References of "Vazquez-Acevedo, Miriam"
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See detailCox2A/Cox2B subunit interaction in Polytomella sp. cytochrome c oxidase: role of the Cox2B subunit extension
Jiménez-Suárez, Alejandra; Vázquez-Acevedo, Miriam; Miranda Astudillo, Héctor Vicente ULiege et al

in Journal of Bioenergetics and Biomembranes (2017), 49(6), 453-461

Subunit II of cytochrome c oxidase (Cox2) is usually encoded in the mitochondrial genome, synthesized in the organelle, inserted co-translationally into the inner mitochondrial membrane, and assembled ... [more ▼]

Subunit II of cytochrome c oxidase (Cox2) is usually encoded in the mitochondrial genome, synthesized in the organelle, inserted co-translationally into the inner mitochondrial membrane, and assembled into the respiratory complex. In chlorophycean algae however, the cox2 gene was split into the cox2a and cox2b genes, and in some algal species like Chlamydomonas reinhardtii and Polytomella sp. both fragmented genes migrated to the nucleus. The corresponding Cox2A and Cox2B subunits are imported into mitochondria forming a heterodimeric Cox2 subunit. When comparing the sequences of chlorophycean Cox2Aand Cox2B proteins with orthodox Cox2 subunits, a C-terminal extension in Cox2A and an N-terminal extension in Cox2B were identified. It was proposed that these extensions favor the Cox2A/Cox2B interaction. In vitro studies carried out in this work suggest that the removal of the Cox2B extension only partially affects binding of Cox2B to Cox2A.We conclude that this extension is dispensable, but when present it weakly reinforces the Cox2A/Cox2B interaction. [less ▲]

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See detailDissecting the peripheral stalk of the mitochondrial ATP synthase of chlorophycean algae
Vázquez-Acevedo, Miriam; Vega-deLuna, Félix; Sánchez-Vásquez, Lorenzo et al

in Biochimica et Biophysica Acta-Bioenergetics (2016), 1857(8), 1183-1190

The algae Chlamydomonas reinhardtii and Polytomella sp., a green and a colorless member of the chlorophycean lineage respectively, exhibit a highly-stable dimeric mitochondrial F1Fo-ATP synthase (complex ... [more ▼]

The algae Chlamydomonas reinhardtii and Polytomella sp., a green and a colorless member of the chlorophycean lineage respectively, exhibit a highly-stable dimeric mitochondrial F1Fo-ATP synthase (complex V), with a molecular mass of 1600kDa. Polytomella, lacking both chloroplasts and a cell wall, has greatly facilitated the purification of the algal ATP-synthase. Each monomer of the enzyme has 17 polypeptides, eight of which are the conserved, main functional components, and nine polypeptides (Asa1 to Asa9) unique to chlorophycean algae. These atypical subunits form the two robust peripheral stalks observed in the highly-stable dimer of the algal ATP synthase in several electron-microscopy studies. The topological disposition of the components of the enzyme has been addressed with cross-linking experiments in the isolated complex; generation of subcomplexes by limited dissociation of complex V; detection of subunit-subunit interactions using recombinant subunits; in vitro reconstitution of subcomplexes; silencing of the expression of Asa subunits; and modeling of the overall structural features of the complex by EM image reconstruction. Here, we report that the amphipathic polymer Amphipol A8-35 partially dissociates the enzyme, giving rise to two discrete dimeric subcomplexes, whose compositions were characterized. An updated model for the topological disposition of the 17 polypeptides that constitute the algal enzyme is suggested. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. [less ▲]

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See detailSubunit Asa1 spans all the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.
Colina-Tenorio, Lilia; Miranda-Astudillo, Hector; Cano-Estrada, Araceli et al

in Biochimica et biophysica acta (2016)

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These ... [more ▼]

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These subunits build the peripheral stalk of the enzyme and stabilize its dimeric structure. The location of the 66.1kDa subunit Asa1 has been debated. On one hand, it was found in a transient subcomplex that contained membrane-bound subunits Asa1/Asa3/Asa5/Asa8/a (Atp6)/c (Atp9). On the other hand, Asa1 was proposed to form the bulky structure of the peripheral stalk that contacts the OSCP subunit in the F1 sector. Here, we overexpressed and purified the recombinant proteins Asa1 and OSCP and explored their interactions in vitro, using immunochemical techniques and affinity chromatography. Asa1 and OSCP interact strongly, and the carboxy-terminal half of OSCP seems to be instrumental for this association. In addition, the algal ATP synthase was partially dissociated at relatively high detergent concentrations, and an Asa1/Asa3/Asa5/Asa8/a/c10 subcomplex was identified. Furthermore, Far-Western analysis suggests an Asa1-Asa8 interaction. Based on these results, a model is proposed in which Asa1 spans the whole peripheral arm of the enzyme, from a region close to the matrix-exposed side of the mitochondrial inner membrane to the F1 region where OSCP is located. 3D models show elongated, helix-rich structures for chlorophycean Asa1 subunits. Asa1 subunit probably plays a scaffolding role in the peripheral stalk analogous to the one of subunit b in orthodox mitochondrial enzymes. [less ▲]

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See detailSubunit Asa1 spans all the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.
Colina-Tenorio, Lilia; Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli et al

in Biochimica et Biophysica Acta-Bioenergetics (2015), 1857(4), 359-369

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These ... [more ▼]

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These subunits build the peripheral stalk of the enzyme and stabilize its dimeric structure. The location of the 66.1 kDa subunit Asa1 has been debated. On one hand, it was found in a transient subcomplex that contained membrane-bound subunits Asa1/Asa3/Asa5/Asa8/a (Atp6)/c (Atp9). On the other hand, Asa1 was proposed to form the bulky structure of the peripheral stalk that contacts the OSCP subunit in the F1 sector. Here, we overexpressed and purified the recombinant proteins Asa1 and OSCP and explored their interactions in vitro, using immunochemical techniques and affinity chromatography. Asa1 and OSCP interact strongly, and the carboxy-terminal half of OSCP seems to be instrumental for this association. In addition, the algal ATP synthase was partially dissociated at relatively high detergent concentrations, and an Asa1/Asa3/Asa5/Asa8/a/c10 subcomplex was identified. Furthermore, Far-Western analysis suggests an Asa1-Asa8 interaction. Based on these results, a model is proposed in which Asa1 spans the whole peripheral arm of the enzyme, from a region close to the matrix-exposed side of the mitochondrial inner membrane to the F1 region where OSCP is located. 3D models show elongated, helix-rich structures for chlorophycean Asa1 subunits. Asa1 subunit probably plays a scaffolding role in the peripheral stalk analogous to the one of subunit b in orthodox mitochondrial enzymes. [less ▲]

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See detailEstudio de las interacciones de la subunidad atípica ASA1 en la ATP sintasa mitocondrial del alga Polytomella sp
Colina-Tenorio, Lilia; Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli et al

Poster (2015, November)

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See detailDissecting the peripheral arm of the mitocondrial ATP synthase of chlorophycean algae.
Gonzalez-Halphen, Diego; Vazquez-Acevedo, Miriam; Miranda Astudillo, Héctor Vicente ULiege et al

Poster (2014, November 06)

Mitochondrial F 1 Fo-ATP synthase (complex V) makes ATP using the electrochemical proton gradient generated by the respiratory chain. It is an oligomeric complex embedded in the inner mitochondrial ... [more ▼]

Mitochondrial F 1 Fo-ATP synthase (complex V) makes ATP using the electrochemical proton gradient generated by the respiratory chain. It is an oligomeric complex embedded in the inner mitochondrial membrane that works like a rotary motor. Chlamydomonas reinhardtii and Polytomella sp., two members of the chlorophycean lineage of unicellular green algae, have a highly-stable dimeric mitochondrial F 1 Fo-ATP synthase, with an estimated molecular mass of 1600 kDa. The chlorophycean enzyme contains the eight conserved polypeptides present in the vast majority of eukaryotes that represent the main components of the proton-driven rotary motor and the catalytic sector of the enzyme: subunits alpha, beta, gamma, delta, epsilon, a (ATP6), c (ATP9), and OSCP. Nevertheless, and in sharp contrast with other mitochondrial F 1 Fo-ATP synthases like the one from beef heart, the algal enzyme seems to lack several classic components: the subunits of the peripheral stalk b, d, f, A6L, and F6, the subunits responsible for dimer formation e and g, and the regulatory polypeptide IF 1 . Instead, the algal enzyme contains nine subunits with molecular masses ranging from 8 to 60 kDa named ASA1 to ASA9 (for ATP Synthase Associated proteins). These polypeptides have no clear orthologs in the databases and seem to be unique to chlorophycean algae. The nine ASA subunits build up a highly-robust peripheral stalk with a unique architecture, as observed on single-particle electron microscopy (EM) images. Our group has found of interest to gain further insights on the close-neighbor relationships between the ASA subunits and their interactions with some of the classical subunits. We have therefore explored with some detail the topological disposition of the components of the algal mitochondrial ATP synthase using different experimental approaches: detection of subunit-subunit interactions based on cross-linking experiments, the yeast two hybrid system or reconstitution with recombinant subunits; generation of sub-complexes after partial dissociation of the dimeric ATP synthase; inference of subunit stoichiometry based on labelling of cysteine residues and modelling of the overall structural features of the complex from small-angle X ray scattering data and EM image reconstruction. Based on the results obtained from these diverse experimental strategies, we suggest a refined model for the topological disposition of the 17 polypeptides that constitute the algal mitochondrial ATP synthase. [less ▲]

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See detailComplex IV heterodimeric Cox2 in Polytomella sp: Import and protein-protein interactions
Jiménez-Suárez, Alejandra; Miranda Astudillo, Héctor Vicente ULiege; Colina-Tenorio, Lilia et al

Poster (2014, November 04)

Gene transfer from the mitochondrion to the nucleus is an ongoing evolutionary process, that explains why extant mitochondrial genomes are highly reduced, encoding only a few set of proteins required for ... [more ▼]

Gene transfer from the mitochondrion to the nucleus is an ongoing evolutionary process, that explains why extant mitochondrial genomes are highly reduced, encoding only a few set of proteins required for oxidative phosphorylation. These proteins exhibit high hydrophobicity, so it is believed that the functional migration of the corresponding genes to the nucleus has been limited by this parameter. However, the chlorophycean algal lineage, that includes the colorless alga Polytomella sp., has a mitochondrial DNA lacking the cox2 gene that encodes subunit II (COX2) of cytochrome c oxidase. In Polytomella sp. this gene was split into two genes, cox2a and cox2b, which encode proteins COX2A and COX2B respectively. A feature of these subunits is an additional sequences located at one of its ends, at the N-terminal region for COX2B and at the C-terminal region for COX2A. These extensions do not have similarity to any other COX2 sequence reported so far. In addition, COX2A has a pre-sequence of 130 amino acids in theN-terminal region. It has been proposed that the extensions are involved in the formation of the heterodimer COX2A/COX2B and in its structural stabilization. In order to characterize the interaction of proteins COX2A and COX2B and to determine the importance of the extensions, interaction assays were performed, such as affinity chromatography and Far-Western blotting with the recombinant proteins. The results indicate that the COX2B extension is necessary but not essential for the association of COX2A/COX2B. Both proteins are synthesized in the cytosol, imported and assembled, so we explored this process in vitro, using isolated Polytomella sp. mitochondria. Our results suggest that COX2B is imported directly into the intermembrane space, while COX2A follows an energy-dependent import pathway. In addition, the MTS of the COX2A precursor is edited. This is the first time that the in vitro import of split COX2 subunits into mitochondria has been achieved. Work supported by CONACyT (128110) and DGAPA-UNAM (IN 203311). [less ▲]

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See detailStudy of the interactions of atypical subunit ASA1 in the ATP synthase of Polytomella sp.
Colina-Tenorio, Lilia; Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli et al

Poster (2014, November 03)

The mitochondrial ATP synthase of chlorophycean algae has a structure different from that of other organisms. All the subunits that typically make up the the peripheral arm and those that are involved in ... [more ▼]

The mitochondrial ATP synthase of chlorophycean algae has a structure different from that of other organisms. All the subunits that typically make up the the peripheral arm and those that are involved in the dimerization of the enzyme are missing. In compensation, it has acquired nine subunits of unknown evolutionary origin that have been named ASA1 to ASA9. These ASA subunits are only present in chlorophycean algae and are not found in others closely related algal lineages, such as ulvophycean, prasynophycean and trebuxophycean green algae. Heat dissociation experiments, cross linking studies and electronic microscopy studies have allowed the proposal of a structural model of the ATP synthase of chlorophycean algae in which ASA subunits make up the peripheral arm and participate in the dimerization of the enzyme; however, the localization of ASA1 subunit remains unclear. The objective of this work is to clone and purify the ASA1 subunit to perform interaction studies in order to know which are its neighboring subunits and to propose its topological disposition in the peripheral arm of the ATP synthase of Polytomella sp. The experimental strategy is based on the cloning of the corresponding gene, the overexpression of the protein in Escherichia coli and the purification of the recombinant protein in order to perform interaction assays. In this work, we report an ASA1-OSCP interaction, which could link a classical protein of the enzyme, such as OSCP, with an atypical subunit unique to the chlorophycean algal lineage. [less ▲]

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See detailOverexpression of a monomeric form of the bovine odorant-binding protein protects Escherichia coli from chemical-induced oxidative stress.
Macedo-Marquez, Alain; Vazquez-Acevedo, Miriam; Ongay-Larios, Laura et al

in Free Radical Research (2014), 48(7), 814-822

Mammalian odorant-binding proteins (OBPs) are soluble lipocalins produced in the nasal mucosa and in other epithelial tissues of several animal species, where they are supposed to serve as scavengers for ... [more ▼]

Mammalian odorant-binding proteins (OBPs) are soluble lipocalins produced in the nasal mucosa and in other epithelial tissues of several animal species, where they are supposed to serve as scavengers for small structurally unrelated hydrophobic molecules. These would include odorants and toxic aldehydes like 4-hydroxy-2-nonenal (HNE), which are end products of lipid peroxidation; therefore OBP might physiologically contribute to preserve the integrity of epithelial tissues under oxidative stress conditions by removing toxic compounds from the environment and, eventually, driving them to the appropriate degradative pathways. With the aim of developing a biological model based on a living organism for the investigation of the antioxidant properties of OBP, here we asked whether the overexpression of the protein could confer protection from chemical-induced oxidative stress in Escherichia coli. To this aim, bacteria were made to overexpress either GCC-bOBP, a redesigned monomeric mutant of bovine OBP, or its amino-terminal 6-histidine-tagged version 6H-GCC-bOBP. After inducing overexpression for 4 h, bacterial cells were diluted in fresh culture media, and their growth curves were followed in the presence of hydrogen peroxide (H2O2) and tert-Butyl hydroperoxide (tBuOOH), two reactive oxygen species whose toxicity is mainly due to lipid peroxidation, and menadione, a redox-cycling drug producing the superoxide ion. GCC-bOBP and 6H-GCC-bOBP were found to protect bacterial cells from the insulting agents H2O2 and tBuOOH but not from menadione. The obtained data led us to hypothesize that the presence of overexpressed OBP may contribute to protect bacterial cells against oxidative stress probably by sequestering toxic compounds locally produced during the first replication cycles by lipid peroxidation, before bacteria activate their appropriate enzyme-based antioxidative mechanisms. [less ▲]

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See detailInteractions of subunits Asa2, Asa4 and Asa7 in the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.
Miranda-Astudillo, Hector; Cano-Estrada, Araceli; Vazquez-Acevedo, Miriam et al

in Biochimica et Biophysica Acta-Bioenergetics (2014), 1837

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See detailInteraciones de las subunidades Asa2, Asa4 y Asa7 en el brazo periférico de la ATP sintasa mitocondrial del alga incolora Polytomella sp
Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli; Vázquez-Acevedo, Miriam et al

Conference (2013, October 23)

La F1Fo-ATP sintasa mitocondrial de las algas clorofíceas es un complejo parcialmente embebido en la membrana interna mitocondrial que se purifica como un dímero estable de 1600 kDa. Se encuentra formado ... [more ▼]

La F1Fo-ATP sintasa mitocondrial de las algas clorofíceas es un complejo parcialmente embebido en la membrana interna mitocondrial que se purifica como un dímero estable de 1600 kDa. Se encuentra formado por 17 polipéptidos, nueve de los cuales (subunidades Asa1 – Asa9) no se encuentran presentes en las ATP sintasas mitocondriales clásicas y parecen ser exclusivos de este grupo de algas. En particular, las subunidades Asa2, Asa4 y Asa7 parecen formar parte de una sección del brazo periférico de la enzima. En el presente trabajo se sobreexpresaron y purificaron las subunidades Asa2, Asa4, Asa7 y los correspondientes fragmentos amino terminal y carboxilo terminal de las subunidades Asa4 y Asa7, con el fin de realizar estudios de interacción in vitro, empleando técnicas inmunoquímicas, electroforesis azul nativa y cromatografía de afinidad. Se determinó que las subunidades Asa4 y Asa7 interaccionan fuertemente principalmente a través de sus regiones carboxilo terminal. Además, la subunidad Asa2 interacciona con Asa4 y Asa7 así como con la subunidad α en el sector F1. Las 3 subunidades Asa forman un subcomplejo Asa2/Asa4/Asa7 con una estequiometría 1:1:1. La subunidad Asa7 y el extremo carboxilo terminal de Asa4 parecen ser necesarios para la interacción con Asa2. Basados en los resultados se generaron modelos estructurales in silico de las tres subunidades. Se propone un modelo de la vecindad topológica de las tres subunidades (subcomplejo Asa2/Asa4/Asa7) así como de su posición en el brazo periférico de la ATP sintasa del alga. [less ▲]

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See detailInteractions of subunits Asa2, Asa4 and Asa7 in the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.
Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Arceli; Vázquez-Acevedo, Miriam et al

in Biochimica et Biophysica Acta-Bioenergetics (2013), 1837(1), 1-13

Mitochondrial F1FO-ATP synthase of chlorophycean algae is a complex partially embedded in the inner mitochondrial membrane that is isolated as a highly stable dimer of 1600 kDa. It comprises 17 ... [more ▼]

Mitochondrial F1FO-ATP synthase of chlorophycean algae is a complex partially embedded in the inner mitochondrial membrane that is isolated as a highly stable dimer of 1600 kDa. It comprises 17 polypeptides, nine of which (subunits Asa1 to 9) are not present in classical mitochondrial ATP synthases and appear to be exclusive of the chlorophycean lineage. In particular, subunits Asa2, Asa4 and Asa7 seem to constitute a section of the peripheral stalk of the enzyme. Here, we over-expressed and purified subunits Asa2, Asa4 and Asa7 and the corresponding amino-terminal and carboxy-terminal halves of Asa4 and Asa7 in order to explore their interactions in vitro, using immunochemical techniques, blue native electrophoresis and affinity chromatography. Asa4 and Asa7 interact strongly, mainly through their carboxy-terminal halves. Asa2 interacts with both Asa7 and Asa4, and also with subunit ?? in the F1 sector. The three Asa proteins form an Asa2/Asa4/Asa7 subcomplex. The entire Asa7 and the carboxy-terminal half of Asa4 seem to be instrumental in the interaction with Asa2. Based on these results and on computer-generated structural models of the three subunits, we propose a model for the Asa2/Asa4/Asa7 subcomplex and for its disposition in the peripheral stalk of the algal ATP synthase. [less ▲]

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See detailDeterminación del estado de agregación de la triosafosfato isomerasa de distintas especies por electroforesis nativa en geles azules
Pérez-Castañeda, Edgar; Vázquez-Acevedo, Miriam; Cabrera-González, Nayelli et al

Poster (2012, November)

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See detailEstudio de las interacciones in vitro de las subunidades que componen el brazo periférico de la ATP sintasa mitocondrial de Polytomella sp
Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli; Colina-Tenorio, Lilia et al

Poster (2012, November)

La ATP sintasa mitocondrial de las algas clorofíceas no contiene las subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de la misma ... [more ▼]

La ATP sintasa mitocondrial de las algas clorofíceas no contiene las subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de la misma. En compensación, ha adquirido 9 subunidades novedosas, de origen evolutivo desconocido, que han sido llamadas ASA1 a ASA9. Estas subunidades ASA solamente están presentes en el grupo de las algas clorofíceas y no se encuentran en otras algas cercanamente relacionadas, como las algas verdes del linaje de las ulvofíceas, de las prasinofíceas o de las trebuxiofíceas. Experimentos de disociación de la enzima, tratamiento con agentes entrecruzadores y estudios de microscopía electrónica llevaron a la propuesta de un modelo estructural de esta ATP sintasa, en el cual las subunidades ASA1 a 9 forman la estructura del estator periférico y participan en la dimerización de la enzima (Cano-Estrada et al., 2010). El objetivo de este trabajo es conocer cómo interactúan las subunidades ASA2, ASA4 y ASA7 del brazo periférico de la ATP sintasa mitocondrial de Polytomella sp. La estrategia experimental está basada en la clonación de los genes completos así como de fragmentos de éstos, la sobre-expresión heteróloga en bacteria y su purificación. Con las proteínas purificadas se realizaron ensayos de interacción que comprenden inmunorréplicas tipo far western, geles azules nativos y copurificacion de subcomplejos en columnas de afinidad. Los resultados obtenidos indican una interacción entre las subunidades ASA4 y ASA7 mediada por los extremos carboxilo de ambas proteínas, así como una asociación de las subunidades ASA2 – ASA4 y ASA2 – ASA7. [less ▲]

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See detailAislamiento de algunas subunidades que integran el brazo periférico de la ATP sintasa mitocondrial de Polytomella sp. y estudio de sus interacciones.
Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli; Vázquez-Acevedo, Miriam et al

Conference (2011, November 14)

La ATP sintasa mitocondrial de las algas clorofíceas ha perdido una serie de subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de ... [more ▼]

La ATP sintasa mitocondrial de las algas clorofíceas ha perdido una serie de subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de la misma. En compensación, ha adquirido 9 subunidades novedosas, de origen evolutivo desconocido, que han sido llamadas ASA1 a ASA9. Estas subunidades ASA solamente están presentes en el grupo de las algas clorofíceas y no se encuentran en otras algas cercanamente relacionadas, como las algas verdes del linaje de las ulvofíceas, de las prasinofíceas o de las trebuxiofíceas. Experimentos de disociación de la enzima y tratamiento con agentes entrecruzadores llevaron a la propuesta de un modelo estructural de esta ATP sintasa, donde se propone que las subunidades ASA1 a 9 participan en la estructura del estator periférico y en la formación de un dímero estable. En el presente trabajo se desea abordar un estudio más detallado de las subunidades ASA, para conocer acerca de las interacciones que establecen entre ellas y como contribuyen a la formación del brazo periférico de la enzima. La estrategia experimental parte de la clonación de los genes de las subunidades en vectores de expresión en bacteria, la sobre-expresión de las subunidades recombinantes, su purificación y ensayos de interacción mediante geles azules nativos e inmunoréplicas tipo Far Western. Los resultados indican una fuerte interacción entre las subunidades ASA4 y ASA7 que involucra el extremo carboxilo terminal de la subunidad ASA4. Los estudios de inmunoréplica indican interacciones entre las subunidades ASA4-ASA7 y ASA4-ASA2. [less ▲]

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See detail¿Es el carboxilo terminal de la subunidad β el inhibidor natural de la F1Fo ATP sintasa de Polytomella sp.?
Villavicencio-Queijeiro, Alexa; Miranda Astudillo, Héctor Vicente ULiege; Figueroa-Martínez, Francisco et al

Poster (2011, November)

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See detailInteracción de las subunidades ASA2, ASA4 y ASA7 de la ATP sintasa mitocondrial del alga incolora Polytomella sp
Cano-Estrada, Araceli; Miranda Astudillo, Héctor Vicente ULiege; Vázquez-Acevedo, Miriam et al

Poster (2011, November)

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See detailLa expresión de una lipocalina en las mitocondrias de la levadura Saccharomyces cerevisiae podría conferir resistencia al estrés oxidativo
Macedo-Márquez, Alain; Miranda Astudillo, Héctor Vicente ULiege; Vázquez-Acevedo, Miriam et al

Poster (2010, November)

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See detailEstudio de las interacciones entre las subunidades ASA del brazo periférico de la ATP sintasa mitocondrial de Polytomella sp
Miranda Astudillo, Héctor Vicente ULiege; Cano-Estrada, Araceli; Vázquez-Acevedo, Miriam et al

Poster (2010, November)

La ATP sintasa mitocondrial de las algas clorofíceas ha perdido una serie de subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de ... [more ▼]

La ATP sintasa mitocondrial de las algas clorofíceas ha perdido una serie de subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de la misma. En compensación, ha adquirido 9 subunidades novedosas, de origen evolutivo desconocido, que han sido llamadas ASA1 a ASA9. Estas subunidades ASA solamente están presentes en el grupo de las algas clorofíceas y no se encuentran en otras algas cercanamente relacionadas, como las algas verdes del linaje de las ulvofíceas, de las prasinofíceas o de las trebuxiofíceas. Experimentos de disociación de la enzima, tratamiento con agentes entrecruzadores y estudios estructurales llevaron a la propuesta de un modelo estructural de esta ATP sintasa, donde se propone que las subunidades ASA1 a 9 participan en la estructura del estator periférico y en la dimerización de la misma. En el presente trabajo se desea abordar un estudio más detallado de las subunidades ASA, para conocer acerca de las interacciones que establecen entre ellas y como contribuyen a la formación del brazo periférico de la enzima. La estrategia experimental parte de la clonación de los genes de las subunidades en vectores de expresión en bacteria, la expresión de las subunidades recombinantes y su purificación. Los estudios de inmunoréplica indican las interacciones entre las subunidades ASA4-ASA7 y ASA4-ASA2. [less ▲]

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See detailSubunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp
Cano-Estrada, Araceli; Vázquez-Acevedo, Miriam; Villavicencio-Queijeiro, Alexa et al

in Biochimica et Biophysica Acta-Bioenergetics (2010), 1797(8), 1439-1448

Mitochondrial F1F0-ATP synthase of chlorophycean algae is a dimeric complex of 1600kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that ... [more ▼]

Mitochondrial F1F0-ATP synthase of chlorophycean algae is a dimeric complex of 1600kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1-9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp. [less ▲]

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