RESUMO

Caloric restriction is known to extend the lifespan and/or improve diverse physiological parameters in a vast array of organisms. In the yeast Saccharomyces cerevisiae, caloric restriction is performed by reducing the glucose concentration in the culture medium, a condition previously associated with increased chronological lifespan and 20S proteasome activity in cell extracts, which was not due to increased proteasome amounts in restricted cells. Herein, we sought to investigate the mechanisms through which glucose restriction improved proteasome activity and whether these activity changes were associated with modifications in the particle conformation. We show that glucose restriction increases the ability of 20S proteasomes, isolated from Saccharomyces cerevisiae cells, to degrade model substrates and whole proteins. In addition, threonine 55 and/or serine 56 of the α5-subunit, were/was consistently found to be phosphorylated in proteasomes isolated from glucose restricted cells, which may be involved in the increased proteolysis capacity of proteasomes from restricted cells. We were not able to observe changes in the gate opening nor in the spatial conformation in 20S proteasome particles isolated from glucose restricted cells, suggesting that the changes in activity were not accompanied by large conformational alterations in the 20S proteasome but involved allosteric activation of proteasome catalytic site.

Assuntos

RESUMO

Staphylococcal exfoliative toxins (ETs) are glutamyl endopeptidases that specifically cleave the Glu381-Gly382 bond in the ectodomains of desmoglein 1 (Dsg1) via complex action mechanisms. To date, four ETs have been identified in different Staphylococcus aureus strains and ETE is the most recently characterized. The unusual properties of ETs have been attributed to a unique structural feature, i.e., the 180° flip of the carbonyl oxygen (O) of the nonconserved residue 192/186 (ETA/ETE numbering), not conducive to the oxyanion hole formation. We report the crystal structure of ETE determined at 1.61 Å resolution, in which P186(O) adopts two conformations displaying a 180° rotation. This finding, together with free energy calculations, supports the existence of a dynamic transition between the conformations under the tested conditions. Moreover, enzymatic assays showed no significant differences in the esterolytic efficiency of ETE and ETE/P186G, a mutant predicted to possess a functional oxyanion hole, thus downplaying the influence of the flip on the activity. Finally, we observed the formation of ETE homodimers in solution and the predicted homodimeric structure revealed the participation of a characteristic nonconserved loop in the interface and the partial occlusion of the protein active site, suggesting that monomerization is required for enzymatic activity.

Assuntos

RESUMO

Candelilla wax (CW) and 12-hydroxystearic acid (12HSA) are classic solid-fiber-matrix organogelators. Despite the high number of studies using those ingredients in oily systems, there is scarce literature using a mixture of oil and antioxidants. Vitamin E (VE) is an important candidate for its lipophilicity and several applications on pharmaceutical, cosmetics, and food industries. In this work, we investigated the influences of mixtures between vegetable oil (VO) and VE on the microstructures and rheological properties of CW and 12HSA organogels. A weak gel (G''/G' > 0.1) with a shear-thinning behavior was observed for all samples. The presence of VE impacted the gel strength and the phase transition temperatures in a dose-dependent pattern. Larger and denser packed crystals were seen for 12HSA samples, while smaller and more dispersed structures were obtained for CW organogels. The results obtained in this work allowed the correlation of the structural and mechanical properties of the organogels, which plays an important role in the physical-chemical characteristics of these materials.

RESUMO

One of the most remarkable examples of cell-penetrating peptides (CPPs) is Penetratin, a 16-mer fragment derived from the Drosophila Antennapedia homeobox. Understanding the structure of Penetratin/DNA complexes is a key factor for the successful design of new vectors for gene delivery and may assist in optimizing molecular carriers based on CPPs. Herein, we present a comprehensive study on the nanoscale structure of noncovalent complexes formed between Penetratin and DNA. The strong cationic nature of the peptide makes it a very efficient agent for condensing DNA strands via electrostatic attraction, and we show for the first time that DNA condensation is accompanied by random-to-ß-sheet transitions of Penetratin secondary structure, demonstrating that nucleic acids behave as a structuring agent upon complexation. For the first time, nanoscale-resolved spectroscopy is used to provide single-particle infrared data from DNA carriers based on CPPs, and they show that the structures are stabilized by Penetratin ß-sheet cores, whereas larger DNA fractions are preferentially located in the periphery of aggregates. In-solution infrared assays indicate that phosphate diester groups are strongly affected upon DNA condensation, presumably as a consequence of charge delocalization induced by the proximity of cationic amide groups in Penetratin. The morphology is characterized by nanoassemblies with surface fractal features, and short-range order is found in the inner structure of the scaffolds. Interestingly, the formation of beads-on-a-string arrays is found, producing nanoscale architectures that resemble structures observed in early steps of chromatin condensation. A complexation pathway where DNA condensation and peptide pairing into ß-sheets are key steps for organization is proposed.

Assuntos