Hierarchical formation of disulphide bonds in the immunoglobulin Fc-Fragment is assisted by Protein Disulphide Isomerase

Antibodies provide an excellent system to study the folding and assembly of all β-sheet proteins and to elucidate the hierarchy of intra/inter chain disulfide bonds formation during the folding process of multimeric and multidomain proteins. Here, the folding process of the Fc fragment of the heavy chain of the antibody MAK33 was investigated. The Fc fragment consists of the CH3 and CH2 domains of the immunoglobulin heavy chain, both containing a single S-S bond. The folding process was investigated both in the absence and presence of the folding catalyst protein-disulfide isomerase (PDI), monitoring the evolution of intermediates by electrospray mass spectrometry. Moreover, the disulfide bonds present at different times in the folding mixture were identified by mass mapping to determine the hierarchy of disulfide bond formation. The analysis of the uncatalyzed folding showed that the species containing one intramolecular disulfide predominated throughout the entire process, whereas the fully oxidized Fc fragment never accumulated in significant amounts. This result suggests the presence of a kinetic trap during the Fc folding, preventing the one-disulfide-containing species (1S2H) to reach the fully oxidized protein (2S). The assignment of disulfide bonds revealed that 1S2H is a homogeneous species characterized by the presence of a single disulfide bond (Cys-130-Cys-188) belonging to the CH3 domain. When the folding experiments were carried out in the presence of PDI, the completely oxidized species accumulated and predominated at later stages of the process. This species contained the two native S-S bonds of the Fc protein. Our results indicate that the two domains of the Fc fragment fold independently, with a precise hierarchy of disulfide formation in which the disulfide bond, especially, of the C H2 domain requires catalysis by PD

Vanadium biomonitoring by using Perna perna (Linnaeus, 1758) mussels transplanted in the coast of the State of So Paulo, Brazil

The increased pollution in the aquatic ecosystem has led to the investigation of toxic elements in sea water by using marine organisms to assess marine pollution from human activities. Among these organisms, the mollusks bivalves have been used as biomonitors since they can accumulate trace elements and other substances, without the occurrence of their death. In this study, Perna perna mussels were transplanted from a mussel farm (reference region) to four sites located in coastal regions of So Paulo State, Brazil, close to anthropic discharge areas. Vanadium was determined in mussel tissues by instrumental neutron activation analysis (INAA). Quality control of V analysis was checked by analyzing biological reference materials and the results obtained were precise and in good agreement with the certified values. Comparisons between the V concentrations obtained in transplanted mussels indicated that those from So Sebastio region, close to an oil terminal presented the highest concentration of this element, during spring.Sao Paulo Research FoundationBrazilian National Council for Scientific and Technological Development from BrazilInternational Atomic Energy Agenc

From grape to wine: Fate of ochratoxin A during red, rose, and white winemaking process and the presence of ochratoxin derivatives in the final products.

The presence of ochratoxin A (OTA) in wine is mainly due to the contamination of grapes by Aspergillus carbonarius and A. niger, still in the vineyard or at stages prior to winemaking. Throughout winemaking process, although there is a reduction in OTA levels, modified mycotoxins may also be formed. In fact, modified mycotoxins are compounds that normally remain undetectable during the conventional analysis used for the parent toxin. In this context, the current study aimed to evaluate the effect of grape variety and winemaking steps on OTA fate as well as the formation of modified ochratoxins. White, rose and red wines were prepared from Muscat Italia, Syrah and Touriga Nacional varieties, respectively. OTA was determined during different steps of winemaking by UHPLC-ESI-MS/MS. Identification of ochratoxin derivatives was performed using tandem MS experiments. A reduction of 90.72, 92.44 and 88.15% in OTA levels was observed for white, rose and red wines, respectively. Among the sought targets, the following ochratoxin-derived candidates were identified: ochratoxin β, ochratoxin α methyl ester, ochratoxin B methyl ester, ochratoxin A methyl ester, ethylamide ochratoxin A, ochratoxin C and ochratoxin A glucose ester. These results indicate that the formation of ochratoxin derivatives leads to an underestimation of total mycotoxin levels in wine and, therefore, the inclusion of techniques for multi-mycotoxins detection should be considered

Incipient Balancing Selection through Adaptive Loss of Aquaporins in Natural Saccharomyces cerevisiae Populations

A major goal in evolutionary biology is to understand how adaptive evolution has influenced natural variation, but identifying loci subject to positive selection has been a challenge. Here we present the adaptive loss of a pair of paralogous genes in specific Saccharomyces cerevisiae subpopulations. We mapped natural variation in freeze-thaw tolerance to two water transporters, AQY1 and AQY2, previously implicated in freeze-thaw survival. However, whereas freeze-thaw–tolerant strains harbor functional aquaporin genes, the set of sensitive strains lost aquaporin function at least 6 independent times. Several genomic signatures at AQY1 and/or AQY2 reveal low variation surrounding these loci within strains of the same haplotype, but high variation between strain groups. This is consistent with recent adaptive loss of aquaporins in subgroups of strains, leading to incipient balancing selection. We show that, although aquaporins are critical for surviving freeze-thaw stress, loss of both genes provides a major fitness advantage on high-sugar substrates common to many strains' natural niche. Strikingly, strains with non-functional alleles have also lost the ancestral requirement for aquaporins during spore formation. Thus, the antagonistic effect of aquaporin function—providing an advantage in freeze-thaw tolerance but a fitness defect for growth in high-sugar environments—contributes to the maintenance of both functional and nonfunctional alleles in S. cerevisiae. This work also shows that gene loss through multiple missense and nonsense mutations, hallmarks of pseudogenization presumed to emerge after loss of constraint, can arise through positive selection

Olives and olive oil are sources of electrophilic fatty acid nitroalkenes

Extra virgin olive oil (EVOO) and olives, key sources of unsaturated fatty acids in the Mediterranean diet, provide health benefits to humans. Nitric oxide (•NO) and nitrite (NO2-)-dependent reactions of unsaturated fatty acids yield electrophilic nitroalkene derivatives (NO 2-FA) that manifest salutary pleiotropic cell signaling responses in mammals. Herein, the endogenous presence of NO2-FA in both EVOO and fresh olives was demonstrated by mass spectrometry. The electrophilic nature of these species was affirmed by the detection of significant levels of protein cysteine adducts of nitro-oleic acid (NO2-OA-cysteine) in fresh olives, especially in the peel. Further nitration of EVOO by NO2- under acidic gastric digestive conditions revealed that human consumption of olive lipids will produce additional nitro-conjugated linoleic acid (NO2-cLA) and nitro-oleic acid (NO2-OA). The presence of free and protein-adducted NO2-FA in both mammalian and plant lipids further affirm a role for these species as signaling mediators. Since NO2-FA instigate adaptive anti-inflammatory gene expression and metabolic responses, these redox-derived metabolites may contribute to the cardiovascular benefits associated with the Mediterranean diet. © 2014 Fazzari et al