody . Antibody binding was detected using the enhanced chemiluminescence technique . Primary antibodies used for immunoblotting were: Cytc, SDS-PAGE was carried out using a 12% Tris-Tricine gel. After protein transfer to a PVDF membrane , Western analysis was carried out as described For Western analysis of isolated Cytc were reconstituted with 40 µL of 100 mM NH4HCO3 (pH 8.9) and then reduced with 10 mM DTT for 1 h at 56 °C, followed by alkylation with 55 mM indoacetamide for 1 h at room temperature in the dark. Proteins were digested with affinity purified and TPCK treated trypsin  at a trypsin:protein ratio of 1∶100 (w/w) overnight at 37 °C. Digests were enriched for phosphopeptides using PhosTio Kit . Briefly, PhosTio Tips were rinsed with Buffer A  and Buffer B . Then samples were diluted with 1.5-fold Buffer B and loaded onto the tips two times. Tips were rinsed with Buffer B once and Buffer A three times. Peptides were eluted from the TiO2 resin using 50 µL of 1% NH4OH in water and 50 µL of 1% NH4OH in 40% acetonitrile sequentially. Eluted peptides were further acidified by adding 4 µL of 50% acetic acid and dried to completeness.Forty µg of purified and desalted CytThe enriched phosphopeptide samples were reconstituted in 10 µL of 0.1 M acetic acid in water and loaded on a 75 µm I.D. precolumn packed with 3 cm of 5 µm Monitor C18 particles and eluted with a reversed-phase gradient (0–70% acetonitrile in 30 min) into the mass spectrometer (Linear Trap Quadrupole-Fourier Transform (LTQ-FT), Thermo Fisher Scientific, Waltham, MA) through an analytical column (360 µm outer diameter ×75 µm inner diameter-fused silica capillary with 12 cm of 5 µm Monitor C18 particles with an integrated 4 µm-ESI emitter tip fritted with polymer). Static peak parking was performed via flow rate reduction from 200 nL/min to 40 nL/min when peptides began to elute as judged from a bovine serum albumin peptide scouting run. Using a split flow configuration, an electrospray voltage of 2.0 kV was applied as described MS/MS spectra were assigned to peptide sequences from the NCBI non-redundant protein database sliced in Bioworks 3.1 for porcine proteins and searched with the SEQUEST algorithm. SEQUEST search parameters designated variable modifications of +79.9663 Da on Ser, Thr, and Tyr (phosphorylation). Identified phosphopeptide spectra of interest were manually verified.c is tyrosine-phosphorylated in vivo in heart and liver tissue, and that these phosphorylations lead to healthy, controlled  cell respiration rates, which may prevent the execution of the apoptotic pathway c.We have previously shown that Cytc from the mitochondria into the cytosol and subsequent cell death in a rat model of global brain ischemia/reperfusion injury. Cytc release was detected by immunoblotting rat hippocampal homogenates fractionated into cytosolic and mitochondrial fractions with an anti-Cytc antibody. Antibodies against cytochrome c oxidase subunit IV (CcOIV) and β-actin were used as loading controls and to determine the purity of mitochondrial and cytosolic fractions. We previously demonstrated that Cytc release following global brain ischemia reaches a maximum level by 24 hours of reperfusion c release at this late reperfusion interval, i.e., 24 hours of reperfusion, in order to determine if Cytc release can be prevented and not just delayed. Western blot of cytosolic fractions showed a significant 4-fold increase in cytosolic Cytc in the untreated controls . A single bolus of 20 U/Kg insulin administered at the onset of reperfusion prevented increased cytosolic Cytc levels, maintaining cytosolic Cytc levels similar to sham-operated controls , an astrocyte marker (GFAP-Magenta), and a microglial marker (Iba-1-Green). Sham-operated control animals exhibit a CA1 hippocampus densely populated with pyramidal neurons positive for NeuN, with few astrocytes and microglia . Cytc was then purified to homogeneity using our protocol that preserves phosphorylation. We performed Western analysis with anti-phospho-Ser/Thr/Tyr-specific antibodies and obtained a strong and specific signal with the anti-phospho-Tyr antibody .c was tyrosine phosphorylated after insulin treatment we determined the specific phosphorylation site. Isolated pig brain Cytc was digested with trypsin and analyzed by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS/MS). This method first enriched phosphorylated peptides via a TiO2 resin, and the eluted peptides were then subjected to tandem-MS for fragment examination. This analysis unambiguously revealed that Tyr97 was phosphorylated on Cytc following insulin treatment on the peptide EDLIApYLKKATNE by fragment ions b6, b8, b9, b10, and y3, y4, y6 . The resultant Cytc was of high purity  electron transfer rates compared to phosphorylated Cytc. Specifically, for Tyr97-phosphorylated Cytc we have shown that the Km of cytochrome c oxidase for Cytc is 5.5 µM with sigmoidal kinetics, whereas dephosphorylation leads to a shift of the Km to 2.5 µM and hyperbolic kinetics c isolated from heart showed a single phosphorylation site at Tyr97, similar to our results in brain reported here after insulin treatment. Both heart and brain are highly aerobic tissues that fully depend on energy provided by OxPhos.Our results are the first to show that Cytin vitro  and in vin vitro . In bothin vitro  and 5. Ic to participate in apoptosis as was suggested for Tyr48-phosphorylated Cytc based on studies with the phosphomimetic mutant Tyr48Glu Cytcc mimicking Tyr48 phosphorylation was incapable of triggering downstream caspase activation, suggesting a safeguard mechanism to regulate apoptosis via cell signaling pathways. Therefore, insulin-triggered phosphorylation of Cytc may confer neuroprotection in two ways: 1) a beneficial reduction of ETC flux, preventing a hyperpolarization of the mitochondrial membrane potential and thus ROS production, and 2) a change in its structure that interferes with apoptosome formation and/or activation. Indeed, preventing the interaction of Apaf-1 with Cytc has been shown to be a potent neuroprotective strategy following brain ischemia c tyrosine nitration, which introduces a negative charge to the tyrosine and can thus be considered a phosphomimetic modification, demonstrated that Tyr97 nitration both reduces the binding affinity of Cytc to Apaf-1 and inhibits the activation of downstream caspases Future work is necessary to reveal if Tyr97 phosphorylation also abolishes the ability of Cytc via phosphorylation expands the role of Cytc from that of a passive electron carrier and apoptotic co-factor to an active player controlling cell fate decisions. Traditional studies attempting to prevent Cytc release by preventing outer mitochondrial membrane permeabilization have yet to yield an effective neuroprotective agent. Specifically targeting Cytc phosphorylation may provide a potent method for preventing cell death after ischemia/reperfusion injury in brain. Further studies are necessary to identify the kinase(s) and phosphatase(s) that act on Cytc. These enzymes could then be specifically targeted to maintain Cytc in the phosphorylated state during conditions of stress that would normally result in cell death.In conclusion, the discovery that cell signaling controls Cyt


Netphos 2.0 and Disphos 1.3 programs were used for potential phosphorylation; NetPhosK 1.0 and KinasePhos for kinase prediction; and YinOYang 1.2 and OGPET to predict possible O-glycosylation sites. We also identified Yin Yang sites that may have potential for O-β-GlcNAc and phosphorylation interplay at same Ser/Thr residues. We for the first time proposed that alternate phosphorylation and O-β-GlcNAc modification on Ser 192, Ser 205, Ser 206; and Thr 191 may provide an on/off switch to regulate assembly of claudin-1 at tight junctions. In addition these phosphorylation sites may be targeted by novel chemotherapeutic agents to prevent phosphorylation lead by HCV viral entry complex.HCV is a leading cause of hepatocellular carcinoma and cirrhosis all over the world. Claudins belong to family of tight junction's proteins that are responsible for establishing barriers for controlling the flow of molecules around cells. For therapeutic strategies, regulation of viral entry into the host cells holds a lot of promise. During HCV infection claudin-1 is highly expressed in liver and believed to be associated with HCV virus entry after HCV binding with or without co-receptor CD81. The claudin-1 assembly with tight junctions is regulated by post translational modifications. During claudins assembly and disassembly with tight junctions, phosphorylation is required at C-terminal tail. In cellular proteins, interplay between phosphorylation and O- HCV, the deadly virus has infected almost 3% of the world population. Most of the infected patients develop chronic infection leading to end stage hepatocellular carcinoma. A better understanding of mechanism of infection and the potential host co-factors facilitating its replication is an urgent need of the hour for the release of disease burden and vaccine development -3.In multicellular organisms, movement of ions, proteins and water is controlled by barriers known as tight junctions (TJs) formed by epithelial and endothelial cell monolayers . While tIn biological systems, protein localization, activity, their interaction with other proteins and overall turnover is determined by post translational modifications (PTMs) . SeveralO-glycosylation is also very important PTM of nuclear and cytoplasmic proteins. During O-glycosylation one molecule of N-acetylglucosamine (O-GlcNAc) is introduced on Ser or Thr residue by enzyme OGT (O-GlcNAc transferase). Addition of O-β-GlcNAc can inhibit phosphorylation on Ser or Thr residue. Interplay between O-β-GlcNAc modification and phosphorylation on the same amino acid residues has been observed in several nuclear and cytoplasmic proteins [β-GlcNAc and phosphorylation compete for each other are known as Yin Yang sites [O-glycosylation and their possible interplay sites which may influence claudin-1 interaction with TJs and their possible effects on HCV entry and future therapeutics.proteins . These Png sites . These YThe FASTA sequence of human claudin-1 was retrieved from the SWISS-PROT sequence database  with entThe claudin-1 sequence used in this study was "MANAGLQLLGFILAFLGWIGAIVSTALPQWRIYSYAGDNIVTAQAMYEGLWMSCVSQSTGQIQCKVFDSLLNLSSTLQATRALMVVGILLGVIAIFVATVGMKCMKCLEDDEVQKMRMAVIGGAIFLLAGLAILVATAWYGNRIVQEFYDPMTPVNARYEFGQALFTGWAAASLCLLGGALLCCSCPRKTTSYPTPRPYPKPAPSSGKDYV".We used more than one bioinformatics tools to access the post-translational modification on claudin-1 to get best results.Phosphorylation potential for human claudin-1 was predicted by using NetPhos 2.0  and DispKinase specific phosphorylation sites in human claudin-1 were predicted by NetPhosK 1.0  and KinaFor the evaluation of experimentally verified phosphorylation sites on human claudin-1, Phospho.ELM database was used . This daO-β-GlcNAc modification potential sites were predicted by YinOYang 1.2 [Yang 1.2 -34 and OYang 1.2 . YinOYanAs there is no template model of claudin-1 available in protein data bank , we desiAll the methods used for predicting post translational modifications like ANNs  or SVM (support vector machine) etc have been extensively used in biological sequence study and predicting the possible potentials for PTMs . The metHuman claudin-1 was aligned with other species. Conserved and semi-conserved substituted Ser and Thr residues within each subtype were determined . A list of Yin Yang and FN-Yin Yang residues is given in Table The Ser and Thr residues which were not predicted to be O-β-GlcNAc modification, five possible Yin Yang sites were proposed  found that claudin-1 was necessary for HCV entry after its binding with CD81 receptors. Recent findings showed that claudin-1 can enable cell to cell transfer of HCV [Among vertebrates claudin-1 has highly conserved globular domain while, less conserved N- and C-terminals. Claudin-1 also showed highly conserved status among mammals , Waqar Ahmad (M Phil Chemistry) and Gull S (MSc Biochemistry) are Research Officer; Shabbiri K is lecturer while Fouzia K is BS (Hons) student at GC University, Lahore. Kausar H, Sawar MT and Shahid I are Phd scholars. Asad S is MPhil scholar, while Sajida Hassan (PhD Molecular Biology) is Principal Investigator at CEMB, University of the Punjab, Lahore