Exploring the Use of a Lipopeptide in Dipalmitoylphosphatidylcholine Monolayers for Enhanced Detection of Glyphosate in Aqueous Environments.

The growing reliance on pesticides for pest management in agriculture highlights the need for new analytical methods to detect these substances in food and water. Our research introduces a SPRWG-(C18H37) lipopeptide (LP) as a functional analog of acetylcholinesterase (AChE) for glyphosate detection in environmental samples using phosphatidylcholine (PC) monolayers. This LP, containing hydrophilic amino acids linked to an 18-carbon aliphatic chain, alters lipid assembly properties, leading to a more flexible system. Changes included reduced molecular area and peak pressure in Langmuir adsorption isotherms. Small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) analyses provided insights into the LP's structural organization within the membrane and its interaction with glyphosate (PNG). Structural and geometric parameters, as derived from in silico molecular dynamics simulations (MD), substantiated the impact of LP on the monolayer structure and the interaction with PNG. Notably, the presence of the LP and glyphosate increased charge transfer resistance, indicating strong adherence of the monolayer to the indium tin oxide (ITO) surface and effective pesticide interaction. A calibration curve for glyphosate concentration adjustment revealed a detection limit (LOD) of 24 nmol L-1, showcasing the high sensitivity of this electrochemical biosensor. This LOD is significantly lower than that of a similar colorimetric biosensor in aqueous media with a detection limit of approximately 0.3 µmol L-1. Such an improvement in sensitivity likely stems from adding a polar residue to the amino acid chain of the LP.

Structural and thermodynamic characterization of a highly amyloidogenic dimer of transthyretin involved in a severe cardiomyopathy.

Transthyretin (TTR) is a homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy (FAC). Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure (HHP). Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of sub-denaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.

pMHC Structural Comparisons as a Pivotal Element to Detect and Validate T-Cell Targets for Vaccine Development and Immunotherapy-A New Methodological Proposal.

The search for epitopes that will effectively trigger an immune response remains the "El Dorado" for immunologists. The development of promising immunotherapeutic approaches requires the appropriate targets to elicit a proper immune response. Considering the high degree of HLA/TCR diversity, as well as the heterogeneity of viral and tumor proteins, this number will invariably be higher than ideal to test. It is known that the recognition of a peptide-MHC (pMHC) by the T-cell receptor is performed entirely in a structural fashion, where the atomic interactions of both structures, pMHC and TCR, dictate the fate of the process. However, epitopes with a similar composition of amino acids can produce dissimilar surfaces. Conversely, sequences with no conspicuous similarities can exhibit similar TCR interaction surfaces. In the last decade, our group developed a database and in silico structural methods to extract molecular fingerprints that trigger T-cell immune responses, mainly referring to physicochemical similarities, which could explain the immunogenic differences presented by different pMHC-I complexes. Here, we propose an immunoinformatic approach that considers a structural level of information, combined with an experimental technology that simulates the presentation of epitopes for a T cell, to improve vaccine production and immunotherapy efficacy.

Nonspecific prion protein-nucleic acid interactions lead to different aggregates and cytotoxic species.

A misfolded form of the prion protein (PrP) is the primary culprit in mammalian prion diseases. It has been shown that nucleic acids catalyze the misfolding of cellular PrP into a scrapie-like conformer. It has also been observed that the interaction of PrP with nucleic acids is nonspecific and that the complex can be toxic to cultured cells. No direct correlation has yet been drawn between changes in PrP structure and toxicity due to nucleic acid binding. Here we asked whether different aggregation, stability, and toxicity effects are detected when nonrelated DNA sequences interact with recombinant PrP. Using spectroscopic techniques to analyze PrP tertiary and secondary structure and cellular assays to assess toxicity, we found that rPrP-DNA interactions lead to different aggregated species, depending on the sequence and size of the oligonucleotide tested. A 21-mer DNA sequence (D67) induced higher levels of aggregation and also dissimilar structural changes in rPrP, compared to binding to oligonucleotides with the same length and different nucleotide sequences or different GC contents. The rPrP-D67 complex induced significant cell dysfunction, which appears to be correlated with the biophysical properties of the complex. Although sequence specificity is not apparent for PrP-nucleic acid interactions, we believe that particular nucleic acid patterns, possibly related to GC content, oligonucleotide length, and structure, govern PrP recognition. Understanding the structural and cellular effects observed for PrP-nucleic acid complexes may shed light on the still mysterious pathology of the prion protein.

Prion protein complexed to N2a cellular RNAs through its N-terminal domain forms aggregates and is toxic to murine neuroblastoma cells.

Conversion of the cellular prion protein (PrP(C)) into its altered conformation, PrP(Sc), is believed to be the major cause of prion diseases. Although PrP is the only identified agent for these diseases, there is increasing evidence that other molecules can modulate the conversion. We have found that interaction of PrP with double-stranded DNA leads to a protein with higher beta-sheet content and characteristics similar to those of PrP(Sc). RNA molecules can also interact with PrP and potentially modulate PrP(C) to PrP(Sc) conversion or even bind differentially to both PrP isoforms. Here, we investigated the interaction of recombinant murine PrP with synthetic RNA sequences and with total RNA extracted from cultured neuroblastoma cells (N2aRNA). We found that PrP interacts with N2aRNA with nanomolar affinity, aggregates upon this interaction, and forms species partially resistant to proteolysis. RNA does not bind to N-terminal deletion mutants of PrP, indicating that the N-terminal region is important for this process. Cell viability assays showed that only the N2aRNA extract induces PrP-RNA aggregates that can alter the homeostasis of cultured cells. Small RNAs bound to PrP give rise to nontoxic small oligomers. Nuclear magnetic resonance measurements of the PrP-RNA complex revealed structural changes in PrP, but most of its native fold is maintained. These results indicate that there is selectivity in the species generated by interaction with different molecules of RNA. The catalytic effect of RNA on the PrP(C)-->PrP(Sc) conversion depends on the RNA sequence, and small RNA molecules may exert a protective effect.