RESUMO
The maintenance of proteome integrity is of primary importance in post-mitotic tissues such as muscle cells; thus, protein quality control mechanisms must be carefully regulated to ensure their optimal efficiency, a failure of these processes being associated with various muscular disorders. Duchenne muscular dystrophy (DMD) is one of the most common and severe forms of muscular dystrophies and is caused by mutations in the dystrophin gene. Protein quality control modulations have been diversely observed in degenerating muscles of patients suffering from DMD or in animal models of the disease. In this study, we investigated whether modulations of protein quality control mechanisms already pre-exist in undifferentiated myoblasts originating from DMD patients. We report for the first time that the absence of dystrophin in human myoblasts is associated with protein aggregation stress characterized by an increase of protein aggregates. This stress is combined with BAG1 to BAG3 switch, NFκB activation and up-regulation of BAG3/HSPB8 complexes that ensure preferential routing of misfolded/aggregated proteins to autophagy rather than to deficient 26S proteasome. In this context, restoration of pre-existing alterations of protein quality control processes might represent an alternative strategy for DMD therapies.
Assuntos
Autofagia , Distrofia Muscular de Duchenne/metabolismo , Mioblastos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Resposta a Proteínas não Dobradas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Humanos , Mioblastos/patologia , NF-kappa B/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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
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.
Assuntos
Proteínas de Choque Térmico/metabolismo , Inflamação/tratamento farmacológico , Chaperonas Moleculares/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Cadeia B de alfa-Cristalina/metabolismo , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/química , Humanos , Inflamação/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Neoplasias/metabolismo , Conformação Proteica , Cadeia B de alfa-Cristalina/antagonistas & inibidores , Cadeia B de alfa-Cristalina/químicaRESUMO
We previously showed that Hsp27 protects against apoptosis through its interaction with cytosolic cytochrome c. We have revisited this protective activity in murine cell lines expressing different levels of Hsp27. We report that Hsp27 also interferes, in a manner dependent on level of expression, with the release of cytochrome c from mitochondria. Moreover, a decreased level of endogenous Hsp27, which sensitized HeLa cells to apoptosis, reduced the delay required for cytochrome c release and procaspase 3 activation. The molecular mechanism regulating this function of Hsp27 is unknown. In our cell systems, Hsp27 is mainly cytosolic and only a small fraction of this protein colocalized with mitochondria. Moreover, we show that only a very small fraction of cytochrome c interacts with Hsp27, hence excluding a role of this interaction in the retention of cytochrome c in mitochondria. We also report that Bid intracellular relocalization was altered by changes in Hsp27 level of expression, suggesting that Hsp27 interferes with apoptotic signals upstream of mitochondria. We therefore investigated if the ability of Hsp27 to act as an expression-dependent modulator of F-actin microfilaments integrity was linked to the retention of cytochrome c in mitochondria. We show here that the F-actin depolymerizing agent cytochalasin D rapidly induced the release of cytochrome c from mitochondria and caspase activation. This phenomenon was delayed in cells pretreated with the F-actin stabilizer phalloidin and in cells expressing a high level of Hsp27. This suggests the existence of an apoptotic signaling pathway linking cytoskeleton damages to mitochondria. This pathway, which induces Bid intracellular redistribution, is negatively regulated by the ability of Hsp27 to protect F-actin network integrity. However, this upstream pathway is probably not the only one to be regulated by Hsp27 since, in staurosporine-treated cells, phalloidin only partially inhibited cytochrome c release and caspase activation. Moreover, in etoposide-treated cells, Hsp27 still delayed the release of cytochrome c from mitochondria and Bid intracellular redistribution in conditions where F-actin was not altered.
Assuntos
Grupo dos Citocromos c/metabolismo , Proteínas de Choque Térmico , Proteínas de Neoplasias/metabolismo , Células 3T3 , Actinas/metabolismo , Animais , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/metabolismo , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Ativação Enzimática , Etoposídeo/farmacologia , Expressão Gênica , Glutationa/metabolismo , Proteínas de Choque Térmico HSP27 , Células HeLa , Humanos , Cinética , Camundongos , Mitocôndrias/metabolismo , Modelos Biológicos , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Estaurosporina/farmacologiaRESUMO
Autophagy is a potent cell autonomous defense mechanism that engages the lysosomal pathway to fight intracellular pathogens. Several autophagy receptors can recognize invading pathogens in order to target them towards autophagy for their degradation after the fusion of pathogen-containing autophagosomes with lysosomes. However, numerous intracellular pathogens can avoid or exploit autophagy, among which is measles virus (MeV). This virus induces a complete autophagy flux, which is required to improve viral replication. We therefore asked how measles virus interferes with autophagy receptors during the course of infection. We report that in addition to NDP52/CALCOCO2 and OPTINEURIN/OPTN, another autophagy receptor, namely T6BP/TAXIBP1, also regulates the maturation of autophagosomes by promoting their fusion with lysosomes, independently of any infection. Surprisingly, only two of these receptors, NDP52 and T6BP, impacted measles virus replication, although independently, and possibly through physical interaction with MeV proteins. Thus, our results suggest that a restricted set of autophagosomes is selectively exploited by measles virus to replicate in the course of infection.
Assuntos
Autofagia/fisiologia , Proteínas de Transporte/fisiologia , Vírus do Sarampo/fisiologia , Sarampo/virologia , Replicação Viral/fisiologia , Proteínas de Ciclo Celular , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Vírus do Sarampo/patogenicidade , Proteínas de Membrana Transportadoras , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fagossomos/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Proteínas Virais/metabolismoRESUMO
Recently, we reported that the transient expression of huntingtin exon1 polypeptide containing polyglutamine tracts of various sizes (httEx1-polyQ) in cell models of Huntington disease generated an oxidative stress whose intensity was CAG repeat expansion-dependent. Here, we have analyzed the intracellular localization of the oxidative events generated by the httEx1-polyQ polypeptides. Analysis of live COS-7 cells as well as neuronal SK-N-SH and PC12 cells incubated with hydroethidine or dichlorofluorescein diacetate revealed oxidation of these probes at the level of the inclusion bodies formed by httEx1-polyQ polypeptides. The intensity and frequency of the oxidative events among the inclusions were CAG repeat expansion-dependent. Electron microscopic analysis of cell sections revealed the presence of oxidation-dependent morphologic alterations in the vicinity of httEx1-polyQ inclusion bodies. Moreover, a high level of oxidized proteins was recovered in partially purified inclusions. We also report that the iron chelator deferroxamine altered the structure, localization and oxidative potential of httEx1-polyQ inclusion bodies. Hence, despite the fact that the formation of inclusion bodies may represent a defense reaction of the cell to eliminate httEx1 mutant polypeptide, this phenomenon appears inherent to the generation of iron-dependent oxidative events that can be deleterious to the cell.
Assuntos
Doença de Huntington/metabolismo , Corpos de Inclusão/metabolismo , Ferro/metabolismo , Acetilcisteína/farmacologia , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Desferroxamina/metabolismo , Éxons , Proteínas de Choque Térmico/metabolismo , Corpos de Inclusão/ultraestrutura , Microscopia Confocal , Oxirredução , Fenantridinas/metabolismo , Ratos , Proteínas Recombinantes de FusãoRESUMO
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
Small stress proteins [small heat shock proteins (sHsps)] are molecular chaperones that modulate the ability of cells to respond to oxidative stress. The current knowledge concerning the protective mechanism generated by the expression of mammalian heat shock protein-27 (Hsp27) that allows cells to increase their resistance to oxidative stress is presented. We describe the effects mediated by Hsp27 expression toward crucial enzymes such as glucose-6-phosphate dehydrogenase and glutathione reductase that uphold glutathione in its reduced form. New data are presented showing that the expression of sHsps correlates with a drastic decrease in the intracellular level of iron, a catalyzer of hydroxyl radical (OH( . )) generation. A decreased ability of sHsps expressing cells to concentrate iron will therefore end up in a decreased level of oxidized proteins. In addition, we propose a role of Hsp27 in the presentation of oxidized proteins to the proteasome degradation machinery. We also present an analysis of several Hsp27 mutants that suggests that the C-terminal part of this stress protein is essential for its protective activity against oxidative stress.
Assuntos
Glutationa/metabolismo , Proteínas de Choque Térmico/fisiologia , Ferro/metabolismo , Estresse Oxidativo , Animais , Regulação para Baixo , Proteínas de Choque Térmico/genética , Homeostase , Camundongos , OxirreduçãoRESUMO
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.
Assuntos
Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Mutação , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismo , Expressão Gênica , Proteínas de Choque Térmico HSP27/química , Células HeLa , Humanos , Estresse Oxidativo/genética , Fosforilação , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Cadeia B de alfa-Cristalina/químicaRESUMO
Hsp27 belongs to the heat shock protein family and displays chaperone properties in stress conditions by holding unfolded polypeptides, hence avoiding their inclination to aggregate. Hsp27 is often referenced as an anti-cancer therapeutic target, but apart from its well-described ability to interfere with different stresses and apoptotic processes, its role in non-stressed conditions is still not well defined. In the present study we report that three polypeptides (histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3) were degraded in human cancerous cells displaying genetically decreased levels of Hsp27. In addition, these proteins interacted with Hsp27 complexes of different native size. Altogether, these findings suggest that HDAC6, STAT2 and procaspase-3 are client proteins of Hsp27. Hence, in non stressed cancerous cells, the structural organization of Hsp27 appears to be a key parameter in the regulation by this chaperone of the level of specific polypeptides through client-chaperone type of interactions.
Assuntos
Proteínas de Choque Térmico HSP27/genética , Proteínas/metabolismo , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Proteínas de Choque Térmico HSP27/metabolismo , Células HeLa , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Transfecção , Células Tumorais CultivadasAssuntos
Apoptose/fisiologia , Proteínas de Choque Térmico/fisiologia , Animais , Transformação Celular Neoplásica , Grupo dos Citocromos c/metabolismo , Citoesqueleto/metabolismo , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/química , Temperatura Alta , Humanos , Mitocôndrias/metabolismo , Modelos Biológicos , Chaperonas Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiologia , Fosforilação , Espécies Reativas de Oxigênio/metabolismoAssuntos
Expressão Gênica , Compostos de Sulfidrila/metabolismo , Transporte Ativo do Núcleo Celular , Antioxidantes/farmacologia , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Corantes Fluorescentes , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Células HeLa , Fatores de Transcrição de Choque Térmico , Humanos , NF-kappa B/metabolismo , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição , Ativação TranscricionalRESUMO
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.
Assuntos
Autofagia , Resposta ao Choque Térmico , NF-kappa B/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Células COS , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Citoproteção/efeitos dos fármacos , Glicerol/farmacologia , Células HeLa , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fator de Transcrição RelA/deficiência , Fator de Transcrição RelA/metabolismoRESUMO
Several viral proteins expressed by DNA or RNA transforming viruses have the particular property of binding via their C-terminal end to various cellular proteins with PDZ domains. This study is focused on the PDZ protein TIP-2/GIPC, which was originally identified in two-hybrid screens performed with two different baits: the human T-cell leukemia virus type 1 Tax oncoprotein and the regulator of G signaling RGS-GAIP. Further studies have shown that TIP-2/GIPC is also able to associate with the cytoplasmic domains of various transmembrane proteins. In this report we show that TIP-2/GIPC interacts with the E6 protein of human papillomavirus type 18 (HPV-18). This event triggers polyubiquitination and proteasome-mediated degradation of the cellular protein. In agreement with this observation, silencing of E6 by RNA interference in HeLa cells causes an increase in the intracellular TIP-2/GIPC level. This PDZ protein has been previously found to be involved in transforming growth factor beta (TGF-beta) signaling by favoring expression of the TGF-beta type III receptor at the cell membrane. In line with this activity of TIP-2/GIPC, we observed that depletion of this protein in HeLa cells hampers induction of the Id3 gene by TGF-beta treatment and also diminishes the antiproliferative effect of this cytokine. Conversely, silencing of E6 increases the expression of Id3 and blocks proliferation of HeLa cells. These results support the notion that HPV-18 E6 renders cells less sensitive to the cytostatic effect of TGF-beta by lowering the intracellular amount of TIP-2/GIPC.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neuropeptídeos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Mapeamento de Interação de Proteínas , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células COS , Linhagem Celular , Proliferação de Células , Inativação Gênica , Células HeLa , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Fator de Crescimento Transformador beta/antagonistas & inibidores , Ubiquitina/metabolismoRESUMO
There is currently great interest in the development of methods to analyze intracellular redox state and the cellular damages generated by oxidative stress. General methods for analyzing reactive oxygen species and glutathione level are presented together with more recently developed protocols to analyze the consequences of oxidative stress on the oxidation of macromolecules. Finally, techniques to study modalities of constitutive expression of Hsp27 in mammalian cells are considered as well as methods used to determine the protective activity of this small heat shock protein against the deleterious effects induced by oxidative stress.
Assuntos
Morte Celular , Proteínas de Choque Térmico HSP27/fisiologia , Estresse Oxidativo , Animais , Anexina A5/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Células Cultivadas , Dimerização , Ativação Enzimática , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Peroxidação de Lipídeos , Oxirredução , Espécies Reativas de OxigênioRESUMO
ATP- and ubiquitin-independent proteolysis by the 20S proteasome is responsible for the selective degradation of oxidized proteins. In vitro, the 20S proteasome shows an increased proteolytic activity toward oxidized polypeptides and the suc-LLVY-MCA peptide specific for its chymotrypsin-like activity. We have analyzed the effect of the intracellular redox status on the chymotrypsin-like activity of the 20S proteasome in human T47D cells overexpressing the detoxifiant enzyme seleno-glutathione peroxidase-1 (GPx-1). We report a 30% decreased activity of the chymotrypsin-like activity in cells overexpressing GPx-1. This phenomenon correlated with a 2-fold increase in IkappaB alpha half-life, a protein whose basal turnover is 20S proteasome-dependent. Following exposure to H2O2, these cells showed a seleno-dependently decreased accumulation of intracellular reactive oxygen species and 20S proteasome chymotrypsin-like activity. Similar results were obtained in HeLa cells transiently overexpressing human GPx-1. Moreover, exposure of HeLa cells to antioxidant compounds reduced the proteasome 20S chymotrypsin-like activity. In contrast, no effects were observed when HeLa cell extracts used to determine proteasome activity were incubated with either reduced or oxidized glutathione. These results suggest that GPx-1 activity or pro-reducing conditions can downregulate basal 20S proteasome activity. Hence, the intracellular redox status, probably through the level of oxidized proteins, is an important element that can either activate or down-regulate the 20S proteasome chymotrypsin-like activity in living cells.