Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Mikhail E. Matlashov, Yulia A. Bogdanova, Galina V. Ermakova, Natalia M. Mishina, Yulia G. Ermakova, Evgeny S. Nikitin, Pavel M. Balaban, Shigeo Okabe, Sergey Lukyanov, Grigori Enikolopov, Andrey G. Zaraisky, Vsevolod V. Belousov

Background SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, the relatively low brightness of the indicator limits its use. Methods Here we designed a new version of pH-sensor called SypHer-2, which has up to three times brighter fluorescence in cultured mammalian cells compared to the SypHer. Results Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent transient neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop that occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate. Conclusions SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo. General significance The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.

Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Francesco Balestri, Mario Cappiello, Roberta Moschini, Rossella Rotondo, Marco Abate, Antonella Del-Corso, Umberto Mura

Background Glucose is considered as one of the main sources of cell damage related to aldose reductase (AR) action in hyperglycemic conditions and a worldwide effort is posed in searching for specific inhibitors of the enzyme. This AR substrate has often been reported as generating non-hyperbolic kinetics, mimicking a negative cooperative behavior. This feature was explained by the simultaneous action of two enzyme forms acting on the same substrate. Methods The reduction of different aldoses and other classical AR substrates was studied using pure preparations of bovine lens and human recombinant AR. Results The apparent cooperative behavior of AR acting on glucose and other hexoses and pentoses, but not on tethroses, glyceraldehyde, 4-hydroxynonenal and 4-nitrobenzaldehyde, is generated by a partial nonclassical competitive inhibition exerted by the aldose hemiacetal on the reduction of the free aldehyde. A kinetic model is proposed and kinetic parameters are determined for the reduction of l-idose. Conclusions Due to the unavoidable presence of the hemiacetal, glucose reduction by AR occurs under different conditions with respect to other relevant AR-substrates, such as alkanals and alkenals, coming from membrane lipid peroxidation. This may have implications in searching for AR inhibitors. The emerging kinetic parameters for the aldoses free aldehyde indicate the remarkable ability of the enzyme to interact and reduce highly hydrophilic and bulky substrates. General significance The discovery of aldose reductase modulation by hemiacetals offers a new perspective in searching for aldose reductase inhibitors to be developed as drugs counteracting the onset of diabetic complications.
Graphical abstract

Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): John J. Stegeman, Lars Behrendt, Bruce R. Woodin, Akira Kubota, Benjamin Lemaire, Denis Pompon, Jared V. Goldstone, Philippe Urban

Background Zebrafish express five cytochrome P450 1 genes: CYP1A, CYP1B1, CYP1C1, CYP1C2, inducible by aryl hydrocarbon receptor agonists, and CYP1D1, a constitutively expressed CYP1A-like gene. We examined substrate selectivity of CYP1s expressed in yeast. Methods CYP1s were expressed in W(R) yeast, engineered to over-express P450 reductase, via pYES/DEST52 and via pYeDP60. Microsomal fractions from transformed yeast were examined for activity with fluorogenic substrates, benzo[a]pyrene and testosterone. Modeling and docking approaches were used to further evaluate sites of oxidation on benzo[a]pyrene and testosterone. Results CYP1s expressed in yeast dealkylated ethoxy-, methoxy-, pentoxy- and benzoxy-resorufin (EROD, MROD, PROD, BROD). CYP1A and CYP1C2 had the highest rates of EROD activity, while PROD and BROD activities were low for all five CYP1s. The relative rates of resorufin dealkylation by CYP1C1, CYP1C2 and CYP1D1 expressed via pYeDP60 were highly similar to relative rates obtained with pYES/DEST52-expressed enzymes. CYP1C1 and CYP1C2 dealkylated substituted coumarins and ethoxy-fluorescein-ethylester, while CYP1D1 did not. The CYP1Cs and CYP1D1 co-expressed with epoxide hydrolase oxidized BaP with different rates and product profiles, and all three produced BaP-7,8,9,10-tetrol. The CYP1Cs but not CYP1D1 metabolized testosterone to 6β-OH-testosterone. However, CYP1D1 formed an unidentified testosterone metabolite better than the CYP1Cs. Testosterone and BaP docked to CYP homology models with poses consistent with differing product profiles. Conclusions Yeast-expressed zebrafish CYP1s will be useful in determining further functionality with endogenous and xenobiotic compounds. General significance Determining the roles of zebrafish CYP1s in physiology and toxicology depends on knowing the substrate selectivity of these enzymes.

Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Xin Qiao, Jaekyun Jeon, Jeff Weber, Fangqiang Zhu, Bo Chen

Background During the maturation process, HIV capsid proteins self-assemble into polymorphic capsids. The strong polymorphism precludes high resolution structural characterization under in vivo conditions. In spite of the determination of structural models for various in vitro assemblies of HIV capsid proteins, the assembly mechanism is still not well-understood. Methods We report 3D simulations of HIV capsid proteins by a novel coarse grain model that captures the backbone of the rigid segments in the protein accurately. The effects of protein dynamics on assembly are emulated by a static ensemble of subunits in conformations derived from molecular dynamics simulation. Results We show that HIV capsid proteins robustly assemble into hexameric lattices in a range of conditions where trimers of dimeric subunits are the dominant oligomeric intermediates. Variations of hexameric lattice curvatures are observed in simulations with subunits of variable inter-domain orientations mimicking the conformation distribution in solution. Simulations with subunits based on pentameric structural models lead to assembly of sharp curved structures resembling the tips of authentic HIV capsids, along a distinct pathway populated by tetramers and pentamers with the characteristic quasi-equivalency of viral capsids. Conclusions Our results suggest that the polymorphism assembly is triggered by the inter-domain dynamics of HIV capsid proteins in solution. The assembly of highly curved structures arises from proteins in conformation with a highly specific inter-domain orientation. Significance Our work proposes a mechanism of HIV capsid assembly based on available structural data, which can be readily verified. Our model can be applied to other large biomolecular assemblies.
Graphical abstract

Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Satyabrata Pany, Joydip Das

Background Alcohol regulates the expression and function of protein kinase C epsilon (PKCε). In a previous study we identified an alcohol binding site in the C1B, one of the twin C1 subdomains of PKCε (Das et al., Biochem. J., 421, 405–13, 2009). Methods In this study, we investigated alcohol binding in the entire C1 domain (combined C1A and C1B) of PKCε. Fluorescent phorbol ester, SAPD and fluorescent diacylglycerol (DAG) analog, dansyl-DAG were used to study the effect of ethanol, butanol, and octanol on the ligand binding using fluorescence resonance energy transfer (FRET). To identify alcohol binding site(s), PKCεC1 was photolabeled with 3-azibutanol and 3-azioctanol, and analyzed by mass spectrometry. The effects of alcohols and the azialcohols on PKCε were studied in NG108-15 cells. Results In the presence of alcohol, SAPD and dansyl-DAG showed different extent of FRET, indicating differential effects of alcohol on the C1A and C1B subdomains. Effects of alcohols and azialcohols on PKCε in NG108-15 cells were comparable. Azialcohols labeled Tyr-176 of C1A and Tyr-250 of C1B. Inspection of the model structure of PKCεC1 reveals that these residues are 40Å apart from each other indicating that these residues form two different alcohol binding sites. Conclusions The present results provide evidence for the presence of multiple alcohol-binding sites on PKCε and underscore the importance of targeting this PKC isoform in developing alcohol antagonists.
Graphical abstract

Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Ying Jai, Kalpit Shah, Robert J. Bridges, Neil A. Bradbury

Background Resveratrol, a natural phenolic compound, has been reported to rescue mutant ΔF508 CFTR in expression systems and primary epithelial cells. Although this implies a therapeutic benefit to patients with CF, investigations were performed using resveratrol concentrations greatly in excess of those achievable in plasma. We evaluated the efficacy of resveratrol as a CFTR corrector in relevant primary airway cells, using physiologically achievable resveratrol concentrations. Methods Cells expressing wt or ΔF508 CFTR were exposed to chronic or acute resveratrol. CFTR mRNA and protein expression were monitored. The effects of resveratrol on primary ΔF508 human airway cells were evaluated by equivalent current analysis using modified Ussing chambers. Results Consistent with previously published data in heterologous expression systems, high doses of resveratrol increased CFTR expression; however physiologically relevant concentrations were without effect. In contrast to heterologous expression systems, resveratrol was unable to increase mutant CFTR channel activity in primary airway cells. Elevated amiloride-sensitive currents, indicative of sodium transport and characteristically elevated in CF airway cells, were also unaffected by resveratrol. Conclusions High concentrations of resveratrol can increase CFTR mRNA and protein in some cell types. In addition, acute resveratrol exposure can stimulate CFTR mediated chloride secretion, probably by increasing cellular cAMP levels. Resveratrol at physiologically achievable levels yielded no benefit in primary ΔF508 airway cells, either in terms of amiloride-sensitive currents of CFTR currents. General significance Taken together, our results do not support the use of resveratrol supplements as a therapy for patients with cystic fibrosis. It is possible that further modifications of the resveratrol backbone would yield a more efficacious compound.

Publication date: November 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 11

Author(s): Deniz Seyhan, Nico Jehmlich, Martin von Bergen, Julia Fersch, Michael Rother

Background Proteins containing selenocysteine (sec) are found in Bacteria, Eukarya, and Archaea. While selenium-dependence of methanogenesis from H2 +CO2 in the archaeon Methanococcus maripaludis JJ is compensated by induction of a set of cysteine-containing homologs, growth on formate is abrogated in the absence of sec due to the dependence of formate dehydrogenase (Fdh) on selenium. Despite this dependence, formate-dependent growth occurs after prolonged incubation of M. maripaludis mutants lacking sec. Methods To study this phenomenon, a M. maripaludis strain with only one Fdh isoform and an FdhA selenoprotein C-terminally tagged for affinity enrichment was constructed. Factors required for sec synthesis were deleted in this strain and translation of UGA in fdhA was analyzed physiologically, enzymatically, immunologically, and via mass spectrometry. Results M. maripaludis JJ mutants lacking sec synthesis grew at least five times more slowly than the wild type on formate due to a 20–35-fold reduction of Fdh activity. The enzyme in the mutant strains lacked sec but was still produced as a full-length protein. Peptide mass spectrometry revealed that both cysteine (cys) and tryptophan (trp) were inserted at the UGA encoding sec without apparent mutations in tRNAcys or tRNAtrp, respectively. Conclusions We demonstrate that M. maripaludis has the inherent capacity to translate UGA with cys and trp; other mechanisms to replace sec with cys in the absence of selenium could thereby be ruled out. General significance This study exemplifies how an organism uses the inherent flexibility in its canonical protein synthesis machinery to recover some activity of an essential selenium-dependent enzyme in the absence of sec.

Publication date: Available online 28 October 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects

Author(s): Anna V. Frontzek (neé Svanidze), Laurent Paccou, Yannick Guinet, Alain Hédoux

Background Recently, it has been revealed that tetragonal lysozyme crystals show a phase transition at 307K upon heating. The underlying mechanisms of the phase transition are still not fully understood. Here we focus on the study of high-frequency vibrational modes arising from the protein and their temperature evolution in the vicinity of T ph as well as on the detailed study of crystalline water dynamics near T ph . Methods Raman experiments have been performed at temperatures 295–323K including T ph . The low-frequency modes and the modes of fingerprint region, CH- and OH-stretching regions have been analyzed. Results and conclusions. In spite of absence of noticeable rearrangements in protein structure, the high-frequency vibrational modes of lysozyme located in the fingerprint region have been found to exhibit the features of critical dynamics near T ph . Pronounced changes in the dynamics of α-helixes and Tyr residues exposed on the protein surface point to the important role of H-bond rearrangements at the phase transition. Additionally the study of T-evolution of OH-stretching modes shows an increase in distortions of tetragonal H-bond network of bulk water, filling in the space between protein molecules, occurs near T ph . These changes in water dynamics could play a crucial role in the mechanisms of the phase transition. General significance. The present results shed light on the mechanisms of the phase transition in lysozyme crystals.

Publication date: Available online 28 October 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects

Author(s): Itzhel García-Torres, Ignacio de la Mora-de la Mora, Jaime Marcial-Quino, Saúl Gómez-Manzo, América Vanoye-Carlo, Gabriel Navarrete-Vázquez, Blanca Colín-Lozano, Pedro Gutiérrez-Castrellón, Edgar Sierra-Palacios, Gabriel López-Velázquez, Sergio Enríquez-Flores

Background Proton pump inhibitors (PPIs) are extensively used in clinical practice because of their effectiveness and safety. Omeprazole is one of the best-selling drugs worldwide and, with other PPIs, has been proposed to be potential drugs for the treatment of several diseases. We demonstrated that omeprazole shows cytotoxic effects in Giardia and concomitantly inactivates giardial triosephosphate isomerase (GlTIM). Therefore, we evaluated the efficiency of commercially available PPIs to inactivate this enzyme. Methods We assayed the effect of PPIs on the GlTIM WT, single Cys mutants and the human counterpart, following enzyme activity, thermal stability, exposure of hydrophobic regions, and susceptibility to limited proteolysis. Results PPIs efficiently inactivated GlTIM; however, rabeprazole was the best inactivating drug and was nearly ten times more effective. The mechanism of inactivation by PPIs was through the modification of the Cys 222 residue. Moreover, there are important changes at the structural level, the thermal stability of inactivated-GlTIM was drastically diminished and the structural rigidity was lost, as observed by the exposure of hydrophobic regions and their susceptibility to limited proteolysis. Conclusions Our results demonstrate that rabeprazole is the most potent PPI for GlTIM inactivation and that all PPIs tested have substantial abilities to alter GITIM at the structural level, causing serious damage. General significance. This is the first report demonstrating the effectiveness of commercial PPIs on a glycolytic parasitic enzyme, with structural features well known. This study is a step forwards in the use and understanding the implicated mechanisms of new antigiardiasic drugs safe in humans.

Publication date: Available online 28 October 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects

Author(s): Flavia M. Michelini, Carlos A. Bueno, Alejandro M. Molinari, Mario D. Galigniana, Lydia R. Galagovsky, Laura E. Alché, Javier A. Ramírez

Background We have previously shown that some synthetic hydroxylated stigmastanes derived from plant sterols inhibit in vitro HSV-1 replication in ocular cell lines and decrease cytokine production in stimulated macrophages, suggesting that these steroids might combine antiviral and immunomodulating properties. In this paper we report the synthesis of some analogues fluorinated at C-6 in order to study the effect of this modification on bioactivity. Methods The following methods were used: organic synthesis of fluorinated analogs, cytotoxicity determination with MTT assays, cytokine production quantification with ELISAs, glucocorticoid activity determination by displacement assays, immunofluorescence and transcriptional activity assays, studies of the activation of signaling pathways by Western blot, antiviral activity evaluation through virus yield reduction assays. Results We report the chemical synthesis of new fluorinated stigmastanes and show that this family of steroidal compounds exerts its immunomodulating activity by inhibiting ERK and Akt signaling pathways, but do not act as glucocorticoids. We also demonstrate that fluorination enhances the antiviral activity. Conclusions Fluorination on C-6 did not enhance the anti-inflammatory effect, however, an increase in the in vitro antiviral activity was observed. Thus, our results suggest that it is possible to introduce chemical modifications on the parent steroids in order to selectively modulate one of the effects. General significance This family of steroids could allow the development of an alternative treatment for ocular immunopathologies triggered by HSV-1, without the undesirable side effects of the currently used drugs.