RESUMO
Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and bacteraemia and is capable of remarkable phenotypic plasticity, responding rapidly to environmental change. Pneumococcus is a nasopharyngeal commensal, but is responsible for severe, acute infections following dissemination within-host. Pneumococcus is adept at utilising host resources, but the airways are compartmentalised and those resources are not evenly distributed. Challenges and opportunities in metabolite acquisition within different airway niches may contribute to the commensal-pathogen switch when pneumococcus moves from nasopharynx into lungs. We used NMR to characterise the metabolic landscape of the mouse airways, in health and during infection. Using paired nasopharynx and lung samples from naïve animals, we identified fundamental differences in metabolite bioavailability between airway niches. Pneumococcal pneumonia was associated with rapid and dramatic shifts in the lung metabolic environment, whilst nasopharyngeal carriage led to only modest change in upper airway metabolite profiles. NMR spectra derived from the nasopharynx of mice infected with closely-related pneumococcal strains that differ in their colonisation potential could be distinguished from one another using multivariate dimensionality reduction methods. The resulting models highlighted that increased branched-chain amino acid (BCAA) bioavailability in nasopharynx is a feature of infection with the high colonisation potential strain. Subsequent analysis revealed increased expression of BCAA transport genes and increased intracellular concentrations of BCAA in that same strain. Movement from upper to lower airway environments is associated with shifting challenges in metabolic resource allocation for pneumococci. Efficient biosynthesis, liberation or acquisition of BCAA is a feature of adaptation to nasopharyngeal colonisation.
Assuntos
Nariz , Infecções Pneumocócicas , Animais , Camundongos , Metabolômica , Streptococcus pneumoniae , Aminoácidos de Cadeia RamificadaRESUMO
NEW FINDINGS: What is the central question of this study? Whole-body substrate utilisation is altered during exercise in hot environments, characterised by increased glycolytic metabolism: does heat stress alter the serum metabolome in response to high intensity exercise? What are the main finding and its importance? Alongside increases in glycolytic metabolite abundance, circulating amino acid concentrations are reduced following exercise under heat stress. Prior research has overlooked the impact of heat stress on protein metabolism during exercise, raising important practical implications for protein intake recommendations in the heat. ABSTRACT: Using untargeted metabolomics, we aimed to characterise the systemic impact of environmental heat stress during exercise. Twenty-three trained male triathletes ( V Ì O 2 peak ${\dot V_{{{\rm{O}}_2}{\rm{peak}}}}$ = 64.8 ± 9.2 ml kg min-1 ) completed a 30-min exercise test in hot (35°C) and temperate (21°C) conditions. Venous blood samples were collected immediately pre- and post-exercise, and the serum fraction was assessed via untargeted 1 H-NMR metabolomics. Data were analysed via uni- and multivariate analyses to identify differences between conditions. Mean power output was higher in temperate (231 ± 36 W) versus hot (223 ± 31 W) conditions (P < 0.001). Mean heart rate (temperate, 162 ± 10 beats min-1 , hot, 167 ± 9 beats min-1 , P < 0.001), peak core temperature (Trec ), core temperature change (ΔTrec ) (P < 0.001) and peak rating of perceived exertion (P = 0.005) were higher in hot versus temperate conditions. Change in metabolite abundance following exercise revealed distinct clustering following multivariate analysis. Six metabolites increased (2-hydroxyvaleric acid, acetate, alanine, glucarate, glucose, lactate) in hot relative to temperate (P < 0.05) conditions. Leucine and lysine decreased in both conditions but to a greater extent in temperate conditions (P < 0.05). Citrate (P = 0.04) was greater in temperate conditions whilst creatinine decreased in hot conditions only (P > 0.05). Environmental heat stress increased glycolytic metabolite abundance and led to distinct alterations in the circulating amino acid availability, including increased alanine, glutamine, leucine and isoleucine. The data highlight the need for additional exercise nutrition and metabolism research, specifically focusing on protein requirements for exercise under heat stress.
Assuntos
Aminoácidos , Resposta ao Choque Térmico , Masculino , Humanos , Leucina , Exercício Físico/fisiologia , Alanina , Temperatura AltaRESUMO
Sole hemorrhage and sole ulcers, referred to as sole lesions, are important causes of lameness in dairy cattle. We aimed to compare the serum metabolome of dairy cows that developed sole lesions in early lactation with that of cows that remained unaffected. We prospectively enrolled a cohort of 1,169 Holstein dairy cows from a single dairy herd and assessed animals at 4 time points: before calving, immediately after calving, early lactation, and late lactation. Sole lesions were recorded by veterinary surgeons at each time point, and serum samples were collected at the first 3 time points. Cases were defined by the presence of sole lesions in early lactation and further subdivided by whether sole lesions had been previously recorded; unaffected controls were randomly selected to match cases. Serum samples from a case-control subset of 228 animals were analyzed with proton nuclear magnetic resonance spectroscopy. Spectral signals, corresponding to 34 provisionally annotated metabolites and 51 unlabeled metabolites, were analyzed in subsets relating to time point, parity cohort, and sole lesion outcome. We used 3 analytic methods (partial least squares discriminant analysis, least absolute shrinkage and selection operator regression, and random forest) to determine the predictive capacity of the serum metabolome and identify informative metabolites. We applied bootstrapped selection stability, triangulation, and permutation to support the inference of variable selection. The average balanced accuracy of class prediction ranged from 50 to 62% depending on the subset. Across all 17 subsets, 20 variables had a high probability of being informative; those with the strongest evidence of being associated with sole lesions corresponded to phenylalanine and 4 unlabeled metabolites. We conclude that the serum metabolome, as characterized by proton nuclear magnetic resonance spectroscopy, does not appear able to predict sole lesion presence or future development of lesions. A small number of metabolites may be associated with sole lesions although, given the poor prediction accuracies, these metabolites are likely to explain only a small proportion of the differences between affected and unaffected animals. Future metabolomic studies may reveal underlying metabolic mechanisms of sole lesion etiopathogenesis in dairy cows; however, the experimental design and analysis need to effectively control for interanimal and extraneous sources of spectral variation.
Assuntos
Doenças dos Bovinos , Doenças do Pé , Casco e Garras , Animais , Bovinos , Feminino , Gravidez , Doenças dos Bovinos/etiologia , Doenças do Pé/veterinária , Lactação , Coxeadura Animal/etiologia , Espectroscopia de Ressonância Magnética , Metabolômica , Prótons , Estudos de Casos e ControlesRESUMO
CBL is rapidly phosphorylated upon insulin receptor activation. Mice whole body CBL depletion improved insulin sensitivity and glucose clearance; however, the precise mechanisms remain unknown. We depleted either CBL or its associated protein SORBS1/CAP independently in myocytes and assessed mitochondrial function and metabolism compared to control cells. CBL- and CAP-depleted cells showed increased mitochondrial mass with greater proton leak. Mitochondrial respiratory complex I activity and assembly into respirasomes were reduced. Proteome profiling revealed alterations in proteins involved in glycolysis and fatty acid degradation. Our findings demonstrate CBL/CAP pathway couples insulin signaling to efficient mitochondrial respiratory function and metabolism in muscle.
Assuntos
Resistência à Insulina , Proteínas Proto-Oncogênicas c-cbl , Animais , Camundongos , Metabolismo Energético , Insulina/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias Musculares/metabolismo , Células Musculares/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Respiração CelularRESUMO
A multi-center prospective cross-sectional and genome-wide association study (GWAS) recruited pregnant women taking low dose aspirin. Objectives were to (i) develop pregnancy-specific 95% reference intervals for a range of laboratory based platelet function tests (PFTs); (ii) select an optimal and acceptable PFT that reflected aspirin's COX-1 inhibition in women with confirmed aspirin adherence in pregnancy; and (iii) identify genomic variants that may influence pregnant women's platelet response to aspirin.The study included two independent cohorts of pregnant women. A range of PFTs and matched phenotyping with urinary 11-dehydrothromboxane B2 (11DTXB2) and nuclear magnetic resonance (NMR) spectroscopy detection of urinary salicyluric acid as a measure of aspirin adherence were performed. Genome-wide data was acquired from the UK Biobank Axiom® (Thermo Fisher Scientific). 11DTXB2 in combination with adherence testing with NMR salicyluric acid was an accurate and acceptable testing strategy for detecting biochemical aspirin responsiveness in pregnant women, with the provision of relevant reference ranges. GWAS meta-analysis found no significant single nucleotide polymorphisms in association with response to aspirin in pregnancy. Further evaluation in relation to effective dosing of aspirin in pregnancy and optimizing the benefits to specific subgroups should now be a priority for future research.
Assuntos
Aspirina , Inibidores da Agregação Plaquetária , Aspirina/farmacologia , Aspirina/uso terapêutico , Estudos Transversais , Feminino , Estudo de Associação Genômica Ampla , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Gravidez , Estudos Prospectivos , Tromboxano B2 , Reino UnidoRESUMO
The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular events during embryogenesis. Induced pluripotent stem cells (IPSCs) are an established model for embryogenesis, germ layer specification and cell lineage differentiation, advancing the study of human embryonic development and the translation of innovations in drug discovery, disease modelling and cell-based therapies. The metabolic regulation of IPSC pluripotency is mediated by balancing glycolysis and oxidative phosphorylation, but there is a paucity of data regarding the influence of individual metabolite changes during cell lineage differentiation. We used 1H NMR metabolite fingerprinting and footprinting to monitor metabolite levels as IPSCs are directed in a three-stage protocol through primitive streak/mesendoderm, mesoderm and chondrogenic populations. Metabolite changes were associated with central metabolism, with aerobic glycolysis predominant in IPSC, elevated oxidative phosphorylation during differentiation and fatty acid oxidation and ketone body use in chondrogenic cells. Metabolites were also implicated in the epigenetic regulation of pluripotency, cell signalling and biosynthetic pathways. Our results show that 1H NMR metabolomics is an effective tool for monitoring metabolite changes during the differentiation of pluripotent cells with implications on optimising media and environmental parameters for the study of embryogenesis and translational applications.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Condrogênese , Epigênese Genética , Humanos , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
Osteoarthritis is an age-related degenerative musculoskeletal disease characterized by loss of articular cartilage, synovitis, and subchondral bone sclerosis. Osteoarthritis pathogenesis is yet to be fully elucidated with no osteoarthritis-specific biomarkers in clinical use. Ex vivo equine cartilage explants (n = 5) were incubated in tumor necrosis factor-α (TNF-α)/interleukin-1ß (IL-1ß)-supplemented culture media for 8 days, with the media removed and replaced at 2, 5, and 8 days. Acetonitrile metabolite extractions of 8 day cartilage explants and media samples at all time points underwent one-dimensional (1D) 1H nuclear magnetic resonance metabolomic analysis, with media samples also undergoing mass spectrometry proteomic analysis. Within the cartilage, glucose and lysine were elevated following TNF-α/IL-1ß treatment, while adenosine, alanine, betaine, creatine, myo-inositol, and uridine decreased. Within the culture media, 4, 4, and 6 differentially abundant metabolites and 154, 138, and 72 differentially abundant proteins were identified at 1-2, 3-5, and 6-8 days, respectively, including reduced alanine and increased isoleucine, enolase 1, vimentin, and lamin A/C following treatment. Nine potential novel osteoarthritis neopeptides were elevated in the treated media. Implicated pathways were dominated by those involved in cellular movement. Our innovative study has provided insightful information on early osteoarthritis pathogenesis, enabling potential translation for clinical markers and possible new therapeutic targets.
Assuntos
Cartilagem Articular , Osteoartrite , Animais , Cavalos , Interleucina-1beta , Metabolômica , Proteômica , Fator de Necrose Tumoral alfaRESUMO
Synovial fluid (SF) is of great interest for the investigation of orthopedic pathologies, as it is in close proximity to various tissues that are primarily altered during these disease processes and can be collected using minimally invasive protocols. Multi-"omic" approaches are commonplace, although little consideration is often given for multiple analysis techniques at sample collection. Nuclear magnetic resonance (NMR) metabolomics and liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics are two complementary techniques particularly suited to the study of SF. However, currently there are no agreed upon standard protocols that are published for SF collection and processing for use with NMR metabolomic analysis. Furthermore, the large protein concentration dynamic range present within SF can mask the detection of lower abundance proteins in proteomics. While combinational ligand libraries (ProteoMiner columns) have been developed to reduce this dynamic range, their reproducibility when used in conjunction with SF, or on-bead protein digestion protocols, has yet to be investigated. Here we employ optimized protocols for the collection, processing, and storage of SF for NMR metabolite analysis and LC-MS/MS proteome analysis, including a Lys-C endopeptidase digestion step prior to tryptic digestion, which increased the number of protein identifications and improved reproducibility for on-bead ProteoMiner digestion.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Espectroscopia de Ressonância Magnética , Metabolômica , Reprodutibilidade dos Testes , Líquido SinovialRESUMO
Osteoarthritis (OA), osteochondrosis (OC), and synovial sepsis in horses cause loss of function and pain. Reliable biomarkers are required to achieve accurate and rapid diagnosis, with synovial fluid (SF) holding a unique source of biochemical information. Nuclear magnetic resonance (NMR) spectroscopy allows global metabolite analysis of a small volume of SF, with minimal sample preprocessing using a noninvasive and nondestructive method. Equine SF metabolic profiles from both nonseptic joints (OA and OC) and septic joints were analyzed using 1D 1H NMR spectroscopy. Univariate and multivariate statistical analyses were used to identify differential metabolite abundance between groups. Metabolites were annotated via 1H NMR using 1D NMR identification software Chenomx, with identities confirmed using 1D 1H and 2D 1H 13C NMR. Multivariate analysis identified separation between septic and nonseptic groups. Acetate, alanine, citrate, creatine phosphate, creatinine, glucose, glutamate, glutamine, glycine, phenylalanine, pyruvate, and valine were higher in the nonseptic group, while glycylproline was higher in sepsis. Multivariate separation was primarily driven by glucose; however, partial-least-squares discriminant analysis plots with glucose excluded demonstrated the remaining metabolites were still able to discriminate the groups. This study demonstrates that a panel of synovial metabolites can distinguish between septic and nonseptic equine SF, with glucose the principal discriminator.
Assuntos
Artropatias/diagnóstico , Metabolômica/métodos , Sepse/diagnóstico , Líquido Sinovial/metabolismo , Animais , Glucose/análise , Cavalos , Artropatias/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Osteoartrite/diagnóstico , Osteoartrite/metabolismo , Osteocondrose/diagnóstico , Osteocondrose/metabolismo , Sepse/metabolismoRESUMO
Despite osteoarthritis (OA) and rheumatoid arthritis (RA) being typically age-related, their underlying etiologies are markedly different. We used 1H nuclear magnetic resonance (NMR) spectroscopy to identify differences in metabolite profiles in low volumes of OA and RA synovial fluid (SF). SF was aspirated from knee joints of 10 OA and 14 RA patients. 100 µL SF was analyzed using a 700 MHz Avance IIIHD Bruker NMR spectrometer with a TCI cryoprobe. Spectra were analyzed by Chenomx, Bruker TopSpin and AMIX software. Statistical analysis was undertaken using Metaboanalyst. 50 metabolites were annotated, including amino acids, saccharides, nucleotides and soluble lipids. Discriminant analysis identified group separation between OA and RA cohorts, with 32 metabolites significantly different between OA and RA SF (false discovery rate (FDR) < 0.05). Metabolites of glycolysis and the tricarboxylic acid cycle were lower in RA compared to OA; these results concur with higher levels of inflammation, synovial proliferation and hypoxia found in RA compared to OA. Elevated taurine in OA may indicate increased subchondral bone sclerosis. We demonstrate that quantifiable differences in metabolite abundance can be measured in low volumes of SF by 1H NMR spectroscopy, which may be clinically useful to aid diagnosis and improve understanding of disease pathogenesis.
Assuntos
Artrite Reumatoide/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Metaboloma , Metabolômica/métodos , Osteoartrite/metabolismo , Líquido Sinovial/química , Idoso , Aminoácidos/química , Aminoácidos/classificação , Aminoácidos/isolamento & purificação , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Ciclo do Ácido Cítrico/imunologia , Estudos de Coortes , Feminino , Glicólise/imunologia , Humanos , Articulação do Joelho/imunologia , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Lipídeos/química , Lipídeos/classificação , Lipídeos/isolamento & purificação , Masculino , Metabolômica/instrumentação , Pessoa de Meia-Idade , Nucleotídeos/química , Nucleotídeos/classificação , Nucleotídeos/isolamento & purificação , Oligossacarídeos/química , Oligossacarídeos/classificação , Oligossacarídeos/isolamento & purificação , Osteoartrite/imunologia , Osteoartrite/patologia , Líquido Sinovial/metabolismoRESUMO
The antiepileptic drug ethosuximide has recently been shown to be neuroprotective in various Caenorhabditis elegans and rodent neurodegeneration models. It is therefore a promising repurposing candidate for the treatment of multiple neurodegenerative diseases. However, high concentrations of the drug are required for its protective effects in animal models, which may impact on its translational potential and impede the identification of its molecular mechanism of action. Therefore, we set out to develop more potent neuroprotective lead compounds based on ethosuximide as a starting scaffold. Chemoinformatic approaches were used to identify compounds with structural similarity to ethosuximide and to prioritise these based on good predicated blood-brain barrier permeability and C. elegans bioaccumulation properties. Selected compounds were initially screened for anti-convulsant activity in a C. elegans pentylenetetrazol-induced seizure assay, as a rapid primary readout of bioactivity; and then assessed for neuroprotective properties in a C. elegans TDP-43 proteinopathy model based on pan-neuronal expression of human A315T mutant TDP-43. The most potent compound screened, α-methyl-α-phenylsuccinimide (MPS), ameliorated the locomotion defects and extended the shortened lifespan of TDP-43 mutant worms. MPS also directly protected against neurodegeneration by reducing the number of neuronal breaks and cell body losses in GFP-labelled GABAergic motor neurons. Importantly, optimal neuroprotection was exhibited by external application of 50⯵M MPS, compared to 8â¯mM for ethosuximide. This greater potency of MPS was not due to bioaccumulation to higher internal levels within the worm, based on 1H-nuclear magnetic resonance analysis. Like ethosuximide, the activity of MPS was abolished by mutation of the evolutionarily conserved FOXO transcription factor, daf-16, suggesting that both compounds act via the same neuroprotective pathway(s). In conclusion, we have revealed a novel neuroprotective activity of MPS that is >100-fold more potent than ethosuximide. This increased potency will facilitate future biochemical studies to identify the direct molecular target(s) of both compounds, as we have shown here that they share a common downstream DAF-16-dependent mechanism of action. Furthermore, MPS is the active metabolite of another approved antiepileptic drug, methsuximide. Therefore, methsuximide may have repurposing potential for treatment of TDP-43 proteinopathies and possibly other human neurodegenerative diseases.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Modelos Animais de Doenças , Succinimidas/uso terapêutico , Proteinopatias TDP-43/tratamento farmacológico , Proteinopatias TDP-43/genética , Animais , Animais Geneticamente Modificados , Anticonvulsivantes/química , Anticonvulsivantes/uso terapêutico , Caenorhabditis elegans , Feminino , Masculino , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Succinimidas/química , Proteinopatias TDP-43/patologiaRESUMO
BACKGROUND: The onset of puberty is influenced by the interplay of stimulating and restraining factors, many of which have a genetic origin. Premature activation of the GnRH secretion in central precocious puberty (CPP) may arise either from gain-of-function mutations of the KISS1 and KISS1R genes or from loss-of-function manner mutations of the MKRN3 gene leading to MKRN3 deficiency. OBJECTIVE: To explore the genetic causes responsible for CPP and the potential role of the RING finger protein 3 (MKRN3) gene. DESIGN AND PATIENTS: We investigated potential sequence variations in the intronless MKRN3 gene by Sanger sequencing of the entire 507 amino acid coding region of exon 1 in a family with two affected girls presented with CPP at the age of 6 and 5·7 years, respectively. RESULTS: A novel heterozygous g.Gly312Asp missense mutation in the MKRN3 gene was identified in these siblings. The imprinted MKRN3 missense mutation was also identified as expected in the unaffected father and followed as expected an imprinted mode of inheritance. In silico analysis of the altered missense variant using the computational algorithms Polyphen2, SIFT and Mutation Taster predicted a damage and pathogenic alteration causing CPP. The pathogenicity of the alteration at the protein level via an in silico structural model is also explored. CONCLUSION: A novel mutation in the MKRN3 gene in two sisters with CPP was identified, supporting the fundamental role of this gene in the suppression of the hypothalamic GnRH neurons.
Assuntos
Simulação por Computador , Predisposição Genética para Doença/genética , Mutação de Sentido Incorreto , Puberdade Precoce/genética , Ribonucleoproteínas/genética , Sequência de Aminoácidos , Criança , Pré-Escolar , Análise Mutacional de DNA , Saúde da Família , Feminino , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Conformação Proteica , Ribonucleoproteínas/química , Irmãos , Ubiquitina-Proteína LigasesRESUMO
Steinfeld syndrome (MIM #184705) was first reported in 1982. It is characterised by holoprosencephaly and limb defects, however other anomalies may also be present. Following the initial description, three further cases have been reported in the literature. We report on a 23-year-old girl, with features of microform holoprosencephaly and bilateral congenital elbow dislocation in association with hypoplastic radial heads. She was identified to have a variant in the CDON gene inherited from her father who had ocular hypotelorism, but no other clinical features. We discuss the clinical features of Steinfeld syndrome, and broaden the phenotypic spectrum of this condition. Structural analysis suggests that this variant could lead to destabilisation of binding of CDON with hedgehog proteins. Further work needs to be done to confirm whether mutations in the CDON gene are the cause of Steinfeld syndrome.
Assuntos
Cardiopatias Congênitas/diagnóstico , Holoprosencefalia/diagnóstico , Deformidades Congênitas dos Membros/diagnóstico , Fenótipo , Sequência de Aminoácidos , Encéfalo/patologia , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Hibridização Genômica Comparativa , Fácies , Feminino , Cardiopatias Congênitas/genética , Heterozigoto , Holoprosencefalia/genética , Humanos , Deformidades Congênitas dos Membros/genética , Imageamento por Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Conformação Proteica , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Adulto JovemRESUMO
The aggregation and fibril deposition of amyloid proteins have been implicated in a range of neurodegenerative and vascular diseases, and yet the underlying molecular mechanisms are poorly understood. Here, we use a combination of cell-based assays, biophysical analysis, and atomic force microscopy to investigate the potential involvement of oxidative stress in aortic medial amyloid (AMA) pathogenesis and deposition. We show that medin, the main constituent of AMA, can induce an environment rich in oxidative species, increasing superoxide and reducing bioavailable nitric oxide in human cells. We investigate the role that this oxidative environment may play in altering the aggregation process of medin and identify potential posttranslational modification sites where site-specific modification and interaction can be unambiguously demonstrated. In an oxidizing environment, medin is nitrated at tyrosine and tryptophan residues, with resultant effects on morphology that lead to longer fibrils with increased toxicity. This provides further motivation to investigate the role of oxidative stress in AMA pathogenicity.
Assuntos
Antígenos de Superfície/toxicidade , Aorta/metabolismo , Proteínas do Leite/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Antígenos de Superfície/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteínas do Leite/metabolismo , Nitratos/metabolismoRESUMO
Type IV pili are polymeric fibers which protrude from the cell surface and play a critical role in adhesion and invasion by pathogenic bacteria. The secretion of pili across the periplasm and outer membrane is mediated by a specialized secretin protein, PilQ, but the way in which this large channel is formed is unknown. Using NMR, we derived the structures of the periplasmic domains from N. meningitidis PilQ: the N-terminus is shown to consist of two ß-domains, which are unique to the type IV pilus-dependent secretins. The structure of the second ß-domain revealed an eight-stranded ß-sandwich structure which is a novel variant of the HSP20-like fold. The central part of PilQ consists of two α/ß fold domains: the structure of the first of these is similar to domains from other secretins, but with an additional α-helix which links it to the second α/ß domain. We also determined the structure of the entire PilQ dodecamer by cryoelectron microscopy: it forms a cage-like structure, enclosing a cavity which is approximately 55 Å in internal diameter at its largest extent. Specific regions were identified in the density map which corresponded to the individual PilQ domains: this allowed us to dock them into the cryoelectron microscopy density map, and hence reconstruct the entire PilQ assembly which spans the periplasm. We also show that the C-terminal domain from the lipoprotein PilP, which is essential for pilus assembly, binds specifically to the first α/ß domain in PilQ and use NMR chemical shift mapping to generate a model for the PilP:PilQ complex. We conclude that passage of the pilus fiber requires disassembly of both the membrane-spanning and the ß-domain regions in PilQ, and that PilP plays an important role in stabilising the PilQ assembly during secretion, through its anchorage in the inner membrane.
Assuntos
Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Neisseria meningitidis/metabolismo , Neisseria meningitidis/ultraestrutura , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Proteínas de Fímbrias/química , Fímbrias Bacterianas/ultraestrutura , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Periplasma/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
MUPs (major urinary proteins) play an important role in chemical signalling in rodents and possibly other animals. In the house mouse (Mus musculus domesticus) MUPs in urine and other bodily fluids trigger a range of behavioural responses that are only partially understood. There are at least 21 Mup genes in the C57BL/6 mouse genome, all located on chromosome 4, encoding sequences of high similarity. Further analysis separates the MUPs into two groups, the 'central' near-identical MUPs with over 97% sequence identity and the 'peripheral' MUPs with a greater degree of heterogeneity and approximately 20-30% non-conserved amino acids. This review focuses on differences between the two MUP sub-groups and categorizes these changes in terms of molecular structure and pheromone binding. As small differences in amino acid sequence can result in marked changes in behavioural response to the signal, we explore the potential of single amino acid changes to affect chemical signalling and protein stabilization. Using analysis of existing molecular structures available in the PDB we compare the chemical and physical properties of the ligand cavities between the MUPs. Furthermore, we identify differences on the solvent exposed surfaces of the proteins, which are characteristic of protein-protein interaction sites. Correlations can be seen between molecular heterogeneity and the specialized roles attributed to some MUPs.
Assuntos
Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Feromônios/química , Feromônios/metabolismo , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
The genomes of rats and mice both contain a cluster of multiple genes that encode small (18-20 kDa) eight-stranded ß-barrel lipocalins that are expressed in multiple secretory tissues, some of which enter urine via hepatic biosynthesis. These proteins have been given different names, but are mostly generically referred to as MUPs (major urinary proteins). The mouse MUP cluster is increasingly well understood, and, in particular, a number of roles for MUPs in chemical communication between conspecifics have been established. By contrast, the literature on the rat orthologues is much less well developed and is fragmented. In the present review, we summarize current knowledge on the MUPs from the Norway (or brown) rat, Rattus norvegicus.
Assuntos
Proteínas/metabolismo , Animais , Feminino , Masculino , Camundongos , Família Multigênica/genética , Proteínas/genética , RatosRESUMO
The three dimensional solution structures of a highly conserved 16mer RNA, endowed with a classic 'GNRA' tetraloop motif, and a 17mer RNA containing a cytosine-rich heptaloop which is predicted to be a potential receptor for the former RNA, of the I-domain of Encephalomyocarditis virus IRES have been determined by NMR spectroscopy. As Mg(2+) plays an important role in the activity of the IRES, the corresponding NMR structures of the Mg(2+) bound RNA complexes have also been determined. These RNA NMR structures, 16mer (21 constraints per residue), 16mer RNA/Mg(2+) (21 constraints per residue), 17mer (17 constraints per residue) and 17mer RNA/Mg(2+) (16 constraints per residue), were calculated to a high degree of precision with low RMSDs and low clash scores represent, to the best our knowledge, the first structures of a type II picornavirus IRES. Conformational analysis of the average structure showed that the RNAs and their Mg(2+) complexes adopt characteristic A-form helical structures, stabilised by canonical and non-canonical interactions in both the stem and loop regions. The GCGA tetraloop of the 16mer folds into a standard GNRA conformation, with the structural role of A550 being in the form of a G547.A550 sheared base-pair made up of two hydrogen bonds. Further, the previously uncharacterised AACCCCA heptaloop present in the 17mer forms a compact tertiary loop motif, held together by strong π-π interactions. Analysis of the NMR structures demonstrates that the role of Mg(2+) is principally to confer enhanced stability to the RNAs whereby the tetra- and heptaloops can achieve optimum conformation for any RNA-RNA interactions which are crucial for understanding the structure-function relationship of the IRES.
Assuntos
Vírus da Encefalomiocardite/química , Magnésio/química , Ressonância Magnética Nuclear Biomolecular , RNA Viral/química , Ribossomos/química , Conformação de Ácido NucleicoRESUMO
SRSF2 is a prototypical SR protein which plays important roles in the alternative splicing of pre-mRNA. It has been shown to be involved in regulatory pathways for maintaining genomic stability and play important roles in regulating key receptors in the heart. We report here the solution structure of the RNA recognition motifs (RRM) domain of free human SRSF2 (residues 9-101). Compared with other members of the SR protein family, SRSF2 structure has a longer L3 loop region. The conserved aromatic residue in the RNP2 motif is absent in SRSF2. Calorimetric titration shows that the RNA sequence 5'AGCAGAGUA3' binds SRSF2 with a K(d) of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical ß1 and ß3 interactions are themselves not sufficient for effective RNA binding; the additional loop L3 is crucial for RNA complex formation. A comparison is made between the structures of SRSF2-RNA complex with other known RNA complexes of SR proteins. We conclude that interactions involving the L3 loop, N- and C-termini of the RRM domain are collectively important for determining selectivity between the protein and RNA.
Assuntos
Proteínas Nucleares/química , RNA/química , Ribonucleoproteínas/química , Sequência de Aminoácidos , Sítios de Ligação , Calorimetria , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Processamento de Serina-ArgininaRESUMO
Human ageing is a normal process and does not necessarily result in the development of frailty. A mix of genetic, environmental, dietary, and lifestyle factors can have an impact on ageing, and whether an individual develops frailty. Frailty is defined as the loss of physiological reserve both at the physical and cellular levels, where systemic processes such as oxidative stress and inflammation contribute to physical decline. The newest "omics" technology and systems biology discipline, metabolomics, enables thorough characterisation of small-molecule metabolites in biological systems at a particular time and condition. In a biological system, metabolites-cellular intermediate products of metabolic reactions-reflect the system's final response to genomic, transcriptomic, proteomic, epigenetic, or environmental alterations. As a relatively newer technique to characterise metabolites and biomarkers in ageing and illness, metabolomics has gained popularity and has a wide range of applications. We will give a comprehensive summary of what is currently known about metabolomics in studies of ageing, with a focus on biomarkers for frailty. Metabolites related to amino acids, lipids, carbohydrates, and redox metabolism may function as biomarkers of ageing and/or frailty development, based on data obtained from human studies. However, there is a complexity that underpins biological ageing, due to both genetic and environmental factors that play a role in orchestrating the ageing process. Therefore, there is a critical need to identify pathways that contribute to functional decline in people with frailty.