Small Molecules Detected by Second-Harmonic Generation Modulate the Conformation of Monomeric {alpha}-Synuclein and Reduce its Aggregation in Cells [Molecular Bases of Disease]

September 22nd, 2015 by

Proteins are structurally dynamic molecules that perform specialized functions through unique conformational changes accessible in physiological environments. An ability to specifically and selectively control protein function via conformational modulation is an important goal for development of novel therapeutics and studies of protein mechanism in biological networks and disease. Here we applied a second-harmonic generation (SHG)-based technique for studying protein conformation in solution and in real time to the intrinsically disordered, Parkinson's disease related protein α-synuclein. From a fragment library, we identified small molecule modulators that bind to monomeric α-synuclein in vitro and significantly reduce α-synuclein aggregation in a neuronal cell culture model. Our results indicate that the conformation of α-synuclein is linked to the protein's aggregation in cells. They also provide support for a therapeutic strategy of targeting specific conformations of the protein to suppress or control its aggregation.
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Discovery of immunodominant B-cell epitopes within surface pneumococcal virulence proteins in paediatric patients with invasive pneumococcal disease [Immunology]

September 22nd, 2015 by

The identification of immunodominant B-cell epitopes within surface pneumococcal virulence proteins (PnVPs) in paediatric patients with invasive pneumococcal disease (IPD) is a valuable approach to define novel vaccine candidates. To this aim, we evaluated sera from children with IPD and age-matched controls against 141 twenty-mer synthetic peptides covering the entire sequence of major antigenic fragments within PnVPs, namely the choline-binding protein D (CbpD), the pneumococcal histidine-triad proteins (PhtD, PhtE), the pneumococcal surface protein A (PspA), the plasminogen and fibronectin binding protein B (PfbB) and the zinc metalloproteinase B (ZmpB). Ten immunodominant B-cell epitopes were identified: CbpD-pep4 [aa291-310], PhtD-pep11 [aa88-107], PhtD-pep17 [aa172-191], PhtD-pep19 [aa200-219], PhtE-pep32 [aa300-319], PhtE-pep40 [aa79-98], PfbB-pep76 [aa180-199], PfbB-pep79 [aa222-241], PfbB-pep90 [aa484-503] and ZmpB-pep125 [aa431-450]. All epitopes were highly conserved among different pneumococcal serotypes and 4 of them were located within the functional zinc-binding domain of histidine triad proteins phtD and PhtE. Peptides CbpD-pep4, PhtD-pep19 and PhtE-pep40 were broadly recognized by IPD patients` sera with prevalence 96.4%, 92.9% and 71.4% respectively, while control sera exhibited only minor reactivities(<10.7%). Their specificity for IPD was 93.3%, 95% and 96.7%, their sensitivity was 96.4%, 92.9% and 71.4% and their positivity likelihood ratio for IPD was 14.5, 18.6 and 21.4 respectively. Furthermore, purified antibodies against CbpD-pep4, PhtD-pep19 and PhtE-pep40 readily bound on the surface of different pneumococcal serotypes, as assessed by FACS and immunofluorescence analysis. The identified immunodominant B-cell epitopes provide a better understanding of immune response in IPD and are worth evaluation in additional studies as potential vaccine candidates.
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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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