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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 detailNon-classical structures and association of the OXPHOS complexes in Euglena gracilis and Polytomella sp.
Miranda Astudillo, Héctor Vicente ULiege; Yadav, Sathish; Colina-Tenorio, Lilia et al

Conference (2017, September 21)

To date, the idea that the OXPHOS complexes can associate with each other in larger structures named supercomplexes [1] is generally accepted. This association can allow a more efficient transport of ... [more ▼]

To date, the idea that the OXPHOS complexes can associate with each other in larger structures named supercomplexes [1] is generally accepted. This association can allow a more efficient transport of electrons to minimize the generation of reactive oxygen species during electron transfer reactions and can also be involved in the regulation of the mitochondrial metabolism in response to different stimuli, carbon sources or stress conditions. Recently, the arising studies outside the classical models yeast and bovine mitochondria brought to light many unusual characteristics in the ATP synthase from a wide variety of organisms. Due to this, the dimer nature of the mitochondrial ATP synthase is no longer a matter of debate. Given the large structural differences among the peripheral stator and dimerization modules of mitochondrial ATP synthases described so far, it is of relevance to study a wider number of species to gain insight into the structural diversity of their OXPHOS complexes. Recently, our group showed that at least 41 of the non-canonical subunits reported in trypanosomes are also present in Euglena complexes along with 48 classical subunits described in other eukaryotes including green plants [2]. Further purification of the complexes I, III, IV and V by liquid chromatography after solubilization with n-β-dodecyl-maltoside and the subsequent analysis by single-particle analysis from transmission electron microscopy revealed some unusual features in Euglena respiratory complexes. In the case of complex V the structures of both the catalytic and central rotor parts are conserved while other structural features are original, including a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring [3]. Complex I also shows an unusually long matricial arm. Complex IV shows an atypical shape compared to that of the bovine one. An unusual association between complexes I and V can be observed when the membranes are extracted with the mild detergent digitonin. Other case of atypical subunit composition is the complex V of chlorophycean algae. 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 this lineage that form the robust peripheral stalk in this complex [4]. This complex presents highly stable dimeric and tetrameric structures, and the respiratory complexes have an unusual capacity to reassociate in vitro and restore the functional respirasome. [less ▲]

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See detailAtypical composition and structure of the mitochondrial dimeric ATP synthase from Euglena gracilis
Miranda Astudillo, Héctor Vicente ULiege; Yadav, Sathish; Colina-Tenorio, Lilia et al

Poster (2017, September 21)

Mitochondrial respiratory-chain complexes fromEuglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena ... [more ▼]

Mitochondrial respiratory-chain complexes fromEuglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Herewe studied the mitochondrial F1FO-ATP synthase (or Complex V) fromthe photosynthetic eukaryote E. gracilis in detail. The enzymewas purified by a two-step chromatographic procedure and its subunit compositionwas resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS).Twenty-two different subunits were identified by mass-spectrometry analyses amongwhich the canonical α, β, γ, δ, ε, and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrierwas also associated to the ATP synthase into a dimericATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning re- gion joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase. [less ▲]

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See detailAtypical composition and structure of the mitochondrial dimeric ATP synthase from Euglena gracilis
Yadav, K.N. Sathish; Miranda Astudillo, Héctor Vicente ULiege; Colina-Tenorio, Lili et al

in Biochimica et Biophysica Acta-Bioenergetics (2017), 1858(4), 267-275

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena ... [more ▼]

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F1FO-ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical alpha, beta, gamma, delta, epsilon and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase. [less ▲]

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See detailAtypical structure and subunit composition of respiratory complexes in Euglena gracilis
Miranda Astudillo, Héctor Vicente ULiege; Yadav, Sathish; Colina-Tenorio, Lilia et al

Conference (2016, August 14)

Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes, has a complex mitochondrial oxidative phosphorylation system constituted by atypical respiratory enzymes (complexes I - V ... [more ▼]

Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes, has a complex mitochondrial oxidative phosphorylation system constituted by atypical respiratory enzymes (complexes I - V). Recently, the analysis of the subunit composition of respiratory complexes by 2D BN/SDS PAGE has shown that at least 41 of the non-canonical subunits reported in trypanosomes are also present in this alga along with 48 classical subunits described in other eukaryotes including green plants. In the present study the complexes I, III, IV and V were further purified from isolated mitochondria using liquid chromatography after solubilization with n-dodecyl-maltoside. Using a 3D BN/SDS/SDS PAGE analysis, we resolved their subunit composition and confirmed the atypical subunit composition of Euglena respiratory complexes. The apparent molecular mass of purified complexes I and V (1.5 and 2.2MDa, respectively) is far above the classical ones. Single-particle analysis from transmission electron microscopy revealed some unusual features for complexes I, IV and V, including smaller angles between monomers and additional membrane extensions for the ATP synthase. Complex I also shows an unusual long matricial arm. Meanwhile the complex IV shows an atypical shape compared with the bovine one. [less ▲]

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See detailDissecting the peripheral stalk of the mitochondrial ATP synthase of chlorophycean algae.
Vázquez-Acevedo, M; Vega de Luna, F; Sánchez-Vásquez, L et al

in Biochimica et Biophysica Acta-Bioenergetics (2016), 1857(8), 1183-90

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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 detailEstructura y composición polipeptídica atípicas de los complejos respiratorios de Euglena gracilis
Miranda Astudillo, Héctor Vicente ULiege; Yadav, Sathish; Colina-Tenorio, Lilia et al

Conference (2015, November 10)

Euglena gracilis, un alga secundaria no parásita relacionada con los tripanosomas, tiene un sistema de fosforilación oxidativa mitocondrial complejo constituido por enzimas respiratorias atípicas ... [more ▼]

Euglena gracilis, un alga secundaria no parásita relacionada con los tripanosomas, tiene un sistema de fosforilación oxidativa mitocondrial complejo constituido por enzimas respiratorias atípicas. Recientemente, el análisis bioinformático y mediante 2D BN/SDS PAGE de la composición de subunidades de estos complejos respiratorios mostró que al menos 41 de las subunidades no canónicas reportadas en tripanosomas están también presentes en esta alga, así como 48 subunidades clásicas descritas en otros eucariotes. En el presente trabajo, los complejos I, III, IV y V fueron purificados a partir de mitocondrias aisladas mediante cromatografía líquida después de ser solubilizados con n-dodecil-maltósido. Utilizando los complejos purificados se realizó un análisis 3D BN/SDS/SDS PAGE y posterior espectrometría de masas, mediante esta estrategia se resolvieron e identificaron las subunidades que los componen, lo que confirmó la composición polipeptídica atípica de los complejos respiratorios de esta alga. La masa molecular aparente de los complejos purificados I y V (1.5 y 2.2 MDa, respectivamente) se encuentra muy por encima de los complejos clásicos. Por su parte, el análisis de partícula única a partir de imágenes de microscopía electrónica de transmisión reveló algunas características estructurales inusuales en el complejo V, estas incluyen ángulos menores entre los monómeros y algunas extensiones adicionales de la región membranal, mientras que el complejo I mostró también una extensión en el brazo extramembranal. [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 detailNative electrophoresis studies of different aggregation states of triosephosphate isomerase.
Toledo-Ibelles, Paola; Pérez-Castañeda, Edgar; Rodríguez-Bolaños, Mónica et al

Poster (2014, November 07)

Protein folding is a process mainly due to weak chemical interactions such as repelling of charges, hydrophobic interactions, and hydrogen bonds, which involve reversible disorder-to-order transitions of ... [more ▼]

Protein folding is a process mainly due to weak chemical interactions such as repelling of charges, hydrophobic interactions, and hydrogen bonds, which involve reversible disorder-to-order transitions of a polypeptide chain. Although changes between partially folded structures, in order to get a native functional protein, are highly efficient, proteins still may have transiently stable conformations, which yield amorphous oligomers and end in a state of aggregation that may or may not be reversible. Even though the amino acid sequence plays a key role in this process, the microenvironment has an important influence as well, given that chemical modifications by reactive oxygen and nitrogen species produce an inadequate folding. Triosephosphate isomerase (TIM) is a protein susceptible to nitrotyrosination after which it becomes prone to aggregation; such aggregates have been observed in Alzheimer patients. This suggests that protein folding, oligomer formation and aggregation of this protein plays a key role in the complex biochemical pathway through its structural transitions, as well as in the development of diseases such as Alzheimer. Other research groups and ourselves have been able to provide evidence of protein aggregation in vitro using native electrophoresis and ionic exchange chromatography. The present work focuses on the characterization of protein aggregates of TIMs from different species. Despite the fact that all TIM proteins described to date have a conserved (α/β) 8 barrel structure, our results show a notable difference in their proclivity to generate one or several aggregation states, when their migration is analyzed using polyacrylamide gradient electrophoresis under native conditions. Supported by grants 167823 from CONACyT and IN221812 from DGAPA-UNAM [less ▲]

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See detailCharacterization of the hydrophobic subunits of the peripheral arm of the ATP synthase from the colorless alga Polytomella sp.
Sánchez-Vásquez, Lorenzo; Jiménez-Suárez, Alejandra; Miranda Astudillo, Héctor Vicente ULiege et al

Poster (2014, November 07)

The F O F1-ATP synthase (EC3.6.3.14) produces most of the cellular ATP in aerobic conditions. This enzyme complex is found in energy transducing membranes such as the mitochondrial inner membrane, the ... [more ▼]

The F O F1-ATP synthase (EC3.6.3.14) produces most of the cellular ATP in aerobic conditions. This enzyme complex is found in energy transducing membranes such as the mitochondrial inner membrane, the thylakoid membrane of the chloroplast, and the bacterial plasma membrane. The Escherichia coli ATP synthase exhibits the simplest known subunit composition, which consists of eight subunits α3 β3 γ1 δ1 ε1 a 1 b 2 c 10-12. Subunits -α, -β, -γ, -δand -εform the F 1 domain, while subunits -a, -b and -c form the F O domain. The mitochondrial F O F 1 -ATP synthase is more complex than the bacterial enzyme and is composed of at least 15 different subunits. Besides having orthologs of the eight essential subunits found in E. coli, it contains several supernumerary subunits (<20 KDa), which are required for the stability or the regulation of the enzymatic complex, while others are involved in its dimerization or oligomerization. In this regard, and in contrast to other known enzyme complexes, the ATPase from the algae Chlamydomonas reinhardtii and Polytomella sp. show unique structural characteristics. The algal enzyme was found to have orthodox subunits of the catalytic domain (-αand-β), as well as those that are part of the central rotary stalk (-γ, -δ, -ε and -c), however, with respect to the subunits which constitute the peripheral arm, only subunits –a and –OSCP were found. In addition, nine subunits of unknown evolutionary origin that do not have clear homologs in the databases were also found. These subunits were named ASA (Mitochondrial ATP Synthase Associated Protein) and were numbered successively as ASA1-9 subunits. It is thought that the ASA subunits replace those involved in the formation of the peripheral arm (-b, -d, -e, -f, -g, -IF1, -A6L and -F6), the dimerization of the complex (-e, -g) and the regulation (-IF1) of the enzyme activity. Therefore, these ASA subunits could be the main components of the peripheral arm (ASA1, ASA2, ASA3, ASA4, and ASA7), others may participate in the dimerization of the complex (ASA6 and ASA9) and others could play a regulatory role (ASA?).Recent work in the laboratory using methodologies such as dissociation of the enzyme into sub-complexes induced by high temperatures, treatment with crosslinking agents or association studies employing recombinant proteins, have addressed the study of the interactions of the ASA subunits in the ATP synthase of Polytomella sp. However, still little is known about subunits -a, -c, ASA5, ASA6, ASA8 and ASA9 that form the membrane fraction of the complex. These subunits probably have a transmembrane domain or highly hydrophobic regions. Therefore, it is our interest to approach the study of the interactions of the hydrophobic subunits of the peripheral arm of the ATP synthase and to obtain more information about the membrane domain of this enzymatic complex. In this work we present the results obtained using recombinant proteins and their purification for the subsequent formation of in vitro sub-complexes, as well as qualitative information obtained from interaction assays using the two hybrid system. For the use of recombinant proteins it is necessary to clone the genes that encode for the mature subunits:ASA6, ASA8 and ASA9 from the alga ATP synthase into vectors for further overexpression in E. coli and purifying them by fast protein liquid chromatography (FPLC). The recombinant proteins were used for interaction assays such as type Far-Western blotting and co-purification by immobilized metal affinity chromatography (IMAC) using nickel-NTA (nitrilotriacetic acid).The in vivo interaction assays were performed using yeast two-hybrid system. For this purpose, the constructions of the genes encoding the ASA6, ASA8 and ASA9 subunits vectors corresponding to the activation domain (pAD) and binding domain (pBD) were obtained. Subsequently, interaction assays were performed in the medium called "low stringency" (-Leu/-Trp/-His) and "high stringency" (-Leu/-Trp/-His/- Ade) observing interactions only in the former conditions. The results suggest an ASA6-ASA6 interaction, and in less degree the interactions ASA9-ASA8 and ASA9-ASA9. We will confirm these data using other methodologies used to determine protein-protein interactions. Work supported by CONACyT (128110) and DGAPA-UNAM (IN 203311). [less ▲]

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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 detailThe mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae.
Perez, Emilie ULiege; Lapaille, Marie; Degand, Herve et al

in Mitochondrion (2014), 19 Pt B

The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential ... [more ▼]

The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential therapeutic target. Recently, numerous additional subunits of the respiratory-chain complexes have been described in Trypanosoma brucei and Trypanosoma cruzi. Since these subunits had apparently no counterparts in other organisms, they were interpreted as potentially associated with the parasitic trypanosome lifestyle. Here we used two complementary approaches to characterise the subunit composition of respiratory complexes in Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes. First, we developed a phylogenetic pipeline aimed at mining sequence databases for identifying homologs to known respiratory-complex subunits with high confidence. Second, we used MS/MS proteomics after two-dimensional separation of the respiratory complexes by Blue Native- and SDS-PAGE to both confirm in silico predictions and to identify further additional subunits. Altogether, we identified 41 subunits that are restricted to E. gracilis, T. brucei and T. cruzi, along with 48 classical subunits described in other eukaryotes (i.e. plants, mammals and fungi). This moreover demonstrates that at least half of the subunits recently reported in T. brucei and T. cruzi are actually not specific to Trypanosomatidae, but extend at least to other Euglenozoa, and that their origin and function are thus not specifically associated with the parasitic lifestyle. Furthermore, preliminary biochemical analyses suggest that some of these additional subunits underlie the peculiarities of the respiratory chain observed in Euglenozoa. [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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