References of "Figueroa, Maximiliano"
     in
Bookmark and Share    
Full Text
Peer Reviewed
See detailThe Basic Property of Lys385 Is Important for Potentiation of the Human alpha1 Glycine Receptor by Ethanol.
Castro, Patricio A; Figueroa, Maximiliano ULg; Yevenes, Gonzalo E et al

in Journal of Pharmacology and Experimental Therapeutics (2012), 340(2), 339-49

Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the alpha1 glycine receptor (GlyR) to ethanol can be ... [more ▼]

Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the alpha1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of Gbetagamma with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patch-clamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC(50) (40 +/- 1 versus 41 +/- 0.5 muM) or the ethanol-induced potentiation (53 +/- 5 versus 46 +/- 5%) of the human alpha1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 +/- 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 +/- 4 and 17 +/- 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with Gbetagamma, is critical for ethanol potentiation of the receptor. [less ▲]

Detailed reference viewed: 53 (8 ULg)
Full Text
See detailRic-8: un nuevo GEF con estructura tipo "armadillo"
Figueroa, Maximiliano ULg

Doctoral thesis (2010)

RIC-8 is a highly conserved cytosolic protein (63 KDa) initially identified in C. elegans as an essential factor in neurotransmitter release and asymmetric cell division. Two different isoforms have been ... [more ▼]

RIC-8 is a highly conserved cytosolic protein (63 KDa) initially identified in C. elegans as an essential factor in neurotransmitter release and asymmetric cell division. Two different isoforms have been described in mammals, RIC-8A and RIC-8B; each possess guanine nucleotide exchange activity (GEF) on heterotrimeric G-proteins, but with different Gα subunits specificities. To gain insight on the mechanisms involved in RIC-8 cellular functions it is essential to obtain some information about its structure. Therefore, the aim of this thesis was to study the relationship between structure and function on RIC-8, using as model RIC-8 from X. laevis. Analysis in its primary structure did not give us information about the function of xRIC-8 and RIC-8 proteins were showed as a single family without similarity with others. For this reason, to obtain a 3D model of xRIC-8, different bioinformatics approaches that include protein folding and structure prediction were used. The RIC-8 structural model is composed of 10 armadillo folding motifs, organized in a right-twisted alpha-alpha super helix. In order to validate the structural model, a His-tag fusion construct of RIC-8 was expressed in E. coli, purified by affinity and anion exchange chromatography and subjected to circular dichroism analysis (CD) and thermostability studies. This model together with the comparison among RIC-8 proteins that shows a high conservation in the carboxy region, deletion mutants that remove the last three armadillo domain were created in order to search a loss of the GEF function. The mutants could not be expressed in bacteria for in vitro assays, but their expression in HEK293T culture showed that all of them preserved the GEF activity. Furthermore, confocal microscopy showed that all the mutants could translocate to plasmatic membrane under stimulation with isoproterenol, and have the capacity to interact with Gs. The results of this thesis has allowed present the first 3D model for a RIC-8 protein, as well as classify RIC-8 as a new member of the protein family with armadillo repeats. Functionally, the carboxi terminal region, the most conserved among RIC-8, did not show has the GEF activity, and did not alter its behavior in HEK293T cell cultures while was stimulated with isoproterenol. [less ▲]

Detailed reference viewed: 36 (7 ULg)
Full Text
Peer Reviewed
See detailMolecular requirements for ethanol differential allosteric modulation of glycine receptors based on selective Gbetagamma modulation.
Yevenes, Gonzalo E; Moraga-Cid, Gustavo; Avila, Ariel et al

in Journal of Biological Chemistry (2010), 285(39), 30203-13

It is now believed that the allosteric modulation produced by ethanol in glycine receptors (GlyRs) depends on alcohol binding to discrete sites within the protein structure. Thus, the differential ethanol ... [more ▼]

It is now believed that the allosteric modulation produced by ethanol in glycine receptors (GlyRs) depends on alcohol binding to discrete sites within the protein structure. Thus, the differential ethanol sensitivity of diverse GlyR isoforms and mutants was explained by the presence of specific residues in putative alcohol pockets. Here, we demonstrate that ethanol sensitivity in two ligand-gated ion receptor members, the GlyR adult alpha(1) and embryonic alpha(2) subunits, can be modified through selective mutations that rescued or impaired Gbetagamma modulation. Even though both isoforms were able to physically interact with Gbetagamma, only the alpha(1) GlyR was functionally modulated by Gbetagamma and pharmacological ethanol concentrations. Remarkably, the simultaneous switching of two transmembrane and a single extracellular residue in alpha(2) GlyRs was enough to generate GlyRs modulated by Gbetagamma and low ethanol concentrations. Interestingly, although we found that these TM residues were different to those in the alcohol binding site, the extracellular residue was recently implicated in conformational changes important to generate a pre-open-activated state that precedes ion channel gating. Thus, these results support the idea that the differential ethanol sensitivity of these two GlyR isoforms rests on conformational changes in transmembrane and extracellular residues within the ion channel structure rather than in differences in alcohol binding pockets. Our results describe the molecular basis for the differential ethanol sensitivity of two ligand-gated ion receptor members based on selective Gbetagamma modulation and provide a new mechanistic framework for allosteric modulations of abuse drugs. [less ▲]

Detailed reference viewed: 11 (2 ULg)
Full Text
Peer Reviewed
See detailBiophysical studies support a predicted superhelical structure with armadillo repeats for Ric-8.
Figueroa, Maximiliano ULg; Hinrichs, Maria Victoria; Bunster, Marta et al

in Protein Science : A Publication of the Protein Society (2009), 18(6), 1139-45

Ric-8 is a highly conserved cytosolic protein (MW 63 KDa) initially identified in C. elegans as an essential factor in neurotransmitter release and asymmetric cell division. Two different isoforms have ... [more ▼]

Ric-8 is a highly conserved cytosolic protein (MW 63 KDa) initially identified in C. elegans as an essential factor in neurotransmitter release and asymmetric cell division. Two different isoforms have been described in mammals, Ric-8A and Ric-8B; each possess guanine nucleotide exchange activity (GEF) on heterotrimeric G-proteins, but with different Galpha subunits specificities. To gain insight on the mechanisms involved in Ric-8 cellular functions it is essential to obtain some information about its structure. Therefore, the aim of this work was to create a structural model for Ric-8. In this case, it was not possible to construct a model based on comparison with a template structure because Ric-8 does not present sequence similarity with any other protein. Consequently, different bioinformatics approaches that include protein folding and structure prediction were used. The Ric-8 structural model is composed of 10 armadillo folding motifs, organized in a right-twisted alpha-alpha super helix. In order to validate the structural model, a His-tag fusion construct of Ric-8 was expressed in E. coli, purified by affinity and anion exchange chromatography and subjected to circular dichroism analysis (CD) and thermostability studies. Ric-8 is approximately 80% alpha helix, with a Tm of 43.1 degrees C, consistent with an armadillo-type structure such as alpha-importin, a protein composed of 10 armadillo repeats. The proposed structural model for Ric-8 is intriguing because armadillo proteins are known to interact with multiple partners and participate in diverse cellular functions. These results open the possibility of finding new protein partners for Ric-8 with new cellular functions. [less ▲]

Detailed reference viewed: 9 (2 ULg)
Full Text
Peer Reviewed
See detailBlockade of ethanol-induced potentiation of glycine receptors by a peptide that interferes with Gbetagamma binding.
Guzman, Leonardo; Moraga-Cid, Gustavo; Avila, Ariel et al

in Journal of Pharmacology and Experimental Therapeutics (2009), 331(3), 933-9

The large intracellular loop (IL) of the glycine receptor (GlyR) interacts with various signaling proteins and plays a fundamental role in trafficking and regulation of several receptor properties ... [more ▼]

The large intracellular loop (IL) of the glycine receptor (GlyR) interacts with various signaling proteins and plays a fundamental role in trafficking and regulation of several receptor properties, including a direct interaction with Gbetagamma. In the present study, we found that mutation of basic residues in the N-terminal region of the IL reduced the binding of Gbetagamma to 21 +/- 10% of control. Two basic residues in the C-terminal region, on the other hand, contributed to a smaller extent to Gbetagamma binding. Using docking analysis, we found that both basic regions of the IL bind in nearby regions to the Gbetagamma dimer, within an area of high density of amino acids having an electronegative character. Thereafter, we generated a 17-amino acid peptide with the N-terminal sequence of the wild-type IL (RQH) that was able to inhibit the in vitro binding of Gbetagamma to GlyRs to 57 +/- 5% of control in glutathione S-transferase pull-down assays using purified proteins. More interestingly, when the peptide was intracellularly applied to human embryonic kidney 293 cells, it inhibited the Gbetagamma-mediated modulations of G protein-coupled inwardly rectifying potassium channel by baclofen (24 +/- 14% of control) and attenuated the GlyR potentiation by ethanol (51 +/- 10% versus 10 +/- 3%). [less ▲]

Detailed reference viewed: 14 (0 ULg)
Full Text
Peer Reviewed
See detailA semiempirical approach to the intra-phycocyanin and inter-phycocyanin fluorescence resonance energy-transfer pathways in phycobilisomes.
Matamala, Adelio R; Almonacid, Daniel E; Figueroa, Maximiliano ULg et al

in Journal of Computational Chemistry (2007), 28(7), 1200-7

A semiempirical methodology to model the intra-phycocyanin and inter-phycocyanin fluorescence resonance energy-transfer (FRET) pathways in the rods of the phycobilisomes (PBSs) from Fremyella diplosiphon ... [more ▼]

A semiempirical methodology to model the intra-phycocyanin and inter-phycocyanin fluorescence resonance energy-transfer (FRET) pathways in the rods of the phycobilisomes (PBSs) from Fremyella diplosiphon is presented. Using the Forster formulation of FRET and combining experimental data and PM3 calculation of the dipole moments of the aromatic portions of the chromophores, transfer constants between pairs of chromophores in the phycocyanin (PC) structure were obtained. Protein docking of two PC hexamers was used to predict the optimal distance and axial rotation angle for the staked PCs in the PBSs' rods. Using the distance obtained by the docking process, transfer constants between pairs of chromophores belonging to different PC hexamers were calculated as a function of the angle of rotation. We show that six preferential FRET pathways within the PC hexameric ring and 15 pathways between hexamers exist, with transfer constants consistent with experimental results. Protein docking predicted the quaternary structure for PCs in rods with inter-phycocyanin distance of 55.6 A and rotation angle of 20.5 degrees . The inter-phycocyanin FRET constant between chromophores at positions beta(155) is maximized at the rotation angle predicted by docking revealing the crucial role of this specific inter-phycocyanin channel in defining the complete set of FRET pathways in the system. [less ▲]

Detailed reference viewed: 5 (0 ULg)
Full Text
Peer Reviewed
See detailThe structure at 2 A resolution of Phycocyanin from Gracilaria chilensis and the energy transfer network in a PC-PC complex.
Contreras-Martel, Carlos; Matamala, Adelio; Bruna, Carola et al

in Biophysical Chemistry (2007), 125(2-3), 388-96

Phycocyanin is a phycobiliprotein involved in light harvesting and conduction of light to the reaction centers in cyanobacteria and red algae. The structure of C-phycocyanin from Gracilaria chilensis was ... [more ▼]

Phycocyanin is a phycobiliprotein involved in light harvesting and conduction of light to the reaction centers in cyanobacteria and red algae. The structure of C-phycocyanin from Gracilaria chilensis was solved by X-ray crystallography at 2.0 A resolution in space group P2(1). An interaction model between two PC heterohexamers was built, followed by molecular dynamic refinement. The best model showed an inter-hexamer rotation of 23 degrees . The coordinates of a PC heterohexamer (alphabeta)(6) and of the PC-PC complex were used to perform energy transfer calculations between chromophores pairs using the fluorescence resonance energy transfer approach (FRET). Two main intra PC ((I)beta(3)(82)-->(I)alpha(1)(84)-->(I)alpha(5)(84)-->(I)beta(6)(82) and (I)beta(3)(153)-->(I)beta(5)(153)) and two main inter PC ((I)beta(6)(82)-->(II)beta(3)(82) and (I)beta(5)(153)-->(II)beta(3)(153)) pathways were proposed based on the values of the energy transfer constants calculated for all the chromophore pairs in the hexamer and in the complex. [less ▲]

Detailed reference viewed: 11 (0 ULg)
Full Text
Peer Reviewed
See detailIn situ photoacoustic spectroscopy of phycobiliproteins in Gracilaria chilensis
Saavedra, R.; Figueroa, Maximiliano ULg; Wandersleben, T. et al

in Journal de Physique IV (2005), 125

Phycobiliproteins, the main polypeptidic components of the phycobilisomes (PBS), are biolog- ical macromolecules arranged in complex interaction systems to perform light harvesting and conduction. The ... [more ▼]

Phycobiliproteins, the main polypeptidic components of the phycobilisomes (PBS), are biolog- ical macromolecules arranged in complex interaction systems to perform light harvesting and conduction. The optical properties of these systems can hardly be studied by conventional spectroscopic techniques. Furthermore this techniques also involve laborious chemical extraction methods. Photoacoustic (PA) spec- troscopy was successfully applied to an in situ study of the phycobiliproteins expression in the eukaryotic red algae: Gracilaria chilensis. [less ▲]

Detailed reference viewed: 5 (0 ULg)