Ciona intestinalis NADH dehydrogenase NDX confers stress-resistance and extended lifespan on Drosophila

An assembled cDNA coding for the putative single-subunit NADH dehydrogenase (NDX) of Ciona intestinalis was introduced into Drosophila melanogaster. The encoded protein was found to localize to mitochondria and to confer rotenone-insensitive substrate oxidation in organello. Transgenic flies exhibited increased resistance to menadione, starvation and temperature stress, and manifested a sex and diet-dependent increase in mean lifespan of 20–50%. However, NDX was able only weakly to complement the phenotypes produced by the knockdown of complex I subunits.     

Stress response factors as hub‐regulators of microRNA biogenesis: implication to the diseased heart

MicroRNAs (miRNAs) are important regulators of heart function and then an intriguing therapeutic target for plenty of diseases. The problem raised is that many data in this area are contradictory, thus limiting the use of miRNA‐based therapy. The goal of this review is to describe the hub‐mechanisms regulating the biogenesis and function of miRNAs, which could help in clarifying some contradictions in the miRNA world. With this scope, we analyse an array of factors, including several known agents of stress response, mediators of epigenetic changes, regulators of alternative splicing, RNA editing, protein synthesis and folding and proteolytic systems. All these factors are important in cardiovascular function and most of them regulate miRNA biogenesis, but their influence on miRNAs was shown for non‐cardiac cells or some specific cardiac pathologies. Finally, we consider that studying the stress response factors, which are upstream regulators of miRNA biogenesis, in the diseased heart could help in (1) explaining some contradictions concerning miRNAs in heart pathology, (2) making the role of miRNAs in pathogenesis of cardiovascular disease more clear, and therefore, (3) getting powerful targets for its molecular therapy. Copyright © 2015 John Wiley & Sons, Ltd.     

Fluorescent dyes undergoing intramolecular proton transfer with improved sensitivity to surface charge in lipid bilayers.

4'-(Dialkylamino)-3-hydroxyflavones are characterized by an excited-state proton transfer reaction between two tautomeric excited states, which results in two emission bands well separated on the wavelength scale. Due to the high sensitivity of the relative intensities of the two emission bands to solvent polarity, hydrogen bonding and local electric fields, these dyes found numerous applications in biomembrane studies. In order to further improve their fluorescence characteristics, we have synthesized new dyes where the 2-phenyl group is substituted with a 2-thienyl group. In organic solvents, the new dyes exhibit red shifted absorption and dual fluorescence. Although they show lower sensitivity to solvent polarity and H-bond donor ability (acidicity) than their parent 3-hydroxyflavone dyes, they exhibit a much higher sensitivity to solvent H-bond acceptor ability (basicity). Moreover, when tested in lipid vesicles of different surface charge, the new dyes show much better resolved dual emission and higher sensitivity to the surface charge of lipid bilayers than the parent dyes. The response of the new dyes to surface charge is probably connected with the H-bond basicity of the membrane surface, which is the highest for negatively charged surfaces. As a consequence, the new dyes appear as prospective fluorophores for the development of new fluorescent probes for biomembranes.     

Mechanism of elongation factor (EF)-Ts-catalyzed nucleotide exchange in EF-Tu. Contribution of contacts at the guanine base.

Nucleotide exchange in elongation factor Tu (EF-Tu) is catalyzed by elongation factor Ts (EF-Ts). Similarly to other GTP-binding proteins, the structural changes in the P loop and the Mg(2+) binding site are known to be important for nucleotide release from EF-Tu. In the present paper, we determine the contribution of the contacts between helix D of EF-Tu at the base side of the nucleotide and the N-terminal domain of EF-Ts to the catalysis. The rate constants of the multistep reaction between Escherichia coli EF-Tu, EF-Ts, and GDP were determined by stopped-flow kinetic analysis monitoring the fluorescence of either Trp-184 in EF-Tu or mant-GDP. Mutational analysis shows that contacts between helix D of EF-Tu and the N-terminal domain of EF-Ts are important for both complex formation and the acceleration of GDP dissociation. The kinetic results suggest that the initial contact of EF-Ts with helix D of EF-Tu weakens binding interactions around the guanine base, whereas contacts of EF-Ts with the phosphate binding side that promotes the release of the phosphate moiety of GDP appear to take place later. This "base-side-first" mechanism of guanine nucleotide release resembles that found for Ran x RCC1 and differs from mechanisms described for other GTPase x GEF complexes where interactions at the phosphate side of the nucleotide are released first.     

Structural transitions in polycytidylic acid: proton buffer capacity data

The pH-dependences of proton buffer capacity of poly(C) were computed on the basis of the literature data. In these curves there were observed four peaks: two narrow and two wide ones. The first narrow peak reflects the process of cooperative formation of double helices, which is induced by protonation of the N3 atom of nucleotide bases. The first wide peak is assigned to noncooperative process of poly(C) double helices protonation at the N3 nitrogen atom. It is proposed that the second wide peak corresponds to noncooperative protonation of the neutral cytosine bases at the oxygen atom. This reaction causes cooperative dissociation of the poly(C) double helices. The second narrow peak reflects the dissociation process.     

OXIDIZED LIPIDS DID NOT REDUCE LIFESPAN IN THE FRUIT FLY,Drosophila melanogaster

Aging is often associated with accumulation of oxidative damage in proteins and lipids. However, some studies do not support this view, raising the question of whether high levels of oxidative damage are associated with lifespan. In the current investigation, Drosophila melanogaster flies were kept on diets with 2 or 10% of either glucose or fructose. The lifespan, fecundity, and feeding as well as amounts of protein carbonyls (PC) and lipid peroxides (LOOH), activities of superoxide dismutase (SOD), catalase, glutathione‐S‐transferase (GST), and glutathione reductase activity of thioredoxin reductase (TrxR) were measured in “young” (10‐day old) and “aged” (50‐day old) flies. Flies maintained on diets with 10% carbohydrate lived longer than those on the 2% diets. However, neither lifespan nor fecundity was affected by the type of carbohydrate. The amount of PC was unaffected by diet and age, whereas flies fed on diets with 10% carbohydrate had about fivefold higher amounts of LOOH compared to flies maintained on the 2% carbohydrate diets. Catalase activity was significantly lower in flies fed on diets with 10% carbohydrates compared to flies on 2% carbohydrate diets. The activities of SOD, GST, and TrxR were not affected by the diet or age of the flies. The higher levels of LOOH in flies maintained on 10% carbohydrate did not reduce their lifespan, from which we infer that oxidative damage to only one class of biomolecules, particularly lipids, is not sufficient to influence lifespan.     

Expression and hypoxia-responsiveness of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 in mammary gland malignant cell lines

Recently, we have shown that PFKFB4 gene which encodes the testis isoenzyme of PFKFB is also expressed in the prostate and hepatoma cancer cell lines. Here we have studied expression and hypoxic regulation of the testis isoenzyme of PFKFB4 in several malignant cell lines from a female organ--the mammary gland. Our studies clearly demonstrated that PFKFB4 mRNA is also expressed in mammary gland malignant cells (MCF-7 and T47D cell lines) in normoxic conditions and that hypoxia strongly induces it expression. To better understand the mechanism of hypoxic regulation of PFKFB4 gene expression, we used dimethyloxalylglycine, a specific inhibitor of HIF-1alpha hydroxylase enzymes, which strongly increases HIF-1alpha levels and mimics the effect of hypoxia. It was observed that PFKFB4 expression in the MCF7 and T47D cell lines was highly responsive to dimethyloxalylglycine, suggesting that the hypoxia responsiveness of PFKFB4 gene in these cell lines is regulated by HIF-1 proteins. Moreover, desferrioxamine and cobalt chloride, which mimic the effect of hypoxia by chelating or substituting for iron, had a similar stimulatory effect on the expression of PFKFB mRNA. In other mammary gland malignant cell lines (BT549, MDA-MB-468, and SKBR-3) hypoxia and hypoxia mimics also induced PFKFB4 mRNA, but to variable degrees. The hypoxic induction of PFKFB4 mRNA was equivalent to the expression of PFKFB3, Glut1, and VEGF, which are known HIF-1-dependent genes. Hypoxia and dimethyloxalylglycine increased the PFKFB4 protein levels in all cell lines studied except MDA-MB-468. Through site-specific mutagenesis in the 5'-flanking region of PFKFB4 gene the hypoxia response could be limited. Thus, this study provides evidence that PFKFB4 gene is also expressed in mammary gland cancer cells and strongly responds to hypoxia via an HIF-1alpha dependent mechanism. Moreover, the PFKFB4 and PFKFB3 gene expression in mammary gland cancer cells has also a significant role in the Warburg effect which is found in all malignant cells.     

Specificity and kinetics of alpha-synuclein binding to model membranes determined with fluorescent excited state intramolecular proton transfer (ESIPT) probe

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.     

Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition

Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.