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1.
Microbiologyopen ; 9(3): e989, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31970933

RESUMEN

Phycobiliproteins (PBPs) are colored fluorescent proteins present in cyanobacteria, red alga, and cryptophyta. These proteins have many potential uses in biotechnology going from food colorants to medical applications. Allophycocyanin, the simplest PBP, is a heterodimer of αß subunits that oligomerizes as a trimer (αß)3 . Each subunit contains a phycocyanobilin, bound to a cysteine residue, which is responsible for its spectroscopic properties. In this article, we are reporting the expression of recombinant allophycocyanin (rAPC) from the eukaryotic red algae Agarophyton chilensis in Escherichia coli, using prokaryotic accessory enzymes to obtain a fully functional rAPC. Three duet vectors were used to include coding sequences of α and ß subunits from A. chilensis and accessorial enzymes (heterodimeric lyase cpc S/U, heme oxygenase 1, phycocyanobilin oxidoreductase) from cyanobacteria Arthrospira maxima. rAPC was purified using several chromatographic steps. The characterization of the pure rAPC indicates very similar spectroscopic properties, λmaxAbs , λmaxEm , fluorescence lifetime, and chromophorylation degree, with native allophycocyanin (nAPC) from A. chilensis. This method, to produce high-quality recombinant allophycocyanin, can be used to express and characterize other macroalga phycobiliproteins, to be used for biotechnological or biomedical purposes.


Asunto(s)
Eucariontes/genética , Ficocianina/biosíntesis , Ficocianina/genética , Células Procariotas/enzimología , Proteínas Recombinantes , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Vectores Genéticos/genética , Peso Molecular , Ficocianina/aislamiento & purificación , Análisis Espectral
2.
Protein Sci ; 18(6): 1139-45, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19472323

RESUMEN

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.


Asunto(s)
Proteínas del Dominio Armadillo/química , Proteínas de Caenorhabditis elegans/química , Proteínas Nucleares/química , Animales , Vectores Genéticos , Factores de Intercambio de Guanina Nucleótido , Modelos Moleculares , Estructura Secundaria de Proteína , Proteínas Recombinantes/química
3.
J Cell Physiol ; 214(2): 483-90, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17654482

RESUMEN

The non-canonical Wnt/Ca2+ signaling pathway has been implicated in the regulation of axis formation and gastrulation movements during early Xenopus laevis embryo development, by antagonizing the canonical Wnt/beta-catenin dorsalizing pathway and specifying ventral cell fate. However, the molecular mechanisms involved in this antagonist crosstalk are not known. Since Galphaq is the main regulator of Ca2+ signaling in vertebrates and from this perspective probably involved in the events elicited by the non-canonical Wnt/Ca2+ pathway, we decided to study the effect of wild-type Xenopus Gq (xGalphaq) in dorso-ventral axis embryo patterning. Overexpression of xGalphaq or its endogenous activation at the dorsal animal region of Xenopus embryo both induced a strong ventralized phenotype and inhibited the expression of dorsal-specific mesoderm markers goosecoid and chordin. Dorsal expression of an xGalphaq dominant-negative mutant reverted the xGalphaq-induced ventralized phenotype. Finally, we observed that the Wnt8-induced secondary axis formation is reverted by endogenous xGalphaq activation, indicating that it is negatively regulating the Wnt/beta-catenin pathway.


Asunto(s)
Tipificación del Cuerpo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Wnt/antagonistas & inhibidores , Xenopus laevis/metabolismo , beta Catenina/antagonistas & inhibidores , Animales , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Gastrulación , Xenopus laevis/embriología
4.
J Cell Physiol ; 214(3): 673-80, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17960561

RESUMEN

Immature stage VI Xenopus oocytes are arrested at the G(2)/M border of meiosis I until exposed to progesterone, which induces meiotic resumption through a non-genomic mechanism. One of the earliest events produced by this hormone is inhibition of the plasma membrane enzyme adenylyl cyclase (AC), with the concomitant drop in intracellular cAMP levels and reinitiation of the cell cycle. Recently Gsalpha and Gbetagamma have been shown to play an important role as positive regulators of Xenopus oocyte AC, maintaining the oocyte in the arrested state. However, a question that still remains unanswered, is how the activated state of Gsalpha and Gbetagamma is achieved in the immature oocyte, since no receptor or ligand have been found to be required. Here we provide evidence that xRic-8 can act in vitro and in vivo as a GEF for Gsalpha. Overexpression of xRic-8, through mRNA injection, greatly inhibits progesterone induced oocyte maturation and endogenous xRic-8 mRNA depletion, through siRNA microinjection, induces spontaneous oocyte maturation. These results suggest that xRic-8 is participating in the immature oocyte by keeping Gsalpha-Gbetagamma-AC signaling complex in an activated state and therefore maintaining G2 arrest.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Meiosis , Oocitos/citología , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/genética
5.
J Cell Physiol ; 211(2): 560-7, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17219407

RESUMEN

During the last decade, considerable evidence is accumulating that supports the view that the classic progesterone receptor (xPR-1) is mediating Xenopus laevis oocyte maturation through a non-genomic mechanism. Overexpression and depletion of oocyte xPR-1 have been shown to accelerate and to block progesterone-induced oocyte maturation, respectively. In addition, rapid inhibition of plasma membrane adenylyl cyclase (AC) by the steroid hormone, supports the idea that xPR-1 should be localized at the oocyte plasma membrane. To test this hypothesis, we transiently transfected xPR-1 cDNA into Cos-7 cells and analyzed its subcellular distribution. Through Western blot and immunofluorescence analysis, we were able to detect xPR-1 associated to the plasma membrane of transfected Cos-7 cells. Additionally, using Progesterone-BSA-FITC, we identified specific progesterone-binding sites at the cell surface of xPR-1 expressing cells. Finally, we found that the receptor ligand-binding domain displayed membrane localization, in contrast to the N-terminal domain, which expressed in similar levels, remained cytosolic. Overall, these results indicate that a fraction of xPR-1 expressed in Cos-7 cells, associates to the plasma membrane through its LBD.


Asunto(s)
Membrana Celular/metabolismo , Receptores de Progesterona/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Sitios de Unión , Western Blotting , Células COS , Chlorocebus aethiops , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Colorantes Fluorescentes/metabolismo , Ligandos , Microscopía Fluorescente , Progesterona/análogos & derivados , Progesterona/metabolismo , Estructura Terciaria de Proteína , Receptores de Progesterona/química , Receptores de Progesterona/genética , Albúmina Sérica Bovina/metabolismo , Transfección , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis
6.
J Cell Biochem ; 99(4): 995-1000, 2006 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-16927375

RESUMEN

1alpha,25-dihydroxy vitamin D3 has a major role in the regulation of the bone metabolism as it promotes the expression of key bone-related proteins in osteoblastic cells. In recent years it has become increasingly evident that in addition to its well-established genomic actions, 1alpha,25-dihydroxy vitamin D3 induces non-genomic responses by acting through a specific plasma membrane-associated receptor. Results from several groups suggest that the classical nuclear 1alpha,25-dihydroxy vitamin D3 receptor (VDR) is also responsible for these non-genomic actions of 1alpha,25-dihydroxy vitamin D3. Here, we have used siRNA to suppress the expression of VDR in osteoblastic cells and assessed the role of VDR in the non-genomic response to 1alpha,25-dihydroxy vitamin D3. We report that expression of the classic VDR in osteoblasts is required to generate a rapid 1alpha,25-dihydroxy vitamin D3-mediated increase in the intracellular Ca(2+) concentration, a hallmark of the non-genomic actions of 1alpha,25-dihydroxy vitamin D3 in these cells.


Asunto(s)
Genoma/genética , Osteosarcoma/patología , Receptores de Calcitriol/metabolismo , Vitamina D/análogos & derivados , Animales , ARN Interferente Pequeño , Ratas , Vitamina D/metabolismo
7.
J Cell Biochem ; 99(3): 853-9, 2006 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-16721828

RESUMEN

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a non-genomic mechanism initiated at the cell membrane. Recently, two Xenopus oocyte progesterone receptors have been cloned; one is the classical progesterone receptor (xPR-1) involved in genomic actions and the other a putative seven-transmembrane-G-protein-couple receptor. Both receptors are postulated to be mediating the steroid-induced maturation process in the frog oocyte. In this study, we tested the hypothesis that the classical progesterone receptor, associated to the oocyte plasma membrane, is participating in the reinitiation of the cell cycle. Addition of a myristoilation and palmytoilation signal at the amino terminus of xPR-1 (mp xPR-1), increased the amount of receptor associated to the oocyte plasma membrane and most importantly, significantly potentiated progesterone-induced oocyte maturation sensitivity. These findings suggest that the classical xPR-1, located at the plasma membrane, is mediating through a non-genomic mechanism, the reinitiation of the meiotic cell cycle in the X. laevis oocyte.


Asunto(s)
Membrana Celular/metabolismo , Oocitos/fisiología , Progesterona/farmacología , Receptores de Progesterona/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Células COS , Ciclo Celular/fisiología , Chlorocebus aethiops , Femenino , Oocitos/citología , Oocitos/efectos de los fármacos , Progesterona/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de Progesterona/genética , Proteínas de Xenopus/genética , Xenopus laevis
8.
J Cell Biochem ; 93(2): 409-17, 2004 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-15368366

RESUMEN

G protein signalling regulates a wide range of cellular processes such as motility, differentiation, secretion, neurotransmission, and cell division. G proteins consist of three subunits organized as a Galpha monomer associated with a Gbetagamma heterodimer. Structural studies have shown that Galpha subunits are constituted by two domains: a Ras-like domain, also called the GTPase domain (GTPaseD), and an helical domain (HD), which is unique to heterotrimeric G-proteins. The HD display significantly higher primary structure diversity than the GTPaseD. Regardless of this diversity, there are small regions of the HD which show high degree of identity with residues that are 100% conserved. One of such regions is the alpha helixD-alpha helixE loop (alphaD-alphaE) in the HD, which contains the consensus aminoacid sequence R*-[RSA]-[RSAN]-E*-[YF]-[QH]-L in all mammalian Galpha subunits. Interestingly, the highly conserved arginine (R*) and glutamic acid (E*) residues form a salt bridge that stabilizes the alphaD-alphaE loop, that is localized in the top of the cleft formed between the GTPaseD and HD. Because the guanine nucleotide binding site is deeply buried in this cleft and those interdomain interactions are playing an important role in regulating the basal GDP/GTP nucleotide exchange rate of Galpha subunits, we studied the role of these highly conserved R and E residues in Galpha function. In the present study, we mutated the human Gsalpha R165 and E168 residues to alanine (A), thus generating the R165--> A, E168--> A, and R165/E168--> A mutants. We expressed these human Gsalpha (hGsalpha) mutants in bacteria as histidine tagged proteins, purified them by niquel-agarose chromatography and studied their nucleotide exchange properties. We show that the double R165/E168--> A mutant exhibited a fivefold increased GTP binding kinetics, a higher GDP dissociation rate, and an augmented capacity to activate adenylyl cyclase. Structure analysis showed that disruption of the salt bridge between R165 and E168 by the introduced mutations, caused important structural changes in the HD at the alphaD-alphaE loop (residues 160-175) and in the GTPaseD at a region required for Gsalpha activation by the receptor (residues 308-315). In addition, other two GTPaseD regions that surround the GTP binding site were also affected.


Asunto(s)
Arginina/metabolismo , Secuencia Conservada/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Ácido Glutámico/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Mutación/genética , Adenilil Ciclasas/metabolismo , Arginina/genética , Cristalografía por Rayos X , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Ácido Glutámico/genética , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Estructura Terciaria de Proteína
9.
J Cell Physiol ; 195(2): 151-7, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12652642

RESUMEN

Heterotrimeric G-proteins transduce signals from heptahelical transmembrane receptors to different effector systems, regulating diverse complex intracellular pathways and functions. In brain, facilitation of depolarization-induced neurotransmitter release for synaptic transmission is mediated by Gsalpha and Gqalpha. To identify effectors for Galpha-proteins, we performed a yeast two-hybrid screening of a human brain cDNA library, using the human Galphas protein as a bait. We identified a protein member of the synembryn family as one of the interacting proteins. Extending the study to other Galpha subunits, we found that Gqalpha also interacts with synembryn, and these interactions were confirmed by in vitro pull down studies and by in vivo confocal laser microscopy analysis. Furthermore, synembryn was shown to translocate to the plasma membrane in response to carbachol and isoproterenol. This study supports recent findings in C. elegans where, through genetic studies, synembryn was shown to act together with Gqalpha regulating neuronal transmitter release. Based on these observations, we propose that synembryn is playing a similar role in human neuronal cells.


Asunto(s)
Encéfalo/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Transporte de Proteínas/fisiología , Agonistas alfa-Adrenérgicos/farmacología , Animales , Encéfalo/efectos de los fármacos , Carbacol/farmacología , Membrana Celular/efectos de los fármacos , Agonistas Colinérgicos/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Isoproterenol/farmacología , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Células PC12 , Transporte de Proteínas/efectos de los fármacos , Ratas , Técnicas del Sistema de Dos Híbridos
10.
J Cell Biochem ; 85(3): 615-20, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-11968001

RESUMEN

G-protein alpha subunits consist of two domains: a Ras-like domain also called GTPase domain (GTPaseD), structurally homologous to monomeric G-proteins, and a more divergent domain, unique to heterotrimeric G-proteins, called helical domain (HD). G-protein activation, requires the exchange of bound GDP for GTP, and since the guanine nucleotide is buried in a deep cleft between both domains, it has been postulated that activation may involve a conformational change that will allow the opening of this cleft. Therefore, it has been proposed, that interdomain interactions are playing an important role in regulating the nucleotide exchange rate of the alpha subunit. While constructing different Gs(alpha) quimeras, we identified a Gs(alpha) random mutant, which was very inefficient in stimulating adenylyl cyclase activity. The introduced mutation corresponded to the substitution of Ser(111) for Asn (S111N), located in the carboxi terminal end of helix A of the HD, a region neither involved in AC interaction nor in the interdomain interface. In order to characterize this mutant, we expressed it in bacteria, purified it by niquel-agarose chromatography, and studied its nucleotide exchange properties. We demonstrated that the recombinant S111N Gs(alpha) was functional since it was able to undergo the characteristic conformational change upon GTP binding, detected by the acquisition of a trypsin-resistant conformation. When the biochemical properties were determined, the mutant protein exhibited a reduced GDP dissociation kinetics and as a consequence a slower GTPgammaS binding rate that was responsible for a diminished adenylyl cyclase activation when GTPgammaS was used as activator. These data provide new evidence that involves the HD as a regulator of Gs(alpha) function, in this case the alphaA helix, which is not directly involved with the nucleotide binding site nor the interdomain interface.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Mutación Puntual , Adenilil Ciclasas/metabolismo , Compuestos de Aluminio/metabolismo , Sustitución de Aminoácidos , Asparagina/genética , Fluoruros/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/aislamiento & purificación , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Receptores de Superficie Celular/metabolismo , Serina/genética , Tripsina/metabolismo
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