Chronic ingestion of a Western diet increases O-linked-β-N-acetylglucosamine (O-GlcNAc) protein modification in the rat heart

AimsProtein O-GlcNAcylation is both a nutrient sensing and cellular stress response that mediates signal transduction in the heart. Chronically elevated O-GlcNAc has been associated with the development of cardiac dysfunction at both the cellular and organ levels in obesity, insulin resistance and diabetes. Development of these pathologies is often attributed to diets high in saturated fat and sugar (a “Western” diet; WES) but a role for O-GlcNAc in diet-induced cardiac dysfunction has not been established. The aims of this study were to examine the effect of chronic consumption of WES on cardiac O-GlcNAcylation and investigate associations of O-GlcNAc with cardiac function and markers of cellular stress.Main methodsYoung male rats received either a control diet (CON; n = 9) or WES (n = 8) diet for 52 weeks.Key findingsThere was no evidence of cardiac dysfunction, advanced glycation endproduct (AGE) accumulation, pathological cardiac hypertrophy, calcium handling impairment, fibrosis or endoplasmic reticulum stress in WES hearts. However, cardiac O-GlcNAc protein, particularly in the higher molecular weight range, was significantly higher in WES hearts compared to CON (P < 0.05). Protein levels of the enzymes that regulate O-GlcNAc attachment were not different between groups; thus, the increased O-GlcNAcylation in WES hearts appears to be due to increased nutrient availability rather than enzymatic regulation of cellular stress.SignificanceThese data suggest that diets high in saturated fat and sugar may contribute to the adverse effects of metabolic syndrome and diabetes by an O-GlcNAc-mediated process and that this may occur in the absence of overt cellular stress.     

A Long Lasting β1 Adrenergic Receptor Stimulation of cAMP/Protein Kinase A (PKA) Signal in Cardiac Myocytes

Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β₁ adrenergic receptor (β₁AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t_½ of 3.9 h) in cardiac myocytes. However, the β₁AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β₁AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β₁AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.     

Regulation of BMAL1 Protein Stability and Circadian Function by GSK3β-Mediated Phosphorylation

Background

Circadian rhythms govern a large array of physiological and metabolic functions. To achieve plasticity in circadian regulation, proteins constituting the molecular clock machinery undergo various post-translational modifications (PTMs), which influence their activity and intracellular localization. The core clock protein BMAL1 undergoes several PTMs. Here we report that the Akt-GSK3β signaling pathway regulates BMAL1 protein stability and activity.

Principal Findings

GSK3β phosphorylates BMAL1 specifically on Ser 17 and Thr 21 and primes it for ubiquitylation. In the absence of GSK3β-mediated phosphorylation, BMAL1 becomes stabilized and BMAL1 dependent circadian gene expression is dampened. Dopamine D2 receptor mediated signaling, known to control the Akt-GSK3β pathway, influences BMAL1 stability and in vivo circadian gene expression in striatal neurons.

Conclusions

These findings uncover a previously unknown mechanism of circadian clock control. The GSK3β kinase phosphorylates BMAL1, an event that controls the stability of the protein and the amplitude of circadian oscillation. BMAL1 phosphorylation appears to be an important regulatory step in maintaining the robustness of the circadian clock.     

Modulation of endogenous prostaglandins by thymosin-α1 in lymphocytes

The effects of thymosin-α₁ on the stimulation of specific release of prostaglandin E₂ (PGE₂) from splenic lymphocytes and thymocytes were studied. Experiments were also performed to study in parallel the absolute levels of thymosin-α₁ in the blood and the induction of serum FTS activity and of azathioprine sensitivity of spleen cells from adult thymectomized (ATx) mice. A significant difference in the release of PGE₂ between normal splenocytes and splenocytes from ATx mice was observed. Thymosin-α₁ at certain concentrations was able to stimulate PGE₂ release from lymphocytes of ATx mice while inhibiting release in lymphocytes of normal mice. Also, thymocytes were stimulated to release PGE₂ after incubation with α₁ in a manner similar to that seen in spleen cells of ATx mice. Approximately the same concentration of α₁ was found to also correct the low azathioprine sensitivity of splenocytes from ATx mice. Determinations of FTS-like activity in the blood and the pharmacokinetics of α₁ after administration of this synthetic molecule show a clear dissociation. A maximum peak of α₁ activity was obtained after 1 hr, while maximal FTS-like activity was observed after 24 hr. The inhibition of the induction by α₁ of FTS-like activity and of Thy 1.2 antigen by indomethacin suggests that the action of α₁ requires prostaglandin biosynthesis.     

β-Arrestin Promotes Wnt-induced Low Density Lipoprotein Receptor-related Protein 6 (Lrp6) Phosphorylation via Increased Membrane Recruitment of Amer1 Protein

β-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/β-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the β-arrestin function in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P₂ production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that β-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that β-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIβ. Importantly, cells lacking β-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P₂, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dishevelled-associated kinases.     

Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-β1

Summary Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation. However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells. In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels. In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed α-smooth muscle (SM) actin mRNA at low levels. α-SM actin mRNA expression is dramatically enhanced by TGF-β₁. In addition, double immunofluorescence staining with anti-vWF and anti-α-SM-1 (a monoclonal antibody to α-SM actin) shows that RFCs co-express the two proteins. In three dimensional cultures, RFCs still expressed vWF, but lost staining for α-SM actin, whereas α-SM actin mRNA became barely detectable. In contrast to two-dimensional cultures, the addition of TGF-β₁ to the culture media did not enhance α-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation. Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-β₁ with a pattern very different from that of RFCs. Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions. This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations. Supported by a Post-Doctoral Fellowship from the Swiss National Science Foundation (OK) and grant HL-RO1-28373 (JAM) from the Department of Human Services, Public Health Service, Washington, D.C.     

Cardiac Function Is Regulated by B56α-mediated Targeting of Protein Phosphatase 2A (PP2A) to Contractile Relevant Substrates

Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]_i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of β-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of −5 mV, the I_Ca,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser¹⁶ was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca²⁺ sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation.     

Regulation of CCAAT/Enhancer-binding Protein Homologous Protein (CHOP) Expression by Interleukin-1β in Pancreatic β Cells

Apoptosis contributes to immune-mediated pancreatic β cell destruction in type I diabetes. Exposure of β cells to interleukin-1β (IL-1β) causes endoplasmic reticulum stress and activates proapoptotic networks. Here, we show that nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways regulate the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), which mediates endoplasmic reticulum stress-induced apoptosis. Both CHOP mRNA and protein increase in β cells treated with IL-1β. In addition, prolonged exposure to high glucose further increases IL-1β-triggered CHOP expression. IL-1β also causes increased expression of C/EBP-β and a reduction of MafA, NFATc2, and Pdx-1 expression in β cells. Inhibition of the NF-κB and MAPK signaling pathways differentially attenuates CHOP expression. Knocking down CHOP by RNA interference protects β cells from IL-1β-induced apoptosis. These studies provide direct mechanistic links between cytokine-induced signaling pathways and CHOP-mediated apoptosis of β cells.     

Cardiac Ankyrin Repeat Protein Attenuates Cardiac Hypertrophy by Inhibition of ERK1/2 and TGF-β Signaling Pathways

Aims

It has been reported that cardiac ankyrin repeat protein is associated with heart development and diseases. This study is aimed to investigate the role of CARP in heart hypertrophy in vivo.

Methods and Results

We generated a cardiac-specific CARP-overexpressing transgenic mouse. Although such animals did not display any overt physiological abnormality, they developed less cardiac hypertrophy in response to pressure overload than did wildtype mice, as indicated by heart weight/body weight ratios, echocardiographic and histological analyses, and expression of hypertrophic markers. These mice also exhibited less cardiac hypertrophy after infusion of isoproterenol. To gain a molecular insight into how CARP attenuated heart hypertrophy, we examined expression of the mitogen-activated protein kinase cascade and found that the concentrations of phosphorylated ERK1/2 and MEK were markedly reduced in the hearts of transgenic mice subjected to pressure overload. In addition, the expressions of TGF-β and phosphorylated Smad3 were significantly downregulated in the hearts of CARP Tg mice in response to pressure overload. Furthermore, addition of human TGF-β1 could reverse the inhibitory effect of CARP on the hypertrophic response induced by phenylephrine in cardiomyocytes. It was also evidenced that the inhibitory effect of CARP on cardiac hypertrophy was not attributed to apoptosis.

Conclusion

CARP attenuates cardiac hypertrophy, in which the ERK and TGF-β pathways may be involved. Our findings highlight the significance of CARP as an anti-hypertrophic factor in therapy of cardiac hypertrophy.     

Modulation of the Protein Kinase Cδ Interaction with the “d” Subunit of F1F0-ATP Synthase in Neonatal Cardiac Myocytes: DEVELOPMENT OF CELL-PERMEABLE,MITOCHONDRIALLY TARGETED INHIBITOR AND FACILITATOR PEPTIDES*

The F₁F₀-ATP synthase provides ∼90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase Cδ (PKCδ) inhibits F₁F₀ activity via an interaction with the “d” subunit of F₁F₀-ATP synthase (dF₁F₀) in neonatal cardiac myocytes (NCMs) (Nguyen, T., Ogbi, M., and Johnson, J. A. (2008) J. Biol. Chem. 283, 29831–29840). We have now identified a dF₁F₀-derived peptide (NH₂-²AGRKLALKTIDWVSF¹⁶-COOH) that inhibits PKCδ binding to dF₁F₀ in overlay assays. We have also identified a second dF₁F₀-derived peptide (NH₂-¹¹¹RVREYEKQLEKIKNMI¹²⁶-COOH) that facilitates PKCδ binding to dF₁F₀. Incubation of NCMs with versions of these peptides containing HIV-Tat protein transduction and mammalian mitochondrial targeting sequences resulted in their delivery into mitochondria. Preincubation of NCMs, with 10 nm extracellular concentrations of the mitochondrially targeted PKCδ-dF₁F₀ interaction inhibitor, decreased 100 nm 4β-phorbol 12-myristate 13-acetate (4β-PMA)-induced co-immunoprecipitation of PKCδ with dF₁F₀ by 50 ± 15% and abolished the 30 nm 4β-PMA-induced inhibition of F₁F₀-ATPase activity. A scrambled sequence (inactive) peptide, which contained HIV-Tat and mitochondrial targeting sequences, was without effect. In contrast, the cell-permeable, mitochondrially targeted PKCδ-dF₁F₀ facilitator peptide by itself induced the PKCδ-dF₁F₀ co-immunoprecipitation and inhibited F₁F₀-ATPase activity. In in vitro PKC add-back experiments, the PKCδ-F₁F₀ inhibitor blocked PKCδ-mediated inhibition of F₁F₀-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially targeted modulators of the PKCδ-dF₁F₀ interaction in NCMs. These novel peptides will improve our understanding of cardiac F₁F₀ regulation and may have potential as therapeutics to attenuate cardiac injury.     

Modulation of G Protein α-Subunit mRNA Levels in Discrete Rat Brain Regions by Cerebroventricular Infusion of Ginsenoside Rc and Rg1

We have investigated the effects of centrally administered ginsenoside Rc and Rg1 on the modulation of G protein expression in the central nervous system in rat brain. The effects of continuous infusion of ginsenosides on the modulation of G protein -subunit mRNA were investigated by using in situ hybridization study. Rats were infused with ginsenoside Rc or Rg1 (10 g/10 l/h, i.c.v.) for 7 days, through preimplanted cannula by osmotic minipumps. The level of Gs mRNA was not changed by the infusion of ginsenoside Rc or Rg1. The level of Gai mRNA was significantly elevated in frontal cortex and hippocampus following treatment with ginsenoside Rc as well as ginsenoside Rg1. However, the level of Go mRNA was significantly decreased in part of the hippocampus and cerebellum after the animals had received ginsenoside Rg1 infusion. These results suggest that prolonged infusion of ginsenosides could differentially modulate the expression of G protein -subunit mRNA in rat brain in a region-specific manner.     

Mathematical Basis of Predicting Dominant Function in Protein Sequences by a Generic HMM–ANN Algorithm

The accurate annotation of an unknown protein sequence depends on extant data of template sequences. This could be empirical or sets of reference sequences, and provides an exhaustive pool of probable functions. Individual methods of predicting dominant function possess shortcomings such as varying degrees of inter-sequence redundancy, arbitrary domain inclusion thresholds, heterogeneous parameterization protocols, and ill-conditioned input channels. Here, I present a rigorous theoretical derivation of various steps of a generic algorithm that integrates and utilizes several statistical methods to predict the dominant function in unknown protein sequences. The accompanying mathematical proofs, interval definitions, analysis, and numerical computations presented are meant to offer insights not only into the specificity and accuracy of predictions, but also provide details of the operatic mechanisms involved in the integration and its ensuing rigor. The algorithm uses numerically modified raw hidden markov model scores of well defined sets of training sequences and clusters them on the basis of known function. The results are then fed into an artificial neural network, the predictions of which can be refined using the available data. This pipeline is trained recursively and can be used to discern the dominant principal function, and thereby, annotate an unknown protein sequence. Whilst, the approach is complex, the specificity of the final predictions can benefit laboratory workers design their experiments with greater confidence.     

Intra-domain Cross-talk Regulates Serine-arginine Protein Kinase 1-dependent Phosphorylation and Splicing Function of Transformer 2β1

Transformer 2β1 (Tra2β1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2β1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2β1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a docking groove in the kinase domain. Although SRPK1 readily phosphorylates RS2 in a splice variant lacking the N-terminal RS domain (Tra2β3), RS1 blocks phosphorylation of these serines in the full-length Tra2β1. Thus, RS2 serves two new functions. First, RS2 positively regulates binding of the central RNA recognition motif to an exonic splicing enhancer sequence, a phenomenon reversed by SRPK1 phosphorylation on RS1. Second, RS2 enhances ligand exchange in the SRPK1 active site allowing highly efficient Tra2β1 phosphorylation. These studies demonstrate that SRPK1 is a regulator of Tra2β1 splicing function and that the individual RS domains engage in considerable cross-talk, assuming novel functions with regard to RNA binding, splicing, and SRPK1 catalysis.     

Increased poly-β-hydroxybutyrate (PHB) chain length by the modulation of PHA synthase activity in recombinant Escherichia coli

A second promoter (P1) was inserted to the PHA (poly--hydroxyalkanoate) operon (pSP2) of Esherichia coli DH5 with an optimal E. coli ribosome binding site and a trc strong promoter (pSJS1) to obtain poly--hydroxybutyrate (PHB) with long chain length. When the inducer, IPTG was added to the culture at 0.4 mM, the average molecular weight was 1.1 × 10⁶ Da. However, an even greater increase of the PHB average molecular weight to 2.5 × 10⁷ Da was observed without IPTG being added.