Identification of fibrillogenic regions in human triosephosphate isomerase

Background. Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology. Methods. This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation. Results. We identified four fibrillogenic regions in the HsTPI corresponding to the β3, β6, β7 y α8 of the TIM barrel. From these, the β3-strand region (residues 59–66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-β structure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis. Discussion. Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, where β-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promoting β-aggregation, as well as the formation of hydrophobic disordered aggregates. Being this last pathway kinetically favored over the thermodynamically more stable fibril aggregation pathway

An ATCUN-like copper site in B2-crystallin plays a protective role in cataract-associated aggregation

Cataracts is the leading cause of blindness worldwide and it is caused by crystallin damage and aggregation. Senile cataractous lenses have relatively high levels of metals, while some metal ions can directly induce aggregation of human -crystallins. Here we evaluated the impact of divalent metal ions in the aggregation of human B2-crystallin, one of the most abundant crystallins in the lens. Turbidity assays showed that Pb2+, Hg2+, Cu2+, and Zn2+ ions induce the aggregation of B2-crystallin. Metal-induced aggregation is partially reverted by a chelating agent, indicating formation of metal-bridged species. Our study focused on the mechanism of copper-induced aggregation of B2-crystallin, finding that it involves metal-bridging, disulfide-bridging, and loss of protein stability. Circular dichroism (CD) and electron paramagnetic resonance (EPR) revealed the presence of at least three Cu2+ binding sites in B2-crystallin; one of them with spectroscopic features typical of Cu2+ bound to an amino-terminal copper and nickel binding motif (ATCUN), a motif found in Cu transport proteins. The ATCUN-like Cu binding site is located at the unstructured N-terminus of B2-crystallin, and it could be modeled by a peptide with the first six residues in the protein sequence (NH2-ASDHQF-). Removal of the N-terminus yields an N-truncated form of B2-crystallin that is more susceptible to Cu-induced aggregation and loss of thermal stability, indicating a protective role for the ATCUN-like site. EPR and X-ray absorption spectroscopy (XAS) studies reveal the presence of a copper redox active site in B2-crystallin that is associated to metal-induced aggregation and formation of disulfide-bridged oligomers. Our study demonstrates metal-induced aggregation of cataract-related B2-crystallin and the presence of putative copper binding sites in the protein. Whether the copper-transport ATCUN-like site in B2-crystallin plays a functional/protective role or constitute a vestige from its evolution as a lens structural protein, remains to be elucidated

Structural characterization of scorpion peptides and their bactericidal activity against clinical isolates of multidrug-resistant bacteria.

Scorpion venom peptides represent a novel source of antimicrobial peptides (AMPs) with broad-spectrum activity. In this study, we determined the minimum bactericidal concentration (MBC) of three scorpion AMPs, Uy234, Uy17, and Uy192, which are found in the venomous glands of the Urodacus yaschenkoi scorpion, against the clinical isolates of multidrug-resistant (MDR) bacteria. In addition, we tested the activity of a consensus AMP designed in our laboratory based on some previously reported IsCT-type (cytotoxic linear peptide) AMPs with the aim of obtaining higher antimicrobial activity. All peptides tested showed high antimicrobial activity against MDR clinical isolates, with the highest activity against β-hemolytic Streptococcus strains. The hemolytic activity was determined against human red blood cells and was significantly lower than that of previously reported AMPs. The α-helical structure of the four AMPs was confirmed by circular dichroism (CD). These results suggest that the four peptides can be valuable tools for the design and development of AMPs for use in the inhibition of MDR pathogenic bacteria. A clear index of synergism and additivity was found for the combination of QnCs-BUAP + Uy234, which makes these peptides the most promising candidates against pathogenic bacteria