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1.
J Cell Sci ; 133(15)2020 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-32661089

RESUMEN

Protein aggregates that result in inclusion formation are a pathological hallmark common to many neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease. Under conditions of cellular stress, activation of the heat shock response (HSR) results in an increase in the levels of molecular chaperones and is a first line of cellular defence against inclusion formation. It remains to be established whether neurodegenerative disease-associated proteins and inclusions are themselves capable of inducing an HSR in neuronal cells. To address this, we generated a neuroblastoma cell line that expresses a fluorescent reporter protein under conditions of heat shock transcription factor 1 (HSF1)-mediated HSR induction. We show that the HSR is not induced by exogenous treatment with aggregated forms of recombinant α-synuclein or the G93A mutant of superoxide dismutase-1 (SOD1G93A) nor intracellular expression of SOD1G93A or a pathogenic form of polyglutamine-expanded huntingtin (Htt72Q). These results suggest that pathogenic proteins evade detection or impair induction of the HSR in neuronal cells. A failure of protein aggregation to induce an HSR might contribute to the development of inclusion pathology in neurodegenerative diseases.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Enfermedades Neurodegenerativas , Factores de Transcripción del Choque Térmico/genética , Respuesta al Choque Térmico/genética , Humanos , Enfermedades Neurodegenerativas/genética , Agregado de Proteínas , Superóxido Dismutasa-1
2.
Neurosci Lett ; 778: 136614, 2022 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-35367314

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, characterised by the loss of motor neurons and subsequent paralysis. Evidence indicates that synaptic alterations are associated with the early stages of ALS pathogenesis. A hallmark of ALS postmortem tissue is the presence of proteinaceous inclusions, indicative of disturbed protein homeostasis, particularly in spinal cord motor neurons. We recently demonstrated that spinal cord motor neurons contain a supersaturated proteome, as they possess proteins at concentrations that exceed their solubility limits, resulting in a metastable proteome conducive to protein misfolding and aggregation. Recent evidence indicates metastable sub-proteomes within neuronal compartments, such as the synapse, may be particularly vulnerable and underlie their involvement in the initial stages of neurodegenerative diseases. To investigate if the motor neuron presynaptic terminal possesses a metastable sub-proteome, we used human and mouse spinal cord motor neuron expression data to calculate supersaturation scores. Here, we found that both the human and mouse presynaptic terminal sub-proteomes have higher supersaturation scores than the entire motor neuron proteome. In addition, we observed that proteins down-regulated in ALS were over-represented in the synapse. These results provide support for the notion that the metastability of the sub-proteome within the motor neuron presynaptic terminal may be particularly susceptible to protein homeostasis disturbances in ALS, and may contribute to explaining the observed synaptic dysfunction in ALS.


Asunto(s)
Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Ratones , Neuronas Motoras/metabolismo , Terminales Presinápticos/metabolismo , Proteoma/metabolismo , Médula Espinal/metabolismo , Sinapsis/metabolismo
3.
Nucleic Acids Res ; 37(20): 6970-83, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19759211

RESUMEN

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon-helix-helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Regiones Operadoras Genéticas , Plásmidos/genética , Regulón , Factores de Transcripción/genética , Transcripción Genética
4.
Stem Cell Res ; 42: 101701, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-32006803

RESUMEN

Dermal fibroblasts were donated by a 43 year old male patient with clinically diagnosed familial amyotrophic lateral sclerosis (ALS), carrying the SOD1E101G mutation. The induced pluripotent stem cell (iPSC) line UOWi007-A was generated using repeated mRNA transfections for pluripotency transcription factors Oct4, Klf4, Sox2, c-Myc, Lin28 and Nanog. The iPSCs carried the SOD1E101G genotype and had a normal karyotype, expressed expected pluripotency markers and were capable of in vitro differentiation into endodermal, mesodermal and ectodermal lineages. This iPSC line may be useful for investigating familial ALS resulting from a SOD1E101G mutation.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Fibroblastos/metabolismo , Superóxido Dismutasa-1/genética , Línea Celular , Humanos , Factor 4 Similar a Kruppel , ARN Mensajero/metabolismo
5.
Br J Pharmacol ; 177(12): 2812-2829, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32017039

RESUMEN

BACKGROUND AND PURPOSE: P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors. EXPERIMENTAL APPROACH: Genomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 receptors tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. In these cells, receptor pharmacology was characterised using nucleotide-induced Fura-2 AM measurements of intracellular Ca2+ and known P2X4 receptor antagonists. P2X4 receptor-mediated inward currents in HEK293 cells were assessed by automated patch clamp. KEY RESULTS: No P2RX4 missense variants were identified in any canine samples. Canine and human P2X4 receptors were localised primarily to lysosomal compartments. ATP was the primary agonist of canine P2X4 receptors with near identical efficacy and potency at human receptors. 2'(3')-O-(4-benzoylbenzoyl)-ATP, but not ADP, was a partial agonist with reduced potency for canine P2X4 receptors compared to the human orthologues. Five antagonists inhibited canine P2X4 receptors, with 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea displaying reduced sensitivity and potency at canine P2X4 receptors. CONCLUSION AND IMPLICATIONS: P2X4 receptors are highly conserved across dog pedigrees and display expression patterns and pharmacological profiles similar to human receptors, supporting validation and use of therapeutic agents for P2X4 receptor-related disease onset and management in dogs and humans.


Asunto(s)
Antagonistas del Receptor Purinérgico P2X , Receptores Purinérgicos P2X4 , Adenosina Trifosfato , Animales , Perros , Células HEK293 , Humanos , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X7
6.
Cells ; 9(9)2020 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-32887382

RESUMEN

The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer's disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch clamp. We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Neuronas Colinérgicas/efectos de los fármacos , Medios de Cultivo/farmacología , Cuerpos Embrioides/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Cultivo Primario de Células/métodos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Prosencéfalo Basal/metabolismo , Prosencéfalo Basal/patología , Benzamidas/farmacología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Línea Celular , Neuronas Colinérgicas/citología , Neuronas Colinérgicas/metabolismo , Medios de Cultivo/química , Dioxoles/farmacología , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Factor 2 de Diferenciación de Crecimiento/farmacología , Proteínas Hedgehog/farmacología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Modelos Biológicos , Factor de Crecimiento Nervioso/farmacología , Técnicas de Placa-Clamp , Pirazoles/farmacología , Pirimidinas/farmacología , Factor de Crecimiento Transformador beta/farmacología
7.
Cell Death Dis ; 10(4): 310, 2019 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-30952836

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a type of motor neuron disease (MND) in which humans lose motor functions due to progressive loss of motoneurons in the cortex, brainstem, and spinal cord. In patients and in animal models of MND it has been observed that there is a change in the properties of motoneurons, termed neuronal hyperexcitability, which is an exaggerated response of the neurons to a stimulus. Previous studies suggested neuronal excitability is one of the leading causes for neuronal loss, however the factors that instigate excitability in neurons over the course of disease onset and progression are not well understood, as these studies have looked mainly at embryonic or early postnatal stages (pre-symptomatic). As hyperexcitability is not a static phenomenon, the aim of this study was to assess the overall excitability of upper motoneurons during disease progression, specifically focusing on their oscillatory behavior and capabilities to fire repetitively. Our results suggest that increases in the intrinsic excitability of motoneurons are a global phenomenon of aging, however the cellular mechanisms that underlie this hyperexcitability are distinct in SOD1G93A ALS mice compared with wild-type controls. The ionic mechanism driving increased excitability involves alterations of the expression levels of HCN and KCNQ channel genes leading to a complex dynamic of H-current and M-current activation. Moreover, we show a negative correlation between the disease onset and disease progression, which correlates with a decrease in the expression level of HCN and KCNQ channels. These findings provide a potential explanation for the increased vulnerability of motoneurons to ALS with aging.


Asunto(s)
Envejecimiento , Esclerosis Amiotrófica Lateral/fisiopatología , Excitabilidad Cortical , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales de Potasio KCNQ/metabolismo , Neuronas Motoras/fisiología , Superóxido Dismutasa-1/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Excitabilidad Cortical/efectos de los fármacos , Excitabilidad Cortical/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales de Potasio KCNQ/genética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Ratones , Ratones Transgénicos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Superóxido Dismutasa-1/metabolismo
8.
Stem Cell Res ; 40: 101530, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31445393

RESUMEN

Dermal fibroblasts from a 59 year old male patient with amyotrophic lateral sclerosis (symptomatic at the time of collection), attributed to a mutation in the cyclin F gene (CCNFS621G), were reprogrammed using mRNA and microRNA-delivered OSKM factors to induced pluripotent stem cells (iPSCs). The generated iPSCs were confirmed pluripotent, expressing typical pluripotency markers and were capable of three germ layer differentiation. This is the first reported reprogramming of cells with a mutation in the cyclin F gene, and represents a novel resource for the study of amyotrophic lateral sclerosis.


Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Ciclinas/genética , Dermis/citología , Células Madre Pluripotentes Inducidas/citología , Esclerosis Amiotrófica Lateral/genética , Diferenciación Celular , Línea Celular , Reprogramación Celular , Fibroblastos/citología , Estratos Germinativos/citología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipo , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple
9.
Stem Cell Res ; 31: 227-230, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-30138848

RESUMEN

The induced pluripotent stem cell (iPSC) lines UOWi002-A and UOWi003-A were reprogrammed from dermal fibroblasts via mRNA transfection. Dermal fibroblasts from a 56 year old female caucasian familial Alzheimer's disease patient carrying A246E mutation in the PSEN1 gene (familial AD3, autopsy confirmed Alzheimer's disease) and a 75 year old female non-demented control from the same family bearing the wild-type PSEN1 A246 genotype were obtained from the Coriell Institute (AG06848 and AG06846, respectively). The generated iPSCs were characterized and pluripotency was confirmed. The PSEN1 genotype was maintained in both iPSC lines. Resource table.


Asunto(s)
Enfermedad de Alzheimer/genética , Células Madre Pluripotentes Inducidas/metabolismo , Presenilina-1/metabolismo , Anciano , Diferenciación Celular , Línea Celular , Femenino , Humanos , Persona de Mediana Edad
10.
Sci Rep ; 7(1): 2387, 2017 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-28539657

RESUMEN

Heat shock proteins (Hsps) are molecular chaperones that prevent the aggregation of client proteins by facilitating their refolding, or trafficking them for degradation. The chaperone activities of Hsps are dependent on dynamic protein-protein interactions, including their oligomerisation into large multi-subunit complexes. Thus, tagging Hsps with fluorescent proteins can interfere with their chaperone activity. To overcome this limitation, we have exploited bicistronic constructs for the concurrent expression of a non-tagged Hsp and fluorescent reporter from a single mRNA in cells. We used the Hsp-encoding bicistronic constructs in a cell-based model of protein aggregation, using a destabilised (mutant) form of firefly luciferase (mFluc) that forms inclusion bodies in cells. Expression of Hsp40, Hsp70, or Hsp40 and Hsp70 in cells expressing mFluc decreased the formation of inclusion bodies by 25-46% compared to controls. Moreover, there was a concentration-dependent decrease in the proportion of cells with inclusions when Hsp70, or Hsp40 and Hsp70 were co-expressed with mFluc in cells. The Hsp-encoding bicistronic constructs enable transfection efficiencies and concentration-dependent effects of Hsp expression to be determined using fluorescence based techniques, without the need to tag the Hsp with a fluorescent protein.


Asunto(s)
Proteínas del Choque Térmico HSP40/genética , Proteínas HSP70 de Choque Térmico/genética , Luciferasas de Luciérnaga/genética , Chaperonas Moleculares/genética , Neuronas/metabolismo , Animales , Sitios de Unión , Línea Celular Tumoral , Clonación Molecular , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Luciferasas de Luciérnaga/metabolismo , Ratones , Chaperonas Moleculares/metabolismo , Neuronas/citología , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección
11.
Chem Biol ; 22(2): 186-95, 2015 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-25699602

RESUMEN

Serine phosphorylation of the mammalian small heat-shock protein Hsp27 at residues 15, 78, and 82 is thought to regulate its structure and chaperone function; however, the site-specific impact has not been established. We used mass spectrometry to assess the combinatorial effect of mutations that mimic phosphorylation upon the oligomeric state of Hsp27. Comprehensive dimerization yielded a relatively uncrowded spectrum, composed solely of even-sized oligomers. Modification at one or two serines decreased the average oligomeric size, while the triple mutant was predominantly a dimer. These changes were reflected in a greater propensity for oligomers to dissociate upon increased modification. The ability of Hsp27 to prevent amorphous or fibrillar aggregation of target proteins was enhanced and correlated with the amount of dissociated species present. We propose that, in vivo, phosphorylation promotes oligomer dissociation, thereby enhancing chaperone activity. Our data support a model in which dimers are the chaperone-active component of Hsp27.


Asunto(s)
Proteínas de Choque Térmico HSP27/metabolismo , Chaperonas Moleculares/metabolismo , Línea Celular , Dicroismo Circular , Dimerización , Células HEK293 , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Humanos , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , Fosforilación , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
12.
Microbiology (Reading) ; 151(Pt 6): 1823-1837, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15941991

RESUMEN

Mature biofilm and planktonic cells of Streptococcus mutans cultured in a neutral pH environment were subjected to comparative proteome analysis. Of the 242 protein spots identified, 48 were significantly altered in their level of expression (P<0.050) or were unique to planktonic or biofilm-grown cells. Among these were four hypothetical proteins as well as proteins known to be associated with the maintenance of competence or found to possess a cin-box-like element upstream of their coding gene. Most notable among the non-responsive genes were those encoding the molecular chaperones DnaK, GroEL and GroES, which are considered to be up-regulated by sessile growth. Analysis of the rest of the proteome indicated that a number of cellular functions associated with carbon uptake and cell division were down-regulated. The data obtained were consistent with the hypothesis that a reduction in the general growth rate of mature biofilms of S. mutans in a neutral pH environment is associated with the maintenance of transformation without the concomitant stress response observed during the transient state of competence in bacterial batch cultures.


Asunto(s)
Proteínas Bacterianas/biosíntesis , Biopelículas , Regulación Bacteriana de la Expresión Génica , Proteoma/análisis , Streptococcus mutans/genética , Streptococcus mutans/fisiología , Adaptación Fisiológica/genética , Proteínas Bacterianas/análisis , Electroforesis en Gel Bidimensional , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Streptococcus mutans/crecimiento & desarrollo
13.
Biochem Biophys Res Commun ; 319(2): 439-47, 2004 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-15178426

RESUMEN

An N-acetyl-beta-D-glucosaminidase (GcnA) from Streptococcus gordonii FSS2 was cloned and sequenced. GcnA had a deduced molecular mass of 72,120 Da. The molecular weight after gel-filtration chromatography was 140,000 Da and by SDS-PAGE was 70,000 Da, indicating that the native protein was a homodimer. The deduced amino acid sequence had significant homology to a glycosyl hydrolase from Streptococcus pneumoniae and the conserved catalytic domain of the Family 20 glycosyl hydrolases. GcnA catalysed the hydrolysis of the synthetic substrates, 4-methylumbelliferyl (4MU)-N-acetyl-beta-D-glucosaminide, 4MU-N-acetyl-beta-D-galactosaminide, 4-MU-beta-D-N,N'-diacetylchitobioside, and 4-MU-beta-D-N,N',N''-chitotrioside as well as the respective chito-oligosaccharides. GcnA was optimally active at pH 6.6 and 42 degrees C. The Km for 4-MU-beta-D-N,N',N''-chitotrioside, 45 microM, was the lowest for all the substrates tested. Hg2+, Cu2+, Fe2+, and Zn2+ completely inhibited while Co2+, Mn2+, and Ni2+ partially inhibited activity. S. gordonii FSS2 and a GcnA negative mutant grew equally well on chito-oligosaccharides as substrates. The S. gordonii sequencing projects indicate two further N-acetyl-beta-D-glucosaminidase activities.


Asunto(s)
Acetilglucosaminidasa/metabolismo , Streptococcus/enzimología , Acetilglucosaminidasa/química , Acetilglucosaminidasa/genética , Acetilglucosaminidasa/aislamiento & purificación , Secuencia de Aminoácidos , Clonación Molecular , Dimerización , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
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