Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies [Microbiology]

September 22nd, 2015 by Herrera, C., Tremblay, J. M., Shoemaker, C. B., Mantis, N. J.

Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50s in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors.

Sickle cell hemoglobin in the ferryl state promotes {beta}Cys93 oxidation and mitochondrial dysfunction in epithelial lung cells (E10) [Protein Structure and Folding]

September 22nd, 2015 by

Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia, in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H2O2) drives a catalytic cycle that includes three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb, 2) autoreduction of the ferryl intermediate to ferric (metHb), and 3) reaction of metHb with an additional H2O2 molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H2O2 at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, DMPO, we found more irreversibly oxidized βCys93 in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate (OCR) and impairment of cellular bioenergetics which was related to the redox state of the iron. Ferryl HbS induced substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial co-localization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation may be central to the evolution of vasculopathy in SCD and suggest therapeutic modalities that interrupt heme-mediated inflammation.
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Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System [Metabolism]

September 22nd, 2015 by

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. While all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, while ruling out significant roles for Erv1 or Mia40 in these pathways.

A Diatom Ferritin Optimized for Iron Oxidation but not Iron Storage [Protein Structure and Folding]

September 22nd, 2015 by

Ferritin from the marine pennate diatom Pseudo-nitzschia multiseries (PmFTN) plays a key role in sustaining growth in iron-limited ocean environments. The di-iron catalytic ferroxidase center of PmFTN (sites A and B) has a nearby third iron site (site C) in an arrangement typically observed in prokaryotic ferritins. Here we demonstrate that Glu44, a site C ligand, and Glu130, a residue that bridges iron bound at sites B and C, limit the rate of post-oxidation reorganization of iron coordination and the rate at which Fe3+ exits the ferroxidase center for storage within the mineral core. The latter, in particular, severely limits the overall rate of iron mineralization. Thus, the diatom ferritin is optimized for initial Fe2+ oxidation but not for mineralization, pointing to a role for this protein in buffering iron availability and facilitating iron-sparing rather than only long-term iron storage.

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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