Small heat-shock proteins interact with a flanking domain to suppress polyglutamine aggregation Amy L. Robertsona, Stephen J. Headeyb, Helen M. Saundersa, Heath Ecroydc, Martin J. Scanlonb, John A. Carverd, and Stephen P. Bottomleya,1 aDepartment of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia; bMedicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia; cSchool of Biological Sciences, University of Wollongong, Wollongong, New South Wales, 2522, Australia; and dSchool of Chemistry and Physics, The University of Adelaide, Adelaide, South Australia, 5005, Australia Edited by George H. Lorimer, University of Maryland, College Park, MD, and approved April 15, 2010 (received for review December 20, 2009) Small heat-shock proteins (sHsps) are molecular chaperones that αB-c and a number of aggregating proteins has been characterized. play an important protective role against cellular protein misfolding Most substrates aggregate through a nucleation-dependent kinetic by interacting with partially unfolded proteins on their off-folding mechanism; however, the mode of interaction with αB-c appears pathway, preventing their aggregation. Polyglutamine (polyQ) to be dependent upon the specific target protein and the type of repeat expansion leads to the formation of fibrillar protein aggre- aggregation—i.e., amorphous or fibrillar (11–13). Complex stabi- gates and neuronal cell death in nine diseases, including Huntington lity is influenced by properties of the target protein, for example, disease and the spinocerebellar ataxias (SCAs). There is evidence the fibril-forming proteins apolipoprotein-CII (11) and amyloid-β that sHsps have a role in suppression of polyQ-induced neurodegen- (14) transiently interact with αB-c whereas α-synuclein (6) and eration; for example, the sHsp alphaB-crystallin (αB-c) has been κ-casein (15) form stable noncovalent complexes. Determining identified as a suppressor of SCA3 toxicity in a Drosophila model. the mechanism by which αB-c interacts with specific aggrega- However, the molecular mechanism for this suppression is un- tion-prone substrates is therefore important and may provide known. In this study we tested the ability of αB-c to suppress the insight into the misfolding pathways leading to toxicity. aggregation of a polyQ protein. We found that αB-c does not inhibit Polyglutamine (polyQ) expansion leads to the formation of the formation of SDS-insoluble polyQ fibrils. We further tested the fibrillar protein aggregates and neuronal cell death in nine dis- effect of αB-c on the aggregation of ataxin-3, a polyQ protein that eases, including HD and the spinocerebellar ataxias (SCAs) 1, aggregates via a two-stage aggregation mechanism. The first stage 2, 3, 6, 7, and 17. PolyQ peptides and proteins form fibrillar ag- involves association of the N-terminal Josephin domain followed by gregates by a nucleation-dependent mechanism that is initiated – polyQ-mediated interactions and the formation of SDS-resistant by a monomeric nucleus (16 18). Recent evidence suggests that mature fibrils. Our data show that αB-c potently inhibits the first polyQ protein misfolding involves not only the polyQ tract but stage of ataxin-3 aggregation; however, the second polyQ-depen- other aggregation-prone regions within the polyQ proteins – dent stage can still proceed. By using NMR spectroscopy, we have (19 21). There is experimental evidence that the proteins atax- determined that αB-c interacts with an extensive region on the in-3 (in SCA3), ataxin-1 (in SCA1), and huntingtin (in HD) form surface of the Josephin domain. These data provide an example fibrillar aggregates by a multidomain misfolding mechanism in of a domain/region flanking an amyloidogenic sequence that has which non-polyQ regions of the protein self-associate before a critical role in modulating aggregation of a polypeptide and plays the polyQ tract (19, 20, 22). Our laboratory has previously shown a role in the interaction with molecular chaperones to prevent this that ataxin-3 has a minimal two-stage aggregation mechanism aggregation. (19). The first stage involves aggregation of the globular N-term- inal Josephin domain that precedes the self-association of fibrillogenesis ∣ α-crystallin expanded polyQ segments (19). sHsp overexpression decreases neuronal toxicity in HD by nonchaperone mechanisms, including suppression of reactive mall heat-shock proteins (sHsps) are important in the main- oxygen species (23) and stimulation of autophagy (24). In disease Stenance of cellular homeostasis. sHsps are induced under models overexpression of αB-c modulates toxicity in a context- stress conditions and interact with partially unfolded proteins dependent manner. αB-c did not alter the HD phenotype in a on their off-folding pathways, thereby providing a protective Drosophila model (25); however, it suppressed SCA3 toxicity mechanism against protein misfolding and aggregation (1). They in a Drosophila model (9). Enhanced suppression was observed are present in many species, and 10 have been identified in the when αB-c was coexpressed with full-length ataxin-3 in compar- human proteome (see reviews in refs. 1 and 2). Alpha-crystallin ison with a C-terminal fragment not containing the Josephin α ( -crystallin) is the most well characterized sHsp. The two isoforms domain (9). These data suggest that non-polyQ protein regions α α α α α of -crystallin, A-crystallin ( A-c) and B-crystallin ( B-c), are a influence sHsp chaperone activity. α large component of the human eye lens, and B-c is expressed in In this study we tested the chaperone ability of αB-c for a α many other cell types including neurons (3, 4). Monomeric B-c is polyQ protein (SpAcQ52) that forms fibrillar aggregates by a approximately 20 kDa in mass, and under native conditions it mechanism involving only the polyQ region. We found that forms a heterogeneous array of multimeric complexes ranging from 160 to 1,000 kDa in mass (2). The activity of αB-c is thought to involve hydrophobic interactions between αB-c and the partially Author contributions: A.L.R., J.A.C., and S.P.B. designed research; A.L.R., S.J.H., H.M.S., and M.J.S. performed research; H.E. contributed new reagents/analytic tools; A.L.R., S.J.H., folded protein. Under stress conditions, such as elevated tempera- H.M.S., M.J.S., J.A.C., and S.P.B. analyzed data; and A.L.R., S.J.H., H.E., M.J.S., J.A.C., and ture, the activity of αB-c is enhanced because of a conformational S.P.B. wrote the paper. change within the multimeric complex (5, 6). Cellular stress is in- The authors declare no conflict of interest. ’ duced in several neurodegenerative diseases, such as Alzheimer s This article is a PNAS Direct Submission. disease (7), prion diseases (8), Parkinson disease (4), and Hunting- 1To whom correspondence should be addressed. E-mail: [email protected]. ton disease (HD) (4), and there is evidence of increased αB-c edu.au. α activity (4). In addition, B-c reduces neurotoxicity in a number This article contains supporting information online at www.pnas.org/lookup/suppl/ of disease models (9, 10). In vitro the interaction between doi:10.1073/pnas.0914773107/-/DCSupplemental. 10424–10429 ∣ PNAS ∣ June 8, 2010 ∣ vol. 107 ∣ no. 23 www.pnas.org/cgi/doi/10.1073/pnas.0914773107 Downloaded by guest on September 25, 2021 αB-c does not inhibit fibrillogenesis in this system. We then tested αB-c Inhibits the Josephin-Dependent Stage of the Two-Stage Ataxin-3 the effect of αB-c on the aggregation of ataxin-3 harboring a Aggregation Pathway. We have previously reported that full-length pathological length polyQ tract [at3(Q64)] and a truncated ataxin-3 forms aggregates by a two-stage mechanism (18). The variant comprising only the Josephin domain. Our data show that aggregates formed in both stages are ThT-reactive, and in the ab- αB-c potently inhibits the initial Josephin-dependent stage of sence of glycerol the two stages are characterized by a biphasic ataxin-3 aggregation. By using NMR spectroscopy, we deter- time course (Fig. 2A). The first stage is dominated by interactions mined that αB-c interacts with specific regions of Josephin. These of the N-terminal Josephin domain, which is an independently data provide an example and characterization of a domain/region aggregation-prone domain (30). Aggregates formed during the flanking an amyloidogenic sequence that has a critical role in mod- first stage appear as short curvilinear structures and can be solu- ulation of aggregation by interacting with molecular chaperones. bilized by SDS (19). These aggregates are stabilized in the second stage by interactions of the polyQ regions. Stage-two aggregates Results can be distinguished from stage-one aggregates because they are αB-c Does Not Inhibit PolyQ-Mediated Aggregation. We first tested SDS-insoluble and morphologically appear as larger fibrillar whether αB-c can prevent polyQ tract-mediated aggregation. We structures. We determined the effect of αB-c on the two-stage coincubated αB-c with a polyQ protein (SpAcQ52), which forms aggregation pathway of at3(Q64). αB-c significantly slows the for- SDS-insoluble aggregates by a mechanism involving only the mation of ThT-reactive at3(Q64) aggregates in a concentration- polyQ tract (26). The rate at which SpAcQ52 forms ThT-reactive dependent manner (Fig. 2A and Table 1). aggregates does not change when incubated with increasing The Josephin domain forms short aggregates with a ribbon-like concentrations of αB-c (Fig. 1A). It has been previously shown morphology (30); however, the aggregation kinetics have not that molecular chaperones can redirect the aggregation pathway been described. Isolated Josephin forms ThT-reactive aggregates of a protein from amyloid to amorphous aggregation (27, 28). at a much slower rate than at3(Q64) (Fig.