Novel mechanism of Hsp70 chaperone-mediated prevention of polyglutamine aggregates in a cellular model of huntington disease

Abstract

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.

Molecular Interaction between the Chaperone Hsc70 and the N-terminal Flank of Huntingtin Exon 1 Modulates Aggregation

“…The guardian activity of chaperones is based on their ability to bind stretches of aggregation-prone hydrophobic residues that are exposed to the solvent in unstructured or misfolded proteins (23,24). An intriguing question in this respect was whether or not the alleviating effect of Hsc70 on the toxicity of the polyQ-expanded huntingtin protein observed in vivo (15,17,18,42,43) reflected a presumably unfavorable direct interaction between the chaperone and the aggregationprone polyQ stretch.…”

Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase

“…Insoluble aggregates of GAPDH are found in many diseases (11)(12)(13)(14)(15)(16)(17)(18)(19). No prior study has ascribed a structural/functional role for Met-46 in GAPDH biology.…”

“…In vivo studies show that GAPDH converts from its native soluble state into a non-native high molecular weight insoluble state during disease (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). For instance, insoluble aggregates of GAPDH have been observed in the affected tissues of patients with Alzheimer disease (13) and alcoholic liver cirrhosis (12).…”

Modulation of Molecular Chaperones in Huntington’s Disease and Other Polyglutamine Disorders

“…Not surprisingly, mutant ataxin-7 induced transcriptional dysregulation in SCA7 transgenic models [187][188][189], and the disease associates with nuclear aggregates of the polyQ-expanded protein [190]. 20 ! TBP, the protein involved in SCA17, is an essential component of the transcriptional initiation complex [191], directing it to DNA via binding to the TATA-box [192].…”

“…3b). In addition to its role in protein folding and degradation, Hsp70 is also involved in protein disaggregation, acting in cooperation with Hsp40 co-chaperones and the Hsp110 NEF in the formation of a chaperone complex with disaggregase activity [70,20] (Fig. 3c).…”