Stable α-Synuclein Oligomers Strongly Inhibit Chaperone Activity of the Hsp70 System by Weak Interactions with J-domain Co-chaperones

Abstract

␣-Synuclein aggregation and accumulation in Lewy bodies are implicated in progressive loss of dopaminergic neurons in Parkinson disease and related disorders. In neurons, the Hsp70s and their Hsp40-like J-domain co-chaperones are the only known components of chaperone network that can use ATP to convert cytotoxic protein aggregates into harmless natively refolded polypeptides. Here we developed a protocol for preparing a homogeneous population of highly stable ␤-sheet enriched toroid-shaped ␣-Syn oligomers with a diameter typical of toxic pore-forming oligomers. These oligomers were partially resistant to in vitro unfolding by the bacterial Hsp70 chaperone system (DnaK, DnaJ, GrpE). Moreover, both bacterial and human Hsp70/Hsp40 unfolding/refolding activities of model chaperone substrates were strongly inhibited by the oligomers but, remarkably, not by unstructured ␣-Syn monomers even in large excess. The oligomers acted as a specific competitive inhibitor of the J-domain co-chaperones, indicating that J-domain co-chaperones may preferably bind to exposed bulky misfolded structures in misfolded proteins and, thus, complement Hsp70s that bind to extended segments. Together, our findings suggest that inhibition of the Hsp70/Hsp40 chaperone system by ␣-Syn oligomers may contribute to the disruption of protein homeostasis in dopaminergic neurons, leading to apoptosis and tissue loss in Parkinson disease and related neurodegenerative diseases.

Physical Interaction between Bacterial Heat Shock Protein (Hsp) 90 and Hsp70 Chaperones Mediates Their Cooperative Action to Refold Denatured Proteins

“…However, we have to point out that the two J-proteins are different from each other in terms of their affinity toward a denatured substrate. CbpA can bind a substrate to prevent its aggre- gation (33), although DnaJ2 does not interact stably with the heat-denatured MDH because it could not suppress the enzyme's aggregation efficiently as compared with HtpG and DnaK2 (Fig. 7B).…”

GroEL and CCT are catalytic unfoldases mediating out-of-cage polypeptide refolding without ATP

“…Further experiments beyond the scope of this work are needed to assess the relative importance of iterative catalytic unfolding/refolding and prevention of aggregation by sequestration as complementary mechanisms to delay the onset of protein misfolding diseases and aging (34).…”

Reactivation of protein aggregates by mortalin and Tid1—the human mitochondrial Hsp70 chaperone system

“…In vitro, DnaK can perform this function when assisted by its two cochaperones, a J-domain-containing protein (either DnaJ, CbpA, or DjlA; Genevaux et al 2007) and the nucleotide exchange factor GrpE (Sharma et al 2009). Specificity of the chaperone system is conferred primarily by the DnaJ co-chaperone, which recognizes and binds to a misfolded polypeptide (Hinault et al 2010), followed by the binding of the polypeptide to a "low-affinity" DnaK, which becomes entrapped upon hydrolyzing ATP. GrpE, acting as an ADP release factor, accelerates discharge of the unfolded polypeptide and spontaneous refolding to the native state .…”