Kluyveromyces wickerhamii killer toxin: purification and activity towards Brettanomyces/Dekkera yeasts in grape must

Brettanomyces/Dekkera yeasts have been identified as part of the grape yeast flora. They are well known for colonizing the cellar environmental and spoiling wines, causing haze, turbidity and strong off-flavours in wines and enhancing the volatile acidity. As the general practices applied to combat Brettanomyces/Dekkera yeasts are not particularly appropriate during wine ageing and storage, a biological alternative to curtailing their growth would be welcomed in winemaking. In this study, we investigated the Kluyveromyces wickerhamii killer toxin (Kwkt) that is active against Brettanomyces/Dekkera spoilage yeasts. Purification procedures allowed the identification of Kwkt as a protein with an apparent molecular mass of 72 kDa and without any glycosyl residue. Interestingly, purified Kwkt has fungicidal effects at low concentrations under the physicochemical conditions of winemaking. The addition of 40 and 80 mg L(-1) purified Kwkt showed efficient antispoilage effects, controlling both growth and metabolic activity of sensitive spoilage yeasts. At these two killer toxin concentrations, compounds known to contribute to the 'Brett' character of wines, such as ethyl phenols, were not produced. Thus, purified Kwkt appears to be a suitable biological strategy to control Brettanomyces/Dekkera yeasts during fermentation, wine ageing and storage.     

Age-related changes in brain mitochondrial DNA deletion and oxidative stress are differentially modulated by dietary fat type and coenzyme Q₁₀

Mitochondria-related oxidative damage is a primary event in aging and age-related neurodegenerative disorders. Some dietary treatments, such as antioxidant supplementation or the enrichment of mitochondrial membranes with less oxidizable fatty acids, reduce lipid peroxidation and lengthen life span in rodents. This study compares life-long feeding on monounsaturated fatty acids (MUFAs), such as virgin olive oil, and n-6 polyunsaturated fatty acids, such as sunflower oil, with or without coenzyme Q₁₀ supplementation, with respect to age-related molecular changes in rat brain mitochondria. The MUFA diet led to diminished age-related phenotypic changes, with lipoxidation-derived protein markers being higher among the older animals, whereas protein carbonyl compounds were lower. It is noteworthy that the MUFA diet prevented the age-related increase in levels of mitochondrial DNA deletions in the brain mitochondria from aged animals. The findings of this study suggest that age-related oxidative stress is related, at the mitochondrial level, to other age-related features such as mitochondrial electron transport and mtDNA alterations, and it can be modulated by selecting an appropriate dietary fat type and/or by suitable supplementation with low levels of the antioxidant/electron carrier molecule coenzyme Q.     

A fast virtual screening approach to identify structurally diverse inhibitors of trypanothione reductase

Trypanothione reductase (TryR) is one of the favorite targets for those designing drugs for the treatment of Chagas disease. We present the application of a fast virtual screening approach for designing hit compounds active against TryR. Our protocol combines information derived from structurally known inhibitors and from the TryR receptor structure. Five structurally diverse hit compounds active against TryR and holding promise for the treatment of Chagas disease are reported.     

Sequence-specific long range networks in PSD-95/discs large/ZO-1 (PDZ) domains tune their binding selectivity

Protein-protein interactions mediated by modular protein domains are critical for cell scaffolding, differentiation, signaling, and ultimately, evolution. Given the vast number of ligands competing for binding to a limited number of domain families, it is often puzzling how specificity can be achieved. Selectivity may be modulated by intradomain allostery, whereby a remote residue is energetically connected to the functional binding site via side chain or backbone interactions. Whereas several energetic pathways, which could mediate intradomain allostery, have been predicted in modular protein domains, there is a paucity of experimental data to validate their existence and roles. Here, we have identified such functional energetic networks in one of the most common protein-protein interaction modules, the PDZ domain. We used double mutant cycles involving site-directed mutagenesis of both the PDZ domain and the peptide ligand, in conjunction with kinetics to capture the fine energetic details of the networks involved in peptide recognition. We performed the analysis on two homologous PDZ-ligand complexes and found that the energetically coupled residues differ for these two complexes. This result demonstrates that amino acid sequence rather than topology dictates the allosteric pathways. Furthermore, our data support a mechanism whereby the whole domain and not only the binding pocket is optimized for a specific ligand. Such cross-talk between binding sites and remote residues may be used to fine tune target selectivity.     

Characterization of inositol phospho-sphingolipid-phospholipase C 1 (Isc1) in Cryptococcus neoformans reveals unique biochemical features

In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.     

Epigallocatechin-3-gallate potently inhibits the in vitro activity of hydroxy-3-methyl-glutaryl-CoA reductase

Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is the rate-controlling enzyme of cholesterol synthesis, and owing to its biological and pharmacological relevance, researchers have investigated several compounds capable of modulating its activity with the hope of developing new hypocholesterolemic drugs. In particular, polyphenol-rich extracts were extensively tested for their cholesterol-lowering effect as alternatives, or adjuvants, to the conventional statin therapies, but a full understanding of the mechanism of their action has yet to be reached. Our work reports on a detailed kinetic and equilibrium study on the modulation of HMGR by the most-abundant catechin in green tea, epigallocatechin-3-gallate (EGCG). Using a concerted approach involving spectrophotometric, optical biosensor, and chromatographic analyses, molecular docking, and site-directed mutagenesis on the cofactor site of HMGR, we have demonstrated that EGCG potently inhibits the in vitro activity of HMGR (K(i) in the nanomolar range) by competitively binding to the cofactor site of the reductase. Finally, we evaluated the effect of combined EGCG-statin administration.     

Autophagosome precursor maturation requires homotypic fusion

Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which can be derived from preautophagosomal structures coming from the plasma membrane and other sites like the endoplasmic reticulum and mitochondria. The mechanisms by which preautophagosomal structures elongate their membranes and mature toward fully formed autophagosomes still remain unknown. Here, we show that the maturation of the early Atg16L1 precursors requires homotypic fusion, which is essential for subsequent autophagosome formation. Atg16L1 precursor homotypic fusion depends on the SNARE protein VAMP7 together with partner SNAREs. Atg16L1 precursor homotypic fusion is a critical event in the early phases of autophagy that couples membrane acquisition and autophagosome biogenesis, as this step regulates the size of the vesicles, which in turn appears to influence their subsequent maturation into LC3-positive autophagosomes.     

ClpX(P) generates mechanical force to unfold and translocate its protein substrates

AAA(+) unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from E. coli, alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.     

Preclinical class 1 integron with a complete Tn402-like transposition module

The presence of integrons was assessed in gut bacteria isolated from wild-caught prawns. A pseudomonad was recovered that contained a Tn402-like class 1 integron with a complete transposition module and two gene cassettes. One cassette was identical to a previously described cassette from a chromosomal class 3 integron in Delftia tsuruhatensis.