Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism

Abstract

Over-expression and aggregation of α-synuclein (ASyn) are linked to the onset and pathology of Parkinson's disease and related synucleinopathies. Elevated levels of the stress induced chaperone, Hsp70, protects against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations and inhibitors, we confirmed that Hsp70 interacts with ASyn via an as yet unidentified, non-canonical interaction site in the C-terminal domain. Finally, a biological role for a non-canonical interaction was observed in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70 canonical actions. Additionally, these results raise the question of whether other misfolded proteins might also engage via the same non-canonical mechanism.KEYWORDS: α-Synuclein, chaperone, Hsp70, protein misfolding, Parkinson's Disease, Lewy body dementia, synucleinopathy Neuropathological, biochemical and genetic evidence strongly implicates α-Synuclein (ASyn) in the onset and progression of Parkinson's disease (PD) and related syncucleinopathies including Lewy body dementias (LBD), multiple systems atrophy and Alzheimer's disease. Aggregated ASyn inclusions are a hallmark of these diseases [1], and ASyn gene mutations or multiplications cause early onset PD or LBD [2,3,4]. The sequential misfolding of ASyn into oligomers and fibrils is central to the pathogenesis of the synucleinopathies. ASyn gene multiplications and mutations causing PD and LBD are associated with enhanced oligomer formation [5,6,7]. Significant evidence supports the hypothesis that prion-like transmission of misfolded ASyn underlies disease progression [8,9]. The severity of disease correlates with the progressive spread of aggregated ASyn in patients [10], and misfolding is associated with toxicity in cell and animal models [11]. As shown for other amyloid misfolding proteins, compelling evidence supports pre-fibrillar ASyn oligomers and not fibrillar deposits as the pathogenic species in disease [5,12,13,14,15,16]. ASyn oligomers are directly toxic to cells [16], and mutations enhancing ASyn oligomer formation increase ASyn toxicity in neurons and rodents [12,13,14].The constitutive (Hsc70) and inducible (Hsp70) forms of the 70-KDa cytosolic heat shock molecular chaperones (Hsp70s) assist a wide variety of folding processes and provide broad protection against protein misfolding in the cell. ...

Hsp70 Inhibits Aggregation of IAPP by Binding to the Heterogeneous Prenucleation Oligomers

“…A recent study has demonstrated the protective role of the secretory Hsp70 in blocking the amyloid-b-induced neurotoxicity in Drosophila (56). Holdase activity of Hsp70 has also been shown to block formation of the toxic oligomers of a-synuclein (57). Our results demonstrate that Hsp70 strongly inhibits aggregation of IAPP under diverse solution conditions via ''holdase'' activity (see Figs.…”

“…Furthermore, a second binding site that is important for the activity of the Hsp70 has been reported recently (70). A recent study suggests involvement of a noncanonical site of Hsp70 in blocking the oligomerization of a-synuclein (57). Hence, multivalent binding might involve the alternative binding sites and/or other protein-protein interaction sites on the surface of Hsp70.…”

Folding or holding?—Hsp70 and Hsp90 chaperoning of misfolded proteins in neurodegenerative disease

“…The interaction between Hsp70 and α-synuclein was initially identified through the co-localization of Hsp70 with α-synuclein in LBs (61). Later studies have shown that Hsp70 inhibits α-synuclein fibril formation in a dose-dependent manner (49,(62)(63)(64). This suppression of α-synuclein fibrillation is nucleotide independent as the presence of ATP does not significantly alter the inhibitory effects (49).…”

“…Furthermore, Hsp70 truncations that lack the NBD are sufficient to inhibit fibril formation, suggesting that the inhibitory effects of Hsp70 are not mediated by an ATPase-dependent folding by Hsp70 (64). It has been well documented that the SBD (residues 386-640) alone is sufficient to inhibit fibril formation (49,62,64), even showing more efficient inhibition than full-length Hsp70 (64). By contrast, truncations of the SBD that lack the C-terminal lid domain (residues 386-543) show reduced suppression of α-synuclein fibrillation.…”

“…By contrast, truncations of the SBD that lack the C-terminal lid domain (residues 386-543) show reduced suppression of α-synuclein fibrillation. Overexpression of full-length Hsp70, as well as the SBD alone, can inhibit the toxicity of α-synuclein accumulation in cells (62), whereas SBD constructs lacking the lid domain show reduced protection against α-synuclein toxicity (64).…”