Hsc70 Protein Interaction with Soluble and Fibrillar α-Synuclein

Abstract

The aggregation of ␣-synuclein (␣-Syn), the primary component of Lewy bodies, into high molecular weight assemblies is strongly associated with Parkinson disease. This event is believed to result from a conformational change within native ␣-Syn. Molecular chaperones exert critical housekeeping functions in vivo including refolding, maintaining in a soluble state, and/or pacifying protein aggregates. The influence of the stressinduced heat shock protein 70 (Hsp70) on ␣-Syn aggregation has been notably investigated. The constitutively expressed chaperone Hsc70 acts as an antiaggregation barrier before cells are overwhelmed with ␣-Syn aggregates and Hsp70 expression induced. Here, we investigate the interaction between Hsc70 and ␣-Syn, the consequences of this interaction, and the role of nucleotides and co-chaperones Hdj1 and Hdj2 as modulators. We show that Hsc70 sequesters soluble ␣-Syn in an assembly incompetent complex in the absence of ATP. The affinity of Hsc70 for soluble ␣-Syn diminishes upon addition of ATP alone or together with its co-chaperones Hdj1 or Hdj2 allowing faster binding and release of client proteins thus abolishing ␣-Syn assembly inhibition by Hsc70. We show that Hsc70 binds ␣-Syn fibrils with a 5-fold tighter affinity compared with soluble ␣-Syn. This suggests that Hsc70 preferentially interacts with high molecular weight ␣-Syn assemblies in vivo. Hsc70 binding certainly has an impact on the physicochemical properties of ␣-Syn assemblies. We show a reduced cellular toxicity of ␣-Syn fibrils coated with Hsc70 compared with "naked" fibrils. Hsc70 may therefore significantly affect the cellular propagation of ␣-Syn aggregates and their spread throughout the central nervous system in Parkinson disease.2 is one of the principal components of intracellular Lewy bodies, whose presence in the central nervous system is a defining feature of Parkinson disease (PD) and other synucleinopathies (1). Symptoms of PD are apparent after more than 70% of dopaminergic terminals and/or neurons have been lost, with some of the remaining neurons containing filamentous ␣-Syn. The precise cellular function of soluble ␣-Syn is unknown. There is, however, evidence that it plays an important role at presynaptic termini (2-4) and it has recently been suggested that the protein is a cellular ferrireductase (5). Various factors, including genetic susceptibility and environmental influences, can induce the aggregation of the naturally unfolded, soluble ␣-Syn (6, 7), leading to PD pathogenesis.Molecular chaperones assist newly synthesized proteins to reach their native-fold and are in charge of refolding misfolded or unfolded proteins (8). As the progression of PD is strongly associated with a change in ␣-Syn solubility, which is widely believed to be the consequence of a conformational change, there has been significant focus on the possible therapeutic role of molecular chaperones in the past 5 years (9). Previous investigations have focused on heat shock protein 70 (Hsp70) (10 -13), a molecular chaperone whose express...

Identification of Protein Interfaces between α-Synuclein, the Principal Component of Lewy Bodies in Parkinson Disease, and the Molecular Chaperones Human Hsc70 and the Yeast Ssa1p

“…This is why cross-linking was performed in the absence of nucleotide. No inhibition ␣-Syn assembly is observed in the presence of ADP (18). Consistent with that no Hsc70-␣-Syn complex with apparent molecular mass of 100 kDa was observed in the presence of BS2G and ADP.…”

“…Interaction between Hsc70 and Soluble ␣-Syn-We recently demonstrated that Hsc70 sequesters ␣-Syn in an assembly incompetent state (18). We show here (Fig.…”

“…The most efficient sequestering activity was observed for Hsc70 in the absence of nucleotide. Hsc70 inhibits to a lesser extent ␣-Syn assembly in the presence of ATP (18). This is why cross-linking was performed in the absence of nucleotide.…”

“…Furthermore, a reduction in the toxicity of fibrillar ␣-Syn upon coating the fibrils with Hsc70 was observed. These findings encouraged us to consider the use of Hsc/p70 as a potential therapeutic tool in PD (18,20).…”

“…The expression of members of the Hsp70 family (13)(14)(15)(16)) is induced when a cellular environment is under stress such as heat shock, ischemia, or other stresses (17). This prompted us and others to document the effect of Hsc/p70 on ␣-Syn assembly in vitro and in vivo (18,19). Hsc/p70 have been shown to inhibit ␣-Syn fibril formation.…”