RESUMEN
The present study aims at isolation, identification, characterization and prediction of three-dimensional molecular architecture of a proteolytic enzyme from the early blight pathogen, Alternaria solani which are hypothesized to be a marker of phytopathogenicity. Maximum enzyme production by A. solani was observed in Czapex's Dox broth amended with 2% (w/v) casein than other inducer amendments. Results indicate that the enzyme remained highly active in a pH range of 7.0-10.0 and a temperature range of 45-50°C. The enzyme was strongly inhibited by EDTA, whereas phenylmethylsulfonyl fluoride and monovalent cations (Na(+), K(+)) had little effect. Metal ions such as MgSO4, CaCl2, KCl at 10 mM concentration showed a stimulatory effect (>85%) on protease activity. Matrix-assisted laser desorption and ionization time of flight/mass spectrometry analysis of partially purified enzyme revealed the presence of protease belonging to a keratinolytic protein (metalloprotease) of exopeptidase nature. Putative A. solani keratinolytic enzyme (AsK) is made up of 216 amino acid residues with molecular weight (MW) 24.5 kDa, having a molecular formula of C1094H1704N290O342S4. Ramachandran plot analysis of the protein residues falling into the most favored secondary structures was observed at 84.2%. The major protein structural blocks, 2-ß-sheets, and 9-α-helices have a greater tendency to be conserved during the evolutionary process than do mere sequences of amino acids. Besides, AsK, model prediction showed the presence of a Zinc atom at helix regions (Helix 3, 6, 7: His(57), His(130), His(169), and Cys(123)). Thus, it can be concluded that the major proteinases of AsK are divalent cation-requiring metalloproteinases and make them potential targets of protease inhibitors designing.
Asunto(s)
Alternaria/enzimología , Metaloproteasas/química , Metaloproteasas/aislamiento & purificación , Secuencia de Aminoácidos , Cationes Bivalentes/metabolismo , Cationes Bivalentes/farmacología , Ácido Edético/farmacología , Concentración de Iones de Hidrógeno , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Modelos Moleculares , Peso Molecular , Fluoruro de Fenilmetilsulfonilo/farmacología , Filogenia , Estructura Secundaria de Proteína , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Temperatura , Zinc/metabolismo , Zinc/farmacologíaRESUMEN
Autophagy is a process that is necessary during starvation, as it replenishes metabolic precursors by eliminating damaged organelles. Autophagy is mediated by more than 35 autophagy-related (Atg) proteins that participate in the nucleation, elongation, and curving of the autophagosome membrane. In a pursuit to address the role of autophagy during development and immune resistance of the mealworm beetle, Tenebrio molitor, we screened ATG gene sequences from the whole-larva transcriptome database. We identified a homolog of ATG13 gene in T. molitor (designated as TmATG13) that comprises a cDNA of 1176 bp open reading frame (ORF) encoding a protein of 391 amino acids. Analyses of the structure-specific features of TmAtg13 showed an intrinsically disordered middle and C-terminal region that was rich in regulatory phosphorylation sites. The N-terminal Atg13 domain had a HORMA (Hop1, Rev7, and Mad2) fold containing amino acid residues conserved across the Atg13 insect orthologs. A quantitative reverse-transcription-polymerase chain reaction analysis revealed that TmATG13 was expressed ubiquitously during all developmental stages of the insect. TmATG13 mRNA expression was high in the fat body and gut of the larval and adult stages of the insect. The TmATG13 transcripts were expressed at a high level until 6 days of ovarian development, followed by a significant decline. Silencing of ATG13 transcripts in T. molitor larvae showed a reduced survivability of 39 and 38% in response to Escherichia coli and Staphylococcus aureus infection. Furthermore, the role of TmAtg13 in initiating autophagy as a part of the host cell autophagic complex of the host cells against the intracellular pathogen Listeria monocytogenes is currently under study and will be critical to unfold the structure-function relationships.
RESUMEN
Aphid saliva is predicted to contain proteins that modulate plant defenses and facilitate feeding. Armet is a well-characterized bifunctional protein in mammalian systems. Here we report a new role of Armet, namely as an effector protein in the pea aphid, Acyrthosiphon pisum. Pea aphid Armet's physical and chemical properties and its intracellular role are comparable to those reported for mammalian Armets. Uniquely, we detected Armet in aphid watery saliva and in the phloem sap of fava beans fed on by aphids. Armet's transcript level is several times higher in the salivary gland when aphids feed on bean plants than when they feed on an artificial diet. Knockdown of the Armet transcript by RNA interference disturbs aphid feeding behavior on fava beans measured by the electrical penetration graph technique and leads to a shortened life span. Inoculation of pea aphid Armet protein into tobacco leaves induced a transcriptional response that included pathogen-responsive genes. The data suggest that Armet is an effector protein mediating aphid-plant interactions.
Asunto(s)
Áfidos/fisiología , Interacciones Huésped-Patógeno/fisiología , Proteínas de Insectos/metabolismo , Saliva/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Vicia faba/parasitología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Dicroismo Circular , Clonación Molecular , Ingestión de Alimentos/fisiología , Estrés del Retículo Endoplásmico , Evolución Molecular , Técnicas para Inmunoenzimas , Inmunoglobulina G/inmunología , Proteínas de Insectos/genética , Proteínas de Insectos/inmunología , Datos de Secuencia Molecular , ARN Mensajero/genética , Conejos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saliva/química , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vicia faba/metabolismoRESUMEN
Serine proteases are involved in an enormous number of biological processes. The present study aims at characterizing three-dimensional (3D) molecular architecture of serine proteases from early blight pathogen, Alternaria solani that are hypothesized to be markers of phytopathogenicity. A serine protease was purified to homogeneity and MALDI-TOF-MS/MS analysis revealed that protease produced by A. solani belongs to alkaline serine proteases (AsP). AsP is made up of 403 amino acid residues with molecular weight of 42.1 kDa (Isoelectric point - 6.51) and its molecular formula was C1859 H2930 N516 O595 S4 . AsP structure model was built based on its comparative homology with serine protease using the program, MODELER. AsP had 16 ß-sheets and 10 α-helices, with Ser(350) (G347-G357), Asp(158) (D158-H169), and His(193) (H193-G203) in separate turn/coil structures. Biological metal binding region situated near 6th-helix and His(193) residue is responsible for metal binding site. Also, calcium ion (Ca(2+)) is coordinated by the carboxyl groups of Lys(84), Ile(85), Lys(86), Asp(87), Phe(88), Ala(89), Ala(90) (K84-A90) for first Ca(2+) binding site and carbonyl oxygen atom of Lys(244), Gly(245), Arg(246), Thr(247), Lys(248), Lys(249), and Ala(250) (K244-A250), for second Ca(2+) binding site. Moreover, Ramachandran plot analysis of protein residues falling into most favored secondary structures were determined (83.3%). The predicted molecular 3D structural model was further verified using PROCHECK, ERRAT, and VADAR servers to confirm the geometry and stereo-chemical parameters of the molecular structural design. The functional analysis of AsP 3D molecular structure predictions familiar in the current study may provide a new perspective in the understanding and identification of antifungal protease inhibitor designing.
Asunto(s)
Alternaria/enzimología , Serina Proteasas/química , Serina Proteasas/metabolismo , Secuencia de Aminoácidos , Simulación por Computador , Punto Isoeléctrico , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Filogenia , Conformación Proteica , Homología de Secuencia , Serina Proteasas/genética , Serina Proteasas/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The brain is one of the major targets of alcohol actions. Most of the excitatory synaptic transmission in the central nervous system is mediated by N-methyl-D-aspartate (NMDA) receptors. However, one of the most devastating effects of alcohol leads to brain shrinkage, loss of nerve cells at specific regions through a mechanism involving excitotoxicity, oxidative stress. Earlier studies have indicated that chronic exposure to ethanol both in vivo and in vitro, increases NR1 and NR2B gene expression and their polypeptide levels. The effect of alcohol and molecular changes on the regulatory process, which modulates NMDAR functions including factors altering transcription, translation, post-translational modifications, and protein expression, as well as those influencing their interactions with different regulatory proteins (downstream effectors) are incessantly increasing at the cellular level. Further, I discuss the various genetically altered mice approaches that have been used to study NMDA receptor subunits and their functional implication. In a recent countable review, epigenetic dimension (i.e., histone modification-induced chromatin remodeling and DNA methylation, in the process of alcohol related neuroadaptation) is one of the key molecular mechanisms in alcohol mediated NMDAR alteration. Here, I provide a recount on what has already been achieved, current trends and how the future research/studies of the NMDA receptor might lead to even greater engagement with many possible new insights into the neurobiology and treatment of alcoholism.
RESUMEN
The accumulation and utilization of storage proteins are prominent events linked to the metamorphosis of holometabolous insects. The female-specific storage protein 1 (SP1) is the major storage protein found in the hemolymph and fat body of female larvae of the groundnut pest, Amsacta albistriga. Here we show SP1 expression and localization in differentiated fat body tissues using biochemical and immunohistochemistry scrutiny. Comparison of A. albistriga SP1 with that of other species with respect to amino acid composition and N-terminal sequences show that SP1 is a methonine-rich protein and its identity was confirmed by means of immunoblot analysis. Northern blot studies revealed that the SP1 gene demonstrates stage- and tissue-specific expression in the peripheral fat body cells during the mid-larval period of fifth instar of A. albistriga. During the larval pupal transformation, SP1 are sequestered mainly by the perivisceral fat body tissues, until they serve the purpose of supplying amino acids for the production of egg yolk proteins. Further, electron microscopic studies using immunogold tracer techniques confirmed the localization of crystalline SP1 reserves, stored in the perivisceral fat body tissues. Hence, the peripheral fat body is responsible for biosynthesis of storage proteins, whereas the perivisceral fat body is a specialized storage organ.