Different ataxin-3 amyloid aggregates induce intracellular Ca2+ deregulation by different mechanisms in cerebellar granule cells

AbstractThis work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca2+ levels and the abnormal Ca2+ signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca2+ responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca2+ response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells

A covalent homodimer probing early oligomers along amyloid aggregation

Early oligomers are crucial in amyloid aggregation; however, due to their transient nature they are among the least structurally characterized species. We focused on the amyloidogenic protein beta2-microglobulin (beta2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing beta2m molecules was repeatedly observed, suggesting that such interface may be relevant for beta2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized beta2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt beta2m under native conditions, it shows enhanced amyloid aggregation propensity. Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity. Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of beta2m aggregation. The present data provide new insight into beta2m early steps of amyloid aggregation

Wild type beta-2 microglobulin and DE loop mutants display a common fibrillar architecture

Beta-2 microglobulin (\u3b22m) is the protein responsible for a pathologic condition known as dialysis related amyloidosis. In recent years an important role has been assigned to the peptide loop linking strands D and E (DE loop) in determining \u3b22m stability and amyloid propensity. Several mutants of the DE loop have been studied, showing a good correlation between DE loop geometrical strain, protein stability and aggregation propensity. However, it remains unclear whether the aggregates formed by wild type (wt) \u3b22m and by the DE loop variants are of the same kind, or whether the mutations open new aggregation pathways. In order to address this question, fibrillar samples of wt and mutated \u3b22m variants have been analysed by means of atomic force microscopy and infrared spectroscopy. The data here reported indicate that the DE loop mutants form aggregates with morphology and structural organisation very similar to the wt protein. Therefore, the main effect of \u3b22m DE loop mutations is proposed to stem from the different stabilities of the native fold. Considerations on the structural role of the DE loop in the free monomeric \u3b22m and as part of the Major Histocompatibility Complex are also presented

Salt anions promote the conversion of HypF-N into amyloid-like oligomers and modulate the structure of the oligomers and the monomeric precursor state

An understanding of the solution factors contributing to the rate of aggregation of a protein into amyloid oligomers, to the modulation of the conformational state populated prior to aggregation and to the structure/morphology of the resulting oligomers is one of the goals of present research in this field. We have studied the influence of six different salts on the conversion of the N-terminal domain of Escherichia coli HypF (HypF-N) into amyloid-like oligomers under conditions of acidic pH. Our results show that salts having different anions (NaCl, NaClO4, NaI, Na 2SO4) accelerate oligomerization with an efficacy that follows the electroselectivity series of the anions (SO4 2- ≥ ClO4 - > I- > Cl-). By contrast, salts with different cations (NaCl, LiCl, KCl) have similar effects. We also investigated the effect of salts on the structure of the final and initial states of HypF-N aggregation. The electroselectivity series does not apply to the effect of anions on the structure of the oligomers. By contrast, it applies to their effect on the content of secondary structure and on the exposure of hydrophobic clusters of the monomeric precursor state. The results therefore indicate that the binding of anions to the positively charged residues of HypF-N at low pH is the mechanism by which salts modulate the rate of oligomerization and the structure of the monomeric precursor state but not the structure of the resulting oligomers. Overall, the data contribute to rationalize the effect of salts on amyloid-like oligomer formation and to explain the role of charged biological macromolecules in protein aggregation processes.Peer Reviewe

Photochromic and photomechanical responses of an amorphous diarylethene-based polymer: a spectroscopic ellipsometry investigation of ultrathin films

This work deals with very thin (14-40 nm) films of a polyester containing diarylethene units in the main chain spin cast on a silicon wafer. By irradiation with UV light the colourless form turns blue due to the appearance of a strong absorption band centred at about 600 nm. The coloured state is thermally stable and the backward conversion can be triggered with visible light. Comparison of broadband (245-1700 nm) spectroscopic ellipsometry data with simulations based on an isotropic, Kramers???Kronig consistent, multiple-resonance model allowed us to determine the complex index of refraction n of the film in its blue and colourless forms. The refractive index of the blue phase neatly exceeds that of the transparent form for wavelengths in the NIR. In particular, out of resonance, at lambda 1700 nm, DeltaRe(n) 0.05. Parallel to the DeltaRe(n) increase, the analysis of ellipsometry data suggests a decrease of the film thickness (about 1.5%) during the transition from the open (transparent) to the closed (coloured) form

Photochromic and photomechanical responses of an amorphous diarylethene-based polymer: a spectroscopic ellipsometry investigation of ultrathin films

This work deals with very thin (14-40 nm) films of a polyester containing diarylethene units in the main chain spin cast on a silicon wafer. By irradiation with UV light the colourless form turns blue due to the appearance of a strong absorption band centred at about 600 nm. The coloured state is thermally stable and the backward conversion can be triggered with visible light. Comparison of broadband (245-1700 nm) spectroscopic ellipsometry data with simulations based on an isotropic, Kramers???Kronig consistent, multiple-resonance model allowed us to determine the complex index of refraction n of the film in its blue and colourless forms. The refractive index of the blue phase neatly exceeds that of the transparent form for wavelengths in the NIR. In particular, out of resonance, at lambda 1700 nm, DeltaRe(n) 0.05. Parallel to the DeltaRe(n) increase, the analysis of ellipsometry data suggests a decrease of the film thickness (about 1.5%) during the transition from the open (transparent) to the closed (coloured) form