RESUMEN
The oncogenic transcriptional corepressor Nac1 contains a conserved POZ protein-protein interaction module that mediates homodimerization or heterodimerization with itself or other POZ proteins. The dimerization has been recognized as an attractive target for cancer therapy. Here, we attempted to (i) discover those potential binding partners of Nac1 in the human genome, (ii) derive key peptide segments from the complex interface of Nac1 with its putative partners, and (iii) improve the peptide binding affinity to Nac1 POZ domain. In the procedure, Nac1 POZ dimerization with 136 human POZ domains was modeled, simulated and analyzed at atomic level to elucidate structural basis, energetic property and dynamics behavior. Two hotspot regions, namely α1-helix and α2/α3-hairpin, at the dimerization interface were identified that are responsible for stabilizing the formed POZ-POZ dimer complexes. The α1-helix and α2/α3-hairpin were stripped from the interface to derive their respective isolated SIP peptides, which, however, exhibited a large flexibility and intrinsic disorder in free state, and thus would incur a considerable penalty upon rebinding to Nac1 POZ domain. By carefully examining the natively folded structures of α1-helix and α2/α3-hairpin in protein context and their interaction modes with the domain, we rationally designed a hydrocarbon bridge and a disulfide bond separately for the two peptides in order to constrain their conformational flexibility in free state, thus largely minimizing the flexibility penalty. Consequently, three α1-helix peptides derived from Nac1, Miz1 and Slx4 were stapled by all-hydrocarbon bridge, while four α2/α3-hairpin peptides derived from Nac1, Bacd1, Klh28 and Mynn were cyclized by disulfide bond. Binding affinity analysis revealed that, as designed, these peptides were converted from non- or weak binders to moderate or good binders of Nac1 POZ domain upon the stapling and cyclization.
Asunto(s)
Proteínas de Neoplasias/metabolismo , Péptidos/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Dominio BTB-POZ , Sitios de Unión , Ciclización , Dimerización , Humanos , Simulación de Dinámica Molecular , Proteínas de Neoplasias/química , Péptidos/química , Unión Proteica , Conformación Proteica en Hélice alfa , Proteínas Represoras/química , TermodinámicaRESUMEN
BACKGROUND: The regulation of gonadotropin synthesis and release by gonadotropin-releasing hormone (GnRH) plays an essential role in the neuroendocrine control of reproduction. However, the mechanisms underlying gonadotropin regulation by GnRH pulse frequency and amplitude are still ambiguous. This study aimed to explore the molecular mechanisms and biological pathways associated with gonadotropin synthesis by GnRH pulse frequencies and amplitudes. METHODS: Using GSE63251 datasets downloaded from the Gene Expression Omnibus (GEO), differentially expressed genes (DEGs) were screened by comparing the RNA expression from the GnRH pulse group, the GnRH tonic group and the control group. Pathway enrichment analyses of DEGs was performed, followed by protein-protein interaction (PPI) network construction. Furthermore, sub-network modules were constructed by ClusterONE and GO function and pathways analysed by DAVID. In addition, the relationship between the metabolic pathways and the GnRH pathway was verified in vitro. RESULTS: In total, 531 common DEGs were identified in GnRH groups, including 290 up-regulated and 241 down-regulated genes. DEGs predominantly enriched in 16 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including 11 up-regulated pathways (signallingsignallingmetabolic pathways, signallingand GnRH signalling pathway) and 5 down-regulated pathways (type II diabetes mellitus). Moreover, FBJ osteosarcoma oncogene (FOS) and jun proto-oncogene (JUN) had higher connectivity degrees in the PPI network. Three modules in the PPI were identified with ClusterONE. The genes in module 1 were significantly enriched in five pathways, including signallingthe insulin resistance and GnRH signalling pathway. The genes in modules 2 and 3 were mainly enriched in metabolic pathways and steroid hormone biosynthesis, respectively. Finally, knockdown leptin receptor (LEPR) and insulin receptor (INSR) reversed the GnRH-modulated metabolic related-gene expression. CONCLUSIONS: The present study revealed the involvement of GnRH in the regulation of gonadotropin biosynthesis and metabolism in the maintenance of reproduction, achieved by bioinformatics analyses. This, indicates that the GnRH signalling pathway played a central linkings role in reproductive function and metabolic balance. In addition, the present study identified the difference response between GnRH pulse and GnRH tone, indicated that abnormal GnRH pulse and amplitude may cause disease, which may provide an improved understanding of the GnRH pathway and a new insight for disease diagnosis and treatment.
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Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/farmacología , Gonadotropinas/genética , Ontología de Genes , Redes Reguladoras de Genes/genética , Humanos , Mapas de Interacción de Proteínas/genética , Proto-Oncogenes Mas , Transducción de Señal/genéticaRESUMEN
Recent reports have shown that preconditioning with the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) protects against cerebral ischemia/reperfusion (I/R) injury. However, it is unclear whether poly(I:C) treatment after cerebral I/R injury is also effective. We used mouse/rat middle cerebral artery occlusion and cell oxygen-glucose deprivation models to evaluate the therapeutic effects and mechanisms of poly(I:C) treatment. Poly(I:C) was i.p. injected 3 h after ischemia (treatment group). Cerebral infarct volumes and brain edemas were significantly reduced, and neurologic scores were significantly increased. TNF-α and IL-1ß levels were markedly decreased, whereas IFN-ß levels were greatly increased, in the ischemic brain tissues, cerebral spinal fluid, and serum. Injuries to hippocampal neurons and mitochondria were greatly reduced. The numbers of TUNEL-positive and Fluoro-Jade B(+) cells also decreased significantly in the ischemic brain tissues. Poly(I:C) treatment increased the levels of Hsp27, Hsp70, and Bcl2 and decreased the level of Bax in the ischemic brain tissues. Moreover, poly(I:C) treatment attenuated the levels of TNF-α and IL-1ß in serum and cerebral spinal fluid of mice stimulated by LPS. However, the protective effects of poly(I:C) against cerebral ischemia were abolished in TLR3(-/-) and TLR4(-/-)mice. Poly(I:C) downregulated TLR4 signaling via TLR3. Poly(I:C) treatment exhibited obvious protective effects 14 d after ischemia and was also effective in the rat permanent middle cerebral artery occlusion model. The results suggest that poly(I:C) exerts therapeutic effects against cerebral I/R injury through the downregulation of TLR4 signaling via TLR3. Poly(I:C) is a promising new drug candidate for the treatment of cerebral infarcts.
Asunto(s)
Antivirales/farmacología , Isquemia Encefálica/tratamiento farmacológico , Regulación hacia Abajo/efectos de los fármacos , Poli I-C/farmacología , Daño por Reperfusión/tratamiento farmacológico , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 4/inmunología , Animales , Isquemia Encefálica/genética , Isquemia Encefálica/inmunología , Isquemia Encefálica/patología , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/inmunología , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Masculino , Ratones , Ratones Noqueados , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/genética , Daño por Reperfusión/inmunología , Daño por Reperfusión/patología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Factores de Tiempo , Receptor Toll-Like 3/genética , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The mammalian gonadotropin-inhibitory hormone (GnIH) ortholog, RFamide-related peptide (RFRP), is considered to act on gonadotropin-releasing hormone (GnRH) neurons and the pituitary to inhibit gonadotropin synthesis and release. However, there is little evidence documenting whether RFamide-related peptide 3 (RFRP-3) plays a primary role in inhibition of the hypothalamo-pituitary-gonadal (HPG) axis prior to the onset of puberty. The present study aimed to understand the functional significance of the neuropeptide on pubertal development. The developmental changes in reproductive-related gene expression at the mRNA level were investigated in the hypothalamus of female mice. The results indicated that RFRP-3 may be an endogenous inhibitory factor for the activation of the HPG axis prior to the onset of puberty. In addition, centrally administered RFRP-3 significantly suppressed plasma luteinizing hormone (LH) levels in prepubertal female mice. Surprisingly, centrally administered RFRP-3 had no effects on plasma LH levels in ovariectomized (OVX) prepubescent female mice. In contrast, RFRP-3 also inhibited plasma LH levels in OVX prepubescent female mice that were treated with 17beta-estradiol replacement. Our study also examined the effects of RFRP-3 on plasma LH release in adult female mice that were ovariectomized at dioestrus, with or without estradiol (E2). Our results showed that the inhibitory effects of RFRP-3 were independent of E2 status. Quantitative real-time PCR and immunohistochemistry analyses showed that RFRP-3 inhibited GnRH expression at both the mRNA and protein levels in the hypothalamus. These data demonstrated that RFRP-3 could effectively suppress pituitary LH release, via the inhibition of GnRH transcription and translation in prepubescent female mice, which is associated with estrogen signaling pathway and developmental stages.
Asunto(s)
Estradiol/fisiología , Hormona Luteinizante/antagonistas & inhibidores , Hormona Luteinizante/metabolismo , Neuropéptidos/farmacología , Maduración Sexual/fisiología , Animales , Estradiol/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/biosíntesis , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Inyecciones Intraventriculares , Ratones , Ovariectomía , Embarazo , ARN Mensajero/biosíntesis , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced secondary brain injury. However, the upstream events that initiate inflammatory responses following ICH remain elusive. Our previous studies suggested that Toll-like receptor 4 (TLR4) may be the upstream signal that triggers inflammatory injury in ICH. In addition, recent clinical findings indicated that both TLR2 and TLR4 may participate in ICH-induced brain injury. However, it is unclear how TLR2 functions in ICH-induced inflammatory injury and how TLR2 interacts with TLR4. METHODS: The role of TLR2 and TLR2/TLR4 heterodimerization in ICH-induced inflammatory injury was investigated in both in vivo and in vitro models of ICH. RESULTS: TLR2 mediated ICH-induced inflammatory injury, which forms a heterodimer with TLR4 in both in vivo and in vitro models of ICH. Hemoglobin (Hb), but not other blood components, triggered inflammatory injury in ICH via assembly of TLR2/TLR4 heterodimers. MyD88 (myeloid differentiation primary response gene 88), but not TRIF (Toll/IR-1 domain-containing adaptor protein inducing interferon-beta), was required for ICH-induced TLR2/TLR4 heterodimerization. Mutation of MyD88 Arg196 abolished the TLR2/TLR4 heterodimerization. INTERPRETATION: Our results suggest that a novel TLR2/TLR4 heterodimer induced by Hb initiates inflammatory injury in ICH. Interfering with the assembly of the TLR2/TLR4 heterodimer may be a novel target for developing effective treatment of ICH.
Asunto(s)
Hemorragia Cerebral/complicaciones , Encefalitis/etiología , Encefalitis/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Edema Encefálico/diagnóstico , Edema Encefálico/etiología , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/etiología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , ARN Interferente Pequeño/farmacología , Transducción de Señal/fisiología , Receptor Toll-Like 2/química , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/química , Receptor Toll-Like 4/genéticaRESUMEN
An accurate method was developed for determining ochratoxin A (OTA) in pig kidney using an immunoaffinity column for cleanup and ultra-HPLC/MS/MS for identification and quantification. Mean recoveries of OTA from kidney samples fortified at 0.10-5 µg/kg levels ranged from 74 to 92%, with RSDs of 4.6-7.5%. The LOD was estimated to be 0.03 µg/kg and the LOQ was 0.10 µg/kg based on an S/N in kidney of 3:1 and 10:1, respectively. The developed method was used for the determination of OTA in pig kidney. A survey of the OTA content of pig kidneys slaughtered in Chongqing, China, revealed that 35 out of 40 analyzed samples were contaminated; the OTA concentration in kidney ranged from trace level (0.03-0.1) to 0.323 µg/kg. This method was shown to be useful for accurately evaluating the intake of OTA from pig kidneys.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Riñón/química , Ocratoxinas/análisis , Animales , Cromatografía de Afinidad/métodos , Límite de Detección , Porcinos , Espectrometría de Masas en TándemRESUMEN
Many important protein-protein interactions in eukaryotic signaling networks are mediated by peptide recognition domains (PRDs), which bind short linear sequence motifs in other proteins. However, high ligand cross-reactivity is observed within most PRD families, rendering a broad specificity for the family members. In the present study, we attempt to explore the molecular mechanism and physicochemical origin of PRD cross-reactivity. In the procedure, a structure-based method called atomic cross-nonbonded interaction analysis (ACNIA) is described to extract atomic-level nonbonded interaction information at domain-peptide interface and to correlate the information with peptide affinity based on a set of structure-solved, affinity-known protein-peptide complex samples compiled from numerous literatures and databases. The ACNIA-derived affinity predictor is tested rigorously with statistical validation approach, which is also demonstrated to be capable of perceiving slight structural change in the interface using three distinct panels of SH3-binding peptide data. Subsequently, with help of the affinity predictor we adopt the human c-Src SH3 domain, one of the most sophisticated PRDs, as a paradigm to investigate the ligand cross-reactivity within SH3 family. It is found that most of the family members have only few non-essential residue differences in their peptide-binding pockets, and thus exhibit a similar peptide recognition profile and high cross-reactivity. The cross-reactivity is even shared by different subclasses of SH3 domains. The findings suggest that inherent binding specificity is not the only factor to select appropriate binders for specific SH3 domains, and other aspects such as cellular context and the rest of the SH3-containing proteins may play important roles in reducing their ligand cross-reactivity.
Asunto(s)
Ligandos , Péptidos/química , Proteínas Proto-Oncogénicas pp60(c-src)/química , Algoritmos , Secuencia de Aminoácidos , Aminoácidos/química , Sitios de Unión , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Mutación Puntual , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Electricidad Estática , Dominios Homologos srcRESUMEN
Atopic dermatitis (AD) is a frequent skin disorder that is associated with immune dysfunction and skin inflammation. Histone deacetylase 3 (HDAC3) possesses strong immune and inflammatory modulatory properties in multiple diseases. However, the role and mechanism of HDAC3 in AD remain unknown. Here, we reported that HDAC3 expression was aberrantly upregulated in 2,4-dinitrochlorobenzene (DNCB)-induced lesional AD skin in mice. Inhibition of HDAC3 by RGFP966 protected against DNCB-induced AD, indicated by improved histological damages, relieved inflammatory and immune dysfunction. Nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling pathway activity in lesional AD skin was significantly decreased and RGFP966 attenuated the decrease. Inhibition of Nrf2/HO-1 signaling pathway via Nrf2 inhibitor ML385 blunted anti-AD effect of RGFP966 in DNCB-treated mice. Mechanistically, RGFP966 promoted Nrf2 expression and upregulated H3K27ac deposition on the promoter region of Nrf2. Collectively, HDAC3 inhibition protects against AD via epigenetically activating Nrf2 transcription to upregulate Nrf2/HO-1 signaling pathway activity. HDAC3 may act as a promising therapeutic target for the treatment of AD.
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Dermatitis Atópica , Animales , Ratones , Dermatitis Atópica/inducido químicamente , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/genética , Dinitroclorobenceno , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Citocinas/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Transducción de Señal , Piel/patología , Ratones Endogámicos BALB CRESUMEN
Protein-peptide interaction is fundamentally important for signal transduction, transcription regulation, protein degradation, cell regeneration, and immune response. Here, we report the use of a fast conformational sampling strategy to improve the prediction of protein-peptide binding affinity. This method generates hundreds of alternative conformers for a protein-peptide complex and then performs classical MM-PB/SA analysis over these conformers to derive a consistent binding energy expression for the complex. We show a proof-of-concept study on vascular endothelial growth factor A (VEGF A) interaction with its peptide ligands. The structures of VEGF A complexed with 13 peptides are modeled with a virtual mutagenesis protocol and their binding energies are subsequently calculated by using the conformational sampling-based method. A good linear correlation between the calculated and experimental values is observed, and we demonstrate that the correlation could be further improved by fitting the decomposed energy terms to experimentally measured affinity. Furthermore, the obtained results are discussed in detail in order to elucidate the structural basis and energetic implication underlying VEGF A-peptide recognition and association. We also give a detailed comparison between the proposed method and other widely used approaches, from which it is suggested that our method exhibits a good compromise between the effectiveness and efficiency in evaluating protein-peptide affinity.
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Péptidos/química , Péptidos/metabolismo , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/metabolismo , Secuencia de Aminoácidos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Puntual/genética , Unión Proteica , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
Thirst and water intake are regulated by the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO), located around the anteroventral third ventricle, which plays a critical role in sensing dynamic changes in sodium and water balance in body fluids. Meanwhile, neural circuits involved in thirst regulation and intracellular mechanisms underlying the osmosensitive function of OVLT and SFO are reviewed. Having specific Nax channels in the glial cells and other channels (such as TRPV1 and TRPV4), the OVLT and SFO detect the increased Na+ concentration or hyperosmolality to orchestrate osmotic stimuli to the insular and cingulate cortex to evoke thirst. Meanwhile, the osmotic stimuli are relayed to the supraoptic nucleus (SON) and paraventricular nucleus of the hypothalamus (PVN) via direct neural projections or the median preoptic nucleus (MnPO) to promote the secretion of vasopressin which plays a vital role in the regulation of body fluid homeostasis. Importantly, the vital role of OVLT in sleep-arousal regulation is discussed, where vasopressin is proposed as the mediator in the regulation when OVLT senses osmotic stimuli.
RESUMEN
BACKGROUND: Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced neurological deficits; however, the signaling pathways are not apparent by which the upstream cellular events trigger innate immune and inflammatory responses that contribute to neurological impairments. Toll-like receptor 4 (TLR4) plays a role in inflammatory damage caused by brain disorders. METHODS: In this study, we investigate the role of TLR4 signaling in ICH-induced inflammation. In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR. Activation of microglia was detected by immunohistochemistry, cytokines were measured by ELISA, MyD88, TRIF and NF-κB were measured by Western blot and EMSA, animal behavior was evaluated by animal behavioristics. RESULTS: Compared to WT mice, TLR4(-/-) mice had restrained ICH-induced brain damage showing in reduced cerebral edema and lower neurological deficit scores. Quantification of cytokines including IL-6, TNF-α and IL-1ß and assessment of macrophage infiltration in perihematoma tissues from TLR4(-/-), MyD88(-/-) and TRIF(-/-) mice showed attenuated inflammatory damage after ICH. TLR4(-/-) mice also exhibited reduced MyD88 and TRIF expression which was accompanied by decreased NF-κB activity. This suggests that after ICH both MyD88 and TRIF pathways might be involved in TLR4-mediated inflammatory injury possibly via NF-κB activation. Exogenous hemin administration significantly increased TLR4 expression and microglial activation in cultures and also exacerbated brain injury in WT mice but not in TLR4(-/-) mice. Anti-TLR4 antibody administration suppressed hemin-induced microglial activation in cultures and in the mice model of ICH. CONCLUSIONS: Our findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately increasing cytokine expression and inflammatory injury in ICH. Targeting TLR4 signaling may be a promising therapeutic strategy for ICH.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Hemorragia Cerebral/complicaciones , Hemo/farmacología , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal/fisiología , Receptor Toll-Like 4/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales/uso terapéutico , Células Cultivadas , Corteza Cerebral/citología , Hemorragia Cerebral/tratamiento farmacológico , Citocinas/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Inflamación/etiología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/efectos de los fármacos , Factor 88 de Diferenciación Mieloide/deficiencia , FN-kappa B/metabolismo , Examen Neurológico , ARN Mensajero/metabolismo , Estadísticas no Paramétricas , Factores de Tiempo , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Ny-Ålesund in Svalbard is a complex area with both continental and tidal glaciers. There are a lot of studies on prokaryotic and eukaryotic communities in coastal water and soil, but without studies in glacial-related waters. We make a distinctive and consolidated study on the structure of the prokaryotic and eukaryotic communities of pure glacial meltwater, glacial melting lake, glacial meltwater flowing via different types of soil at various elevations, estuarine glacial water and marine water. Moreover, we analyze the environmental-microbial relationships of the prokaryotic and eukaryotic communities via a canonical correspondence analysis and redundant analysis compared by a Pearson analysis. We found that there were distinct microbes in different environments. Altitude had significant correlations with prokaryotic and eukaryotic species in the 12 water samples (ppro = 0.001, npro = 1010, and peuk = 0.012, npro = 1651) (Pearson analysis). Altitude, temperature and salinity, respectively, accounted for 28.27%, 10.86% and 8.24% in the prokaryotic community structure and 25.77%, 17.72% and 3.46% in the eukaryotic, respectively, in water. Nitrogen, silicate and pH accounted for 38.15%, 6.15% and 2.48% in the prokaryotic community structure in soil and 26.65%, 12.78% and 8.66% in the eukaryotic. Eukaryotes were more stable than prokaryotes in changing environments. Cyanobacteria and dinoflagellates better adapt to a warming environment. Gammaproteobacteria and Chrysophysceae were most abundant in soil. Alphaproteobacteria, Bacteroidia, Mamiellophyceae and Prasinophytae were most abundant in water. Within these microbes, Bacilli and Chlorophyceae were only found in glaciers; Actinobacteria, KD94-96, Thermleophilia, Embryophyta, Trebouxiophyceae and Sordariomycetes were unique to soil.
RESUMEN
Periplasmic oligopeptide-binding protein (OppA) is the initial receptor in the ATP-binding cassette (ABC) system of bacteria, which exhibits a broad specificity in binding oligopeptides without regard to sequence. Here, we present a computational study on the structural properties and energetic landscapes of OppA protein interacting with its cognate ligands on the basis of 28 structure/affinity-known OppA-tripeptide complexes. By employing a well-designed protocol that couples the hybrid quantum mechanical/molecular mechanical (QM/MM) scheme and the sophisticated Poisson-Boltzmann/surface area (PB/SA) solvent model together to analyze and decompose the energy components associated with the OppA-peptide binding, we demonstrate that the broad specificity of OppA-recognizing peptides is originated from a series of exquisite balances between the free energy contributions from, for example, the direct nonbonded interactions and indirect desolvation effects, the main chains and side chains, and the different residue positions of the tripeptide ligands. We also show that, in a framework of structure-based quantitative structure-activity relationship (SB-QSAR) methodology, the QM/MM-PB/SA-derived energy terms could be used as a good descriptor to characterize the interaction profile of OppA with peptides and correlate pretty well with the experimentally measured affinities of the binding.
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Proteínas Bacterianas/química , Proteínas Portadoras/química , Lipoproteínas/química , Péptidos/química , Relación Estructura-Actividad Cuantitativa , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , TermodinámicaRESUMEN
Human transcriptional enhanced associate domain (TEAD) family consists of four paralogous transcription factors that function to modulate gene expression by interacting with YAP-like coactivators and have been recognized as potential therapeutic targets of diverse diseases including lung cancer and gastric tumor. Here, we attempt to explore the systematic interaction profile between the 4 TEAD proteins and the peptides derived from the binding sites of 8 known YAP-like coactivators, in order to analyze the binding affinity and recognition specificity of these peptides toward the TEAD family, and to design hydrocarbon-stapled/cyclized peptides that can target the specific interaction profile for each coactivator. Structural, energetic, and dynamic investigations of TEAD-coactivator interactions reveal that the coactivators adopt three independent secondary structure regions (ß-strand, α-helix, and Ω-loop) to surround on the surface of TEAD proteins, in which the α-helical and Ω-loop regions are primarily responsible for the interactions. Five α-helical peptides and four Ω-loop peptides are derived from the 8 YAP-like coactivators, and their systematic binding profile toward the 4 TEAD proteins is created, and hydrocarbon stapling and cyclization strategies are employed to constrain the free α-helical and Ω-loop peptides into their native conformations, respectively, thus effectively promoting peptide binding to TEADs. The all-hydrocarbon and disulfide bridges are designed to point out the TEAD-peptide complex interface, which would not disrupt the direct intermolecular interaction between the TEAD and peptide. Therefore, the stapling and cyclization only improve peptide binding affinity to these TEADs, but do not alter peptide recognition specificity over different TEADs.
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Proteínas de Ciclo Celular/química , Hidrocarburos/química , Péptidos/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Ciclización , Polarización de Fluorescencia , Humanos , Simulación de Dinámica Molecular , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/metabolismo , Factores de Transcripción/agonistas , Factores de Transcripción/químicaRESUMEN
AIMS: The aim of this study was to investigate the role of TRPA1 in the pathogenesis of AD. MAIN METHODS: The experimental atopic dermatitis (AD)-like skin lesions were established using 2,4-dinitrochlorobenzene (DNCB). Mice were divided into three groups: TRPA1-/- and WT groups were treated with DNCB dissolved in a 3:1 mixture of acetone and olive oil; the negative control group was treated with 3:1 mixture of acetone and olive oil without DNCB. The treatment lasted for 21 days, after which the animals were sacrificed and their blood, ears and dorsal skin tissue samples were collected for analysis. KEY FINDINGS: Lower dermatitis score, ear thickness, pruritus score, and epidermal hyperplasia were observed in mice in TRPA1-/- mice compared to the WT group. Besides, lower dermal mast cell infiltration, proinflammatory cytokines, Th2 cytokines and the infiltration of macrophages were observed in the TRPA1-/- mice compared to the WT group. Furthermore, we demonstrated that TRPA1 antagonist HC-030031 could alleviate AD-like symptoms and reduce the degree of epidermal hyperplasia in mice. SIGNIFICANCE: TRPA1 has a crucial role during the AD pathogenesis in mice, thus may be used as a potential new target for treating patients with chronic skin inflammatory disease.
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Dermatitis Atópica/complicaciones , Inflamación/prevención & control , Macrófagos/inmunología , Mastocitos/inmunología , Prurito/prevención & control , Canal Catiónico TRPA1/fisiología , Acetanilidas/farmacología , Animales , Dermatitis Atópica/inducido químicamente , Dermatitis Atópica/patología , Dinitroclorobenceno/toxicidad , Inflamación/etiología , Inflamación/patología , Macrófagos/efectos de los fármacos , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Prurito/etiología , Prurito/patología , Purinas/farmacología , Canal Catiónico TRPA1/antagonistas & inhibidoresRESUMEN
Sulfur atoms have been known to participate in hydrogen bonds (H-bonds) and these sulfur-containing H-bonds (SCHBs) are suggested to play important roles in certain biological processes. This study aims to comprehensively characterize all the SCHBs in 500 high-resolution protein structures (< or =1.8 A). We categorized SCHBs into six types according to donor/acceptor behaviors and used explicit hydrogen approach to distinguish SCHBs from those of nonhydrogen bonding interactions. It is revealed that sulfur atom is a very poor H-bond acceptor, but a moderately good H-bond donor. In alpha-helix, considerable SCHBs were found between the sulphydryl group of cysteine residue i and the carbonyl oxygen of residue i-4, and these SCHBs exert effects in stabilizing helices. Although for other SCHBs, they possess no specific secondary structural preference, their geometric characteristics in proteins and in free small compounds are significantly distinct, indicating the protein SCHBs are geometrically distorted. Interestingly, sulfur atom in the disulfide bond tends to form bifurcated H-bond whereas in cysteine-cysteine pairs prefer to form dual H-bond. These special H-bonds remarkably boost the interaction between H-bond donor and acceptor. By oxidation/reduction manner, the mutual transformation between the dual H-bonds and disulfide bonds for cysteine-cysteine pairs can accurately adjust the structural stability and biological function of proteins in different environments. Furthermore, few loose H-bonds were observed to form between the sulphydryl groups and aromatic rings, and in these cases the donor H is almost over against the rim rather than the center of the aromatic ring.
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Proteínas/química , Azufre/química , Cristalografía por Rayos X , Cisteína/química , Bases de Datos de Proteínas , Enlace de Hidrógeno , Metionina/química , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
Two kinds of structural characterization method as local descriptors and global descriptors were used to parameterize peptide structures, and several quantitative structure-retention relationship models were then constructed using partial least square (PLS), least-squares support vector machine (LS-SVM) and Gaussian process (GP) coupled with genetic algorithm-variable selection. These models were validated rigorously and investigated systematically by Tropsha et al. criteria, Monte Carlo cross-validation and one-way analysis of variance. Results show that regression models constructed using nonlinear approaches such as LS-SVM and GP are more robust and predictable than those by linear PLS method. By including linear and nonlinear terms in the covariance function, the GP is capable of handling both linear and nonlinear-mixed relationship, and thus presents a better performance than LS-SVM. Investigation of the optimal GP model revealed that diversified properties contribute to the retention behavior of peptides in immobilized metal-affinity chromatography. Particularly, coordination interaction, electrostatic factor, sovlation effect and hydrogen bonding are correlated significantly with the peptide retention ability.
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Cromatografía de Afinidad , Histidina/química , Metales/química , Modelos Químicos , Péptidos/química , Algoritmos , Simulación por Computador , Análisis de los Mínimos Cuadrados , Modelos Moleculares , Relación Estructura-Actividad Cuantitativa , Análisis de Regresión , Reproducibilidad de los Resultados , Programas Informáticos , Solventes/químicaRESUMEN
OBJECTIVE: To investigate the effect of C5a on the expression of thrombomodulin (TM) in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs cultured in vitro were stimulated with C5a for 8, 12, 16, 20 hours in a concentration of 200 microg/L, and also with different concentrations of 100, 200, 300 microg/L for 12 hours respectively. The levels of both mRNA and protein expression of TM was detected by real-time polymerase chain reaction (PCR) and Western blotting respectively, and the dose and time dependent effects of C5a on the expression of TM were evaluated. RESULTS: C5a down-regulated the TM expression at both protein and mRNA level. The down-regulation was time-dependent [(93.11+/- 1.57) x10(-2) , (71.05+/-3.39)x10(-2), (65.48+/-4.28)x10(-2), (62.69+/-4.03)x10(-2)at protein level and (301.71+/-80.40)x10(-6), (38.29+/-20.24)x10(-6), (8.82+/-2.66)x10(-6), (7.05+/-0.80)x10(-6) at mRNA level, all P<0.05] and dose-dependent [(113.25+/-3.97)x10(-2), (80.18+/-2.56)x10(-2), (73.22+/- 4.36)x10(-2) at protein level and (401.77+/-20.46)x10(-6), (31.12+/-3.51)x10(-6), (18.19+/-1.46)x10(-6) at mRNA level , all P<0.05]. When concentration of C5a at 300 microg/L was used to stimulate HUVECs for longer than 12 hours, the lowering of TM at protein level was slowed down obviously. And concentration of C5a at 200 microg/L was used to stimulate HUVECs for 12 hours, the lowering of TM at mRNA level was slowed down obviously. CONCLUSION: C5a can depress the gene expression of TM, and then affect the protein's translation. By this means, C5a can lead to hypercoagulability and inflammatory injuries in sepsis.
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Complemento C5a/farmacología , Células Endoteliales/metabolismo , Trombomodulina/metabolismo , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Humanos , ARN Mensajero/genética , Trombomodulina/genética , Venas Umbilicales/citologíaRESUMEN
The atypical orphan receptors DAX1 and SHP constitute the NR0B subgroup of human nuclear receptor (hNR) family; they play key roles in metabolism, reproduction, nutrition and steroidogenesis, and are involved in the pathogenesis of a variety of diseases such as cancer and adrenal hypoplasia. The two receptors lack the classical DNA-binding domain and act as the corepressors of other hNRs. The DAX1 and SHP contains three and two conserved LXXLL motifs, respectively, which can be recognized and bound by the activation function-2 (AF-2) domain of hNR proteins in agonist conformation. Here, we attempt to explore the systematic interaction profile between the five DAX1/SHP LXXLL motifs and all the 48 hNR AF-2 domains found in the human genome, to analyze the binding affinity and specificity of these motifs towards the complete domain array, and to design LXXLL-based, hydrocarbon-stapled peptides that can target the specific interaction profile for each motif. A weighted source-target network from motifs to domains is created based on the modeled domain-motif complex structures and calculated binding potencies, from which the specific interaction profile of each motif against the whole hNR array is depicted and clustered to measure the binding similarity and relationship among these motifs. Dynamics simulations reveal that the LXXLL-based peptides are highly flexible in free unbound state, thus unfavorable to be recognized and bound by AF-2 domains. Hydrocarbon-stapling technique is employed to help the constraint of these unstructured peptides to active helical conformation, thus largely improving their binding affinity to the hNR array. The hydrocarbon bridge is designed to point out of the domain's active pocket, which would not disrupt the direct interaction between the domain and peptide. Energetic decomposition imparts that the stapling has only a very modest influence on the interaction enthalpy and desolvation effect of domain-peptide binding, but can substantially reduce entropy penalty upon the binding. For a peptide ligand, the entropic reduction can be roughly regarded as a constant, which only improves (absolute) peptide binding affinity towards the whole domain array, but does not alter (relative) peptide binding specificity over different domains in the array. Overall, the stapled peptides can be considered as potent competitors to selectively target the specific interaction networks mediated by their parent LXXLL motifs in DAX1 and SHP proteins.
Asunto(s)
Secuencias de Aminoácidos , Receptor Nuclear Huérfano DAX-1/metabolismo , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Análisis por Conglomerados , Receptor Nuclear Huérfano DAX-1/química , Receptor Nuclear Huérfano DAX-1/genética , Diseño de Fármacos , Estudio de Asociación del Genoma Completo , Humanos , Modelos Moleculares , Péptidos/química , Péptidos/farmacología , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad Cuantitativa , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
BACKGROUND: A promising arsenal of histone deacetylase (HDAC)-targeted treatment has emerged in the past decade, as the abnormal targeting or retention of HDACs to DNA regulatory regions often occurs in many cancers. Head and neck squamous cell carcinoma (HNSCC) is one of the most aggressive malignancies worldwide associated with poor overall survival in late-stage patients. HDAC inhibitors have great potential to treat this devastating disease; however, few has been studied regarding the beneficial role of HDAC inhibition in anti-HNSCC therapy and the underlying molecular mechanisms remain elusive. METHODS: Cell migration and invasion were examined by wound closure and Transwell assays. Protein levels and interactions were assessed by Western blotting and immunoprecipitation. HDAC activity was measured with the fluorometric HDAC Activity Assay. Phospho-receptor tyrosine kinase (RTK) profiling was determined by the Proteome Profiler Human Phospho-RTK Array. RESULTS: ADP-ribosylation factor 1 (Arf1), a small GTPase coordinating vesicle-mediated intracellular trafficking, can be inactivated by HDAC inhibitors through histone acetylation-independent degradation of epidermal growth factor receptor (EGFR) in HNSCC cells. Mechanistically, high levels of Arf1 activity are maintained by binding to phosphorylated EGFR which is localized on HNSCC cell plasma membrane. Decreased EGFR phosphorylation is associated with reduced EGFR protein levels in the presence of TSA, which inactivates Arf1 and eventually inhibits invasion in HNSCC cells. CONCLUSIONS: Our insights explore the critical role of EGFR-Arf1 complex in driving HNSCC progression, and demonstrate the selective action of HDAC inhibitors on this specific axis for suppressing HNSCC invasion. This novel finding represents the first example of modulating the EGFR-Arf1 complex in HNSCC by small molecule agents.