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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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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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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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.

Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties and Modulation by PKA-mediated Phosphorylation [Molecular Biophysics]

September 21st, 2015 by

Two HCM-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory-peptide and the cardiac-specific N-terminus. We recently reported these regions may interact when Ser23/Ser24 are phosphorylated, weakening cTnI interaction with cardiac TnC (cTnC). Little is known about how these mutations influence the affinity of cTnC for cTnI (KC-I) or contractile kinetics during β-adrenergic stimulation. Here, we tested how cTnIR146G or cTnIR21C influence contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca2+ binding affinity to cTn (KCa) and KC-I. PKA phosphorylation resulted in a similar reduction of KCa for all complexes, but KC-I was reduced only with cTnIWT. cTnIWT, cTnIR146G and cTnIR21C were complexed into cTn and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured ± PKA phosphorylation. Maximal tension (TMAX) was maintained for cTnIR146G and cTnIR21C exchanged myofibrils, and Ca2+ sensitivity of tension (pCa50) was increased. PKA phosphorylation decreased pCa50 for cTnIWT exchanged myofibrils, but not for either mutation. PKA phosphorylation accelerated the early, slow-phase relaxation for cTnIWT myofibrils, especially at Ca2+-levels that the heart operates in vivo. Importantly, this effect was blunted for cTnIR146G and cTnIR21C exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N-terminus and the inhibitory-peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together our results suggest that cTnIR146G and cTnIR21C blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory-peptide.
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Phosphatidylcholine affects the role of the sorting and assembly machinery in the biogenesis of mitochondrial {beta}-barrel proteins [Protein Synthesis and Degradation]

September 18th, 2015 by

Two protein translocases drive the import of β-barrel precursor proteins into the mitochondrial outer membrane: The translocase of the outer membrane (TOM complex) promotes transport of the precursor to the intermembrane space, whereas the sorting and assembly machinery (SAM complex) mediates subsequent folding of the β-barrel and its integration into the target membrane. The non-bilayer forming phospholipids phosphatidylethanolamine (PE) and cardiolipin (CL) are required for the biogenesis of β-barrel proteins. Whether bilayer-forming phospholipids such as phosphatidylcholine (PC), the most abundant phospholipid of the mitochondrial outer membrane, play a role in the import of β-barrel precursors is unclear. In this study we show that PC is required for stability and function of the SAM complex during the biogenesis of β-barrel proteins. PC further promotes the SAM-dependent assembly of the TOM complex, indicating a general role of PC for the function of the SAM complex. In contrast to PE-deficient mitochondria precursor accumulation at the TOM complex is not affected by depletion of PC. We conclude that PC and PE affect the function of distinct protein translocases in mitochondrial β-barrel biogenesis.
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Novel {beta}-1,4-mannanase belonging to a new glycoside hydrolase family in Aspergillus nidulans [Enzymology]

September 18th, 2015 by

Many filamentous fungi produce β-mannan-degrading β-1,4-mannanases that belong to the glycoside hydrolase (GH) 5 and GH26 families. Here we identified a novel β-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M2), mannotriose (M3), and mannotetraose (M4) but not mannopentaose (M5) or higher manno-oligosaccharides when galactose-free β-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with β-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (kcat/Km) towards mannohexaose (M6) compared with the endo-β-1,4-mannanase Man5C, and notably converted M6 to M2, M3 and M4, with M3 being the predominant reaction product. The action of Man5C towards β-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when β-mannans comprised the sole carbon source, indicating that Man134A is involved in β-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of β-mannan degradation that is enhanced by the novel β-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-β-1,4-mannanases.

Klotho reduction in alveolar macrophages contributes to CSE-induced inflammation in chronic obstructive pulmonary disease [Metabolism]

September 18th, 2015 by

Abnormal inflammation and accelerated decline in lung function occur in patients with chronic obstructive pulmonary disease (COPD).Klotho, an anti-aging protein, has an anti-inflammatory function. However, the role of Klotho has never been investigated in COPD. The aim of this study is to investigate the possible role of Klotho by alveolar macrophages in airway inflammation in COPD.Klotho levels were assessed in the lung samples and peripheral blood mononuclear cells (PBMCs) of non-smokers, smokers and patients with COPD.The regulation of Klotho expression by cigarette smoke extract (CSE) was studied in vitro, and small interfering RNA (siRNA) and recombinant Klotho were employed to investigate the role of Klotho on CSE-induced inflammation.Klotho expression was reduced in alveolar macrophages in the lungs and PBMCs of COPD patients.CSE decreased Klotho expression and release from alveolar macrophages. Knockdown of endogenous Klotho augmented the expression of the inflammatory mediators, such as MMP-9, IL-6 and TNF-alpha by alveolar macrophages.Exogenous Klotho inhibited the expression of CSE-induced the inflammatory mediators. Furthermore, we showed that Klotho interacts with IkappaBalpha of the NF-kappaB pathway.Dexamethasone treatment increased expression and release level of Klotho in alveolar macrophages. Our findings suggest that Klotho plays a role in sustained inflammation of the lungs, which in turn may have therapeutic implications in COPD.