Agata Rekas

Small heat-shock proteins and clusterin: intra- and extracellular molecular chaperones with a common mechanism of action and function?

Small heat‐shock proteins (sHsps) and clusterin are molecular chaperones that share many functional similarities despite their lack of significant sequence similarity. These functional similarities, and some differences, are discussed. sHsps are ubiquitous intracellular proteins whereas clusterin is generally found extracellularly. Both chaperones potently prevent the amorphous aggregation and precipitation of target proteins under stress conditions such as elevated temperature, reduction and oxidation. In doing so, they act on the slow, off‐folding protein pathway. The conformational dynamism and aggregated state of both proteins may be crucial for their chaperone function. Subunit exchange is likely to be important in regulating chaperone action; the dissociated form of the protein is probably the chaperone‐active species rather than the aggregated state. They both exert their chaperone action without the need for hydrolysis of ATP and have little ability to refold target proteins. Increased expression of sHsps and clusterin accompanies a range of diseases that arise from protein misfolding and deposition of highly structured protein aggregates known as amyloid fibrils, e.g., Alzheimers, Creutzfeldt‐Jakob and Parkinsons diseases. The interaction of sHsps and clusterin with fibril‐forming species is discussed along with their ability to prevent fibril formation. IUBMB Life, 55: 661‐668, 2003

The structure of dopamine induced α-synuclein oligomers

Inclusions of aggregated α-synuclein (α-syn) in dopaminergic neurons are a characteristic histological marker of Parkinson’s disease (PD). In vitro, α-syn in the presence of dopamine (DA) at physiological pH forms SDS-resistant non-amyloidogenic oligomers. We used a combination of biophysical techniques, including sedimentation velocity analysis, small angle X-ray scattering (SAXS) and circular dichroism spectroscopy to study the characteristics of α-syn oligomers formed in the presence of DA. Our SAXS data show that the trimers formed by the action of DA on α-syn consist of overlapping worm-like monomers, with no end-to-end associations. This lack of structure contrasts with the well-established, extensive β-sheet structure of the amyloid fibril form of the protein and its pre-fibrillar oligomers. We propose on the basis of these and earlier data that oxidation of the four methionine residues at the C- and N-terminal ends of α-syn molecules prevents their end-to-end association and stabilises oligomers formed by cross linking with DA-quinone/DA-melanin, which are formed as a result of the redox process, thus inhibiting formation of the β-sheet structure found in other pre-fibrillar forms of α-syn.

R120G αB-crystallin promotes the unfolding of reduced α-lactalbumin and is inherently unstable

α‐Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, αB‐crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin‐related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92–95]. In the present study, real‐time 1H‐NMR spectroscopy showed that the ability of R120G αB‐crystallin to stabilize the partially folded, molten globule state of α‐lactalbumin was significantly reduced in comparison with wild‐type αB‐crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced α‐lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild‐type protein, R120G αB‐crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C‐terminus. Light scattering, MS, and size‐exclusion chromatography data indicated that R120G αB‐crystallin exists as a larger oligomer than wild‐type αB‐crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of αB‐crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G αB‐crystallin with human disease states.

Monitoring the prevention of amyloid fibril formation by α‐crystallin

The molecular chaperone, α‐crystallin, has the ability to prevent the fibrillar aggregation of proteins implicated in human diseases, for example, amyloid β peptide and α‐synuclein. In this study, we examine, in detail, two aspects of α‐crystallins fibril‐suppressing ability: (a) its temperature dependence, and (b) the nature of the aggregating species with which it interacts. First, the efficiency of α‐crystallin to suppress fibril formation in κ‐casein and α‐synuclein increases with temperature, despite their rate of fibrillation also increasing in the absence of α‐crystallin. This is consistent with an increased chaperone ability of α‐crystallin at higher temperatures to protect target proteins from amorphous aggregation [GB Reddy, KP Das, JM Petrash & WK Surewicz (2000) J Biol Chem275, 4565–4570]. Second, dual polarization interferometry was used to monitor real‐time α‐synuclein aggregation in the presence and absence of αB‐crystallin. In contrast to more common methods for monitoring the time‐dependent formation of amyloid fibrils (e.g. the binding of dyes like thioflavin T), dual polarization interferometry data did not reveal any initial lag phase, generally attributed to the formation of prefibrillar aggregates. It was shown that αB‐crystallin interrupted α‐synuclein aggregation at its earliest stages, most likely by binding to partially folded monomers and thereby preventing their aggregation into fibrillar structures.

PAMAM Dendrimers as Potential Agents against Fibrillation of α-Synuclein, a Parkinson's Disease-Related Protein

The effect of PAMAM dendrimers (generations G3, G4 and G5) on the fibrillation of alpha-synuclein was examined by fluorescence and CD spectroscopy, TEM and SANS. PAMAM dendrimers inhibited fibrillation of alpha-synuclein and this effect increased both with generation number and PAMAM concentration. SANS showed structural changes in the formed aggregates of alpha-synuclein--from cylindrical to dense three-dimensional ones--as the PAMAM concentration increased, on account of the inhibitory effect. PAMAM also effectively promoted the breaking down of pre-existing fibrils of alpha-synuclein. In both processes--that is, inhibition and disassociation of fibrils--PAMAM redirected alpha-synuclein to an amorphous aggregation pathway.

The Quaternary Organization and Dynamics of the Molecular Chaperone HSP26 Are Thermally Regulated

The function of ScHSP26 is thermally controlled: the heat shock that causes the destabilization of target proteins leads to its activation as a molecular chaperone. We investigate the structural and dynamical properties of ScHSP26 oligomers through a combination of multiangle light scattering, fluorescence spectroscopy, NMR spectroscopy, and mass spectrometry. We show that ScHSP26 exists as a heterogeneous oligomeric ensemble at room temperature. At heat-shock temperatures, two shifts in equilibria are observed: toward dissociation and to larger oligomers. We examine the quaternary dynamics of these oligomers by investigating the rate of exchange of subunits between them and find that this not only increases with temperature but proceeds via two separate processes. This is consistent with a conformational change of the oligomers at elevated temperatures which regulates the disassembly rates of this thermally activated protein.

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS

Background: β2-Microglobulin (β2m) is a paradigmatic amyloidogenic protein. Results: In vitro, the molecular chaperone αB-crystallin affects the oligomerization and the fibrillogenesis of β2m and its R3A mutant. Conclusion: αB-crystallin prevents β2m aggregation at various stages of its aggregation pathway. Significance: Molecular chaperones may be relevant to amyloid formation in vivo. The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β2-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.

The chaperone activity of α-synuclein: Utilizing deletion mutants to map its interaction with target proteins

α‐Synuclein is the principal component of the Lewy body deposits that are characteristic of Parkinsons disease. In vivo, and under physiological conditions in vitro, α‐synuclein aggregates to form amyloid fibrils, a process that is likely to be associated with the development of Parkinsons disease. α‐Synuclein also possesses chaperone activity to prevent the precipitation of amorphously aggregating target proteins, as demonstrated in vitro. α‐Synuclein is an intrinsically disordered (i.e., unstructured) protein of 140 amino acids in length, and therefore studies on its fragments can be correlated directly to the functional role of these regions in the intact protein. In this study, the fragment containing residues 61–140 [α‐syn(61–140)] was observed to be highly amyloidogenic and was as effective a chaperone in vitro as the full‐length protein, while the N‐ and C‐terminal fragments α‐syn(1–60) and α‐syn(96–140) had no intrinsic chaperone activity. Interestingly, full‐length fibrillar α‐synuclein had greater chaperone activity than nonfibrillar α‐synuclein. It is concluded that the amyloidogenic NAC region (residues 61–95) contains the chaperone‐binding site which is optimized for target protein binding as a result of its β‐sheet formation and/or ordered aggregation by α‐synuclein. On the other hand, the first 60 residues of α‐synuclein modulate the proteins chaperone‐active site, while at the same time protecting α‐synuclein from fibrillation. On its own, however, this fragment [α‐syn(1–60)] had a tendency to aggregate amorphously. As a result of this study, the functional roles of the various regions of α‐synuclein in its chaperone activity have been delineated. Proteins 2012;

Guanidine hydrochloride denaturation of dopamine-induced α-synuclein oligomers: A small-angle X-ray scattering study

Alpha‐synuclein (α‐syn) forms the amyloid‐containing Lewy bodies found in the brain in Parkinsons disease. The neurotransmitter dopamine (DA) reacts with α‐syn to form SDS‐resistant soluble, non‐amyloid, and melanin‐containing oligomers. Their toxicity is debated, as is the nature of their structure and their relation to amyloid‐forming conformers of α‐syn. The small‐angle X‐ray scattering technique in combination with modeling by the ensemble optimization method showed that the un‐reacted native protein populated three broad classes of conformer, while reaction with DA gave a restricted ensemble range suggesting that the rigid melanin molecule played an important part in their structure. We found that 6 M guanidine hydrochloride did not dissociate α‐syn DA‐reacted dimers and trimers, suggesting covalent linkages. The pathological significance of covalent association is that if they are non‐toxic, the oligomers would act as a sink for toxic excess DA and α‐syn; if toxic, their stability could enhance their toxicity. We argue it is essential, therefore, to resolve the question of whether they are toxic or not. Proteins 2014; 82:10–21.

Interrogating protonated/deuterated fibronectin fragment layers adsorbed to titania by neutron reflectivity and their concomitant control over cell adhesion.

The fibronectin fragment, 9th–10th-type III domains (FIII9–10), mediates cell attachment and spreading and is commonly investigated as a bioadhesive interface for implant materials such as titania (TiO2). How the extent of the cell attachment–spreading response is related to the nature of the adsorbed protein layer is largely unknown. Here, the layer thickness and surface fraction of two FIII9–10 mutants (both protonated and deuterated) adsorbed to TiO2 were determined over concentrations used in cell adhesion assays. Unexpectedly, the isotopic forms had different adsorption behaviours. At solution concentrations of 10 mg l−1, the surface fraction of the less conformationally stable mutant (FIII9′10) was 42% for the deuterated form and 19% for the protonated form (fitted to the same monolayer thickness). Similarly, the surface fraction of the more stable mutant (FIII9′10–H2P) was 34% and 18% for the deuterated and protonated forms, respectively. All proteins showed a transition from monolayer to bilayer between 30 and 100 mg l−1, with the protein longitudinal orientation moving away from the plane of the TiO2 surface at high concentrations. Baby hamster kidney cells adherent to TiO2 surfaces coated with the proteins (100 mg l−1) showed a strong spreading response, irrespective of protein conformational stability. After surface washing, FIII9′10 and FIII9′10–H2P bilayer surface fractions were 30/25% and 42/39% for the lower/upper layers, respectively, implying that the cell spreading response requires only a partial protein surface fraction. Thus, we can use neutron reflectivity to inform the coating process for generating bioadhesive TiO2 surfaces.