Secreted frizzled-related protein 5 diminishes cardiac inflammation and protects the heart from ischemia-reperfusion injury [Signal Transduction]

December 2nd, 2015 by

Wnt signaling has diverse actions in cardiovascular development and disease processes. Secreted frizzled-related protein 5 (Sfrp5) has been shown to function as an extracellular inhibitor of non-canonical Wnt signaling that is expressed at relatively high levels in white adipose tissue. The aim of this study was to investigate the role of Sfrp5 in the heart under ischemic stress. Sfrp5 knock-out (KO) and wild type (WT) mice were subjected to ischemia/reperfusion (I/R). Whereas Sfrp5-KO mice exhibited no detectable phenotype compared to WT control at baseline, they displayed larger infarct sizes, enhanced cardiac myocyte apoptosis and diminished cardiac function following I/R. The ischemic lesions of Sfrp5-KO mice had greater infiltration of Wnt5a-positive macrophages and greater inflammatory cytokine and chemokine gene expression compared to WT mice. In bone marrow-derived macrophages, Wnt5a promoted JNK activation and increased inflammatory gene expression, whereas treatment with Sfrp5 blocked these effects. These results indicate that Sfrp5 functions to antagonize inflammatory responses after I/R in the heart, possibly through a mechanism involving non-canonical Wnt5a/JNK signaling.
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DndEi exhibits helicase activity essential for DNA phosphorothioate modification and ATPase activity strongly stimulated by DNA substrate with a GAAC/GTTC motif [Microbiology]

December 2nd, 2015 by

Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen of the backbone phosphate group is replaced by sulfur, is governed by the DndA-E proteins in prokaryotes. To better understand the biochemical mechanism of PT modification, functional analysis of the recently found PT-modifying enzyme DndEi, which has an additional domain compared to canonical DndE, from Riemerella anatipestifer is performed in this study. The additional domain is identified as an ATP-dependent DNA helicase, and functional deletion of this domain in vivo leads to PT modification deficiency, indicating an essential role of helicase activity in PT modification. Subsequent analysis reveals that the additional domain has an ATPase activity. Intriguingly, the ATPase activity is strongly stimulated by DNA substrate containing a GAAC/GTTC motif (i.e., the motif at which PT modifications occur in R. anatipestifer) when the additional domain and the other domain (homologous to canonical DndE) are co-expressed as a full-length DndEi. These results reveal that PT modification is a biochemical process with DNA strands separation and intense ATP hydrolysis.
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Interaction between RING1 (R1) and Ubiquitin-Like (UBL) Domain Is Critical for the Regulation of Parkin Activity [Neurobiology]

December 2nd, 2015 by Ham, S. J., Lee, S. Y., Song, S., Chung, J.-R., Choi, S., Chung, J.

Parkin is an E3 ligase that contains an UBL domain in the N-terminus and an R1-in-between-ring (IBR)-RING2 (R2) motif in the C-terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at S65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at S65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on the interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, is also suppressed by competitive binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations-associated with Parkinson′s disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by CCCP treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1, and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis.

Astrocyte Resilience to Oxidative Stress Induced by Insulin Like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity [Molecular Bases of Disease]

December 2nd, 2015 by Davila, D., Fernandez, S., Torres–Aleman, I.

Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the pro-survival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases P38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that in astrocytes exposed to oxidative stress by hydrogen peroxide (H2O2), P38 activation did not inhibit AKT (Protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and lasted more in astrocytes than in neurons, either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H2O2-exposed astrocytes, boosting their neuro-protective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cyto-protective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury.
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Reciprocal changes in phosphoenolpyruvate carboxykinase and pyruvate kinase with age are a determinant of aging in C. elegans [Metabolism]

December 2nd, 2015 by

Aging involves progressive loss of cellular function and integrity, presumably caused by accumulated stochastic damage to cells. Alterations in energy metabolism contribute to aging, but how energy metabolism changes with age, how these changes affect aging, and whether they can be modified to modulate aging remain unclear. In locomotory muscle of post-fertile C. elegans, we identified a progressive decrease in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), a longevity-associated metabolic enzyme, and a reciprocal increase in glycolytic pyruvate kinase (PK) that were necessary and sufficient to limit lifespan. Decline in PEPCK-C with age also led to loss of cellular function and integrity including muscle activity, and cellular senescence. Genetic and pharmacologic interventions of PEPCK-C, muscle activity and AMPK signaling demonstrate that declines in PEPCK-C and muscle function with age interacted to limit reproductive life and lifespan via disrupted energy homeostasis. Quantifications of metabolic flux show that reciprocal changes in PEPCK-C and PK with age shunted energy metabolism toward glycolysis, reducing mitochondrial bioenergetics. Lastly, calorie restriction countered changes in PEPCK-C and PK with age to elicit anti-aging effects via TOR inhibition. Thus, a programmed metabolic event involving PEPCK-C and PK is a determinant of aging that can be modified to modulate aging.

Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1) Mutation and TRAP1 Inhibitor Gamitrinib-triphenylphosphonium (G-TPP) Induce a Forkhead box O (FOXO)-dependent Cell Protective Signal from Mitochondria [Neurobiology]

December 2nd, 2015 by

Tumor necrosis factor receptor (TNFR)-associated protein 1 (TRAP1), a mitochondrial Hsp90 family chaperone, has been identified as a critical regulator of cell survival and bioenergetics in tumor cells. To discover novel signaling networks regulated by TRAP1, we generated Drosophila TRAP1 mutants. The mutants successfully developed into adults and produced fertile progeny, showing that TRAP1 is dispensable in development and reproduction. Surprisingly, mutation or knock-down of TRAP1 markedly enhanced Drosophila survival under oxidative stress. Moreover, TRAP1 mutation ameliorated mitochondrial dysfunction and dopaminergic (DA) neuron loss induced by deletion of a familial Parkinson disease (PD) gene Pten-induced kinase 1 (PINK1) in Drosophila. Gamitrinib-triphenylphosphonium (G-TPP), a mitochondria-targeted Hsp90 inhibitor that increases cell death in HeLa and MCF7 cells, consistently inhibited cell death induced by oxidative stress and mitochondrial dysfunction induced by PINK1 loss in mouse embryonic fibroblast (MEF) cells and DA cell models such as SH-SY5Y and SN4741 cells. Additionally, G-TPP also suppressed the defective locomotive activity and DA neuron loss in Drosophila PINK1 null mutants. In further genetic analyses, we showed enhanced expression of Thor, a downstream target gene of transcription factor FOXO, in TRAP1 mutants. Furthermore, deletion of FOXO almost nullified the protective roles of TRAP1 mutation against oxidative stress and PINK1 mutation. These results strongly suggest that inhibition of the mitochondrial chaperone TRAP1 generates a retrograde cell protective signal from mitochondria to the nucleus in a FOXO-dependent manner.
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Visualization of NO3-/NO2- Dynamics in Living cells by Fluorescence Resonance Energy Transfer (FRET) Imaging Employing a Rhizobial Two-Component Regulatory System [Molecular Biophysics]

December 2nd, 2015 by

Nitrate (NO3−) and nitrite (NO2−) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO3− /NO2− exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO3−/NO2− for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO3−/NO2− in physiology), by employing NO3−/NO2−- induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO3− and NO2−, and its FRET signal is changed in response to NO3− /NO2− in the low micromolar range. Furthermore, both an increase and decrease in cellular NO3− concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO3−/NO2− dynamics in various organisms, including plants and animals.
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Characterization of Tiki, a new family of WNT-specific metalloproteases [Enzymology]

December 2nd, 2015 by

The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family with dependence on Mn2+/Co2+ but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in inhibition of mating pheromones. The TIKI/TRAB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core β sheet within an α+β fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that resides outside the β sheet catalytic core but likely assists the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing hydrophobic nature of TIKI cleavage sites. Our study provides insights into the TIKI family of proteases and its WNT substrates.

Multidrug Transporter LmrP allows Relocation of Catalytic Carboxylates [Membrane Biology]

December 2nd, 2015 by Tong, Z., Ding, N., Neuberger, A., van Veen, H. W.

One of the important mechanisms of drug resistance in cells is based on active drug extrusion by multidrug efflux transporters. LmrP is a secondary-active major facilitator superfamily multidrug transporter from the non-pathogenic, food-grade, Gram-positive bacterium Lactococcus lactis, which is similar to major facilitator superfamily multidrug transporters in pathogenic bacteria. LmrP mediates multidrug efflux in a proton motive force-dependent fashion by catalyzing electrogenic substrate/proton antiport. For this purpose, LmrP contains a number of carboxyl residues which, together with polar and aromatic residues, are organized in two clusters on the surface of a large interior chamber. To further investigate the functional role of these catalytic carboxylates and clusters in LmrP, we changed the composition of Cluster 1 by removal of Glu-327 and relocation of this carboxyl to 6 positions in the interior chamber. Among the tested positions, we found that the reinsertion at positions Thr-331 and Ala-355 in proximity of Cluster 2 restored wild-type energetics and kinetics of propidium and/or ethidium transport. Moreover, the introduction of T331E in wild-type LmrP enhanced the rate of active propidium efflux due to the ability of T331E to act as an additional proton-binding group. This behavior is very different from substrate-dedicated transporters in which the location of catalytic carboxylates in the proton coupling reaction is very precise and crucial for enzyme activity. Our data uncover an intrinsic flexibility in the location of the key residues in LmrP that are responsible for proton/drug exchange.

Replacing the promoter of the murine gene encoding P-selectin with the human promoter confers human-like basal and inducible expression in mice [Gene Regulation]

December 2nd, 2015 by Liu, Z., Zhang, N., Shao, B., Panicker, S. R., Fu, J., McEver, R. P.

In humans and mice, megakaryocytes/platelets and endothelial cells constitutively synthesize P-selectin and mobilize it to the plasma membrane to mediate leukocyte rolling during inflammation. Tumor necrosis factor (TNF)-α, interleukin-1β, and lipopolysaccharide (LPS) markedly increase P-selectin mRNA in mice but decrease P-selectin mRNA in humans. Transgenic mice bearing the entire human SELP gene recapitulate basal and inducible expression of human P-selectin, and reveal human-specific differences in P-selectin function. Differences in the human SELP and murine Selp promoters account for divergent expression in vitro, but their significance in vivo is not known. Here, we generated knock-in mice that replace the 1.4-kb proximal Selp promoter with the corresponding SELP sequence (SelpKI). SelpKI/KI mice constitutively expressed more P-selectin on platelets and more P-selectin mRNA in tissues, but only slightly increased P-selectin mRNA after injection of TNF-α or LPS. Consistent with higher basal expression, leukocytes rolled slower on P-selectin in trauma-stimulated venules of SelpKI/KI mice. However, TNF-α did not further reduce P-selectin-dependent rolling velocities. Blunted up-regulation of P-selectin mRNA during contact hypersensitivity reduced P-selectin-dependent inflammation in SelpKI/- mice. Higher basal P-selectin in SelpKI/KI mice compensated for this defect. Thus, divergent sequences in a short promoter mediate most of the functionally significant differences in expression of human and murine P-selectin in vivo.
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