RESUMO
Optic atrophy 1(OPA1) is a GTPase protein that controls mitochondrial fusion, cristae integrity, and mtDNA maintenance. In neurodegenerative diseases such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), the mitochondrial network morphology is compromised. Studies on TAR-DNA binding protein 43 (TDP-43) has been the focus in our lab. OPA1 and TDP-43 interaction may shed a light on how aberrant TDP-43 interacts with OPA1, which will lead to mitochondrial dysfunction. The preliminary study tested the idea of whether OPA1 and TDP-43 are physically interacting in human platelet derived mitochondria obtained from healthy human subjects.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Ligação a DNA , GTP Fosfo-Hidrolases , Doenças Neurodegenerativas , Humanos , Esclerose Lateral Amiotrófica/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , Isoformas de Proteínas/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismoRESUMO
Impaired interactions between Calcineurin (Cn) and (Cu/Zn) superoxide dismutase (SOD1) are suspected to be responsible for the formation of hyperphosphorylated protein aggregation in amyotrophic lateral sclerosis (ALS). Serine (Ser)- enriched phosphorylated TDP-43 protein aggregation appears in the spinal cord of ALS animal models, and may be linked to the reduced phosphatase activity of Cn. The mutant overexpressed SOD1G93A protein does not properly bind zinc (Zn) in animal models; hence, mutant SOD1G93A-Cn interaction weakens. Consequently, unstable Cn fails to dephosphorylate TDP-43 that yields hyperphosphorylated TDP-43 aggregates. Our previous studies had suggested that Cn and SOD1 interaction was necessary to keep Cn enzyme functional. We have observed low Cn level, increased Zn concentrations, and increased TDP-43 protein levels in cervical, thoracic, lumbar, and sacral regions of the spinal cord tissue homogenates. This study further supports our previously published work indicating that Cn stability depends on functional Cn-SOD1 interaction because Zn is crucial for maintaining the Cn stability. Less active Cn did not efficiently dephosphorylate TDP-43; hence TDP-43 aggregations appeared in the spinal cord tissue.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Calcineurina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Medula Espinal/metabolismo , Superóxido Dismutase/metabolismo , Zinco/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Calcineurina/genética , Proteínas de Ligação a DNA/genética , Ratos , Ratos Transgênicos , Superóxido Dismutase/genéticaRESUMO
INTRODUCTION: Rasagiline is a monoamine oxidase B (MAO-B) inhibitor with possible neuroprotective effects in patients with amyotrophic lateral sclerosis (ALS). METHODS: We performed a randomized, double-blind, placebo-controlled trial of 80 ALS participants with enrichment of the placebo group with historical controls (n = 177) at 10 centers in the United States. Participants were randomized in a 3:1 ratio to 2 mg/day rasagiline or placebo. The primary outcome was average slope of decline on the ALS Functional Rating Scale-Revised (ALSFRS-R). Secondary measures included slow vital capacity, survival, mitochondrial and molecular biomarkers, and adverse-event reporting. RESULTS: There was no difference in the average 12-month ALSFRS-R slope between rasagiline and the mixed placebo and historical control cohorts. Rasagiline did not show signs of drug-target engagement in urine and blood biomarkers. Rasagiline was well tolerated with no serious adverse events. DISCUSSION: Rasagiline did not alter disease progression compared with controls over 12 months of treatment. Muscle Nerve 59:201-207, 2019.
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Esclerose Lateral Amiotrófica/tratamento farmacológico , Indanos/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/psicologia , Proteínas de Ligação a DNA/metabolismo , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Qualidade de Vida , Estudos Retrospectivos , Índice de Gravidade de Doença , Resultado do Tratamento , Estados Unidos , Adulto JovemRESUMO
OBJECTIVE: Blood-based biomarkers provide a crucial information in the progress of neurodegenerative diseases with a minimally invasive sampling method. Validated blood-based biomarker application in people with amyotrophic lateral sclerosis would derive numerous benefits. Canine degenerative myelopathy is a naturally occurring animal disease model to study the biology of human amyotrophic lateral sclerosis. Serum derived exosomes are potential carriers for cell-specific cargoes making them ideal venue to study biomarkers for a variety of diseases and biological processes. This study assessed the exosomal proteins that may be assigned as surrogate biomarker that may reflect biochemical changes in the central nervous system. METHODS: Exosomes were isolated from canine serum using commercial exosome isolation reagents. Exosomes target proteins contents were analyzed by the Western blotting method. RESULTS: The profiles of potential biomarker candidates in spinal cord homogenate and that of serum-derived exosomes were found elevated in dogs with degenerative myelopathy as compared to control subjects. CONCLUSIONS: Serum-derived exosomal biomolecules can serve as surrogate biomarkers in neurodegenerative diseases.KEY MESSAGESA canine with degenerative myelopathy can serve as a model animal to study human amyotrophic lateral sclerosis.Serum-derived exosomes contain Transactive Response DNA Binding Protein 43 (TDP-43), a potential biomarker candidate.The levels of spinal cord TDP-43 proteins and that of serum-derived exosomes exhibited similar profiling. Therefore, serum derived exosomes may be used as a venue for establishing blood-based biomarkers for neurodegenerative diseases.
Assuntos
Esclerose Lateral Amiotrófica , Exossomos , Doenças Neurodegenerativas , Cães , Humanos , Animais , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/genética , Doenças Neurodegenerativas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Biomarcadores , Exossomos/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) remains a devastating motor neuron disease with limited treatment options. Oxaloacetate treatment has a neuroprotective effect in rodent models of seizure and neurodegeneration. Therefore, we treated the ALS model superoxide dismutase 1 (SOD1) G93A mice with oxaloacetate and evaluated their neuromuscular function and lifespan. Treatment with oxaloacetate beginning in the presymptomatic stage significantly improved neuromuscular strength measured during the symptomatic stage in the injected mice compared to the non-treated group. Oxaloacetate treatment starting in the symptomatic stage significantly delayed limb paralysis compared with the non-treated group. For lifespan analysis, oxaloacetate treatment did not show a statistically significant positive effect, but the treatment did not shorten the lifespan. Mechanistically, SOD1G93A mice showed increased levels of tumor necrosis factor-α (TNFα) and peroxisome proliferative activated receptor gamma coactivator 1α (PGC-1α) mRNAs in the spinal cord. However, oxaloacetate treatment reverted these abnormal levels to that of wild-type mice. Similarly, the altered expression level of total NF-κB protein returned to that of wild-type mice with oxaloacetate treatment. These results suggest that the beneficial effects of oxaloacetate treatment in SOD1G93A mice may reflect the effects on neuroinflammation or bioenergetic stress.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Atividade Motora/efeitos dos fármacos , Ácido Oxaloacético/farmacologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Medula Espinal/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Longevidade/efeitos dos fármacos , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Ácido Oxaloacético/uso terapêutico , Medula Espinal/metabolismo , Superóxido Dismutase/metabolismoRESUMO
The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.
Assuntos
Elétrons , Mitocôndrias , Animais , Transporte de Elétrons , Metabolismo Energético , Feminino , Coração , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Gravidez , SuínosRESUMO
The effects of lifelong, moderate excess release of glutamate (Glu) in the CNS have not been previously characterized. We created a transgenic (Tg) mouse model of lifelong excess synaptic Glu release in the CNS by introducing the gene for glutamate dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. Glud1 is, potentially, an important enzyme in the pathway of Glu synthesis in nerve terminals. Increased levels of GLUD protein and activity in CNS neurons of hemizygous Tg mice were associated with increases in the in vivo release of Glu after neuronal depolarization in striatum and in the frequency and amplitude of miniature EPSCs in the CA1 region of the hippocampus. Despite overexpression of Glud1 in all neurons of the CNS, the Tg mice suffered neuronal losses in select brain regions (e.g., the CA1 but not the CA3 region). In vulnerable regions, Tg mice had decreases in MAP2A labeling of dendrites and in synaptophysin labeling of presynaptic terminals; the decreases in neuronal numbers and dendrite and presynaptic terminal labeling increased with advancing age. In addition, the Tg mice exhibited decreases in long-term potentiation of synaptic activity and in spine density in dendrites of CA1 neurons. Behaviorally, the Tg mice were significantly more resistant than wild-type mice to induction and duration of anesthesia produced by anesthetics that suppress Glu neurotransmission. The Glud1 mouse might be a useful model for the effects of lifelong excess synaptic Glu release on CNS neurons and for age-associated neurodegenerative processes.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glutamato Desidrogenase/biossíntese , Ácido Glutâmico/metabolismo , Plasticidade Neuronal/genética , Neurônios/enzimologia , Neurônios/metabolismo , Sinapses/enzimologia , Sinapses/metabolismo , Animais , Animais Geneticamente Modificados , Sobrevivência Celular/genética , Glutamato Desidrogenase/genética , Potenciação de Longa Duração/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Sinapses/genéticaRESUMO
BACKGROUND: Increases during aging in extracellular levels of glutamate (Glu), the major excitatory neurotransmitter in the brain, may be linked to chronic neurodegenerative diseases. Little is known about the molecular responses of neurons to chronic, moderate increases in Glu levels. Genome-wide gene expression in brain hippocampus was examined in a unique transgenic (Tg) mouse model that exhibits moderate Glu hyperactivity throughout the lifespan, the neuronal Glutamate dehydrogenase (Glud1) mouse, and littermate 9 month-old wild type mice. RESULTS: Integrated bioinformatic analyses on transcriptomic data were used to identify bio-functions, pathways and gene networks underlying neuronal responses to increased Glu synaptic release. Bio-functions and pathways up-regulated in Tg mice were those associated with oxidative stress, cell injury, inflammation, nervous system development, neuronal growth, and synaptic transmission. Increased gene expression in these functions and pathways indicated apparent compensatory responses offering protection against stress, promoting growth of neuronal processes (neurites) and re-establishment of synapses. The transcription of a key gene in the neurite growth network, the kinase Ptk2b, was significantly up-regulated in Tg mice as was the activated (phosphorylated) form of the protein. In addition to genes related to neurite growth and synaptic development, those associated with neuronal vesicle trafficking in the Huntington's disease signalling pathway, were also up-regulated. CONCLUSIONS: This is the first study attempting to define neuronal gene expression patterns in response to chronic, endogenous Glu hyperactivity at brain synapses. The patterns observed were characterized by a combination of responses to stress and stimulation of nerve growth, intracellular transport and recovery.
Assuntos
Encéfalo/metabolismo , Perfilação da Expressão Gênica , Ácido Glutâmico/metabolismo , Neurotransmissores/metabolismo , Aminoácidos/metabolismo , Animais , Transporte Biológico , Encéfalo/citologia , Encéfalo/fisiologia , Sinalização do Cálcio/genética , Quimiocinas/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Regulação Enzimológica da Expressão Gênica , Redes Reguladoras de Genes/genética , Genômica , Glutamato Desidrogenase/genética , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/fisiologia , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Espaço Intracelular/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neuritos/metabolismo , Neuroglia/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/metabolismo , Dobramento de Proteína , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Estresse Fisiológico/genética , Sinapses/metabolismo , Fatores de Tempo , Receptores Toll-Like/metabolismoRESUMO
Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%-20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.
Assuntos
Esclerose Lateral Amiotrófica/sangue , Esclerose Lateral Amiotrófica/diagnóstico , Plaquetas/metabolismo , Técnicas de Química Analítica/métodos , Proteínas de Ligação a DNA/sangue , Anticorpos/sangue , Anticorpos/imunologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Western Blotting/métodos , Proteínas de Ligação a DNA/metabolismo , Eletroforese Capilar/métodos , Humanos , Imunoensaio/métodos , Reprodutibilidade dos TestesRESUMO
Measurement of the electron transfer cascade (ETC) enzyme activities and their kinetic profiles is important in assessing mitochondrial function in the nervous system in health and disease or following exposure to toxic agents. The optimization of enzymatic assays for brain tissues and neurons is critical to the development of high-throughput assay formats. This article describes a step-by-step protocol for reliable and reproducible assessment of ETC enzyme kinetics (Complex I-IV) for mitochondria from small quantities of tissue from different brain regions, such as the hippocampus, cerebellum, and frontal cortex, or from neurons in culture. Methods for differential and density gradient centrifugation are detailed for isolating cell body and synaptic mitochondria from brain, as well as measurement of ETC activities in microwell plate or single-cuvette format using spectrophotometric methods. Easy-to follow assay layouts and useful tips are presented, allowing the user to perform these assays in under 3 hr. © 2019 by John Wiley & Sons, Inc.
Assuntos
Encéfalo/citologia , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Neurônios/enzimologia , Animais , Encéfalo/enzimologia , Células Cultivadas , Neurônios/citologia , RoedoresRESUMO
Cn (calcineurin) activity is stabilized by SOD1 (Cu-Zn superoxide dismutase), a phenomenon attributed to protection from superoxide (O2*-). The effects of O2*- on Cn are still controversial. We found that O2*-, generated either in vitro or in vivo did not affect Cn activity. Yet native bovine, recombinant human or rat, and two chimaeras of human SOD1-rat SOD1, all activated Cn, but SOD2 (Mn-superoxide dismutase) did not affect Cn activity. There was also a poor correlation between SOD1 dismutase activity and Cn activation. A chimaera of human N-terminal SOD1 and rat C-terminal SOD1 had little detectable dismutase activity, yet stimulated Cn activity the same as full-length human or rat SOD1. Nevertheless, there was evidence that the active site of SOD1 was involved in Cn activation based on the loss of activation following chelation of Cu from the active site of SOD1. Also, SOD1 engaged in the catalysis of O2*- dismutation was ineffective in activating Cn. SOD1 activation of Cn resulted from a 90-fold decrease in phosphatase K(m) without a change in V(max). A possible mechanism for the activation of Cn was identified in our studies as the prevention of Fe and Zn losses from the active site of Cn, suggesting a conformation-dependent SOD1-Cn interaction. In neurons, SOD1 and Cn were co-localized in cytoplasm and membranes, and SOD1 co-immunoprecipitated with Cn from homogenates of brain hippocampus and was present in immunoprecipitates as large multimers. Pre-incubation of pure SOD1 with Cn caused SOD1 multimer formation, an indication of an altered conformational state in SOD1 upon interaction with Cn.
Assuntos
Encéfalo/metabolismo , Calcineurina/metabolismo , Superóxido Dismutase/metabolismo , Animais , Calcineurina/genética , Catalase/metabolismo , Bovinos , Células Cultivadas , Quelantes/metabolismo , Compostos Cromogênicos/metabolismo , Ativação Enzimática , Herbicidas/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Neurônios/citologia , Neurônios/metabolismo , Oxidantes/metabolismo , Paraquat , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes , Resorcinóis/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Zinco/metabolismoRESUMO
AIM: Alzheimer's disease (AD) and other forms of dementia create a noncurable disease population in world's societies. To develop a blood-based biomarker is important so that the remedial or disease-altering therapeutic intervention for AD patients would be available at the early stage. MATERIALS & METHODS: TDP-43 levels were analyzed in postmortem brain tissue and platelets of AD and control subjects. RESULTS: We observed an increased TDP-43 (<60%) in postmortem AD brain regions and similar trends were also observed in patient's platelets. CONCLUSION: Platelet TDP-43 could be used as a surrogate biomarker that is measurable, reproducible and sensitive for screening the patients with some early clinical signs of AD and can be used to monitor disease prognosis.
RESUMO
Age-related decline in strength of synaptic transmission and memory formation has been attributed to age-associated increases in the activity of calcineurin (Cn) in hippocampus neurons. In the present study, we examined how brain Cn activity, Cn subunit levels, and Cn protein oxidation were changing during the aging process. Cn activity decreased with advancing age in three brain subcellular fractions, homogenate, cytosol, and synaptic membranes, obtained from F344/BNF1 rats of 5-6, 22-24, and 34-36 months of age. Cn activity also decreased during aging in homogenate, cytosol, and a nerve ending-enriched fraction from the hippocampus. Cn protein levels in homogenate and cytosol, as determined by the immune reactivity of its subunits A and B, were not altered during aging. But, in synaptic membranes, there was an age-related decrease in CnA levels, but not of CnB. Another important observation was that of an oxidative modification of CnA, not CnB, with increasing age. Such modification caused the formation of large aggregates of CnA. Aggregate formation was due to SH-group oxidation as the monomeric form of CnA was recovered upon disulfide reduction of the proteins with dithiothreitol. The age-related formation of aggregates of the catalytic subunit of Cn was suggestive of a correlation between aggregate formation and diminished enzyme activity. The loss of Cn activity may alter signal transduction at synapses during the aging process.
Assuntos
Envelhecimento/metabolismo , Calcineurina/metabolismo , Hipocampo/metabolismo , Estresse Oxidativo/fisiologia , Membranas Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Envelhecimento/patologia , Animais , Domínio Catalítico/fisiologia , Citosol/metabolismo , Regulação para Baixo/fisiologia , Ativação Enzimática/fisiologia , Hipocampo/química , Hipocampo/fisiopatologia , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Oxirredução , Subunidades Proteicas , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais/fisiologia , Frações SubcelularesRESUMO
Both protein and mRNA for the NR1 subunit of N-methyl-D-aspartate receptors are present in neuronal dendrites and undergo changes in distribution following synaptic excitation. However, the expression of all exonic splice variants of NR1 in dendrites has not been determined. In the present study, antibodies against the exon 5 (ex5) peptide sequence labeled proteins mostly in the soma of hippocampus neurons, whereas antibodies against ex21 or ex22 labeled cell bodies and dendrites. Antisense cRNAs for ex5 hybridized with mRNAs in cell bodies, whereas cRNAs for ex21 with mRNAs in both cell bodies and dendrites. Antisense DNA to a 24-base sequence identified as being present only in the 5'-UTR of cDNAs lacking ex5 (ex5(-)), hybridized with mRNAs in soma and dendrites and this labeling was coincident, mostly, with RNA granules. Insertion of the 24-base DNA ahead of that for enhanced green fluorescent protein (EGFP) increased the transport of EGFP mRNA and the expression of EGFP in neurites of neurons in culture. Fluorescent sense mRNA that contained the 24-base sequence bound to proteins in dendrites and to two proteins, 60 and 70 kDa, in brain microsomes. Proteins of similar size were also labeled by [32P]CTP-mRNA for NR1-(1a), which contains the 24-base 5'-UTR sequence, but not for NR1-(2b), which does not. Biotinylated 24-base sense mRNA was used to purify from brain microsomes two RNA-binding proteins (60 and 70 kDa). We concluded that the 24-base sequence in 5'-UTR of ex5(-) mRNA functioned as a cis-acting, dendrite-targeting element recognized selectively by two microsome proteins.
Assuntos
Dendritos/metabolismo , Éxons/fisiologia , Sítios de Splice de RNA/fisiologia , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Dendritos/genética , Ligação Proteica/fisiologia , RNA Mensageiro/genética , Ratos , Receptores de N-Metil-D-Aspartato/genética , EstereoisomerismoRESUMO
Methionine oxidation by reactive oxygen species and reduction mediated by the methionine sulfoxide reductase (Msr) system may attenuate protein function in signal transduction pathways. This review will focus on two potential protein targets for methionine oxidation involved in signal transduction of the immune response: Ca(2+)/calmodulin-regulated phosphatase calcineurin (Cn) and inhibitor of kappa B-alpha (IkBα). The major known function of Cn is to regulate nuclear localization of the nuclear factor of activated T cells (NFAT), a family of transcription factors during immune stimulus. Like wise, IκBα inhibits the activity of nuclear factor kappa B (NFkB), which is known to regulate the transcription of various genes participating in immunological and oxidative stress response. Modification of Met (45) in IκBα enhances its resistance to protein-degredation; thereby, preventing NFkB from activating transcription in cells of the immune system. Similarly, the human Cn molecule contains several methionine residues that are either located next to a cysteine residue or a methionine residue. Accordingly, it is suggested that oxidation of a specific Cn-methionine may interfere with the proper NFAT nuclear-localization and transcriptional activation in T-cell. Thus, the roles of oxidized-methionine residues and their reduction, by the Msr system, are discussed as potential regulators of cellular immune response.
RESUMO
The cloning and characterization of the gene for the fourth subunit of a glutamate-binding protein complex in rat brain synaptic membranes are described. The cloned rat brain cDNA contained two open reading frames (ORFs) encoding 8.9- (PRO1) and 9.5-kDa (PRO2) proteins. The cDNA sequence matched contiguous genomic DNA sequences in rat chromosome 17. Both ORFs were expressed within the structure of a single brain mRNA and antibodies against unique sequences in PRO1- and PRO2-labeled brain neurons in situ, indicative of bicistronic gene expression. Dicistronic vectors in which ORF1 and ORF2 were substituted by either two different fluorescent proteins or two luciferases indicated concurrent, yet independent translation of the two ORFs. Transfection with noncapped mRNA led to cap-independent translation of only ORF2 through an internal ribosome entry sequence preceding ORF2. In vitro or cell expression of the cloned cDNA led to the formation of multimeric protein complexes containing both PRO1 and PRO2. These complexes had low affinity (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801)-sensitive phencyclidine-binding sites. Overexpression of PRO1 and PRO2 in CHO cells, but not neuroblastoma cells, caused cell death within 24-48 h. The cytotoxicity was blocked by concurrent treatment with MK-801 or by two tetrahydroisoquinolines that bind to phencyclidine sites in neuronal membranes. Co-expression of two of the other subunits of the protein complex together with PRO1/PRO2 abrogated the cytotoxic effect without altering PRO1/PRO2 protein levels. Thus, this rare mammalian bicistronic gene coded for two tightly interacting brain proteins forming a low affinity phencyclidine-binding entity in a synaptic membrane complex.
Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Citotoxinas/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Fenciclidina/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Morte Celular , Linhagem Celular , Clonagem Molecular , Cricetinae , Citotoxinas/química , Citotoxinas/genética , DNA Complementar/genética , Bases de Dados de Ácidos Nucleicos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fases de Leitura Aberta/genética , Ligação Proteica , Biossíntese de Proteínas/genética , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Transcrição Gênica/genéticaRESUMO
As a major component of the extracellular matrix (ECM), laminin has been found in many vertebrate and invertebrate organisms. Its molecular structure is very similar across species lines and its biological function in the ECM has been extensively studied. In an effort to study ECM structure and function in hydra, we have cloned a partial hydra laminin alpha chain and the full-length hydra laminin beta chain using ECM-enriched cDNA libraries. Analysis of deduced amino acid sequences indicated that both polypeptides have high sequence similarity to a number of invertebrate and vertebrate laminin alpha and beta subunits. Rotary shadow analysis of isolated hydra laminin indicates it has a heterotrimeric organization that is characteristic of vertebrate laminins. A putative integrin-class protein was also identified using a cell-binding peptide sequence from the laminin beta chain as an affinity probe, indicating that integrins are possible cell surface receptors in hydra. In agreement with previous results for the hydra laminin beta chain, in situ hybridization experiments revealed that hydra laminin alpha chain mRNA is restricted to endodermal cells. As with a number of other hydra ECM components, higher levels of laminin alpha chain mRNA are localized to regions where cell migration and differentiation are actively undertaken such as the base of tentacles, the peduncle region, buds, regenerating tentacles, and at the head end during regeneration. The role of laminin in morphogenesis was studied using an antisense approach and the results indicated that translation of the laminin alpha chain is required for head regeneration.