Correlations between photodegradation of bisretinoid constituents of retina and dicarbonyl-adduct deposition [Glycobiology and Extracellular Matrices]

September 22nd, 2015 by Zhou, J., Ueda, K., Zhao, J., Sparrow, J. R.

Non-enzymatic collagen cross-linking and carbonyl-adduct deposition are features of Bruch's membrane aging in the eye and disturbances in extracellular matrix turnover are considered to contribute to Bruch′s membrane thickening. Since bisretinoid constitutents of the lipofuscin of retinal pigment epithelial cells (RPE) are known to photodegrade to mixtures of aldehyde-bearing fragments and small dicarbonyls (glyoxal, GO and methylglyoxal, MG) we investigated RPE lipofuscin as a source of the reactive species that covalently modify protein side−chains. Abca4-/- and Rdh8-/-/Abca4-/- mice that are models of accelerated bisretinoid formation were studied and pre-exposure of mice to 430 nm light enriched for dicarbonyl release by bisretinoid photodegradation. MG protein adducts were elevated in posterior eyecups of mutant mice while carbonylation of an RPE specific protein was observed in Abca4-/- but not in wild-type mice under the same conditions. Immunolabeling of cryostat sectioned eyes harvested from Abca4-/- mice revealed that carbonyl adduct deposition in Bruch′s membrane was accentuated. Cell-based assays corroborated these findings in mice. Moreover, receptor for advanced glycation end-products (RAGE) that recognizes MG and GO adducts and glyoxylase 1 that metabolizes MG and GO, were upregulated in Abca4-/- mice. Additionally, in acellular assays, peptides were cross−linked in the presence of A2E photodegradation products and in a zymography assay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the matrix metalloproteinases MMP2 and MMP9. In conclusion, these mechanistic studies demonstrate a link between the photodegradation of RPE bisretinoid fluorophores and aging changes in underlying Bruch′s membrane that can confer risk of age-related macular degeneration.
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Structural Basis for the Regulation of the MmpL Transporters of Mycobacterium tuberculosis [Gene Regulation]

September 22nd, 2015 by

The mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complex regulatory network, including the TetR-family transcriptional regulators Rv3249c and Rv1816. Here we report the crystal structures of these two regulators, revealing dimeric, two-domain molecules with architecture consistent with the TetR family of regulators. Buried extensively within the C-terminal regulatory domains of Rv3249c and Rv1816 we found fortuitous bound ligands, which were identified as palmitic acid (a fatty acid) and isopropyl laurate (a fatty acid ester), respectively. Our results suggest that fatty acids may be the natural ligands of these regulatory proteins. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by these proteins. Binding of palmitic acid renders these regulators incapable of interacting with their respective operator DNAs, which will result in derepression of the corresponding mmpL genes. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.

Feedback Mechanisms Regulate Ets variant 2 (Etv2) Gene Expression and Hematoendothelial Lineages [Developmental Biology]

September 22nd, 2015 by

Etv2 is an essential transcriptional regulator of hematoendothelial lineages during embryogenesis. While Etv2 downstream targets have been identified, little is known regarding the upstream transcriptional regulation of Etv2 gene expression. In the present study, we have established a novel methodology that utilizes the differentiating embryonic stem cell and differentiating embryoid body (ES/EB) system, to define the modules and enhancers embedded within the Etv2 promoter. Using this system, we defined an autoactivating role for Etv2, which is mediated by two adjacent Ets motifs in the proximal promoter. In addition, we have defined the role of VEGF/Flk1-Calcineurin-NFAT signaling cascade in the transcriptional regulation of Etv2. Furthermore, we have defined an Etv2-Flt1-Flk1 cascade, which serves as a negative feedback mechanism to regulate Etv2 gene expression. To complement and extend these studies, we have demonstrated that the Flt1 null embryonic phenotype was partially rescued in the Etv2 conditional knockout background. In summary, these studies define upstream and downstream networks that serve as a transcriptional rheostat to regulate Etv2 gene expression.

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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Natural polymorphisms and oligomerization of human APOBEC3H contribute to single-stranded DNA scanning ability [Immunology]

September 22nd, 2015 by

APOBEC3H is a deoxycytidine deaminase that can restrict the replication of HIV-1 in the absence of the viral protein Vif that induces APOBEC3H degradation in cells. APOBEC3H exists in humans as seven haplotypes (I-VII) with different cellular stabilities. Of the three stable APOBEC3H haplotypes (II, V, and VII), haplotypes II and V occur most frequently in the population. Despite APOBEC3H being a bona fide restriction factor, there has been no comparative biochemical characterization of APOBEC3H haplotypes. We characterized the single-stranded (ss)DNA scanning mechanisms that haplotypes II and V use to search their ssDNA substrate for cytosine containing deamination motifs. APOBEC3H haplotype II was able to processively deaminate multiple cytosines in a single enzyme-substrate encounter by using sliding, jumping, and intersegmental transfer movements. In contrast, APOBEC3H haplotype V exhibited diminished sliding and intersegmental transfer abilities, but was able to jump along ssDNA. Due to an Asp or Glu at amino acid 178 differentiating these APOBEC3H haplotypes, the data indicated that this amino acid on helix 6 contributes to processivity. The diminished processivity of APOBEC3H haplotype V did not result in a reduced efficiency to restrict HIV-1 replication in single-cycle infectivity assays, suggesting a redundancy in the contributions of jumping and intersegmental transfer to mutagenic efficiency. Optimal processivity on ssDNA also required dimerization of APOBEC3H through the β2 strands. The findings support a model in which jumping can compensate for deficiencies in intersegmental transfer and suggest that APOBEC3H haplotypes II and V induce HIV-1 mutagenesis efficiently, but by different mechanisms.

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.