Dual Regulatory Role of Polyamines in Adipogenesis [Signal Transduction]

September 22nd, 2015 by Brenner, S., Bercovich, Z., Feiler, Y., Keshet, R., Kahana, C.

Adipogenesis is a complex process, accompanied by a chain of interdependent events. Disruption of key events in this cascade may interfere with the correct formation of adipose tissue. Polyamines were demonstrated necessary for adipogenesis; however, the underlying mechanism by which they act has not been established. Here, we examined the effect of polyamine depletion on the differentiation of 3T3-L1 preadipocytes. Our results demonstrate that polyamines are required early in the adipogenic process. Polyamine depletion inhibited the second division of the mitotic clonal expansion (MCE), and inhibited the expression of PPARγ and C/EBPα, the master regulators of adipogenesis. However, it did not affect the expression of their transcriptional activator, C/EBPβ. Additionally, polyamine depletion resulted in elevation of mRNA and protein levels of the stress induced C/EBP homologous protein (CHOP), whose dominant negative function is known to inhibit C/EBPβ DNA binding activity. Conditional knockdown of CHOP in polyamine depleted preadipocytes restored PPARγ and C/EBPα expression, but failed to recover MCE and differentiation. Thus, our results suggest that the need for MCE in the adipogenic process is independent from the requirement for PPARγ and C/EBPα expression. We conclude that de-novo synthesis of polyamines during adipogenesis is required for down regulation of CHOP to allow C/EBPβ activation, and for promoting MCE.

Regulation of E2 Promoter Binding Factor 1 (E2F1) transcriptional activity through a deubiquitinating enzyme, UCH37 [Signal Transduction]

September 22nd, 2015 by Mahanic, C. S., Budhavarapu, V., Graves, J. D., Li, G., Lin, W.-C.

E2F1 is tightly controlled by multiple mechanisms, but whether ubiquitination regulates its transcriptional activity remains unknown. Here, we identify UCH37 as the first, to our knowledge, deubiquitinating enzyme for E2F1. UCH37 does not deubiquitinate UbK48 chains or affect E2F1 protein stability. Instead UCH37, but not a catalytically dead mutant, decreases the K63-linked ubiquitination of E2F1 and activates its transcriptional activity. UCH37 depletion reduces gene expression of both proliferative and pro-apoptotic E2F1 target genes. UCH37 depletion also decreases both cell proliferation and apoptosis induction in functional assays. Interestingly, UCH37 expression is induced by E2F1 and its level rises in G1/S transition and S phase, suggesting a positive feedback loop between UCH37 and E2F1. UCH37 protein and mRNA levels are also induced after DNA damage. UCH37 localizes to the promoters of E2F1 pro-apoptotic target genes such as caspase-3, caspase-7, PARP1 and Apaf-1 and activates their expression after DNA damage. Moreover, the expressions of E2F1 proliferative and pro-apoptotic genes are correlated with the levels of UCH37 in many primary tumors. These results uncover a novel mechanism for E2F1 transcriptional activation through removal of its K63-linked ubiquitination by UCH37.

Nanoscale Landscape of Phosphoinositides Revealed by the Specific PH-domains Using Single-molecule Super-resolution Imaging in the Plasma Membrane [Cell Biology]

September 22nd, 2015 by Ji, C., Zhang, Y., Xu, P., Xu, T., Lou, X.

Both PI4P and PI(4,5)P2 are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. However, their nano-scale spatial organization in the PM remains elusive. Using single-molecule super-resolution microscopy and new photoactivatable fluorescence probes based on the PH-domains that specifically recognize phosphatidylinositides in insulin-secreting INS-1 cells, we reported that the PI(4,5)P2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI(4,5)P2 enriched membrane patches/domains of diverse sizes (383 ± 14 nm in average). Quantitative analysis revealed a modest concentration gradient which was much less steep than previously thought, and no densely packed PI(4,5)P2 nano-domains were observed. Live-cell super-resolution imaging further demonstrated the dynamic structural changes of those domains in the flat PM and membrane protrusions. PI4P and PI(3,4,5)P3 showed the similar spatial distributions as PI(4,5)P2. These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at nanometer scale.
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Routes of Ca2+ Shuttling during Ca2+ Oscillations; Focus on the Role of Mitochondrial Ca2+ Handling and Cytosolic Ca2+ Buffers [Computational Biology]

September 22nd, 2015 by Pecze, L., Blum, W., Schwaller, B.

In some cell types, Ca2+ oscillations are strictly dependent on Ca2+ influx across the plasma membrane, while in others oscillations also persist in the absence of Ca2+ influx. We observed that in primary mesothelial cells, the plasmalemmal Ca2+ influx played a pivotal role. However, when the Ca2+ transport across the plasma membrane by the "lanthanum insulation method" was blocked prior to the induction of the serum-induced Ca2+ oscillations, mitochondrial Ca2+ transport was found to be able to substitute for the plasmalemmal Ca2+ exchange function, thus rendering the oscillations independent of extracellular Ca2+. However, in a physiological situation, the Ca2+-buffering capacity of mitochondria was found not to be essential for Ca2+ oscillations. Moreover, brief spontaneous Ca2+ changes were observed in the mitochondrial Ca2+ concentration without apparent changes in the cytosolic Ca2+ concentration indicating the presence of a mitochondrial autonomous Ca2+ signaling mechanism. In the presence of calretinin, a Ca2+-buffering protein, the amplitude of cytosolic spikes during oscillations was decreased and the amount of Ca2+ ions taken up by mitochondria was reduced. Thus, the increased calretinin expression observed in mesothelioma cells and in certain colon cancer might be correlated to the increased resistance of these tumor cells to pro-apoptotic/pro-necrotic signals. We identified and characterized (experimentally and by modeling) three Ca2+ shuttling pathways in primary mesothelial cells during Ca2+ oscillations: Ca2+ shuttled between I) the ER and mitochondria II) the ER and the extracellular space and III) the ER and cytoplasmic Ca2+ buffers.
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The structure of a full-length membrane-embedded integrin bound to a physiological ligand [Signal Transduction]

September 21st, 2015 by Dai, A., Ye, F., Taylor, D. W., Hu, G., Ginsberg, M. H., Taylor, K. A.

Increased ligand binding to integrin ("activation") underpins many biological processes, such as leukocyte trafficking, cell migration, host-pathogen interaction, and hemostasis. Integrins exist in several conformations, ranging from compact and bent to extended and open. However, the exact conformation of membrane-embedded, full-length integrin bound to its physiological macromolecular ligand is still unclear. Integrin α11bβ3, the most abundant integrin in platelets, has been a prototype for integrin activation studies. Using negative stain electron microscopy and nanodisc-embedding to provide a membrane-like environment, we visualized the conformation of full-length α11bβ3 in both a Mn2+-activated, ligand-free state and a Mn2+-activated, fibrin-bound state. Activated but ligand-free integrins exist mainly in the compact conformation; whereas fibrin-bound α11bβ3 predominantly exists in a fully extended, headpiece open conformation. Our results show that membrane-embedded, full-length integrin adopts an extended and open conformation when bound to its physiological macromolecular ligand.

Disruption of Heat Shock Protein 90 (Hsp90)-Protein Kinase C{delta} (PKC{delta}) Interaction by (-)-Maackiain Suppresses Histamine H1 Receptor Gene Transcription in HeLa Cells [Signal Transduction]

September 21st, 2015 by

Histamine H1 receptor (H1R) gene is an allergic disease sensitive gene and its expression level is strongly correlated with the severity of allergic symptoms. (-)-Maackiain was identified as a Kujin-derived anti-allergic compound that suppresses the up-regulation of H1R gene. However, the underlying mechanism of H1R gene suppression remains unknown. Here, we sought to identify a target protein of (-)-maackiain and investigate its mechanism of action. Fluorescence quenching assay and immunoblot analysis identified heat shock protein 90 (Hsp90) as a target protein of (-)-maackiain. Pull down assay revealed that (-)-maackiain disrupted the interaction of Hsp90 with protein kinase C-δ (PKCδ), resulting in the suppression of phorbol 12-myristate 13-acetate (PMA)-induced up-regulation of H1R gene expression in HeLa cells. Additional Hsp90 inhibitors including 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), celastrol, and novobiocin also suppressed PMA-induced H1R gene up-regulation. 17-AAG inhibited PKCδ translocation to the Golgi and phosphorylation of Tyr311 on PKCδ. These data suggest that (-)-maackiain is a novel Hsp90 pathway inhibitor. The underlying mechanism of the suppression of PMA-induced up-regulation of H1R gene expression by (-)-maackiain and Hsp90 inhibitors are the inhibition of PKCδ activation through the disruption Hsp90-PKCδ interaction. Involvement of Hsp90 in H1R gene up-regulation suggests that suppression of Hsp90 pathway could be a novel therapeutic strategy for allergic rhinitis.
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Structural and Biophysical Characterization of the Interactions Between Calmodulin and the Pleckstrin Homology Domain of Akt [Molecular Biophysics]

September 21st, 2015 by Agamasu, C., Ghanam, R. H., Saad, J. S.

The translocation of Akt, a serine/threonine kinase, to the plasma membrane (PM) is a critical step in the Akt activation pathway. It is established that membrane binding of Akt is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3). There is now evidence that Akt activation in many breast cancer cells is also modulated by the calcium-binding protein, calmodulin (CaM). Upon epidermal growth factor (EGF) stimulation of breast cancer cells, CaM co-localizes with Akt at the PM to enhance activation. However, the molecular details of Akt(PHD) interaction with CaM are not known. In this study, we employed nuclear magnetic resonance (NMR), biochemical, and biophysical techniques to characterize CaM binding to Akt(PHD). Our data show that CaM forms a tight complex with the PH domain of Akt (dissociation constant = 100 nM). The interaction between CaM and Akt(PHD) is enthalpically driven and the affinity is greatly dependent on salt concentration, indicating that electrostatic interactions are important for the interaction. The CaM-binding interface in Akt(PHD) was mapped to two loops adjacent to the PI(3,4,5)P3 binding site, which represents a rare CaM-binding motif and suggests a synergistic relationship between CaM and PI(3,4,5)P3 upon Akt activation. Elucidation of the mechanism by which Akt interacts with CaM will help in understanding the activation mechanism, which may provide insights for new potential targets to control the pathophysiological processes of cell survival.
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Ac2PIM-responsive miR-150 and miR-143 target receptor-interacting protein kinase 2 and transforming growth factor beta-activated kinase 1 to suppress NOD2-induced immunomodulators [Signal Transduction]

September 21st, 2015 by

Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac2PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac2PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3 and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-α, VEGF-A and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac2PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1 respectively. Interestingly, Ac2PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac2PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKCδ-MAPK pathway to suppress β-CATENIN-mediated expression of COX-2, SOCS-3 and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac2PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac2PIM and/or MDP.
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Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice [Enzymology]

September 21st, 2015 by

Regeneration of the visual chromophore, 11-cis-retinal, is a crucial step in the visual cycle required to sustain vision. This cycle consists of sequential biochemical reactions that occur in photoreceptor cells and the retinal pigmented epithelium (RPE). Oxidation of 11-cis-retinol to 11-cis-retinal is accomplished by a family of enzymes termed 11-cis-retinol dehydrogenases, including RDH5 and RDH11. Double deletion of Rdh5 and Rdh11 does not limit the production of 11-cis-retinal in mice. Here we describe a third retinol dehydrogenase in the RPE, RDH10, which can produce 11-cis-retinal. Mice with a conditional knockout of Rdh10 in RPE cells (Rdh10 cKO) displayed delayed 11-cis-retinal regeneration and dark adaption after bright light illumination. Retinal function measured by ERG after light exposure was also delayed in Rdh10 cKO mice as compared with controls. Double deletion of Rdh5 and Rdh10 (cDKO) in mice caused elevated 11/13-cis-retinyl ester content also seen in Rdh5-/-Rdh11-/- mice when compared to Rdh5-/- mice. Normal retinal morphology was observed in 6-month-old Rdh10 cKO and cDKO mice suggesting loss of Rdh10 in the RPE does not negatively affect the health of the retina. Compensatory expression of other retinol dehydrogenases was observed in both Rdh5-/- and Rdh10 cKO mice. These results indicate that RDH10 acts in cooperation with other RDH isoforms to produce the 11-cis-retinal chromophore needed for vision.

AMPK control of mTORC1 is p53- and TSC2-independent in pemetrexed-treated carcinoma cells [Molecular Bases of Disease]

September 21st, 2015 by Agarwal, S., Bell, C. M., Rothbart, S. B., Moran, R. G.

The key sensor of energy status in mammalian cells, AMP-activated protein kinase (AMPK), can also be activated by the AMP analog ZMP generated directly from aminoimidazolecarboxamide ribonucleoside (AICAR) or from inhibition of purine synthesis by the antifolate pemetrexed (PTX), a drug used extensively in the treatment of lung cancers. In spite of this common mechanism, signaling downstream of AMPK activated by PTX or AICAR differed. AICAR-activated AMPK inhibited mTORC1 both directly by phosphorylation of the mTORC1 subunit Raptor and indirectly by phosphorylation of the regulator TSC2. In contrast, PTX-activated AMPK inhibited mTORC1 solely through Raptor phosphorylation. This dichotomy was due to p53 function. Transcription of p53 target genes, including TSC2, was activated by AICAR but not by PTX. While both PTX and AICAR stabilized p53, only AICAR activated Chk2 phosphorylation, stimulating p53-dependent transcription. However, Raptor phosphorylation by AMPK was independent of p53 and was sufficient, after PTX treatment, to inhibit mTORC1. We concluded that PTX effects on mTORC1 were independent of TSC2 and of p53, and that the activation of a p53 transcriptional response by AICAR was due to an activation of Chk2 that was not elicited by PTX.