RESUMO
Per- and polyfluoroalkyl substances (PFAS) are recalcitrant synthetic organohalides known to negatively impact human health. Short-chain fluorotelomer alcohols are considered the precursor of various perfluorocarboxylic acids (PFCAs) in the environment. Their ongoing production and widespread detection motivate investigations of their biological transformation. Dietzia aurantiaca strain J3 was isolated from PFAS-contaminated landfill leachate using 6:2 fluorotelomer sulphonate (6:2 FTS) as a sulphur source. Resting cell experiments were used to test if strain J3 could transform fluorotelomer alcohols (6:2 and 4:2 FTOH). Strain J3 transformed fluorotelomer alcohols into PFCAs, polyfluorocarboxylic acids and transient intermediates. Over 6 days, 80 % and 58 % of 6:2 FTOH (0.1 mM) and 4:2 FTOH (0.12 mM) were degraded with 6.4 % and 14 % fluoride recovery respectively. Fluorotelomer unsaturated carboxylic acid (6:2 FTUCA) was the most abundant metabolite, accounting for 21 to 30 mol% of 6:2 FTOH (0.015 mM) applied on day zero. Glutathione (GSH) conjugates of 6:2/4:2 FTOH and 5:3 FTCA adducts were also structurally identified. Proteomics studies conducted to identify enzymes in the biotransformation pathway have revealed the role of various enzymes involved in ß oxidation. This is the first report of 6:2/4:2 FTOH glutathione conjugates and 5:3 FTCA adducts in prokaryotes, and the first study to explore the biotransformation of 4:2 FTOH by pure bacterial strain.
RESUMO
Polychlorinated dibenzo-p-dioxins and furans (PCDD/F) are some of the most environmentally recalcitrant and toxic compounds. They occur naturally and as by-products of anthropogenic activity. Sydney Harbour Estuary (Sydney, Australia), is heavily contaminated with PCDD/F. Analysis of sediment cores revealed that the contamination source area in Homebush Bay continues to have one of the highest levels of PCDD/F contamination in the world (5207 pg WHO-TEQ g-1) with >50% of the toxicity attributed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), the most toxic PCDD/F congener. Comparison of congener profiles at the contamination source area with surrounding bays and historical data provided evidence for the attenuation of 2,3,7,8-TCDD and other congeners at the source area. This finding was supported by the detection of di-, mono- and unchlorinated dibenzo-p-dioxin. Microbial community analysis of sediments by 16S rRNA amplicon sequencing revealed an abundance of lineages from the class Dehalococcoidia (up to 15% of the community), including the genus Dehalobium (up to 0.5%). Anaerobic seawater enrichment cultures using perchloroethene as more biologically available growth substrate enriched the Dehalobium population by more than six-fold. The enrichment culture then proved capable of reductively dechlorinating 2,3,7,8-TCDD to 2,3,7-TriCDD and octachlorodibenzo-p-dibenzodioxin (OCDD) to hepta and hexa congeners. This work is the first to show microbial reductive dehalogenation of 2,3,7,8-TCDD with a bacterium from outside the Dehalococcoides genus, and one of only a few that demonstrates PCDD/F dechlorination in a marine environment.
Assuntos
Benzofuranos , Dioxinas , Dibenzodioxinas Policloradas , Baías , Benzofuranos/análise , Dibenzofuranos Policlorados/análise , Dioxinas/análise , Estuários , Dibenzodioxinas Policloradas/análise , RNA Ribossômico 16S/genéticaRESUMO
The polyfluorinated alkyl substance 6:2 fluorotelomer sulfonate (6:2 FTS) has been detected in diverse environments impacted by aqueous film-forming foams used for firefighting. In this study, a bacterial strain (J3) using 6:2 FTS as a sulfur source was isolated from landfill leachate previously exposed to polyfluoroalkyl substances in New South Wales, Australia. Strain J3 shares 99.9% similarity with the 16S rRNA gene of Dietzia aurantiaca CCUG 35676T. Genome sequencing yielded a draft genome sequence of 37 contigs with a G + C content of 69.7%. A gene cluster related to organic sulfur utilisation and assimilation was identified, that included an alkanesulfonate monooxygenase component B (ssuD), an alkanesulfonate permease protein (ssuC), an ABC transporter (ssuB), and an alkanesulfonate-binding protein (ssuA). Proteomic analyses comparing strain J3 cultures using sulfate and 6:2 FTS as sulfur source indicated that the ssu gene cluster was involved in 6:2 FTS biodegradation. Upregulated proteins included the SsuD monooxygenase, the SsuB transporter, the ABC transporter permease (SsuC), an alkanesulfonate-binding protein (SsuA), and a nitrilotriacetate monooxygenase component B. 6:2 Fluorotelomer carboxylic acid (6:2 FTCA) and 6:2 fluorotelomer unsaturated acid (6:2 FTUA) were detected as early degradation products in cultures (after 72 h) while 5:3 fluorotelomer acid (5:3 FTCA), perfluorohexanoic acid (PFHxA) and perfluoropentanoic acid (PFPeA) were detected as later degradation products (after 168 h). This work provides biochemical and metabolic insights into 6:2 FTS biodegradation by the Actinobacterium D. aurantiaca J3, informing the fate of PFAS in the environment.
Assuntos
Fluorocarbonos , Poluentes Químicos da Água , Transportadores de Cassetes de Ligação de ATP/metabolismo , Actinobacteria , Alcanossulfonatos/análise , Biotransformação , Fluorocarbonos/análise , Proteínas de Membrana Transportadoras/metabolismo , Oxigenases de Função Mista/metabolismo , Proteômica , RNA Ribossômico 16S/genética , Enxofre/metabolismo , Poluentes Químicos da Água/análiseRESUMO
One hypothesis for the etiology of Parkinson's disease (PD) is that subsets of neurons are vulnerable to a failure in proteasome-mediated protein turnover. Here we show that overexpression of mutant alpha-synuclein increases sensitivity to proteasome inhibitors by decreasing proteasome function. Overexpression of parkin decreases sensitivity to proteasome inhibitors in a manner dependent on parkin's ubiquitin-protein E3 ligase activity, and antisense knockdown of parkin increases sensitivity to proteasome inhibitors. Mutant alpha-synuclein also causes selective toxicity to catecholaminergic neurons in primary midbrain cultures, an effect that can be mimicked by the application of proteasome inhibitors. Parkin is capable of rescuing the toxic effects of mutant alpha-synuclein or proteasome inhibition in these cells. Therefore, parkin and alpha-synuclein are linked by common effects on a pathway associated with selective cell death in catecholaminergic neurons.
Assuntos
Catecolaminas/metabolismo , Cisteína Endopeptidases/metabolismo , Ligases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Doença de Parkinson/genética , Substância Negra/metabolismo , Ubiquitina-Proteína Ligases , Animais , Animais Recém-Nascidos , Contagem de Células , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisteína Endopeptidases/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Humanos , Imuno-Histoquímica , Ligases/genética , Camundongos , Complexos Multienzimáticos/efeitos dos fármacos , Mutação/genética , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Doença de Parkinson/metabolismo , Complexo de Endopeptidases do Proteassoma , Substância Negra/fisiopatologia , Sinucleínas , Células Tumorais Cultivadas , Tirosina 3-Mono-Oxigenase/metabolismo , Regulação para Cima/genética , alfa-SinucleínaRESUMO
Mutations in torsinA, a member of the AAA+ family of ATPases, are associated with early onset-dystonia. A closely related homologue, torsinB, has also been described but the significance of this second form is not clear. Here, we demonstrate that in transfected cells, torsinB has similar electrophoretic mobility to torsinA but is more basic consistent with predictions from the cDNA sequence. Like torsinA, torsinB is glycosylated and localized to PDI-positive structures in cells. However, torsinB unlike torsinA has a tendency to form intracellular inclusions when expressed at similar levels. We were able to confirm previous reports that torsinA is present in brainstem Lewy bodies, but we saw no torsinB-like immunoreactivity in the same structures. These results show that torsins A and B are similar proteins, although there are differences in the abundance of the two homologues and in their recruitment into Lewy bodies.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Sequência de Aminoácidos , Northern Blotting/métodos , Encéfalo/metabolismo , Química Encefálica , Proteínas de Transporte/genética , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Embrião de Mamíferos , Expressão Gênica , Glicosilação , Humanos , Imuno-Histoquímica/métodos , Isocianatos , Corpos de Lewy/metabolismo , Microscopia Confocal , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , TransfecçãoRESUMO
An in-frame deletion (Delta E302/303) in the TorsinA gene has been demonstrated to be responsible for primary torsion dystonia, showing dominant inheritance with reduced penetrance. The Delta E302/303 torsinA mutation forms intracellular ER derived inclusions in a variety of cultured cells, which may suggest that the mutations might evoke ER stress. We used microarray analysis of human derived cell lines expressing the Delta E302/303 torsinA mutation in order to reveal alterations in gene expression in the hope of identifying genetic modifying loci or novel markers for disease pathogenesis. We identified transcriptional changes in multiple members of the heat shock protein family of genes, confirmed by reverse transcription-polymerase chain reaction, which could be indicative of ER stress. However, both wild type and mutant torsinA were affected to a similar extent, suggesting that this is not related to either disease state or the formation of ER-derived inclusions.
Assuntos
Proteínas de Transporte/genética , Distonia Muscular Deformante/genética , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Chaperonas Moleculares , RNA Mensageiro/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Distonia Muscular Deformante/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Humanos , Rim/embriologia , Rim/metabolismo , Mutagênese Sítio-Dirigida , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/metabolismo , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , TransfecçãoRESUMO
Two mutations in torsinA have been identified to date, both of which are associated with an autosomal dominant form of early onset-dystonia. It has been reported previously that expression of the more common mutation, a deletion of one of a pair of glutamates (deltaE302/303) produces intracellular, endoplasmic reticulum-derived inclusions in cultured cells. In this study we have replicated these previous results and have additionally looked at the localization of the more recently described deltaF323-Y328 mutation. We show that the localization of this latter mutation is similar to wild type torsinA and unlike the deltaE302/303 mutation. This data suggests that the formation of intracellular inclusions is specific to deltaE302/303 and not a property shared by deltaF323-Y328.
Assuntos
Proteínas de Transporte/genética , Distonia Muscular Deformante/genética , Chaperonas Moleculares , Adulto , Sequência de Aminoácidos , Proteínas de Transporte/análise , Células Cultivadas , Citoplasma/química , DNA Complementar/genética , Expressão Gênica , Humanos , Corpos de Inclusão/química , Rim/citologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , TransfecçãoRESUMO
Mutations in the parkin gene cause the majority of cases of familial-linked Parkinson's disease, and mounting evidence suggests that parkin may play a role in idiopathic disease. Previous reports suggest that parkin may respond to and relieve, via E3-ligase activity, cellular stress at the endoplasmic reticulum caused by the accumulation of unfolded proteins. However, parkin's relationship to the mammalian unfolded protein response is unclear. Here, we comprehensively evaluate endogenous parkin in SH-SY5Y neuroblastomas at the promoter, RNA, and protein levels in response to unfolded protein stress induced by tunicamycin. While we find strong up-regulation of genes linked to the unfolded protein stress pathway, we detect no significant changes in parkin. These data suggest a lack of association between parkin and the unfolded protein response in SH-SY5Y cells.
Assuntos
Proteínas de Choque Térmico , Ligases/metabolismo , Neuroblastoma/metabolismo , Dobramento de Proteína , Antibacterianos/farmacologia , Western Blotting , Proteínas de Transporte/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica , Humanos , Ligases/química , Ligases/efeitos dos fármacos , Chaperonas Moleculares/metabolismo , Neuroblastoma/patologia , Fragmentos de Peptídeos/metabolismo , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Tempo , Transfecção/métodos , Tunicamicina/farmacologia , Ubiquitina-Proteína LigasesRESUMO
Synphilin-1 has been shown to interact with alpha-synuclein, which in turn is associated with Parkinson's disease. However, the function of synphilin-1 is unknown. We have cloned mouse synphilin in an attempt to describe conserved and therefore likely functional domains. The deduced amino acid sequence of the protein shows extensive homology with its human counterpart, with greatest similarities in those regions that contain ankyrin-like motifs and the coiled-coil domain. Expression of mouse synphilin-1 across tissues is similar to its human counterpart and not limited to brain. The results show that the synphilin-1 sequence and expression patterns are conserved across species.
Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Animais , Repetição de Anquirina/genética , Sítios de Ligação/genética , Encéfalo/fisiopatologia , Proteínas de Transporte/genética , Clonagem Molecular , Regulação da Expressão Gênica/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Sinucleínas , alfa-SinucleínaAssuntos
Proteínas de Transporte/genética , Distonia/genética , Genes Dominantes , Chaperonas Moleculares , Mutação , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Distonia/metabolismo , Humanos , Imuno-Histoquímica , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , TransfecçãoRESUMO
Mutations in the parkin gene (PRKN) are the commonest cause of juvenile and early-onset parkinsonism. However, the pathogenic mechanism by which loss of parkin protein results in degeneration of dopaminergic neurons remains elusive. Animal models provide a useful tool for the study of development and disease, and the recent production of transgenic fly and mouse parkin deficient models allows investigation of the molecular role of parkin in dopamine regulation and nigrostriatal function. We have identified the mouse mutant Quaking as a spontaneously occurring PRKN knockout. The quaking mutation is a deletion of approximately 1.17 Mb of mouse chromosome 17, resulting in the deletion of the entire promoter and first five coding exons of PRKN In addition, the recently described Parkin Co-Regulated Gene (PACRG) is completely deleted. Homozygous Quaking mice show a complete loss of PRKN and PACRG mRNA and protein. These mice will constitute a useful additional model for studies of the molecular role of parkin and PACRG in neurodegeneration.
Assuntos
Deleção Cromossômica , Camundongos Knockout/genética , Camundongos Quaking/genética , Proteínas/genética , Ubiquitina-Proteína Ligases/genética , Animais , Mapeamento Cromossômico , Cromossomos de Mamíferos , Modelos Animais de Doenças , Éxons/genética , Genótipo , Homozigoto , Camundongos , Proteínas dos Microfilamentos , Chaperonas Moleculares , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genéticaRESUMO
The Parkin gene (PRKN) encodes an E3 protein-ubiquitin ligase for which loss of function is associated with autosomal-recessive juvenile (<20 years) and early-onset Parkinsonism (<45 years). Although detailed pathological reports are scarce, brains from patients with homozygous exonic deletions demonstrate neuronal loss in the substantia nigra, albeit without the Lewy body pathology characteristic of idiopathic Parkinson's disease. However, there are rare descriptions of more florid pathology, including Lewy bodies and tau positive astrocytes in individuals with compound heterozygous mutations. In the present study we examined whether PRKN point mutations, leading to amino acid substitutions, may alter the cellular distribution of the protein produced. Wild-type Parkin was homogeneously distributed throughout the cytoplasm with a small amount of protein in the nucleus after transfection into human embryonic kidney cells. Mutant isoforms with A82E, G328E and C431F amino acid substitutions were also normally distributed. However, two mutant isoforms, R256C and R275W, within RING finger 1 of the Parkin protein (238-293 amino acids), produced an unusual distribution of the protein, with large cytoplasmic and nuclear inclusions. We have replicated this observation in primary cultured neurons and demonstrate, by the accumulation/co-localization of cytoskeletal protein vimentin, that the inclusion bodies are aggresomes, a cellular response to misfolded protein.
Assuntos
Mutação Puntual , Ubiquitina-Proteína Ligases/genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Núcleo Celular/química , Células Cultivadas , Clonagem Molecular , Citoplasma/química , Hipocampo/citologia , Humanos , Corpos de Lewy/metabolismo , Mutagênese Sítio-Dirigida , Neurônios/citologia , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Vimentina/biossínteseRESUMO
Mutations in the parkin gene are common in early-onset and familial Parkinson's disease (PD), and the parkin protein interacts in the ubiquitin-proteasome system as an E3 ligase. However, the regulatory pathways that govern parkin expression are unknown. In this study, we showed that a phylogenetically conserved N-myc binding site in the bi-directional parkin promoter interacted with myc-family transcription factors in reporter assays, and N-myc bound to the parkin promoter in chromatin immunoprecipitation assays and repressed transcription activity. Parkin expression was inversely correlated with N-myc levels in the developing mouse and human brain, in human neuroblastoma cell lines with various levels of n-myc amplification, and in an inducible N-myc cell line. Although parkin and N-myc expression were dramatically altered upon retinoic acid-induced differentiation of a human neuroblastoma cell line, modulation of parkin expression did not significantly affect either rates of cellular proliferation or levels of cyclin E. Analysis of additional genes associated with familial PD revealed a shared basis of transcription regulation mediated by N-myc and the cell cycle. Our results, in combination with functional knowledge of the proteins encoded by these genes, suggest a common pathway linking together PD, the ubiquitin-proteasome system, and cell cycle control.
Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Ciclina E/metabolismo , Evolução Molecular , Genes Reporter , Humanos , Camundongos , Doença de Parkinson/genética , Ligação Proteica , Tretinoína/metabolismoRESUMO
Abnormal accumulation of alpha-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) alpha-synuclein using microarrays, with confirmation of selected genes by quantitative RT-PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis-inducing molecules, transcription factors and membrane-bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co-ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild-type but not mutant alpha-synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co-ordinate regulation of dopamine synthesis is regulated through this transcription factor.