RESUMO
Mutations in LRRK2 cause autosomal dominant and sporadic Parkinson's disease, but the mechanisms involved in LRRK2 toxicity in PD are yet to be fully understood. We found that LRRK2 translocates to the nucleus by binding to seven in absentia homolog (SIAH-1), and in the nucleus it directly interacts with lamin A/C, independent of its kinase activity. LRRK2 knockdown caused nuclear lamina abnormalities and nuclear disruption. LRRK2 disease mutations mostly abolish the interaction with lamin A/C and, similar to LRRK2 knockdown, cause disorganization of lamin A/C and leakage of nuclear proteins. Dopaminergic neurons of LRRK2 G2019S transgenic and LRRK2 -/- mice display decreased circularity of the nuclear lamina and leakage of the nuclear protein 53BP1 to the cytosol. Dopaminergic nigral and cortical neurons of both LRRK2 G2019S and idiopathic PD patients exhibit abnormalities of the nuclear lamina. Our data indicate that LRRK2 plays an essential role in maintaining nuclear envelope integrity. Disruption of this function by disease mutations suggests a novel phosphorylation-independent loss-of-function mechanism that may synergize with other neurotoxic effects caused by LRRK2 mutations.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Membrana Nuclear/metabolismo , Doença de Parkinson/genética , Animais , Células Cultivadas , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Células HEK293 , Humanos , Lamina Tipo A/metabolismo , Mutação com Perda de Função , Camundongos , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação , Ratos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
α-Synuclein accumulation is a pathological hallmark of Parkinson's disease (PD). Ubiquitinated α-synuclein is targeted to proteasomal or lysosomal degradation. Here, we identify SUMOylation as a major mechanism that counteracts ubiquitination by different E3 ubiquitin ligases and regulates α-synuclein degradation. We report that PIAS2 promotes SUMOylation of α-synuclein, leading to a decrease in α-synuclein ubiquitination by SIAH and Nedd4 ubiquitin ligases, and causing its accumulation and aggregation into inclusions. This was associated with an increase in α-synuclein release from the cells. A SUMO E1 inhibitor, ginkgolic acid, decreases α-synuclein levels by relieving the inhibition exerted on α-synuclein proteasomal degradation. α-Synuclein disease mutants are more SUMOylated compared with the wild-type protein, and this is associated with increased aggregation and inclusion formation. We detected a marked increase in PIAS2 expression along with SUMOylated α-synuclein in PD brains, providing a causal mechanism underlying the up-regulation of α-synuclein SUMOylation in the disease. We also found a significant proportion of Lewy bodies in nigral neurons containing SUMO1 and PIAS2. Our observations suggest that SUMOylation of α-synuclein by PIAS2 promotes α-synuclein aggregation by two mutually reinforcing mechanisms. First, it has a direct proaggregatory effect on α-synuclein. Second, SUMOylation facilitates α-synuclein aggregation by blocking its ubiquitin-dependent degradation pathways and promoting its accumulation. Therefore, inhibitors of α-synuclein SUMOylation provide a strategy to reduce α-synuclein levels and possibly aggregation in PD.
Assuntos
Doença de Parkinson/metabolismo , Proteólise , Sumoilação , alfa-Sinucleína/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Ratos Sprague-Dawley , Salicilatos/farmacologia , Substância Negra/metabolismoRESUMO
PTEN-induced putative kinase 1 (PINK1) and parkin are mutated in familial forms of Parkinson's disease and are important in promoting the mitophagy of damaged mitochondria. In this study, we showed that synphilin-1 interacted with PINK1 and was recruited to the mitochondria. Once in the mitochondria, it promoted PINK1-dependent mitophagy, as revealed by Atg5 knockdown experiments and the recruitment of LC3 and Lamp1 to the mitochondria. PINK1-synphilin-1 mitophagy did not depend on PINK1-mediated phosphorylation of synphilin-1 and occurred in the absence of parkin. Synphilin-1 itself caused depolarization of the mitochondria and increased the amount of uncleaved PINK1 at the organelle. Furthermore, synphilin-1 recruited seven in absentia homolog (SIAH)-1 to the mitochondria where it promoted mitochondrial protein ubiquitination and subsequent mitophagy. Mitophagy via this pathway was impaired by synphilin-1 knockdown or by the use of a synphilin-1 mutant that is unable to recruit SIAH-1 to the mitochondria. Likewise, knockdown of SIAH-1 or the use of a catalytically inactive SIAH-1 mutant abrogated mitophagy. PINK1 disease mutants failed to recruit synphilin-1 and did not activate mitophagy, indicating that PINK1-synphilin-1-SIAH-1 represents a new parkin-independent mitophagy pathway. Drugs that activate this pathway will provide a novel strategy to promote the clearance of damaged mitochondria in Parkinson's disease.
Assuntos
Proteínas de Transporte/genética , Mitofagia/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Doença de Parkinson/genética , Proteínas Quinases/genética , Ubiquitina-Proteína Ligases/genética , Proteína 5 Relacionada à Autofagia/genética , Proteínas de Transporte/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação , Ligação Proteica , Proteínas Quinases/metabolismo , Transdução de Sinais , Ubiquitina , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Accumulation of α-synuclein is key to the pathogenesis of Parkinson's disease (PD), though the exact mechanisms involved in its toxicity are still subject to debate. Increased α-synuclein expression or reduced degradation may play a role in the proteotoxicity observed in PD. Here we review the mechanisms of α-synuclein ubiquitination by different E3 ubiquitin-ligases, and its degradation via the proteasome, autophagy and lysosomes. Activators of α- synuclein ubiquitination and degradation pathways represent a plausible strategy to decrease α-synuclein burden in the disease. Nevertheless, since proteasomes and autophagy might be impaired in the disease, and because proteolytic impairment causes the accumulation of monoubiquitinated α-synuclein and the formation of toxic inclusions, compounds that promote α-synuclein monoubiquitination should be used in concert with compounds that boost these proteolytic pathways. This combined approach may therefore ease the accumulation of α-synuclein in PD and may represent a promising new avenue for the development of novel treatments for the disease.
Assuntos
Antiparkinsonianos/farmacologia , Doença de Parkinson/tratamento farmacológico , Ubiquitinação/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Animais , Humanos , Doença de Parkinson/metabolismoRESUMO
Parkin E3 ubiquitin-ligase activity and its role in mitochondria homeostasis are thought to play a role in Parkinson's disease (PD). We now report that AF-6 is a novel parkin interacting protein that modulates parkin ubiquitin-ligase activity and mitochondrial roles. Parkin interacts with the AF-6 PDZ region through its C-terminus. This leads to ubiquitination of cytosolic AF-6 and its degradation by the proteasome. On the other hand, endogenous AF-6 robustly increases parkin translocation and ubiquitin-ligase activity at the mitochondria. Mitochondrial AF-6 is not a parkin substrate, but rather co-localizes with parkin and enhances mitochondria degradation through PINK1/parkin-mediated mitophagy. On the other hand, several parkin and PINK1 juvenile disease-mutants are insensitive to AF-6 effects. AF-6 is present in Lewy bodies and its soluble levels are strikingly decreased in the caudate/putamen and substantia nigra of sporadic PD patients, suggesting that decreased AF-6 levels may contribute to the accumulation of dysfunctional mitochondria in the disease. The identification of AF-6 as a positive modulator of parkin translocation to the mitochondria sheds light on the mechanisms involved in PD and underscores AF-6 as a novel target for future therapeutics.
Assuntos
Cinesinas/metabolismo , Mitocôndrias/metabolismo , Mutação , Miosinas/metabolismo , Doença de Parkinson/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Núcleo Caudado/metabolismo , Núcleo Caudado/patologia , Células HEK293 , Humanos , Cinesinas/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Miosinas/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteínas Quinases/genética , Transporte Proteico/genética , Proteólise , Substância Negra/metabolismo , Substância Negra/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genéticaRESUMO
α-Synuclein is central to the pathogenesis of Parkinson disease (PD). Mutations as well as accumulation of α-synuclein promote the death of dopaminergic neurons and the formation of Lewy bodies. α-Synuclein is monoubiquitinated by SIAH, but the regulation and roles of monoubiquitination in α-synuclein biology are poorly understood. We now report that the deubiquitinase USP9X interacts in vivo with and deubiquitinates α-synuclein. USP9X levels are significantly lower in cytosolic fractions of PD substantia nigra and Diffuse Lewy Body disease (DLBD) cortices compared to controls. This was associated to lower deubiquitinase activity toward monoubiquitinated α-synuclein in DLBD cortical extracts. A fraction of USP9X seems to be aggregated in PD and DLBD, as USP9X immunoreactivity is detected in Lewy bodies. Knockdown of USP9X expression promotes accumulation of monoubiquitinated α-synuclein species and enhances the formation of toxic α-synuclein inclusions upon proteolytic inhibition. On the other hand, by manipulating USP9X expression levels in the absence of proteolytic impairment, we demonstrate that monoubiquitination controls the partition of α-synuclein between different protein degradation systems. Deubiquitinated α-synuclein is mostly degraded by autophagy, while monoubiquitinated α-synuclein is preferentially degraded by the proteasome. Moreover, monoubiquitination promotes the degradation of α-synuclein, whereas deubiquitination leads to its accumulation, suggesting that the degradation of deubiquitinated α-synuclein by the autophagy pathway is less efficient than the proteasomal one. Lower levels of cytosolic USP9X and deubiquitinase activity in α-synucleinopathies may contribute to the accumulation and aggregation of monoubiquitinated α-synuclein in Lewy bodies. Our data indicate that monoubiquitination is a key determinant of α-synuclein fate.
Assuntos
Regulação da Expressão Gênica , Ubiquitina Tiolesterase/química , Ubiquitina/química , alfa-Sinucleína/química , Autofagia , Linhagem Celular Tumoral , Córtex Cerebral/metabolismo , Neurônios Dopaminérgicos/metabolismo , Humanos , Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Interferente Pequeno/metabolismoRESUMO
Parkinson disease (PD) is characterized by the presence of ubiquitylated inclusions and the death of dopaminergic neurons. Seven in absentia homolog (SIAH) is a ubiquitin-ligase that ubiquitylates alpha-synuclein and synphilin-1 and is present in Lewy bodies of PD patients. Understanding the mechanisms that regulate the ubiquitylation of PD-related proteins might shed light on the events involved in the formation of Lewy bodies and death of neurons. We show in this study that the recently described synphilin-1 isoform, synphilin-1A, interacts in vitro and in vivo with the ubiquitin-protein isopeptide ligase SIAH and regulates its activity toward alpha-synuclein and synphilin-1. SIAH promotes limited ubiquitylation of synphilin-1A that does not lead to its degradation by the proteasome. SIAH also increases the formation of synphilin-1A inclusions in the presence of proteasome inhibitors, supporting the participation of ubiquitylated synphilin-1A in the formation of Lewy body-like inclusions. Synphilin-1A/SIAH inclusions recruit PD-related proteins, such as alpha-synuclein, synphilin-1, Parkin, PINK1, and UCH-L1. We found that synphilin-1A robustly increases the steady-state levels of SIAH by decreasing its auto-ubiquitylation and degradation. In addition, synphilin-1A blocks the ubiquitylation and degradation of the SIAH substrates synphilin-1 and deleted in colon cancer protein. Furthermore, synphilin-1A strongly decreases the monoubiquitylation of alpha-synuclein by SIAH and the formation of alpha-synuclein inclusions, supporting a role for monoubiquitylation in alpha-synuclein inclusion formation. Our results suggest a novel function for synphilin-1A as a regulator of SIAH activity and formation of Lewy body-like inclusions.
Assuntos
Proteínas de Transporte/fisiologia , Corpos de Lewy/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Proteínas Nucleares/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina/química , alfa-Sinucleína/química , Animais , Bioquímica/métodos , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Humanos , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , TransfecçãoRESUMO
alpha-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of alpha-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. alpha-Synuclein is ubiquitylated in Lewy bodies, but the role of alpha-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates alpha-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates alpha-synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished alpha-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates alpha-synuclein. Moreover, SIAH co-immunoprecipitated with alpha-synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of alpha-synuclein by SIAH. Monoubiquitylation increased the aggregation of alpha-synuclein in vitro. At the electron microscopy level, monoubiquitylated alpha-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased alpha-synuclein aggregation in vivo as observed by increased formation of alpha-synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for alpha-synuclein aggregation and Lewy body formation.
Assuntos
Dopamina/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitina/química , alfa-Sinucleína/química , Linhagem Celular Tumoral , Humanos , Corpos de Inclusão/metabolismo , Corpos de Lewy/metabolismo , Lisina/química , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Ligação Proteica , Ubiquitina-Proteína Ligases/química , alfa-Sinucleína/metabolismoRESUMO
Mutations in Parkin are responsible for a large percentage of autosomal recessive juvenile parkinsonism cases. Parkin displays ubiquitin-ligase activity and protects against cell death promoted by several insults. Therefore, regulation of Parkin activities is important for understanding the dopaminergic cell death observed in Parkinson disease. We now report that cyclin-dependent kinase 5 (Cdk5) phosphorylates Parkin both in vitro and in vivo. We found that highly specific Cdk5 inhibitors and a dominant negative Cdk5 construct inhibited Parkin phosphorylation, suggesting that a significant portion of Parkin is phosphorylated by Cdk5. Parkin interacts with Cdk5 as observed by co-immunoprecipitation experiments of transfected cells and rat brains. Phosphorylation by Cdk5 decreased the auto-ubiquitylation of Parkin both in vitro and in vivo. We identified Ser-131 located at the linker region of Parkin as the major Cdk5 phosphorylation site. The Cdk5 phosphorylation-deficient S131A Parkin mutant displayed a higher auto-ubiquitylation level and increased ubiquitylation activity toward its substrates synphilin-1 and p38. Additionally, the S131A Parkin mutant more significantly accumulated into inclusions in human dopaminergic cells when compared with the wild-type Parkin. Furthermore, S131A Parkin mutant increased the formation of synphilin-1/alpha-synuclein inclusions, suggesting that the levels of Parkin phosphorylation and ubiquitylation may modulate the formation of inclusion bodies relevant to the disease. The data indicate that Cdk5 is a new regulator of the Parkin ubiquitin-ligase activity and modulates its ability to accumulate into and modify inclusions. Phosphorylation by Cdk5 may contribute to the accumulation of toxic Parkin substrates and decrease the ability of dopaminergic cells to cope with toxic insults in Parkinson disease.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Mutação de Sentido Incorreto , Doença de Parkinson/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/metabolismo , Substituição de Aminoácidos , Animais , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Quinase 5 Dependente de Ciclina/genética , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/genética , Fosforilação , Processamento de Proteína Pós-Traducional/genética , Ratos , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
alpha-Synucleinopathies are a group of neurological disorders characterized by the presence of intracellular inclusion bodies containing alpha-synuclein. We previously demonstrated that synphilin-1 interacts with alpha-synuclein, implying a role in Parkinson's disease. We now report the identification and characterization of synphilin-1A, an isoform of synphilin-1, which has enhanced aggregatory properties and causes neurotoxicity. The two transcripts encoding synphilin-1A and synphilin-1 originate from the SNCAIP gene but differ in both their exon organization and initial reading frames used for translation. Synphilin-1A binds to alpha-synuclein and induces the formation of intracellular aggregates in human embryonic kidney 293 cells, primary neuronal cultures, and human dopaminergic cells. Overexpression of synphilin-1A in neurons results in striking cellular toxicity that is attenuated by the formation of synphilin-1A inclusions, which recruit alpha-synuclein. Synphilin-1A is present in Lewy bodies of patients with Parkinson's disease and Diffuse Lewy Body disease, and is observed in detergent-insoluble fractions of brain protein samples obtained from Diffuse Lewy Body disease patients. These findings suggest that synphilin-1A may contribute to neuronal degeneration in alpha-synucleinopathies and also provide important insights into the role of inclusion bodies in neurodegenerative disorders.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/patologia , Doença de Parkinson/genética , alfa-Sinucleína/genética , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Morte Celular , Linhagem Celular , Clonagem Molecular , Códon , Éxons , Humanos , Rim , Doença por Corpos de Lewy/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/fisiologia , alfa-Sinucleína/metabolismoRESUMO
alpha-Synuclein is known to play a major role in the pathogenesis of Parkinson disease. We previously identified synphilin-1 as an alpha-synuclein-interacting protein and more recently found that synphilin-1 also interacts with the E3 ubiquitin ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 and promote its degradation through the ubiquitin proteasome system. Inability of the proteasome to degrade synphilin-1 promotes the formation of ubiquitylated inclusion bodies. We now show that synphilin-1 is phosphorylated by GSK3beta within amino acids 550-659 and that this phosphorylation is significantly decreased by pharmacological inhibition of GSK3beta and suppression of GSK3beta expression by small interfering RNA duplex. Mutation analysis showed that Ser556 is a major GSK3beta phosphorylation site in synphilin-1. GSK3beta co-immunoprecipitated with synphilin-1, and protein 14-3-3, an activator of GSK3beta activity, increased synphilin-1 phosphorylation. GSK3beta decreased the in vitro and in vivo ubiquitylation of synphilin-1 as well as its degradation promoted by SIAH. Pharmacological inhibition and small interfering RNA suppression of GSK3beta greatly increased ubiquitylation and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant, which is less phosphorylated by GSK3beta, formed more inclusion bodies than wild type synphilin-1. Inhibition of GSK3beta in primary neuronal cultures decreased the levels of endogenous synphilin-1, indicating that synphilin-1 is a physiologic substrate of GSK3beta. Using GFPu as a reporter to measure proteasome function in vivo, we found that synphilin-1 S556A is more efficient in inhibiting the proteasome than wild type synphilin-1, raising the possibility that the degree of synphilin-1 phosphorylation may regulate the proteasome function. Activation of GSK3beta during endoplasmic reticulum stress and the specific phosphorylation of synphilin-1 by GSK3beta place synphilin-1 as a possible mediator of endoplasmic reticulum stress and proteasomal dysfunction observed in Parkinson disease.
Assuntos
Proteínas de Transporte/química , Quinase 3 da Glicogênio Sintase/fisiologia , Corpos de Lewy/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina/química , Proteínas 14-3-3/química , Sítios de Ligação , Western Blotting , Linhagem Celular , Citoplasma/metabolismo , Análise Mutacional de DNA , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Genes Reporter , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Microscopia de Fluorescência , Mutação , Fosforilação , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Serina/química , Fatores de Tempo , Transfecção , Ubiquitina/metabolismoRESUMO
Parkinson's disease (PD) is a neurodegenerative disease characterized by Lewy body formation and death of dopaminergic neurons. Mutations in alpha-synuclein and parkin cause familial forms of PD. Synphilin-1 was shown to interact with alpha-synuclein and to promote the formation of cytosolic inclusions. We now report that synphilin-1 interacts with the E3 ubiquitin-ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 both in vitro and in vivo, promoting its degradation by the ubiquitin-proteasome system. Inability of the proteasome to degrade synphilin-1/SIAH complex leads to a robust formation of ubiquitylated cytosolic inclusions. Ubiquitylation is required for inclusion formation, because a catalytically inactive mutant of SIAH-1, which still binds to synphilin-1, fails to promote inclusions. Like synphilin-1, alpha-synuclein associates with SIAH in intact cells, but the interaction with SIAH-2 was much stronger that with SIAH-1. In vitro experiments show that SIAH-2 monoubiquitylates alpha-synuclein. Further evidence that SIAH proteins may play a role in inclusion formation comes from the demonstration of SIAH immunoreactivity in Lewy bodies of PD patients.
Assuntos
Proteínas de Transporte/metabolismo , Corpos de Inclusão/metabolismo , Corpos de Lewy/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Doença de Parkinson/metabolismo , Ubiquitina/metabolismo , Animais , Encéfalo/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Ligação Proteica , Proteínas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sinucleínas , Fatores de Transcrição/metabolismo , Transfecção , Ubiquitina-Proteína Ligases , alfa-SinucleínaRESUMO
BACKGROUND: Gitelman syndrome (GS) and Bartter syndrome (BS) are hereditary hypokalemic tubulopathies with distinct phenotypic features. GS has been considered a genetically homogeneous disorder caused by mutation in the gene encoding the NaCl cotransporter (TSC) of the distal convoluted tubule. In contrast, BS is caused by mutations in the genes encoding either the Na-K-2Cl cotransporter (NKCC2), the K+ channel (ROMK) or the Cl- channel (ClC-Kb) of the thick ascending limb. The purpose of this study was to examine the clinical, biochemical and genetic characteristics of a very large inbred Bedouin kindred in Northern Israel with hereditary hypokalemic tubulopathy. METHODS: Twelve family members affected with hypokalemic tubulopathy, as well as 26 close relatives were clinically and biochemically evaluated. All study participants underwent genetic linkage analysis. Mutation analysis was performed in affected individuals. RESULTS: Evaluation of affected family members (age range 3 to 36 years) revealed phenotypic features of both GS and classic Bartter syndrome (CBS). Features typical of GS included late age of presentation (>15 years) in 7 patients (58%), normal growth in 9 (75%), hypomagnesemia (SMg <0.7mmol/L) in 5 (42%), hypermagnesiuria (FEMg>5%) in 6 (50%) and hypocalciuria (urinary calcium/creatinine mmol/mmol <0.15) in 5 (42%). Features typical of CBS included early age of presentation (<1 year) in 3 (25%), polyuria/dehydration in 4 (33%), growth retardation in 3 (25%), hypercalciuria (urinary calcium/creatinine mmol/mmoverline>0.55) in 4 (33%) and nephrolithiasis in 1 (8%). Linkage analysis in affected patients excluded the TSC gene, SLC12A3, as the mutated gene, but demonstrated linkage to the Cl- channel gene, CLCNKB, on chromosome 1p36. Mutation analysis by direct sequencing revealed a novel homozygous missense mutation, arginine 438 to histidine (R438H), in exon 13 of CLCNKB in all patients. A restriction fragment length polymorphism (RFLP) analysis has been developed to aid in genotyping of family members. CONCLUSIONS: Our findings demonstrate intrafamilial heterogeneity, namely the presence of GS and CBS phenotypes, in a kindred with the CLCNKB R438H mutation. We conclude that GS can be caused by a mutation in a gene other than SLC12A3. The exact role of the CLCNKB R438H mutation in the pathogenesis of the electrolyte and mineral abnormalities in GS and CBS remains to be established.