RÉSUMÉ
Expansion of a polymorphic polyglutamine segment is the common denominator of neurodegenerative polyglutamine diseases. The expanded proteins typically accumulate in large intranuclear inclusions and induce neurodegeneration. However, the mechanisms that determine the subcellular site and rate of inclusion formation are largely unknown. We found that the conserved putative nuclear localization sequence Arg-Lys-Arg-Arg, which is retained in a highly aggregation-prone fragment of ataxin-3, did not affect the site and degree of inclusion formation in a cell culture model of spinocerebellar ataxia type 3. Addition of synthetic nuclear export or import signals led to the expected localization of ataxin-3 and determined the subcellular site of aggregate formation. Triggering a cellular stress response by heat shock transcription factor DeltaHSF1 coexpression abrogated aggregation in the cytoplasm but not in the nucleus. These findings indicate that native aggregation-prone fragments derived from expanded ataxin-3 may eventually escape the cytoplasmic quality control, resulting in aggregation in the nuclear compartment.
Sujet(s)
Noyau de la cellule/métabolisme , Protéines de tissu nerveux/métabolisme , Protéines nucléaires/métabolisme , Fragments peptidiques/métabolisme , Protéines de répression/métabolisme , Facteurs de transcription/métabolisme , Transport nucléaire actif , Séquence d'acides aminés , Animaux , Ataxine-3 , Lignée cellulaire , Cytoplasme , Réaction de choc thermique , Humains , Corps d'inclusion , Maladie de Machado-Joseph , Souris , Maladies neurodégénératives , Signaux de localisation nucléaire , Peptides , Multimérisation de protéines , RatsRÉSUMÉ
The recombinant expression of eukaryotic proteins in Escherichia coli often results in protein aggregation. Several articles report on improved solubility and increased purification yields of individual proteins upon over-expression of E. coli chaperones but this effect might potentially be protein-specific. To find out whether chaperone over-expression is a generally applicable strategy for the production of human protein kinases in E. coli, we analyzed 10 kinases, mainly as catalytic domain constructs. The kinases studied, namely c-Src, c-Abl, Hck, Lck, Igf1R, InsR, KDR, c-Met, b-Raf and Irak4, belong to the tyrosine and tyrosine kinase-like groups of kinases. Upon over-expression of the E. coli chaperones DnaK/DnaJ/GrpE and GroEL/GroES, the yields of 7 from 10 polyhistidine-tagged kinases were increased up to 5-fold after nickel-affinity purification (IMAC). Additive over-expression of the chaperones ClpB and/or trigger factor showed no further improvement. Co-purification of DnaJ and GroEL indicated incomplete kinase folding, therefore, the oligomerization state of the kinases was determined by size-exclusion chromatography. In our study, kinases behave in three different ways. Kinases where yields are not affected by E. coli chaperone over-expression e.g. c-Src elute in the monomeric fraction (category I). Although IMAC yields increase upon chaperone over-expression, InsR and b-Raf kinase are present as soluble aggregates (category II). Igf1R and c-Met kinase catalytic domains are partially complexed with E. coli chaperones upon over-expression; however, they show approximately 2-fold increased yields of monomer (category III). Together, our results suggest that the benefits of chaperone over-expression on the production of protein kinases in E. coli are indeed case-specific.
Sujet(s)
Escherichia coli/métabolisme , Chaperons moléculaires/biosynthèse , Phosphotransferases/biosynthèse , Protéines recombinantes/biosynthèse , Chromatographie sur gel , Escherichia coli/génétique , Spectrométrie de masse , Chaperons moléculaires/génétique , Chaperons moléculaires/isolement et purification , Phosphotransferases/génétique , Phosphotransferases/isolement et purification , Ingénierie des protéines , Pliage des protéines , Structure tertiaire des protéines , Protéines recombinantes/génétique , Protéines recombinantes/isolement et purification , SolubilitéRÉSUMÉ
The formation of intraneuronal inclusions is a common feature of neurodegenerative polyglutamine disorders, including Spinocerebellar ataxia type 3. The mechanism that triggers inclusion formation in these typically late onset diseases has remained elusive. However, there is increasing evidence that proteolytic fragments containing the expanded polyglutamine segment are critically required to initiate the aggregation process. We analyzed ataxin-3 proteolysis in neuroblastoma cells and in vitro and show that calcium-dependent calpain proteases generate aggregation-competent ataxin-3 fragments. Co-expression of the highly specific cellular calpain inhibitor calpastatin abrogated fragmentation and the formation of inclusions in cells expressing pathological ataxin-3. These findings suggest a critical role of calpains in the pathogenesis of Spinocerebellar ataxia type 3.
Sujet(s)
Calpain/antagonistes et inhibiteurs , Maladie de Machado-Joseph/génétique , Maladie de Machado-Joseph/métabolisme , Protéines de tissu nerveux/métabolisme , Protéines nucléaires/métabolisme , Peptides/génétique , Protéines de répression/métabolisme , Animaux , Anticorps , Ataxine-3 , Calpain/métabolisme , Lignée cellulaire tumorale , Antienzymes/pharmacologie , Humains , Ionomycine/pharmacologie , Ionophores/pharmacologie , Rein/cytologie , Maladie de Machado-Joseph/anatomopathologie , Souris , Protéines de tissu nerveux/génétique , Protéines de tissu nerveux/immunologie , Neuroblastome , Protéines nucléaires/génétique , Protéines nucléaires/immunologie , Lapins , Rats , Protéines de répression/génétique , Protéines de répression/immunologie , Facteurs de transcription/génétique , Facteurs de transcription/immunologie , Facteurs de transcription/métabolismeRÉSUMÉ
Arginine/lysine-rich motifs typically function as targeting signals for the translocation of proteins to the nucleus. Here, we demonstrate that such a motif consisting of four basic amino acids in the polyglutamine protein ataxin-3 (Atx-3) serves as a recognition site for the interaction with the molecular chaperone VCP. Through this interaction, VCP modulates the fibrillogenesis of pathogenic forms of Atx-3 in a concentration-dependent manner, with low concentrations of VCP stimulating fibrillogenesis and excess concentrations suppressing it. No such effect was observed with a mutant Atx-3 variant, which does not contain a functional VCP interaction motif. Strikingly, a stretch of four basic amino acids in the ubiquitin chain assembly factor E4B was also discovered to be critical for VCP binding, indicating that arginine/lysine-rich motifs might be generally utilized by VCP for the targeting of proteins. In vivo studies with Drosophila models confirmed that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic Atx-3. Together, these results define the VCP-Atx-3 association as a potential target for therapeutic intervention and suggest that it might influence the progression of spinocerebellar ataxia type 3.
Sujet(s)
Arginine/génétique , Encéphale/métabolisme , Protéines du cycle cellulaire/métabolisme , Lysine/génétique , Protéines de tissu nerveux/métabolisme , Adenosine triphosphatases , Adénosine triphosphate/métabolisme , Motifs d'acides aminés , Séquence d'acides aminés , Animaux , Ataxine-3 , Encéphale/anatomopathologie , Cellules COS , Protéines du cycle cellulaire/génétique , Chlorocebus aethiops , Drosophila/cytologie , Drosophila/génétique , Drosophila/métabolisme , Protéine huntingtine , Corps d'inclusion/métabolisme , Données de séquences moléculaires , Mutation , Protéines de tissu nerveux/génétique , Neurones/cytologie , Neurones/métabolisme , Signaux de localisation nucléaire/physiologie , Protéines nucléaires/métabolisme , Cellules photoréceptrices d'invertébré/métabolisme , Liaison aux protéines , Protéines de répression , Similitude de séquences d'acides aminés , Protéine contenant la valosineRÉSUMÉ
Spinocerebellar ataxia type 3 (SCA3), like other polyglutamine (polyQ) diseases, is characterized by the formation of intraneuronal inclusions, but the mechanism underlying their formation is poorly understood. Here, we tested the "toxic fragment hypothesis", which predicts that proteolytic production of polyQ-containing fragments from the full-length disease protein initiates the aggregation process associated with inclusion formation and cellular dysfunction. We demonstrate that the removal of the N-terminus of polyQ-expanded ataxin-3 (AT3) is required for aggregation in vitro and in vivo. Consistently, proteolytic cleavage of full-length, pathogenic AT3 initiates the formation of sodium dodecylsulfate-resistant aggregates in neuroblastoma cells. Although full-length AT3 does not readily aggregate on its own, it is susceptible to co-aggregation with polyQ-expanded AT3 fragments. Interestingly, interaction with soluble polyQ-elongated fragments causes a structural distortion of wild-type AT3 prior to the formation of stable co-aggregates. These results establish the critical role of C-terminal, proteolytic fragments of AT3 in the molecular pathomechanism of SCA3, in strong support of the toxic fragment hypothesis.