Browsing by Author "Monasterio, Octavio"

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    Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation
    (OXFORD UNIV PRESS, 2009) Ines Barria, Maria; Gonzalez, Angel; Vera Otarola, Jorge; Leon, Ursula; Vollrath, Valeska; Marsac, Delphine; Monasterio, Octavio; Perez Acle, Tomas; Soza, Alejandro; Lopez Lastra, Marcelo
    The HCV internal ribosome entry site (IRES) spans a region of similar to 340 nt that encompasses most of the 5' untranslated region (5'UTR) of the viral mRNA and the first 24-40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5'UTR on IRES activity, naturally occurring variants of the 5'UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg2+ ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation.
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    Binding of dihydroxynaphthyl aryl ketones to tubulin colchicine site inhibits microtubule assembly
    (2015) Gutierrez, Eunices; Benites, Julio; Valderrama Guerrero, Jaime Adolfo; Calderon, Pedro Buc; Verrax, Julien; Nova, Esteban; Villanelo, Felipe; Maturana, Daniel; Escobar, Cristian; Lagos, Rosalba; Monasterio, Octavio
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    M. jannaschii FtsZ, a key protein in bacterial cell division, is inactivated by peroxyl radical-mediated methionine oxidation
    (Elsevier, 2021) Reyes Valenzuela, Juan Sebastian; Fuentes-Lemus, Eduardo; Aspée, Alexis; Davies, Michael J.; Monasterio, Octavio; López Alarcón, Camilo Ignacio
    Oxidation and inactivation of FtsZ is of interest due to the key role of this protein in bacterial cell division. In the present work, we studied peroxyl radical (from AAPH, 2,2′-azobis(2-methylpropionamidine)dihydrochloride) mediated oxidation of the highly stable FtsZ protein (MjFtsZ) from M. jannaschii, a thermophilic microorganism. MjFtsZ contains eleven Met, and single Tyr and Trp residues which would be expected to be susceptible to oxidation. We hypothesized that exposure of MjFtsZ to AAPH-derived radicals would induce Met oxidation, and cross-linking (via di-Tyr and di-Trp formation), with concomitant loss of its functional polymerization and depolymerization (GTPase) activities. Solutions containing MjFtsZ and AAPH (10 or 100 mM) were incubated at 37 °C for 3 h. Polymerization/depolymerization were assessed by light scattering, while changes in mass were analyzed by SDS-PAGE. Amino acid consumption was quantified by HPLC with fluorescence detection, or direct fluorescence (Trp). Oxidation products and modifications at individual Met residues were quantified by UPLC with mass detection. Oxidation inhibited polymerization-depolymerization activity, and yielded low levels of irreversible protein dimers. With 10 mM AAPH only Trp and Met were consumed giving di-alcohols, kynurenine and di-Trp (from Trp) and the sulfoxide (from Met). With 100 mM AAPH low levels of Tyr oxidation (but not di-Tyr formation) were also observed. Correlation with the functional analyses indicates that Met oxidation, and particularly Met164 is the key driver of MjFtsZ inactivation, probably as a result of the position of this residue at the protein-protein interface of longitudinal interactions and in close proximity to the GTP binding site.
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    The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function.
    (2017) Escobar Álvarez, Elizabeth; Leinisch, Fabian; Araya, Gissela; Monasterio, Octavio; Lorentzen, Lasse G.; Silva, Eduardo; Davies, Michael J.; López Alarcón, Camilo Ignacio
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    The Synaptic Protein Neuroligin-1 Interacts with the Amyloid β-Peptide. Is There a Role in Alzheimer's Disease?
    (2011) Dinamarca, Margarita C.; Weinstein, David; Monasterio, Octavio; Inestrosa, Nibaldo C.
    Amyloid beta-peptide (A beta) is the main component of the amyloid plaques associated with Alzheimer's disease (AD). In the early steps of the disease soluble A beta oligomers are produced. According to the current "amyloid hypothesis" these oligomers can accumulate over time, leading progressively to the loss of synaptic function and the cognitive failure characteristic of AD. To understand the role of oligomeric A beta species in AD pathology, it is important to understand the mechanism by which A beta oligomers are targeted to synaptic junction. We report here the interaction between A beta with neuroligin-1 (NL-1), a postsynaptic cell-adhesion protein specific for excitatory synapses, which shares a high degree of similarity with acetylcholinesterase, the first synaptic protein described to interact with A beta. Using intrinsic fluorescence and surface plasmon resonance, we found that A beta binds to the extracellular domain of NL-1 with a K(d) in the nanomolar range. In the case of NL-2, a postsynaptic cell-adhesion protein specific for inhibitory synapses, just a very weak interaction with A beta was observed. A beta polymerization analysis-studied by thioflavin-T assay and electron microscopy-indicated that NL-1 stabilized A beta aggregates in vitro. Moreover, NL-1 acts as a nucleating factor during the A beta aggregation process, stimulating the formation of A beta oligomers. Besides, irnmunoprecipitation assays confirm that A beta oligomers interact with NL-1 but not with NL-2. In conclusion, our results show that NL-1 interacts with A beta increasing the formation of A beta oligomers, suggesting that this interaction could triggers the targeting of A beta oligomer to the postsynaptic regions of excitatory synapses.