RESUMEN

A short peptide, FHHF-11, was designed to change stiffness as a function of pH due to changing degree of protonation of histidines. As pH changes in the physiologically relevant range, G' was measured at 0 Pa (pH 6) and 50,000 Pa (pH 8). This peptide-based hydrogel is antimicrobial and cytocompatible with skin cells (fibroblasts). It was demonstrated that the incorporation of unnatural AzAla tryptophan analog residue improves the antimicrobial properties of the hydrogel. The material developed can have a practical application and be a paradigm shift in the approach to wound treatment, and it will improve healing outcomes for millions of patients each year.

Asunto(s)

Hidrogeles , Piel , Humanos , Hidrogeles/farmacología , Hidrogeles/química , Péptidos/farmacología , Antibacterianos/química , Concentración de Iones de Hidrógeno

RESUMEN

Activity-regulated cytoskeleton-associated protein, Arc, is a major regulator of long-term synaptic plasticity and memory formation. Here we reveal a novel interaction partner of Arc, a resident endoplasmic reticulum transmembrane protein, calnexin. We show an interaction between recombinantly-expressed GST-tagged Arc and endogenous calnexin in HEK293, SH-SY5Y neuroblastoma and PC12 cells. The interaction was dependent on the central linker region of the Arc protein that is also required for endocytosis of AMPA-type glutamate receptors. High-resolution proximity-ligation assays (PLAs) demonstrate molecular proximity of endogenous Arc with the cytosolic C-terminus, but not the lumenal N-terminus of calnexin. In hippocampal neuronal cultures treated with brain-derived neurotrophic factor (BDNF), Arc interacted with calnexin in the perinuclear cytoplasm and dendritic shaft. Arc also interacted with C-terminal calnexin in the adult rat dentate gyrus (DG). After induction of long-term potentiation (LTP) in the perforant path projection to the DG of adult anesthetized rats, enhanced interaction between Arc and calnexin was obtained in the dentate granule cell layer (GCL). Although Arc and calnexin are both implicated in the regulation of receptor endocytosis, no modulation of endocytosis was detected in transferrin uptake assays. Previous work showed that Arc interacts with multiple protein partners to regulate synaptic transmission and nuclear signaling. The identification of calnexin as a binding partner further supports the role of Arc as a hub protein and extends the range of Arc function to the endoplasmic reticulum, though the function of the Arc/calnexin interaction remains to be defined.

RESUMEN

This study investigated adult rat behaviour in three early life conditions, and how behaviour was affected after exposure to chronic mild stressors in later life. During postnatal days 2-14, male Wistar rats were exposed daily to either long or brief maternal separation, or were left undisturbed with their mothers (non-handled). As adults, non-handled and long maternally separated offspring demonstrated less object exploration than brief maternally separated offspring. Non-handled offspring also showed lower pre-pulse inhibition compared to both long and brief maternally separated offspring. Sucrose preference and open field behaviour as adults did not differ between the early life conditions. Exposure to four weeks of chronic mild stress in adulthood (mimicking daily hassles in humans) increased object exploration, increased pre-pulse inhibition and induced habituation of acoustic startle in non-handled offspring, similar to brief maternally separated offspring. Long maternally separated offspring exposed to chronic mild stress failed to show an increase in object exploration and enhanced pre-pulse inhibition, and did not show habituation of acoustic startle. In conclusion, different early life conditions have a different long-term impact on behaviour. Offspring from all three conditions differed from each other in terms of adult behaviour. Mild daily stressors in the adulthood counteracted the effects observed in the non-handled condition.

Asunto(s)

Privación Materna , Estrés Psicológico/fisiopatología , Estimulación Acústica , Factores de Edad , Análisis de Varianza , Animales , Animales Recién Nacidos , Conducta Exploratoria/fisiología , Femenino , Preferencias Alimentarias , Masculino , Inhibición Prepulso/fisiología , Ratas , Ratas Wistar , Sacarosa/administración & dosificación

RESUMEN

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes.

Asunto(s)

Proteínas del Citoesqueleto/química , Proteínas del Tejido Nervioso/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Fenómenos Biofísicos , Línea Celular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Variación Genética , Humanos , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Plasticidad Neuronal/fisiología , Presenilina-1/metabolismo , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido

RESUMEN

Sleep has been ascribed a critical role in cognitive functioning. Several lines of evidence implicate sleep in the consolidation of synaptic plasticity and long-term memory. Stress disrupts sleep while impairing synaptic plasticity and cognitive performance. Here, we discuss evidence linking sleep to mechanisms of protein synthesis-dependent synaptic plasticity and synaptic scaling. We then consider how disruption of sleep by acute and chronic stress may impair these mechanisms and degrade sleep function.

RESUMEN

Cholinergic signaling induces Arc/Arg3.1, an immediate early gene crucial for synaptic plasticity. However, the molecular mechanisms that dictate Arc mRNA and protein dynamics during and after cholinergic epochs are little understood. Using human SH-SY5Y neuroblastoma cells, we show that muscarinic cholinergic receptor (mAchR) stimulation triggers Arc synthesis, whereas translation-dependent RNA decay and proteasomal degradation strictly limit the amount and duration of Arc expression. Chronic application of the mAchR agonist, carbachol (Cch), induces Arc transcription via ERK signaling and release of calcium from IP(3)-sensitive stores. Arc translation requires ERK activation, but not changes in intracellular calcium. Proteasomal degradation of Arc (half-life ∼37 min) was enhanced by thapsigargin, an inhibitor of the endoplasmic calcium-ATPase pump. Similar mechanisms of Arc protein regulation were observed in cultured rat hippocampal slices. Functionally, we studied the impact of cholinergic epoch duration and temporal pattern on Arc protein expression. Acute Cch treatment (as short as 2 min) induces transient, moderate Arc expression, whereas continuous treatment of more than 30 min induces maximal expression, followed by rapid decline. Cholinergic activity associated with rapid eye movement sleep may function to facilitate long term synaptic plasticity and memory. Employing a paradigm designed to mimic intermittent rapid eye movement sleep epochs, we show that application of Cch in a series of short bursts generates persistent and maximal Arc protein expression. The results demonstrate dynamic, multifaceted control of Arc synthesis during mAchR signaling, and implicate cholinergic epoch duration and repetition as critical determinants of Arc expression and function in synaptic plasticity and behavior.

Asunto(s)

Proteínas del Citoesqueleto/metabolismo , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero/metabolismo , Sinapsis/metabolismo , Animales , Carbacol/metabolismo , Carbacol/farmacología , Línea Celular , Línea Celular Tumoral , Hipocampo/metabolismo , Humanos , Memoria , Modelos Biológicos , Plasticidad Neuronal , Ratas , Ratas Wistar , Receptores Colinérgicos/metabolismo , Transducción de Señal , Sueño , Sueño REM , Factores de Tiempo

RESUMEN

The immediate early gene Arc is emerging as a versatile, finely tuned system capable of coupling changes in neuronal activity patterns to synaptic plasticity, thereby optimizing information storage in the nervous system. Here, we attempt to overview the Arc system spanning from transcriptional regulation of the Arc gene, to dendritic transport, metabolism, and translation of Arc mRNA, to post-translational modification, localization, and degradation of Arc protein. Within this framework we discuss the function of Arc in regulation of actin cytoskeletal dynamics underlying consolidation of long-term potentiation (LTP) and regulation of AMPA-type glutamate receptor endocytosis underlying long-term depression (LTD) and homeostatic plasticity. Behaviorally, Arc has a key role in consolidation of explicit and implicit forms of memory, with recent work implicating Arc in adaptation to stress as well as maladaptive plasticity connected to drug addiction. Arc holds considerable promise as a "master regulator" of protein synthesis-dependent forms of synaptic plasticity, but the mechanisms that modulate and switch Arc function are only beginning to be elucidated.

Asunto(s)

Proteínas del Citoesqueleto/metabolismo , Memoria/fisiología , Proteínas del Tejido Nervioso/metabolismo , Sinapsis/fisiología , Animales , Humanos , Modelos Neurológicos , Plasticidad Neuronal/fisiología , Neuronas/fisiología

RESUMEN

Progenitor cells in the adult dentate gyrus provide a constant supply of neuronal precursors, yet only a small fraction of these cells survive and develop into mature dentate granule cells (DGCs). A major challenge of current research is thus to understand the stringent selection process that governs the maturation and functional integration of adult-born DGCs. In mature DGCs, high-frequency stimulation (HFS) of the perforant path input elicits robust expression of the immediate early gene Arc/Arg3.1, trafficking of its mRNA to dendrites, and local synthesis of the protein necessary for consolidation of long-term potentiation (LTP). Given the synaptic commitment inherent in LTP consolidation, we considered that HFS-evoked expression of Arc could be used to timemap the functional integration of newborn DGCs. Dividing cells were birthmarked by BrdU-labeling at 1, 7, 14, 21, or 28 days prior to induction of LTP and expression of Arc was examined by confocal microscopy. Contrary to expectation, LTP did not induce Arc expression in newborn cells at any age, suggesting they might be refractory to synaptically-evoked Arc expression for at least one month. Importantly, however, spontaneous expression of Arc was detected in BrdU-labeled cells and strongly associated with the survival and maturation of NeuN-positive DGCs. Moreover, Arc expression at the earliest ages (1 and 7 days), clearly precedes the formation of glutamatergic synapses on new neurons. These results suggest an unexpected early role for Arc in adult-born DGCs, distinct from its functions in LTP, LTD, and homeostatic synaptic plasticity.

Asunto(s)

Supervivencia Celular , Gránulos Citoplasmáticos/metabolismo , Proteínas del Citoesqueleto/genética , Giro Dentado/metabolismo , Genes Inmediatos-Precoces , Proteínas del Tejido Nervioso/genética , Animales , Animales Recién Nacidos , Movimiento Celular , Proliferación Celular , Giro Dentado/citología , Giro Dentado/fisiología , Inmunohistoquímica , Hibridación in Situ , Potenciación a Largo Plazo , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

Long-term recognition memory requires protein synthesis, but little is known about the coordinate regulation of specific genes. Here, we examined expression of the plasticity-associated immediate early genes (Arc, Zif268, and Narp) in the dentate gyrus following long-term object-place recognition learning in rats. RT-PCR analysis from dentate gyrus tissue collected shortly after training did not reveal learning-specific changes in Arc mRNA expression. In situ hybridization and immunohistochemistry were therefore used to assess possible sparse effects on gene expression. Learning about objects increased the density of granule cells expressing Arc, and to a lesser extent Narp, specifically in the dorsal blade of the dentate gyrus, while Zif268 expression was elevated across both blades. Thus, object-place recognition triggers rapid, blade-specific upregulation of plasticity-associated immediate early genes. Furthermore, Western blot analysis of dentate gyrus homogenates demonstrated concomitant upregulation of three postsynaptic density proteins (Arc, PSD-95, and alpha-CaMKII) with key roles in long-term synaptic plasticity and long-term memory.

Asunto(s)

Giro Dentado/metabolismo , Genes Inmediatos-Precoces , Aprendizaje/fisiología , Plasticidad Neuronal/genética , Reconocimiento en Psicología/fisiología , Animales , Proteína C-Reactiva/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Homólogo 4 de la Proteína Discs Large , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Ambiente , Conducta Exploratoria , Regulación de la Expresión Génica , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

New gene expression is necessary for long-term potentiation (LTP) consolidation, yet roles for specific activity-induced mRNAs have not been defined. Here we probed the dynamic function of activity-induced Arc (activity-regulated cytoskeletal-associated protein)/Arg3.1 (activity-regulated gene 3.1 protein homolog) mRNA using brief, local infusions of antisense (AS) oligodeoxynucleotides at multiple time points during dentate gyrus LTP in vivo. Surprisingly, early Arc synthesis is necessary for early expression of LTP, whereas sustained synthesis is required to generate stably modified synapses. AS application 2 h after LTP induction results in a rapid and permanent reversal of LTP. This reversal is associated with rapid knockdown of upregulated Arc, dephosphorylation of actin depolymerization factor/cofilin, and loss of nascent filamentous actin (F-actin) at synaptic sites. Infusion of the F-actin stabilizing drug jasplakinolide during LTP maintenance blocks the ability of AS to reverse LTP. These results couple activity-induced expression of Arc to expansion of the actin cytoskeleton underlying enduring LTP. Furthermore, Arc synthesis is required for both the induction and consolidation of LTP elicited by local BDNF infusion, thus identifying Arc as a key molecular effector of BDNF in synaptic plasticity.

Asunto(s)

Actinas/metabolismo , Proteínas del Citoesqueleto/biosíntesis , Giro Dentado/fisiología , Potenciación a Largo Plazo/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Factores Despolimerizantes de la Actina/fisiología , Animales , Biopolímeros/fisiología , Factor Neurotrófico Derivado del Encéfalo/fisiología , Masculino , Modelos Animales , Plasticidad Neuronal/fisiología , Unión Proteica , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/fisiología

RESUMEN

In Ciona intestinalis, the elimination of extra-embryonic test cells during early stage of development is delayed by a fertilization signal. Test cells undergo a caspase-dependent apoptosis event repressed by thyroxine (T4)-activated NF-kappaB. When apoptosis was experimentally blocked, the hatching stage was delayed. The incubation of unfertilized eggs with a 1-h-fertilized egg extract or purified T4 restored apoptosis in test cells at a similar timing than found in fertilized eggs. Ciona expresses specific genes forming a functional IkappaB/NF-kappaB pathway. One, Ci-p65, was transiently induced upon fertilization via T4 and found to exert its anti-apoptotic role in test cells nuclei as well as in a reconstituted cell system. Blocking NF-kappaB activity by dexamethasone-induced overexpression of Ci-IkappaB abrogated the repression of apoptosis in test cells. Overall, the data are consistent for defining a central coupling role of both T4 and NF-kappaB during early embryo development.

Asunto(s)

Apoptosis , Ciona intestinalis/embriología , Fertilización , FN-kappa B/metabolismo , Tiroxina/metabolismo , Cigoto/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Inhibidores de Caspasas , Caspasas/metabolismo , Extractos Celulares/farmacología , Núcleo Celular/química , Ciona intestinalis/citología , Ciona intestinalis/genética , Dexametasona/farmacología , Desarrollo Embrionario , Expresión Génica , Datos de Secuencia Molecular , FN-kappa B/análisis , FN-kappa B/genética , Óvulo/efectos de los fármacos , Óvulo/metabolismo , Transducción de Señal , Tiroxina/farmacología , Cigoto/química , Cigoto/citología

RESUMEN

This article reports the genetic interaction of two F-box genes, SLEEPY1 (SLY1) and SNEEZY (SNE), in Arabidopsis thaliana gibberellin (GA) signaling. The SLY1 gene encodes an F-box subunit of a Skp1-cullin-F-box (SCF) E3 ubiquitin ligase complex that positively regulates GA signaling. The sly1-2 and sly1-10 mutants have recessive, GA-insensitive phenotypes including delayed germination, dwarfism, reduced fertility, and overaccumulation of the DELLA proteins RGA (Repressor of ga1-3), GAI (GA-Insensitive), and RGL2 (RGA-Like 2). The DELLA domain proteins are putative transcription factors that negatively regulate GA signaling. The requirement for SLY1 in GA-stimulated disappearance of DELLA proteins suggests that GA targets DELLA proteins for destruction via SCF(SLY1)-mediated ubiquitylation. Overexpression of SLY1 in sly1-2 and sly1-10 plants rescues the recessive GA-insensitive phenotype of these mutants. Surprisingly, antisense expression of SLY1 also suppresses these mutants. This result caused us to hypothesize that the SLY1 homologue SNE can functionally replace SLY1 in the absence of the recessive interfering sly1-2 or sly1-10 genes. This hypothesis was supported because overexpression of SNE in sly1-10 rescues the dwarf phenotype. In addition to rescuing the sly1-10 dwarf phenotype, SNE overexpression also restored normal RGA protein levels, suggesting that the SNE F-box protein can replace SLY1 in the GA-induced proteolysis of RGA. If the C-terminal truncation in the sly1-2 and sly1-10 alleles interferes with SNE rescue, we reasoned that overexpression of sly1-2 might interfere with wild-type SLY1 function. Indeed, overexpression of sly1-2 in wild-type Ler (Landsberg erecta) yields dwarf plants.

Asunto(s)

Transferasas Alquil y Aril , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas F-Box/metabolismo , Giberelinas/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas F-Box/genética , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Mutación , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Fenotipo , Plantas Modificadas Genéticamente , ARN Mensajero/metabolismo , Alineación de Secuencia

RESUMEN

Drosophila thoracic mechanosensory bristles originate from cells that are singled out from 'proneural' groups of competent epithelial cells. Neural competence is restricted to individual sensory organ precursors (SOPs) by Delta/Notch-mediated 'lateral inhibition', whereas other cells in the proneural field adopt an epidermal fate. The precursors of the large macrochaetes differentiate separately from individual proneural clusters that comprise about 20-30 cells or as heterochronic pairs from groups of more than 100 cells, whereas the precursors of the small regularly spaced microchaetes emerge from even larger proneural fields. This indicates that lateral inhibition might act over several cell diameters; it was difficult to reconcile with the fact that the inhibitory ligand Delta is membrane-bound until the observation that SOPs frequently extend thin processes offered an attractive hypothesis. Here we show that the extension of these planar filopodia--a common attribute of wing imaginal disc cells--is promoted by Delta and that their experimental suppression reduces Notch signalling in distant cells and increases bristle density in large proneural groups, showing that these membrane specializations mediate long-range lateral inhibition.

Asunto(s)

Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/citología , Neuronas/metabolismo , Seudópodos/metabolismo , Animales , Diferenciación Celular , Proteínas del Citoesqueleto , Proteínas de Drosophila , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Proteínas de la Membrana/genética , Fenotipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores Notch , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo

RESUMEN

Two types of necrosis-inducing lipodepsipeptide toxins, called syringomycin and syringopeptin, are major virulence factors of Pseudomonas syringae pv. syringae strain B301D. A previous study showed that a locus, called syrA, was required for both syringomycin production and plant pathogenicity, and the syrA locus was speculated to encode a regulator of toxin production. In this study, sequence analysis of the 8-kb genomic DNA fragment that complements the syrA phenotype revealed high conservation among a broad spectrum of fluorescent pseudomonads. The putative protein encoded by open reading frame 4 (ORF4) (1,299 bp) in the syrA locus region exhibited 85% identity to ArgA, which is involved in arginine biosynthesis in Pseudomonas aeruginosa. Growth of strain W4S2545, the syrA mutant, required supplementation of N minimal medium with arginine. Similarly, syringomycin production of syrA mutant W4S2545 was restored by the addition of arginine to culture media. Furthermore, the insertion of Tn5 in the genome of the syrA mutant W4S2545 was localized between nucleotides 146 and 147 in ORF4, and syringomycin production was complemented in trans with the wild-type DNA fragment containing intact ORF4. These results demonstrate that the syrA locus is the argA gene of P. syringae pv. syringae and that argA is directly involved in arginine biosynthesis and therefore indirectly affects syringomycin production because of arginine deficiency.

Asunto(s)

Arginina/biosíntesis , Proteínas Bacterianas/biosíntesis , Pseudomonas syringae/enzimología , Elementos Transponibles de ADN , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Mutagénesis Insercional , Pseudomonas syringae/genética , Pseudomonas syringae/crecimiento & desarrollo , Pseudomonas syringae/metabolismo , Análisis de Secuencia de ADN

RESUMEN

The Arabidopsis SLY1 (SLEEPY1) gene positively regulates gibberellin (GA) signaling. Positional cloning of SLY1 revealed that it encodes a putative F-box protein. This result suggests that SLY1 is the F-box subunit of an SCF E3 ubiquitin ligase that regulates GA responses. The DELLA domain protein RGA (repressor of ga1-3) is a repressor of GA response that appears to undergo GA-stimulated protein degradation. RGA is a potential substrate of SLY1, because sly1 mutations cause a significant increase in RGA protein accumulation even after GA treatment. This result suggests SCF(SLY1)-targeted degradation of RGA through the 26S proteasome pathway. Further support for this model is provided by the observation that an rga null allele partially suppresses the sly1-10 mutant phenotype. The predicted SLY1 amino acid sequence is highly conserved among plants, indicating a key role in GA response.

Asunto(s)

Transferasas Alquil y Aril , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Péptido Sintasas/genética , Subunidades de Proteína/genética , Secuencia de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Giberelinas/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Mutación , Péptido Sintasas/metabolismo , Proteínas de Plantas , Subunidades de Proteína/metabolismo , Proteínas Ligasas SKP Cullina F-box , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Syringopeptin is a necrosis-inducing phytotoxin, composed of 22 amino acids attached to a 3-hydroxy fatty acid tail. Syringopeptin, produced by Pseudomonas syringae pv. syringae, functions as a virulence determinant in the plant-pathogen interaction. A 73,800-bp DNA region was sequenced, and analysis identified three large open reading frames, sypA, sypB, and sypC, that are 16.1, 16.3, and 40.6 kb in size. Sequence analysis of the putative SypA, SypB, and SypC sequences determined that they are homologous to peptide synthetases, containing five, five, and twelve amino acid activation modules, respectively. Each module exhibited characteristic domains for condensation, aminoacyl adenylation, and thiolation. Within the aminoacyl adenylation domain is a region responsible for substrate specificity. Phylogenetic analysis of the substrate-binding pockets resulted in clustering of the 22 syringopeptin modules into nine groups. This clustering reflects the substrate amino acids predicted to be recognized by each of the respective modules based on placement of the syringopeptin NRPS (nonribosomal peptide synthetase) system in the linear (type A) group. Finally, SypC contains two C-terminal thioesterase domains predicted to catalyze the release of syringopeptin from the synthetase and peptide cyclization to form the lactone ring. The syringopeptin synthetases, which carry 22 NRPS modules, represent the largest linear NRPS system described for a prokaryote.

Asunto(s)

Proteínas Bacterianas/biosíntesis , Lipoproteínas/biosíntesis , Péptido Sintasas/genética , Péptidos Cíclicos/biosíntesis , Pseudomonas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Clonación Molecular , Escherichia coli/genética , Datos de Secuencia Molecular , Péptidos Cíclicos/genética , Filogenia , Plásmidos , Pseudomonas/enzimología , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato

RESUMEN

Two apoptotic events take place during embryonic development of Ciona intestinalis. The first concerns extra-embryonic cells and precedes hatching. The second controls tail regression at metamorphosis, occurs through a polarized wave originating from tail extremity, and is caspase dependent. This was shown by: (1) in vivo incorporation of a fluorescent marker of caspase activation in different cell types of the tail; (2) detection of an activated form of caspase 3-like protein by western blotting; and (3) failure of 30% of larvae to undergo metamorphosis after treatment of fertilized eggs with a pan-caspase inhibitor. In addition, Ciona embryos express a single ERK protein, specifically phosphorylated at metamorphosis. ERK activation was shown to be located in cells of the tail. Addition of MEK inhibitor in the culture medium prevented ERK activation and metamorphosis. In silico analysis of Ciona genome pointed to 15 caspases with high homology with humans, and a single ERK gene with high homology to both mammalian ERK1 and ERK2. It is concluded that the sequence of events leading to metamorphosis includes ERK phosphorylation followed by caspase-dependent apoptosis and tail regression.

Asunto(s)

Caspasas/metabolismo , Ciona intestinalis/crecimiento & desarrollo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Cola (estructura animal)/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Caspasa 3 , Inhibidores de Caspasas , Ciona intestinalis/embriología , Ciona intestinalis/genética , Inhibidores de Cisteína Proteinasa/farmacología , Embrión no Mamífero , Femenino , Humanos , Larva/efectos de los fármacos , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Datos de Secuencia Molecular , Fosforilación , Homología de Secuencia de Aminoácido , Cola (estructura animal)/embriología , Cola (estructura animal)/patología , Tubulina (Proteína)/metabolismo