RESUMO
Abnormal phosphorylation of the microtubule-associated protein tau in neurodegenerative disorders, including Alzheimer's disease (AD) and frontotemporal lobar degeneration, is associated with disrupted axonal transport and synaptic dysfunction ultimately manifesting as histopathological lesions of protein aggregates. Glycogen synthase kinase 3ß (GSK3ß) may be critical for the pathological hyperphosphorylation of tau. Here, we examined the role of the proteasome-associated protein Nedd8 ultimate buster 1 (NUB1) in the neuropathogenic phosphorylation and aggregation of tau. We reveal that NUB1 interacted with both tau and GSK3ß to disrupt their interaction, and abolished recruitment of GSK3ß to tau inclusions. Moreover, NUB1 reduced GSK3ß-mediated phosphorylation of tau and aggregation of tau in intracellular inclusions. Strikingly, NUB1 induced GSK3ß degradation. Deletion of the NUB1 ubiquitin-like (UBL) domain did not impair the interaction with tau and GSK3ß, and the ability to suppress the phosphorylation and aggregation of tau was not affected. However, the UBL motif was necessary for GSK3ß degradation. Deletion of the NUB1 ubiquitin-associated (UBA) domain abrogated the ability of NUB1 to interact with and degrade GSK3ß. Moreover, the UBA domain was required to suppress the aggregation of tau. Silencing of NUB1 in cells stabilized endogenous GSK3ß and exacerbated tau phosphorylation. Thus, we propose that NUB1, by regulating GSK3ß levels, modulates tau phosphorylation and aggregation, and is a key player in neurodegeneration associated with tau pathology. Moreover, NUB1 regulation of GSK3ß could modulate numerous signalling pathways in which GSK3ß is a centrally important effector.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas tau/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Imuno-Histoquímica , Imunoprecipitação , Fosforilação/genética , Fosforilação/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Interferência de RNA , Ratos , Proteínas tau/genéticaRESUMO
We previously found that the NF-κB transcription factor is activated during the recovery period after heat shock; moreover, we demonstrated that NF-κB is essential for cell survival after heat shock by activating autophagy, a mechanism that probably helps the cell to cope with hyperthermic stress through clearance of damaged proteins. In this study, we analyze the involvement of NF-κB in basal and heat-stress-induced protein quality control, by comparing the level of multiubiquitylated and/or aggregated proteins, and proteasome and autophagic activity in NF-κB-competent and NF-κB-incompetent cells. We show that NF-κB has only a minor role in basal protein quality control, where it modulates autophagosome maturation. By contrast, NF-κB is shown to be a key player in protein quality control after hyperthermia. Indeed, NF-κB-incompetent cells show highly increased levels of multiubiquitylated and/or aggregated proteins and aggresome clearance defects; a phenotype that disappears when NF-κB activity is restored to normal. We demonstrate that during heat shock recovery NF-κB activates selective removal of misfolded or aggregated proteins--a process also called 'aggrephagy'--by controlling the expression of BAG3 and HSPB8 and by modulating the level of the BAG3-HspB8 complex. Thus NF-κB-mediated increase in the level of the BAG3-HspB8 complex leads to upregulation of aggrephagy and clearance of irreversibly damaged proteins and might increase cell survival in conditions of hyperthermia.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia/fisiologia , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição RelA/metabolismo , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Células HeLa , Humanos , Chaperonas Moleculares , NF-kappa B/genética , Dobramento de Proteína , Fator de Transcrição RelA/deficiência , Fator de Transcrição RelA/genética , UbiquitinaçãoRESUMO
During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called "protein conformational diseases," such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NFκB transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NFκB triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NFκB-dependent autophagy allows the clearance of protein aggregates. Thus NFκB appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases.
Assuntos
Autofagia/fisiologia , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Células HeLa , Humanos , Neurônios Motores/metabolismo , Conformação Proteica , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Ativação Transcricional , Regulação para Cima , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismoRESUMO
Mutations in AIPL1 cause the inherited blindness Leber congenital amaurosis (LCA). AIPL1 has previously been shown to interact with NUB1, which facilitates the proteasomal degradation of proteins modified with the ubiquitin-like protein FAT10. Here we report that AIPL1 binds non-covalently to free FAT10 and FAT10ylated proteins and can form a ternary complex with FAT10 and NUB1. In addition, AIPL1 antagonised the NUB1-mediated degradation of the model FAT10 conjugate, FAT10-DHFR, and pathogenic mutations of AIPL1 were defective in inhibiting this degradation. While all AIPL1 mutants tested still bound FAT10-DHFR, there was a close correlation between the ability of the mutants to interact with NUB1 and their ability to prevent NUB1-mediated degradation. Interestingly, AIPL1 also co-immunoprecipitated the E1 activating enzyme for FAT10, UBA6, suggesting AIPL1 may have a role in directly regulating the FAT10 conjugation machinery. These studies are the first to implicate FAT10 in retinal cell biology and LCA pathogenesis, and reveal a new role of AIPL1 in regulating the FAT10 pathway.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas do Olho/fisiologia , Ubiquitinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Humanos , Amaurose Congênita de Leber , Mutação , Retina/patologia , Fatores de TranscriçãoRESUMO
The heat shock response is a widely described defense mechanism during which the preferential expression of heat shock proteins (Hsps) helps the cell to recover from thermal damages such as protein denaturation/aggregation. We have previously reported that NFkappaB transcription factor is activated during the recovery period after heat shock. In this study, we analyze the consequences of NFkappaB activation during heat shock recovery, by comparing the heat shock response of NFkappaB competent and incompetent (p65/RelA-depleted) cells. We demonstrate for the first time that NFkappaB plays a major and crucial role during the heat shock response by activating autophagy, which increases survival of heat-treated cells. Indeed, we observed that autophagy is not activated during heat shock recovery and cell death is strongly increased in NFkappaB incompetent cells. Moreover, if autophagy is artificially induced in these cells, the cytotoxicity of heat shock is turned back to normal. We show that despite a post-heat shock increase of Beclin 1 level in NFkappaB competent cells, neither Beclin 1/class III PI3K complex, Bcl(2)/Bcl-X(L) nor mTOR kinase are NFkappaB targets whose modulation of expression could be responsible for NFkappaB activation of autophagy during heat shock recovery. In contrast, we demonstrate that aberrantly folded/aggregated proteins are prime events in the signaling pathway leading to NFkappaB mediated autophagy after heat shock. Hence, our findings demonstrate that NFkappaB-induced autophagy during heat shock recovery is an additional cell response to HS-induced protein denaturation/aggregation; this mechanism increases cell survival, probably through clearance of irreversibly damaged proteins.