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Hsc70 protein interaction with soluble and fibrillar alpha-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 stress-induced 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.

SUBMITTER: Pemberton S

PROVIDER: S-EPMC3186418 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Hsc70 protein interaction with soluble and fibrillar alpha-synuclein.

Pemberton Samantha S Madiona Karine K Pieri Laura L Kabani Mehdi M Bousset Luc L Melki Ronald R

The Journal of biological chemistry 20110810 40

The aggregation of α-synuclein (α-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 α-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 stress-induced heat shock protein 70 (Hsp70) on α-Syn aggregation has been n ...[more]

PMID: 21832061

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Project description:Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E. coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs.

Dataset Information

Hsc70 protein interaction with soluble and fibrillar alpha-synuclein.

Publications

Hsc70 protein interaction with soluble and fibrillar alpha-synuclein.

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