RESUMEN
STUDY DESIGN: Explanatory and mechanistic study. OBJECTIVES: A better understanding of the 'whole-body' response following spinal cord injury (SCI) is needed to guide future research aimed at developing novel therapeutic interventions and identifying prognostic indicators for SCI. This study aimed to characterise the blood proteome following contusion or complete SCI compared to a sham injury in rat models. SETTING: United Kingdom. METHODS: Pooled blood samples from one and seven days after a contusion (serum; n = 5) or from 14 days and 112 days post-complete transection SCI (plasma; n = 8) and their sham-injured counterparts were subjected to independent iTRAQ nanoflow liquid chromatography tandem mass-spectrometry proteomic analyses. Pathway analyses of the proteins that were differentially abundant between SCI and their matched sham injured counterparts were completed to indicate biological pathways that may be changed in response to SCI. RESULTS: Eleven and 42 proteins were differentially abundant (≥±2.0 FC; p ≤ 0.05) between the contusion SCI and sham injured animals at 24 h and seven days post-injury, respectively. Seven and tweleve proteins were differentially abundant between complete and sham injured rats at 14 and 112 days post-injury, respectively. Acute-phase response signalling and Liver X Receptor/Retinoic X Receptor activation were identified as differentially regulated pathways in both models of SCI. CONCLUSIONS: We have utilised longitudinal preclinical SCI models to provide an insight into the blood proteome changes that result following SCI and to highlight a number of biological pathways of interest for future studies.
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Contusiones , Proteoma , Traumatismos de la Médula Espinal , Animales , Contusiones/sangre , Proteómica/métodos , Ratas , Médula Espinal , Traumatismos de la Médula Espinal/sangreRESUMEN
The evolution of human diets led to preferences toward polyunsaturated fatty acid (PUFA) content with 'Western' diets enriched in ω-6 PUFAs. Mounting evidence points to ω-6 PUFA excess limiting metabolic and cognitive processes that define longevity in humans. When chosen during pregnancy, ω-6 PUFA-enriched 'Western' diets can reprogram maternal bodily metabolism with maternal nutrient supply precipitating the body-wide imprinting of molecular and cellular adaptations at the level of long-range intercellular signaling networks in the unborn fetus. Even though unfavorable neurological outcomes are amongst the most common complications of intrauterine ω-6 PUFA excess, cellular underpinnings of life-long modifications to brain architecture remain unknown. Here, we show that nutritional ω-6 PUFA-derived endocannabinoids desensitize CB1 cannabinoid receptors, thus inducing epigenetic repression of transcriptional regulatory networks controlling neuronal differentiation. We found that cortical neurons lose their positional identity and axonal selectivity when mouse fetuses are exposed to excess ω-6 PUFAs in utero. Conversion of ω-6 PUFAs into endocannabinoids disrupted the temporal precision of signaling at neuronal CB1 cannabinoid receptors, chiefly deregulating Stat3-dependent transcriptional cascades otherwise required to execute neuronal differentiation programs. Global proteomics identified the immunoglobulin family of cell adhesion molecules (IgCAMs) as direct substrates, with DNA methylation and chromatin accessibility profiling uncovering epigenetic reprogramming at >1400 sites in neurons after prolonged cannabinoid exposure. We found anxiety and depression-like behavioral traits to manifest in adult offspring, which is consistent with genetic models of reduced IgCAM expression, to suggest causality for cortical wiring defects. Overall, our data uncover a regulatory mechanism whose disruption by maternal food choices could limit an offspring's brain function for life.
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Encéfalo/efectos de los fármacos , Dieta Occidental/efectos adversos , Epigénesis Genética/efectos de los fármacos , Animales , Ansiedad , Encéfalo/metabolismo , Metilación de ADN/efectos de los fármacos , Depresión , Dieta , Suplementos Dietéticos , Endocannabinoides/metabolismo , Epigénesis Genética/genética , Epigenómica/métodos , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-6/metabolismo , Ácidos Grasos Insaturados/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Embarazo , Receptor Cannabinoide CB1/efectos de los fármacosRESUMEN
Spinal muscular atrophy (SMA) is an inherited neurodegenerative condition caused by a reduction in the amount of functional survival motor neuron (SMN) protein. SMN has been implicated in transport of mRNA in neural cells for local translation. We previously identified microtubule-dependent mobile vesicles rich in SMN and SNRPB, a member of the Sm family of small nuclear ribonucleoprotein (snRNP)-associated proteins, in neural cells. By comparing the interactomes of SNRPB and SNRPN, a neural-specific Sm protein, we now show that the essential neural protein neurochondrin (NCDN) interacts with Sm proteins and SMN in the context of mobile vesicles in neurites. NCDN has roles in protein localisation in neural cells and in maintenance of cell polarity. NCDN is required for the correct localisation of SMN, suggesting they may both be required for formation and transport of trafficking vesicles. NCDN may have potential as a therapeutic target for SMA together with, or in place of the targeting of SMN expression.This article has an associated First Person interview with the first author of the paper.
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Atrofia Muscular Espinal/patología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Complejo SMN/metabolismo , Células Cultivadas , HumanosRESUMEN
Cyanobactin heterocyclases share the same catalytic domain (YcaO) as heterocyclases/cyclodehydratases from other ribosomal peptide (RiPPs) biosynthetic pathways. These enzymes process multiple residues (Cys/Thr/Ser) within the same substrate. The processing of cysteine residues proceeds with a known order. We show the order of reaction for threonines is different and depends in part on a leader peptide within the substrate. In contrast to other YcaO domains, which have been reported to exclusively break down ATP into ADP and inorganic phosphate, cyanobactin heterocyclases have been observed to produce AMP and inorganic pyrophosphate during catalysis. We dissect the nucleotide profiles associated with heterocyclization and propose a unifying mechanism, where the γ-phosphate of ATP is transferred in a kinase mechanism to the substrate to yield a phosphorylated intermediate common to all YcaO domains. In cyanobactin heterocyclases, this phosphorylated intermediate, in a proportion of turnovers, reacts with ADP to yield AMP and pyrophosphate.
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Adenilil Ciclasas/metabolismo , Proteínas Bacterianas/metabolismo , Péptidos Cíclicos/metabolismo , Prochloron/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Ciclización , Cisteína/química , Cisteína/metabolismo , Difosfatos/metabolismo , Modelos Químicos , Estructura Molecular , Péptidos Cíclicos/química , Prochloron/fisiología , Treonina/química , Treonina/metabolismo , Urocordados/microbiologíaRESUMEN
A hierarchical hormonal cascade along the hypothalamic-pituitary-adrenal axis orchestrates bodily responses to stress. Although corticotropin-releasing hormone (CRH), produced by parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and released into the portal circulation at the median eminence, is known to prime downstream hormone release, the molecular mechanism regulating phasic CRH release remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single-cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH-releasing neuron population reliant on secretagogin's Ca(2+) sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. Pharmacological tools combined with RNA interference demonstrate that secretagogin's loss of function occludes adrenocorticotropic hormone release from the pituitary and lowers peripheral corticosterone levels in response to acute stress. Cumulatively, these data define a novel secretagogin neuronal locus and molecular axis underpinning stress responsiveness.
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Corticosterona/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Neuronas/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Secretagoginas/metabolismo , Estrés Fisiológico/fisiología , Animales , Corticosterona/genética , Hormona Liberadora de Corticotropina/genética , Masculino , Ratones , Neuronas/citología , Núcleo Hipotalámico Paraventricular/citología , Hipófisis/citología , Hipófisis/metabolismo , Interferencia de ARN , Secretagoginas/genética , Transcriptoma/fisiologíaRESUMEN
The M genome segment of Bunyamwera virus (BUNV)-the prototype of both the Bunyaviridae family and the Orthobunyavirus genus-encodes the glycoprotein precursor (GPC) that is proteolytically cleaved to yield two viral structural glycoproteins, Gn and Gc, and a nonstructural protein, NSm. The cleavage mechanism of orthobunyavirus GPCs and the host proteases involved have not been clarified. In this study, we investigated the processing of BUNV GPC and found that both NSm and Gc proteins were cleaved at their own internal signal peptides (SPs), in which NSm domain I functions as SP(NSm) and NSm domain V as SP(Gc) Moreover, the domain I was further processed by a host intramembrane-cleaving protease, signal peptide peptidase, and is required for cell fusion activities. Meanwhile, the NSm domain V (SP(Gc)) remains integral to NSm, rendering the NSm topology as a two-membrane-spanning integral membrane protein. We defined the cleavage sites and boundaries between the processed proteins as follows: Gn, from residue 17-312 or nearby residues; NSm, 332-477; and Gc, 478-1433. Our data clarified the mechanism of the precursor cleavage process, which is important for our understanding of viral glycoprotein biogenesis in the genus Orthobunyavirus and thus presents a useful target for intervention strategies.
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Ácido Aspártico Endopeptidasas/metabolismo , Virus Bunyamwera/metabolismo , Glicoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Precursores de Proteínas/metabolismo , Serina Endopeptidasas/metabolismo , Células A549 , Animales , Sitios de Unión/genética , Virus Bunyamwera/genética , Virus Bunyamwera/fisiología , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Glicoproteínas/genética , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Precursores de Proteínas/genética , Proteolisis , Células Vero , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Extracellular vesicles (EVs) are released by most cell types and have been associated with multiple immunomodulatory functions. MHC class I molecules have crucial roles in antigen presentation and in eliciting immune responses and are known to be incorporated into EVs. However, the MHC class I immunopeptidome of EVs has not been established. Here, using a small-scale immunoisolation of the antigen serotypes HLA-A*02:01 and HLA-B*27:05 expressed on the Epstein-Barr virus-transformed B cell line Jesthom and MS of the eluted peptides from both cells and EVs, we identified 516 peptides that bind either HLA-A*02:01 or HLA-B*27:05. Of importance, the predicted serotype-binding affinities and peptide-anchor motifs did not significantly differ between the peptide pools isolated from cells or EVs, indicating that during EV biogenesis, no obvious editing of the MHC class I immunopeptidome occurs. These results, for the first time, establish EVs as a source of MHC class I peptides that can be used for the study of the immunopeptidome and in the discovery of potential neoantigens for immunotherapies.
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Antígenos/química , Linfocitos B/química , Antígeno HLA-A2/química , Antígeno HLA-B27/química , Péptidos/química , Antígenos/inmunología , Linfocitos B/inmunología , Línea Celular Transformada , Antígeno HLA-A2/inmunología , Antígeno HLA-B27/inmunología , Humanos , Péptidos/inmunologíaRESUMEN
Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly Δ(9)-tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor-driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB1 cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC-sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin-2, a microtubule-binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis-exposed fetuses, defining SCG10 as the first cannabis-driven molecular effector in the developing cerebrum. CB1 cannabinoid receptor activation recruits c-Jun N-terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring.
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Corteza Cerebral/efectos de los fármacos , Dronabinol/farmacología , Hipocampo/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Psicotrópicos/farmacología , Receptor Cannabinoide CB1/efectos de los fármacos , Animales , Axones/efectos de los fármacos , Proteínas de Unión al Calcio , Diferenciación Celular , Corteza Cerebral/citología , Corteza Cerebral/embriología , Femenino , Feto/anomalías , Feto/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hipocampo/citología , Hipocampo/embriología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Exposición Materna/efectos adversos , Ratones , Ratones Endogámicos C57BL , Fosforilación , Embarazo , Proteómica , ARN Mensajero/genética , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Estatmina , Factores de TiempoRESUMEN
Age-related macular degeneration (AMD) is associated with the formation of sub-retinal pigment epithelial (RPE) deposits that block circulatory exchange with the retina. The factors that contribute to deposit formation are not well understood. Recently, we identified the presence of spherular hydroxyapatite (HAP) structures within sub-RPE deposits to which several AMD-associated proteins were bound. This suggested that protein binding to HAP represents a potential mechanism for the retention of proteins in the sub-RPE space. Here we performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on plasma samples from 23 patients with late-stage neovascular AMD following HAP-binding. Individuals were genotyped for the high risk CFH variant (T1277C) and binding to HAP was compared between wild type and risk variants. From a library of 242 HAP binding plasma proteins (1% false discovery rate), SWATH-MS revealed significant quantitative differences in the abundance of 32 HAP-binding proteins (pâ¯<â¯0.05) between the two homozygous groups. The concentrations of six proteins (FHR1, FHR3, APOC4, C4A, C4B and PZP) in the HAP eluted fractions and whole plasma were further analysed using ELISA and their presence in sections from human cadaver eyes was examined using immunofluorescence. All six proteins were found to be present in the RPE/choroid interface, and four of these (FHR1, FHR3, APOC4 and PZP) were associated with spherules in sub-RPE space. This study provides qualitative and quantitative information relating to the degree by which plasma proteins may contribute to sub-RPE deposit formation through binding to HAP spherules and how genetic differences might contribute to deposit formation.
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Proteínas Sanguíneas/metabolismo , Durapatita/metabolismo , Degeneración Macular Húmeda/sangre , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Proteínas Sanguíneas/genética , Factor H de Complemento/genética , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Técnicas de Genotipaje , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Unión Proteica , Proteómica , Degeneración Macular Húmeda/genéticaRESUMEN
NS1 proteins of influenza A and B viruses share limited sequence homology, yet both are potent manipulators of host cell processes, particularly interferon (IFN) induction. Although many cellular partners are reported for A/NS1, only a few (e.g. PKR and ISG15) have been identified for B/NS1. Here, affinity-purification and mass spectrometry were used to expand the known host interactome of B/NS1. We identified 22 human proteins as new putative targets for B/NS1, validating several, including DHX9, ILF3, YBX1 and HNRNPC. Consistent with two RNA-binding domains in B/NS1, many of the identified factors bind RNA and some interact with B/NS1 in an RNA-dependent manner. Functional characterization of several B/NS1 interactors identified SNRNP200 as a potential positive regulator of host IFN responses, while ILF3 exhibited dual roles in both IFN induction and influenza B virus replication. These data provide a resource for future investigations into the mechanisms underpinning host cell modulation by influenza B virus NS1.
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Virus de la Influenza B/aislamiento & purificación , Gripe Humana/metabolismo , Proteínas no Estructurales Virales/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Humanos , Virus de la Influenza B/genética , Virus de la Influenza B/metabolismo , Gripe Humana/genética , Gripe Humana/virología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismo , Unión Proteica , Proteínas no Estructurales Virales/genética , Proteína 1 de Unión a la Caja Y/genética , Proteína 1 de Unión a la Caja Y/metabolismoRESUMEN
Geobacter sulfurreducens is a dissimilatory metal-reducing bacterium capable of forming thick electron-conducting biofilms on solid electrodes. Here, we employ for the first time comparative proteomics to identify key physiological changes involved in G. sulfurreducens adaptation from fumarate-respiring planktonic cells to electron-conducting biofilms. Increased levels of proteins involved in outer membrane biogenesis, cell motility and secretion are expressed in biofilms. Of particular importance to the electron-conducting biofilms are proteins associated with secretion systems of Type I, II, V and Type IV pili. Furthermore, enzymes involved in lipopolysaccharide and peptidoglycan biosynthesis show increased levels of expression in electron-conducting biofilms compared to planktonic cells. These observations point to similarities in long-range electron transfer mechanisms between G. sulfurreducens and Shewanella oneidensis, while highlighting the wider significance of secretion systems beyond that of Type IV pili identified to date in the adaptation of G. sulfurreducens to electrode respiration.
RESUMEN
Histidine-rich glycoprotein (HRG) is a plasma protein consisting of 6 distinct functional domains and is an important regulator of key cardiovascular processes, including angiogenesis and coagulation. The protein is composed of 2 N-terminal domains (N1 and N2), 2 proline-rich regions (PRR1 and PRR2) that flank a histidine-rich region (HRR), and a C-terminal domain. To date, structural information of HRG has largely come from sequence analysis and spectroscopic studies. It is thought that an HRG fragment containing the HRR, released via plasmin-mediated cleavage, acts as a negative regulator of angiogenesis in vivo. However, its release also requires cleavage of a disulphide bond suggesting that its activity is mediated by a redox process. Here, we present a 1.93 Å resolution crystal structure of the N2 domain of serum-purified rabbit HRG. The structure confirms that the N2 domain, which along with the N1 domain, forms an important molecular interaction site on HRG, possesses a cystatin-like fold composed of a 5-stranded antiparallel ß-sheet wrapped around a 5-turn α-helix. A native N-linked glycosylation site was identified at Asn184. Moreover, the structure reveals the presence of an S-glutathionyl adduct at Cys185, which has implications for the redox-mediated release of the antiangiogenic cleavage product from HRG.
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Neovascularización Fisiológica , Proteínas/química , Proteínas/fisiología , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Disulfuros/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/genética , Conejos , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis. The catalytic longevity of the engineered biofilm is striking, and we had postulated that this was likely to largely result from protection conferred to recombinant enzymes by biofilm's extracellular matrix. SILAC (stable isotopic labelled amino acids in cell cultures), and in particular dynamic SILAC, in which pulses of different isotopically labelled amino acids are administered to cells over a time course, has been used to follow the fate of proteins. To explore within our spin coated biofilm, whether the recombinant enzyme's longevity might be in part due to its regeneration, we introduced pulses of isotopically labelled lysine and phenylalanine into medium overlaying the biofilm and followed their incorporation over the course of biofilm development. RESULTS: Through SILAC analysis, we reveal that constant and complete regeneration of recombinant enzymes occurs within spin coated biofilms. The striking catalytic longevity within the biofilm results from more than just simple protection of active enzyme by the biofilm and its associated extracellular matrix. The replenishment of recombinant enzyme is likely to contribute significantly to the catalytic longevity observed for the engineered biofilm system. CONCLUSIONS: Here we provide the first evidence of a recombinant enzyme's regeneration in an engineered biofilm. The recombinant enzyme was constantly replenished over time as evidenced by dynamic SILAC, which suggests that the engineered E. coli biofilms are highly metabolically active, having a not inconsiderable energetic demand. The constant renewal of recombinant enzyme highlights the attractive possibility of utilising this biofilm system as a dynamic platform into which enzymes of interest can be introduced in a "plug-and-play" fashion and potentially be controlled through promoter switching for production of a series of desired fine chemicals.
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Biopelículas , Enzimas/metabolismo , Ingeniería Genética/métodos , Biocatálisis , Catálisis , Cromatografía Liquida , Enzimas/biosíntesis , Enzimas/genética , Espectrometría de Masas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Understanding the intra- and extracellular proteins involved in the development of the corticospinal tract (CST) may offer insights into how the pathway could be regenerated following traumatic spinal cord injury. Currently, however, little is known about the proteome of the developing corticospinal system. The present study, therefore, has used quantitative proteomics and bioinformatics to detail the protein profile of the rat CST during its formation in the spinal cord. This analysis identified increased expression of 65 proteins during the early ingrowth of corticospinal axons into the spinal cord, and 36 proteins at the period of heightened CST growth. A majority of these proteins were involved in cellular assembly and organization, with annotations being most highly associated with cytoskeletal organization, microtubule dynamics, neurite outgrowth, and the formation, polymerization and quantity of microtubules. In addition, 22 proteins were more highly expressed within the developing CST in comparison to other developing white matter tracts of the spinal cord of age-matched animals. Of these differentially expressed proteins, only one, stathmin 1 (a protein known to be involved in microtubule dynamics), was both highly enriched in the developing CST and relatively sparse in other developing descending and ascending spinal tracts. Immunohistochemical analyses of the developing rat spinal cord and fetal human brain stem confirmed the enriched pattern of stathmin expression along the developing CST, and in vitro growth assays of rat corticospinal neurons showed a reduced length of neurite processes in response to pharmacological perturbation of stathmin activity. Combined, these findings suggest that stathmin activity may modulate axonal growth during development of the corticospinal projection, and reinforces the notion that microtubule dynamics could play an important role in the generation and regeneration of the CST.
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Axones/metabolismo , Regeneración Nerviosa/fisiología , Neuritas/metabolismo , Neuronas/citología , Tractos Piramidales/metabolismo , Estatmina/metabolismo , Animales , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/metabolismoRESUMEN
An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I+III2+IV (S1) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.
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Complejo I de Transporte de Electrón/química , Secuencia de Aminoácidos , Animales , Bovinos , Cisteína/química , Complejo I de Transporte de Electrón/metabolismo , Electroforesis en Gel de Poliacrilamida , Fluorescencia , Lisina/química , Espectrometría de Masas , Datos de Secuencia Molecular , NAD/química , Oxidación-ReducciónRESUMEN
ssDNA-binding proteins (SSBs) based on the oligonucleotide-binding fold are considered ubiquitous in nature and play a central role in many DNA transactions including replication, recombination, and repair. We demonstrate that the Thermoproteales, a clade of hyperthermophilic Crenarchaea, lack a canonical SSB. Instead, they encode a distinct ssDNA-binding protein that we term "ThermoDBP," exemplified by the protein Ttx1576 from Thermoproteus tenax. ThermoDBP binds specifically to ssDNA with low sequence specificity. The crystal structure of Ttx1576 reveals a unique fold and a mechanism for ssDNA binding, consisting of an extended cleft lined with hydrophobic phenylalanine residues and flanked by basic amino acids. Two ssDNA-binding domains are linked by a coiled-coil leucine zipper. ThermoDBP appears to have displaced the canonical SSB during the diversification of the Thermoproteales, a highly unusual example of the loss of a "ubiquitous" protein during evolution.
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Proteínas de Unión al ADN/metabolismo , Thermoproteales/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Unión Proteica , Pliegue de ProteínaRESUMEN
Brevibacterium linens (B. linens) DSM 20158 with an unsequenced genome can be used as a non-pathogenic model to study features it has in common with other unsequenced pathogens of the same genus on the basis of comparative proteome analysis. The most efficient way to kill a pathogen is to target its energy transduction mechanism. In the present study, we have identified the redox protein complexes involved in the electron transport chain of B. linens DSM 20158 from their clear homology with the shot-gun genome sequenced strain BL2 of B. linens by using the SDS-Polyacrylamide gel electrophoresis coupled with nano LC-MS/MS mass spectrometry. B. linens is found to have a branched electron transport chain (Respiratory chain), in which electrons can enter the respiratory chain either at NADH (Complex I) or at Complex II level or at the cytochrome level. Moreover, we are able to isolate, purify, and characterize the membrane bound Complex II (succinate dehydrogenase), Complex III (menaquinone cytochrome c reductase cytochrome c subunit, Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) of B. linens strain DSM 20158.
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Proteínas Bacterianas/química , Brevibacterium/química , Proteínas del Complejo de Cadena de Transporte de Electrón/química , Complejos de ATP Sintetasa/química , Complejos de ATP Sintetasa/aislamiento & purificación , Adenosina Difosfato/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Brevibacterium/genética , Citocromos c/química , Citocromos c/aislamiento & purificación , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/aislamiento & purificación , Transferencia de Energía , Genoma Bacteriano , Cinética , Oxidación-Reducción , Fosfatos/química , Vitamina K 2/química , Vitamina K 2/aislamiento & purificaciónRESUMEN
Deficient energy metabolism and network hyperactivity are the early symptoms of Alzheimer's disease (AD). In this study, we show that administration of exogenous oxidative energy substrates (OES) corrects neuronal energy supply deficiency that reduces the amyloid-beta-induced abnormal neuronal activity in vitro and the epileptic phenotype in AD model in vivo. In vitro, acute application of protofibrillar amyloid-ß1-42 (Aß1-42) induced aberrant network activity in wild-type hippocampal slices that was underlain by depolarization of both the neuronal resting membrane potential and GABA-mediated current reversal potential. Aß1-42 also impaired synaptic function and long-term potentiation. These changes were paralleled by clear indications of impaired energy metabolism, as indicated by abnormal NAD(P)H signaling induced by network activity. However, when glucose was supplemented with OES pyruvate and 3-beta-hydroxybutyrate, Aß1-42 failed to induce detrimental changes in any of the above parameters. We administered the same OES as chronic supplementation to a standard diet to APPswe/PS1dE9 transgenic mice displaying AD-related epilepsy phenotype. In the ex-vivo slices, we found neuronal subpopulations with significantly depolarized resting and GABA-mediated current reversal potentials, mirroring abnormalities we observed under acute Aß1-42 application. Ex-vivo cortex of transgenic mice fed with standard diet displayed signs of impaired energy metabolism, such as abnormal NAD(P)H signaling and strongly reduced tolerance to hypoglycemia. Transgenic mice also possessed brain glycogen levels twofold lower than those of wild-type mice. However, none of the above neuronal and metabolic dysfunctions were observed in transgenic mice fed with the OES-enriched diet. In vivo, dietary OES supplementation abated neuronal hyperexcitability, as the frequency of both epileptiform discharges and spikes was strongly decreased in the APPswe/PS1dE9 mice placed on the diet. Altogether, our results suggest that early AD-related neuronal malfunctions underlying hyperexcitability and energy metabolism deficiency can be prevented by dietary supplementation with native energy substrates.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Precursor de Proteína beta-Amiloide/genética , Neuronas/fisiología , Presenilina-1/genética , Ácido 3-Hidroxibutírico/administración & dosificación , Ácido 3-Hidroxibutírico/farmacología , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Líquido Cefalorraquídeo , Dieta , Metabolismo Energético , Femenino , Hipocampo/metabolismo , Hipocampo/fisiopatología , Técnicas In Vitro , Masculino , Ratones , Ratones Transgénicos , NADP/metabolismo , Ácido Pirúvico/administración & dosificación , Ácido Pirúvico/farmacología , Transducción de Señal , Sinapsis/fisiologíaRESUMEN
Ringing the changes: Selenazolines have applications in medicinal chemistry, but their synthesis is challenging. We report a new convenient and less toxic route to these heterocycles that starts from commercially available selenocysteine. The new route depends on a heterocyclase enzyme that creates oxazolines and thiazolines from serines/threonines and cysteines.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Selenio/química , Secuencia de Aminoácidos , Cisteína/química , Cisteína/metabolismo , Yodoacetamida/química , Oxazoles/química , Oxazoles/metabolismo , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Selenio/metabolismo , Selenocisteína/química , Selenocisteína/metabolismo , Serina/química , Serina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tiazoles/química , Tiazoles/metabolismo , Treonina/química , Treonina/metabolismoRESUMEN
Low-temperature anaerobic digestion (LTAD) technology is underpinned by a diverse microbial community. The methanogenic archaea represent a key functional group in these consortia, undertaking CO2 reduction as well as acetate and methylated C1 metabolism with subsequent biogas (40 to 60% CH4 and 30 to 50% CO2) formation. However, the cold adaptation strategies, which allow methanogens to function efficiently in LTAD, remain unclear. Here, a pure-culture proteomic approach was employed to study the functional characteristics of Methanosarcina barkeri (optimum growth temperature, 37°C), which has been detected in LTAD bioreactors. Two experimental approaches were undertaken. The first approach aimed to characterize a low-temperature shock response (LTSR) of M. barkeri DSMZ 800(T) grown at 37°C with a temperature drop to 15°C, while the second experimental approach aimed to examine the low-temperature adaptation strategies (LTAS) of the same strain when it was grown at 15°C. The latter experiment employed cell viability and growth measurements (optical density at 600 nm [OD600]), which directly compared M. barkeri cells grown at 15°C with those grown at 37°C. During the LTSR experiment, a total of 127 proteins were detected in 37°C and 15°C samples, with 20 proteins differentially expressed with respect to temperature, while in the LTAS experiment 39% of proteins identified were differentially expressed between phases of growth. Functional categories included methanogenesis, cellular information processing, and chaperones. By applying a polyphasic approach (proteomics and growth studies), insights into the low-temperature adaptation capacity of this mesophilically characterized methanogen were obtained which suggest that the metabolically diverse Methanosarcinaceae could be functionally relevant for LTAD systems.