RESUMO
Parkinson disease (PD) is a neurodegenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. Post-mortem examination shows loss of neurons and Lewy bodies, which are cytoplasmic eosinophilic inclusions, in the substantia nigra and other brain regions. A few families have PD caused by mutations (A53T or A30P) in the gene SNCA (encoding alpha-synuclein). Alpha-synuclein is present in Lewy bodies of patients with sporadic PD, suggesting that alpha-synuclein may be involved in the pathogenesis of PD. It is unknown how alpha-synuclein contributes to the cellular and biochemical mechanisms of PD, and its normal functions and biochemical properties are poorly understood. To determine the protein-interaction partners of alpha-synuclein, we performed a yeast two-hybrid screen. We identified a novel interacting protein, which we term synphilin-1 (encoded by the gene SNCAIP). We found that alpha-synuclein interacts in vivo with synphilin-1 in neurons. Co-transfection of both proteins (but not control proteins) in HEK 293 cells yields cytoplasmic eosinophilic inclusions.
Assuntos
Proteínas de Transporte/metabolismo , Corpos de Inclusão/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Química Encefálica , Proteínas de Transporte/genética , Linhagem Celular , Cromossomos Humanos Par 5/genética , Feminino , Humanos , Corpos de Lewy/metabolismo , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Plasmídeos/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Sinucleínas , Distribuição Tecidual , Extratos de Tecidos/metabolismo , Transfecção , alfa-SinucleínaRESUMO
Each of the glutamine repeat neurodegenerative diseases has a particular pattern of pathology largely restricted to the CNS. However, there is considerable overlap among the regions affected, suggesting that the diseases share pathogenic mechanisms, presumably involving the glutamine repeats. We focus on Huntington's disease (HD) and Dentatorubral-pallidoluysian atrophy (DRPLA) as models for this family of diseases, since they have striking similarities and also notable differences in their clinical features and pathology. We review the pattern of pathology in adult and juvenile onset cases. Despite selective pathology, the disease genes and their protein products (huntingtin and atrophin-1) are widely expressed. This presents a central problem for all the glutamine repeat diseases-how do widely expressed gene products give rise to restricted pathology? The pathogenic effects are believed to occur via a "gain of function" mechanism at the protein level. Mechanisms of cell death may include excitotoxicity, metabolic toxicity, apoptosis, and free radical stress. Emerging data indicate that huntingtin and atrophin-1 may have distinct protein interactions. The specific interaction partners may help explain the selective pathology of these diseases.
Assuntos
Giro Denteado/patologia , Globo Pálido/patologia , Doença de Huntington/patologia , Doenças do Sistema Nervoso/patologia , Núcleo Rubro/patologia , Adulto , Morte Celular , Criança , Humanos , Doença de Huntington/etiologia , Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/genética , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/metabolismo , Neurônios/patologiaRESUMO
High levels of D-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl D-aspartate type of glutamate receptors. D-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from L-serine. We now report cloning and expression of human serine racemase, an enzyme that catalyses the synthesis of D-serine from L-serine. The enzyme displays a high homology to the murine serine racemase. It contains a pyridoxal 5'-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine racemase in areas known to contain large amounts of endogenous D-serine, such as hippocampus and corpus callosum. Robust synthesis of D-serine was detected in cells transfected with human serine racemase, demonstrating the conservation of D-amino acid metabolism in humans. Serine racemase may be a therapeutic target in psychiatric diseases as supplementation of D-serine greatly improves schizophrenia symptoms. We identify the human serine racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron-exon boundaries of the human serine racemase gene will be useful to search for mutations in neuropsychiatric disorders.
Assuntos
Racemases e Epimerases/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Encéfalo/enzimologia , Linhagem Celular , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Éxons , Genes/genética , Humanos , Íntrons , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Racemases e Epimerases/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The huntingtin-associated protein (HAP-1) interacts with the Huntington disease gene product, huntingtin. It is predominantly expressed in the brain and shows an increased affinity for mutant huntingtin. We have sequenced an 18,656bp genomic region encompassing the entire human HAP-1 gene and determined its genomic organisation, with 11 exons spanning 12.1kb. We have also found an intragenic polymorphism within intron 6 of HAP-1. We have recently shown that HAP-1 maps to a region of the genome which has been implicated in a variety of neurological conditions, including progressive supranuclear palsy (PSP), a late-onset atypical parkinsonian disorder. The detailed characterisation of the genomic organisation of HAP-1 and the presence of an intragenic polymorphism will be helpful in evaluating its role in different disorders, using candidate gene approaches.
Assuntos
Genes/genética , Proteínas do Tecido Nervoso/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , DNA/química , DNA/genética , DNA Intergênico/genética , Éxons , Humanos , Íntrons , Dados de Sequência Molecular , Polimorfismo Genético , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNARESUMO
The sarcoplasmic reticulum-bound creatine kinase from rabbit skeletal muscle was inhibited by the nitric oxide donor S-nitrosoglutathione (GSNO). This led to a decrease in Ca2+ uptake in sarcoplasmic reticulum vesicles when the transport was driven by ATP generated from phosphocreatine and ADP. In contrast, the Ca 2+ transport measured using 2 mM ATP as substrate was unaffected by GSNO up to 200 microM. GSNO (5-20 microM) inhibited the activity of both soluble and membrane-bound creatine kinase. Oxyhemoglobin (15-40 microM) protected creatine kinase against inactivation by GSNO. The inhibition by 10 microM GSNO was reversed by the addition of dithiothreitol (2 mM). The results indicate that nitric oxide (NO, including NO+, NO and NO-) inactivates creatine kinase in vitro by promoting nitrosylation of critical sulphydryl groups of the enzyme.
Assuntos
Cálcio/metabolismo , Creatina Quinase/antagonistas & inibidores , Glutationa/análogos & derivados , Compostos Nitrosos/farmacologia , Retículo Sarcoplasmático/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Cálcio/farmacocinética , Glutationa/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Músculo Esquelético/enzimologia , Óxido Nítrico/metabolismo , Oxiemoglobinas/metabolismo , Oxiemoglobinas/farmacologia , Coelhos , S-Nitrosoglutationa , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
Eight diseases are now known to be caused by an expansion mutation of the trinucleotide repeat CAG encoding glutamine. Each disease is caused by a CAG expansion in a different gene, and the genes bear no similarity to each other except for the presence of the repeat. Nonetheless, the essential feature of all of these disorders is neurodegeneration in a set of overlapping cortical and subcortical regions. Disease age of onset, and in some cases severity, is correlated with repeat length. These and other observations have led to the hypothesis that CAG expansion causes disease by a toxic gain-of-function of the encoded stretch of polyglutamine residues. Expansion-induced abnormalities of cytoskeletal function or neuronal signalling processes may contribute to the pathogenic process. In addition, theoretical and experimental analysis of the chemistry of uninterrupted stretches of glutamine residues suggest that polyglutamine-containing proteins or protein fragments may aggregate, via a "polar zipper", into beta pleated sheets. Recent findings have now established the presence of such aggregates in selected regions of brain from affected individuals, in transgenic mice expressing expanded repeats, and in isolated cells transfected with expanded repeats. The aggregates are most prominently manifest as neuronal intranuclear inclusion bodies. As the investigation of the link between these inclusions and cell dysfunction and death continues, it is possible that new avenues for therapeutic intervention will emerge.
Assuntos
Encéfalo/patologia , Deleção de Genes , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Peptídeos/genética , Sequências Repetitivas de Aminoácidos/genética , Animais , Encéfalo/metabolismo , Humanos , Camundongos , Camundongos TransgênicosRESUMO
alpha-Synuclein is mutated in rare autosomal dominant forms of Parkinson's disease and is a major component of Lewy bodies and neurites. Synphilin-1, a novel protein interacts in vivo and co-localises with alpha-synuclein in Lewy bodies. We analysed the synphilin-1 gene in familial Parkinson's disease by single-strand conformation polymorphism (SSCP) and automated sequencing but found no coding mutations. However, we identified two novel intronic polymorphisms; an A/T polymorphism in intron 2, resulting in the introduction of an Alu1 site and a second G/T polymorphism in intron 4. We analysed the intron 2 polymorphism for allelic association as it was conducive to rapid screening but observed no changes in frequency between Parkinson's disease cases and controls.
Assuntos
Proteínas de Transporte/genética , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Idoso , Alelos , Éxons/genética , Feminino , Testes Genéticos , Genótipo , Humanos , Íntrons/fisiologia , Corpos de Lewy/genética , Mutação/genética , Mutação/fisiologia , Doença de Parkinson/fisiopatologia , Polimorfismo Conformacional de Fita SimplesRESUMO
Parkinson's disease (PD) is characterized by the death of dopaminergic neurons and the presence of Lewy bodies in the substantia nigra pars compacta. The mechanisms involved in the death of neurons as well as the role of Lewy bodies in the pathogenesis of the disease are still unclear. Lewy bodies are made of aggregated proteins, in which alpha-synuclein represents their major component. Alpha-synuclein interacts with synphilin-1, a protein that is also present in Lewy bodies. When expressed in cells, synphilin-1 forms inclusions together with alpha-synuclein that resemble Lewy bodies. Synphilin-1 is ubiquitylated by various E3 ubiquitin-ligases, such as SIAH, parkin and dorfin. Ubiquitylation of synphilin-1 by SIAH is essential for its aggregation into inclusions. We recently identified a new synphilin-1 isoform, synphilin-1A, that is toxic to neurons, aggregation-prone and accumulates in detergent-insoluble fractions of brains from alpha-synucleinopathy patients. Synphilin-1A inclusions recruit both alpha-synuclein and synphilin-1. Aggregation of synphilin-1 and synphilin-1A seems to be protective to cells. We now discuss several aspects of the neurobiology and pathology of synphilin-1 isoforms, focusing on possible implications for PD.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/etiologia , alfa-Sinucleína/metabolismo , Humanos , Corpos de Lewy , Fosforilação , Isoformas de Proteínas/metabolismo , UbiquitinaçãoRESUMO
Seven in absentia homolog 1 (SIAH-1) is a member of the RING-finger-containing E3 ubiquitin ligases. Two substrates of SIAH-1 are alpha-synuclein and synphilin-1, both of these proteins are involved in Parkinson's disease (PD). Recently, mutations in Parkin, another E3 ubiquitin ligase which ubiquinates synphilin-1 and glycosylated alpha-synuclein, have been defined as a major cause of autosomal recessive PD. The potential role of SIAH-1 in PD is further underlined as SIAH-1 protein is a component of the Lewy bodies and as it plays a role in apoptosis caused by nitric oxide (NO) induced oxidative stress. Thus, we performed a mutation screening of the SIAH-1 gene in PD patients. However, screening a large sample of 209 familial and sporadic PD patients we could not find any disease causing mutation. We therefore conclude that genetic alterations of SIAH-1 do not significantly contribute to the pathogenesis of PD.
Assuntos
DNA/genética , Proteínas Nucleares/genética , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases/genética , Idoso , Cromatografia Líquida de Alta Pressão , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Desnaturação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Protein extracts obtained from male and female schistosomes were incubated with a gender-specific gene, F-10, transcribed only in adult females and encoding a major egg-shell protein. The protein/DNA interaction was measured using the band shift, DNase-I-footprinting and UV cross-linking techniques. The results showed a clear band shift when a 302 bp restriction fragment containing the 3' end of the gene was incubated with either female or male proteins. This fragment also contained a putative steroid hormone regulatory element (HRE). In contrast, only the male proteins produced a shift with the 495 bp fragment corresponding to the middle region of the gene. DNase I footprinting showed that proteins from males and females interacted with the F-10 gene by binding to multiple adjacent sites along the DNA, thus generating relatively long protected fragments of approximately 100 bp. This result suggested that the adjacent binding of several moles of protein occurred at the 5' end of the gene. UV cross-linking between schistosome proteins and a 21 bp synthetic oligonucleotide containing the F-10 HRE, evidenced proteins having MWS of 30, 45 and 65 kDa. These proteins are presumably involved in the regulation of transcription of the F-10 gene.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas do Ovo/genética , Genes de Helmintos , Proteínas de Helminto/metabolismo , Schistosoma mansoni/genética , Animais , Sequência de Bases , Sítios de Ligação , Cricetinae , DNA/genética , DNA/efeitos da radiação , Desoxirribonuclease I , Feminino , Regulação da Expressão Gênica , Masculino , Dados de Sequência Molecular , Ligação Proteica , Maturidade Sexual/genética , Transcrição Gênica , Raios UltravioletaRESUMO
We studied the Ca(2+)-ATPase isoforms of sarco/endoplasmic reticulum (SERCA) derived from cerebellum, cardiac muscle, and skeletal muscle. The Mg2+ dependence varied among the three enzyme preparations. The Ca2+ transport in skeletal muscle vesicles, but not in cerebellar or cardiac vesicles, was activated by free Mg2+ concentrations varying from 0.1 to 0.3 mM. Concentrations of Mg2+ of > 1 mM inhibited Ca2+ transport in all three vesicle preparations but with more pronounced effect in cerebellar and cardiac vesicles. At 10-80 microM, trifluoperazine activated Ca2+ uptake in cerebellar and cardiac vesicles but not in skeletal muscle vesicles. The activation was due to an increase in the coupling ratio between Ca2+ transport and ATP hydrolysis and was observed only in the presence of ATP concentrations of > 100 microM. The Ca2+ transport in all three vesicle preparations was inhibited by trifluoperazine concentrations of > 100 microM. The inhibition promoted by trifluoperazine was prevented by the addition of dimethylsulfoxide (10% v/v) to the medium. The Ca2+ efflux from loaded vesicles was increased by arsenate and even more by trifluoperazine. In skeletal muscle vesicles, the efflux promoted by arsenate was several-fold faster than that promoted in vesicles derived from cerebellum or cardiac muscle. In skeletal muscle, the enhancement of Ca2+ efflux promoted by both arsenate and trifluoperazine was antagonized by thapsigargin, Ca2+, Mg2+, and K+. These agents partly antagonized the enhancement of Ca2+ efflux promoted by trifluoperazine in cardiac vesicles but had little or no effect in the cerebellar vesicles. Finally, Mg.Pi and Mg.ATP, the two substrates that phosphorylate the Ca(2+)-ATPase, antagonized the effect of trifluoperazine in all of the preparations tested. The concentration of ATP needed was in the same range as that of the second K(m) value for ATP (50-300 microM) of the SERCA isoforms. The results indicate that the effect of the drugs on the cytosolic Ca2+ homeostasis may vary depending on the target tissue.
Assuntos
ATPases Transportadoras de Cálcio/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Isoenzimas/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologia , Animais , Transporte Biológico , Cálcio/metabolismo , Cálcio/farmacocinética , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Cerebelo/enzimologia , Cerebelo/ultraestrutura , Dimetil Sulfóxido/farmacologia , Cães , Retículo Endoplasmático/efeitos dos fármacos , Hidrólise , Técnicas In Vitro , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Cinética , Magnésio/farmacologia , Músculo Esquelético/enzimologia , Músculo Esquelético/ultraestrutura , Miocárdio/enzimologia , Miocárdio/ultraestrutura , Coelhos , Ratos , Retículo Sarcoplasmático/efeitos dos fármacos , Trifluoperazina/farmacologiaRESUMO
A schistosome gender specific gene (F-10), was used as a probe to characterize DNA binding proteins from adult male and female Schistoma mansoni. Using the band-shift and DNase I footprinting methods, it was found that proteins from male and female worms bound to the intact F-10 gene and to restriction fragments corresponding to different domains of the gene, generating relatively long protected sites. Clear differences between male and female proteins were only observed when nuclear proteins were tested. Thus, gender-specific binding was detected in fragments corresponding to the 5' and 3' ends. UV-induced cross-linking between schistosome proteins and a synthetic oligonucleotide bearing a steroid response element present in the 3' untranslated end of the F-10 gene, revealed a major DNA binding protein with a molecular mass of 30 kDa, in both male and female worms. These results suggested that the activation of transcription of the F-10 gene may depend essentially on nuclear proteins.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA/genética , Proteínas do Ovo/genética , Genes de Helmintos , Proteínas de Helminto/metabolismo , Schistosoma mansoni/genética , Caracteres Sexuais , Animais , Sequência de Bases , Sítios de Ligação , Cricetinae , DNA/metabolismo , DNA/efeitos da radiação , Proteínas de Ligação a DNA/efeitos da radiação , Desoxirribonuclease I , Feminino , Proteínas de Helminto/efeitos da radiação , Masculino , Mesocricetus , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Transcrição Gênica , Raios UltravioletaRESUMO
Vesicles derived from the dense tubular system of platelets possess a Ca(2+)-ATPase that can use either ATP or acetyl phosphate as a substrate. In the presence of phosphate as a precipitating anion, the maximum amount of Ca2+ accumulated by the vesicles with the use of acetyl phosphate was only one-third of that accumulated with the use of ATP. Vesicles derived from the sarcoplasmic reticulum of skeletal muscle accumulated equal amounts of Ca2+ regardless of the substrate used. When acetyl phosphate was used in platelet vesicles, the transport of Ca2+ was inhibited by Na+, Li+, and K+; in sarcoplasmic reticulum vesicles, only Na+ caused inhibition. When ATP was used as substrate, the different monovalent cation had no effect on either sarcoplasmic reticulum or platelet vesicles. The catalytic cycle of the Ca(2+)-ATPase is reversed when a Ca2+ gradient is formed across the vesicle membrane. The stoichiometry between active Ca2+ efflux and ATP synthesis was one in platelet vesicles and two in sarcoplasmic reticulum vesicles. The coupling between ATP synthesis and Ca2+ efflux in sarcoplasmic reticulum vesicles was abolished by arsenate regardless of whether the vesicles were loaded with Ca2+ using acetyl phosphate or ATP. In platelets, uncoupling was observed only when the vesicles were loaded using acetyl phosphate. In both sarcoplasmic reticulum and platelet vesicles, the effect of arsenate was antagonized by thapsigargin (2 microM), micromolar Ca2+ concentrations, P(i) (5-20 mM), and MgATP (10-100 microM). Trifluoperazine also uncoupled the platelet Ca2+ pump but, different from arsenate, this drug was effective in vesicles that were loaded using either ATP or acetyl phosphate. Trifluoperazine enhanced Ca2+ efflux from both sarcoplasmic reticulum and platelet vesicles; thapsigargin, Ca2+, Mg2+, or K+ antagonized this effect in sarcoplasmic reticulum but not in platelet vesicles. The data indicate that the Ca(2+)-transport isoforms found in sarcoplasmic reticulum and in platelets have different kinetic properties.
Assuntos
Plaquetas/enzimologia , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Isoenzimas/metabolismo , Músculo Esquelético/enzimologia , Arseniatos/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Retículo Endoplasmático/enzimologia , Heparina/farmacologia , Humanos , Inositol 1,4,5-Trifosfato/farmacologia , Cinética , Especificidade por Substrato , Trifluoperazina/farmacologiaRESUMO
Serine racemase is a brain-enriched enzyme that synthesizes d-serine, an endogenous modulator of the glycine site of N-methyl-d-aspartate (NMDA) receptors. We now report that serine racemase catalyzes an elimination reaction toward a nonphysiological substrate that provides a powerful tool to study its neurobiological role and will be useful to develop selective enzyme inhibitors. Serine racemase catalyzes robust elimination of l-serine O-sulfate that is 500 times faster than the physiological racemization reaction, generating sulfate, ammonia, and pyruvate. This reaction provides the most simple and sensitive assay to detect the enzyme activity so far. We establish stable cell lines expressing serine racemase and show that serine racemase can also be converted into a powerful eliminase in cultured cells, while the racemization of l-serine is inhibited. Likewise, l-serine O-sulfate inhibits the synthesis of d-serine in primary astrocyte cultures. We conclude that the synthetic compound l-serine O-sulfate is a better substrate than l-serine as well as an inhibitor of d-serine synthesis. Inhibition of serine racemase provides a new strategy to selectively decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.
Assuntos
Racemases e Epimerases/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/análogos & derivados , Serina/metabolismo , Amônia/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Camundongos , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Racemases e Epimerases/antagonistas & inibidores , Racemases e Epimerases/genética , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/metabolismo , Serina/biossíntese , Serina/química , Estereoisomerismo , Especificidade por SubstratoRESUMO
Alpha-synuclein is believed to play an important role in Parkinson's disease (PD). Mutations in the alpha-synuclein gene are responsible for familial forms of PD and alpha-synuclein protein is a major component of Lewy bodies in patients with sporadic PD. Synphilin-1 is a novel protein that we have previously found to associate in vivo with alpha-synuclein. We now show that synphilin-1 is present in Lewy bodies of patients with PD. Our data suggest that synphilin-1 could play a role in Lewy body formation and the pathogenesis of PD.
Assuntos
Proteínas de Transporte/análise , Corpos de Lewy/química , Corpos de Lewy/patologia , Proteínas do Tecido Nervoso/análise , Doença de Parkinson/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos , Western Blotting , Proteínas de Transporte/imunologia , Humanos , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/imunologia , Neurônios/patologia , Coelhos , Substância Negra/patologia , Sinucleínas , alfa-SinucleínaRESUMO
The effects of acidic pH on the kinetics of Ca2+-ATPase isoforms from intracellular membranes of skeletal muscle, cardiac muscle, cerebellum and blood platelets were studied. At neutral pH, all four Ca2+-ATPase isoforms exhibited similar Ca2+-concentration requirements for half-maximal rates of Ca2+ uptake and ATP hydrolysis. A decrease in the pH from 7.0 to 6.0 promoted a decrease in both the apparent affinity for Ca2+ [increasing half-maximal activation (K0.5)] and the maximal velocity (Vmax) of Ca2+ uptake. With skeletal muscle vesicles these effect were 5 to 10 times smaller than those observed with all the other isoforms. Acidification of the medium from pH 7.0 to 6.5 caused the release of Ca2+ from loaded vesicles and a decrease in the amount of Ca2+ retained by the vesicles at the steady state. With the vesicles derived from skeletal muscle these effects were smaller than for vesicles derived from other tissues. The rate of passive Ca2+ efflux from skeletal and cardiac muscle vesicles, loaded with Ca2+ and diluted in a medium containing none of the ligands of Ca2+-ATPase, was the same at pH 7.0 and 6.0. In contrast, the rate of Ca2+ efflux from cerebellar and platelet vesicles increased 2-fold after acidification of the medium. The effects of DMSO, Mg2+ with Pi and arsenate on the rate of Ca2+ efflux varied among the different preparations tested. The differences became more pronounced when the pH of the medium was decreased from 7.0 to 6.0. It is proposed that the kinetic differences among the Ca2+-ATPase isoforms may reflect different adaptations to cellular acidosis, such as that which occurs during ischaemia.
Assuntos
Acidose/enzimologia , ATPases Transportadoras de Cálcio/metabolismo , Isoenzimas/metabolismo , Retículo Sarcoplasmático/enzimologia , Animais , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Concentração de Íons de Hidrogênio , Líquido Intracelular/metabolismo , Líquido Intracelular/fisiologia , Fosfatos/metabolismo , Fosfatos/fisiologia , CoelhosRESUMO
Parkinson's disease (PD) is a common progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic neural cell death occurs remains unknown. Proteins encoded by two other genes in which mutations cause familial PD, parkin and UCH-L1, are involved in regulation of the ubiquitin-proteasome pathway, suggesting that dysregulation of the ubiquitin-proteasome pathway is involved in the mechanism by which these mutations cause PD. We established inducible PC12 cell lines in which wild-type or mutant alpha-synuclein can be de-repressed by removing doxycycline. Differentiated PC12 cell lines expressing mutant alpha-synuclein showed decreased activity of proteasomes without direct toxicity. Cells expressing mutant alpha-synuclein showed increased sensitivity to apoptotic cell death when treated with sub-toxic concentrations of an exogenous proteasome inhibitor. Apoptosis was accompanied by mitochondrial depolarization and elevation of caspase-3 and -9, and was blocked by cyclosporin A. These data suggest that expression of mutant alpha-synuclein results in sensitivity to impairment of proteasome activity, leading to mitochondrial abnormalities and neuronal cell death.
Assuntos
Acetilcisteína/análogos & derivados , Apoptose , Cisteína Endopeptidases/metabolismo , Mitocôndrias/fisiologia , Complexos Multienzimáticos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Células PC12/fisiologia , Acetilcisteína/farmacologia , Animais , Western Blotting , Caspase 3 , Caspase 9 , Caspases/metabolismo , Ciclosporina/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Regulação da Expressão Gênica , Técnicas Imunoenzimáticas , Potenciais da Membrana , Mutação , Proteínas do Tecido Nervoso/genética , Células PC12/efeitos dos fármacos , Doença de Parkinson/patologia , Complexo de Endopeptidases do Proteassoma , Ratos , Sinucleínas , Tetraciclina/farmacologia , Transfecção , alfa-SinucleínaRESUMO
We have recently identified a protein we called synphilin-1, which interacts in vivo with alpha-synuclein. Mutations in alpha-synuclein cause familial Parkinson's disease (PD). Alpha-synuclein protein is present in the pathologic lesions of familial and sporadic PD, and diffuse Lewy body disease, indicating an important pathogenic role for alpha-synuclein. Here we describe the structure of the human synphilin-1 gene (SNCAIP). The open reading frame of this gene is contained within ten exons. We have designed primers to amplify each SNCAIP exon, so these primers can now be used to screen for mutations or polymorphisms in patients with Parkinson's disease or related diseases. We found a highly polymorphic GT repeat within intron 5 of SNCAIP, suitable for linkage analysis of families with PD. We have mapped SNCAIP locus to Chromosome (Chr) 5q23.1-23.3 near markers WI-4673 and AFMB352XH5. In addition, using immunohistochemistry in human postmortem brain tissue, we found that synphilin-1 protein is present in neuropil, similar to alpha-synuclein protein. Because of its association with alpha-synuclein, synphilin-1 may be a candidate for involvement in Parkinson's disease or other related disorders.
Assuntos
Proteínas de Transporte/genética , Genes/genética , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Sequência de Bases , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Transporte/metabolismo , Mapeamento Cromossômico , Cromossomos Humanos Par 5/genética , DNA/química , DNA/genética , Éxons , Predisposição Genética para Doença , Humanos , Células Híbridas , Imuno-Histoquímica , Íntrons , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Polimorfismo Genético , Análise de Sequência de DNARESUMO
Huntington's disease (HD) occurs when the widely expressed protein huntingtin contains an expanded glutamine repeat. The selective degeneration and neuronal morphologic abnormalities of HD may involve interactions with proteins that bind to huntingtin, such as HAP1. The biological significance of this interaction is unclear because neither HAP1 nor huntingtin have significant homology to known proteins. Therefore, we sought to identify HAP1-binding proteins. Using the yeast two-hybrid system, we isolated a rat cDNA encoding part of a protein that interacts with HAP1, and we confirmed the specificity of this interaction using an in vitro protein-binding assay. We called the protein Duo because it is closely related to the human protein Trio but is shorter. Northern blot analysis indicates brain-specific expression of Duo. Human Duo contains a guanine nucleotide exchange factor (GEF) domain that is likely to be rac1-specific, a pleckstrin homology (PH) domain and spectrin-like repeat units. These data support the hypothesis that huntingtin is involved in vesicle trafficking and cytoskeletal functions, and raise the possibility of a role for huntingtin in the regulation of a ras-related signaling pathway.
Assuntos
Proteínas de Transporte/metabolismo , Doença de Huntington/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Encéfalo/metabolismo , Proteínas de Transporte/genética , Fatores de Troca do Nucleotídeo Guanina , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas/química , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores ras de Troca de Nucleotídeo GuaninaRESUMO
Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine repeat in the HD protein huntingtin. Huntingtin's localization within the cell includes an association with cytoskeletal elements and vesicles. We previously identified a protein (HAP1) which binds to huntingtin in a glutamine repeat length-dependent manner. We now report that HAP1 interacts with cytoskeletal proteins, namely the p150 Glued subunit of dynactin and the pericentriolar protein PCM-1. Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils. We examined the interaction of HAP1 with p150 Glued . Binding of HAP1 to p150 Glued (amino acids 879-1150) was confirmed in vitro by binding of p150 Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads. Also, HAP1 co-immunoprecipitated with p150 Glued from brain extracts, indicating that the interaction occurs in vivo . Like HAP1, p150 Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction. Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150 Glued partially co-localize. These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins. Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD.