ABSTRACT
Phosphatidylinositol (PtdIns) is a major phospholipid in eukaryotic cells. Many studies have revealed that the phosphoinositide (PI) signaling pathway plays an important role in plant growth and development. Phospholipase C (PLC) is reported to have a crucial role in the PI pathway. This work focuses on the isolation and investigation of PLC in response to abiotic stress factors in green gram. The PLC cDNA, designated VrPLC, encoding a protein of 591 amino acids was cloned and expressed in E. coli.The predicted isoelectric point (pI) and molecular weight were 5.96 and 67.3 kDa, respectively. The tertiary structure of the PLC was also predicted and found to be mainly composed of random coils. In addition, VrPLC expression analysis was performed under environmental stress and the results showed that the expression of VrPLC was rapidly induced in an abscisic acid independent manner in response to drought and salt stress. PLC expression was found to be up-regulated by SA and down-regulated by wound in leaf tissues; however, there was no significant difference in the expression of PLC in plants subjected to high temperature and H2O2. Our results suggest that a close link/relationship between PLC expression and stress responses in green gram.
Subject(s)
Fabaceae/enzymology , Fabaceae/physiology , Gene Expression/genetics , Gene Expression/genetics , Phosphatidylinositols/physiology , Phosphoinositide Phospholipase C/genetics , Phosphoinositide Phospholipase C/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Stress, MechanicalABSTRACT
Introducción. La leishmaniasis es una enfermedad de gran impacto en la salud pública. La Organización Mundial de la Salud considera prioritaria la investigación orientada al desarrollo de medicamentos para su tratamiento. La exploración de la ruta del fosfatidil-inositol es interesante, ya que está implicada en la supervivencia del parásito mediante el control de la osmorregulación, el transporte a través de las membranas y la activación de diversos factores de transcripción. Objetivo. Proponer blancos para el desarrollo de medicamentos contra la leishmaniasis mediante el análisis bioinformático y el modelado matemático de esta ruta. Materiales y métodos. Se caracterizaron las proteínas pertenecientes a la ruta del fosfatidil-inositol en las bases de datos TriTrypDB y Pfam. Posteriormente, se hizo un análisis de similitud con las proteínas humanas mediante las herramientas InParanoid7 y OrthoMCL. Finalmente, se propuso un modelo booleano de la ruta, utilizando los programas PROMOT y CellNetAnalyzer. Resultados. Se reconstruyó y se describió la ruta de señalización del fosfatidil-inositol en Leishmania spp. El análisis de similitud con proteínas humanas determinó la viabilidad de las proteínas pertenecientes a la ruta del fosfatidil-inositol como potenciales blancos moleculares. Los modelos matemáticos permitieron integrar los elementos de la ruta y predecir un efecto inhibidor. Se propusieron los siguientes blancos para el desarrollo de medicamentos: inositol-3-fosfato-5-fosfatasa, fosfatidil-inositol-4-cinasa, fosfatidil-inositol-3,4,5-trisfosfato-3-fosfatasa, e inositol-polifosfato1P-fosfatasa. Conclusiones. La ruta de señalización del fosfatidil-inositol aparece como una alternativa sólida desde el punto de vista del modelo cualitativo y a partir de las proteínas encontradas. Se identificaron posibles blancos de medicamentos contra la leishmaniasis. Posteriormente, se buscarán medicamentos contra las proteínas detectadas y se hará la validación experimental.
Introduction: Leishmaniasis is a disease of high impact on public health. Research on drugs for its treatment is considered a priority by the World Health Organization. The phosphatidyl-inositol signaling pathway is interesting to explore because it is involved in the survival of the parasite, by controlling osmoregulation, transport through membranes, and activation of transcription factors. Objective: To propose drug targets against the disease through bioinformatic analysis and mathematical modeling of this signaling pathway. Materials and methods: The phosphatidyl-inositol pathway proteins were characterized through Pfam and TriTrypDB databases. Subsequently, a similarity analysis with human proteins was performed using the OrthoMCL and InParanoid7 tools. Finally, a boolean model of the pathway was proposed using PROMOT and CellNetAnalyzer softwares. Results: The phosphatidyl-inositol signaling pathway in Leishmania spp. was reconstructed and described. The similarity analysis determined the feasibility of the phosphatidyl-inositol pathway proteins as molecular targets. Mathematical models allowed integrating the elements of the path and predicted an inhibitor effect. The following were proposed as drug targets: inositol-3-phosphate-5-phosphatase, phosphatidylinositol-4-kinase, phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and Inositol-1P-polyphosphate phosphatase. Conclusion: The phosphatidyl-inositol signaling pathway is robust from the point of view of the qualitative model and the proteins found. Thus, potential drug targets against leishmaniasis were identified. Subsequently we will seek to detect drugs against this set of proteins and validate them experimentally .
Subject(s)
Humans , Computational Biology , Leishmania/drug effects , Models, Theoretical , Phosphatidylinositols/antagonists & inhibitors , Signal Transduction/drug effects , Leishmaniasis/drug therapy , Molecular Targeted Therapy , Phosphatidylinositols/physiologyABSTRACT
Cuando un agonista se une a su receptor específico sobre la membrana plaquetaria se inician una serie de cambios morfológicosy metabólicos que llevan al cambio de forma, agregación y secreción de contenidos granulares. La trombina, serinoproteasa multifuncional y fuerte agonosta plaquetario, tiene dos tipos de receptores sobre la membrana plaquetaria: de alta y de moderada afinidad. Este último pertenece a la familia de receptores ß2 adrenérgicos que presentan siete dominios de intramembrana, e inician la activación a través de G proteínas específicas. De esta manera se desencadenan diversos pasos metabólicos a través de varias enzimas claves. La actividad de la fosfolipasa Cß (PLCß) origina dos segundos mensajeros: Inositol 3 fosfato (IP3) que promueve la movilización de calcio del sistema tubular denso al citosol y el diacilglicerol (DG) que activa proteína quinasa C (PKC). Si bien la plaqueta no prolifera se han detectado enzimas relacionadas a oncogenes. De esta manera se han estudiado y comprendido nuevos caminos de activación. La familia de la tirosina quinasas, relacionas a la proliferación celular y oncogenes, fosforilan residuos tirosinas; en su mayoría son quinasas del tipo no receptor que se encuentran en el citosol como ser: Scr, Syk y FAK. La fosfolipasa Cy necesita la presencia de RasGAP, Rap 1b para hidrolizar fosfoinosítidos de membrana. La formación de este complejo trimérico se induce por trombina. La fosfoinositol-3-quinasa fosforila la posición 3 del anillo del inositol generando nuevos compuestos. La regulación completa de estos mecanismos de activación llevan a la respuesta hemostática plaquetaria. Su conocimiento hace posible el desarrollo de moléculas inhibitorias como terapéutica en los procesos trombóticos y tromboembólicos
Subject(s)
Humans , Platelet Activation , Antibodies, Monoclonal/therapeutic use , In Vitro Techniques , Receptors, Thrombin/drug effects , Thrombin/physiology , Thrombosis/physiopathology , Platelet Activation/physiology , Platelet Aggregation , Antibodies/therapeutic use , Blood Platelets/drug effects , Blood Coagulation/physiology , Phosphatidylinositols/metabolism , Phosphatidylinositols/physiology , Phosphorylase Kinase , Receptors, Thrombin/antagonists & inhibitors , Receptors, Thrombin/classification , Second Messenger Systems , Thrombin/chemistry , Thrombosis/drug therapy , Thrombosis/therapyABSTRACT
We have developed an experimental system that utilizes purified Golgi fractions obtained from virus infected infected MDCK cells to reproduce in vitro the process of vesicle generation in the trans Golgi network, an important site for the sorting of proteins addressed to the plasma membrane, secretory vesicles, or lysosomes. Using an integrated biochemical and electron microscopic approach, we have shown that the formation of post Golgi vesicles carrying proteins destined to both plasma membrane domains of epithelial cells requires the activation of an ArF-like GTP-binding protein that serves to promote the assembly of the protein coat necessary to deform the donor membrane and generate a vesicle. The formation of the post Golgi vesicles also requires the participation of a Golgi membrane-associated Protein Kinase C, but not its phosphorylating activity. Other authors have shown that this is also the case for the PKC activation of the enzyme phospholipase D, which generates phosphatidic acid from phosphatidyl choline and may be involved in remodeling of membranes. We have been able to dissect the process of post Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and a subsequent one of vesicle scission. The first stage can occur at 20 degrees C and requires the activation of the Arf protein necessary for coat assembly. The second stage does not require nucleotides or an energy supply, but requires cytosolic proteins, and in particular, an NEM sensitive membrane scission promoting activity that operates only at a higher temperature of incubation. Because various PKC inhibitors blocked vesicle scission without preventing bud formation, we propose that the PKC is required for the activation of a PLD in the TGN, which leads to remodeling of the donor membrane and the severing of connections between the emerging vesicles and the membranes.
Subject(s)
Animals , Dogs , Golgi Apparatus , Intracellular Membranes , Protein Kinase C/physiology , Viral Proteins/physiology , Coated Vesicles/physiology , Biological Transport , Cell Line , Cell-Free System , Coatomer Protein , Phosphatidylinositols/physiology , Guanosine Triphosphate , Kidney , Lysosomes , Phospholipase D , Membrane Proteins/metabolismABSTRACT
While proteins modified at their COOH-terminal end by a glycosylphosphatidylinositol (GPI) membrane anchor have been found as minor components in many eukaryotic cells, they dominate surface constituents of several parasitic protozoa. In this article, GPI-anchored proteins of Trypanosoma brucei are discussed
Subject(s)
Carrier Proteins , Phosphatidylinositols/physiology , Phosphatidylinositols/chemistry , Glycolipids/chemistry , Glycolipids/physiology , Transferrin , Trypanosoma brucei brucei , Variant Surface Glycoproteins, Trypanosoma , Eukaryotic CellsABSTRACT
The glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from trypanosoma brucei exhibits exquisite specificity for the GPI-anchor of the variant specific glycoprotein (VSG). However the evidence that it is involved in VSG metabolism in the living trypanosome is circunstantial; it shows the same life cycle stage regulated expression as the VSG, no feasible alternative substrate has been identified, and it metabolises the VSG efficiently in vitro and in vivo on hypotomic lysis. Against these considerations are the observations that the GPI-PLC is found on the cytoplasmic face of vesicles so it could not gain access to the VSG through normal vesicle fusion and that the accelerated loss of VSG from bloodstream forms on differentiation to procyclic forms occurs through the action of a protease. To try to determine the role of the GPI-PLC, a homozygous mull mutant T. brucei has been constructed. The null mutant was created by replacement of the entire gene at both alleles with selectable antibiotic resistance markers in procyclic form trypanosomes. The GPI-PLC gene is not usually expressed in procyclic forms and so, as would be expected, the null procyclics display no obvious phenotype. The null procyclics have been used to infect tsetse flies and it remains to be seen whether it is possible for them to differentiate to bloodstream forms and, if so, what the antigenic variation phenotype of the null bloodstream forms would be