Fundación Instituto Leloir

About: Fundación Instituto Leloir is a facility organization based out in Buenos Aires, Argentina. It is known for research contribution in the topics: Dentate gyrus & Neurogenesis. The organization has 702 authors who have published 1052 publications receiving 39299 citations.

Fine modulation of the respiratory syncytial virus M2-1 protein quaternary structure by reversible zinc removal from its Cys(3)-His(1) motif.

Abstract: Human respiratory syncytial virus (hRSV) is a worldwide distributed pathogen that causes respiratory disease mostly in infants and the elderly. The M2-1 protein of hRSV functions as a transcription antiterminator and partakes in virus particle budding. It is present only in Pneumovirinae, namely, Pneumovirus (RSV) and Metapneumovirus, making it an interesting target for specific antivirals. hRSV M2-1 is a tight tetramer bearing a Cys3-His1 zinc-binding motif, present in Ebola VP30 protein and some eukaryotic proteins, whose integrity was shown to be essential for protein function but without a biochemical mechanistic basis. We showed that removal of the zinc atom causes dissociation to a monomeric apo-M2-1 species. Surprisingly, the secondary structure and stability of the apo-monomer is indistinguishable from that of the M2-1 tetramer. Dissociation reported by a highly sensitive tryptophan residue is much increased at pH 5.0 compared to pH 7.0, suggesting a histidine protonation cooperating in zinc removal. The monomeric apo form binds RNA at least as well as the tetramer, and this interaction is outcompeted by the phosphoprotein P, the RNA polymerase cofactor. The role of zinc goes beyond stabilization of local structure, finely tuning dissociation to a fully folded and binding competent monomer. Removal of zinc is equivalent to the disruption of the motif by mutation, only that the former is potentially reversible in the cellular context. Thus, this process could be triggered by a natural chelator such as glutathione or thioneins, where reversibility strongly suggests a modulatory role in the participation of M2-1 in the assembly of the polymerase complex or in virion budding.

A helix–coil transition induced by the metal ion interaction with a grafted iron-binding site of the CyaY protein family

Abstract: Iron–protein interactions are involved in electron transfer reactions. Alterations of these processes are present in a number of human pathologies; among them, in Friedreich's ataxia, in which a deficiency of functional frataxin, an iron-binding protein, leads to progressive neuromuscular degenerative disease. The putative iron-binding motif of acidic residues EExxED was selected from the first α-helical stretch of the frataxin protein family and grafted onto a foreign peptide scaffold corresponding to the C-terminal α-helix from E. coli thioredoxin. The resulting grafted peptide named GRAP was studied by applying experimental (circular dichroism, isothermal titration calorimetry, capillary zone electrophoresis, thermal denaturation, NMR) and computational approaches (docking, molecular dynamics simulations). Although isolated GRAP lacks a stable secondary structure in solution, when iron is added, the peptide acquires an α-helical structure. Here we have shown that the designed peptide is able to specifically bind Fe3+ with a moderate affinity (KD = 1.9 ± 0.2 μM) and a 1 : 1 stoichiometry. Remarkably, the GRAP/Fe3+ interaction is entropically driven (ΔH° = −1.53 ± 0.03 kcal mol−1 and TΔS° = 6.26 kcal mol−1). Experiments and simulations indicate that Fe3+ interacts with the peptide through three acidic side chains, inducing an α-helical conformation of the grafted motif. In addition, the acidic side chains involved undergo significant conformational rearrangements upon binding, as judged by the analysis of MDs. Altogether, these results contribute to an understanding of the iron-binding mechanisms in proteins and, in particular, in the case of human frataxin.

Regulation of Tacaribe Mammarenavirus Translation: Positive 5' and Negative 3' Elements and Role of Key Cellular Factors.

Abstract: Mammarenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome that encodes the nucleocapsid protein (NP), the envelope glycoprotein precursor (GPC), the RNA polymerase (L), and a RING matrix protein (Z). Viral proteins are synthesized from subgenomic mRNAs bearing a capped 5' untranslated region (UTR) and lacking 3' poly(A) tail. We analyzed the translation strategy of Tacaribe virus (TCRV), a prototype of the New World mammarenaviruses. A virus-like transcript that carries a reporter gene in place of the NP open reading frame and transcripts bearing modified 5' and/or 3' UTR were evaluated in a cell-based translation assay. We found that the presence of the cap structure at the 5' end dramatically increases translation efficiency and that the viral 5' UTR comprises stimulatory signals while the 3' UTR,specifically the presence of a terminal C+G-rich sequence and/or a stem-loop structure, down-modulates translation. Additionally, translation was profoundly reduced in eukaryotic initiation factor (eIF) 4G-inactivated cells, whereas depletion of intracellular levels of eIF4E had less impact on virus-like mRNA translation than on a cell-like transcript. Translation efficiency was independent of NP expression or TCRV infection. Our results indicate that TCRV mRNAs are translated using a cap-dependent mechanism, whose efficiency relies on the interplay between stimulatory signals in the 5' UTR and a negative modulatory element in the 3' UTR. The low dependence on eIF4E suggests that viral mRNAs may engage yet-unknown noncanonical host factors for a cap-dependent initiation mechanism.IMPORTANCE Several members of the Arenaviridae family cause serious hemorrhagic fevers in humans. In the present report, we describe the mechanism by which Tacaribe virus, a prototypic nonpathogenic New World mammarenavirus, regulates viral mRNA translation. Our results highlight the impact of untranslated sequences and key host translation factors on this process. We propose a model that explains how viral mRNAs outcompete cellular mRNAs for the translation machinery. A better understanding of the mechanism of translation regulation of this virus can provide the bases for the rational design of new antiviral tools directed to pathogenic arenaviruses.

Dissecting the Immune Stimulation Promoted by CSF-470 Vaccine Plus Adjuvants in Cutaneous Melanoma Patients: Long Term Antitumor Immunity and Short Term Release of Acute Inflammatory Reactants.

Abstract: As cutaneous melanoma (CM) currently remains with a bleak prognosis, thorough investigation of new treatment options are of utmost relevance. In the phase II/III randomized clinical trial (CASVAC-0401), the repeated immunization of stages IIB-III CM patients with the irradiated, allogeneic cellular CSF-470 vaccine plus the adjuvants bacillus Calmette-Guerin (BCG) and recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) demonstrated a significant benefit over IFN-alpha2B treatment in distant metastasis-free survival. Here we present on the short and long term immune monitoring results after completing the 2-year protocol; a continuation of the previous report by Mordoh et al. (1). We demonstrate that the repeated CSF-470 vaccinations stimulated a long term cellular and humoral immunity response directed against the vaccine antigens. In the case of 2 patients, we are able to show that a similar immune response was generated against autologous antigens. Evaluation of inhibitory receptor co-expression on patient's T cells indicates that the vaccination protocol did not stimulate T cell exhaustion. In order to better understand the basis for the efficacious vaccine responses observed, we investigated the short term immune events following vaccine injection. A significant increase in C-reactive protein (CRP) and IL-6 was observed 24 h after vaccination, with in vitro studies suggesting IL-6 production occurs in the vaccine site. We demonstrate that CRP enhances the cytotoxicity of peripheral blood mononuclear cells (PBMC) against melanoma cells in an in vitro model. Additionally, CRP stimulates the release of pro and anti-inflammatory cytokines from PBMC. As our results demonstrate that successive vaccinations with CSF-470 plus adjuvants promoted an increase in both anti-tumor innate and adaptive immunity, we propose a subsequent model of action.