Interaction of the Molecular Chaperone αB-Crystallin with α-Synuclein: Effects on Amyloid Fibril Formation and Chaperone Activity

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Abstract

α-Synuclein is a pre-synaptic protein, the function of which is not completely understood, but its pathological form is involved in neurodegenerative diseases. In vitro, α-synuclein spontaneously forms amyloid fibrils. Here, we report that αB-crystallin, a molecular chaperone found in Lewy bodies that are characteristic of Parkinson's disease (PD), is a potent in vitro inhibitor of α-synuclein fibrillization, both of wild-type and the two mutant forms (A30P and A53T) that cause familial, early onset PD. In doing so, large irregular aggregates of α-synuclein and αB-crystallin are formed implying that αB-crystallin redirects α-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway, thus reducing the amount of physiologically stable amyloid deposits in favor of easily degradable amorphous aggregates. α-Synuclein acts as a molecular chaperone to prevent the stress-induced, amorphous aggregation of target proteins. Compared to wild-type α-synuclein, both mutant forms have decreased chaperone activity in vitro against the aggregation of reduced insulin at 37 °C and the thermally induced aggregation of βL-crystallin at 60 °C. Wild-type α-synuclein abrogates the chaperone activity of αB-crystallin to prevent the precipitation of reduced insulin. Interaction between these two chaperones and formation of a complex are also indicated by NMR spectroscopy, size-exclusion chromatography and mass spectrometry. In summary, α-synuclein and αB-crystallin interact readily with each other and affect each other's properties, in particular α-synuclein fibril formation and αB-crystallin chaperone action.

Introduction

Lewy bodies (LBs) are aggregated protein deposits found in neuronal and glial cytoplasm. Their presence is associated with neurodegenerative disorders such as sporadic and familial Parkinson's disease (PD), dementia with LBs and the LB variant of Alzheimer's disease.1., 2., 3. The principal component of LBs is the protein α-synuclein, but they also contain various amounts of other proteins, including the molecular chaperones, αB-crystallin, clusterin, torsin A, Hsp27 and Hsp70.4., 5., 6., 7., 8. αB-crystallin and Hsp27 are members of the small heat-shock protein (sHsp) family of molecular chaperones and have a similar structure and chaperone ability. αB-crystallin, and the closely related sHsp, αA-crystallin (collectively known as α-crystallin) are the major components of the vertebrate lens, with αB-crystallin being present in many other tissues. sHsps act as molecular chaperones by preventing the aggregation of other proteins under conditions of physiological stress (e.g. heat, changes in pH, osmotic shock, reducing environment), but unlike many other chaperones, have very limited ability to refold stressed proteins.9., 10. The mechanism of sHsp chaperone action is not fully understood; however, sHsps interact with aggregation-prone, non-native, intermediately folded forms of proteins (e.g. molten-globule states).11., 12. sHsps occur in large aggregates, which at higher temperatures undergo dynamic subunit exchange, a process that is believed to be important in their chaperone action.13

α-Synuclein was first identified as a minor fibrillar component of senile plaques in brains of patients with Alzheimer's disease, and was named precursor of non-amyloid β component protein (NACP).14 Initially, this protein was thought to be localized in both neuronal synapses and nuclei, and was therefore named “synuclein”; however, later it was confirmed only as a presynaptic protein.15 A major role for α-synuclein in neurodegeneration was recognized in PD when a mutation in the α-synuclein gene was associated with autosomal dominant inheritance of the disease.16 Two autosomal dominant mutations in the α-synuclein gene were linked to familial early onset PD: A53T was found in Italian and Greek families,16 and A30P in a German pedigree.17 The fibrillar morphology, diagnostic staining characteristics and underlying β-sheet structure of the α-synuclein deposits in LBs led to them being classified as amyloid fibrils, and the disease is now regarded as a protein misfolding disease like Alzheimer's, Creutzfeldt–Jakobs and Huntington's.18

In vitro, α-synuclein forms amyloid fibrils which have a morphology similar to those isolated from LBs.18., 19. Although it is not clear what turns this natively unfolded protein20 into cross-β-sheet containing fibrils, evidence suggests that the central hydrophobic region of α-synuclein (NAC) is amyloidogenic.19 The enhancement of the fibrillization rate by the two mutant forms21 may lead to LB deposits that can contribute to the severity of PD. However, the accumulation of cytotoxic soluble forms of α-synuclein, due to impairment of protein catabolism may represent the primary cause of neuronal loss.22 The overexpression of α-synuclein, especially its A53T or A30P forms, leads to death of dopaminergic neurons in culture or transgenic rat brains.23., 24. The lethal effect of these forms is based on oxidative damage, and cells expressing C-terminally truncated α-synuclein and mutants are more vulnerable.25 Wild-type, but not A53T, can modulate neuronal response to apoptotic stimuli.26

The physiological function of α-synuclein is not well understood. Studies have suggested a role in the learning process20 most likely by regulating neuroplasticity,27 in neuronal cell injury,28., 29. in modulating dopamine neurotransmission by inhibiting tyrosine hydroxylase activity (a rate-limiting enzyme in dopamine biosynthesis),30 and up-regulation of dopamine transporter molecules in the plasma membrane.29 The overexpression of α-synuclein has been directly associated with neuronal loss in PD,31 but a protective role of α-synuclein against chemically induced neurodegeneration has been shown.32

α-Synuclein can interact with other proteins, such as 14-3-3 proteins, PKC, BAD, ERK and microtubule-associated protein tau.33., 34. Being homologous to the ubiquitous molecular chaperones, the 14-3-3 proteins, it was postulated that α-synuclein could have chaperone activity.33 α-Synuclein and its related proteins, β and γ-synucleins can act as molecular chaperones in vitro by preventing aggregation of non-native conformations of proteins under stress conditions such as heat or chemical denaturation.35., 36. However, the ability to refold aggregated or incorrectly folded proteins was not observed. This suggests α-synuclein has chaperone activity that is similar to sHsps.9., 10. α-Synuclein is natively unstructured;20., 37. however, its conserved N-terminal region can shift into α-helical conformation upon weak association with pre-synaptic vesicles.38 The N-terminal domain is also responsible for binding target proteins during chaperone action, while the C-terminal acidic extension solubilizes the resultant high molecular mass complexes.39 sHsps have a similar structural arrangement, in particular the presence of a solubilizing, flexible C-terminal extension.10 Although some evidence suggested a correlation between partial structural changes in α-synuclein and its propensity to fibrillate, as well as binding other proteins,36 it is not clear whether the chaperone function of α-synuclein is involved in PD.

α-Crystallin potently inhibits the in vitro aggregation of an amyloid-forming protein, apolipoprotein C-II.40 Furthermore, αB-crystallin stabilizes pre-fibrillar cytotoxic forms of Aβ-peptide, the putative causative agent in Alzheimer's disease, and prevents their progression to mature fibrils.41., 42. In addition, fibrillization of α-synuclein can be prevented by molecular chaperones such as torsin A and Hsp70,8 as well as by β and γ-synucleins.35., 43.

In this study, we examined in detail the in vitro interaction of αB-crystallin with α-synuclein, both from its fibril-forming characteristics and its chaperone ability. The overall goal was to gain insight into the role of sHsps (and potentially other chaperones) in the development of PD.

Section snippets

Assembly of α-synuclein into fibrils and the effect of αB-crystallin on fibril formation

Concentrated solutions (0.25–0.50 mM) of wild-type α-synuclein, as well as its mutant forms, A30P and A53T, at physiological temperature and pH, underwent slow polymerization resulting in formation of fibrils over a period of one to two weeks. Fibril formation was assessed by electron microscopy (EM), dye binding and X-ray fibre diffraction. Fibrils of α-synuclein bound Congo red and produced apple-green birefringence under crossed polarized light (data not shown) and the X-ray diffraction

Discussion

Here, the mutual effects of the interaction between α-synuclein and αB-crystallin were investigated under slow aggregation conditions (amyloid fibril formation by α-synuclein itself) and fast, amorphously aggregating systems (reduction and heating of target proteins).

Materials

Bovine pancreas insulin, dithiothreitol (DTT), thioflavin T and dextran 68,800 were purchased from Sigma. [15N]–NH4Cl was from Cambridge Isotopes, Inc., USA.

Protein expression and purification

pRSET plasmids containing human α-synuclein sequence (wild-type, A30P or A53T) were used to overexpress α-synucleins in the Escherichia coli strain BL21(DE3). Transformed cells were grown on ampicillin-containing (100 μg/ml) LB media and protein expression was induced with 1 mM IPTG. Cells were harvested by centrifugation and pellets lysed

Acknowledgements

This work was supported by a grant from the National Health and Medical Research Council of Australia (to J.A.C.) (213112). We thank Professor W. W. de Jong and Dr W. Boelens, University of Nijmegen, The Netherlands, for provision of the B-crystallin plasmid, and the Wollondilly Abattoirs, NSW, for donating calf lenses. We are grateful to Dr M. Guss for help with X-ray image collection.

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    Present address: J. A. Carver, School of Chemistry and Physics, The University of Adelaide, Adelaide, SA 5005, Australia.

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