RESUMEN
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.
Asunto(s)
Autofagia/fisiología , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Células HeLa , Humanos , Neuronas Motoras/metabolismo , Conformación Proteica , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Estrés Fisiológico/genética , Estrés Fisiológico/fisiología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Activación Transcripcional , Regulación hacia Arriba , Cadena B de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Several human small heat shock proteins (sHsps) are phosphorylated oligomeric chaperones that enhance stress resistance. They are characterized by their ability to interact and form polydispersed hetero-oligomeric complexes. We have analyzed the cellular consequences of the stable expression of either wild type HspB5 or its cataracts and myopathies inducing R120G mutant in growing and oxidative stress treated HeLa cells that originally express only HspB1. Here, we describe that wild type and mutant HspB5 induce drastic and opposite effects on cell morphology and oxidative stress resistance. The cellular distribution and phosphorylation of these polypeptides as well as the oligomerization profile of the resulting hetero-oligomeric complexes formed by HspB1 with the two types of exogenous polypeptides revealed the dominant effects induced by HspB5 polypeptides towards HspB1. The R120G mutation enhanced the native size and salt resistance of HspB1-HspB5 complex. However, in oxidative conditions the interaction between HspB1 and mutant HspB5 was drastically modified resulting in the aggregation of both partners. The mutation also induced the redistribution of HspB1 phosphorylated at serine 15, originally observed at the level of the small oligomers that do not interact with wild type HspB5, to the large oligomeric complex formed with mutant HspB5. This phosphorylation stabilized the interaction of HspB1 with mutant HspB5. A dominant negative effect towards HspB1 appears therefore as an important event in the cellular sensitivity to oxidative stress mediated by mutated HspB5 expression. These observations provide novel data that describe how a mutated sHsp can alter the protective activity of another member of this family of chaperones.
Asunto(s)
Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Mutación , Cadena B de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/metabolismo , Expresión Génica , Proteínas de Choque Térmico HSP27/química , Células HeLa , Humanos , Estrés Oxidativo/genética , Fosforilación , Unión Proteica , Multimerización de Proteína , Transporte de Proteínas , Cadena B de alfa-Cristalina/químicaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia/fisiología , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/fisiología , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Células HeLa , Humanos , Chaperonas Moleculares , FN-kappa B/genética , Pliegue de Proteína , Factor de Transcripción ReIA/deficiencia , Factor de Transcripción ReIA/genética , UbiquitinaciónRESUMEN
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.
Asunto(s)
Autofagia , Respuesta al Choque Térmico , FN-kappa B/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Células COS , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Citoprotección/efectos de los fármacos , Glicerol/farmacología , Células HeLa , Respuesta al Choque Térmico/efectos de los fármacos , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfatidiletanolaminas/metabolismo , Factor de Transcripción ReIA/deficiencia , Factor de Transcripción ReIA/metabolismoRESUMEN
Hsp27 and alphaB-crystallin are molecular chaperones that are constitutively expressed in several mammalian cells, particularly in pathological conditions. These proteins share functions as diverse as protection against toxicity mediated by aberrantly folded proteins or oxidative-inflammation conditions. In addition, these proteins share anti-apoptotic properties and are tumorigenic when expressed in cancer cells. This review summarizes the current knowledge about Hsp27 and alphaB-crystallin and the implications, either positive or deleterious, of these proteins in pathologies such as neurodegenerative diseases, myopathies, asthma, cataracts and cancers. Approaches towards therapeutic strategies aimed at modulating the expression and/or the activities of Hsp27 and alphaB-crystallin are presented.