The key feature of polyglutamine aggregates accumulating in the course of Huntington disease (HD) is their resistance to protein denaturants, and to date only chaperones are proved to prevent mutant protein aggregation. It was suggested that expanded polyglutamine chains (polyQ) of mutant huntingtin are cross-linked to other proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Here we clarify the roles of GAPDH and molecular chaperone Hsp70 in the formation of sodium dodecyl sulfate (SDS)-insoluble polyQ aggregates. First, the addition of pure GAPDH was found to enhance the aggregation of polyQ in a cell-free model of HD. Secondly, the immunodepletion of GAPDH dose-dependently decreased polyQ aggregation. Finally, siRNA-mediated inhibition of GAPDH protein in SK-N-SH neuroblastoma cells has also reduced the aggregation of cellular polyQ. Regulated over-expression of Hsp70 decreased the amount of GAPDH associated with SDS-insoluble polyQ aggregates. Physical association of Hsp70 and GAPDH in SK-N-SH cells was shown by reciprocal immunoprecipitation and confocal microscopy. Pure Hsp70 dose-dependently inhibited the formation of polyQ aggregates in cell-free model of HD by sequestering both GAPDH and polyQ. We demonstrated that Hsp70 binds to polyQ in adenosine triphosphate-dependent manner, which suggests that Hsp70 exerts a chaperoning activity in the course of this interaction. Binding of Hsp70 to GAPDH was nicotinamide adenine dinucleotide-dependent suggesting another type of association. Based on our findings, we conclude that Hsp70 protects cells in HD by removing/sequestering two intrinsic components of protein aggregates: the polyQ itself and GAPDH. We propose that GAPDH might be an important target for pharmacological treatment of HD and other polyglutamine expansion-related diseases.
At present, neurodegenerative pathologies are the object of active research. Aggregates made of misfolded, mutant proteins causing neuronal death are the reason of the majority of such diseases. One of these illnesses is the Huntington's disease (HD) that is provoked by expanded repeats of glutamine in N-terminal part of huntingtin. The development of the disease stems from the formation and accumulation of SDSinsoluble protein aggregates in neurons of striatum and cerebral cortex. The aggregates are formed by tissue transglutaminase, which catalyzes the ligation of glutamine residues of mutant huntingtin to lysines of other cellular proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results of our experiments proved that GAPDH is implicated in the formation of SDS-insoluble aggregates because the factors tightly binding the enzyme reduce both the size and amount of aggregates in the cells over-expressing the gene of mutant huntingtin with Q103 repeat. Conversely, the addition of pure GAPDH to ex vivo system of Q103 aggregate formation significantly increased the amount of aggregating Q103.To explore the mechanism of anti-aggregate activity of Hsp70 in HD model we generated human neuroblastoma cell line with metal-dependent expression of Hsp70. The elevation of the Hsp70 level lead to the reduction of both number and size of aggregates. These data were confirmed by using ex-vivo system of aggregate formation: the addition of pure Hsp70 elevated the amount of soluble polyglutamine-containing protein.Using the method of immunoprecipitation we showed that Hsp70 was able to bind GAPDH in dose-dependent manner: the more Hsp70, the lesser content of GAPDH in insoluble polyglutamine aggregates and the lower amount of the latter. It is concluded that a novel mechanism exists, through which Hsp70 sequesters GAPDH from the control of transglutaminase and by this diminishes cell mortality from cytotoxicity of the aggregates.
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