Sérgio Oyama

Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement: Structural and functional characterization of a vaccine candidate☆☆☆

The Schistosoma mansoni fatty acid binding protein (FABP), Sm14, is a vaccine candidate against, S. mansoni and F. hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751.]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14-M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine.

Looking over Toxin–K+ Channel Interactions. Clues from the Structural and Functional Characterization of α-KTx Toxin Tc32, a Kv1.3 Channel Blocker

α-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif, including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an α/β scaffold typical of the members of the α-KTx family. TdK2 and TdK3, all belonging to the same α-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.

Mitogenic activity of peptides related to the sequence of human Fibroblast Growth Factor‐1

Linear synthetic peptides related to the human Fibroblast Growth Factor‐1 (hFGF‐1) segment [112‐147] were tested for their capacity of mimicking FGF mitogenic activity, binding to heparin‐Sepharose columns, stimulating DNA synthesis and competing with hFGF‐1 for the cellular receptors. The results obtained indicated that the activity of these compounds is dependent on the presence of the sequence WFVGLK in their structures. The affinity for the cellular receptors increased when this sequence was elongated in order to incorporate amino acid residues that are important for FGF‐heparin binding.