Biophysical studies of protein misfolding and aggregation in in vivo models of Alzheimer's and Parkinson's diseases.

Neurodegenerative disorders, including Alzheimer's (AD) and Parkinson's diseases (PD), are characterised by the formation of aberrant assemblies of misfolded proteins. The discovery of disease-modifying drugs for these disorders is challenging, in part because we still have a limited understanding of their molecular origins. In this review, we discuss how biophysical approaches can help explain the formation of the aberrant conformational states of proteins whose neurotoxic effects underlie these diseases. We discuss in particular models based on the transgenic expression of amyloid-ß (Aß) and tau in AD, and α-synuclein in PD. Because biophysical methods have enabled an accurate quantification and a detailed understanding of the molecular mechanisms underlying protein misfolding and aggregation in vitro, we expect that the further development of these methods to probe directly the corresponding mechanisms in vivo will open effective routes for diagnostic and therapeutic interventions.

Amyloid-like Fibrils from an α-Helical Transmembrane Protein.

The propensity to misfold and self-assemble into stable aggregates is increasingly being recognized as a common feature of protein molecules. Our understanding of this phenomenon and of its links with human disease has improved substantially over the past two decades. Studies thus far, however, have been almost exclusively focused on cytosolic proteins, resulting in a lack of detailed information about the misfolding and aggregation of membrane proteins. As a consequence, although such proteins make up approximately 30% of the human proteome and have high propensities to aggregate, relatively little is known about the biophysical nature of their assemblies. To shed light on this issue, we have studied as a model system an archetypical representative of the ubiquitous major facilitator superfamily, the Escherichia coli lactose permease (LacY). By using a combination of established indicators of cross-ß structure and morphology, including the amyloid diagnostic dye thioflavin-T, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, X-ray fiber diffraction, and transmission electron microscopy, we show that LacY can form amyloid-like fibrils under destabilizing conditions. These results indicate that transmembrane α-helical proteins, similarly to cytosolic proteins, have the ability to adopt this generic state.

Probing Polyoxometalate-Protein Interactions Using Molecular Dynamics Simulations.

The molecular interactions between the CeIV -substituted Keggin anion [PW11 O39 Ce(OH2 )4 ]3- (CeK) and hen egg-white lysozyme (HEWL) were investigated by molecular dynamics simulations. The analysis of CeK was compared with the CeIV -substituted Keggin dimer [(PW11 O39 )2 Ce]10- (CeK2 ) and the ZrIV -substituted Lindqvist anion [W5 O18 Zr(OH2 )(OH)]3- (ZrL) to understand how POM features such as shape, size, charge, or type of incorporated metal ion influence the POM⋅⋅⋅protein interactions. Simulations revealed two regions of the protein in which the CeK anion interacts strongly: cationic sites formed by Arg21 and by Arg45 and Arg68. The POMs chiefly interact with the side chains of the positively charged (arginines, lysines) and the polar uncharged residues (tyrosines, serines, aspargines) via electrostatic attraction and hydrogen bonding with the oxygen atoms of the POM framework. The CeK anion shows higher protein affinity than the CeK2 and ZrL anions, because it is less hydrophilic and it has the right size and shape for establishing interactions with several residues simultaneously. The larger, more negatively charged CeK2 anion has a high solvent-accessible surface, which is sub-optimal for the interaction, while the smaller ZrL anion is highly hydrophilic and cannot efficiently interact with several residues simultaneously.

Regioselective hydrolysis of human serum albumin by Zr(IV)-substituted polyoxotungstates at the interface of positively charged protein surface patches and negatively charged amino acid residues.

Complexes comprising the Lewis acidic Zr(IV) metal and protein binding polyoxotungstate ligands of Lindqvist-, Keggin- and Wells-Dawson-type were found to region selectively hydrolyze human serum albumin at four distinct positions. Higher reactivities were found for structures with higher polyoxometalate charges and the cleavage positions were found in protein regions of mixed charge. Both findings suggest an electrostatic nature of the observed reactivity.

Molecular origin of the hydrolytic activity and fixed regioselectivity of a Zr(IV) -substituted polyoxotungstate as artificial protease.

A multitechnique approach has been applied in order to identify the thermodynamic and kinetic parameters related to the regioselective hydrolysis of human serum albumin (HSA) promoted by the Wells-Dawson polyoxometalate (POM), K15 H[Zr(α2 -P2 W17 O61 )2 ]. Isothermal titration calorimetry (ITC) studies indicate that up to four POM molecules interact with HSA. While the first interaction site is characterized by a 1:1 binding and an affinity constant of 2×10(8) M(-1) , the three remaining sites are characterized by a lower global affinity constant of 7×10(5) M(-1) . The higher affinity constant at the first site is in accordance with a high quenching constant of 2.2×10(8) M(-1) obtained for fluorescence quenching of the Trp214 residue located in the only positively charged cleft of HSA, in the presence of K15 H[Zr(α2 -P2 W17 O61 )2 ]. In addition, Eu(III) luminescence experiments with an Eu(III) -substituted POM analogue have shown the replacement of water molecules in the first coordination sphere of Eu(III) due to binding of the metal ion to amino acid side chain residues of HSA. All three interaction studies are in accordance with a stronger POM dominated binding at the positive cleft on the one hand, and interaction mainly governed by metal anchoring at the three remaining positions, on the other hand. Hydrolysis experiments in the presence of K15 H[Zr(α2 -P2 W17 O61 )2 ] have demonstrated regioselective cleavage of HSA at the Arg114Leu115, Ala257Asp258, Lys313Asp314 or Cys392Glu393 peptide bonds. This is in agreement with the interaction studies as the Arg114Leu115 peptide bond is located in the positive cleft of HSA and the three remaining peptide bonds are each located near an upstream acidic residue, which can be expected to coordinate to the metal ion. A detailed kinetic study has evidenced the formation of additional fragments upon prolonged reaction times. Edman degradation of the additional reaction products has shown that these fragments result from further hydrolysis at the initially observed cleavage positions, indicating a fixed selectivity for K15 H[Zr(α2 -P2 W17 O61 )2 ].

Polyoxometalates as a novel class of artificial proteases: selective hydrolysis of lysozyme under physiological pH and temperature promoted by a cerium(IV) Keggin-type polyoxometalate.

Hen-egg-white lysozyme (HEWL) is specifically cleaved at the Trp28-Val29 and Asn44-Arg45 peptide bonds in the presence of a Keggin-type [Ce(α-PW(11)O(39))(2)](10-) polyoxometalate (POM; 1) at pH 7.4 and 37 °C. The reactivity of 1 towards a range of dipeptides was also examined and the calculated reaction rates were comparable to those observed for the hydrolysis of HEWL. Experiments with α-lactalbumin (α-LA), a protein that is structurally highly homologous to HEWL but has a different surface potential, showed no evidence of hydrolysis, which indicates the importance of electrostatic interactions between 1 and the protein surface for the hydrolytic reaction to occur. A combination of spectroscopic techniques was used to reveal the molecular interactions between HEWL and 1 that lead to hydrolysis. NMR spectroscopy titration experiments showed that on protein addition the intensity of the (31)P NMR signal of 1 gradually decreased due to the formation of a large protein/polyoxometalate complex and completely disappeared when the HEWL/1 ratio reached 1:2. Circular dichroism (CD) measurements of HEWL indicate that addition of 1 results in a clear decrease in the signal at λ=208 nm, which is attributed to changes in the α-helical content of the protein. (15)N-(1)H heteronuclear single quantum coherence (HSQC) NMR measurements of HEWL in the presence of 1 reveal that the interaction is mainly observed for residues that are located in close proximity to the first site in the α-helical part of the structure (Trp28-Val29). The less pronounced NMR spectroscopic shifts around the second cleavage site (Asn44-Arg45), which is found in the ß-strand region of the protein, might be caused by weaker metal-directed binding, compared with strong POM-directed binding at the first site.

Molecular interactions between serum albumin proteins and Keggin type polyoxometalates studied using luminescence spectroscopy.

The interaction between the plenary Keggin H3PW12O40, lacunary Keggin K7PW11O39 and the Eu(III)-substituted Keggin K4EuPW11O39 (Eu-Keggin) type polyoxometalates (POMs), and the proteins human and bovine serum albumin (HSA and BSA) was studied using steady state and time-resolved Eu(III) luminescence and tryptophan (Trp) fluorescence spectroscopy. The excitation spectrum of the Eu-Keggin POM is dominated by a ligand-to-metal charge transfer band at 291 nm. In the absence of proteins, the number of water molecules coordinated in the first coordination sphere of the Eu(III) center of Eu-Keggin was determined to be 4, indicating that Eu(III) occurs as a 1 : 1 isomer in solution. In the presence of HSA or BSA, the number of coordinated water molecules decreased to 0 and 1, respectively, suggesting interaction between the Eu-Keggin POM and the protein surface. As a result of this interaction, a five-fold increase of the hypersensitive (5)D0 → (7)F2 transition in the luminescence intensity was observed for the Eu-Keggin-HSA complex. The association constants were calculated to be 1.5 × 10(2) M(-1) and 2.0 × 10(3) M(-1) for the Eu-Keggin-HSA and Eu-Keggin-BSA complexes, respectively. Tryptophan fluorescence quenching studies were performed and the quenching constants were calculated using a Stern-Volmer analysis. The obtained values of the quenching constants were 6.1 × 10(4) M(-1) and 2.0 × 10(6) M(-1) for the Eu-Keggin-HSA and Eu-Keggin-BSA complexes, respectively. The surface map of both proteins shows that the cavity containing the tryptophan has a positive surface potential, providing a specific binding site at the surface of albumin proteins for the negatively charged POM.

Hydrolysis of serine-containing peptides at neutral pH promoted by [MoO4]2- oxyanion.

Hydrolysis of the dipeptides glycylserine (GlySer), leucylserine (LeuSer), histidylserine (HisSer), glycylalanine (GlyAla), and serylglycine (SerGly) was examined in oxomolybdate solutions by means of (1)H, (13)C, and (95)Mo NMR spectroscopy. In the presence of a mixture of oxomolybdates, the hydrolysis of the peptide bond in GlySer proceeded under neutral pD conditions (pD = 7.0, 60 °C) with a rate constant of k(obs) = 5.9 × 10(-6) s(-1). NMR spectra did not show evidence of the formation of paramagnetic species, excluding the possibility of Mo(VI) reduction to Mo(V), indicating that the cleavage of the peptide bond is purely hydrolytic. The pD dependence of k(obs) exhibits a bell-shaped profile, with the fastest cleavage observed at pD 7.0. Comparison of the rate profile with the concentration profile of oxomolybdate species implicated monomolybdate MoO(4)(2-) as the kinetically active complex. Kinetics experiments at pD 7.0 using a fixed amount of GlySer and increasing amounts of MoO(4)(2-) allowed for calculation of the catalytic rate constant (k(2) = 9.25 × 10(-6) s(-1)) and the formation constant for the GlySer-MoO(4)(2-) complex (K(f) = 15.25 M(-1)). The origin of the hydrolytic activity of molybdate is most likely a combination of the polarization of amide oxygen in GlySer due to the binding to molybdate, followed by the intramolecular attack of the Ser hydroxyl group.

Cytosolic aggregation of mitochondrial proteins disrupts cellular homeostasis by stimulating the aggregation of other proteins.

Mitochondria are organelles with their own genomes, but they rely on the import of nuclear-encoded proteins that are translated by cytosolic ribosomes. Therefore, it is important to understand whether failures in the mitochondrial uptake of these nuclear-encoded proteins can cause proteotoxic stress and identify response mechanisms that may counteract it. Here, we report that upon impairments in mitochondrial protein import, high-risk precursor and immature forms of mitochondrial proteins form aberrant deposits in the cytosol. These deposits then cause further cytosolic accumulation and consequently aggregation of other mitochondrial proteins and disease-related proteins, including α-synuclein and amyloid ß. This aggregation triggers a cytosolic protein homeostasis imbalance that is accompanied by specific molecular chaperone responses at both the transcriptomic and protein levels. Altogether, our results provide evidence that mitochondrial dysfunction, specifically protein import defects, contributes to impairments in protein homeostasis, thus revealing a possible molecular mechanism by which mitochondria are involved in neurodegenerative diseases.

Eu(III) luminescence and tryptophan fluorescence spectroscopy as a tool for understanding interactions between hen egg white lysozyme and metal-substituted Keggin type polyoxometalates.

The interaction between the lacunary Keggin K7PW11O39, the Eu(III)-substituted Keggin K4EuPW11O39 (Eu-Keggin) and the Ce(IV)-substituted Keggin [Me2NH2]10[Ce(PW11O39)2] (Ce-Keggin) polyoxometalates (POMs), and the proteins hen egg white lysozyme (HEWL) and the structurally homologous α-lactalbumin (α-LA) was studied by steady state and time-resolved Eu(III) luminescence and tryptophan (Trp) fluorescence spectroscopy. The excitation spectrum of Eu-Keggin at lower concentrations ([Eu-Keggin]<100 µM) is dominated by a ligand-to-metal charge transfer band (291 nm). For higher concentrations ([Eu-Keggin]>250 µM) the (5)L6←(7)F0 transition becomes the most intense peak. In the absence of protein, the number of coordinated water molecules to the Eu(III) centre of Eu-Keggin is 4, indicating a 1:1 Eu(III):POM species. In the presence of phosphate buffer this number linearly decreases from 4 to 2 upon increasing phosphate buffer concentration. Upon addition of HEWL, there are no coordinated water molecules, suggesting interaction between Eu-Keggin and the protein surface. In addition, this interaction results in a more than threefold increase of the hypersensitive (5)D0→(7)F2 transition for the Eu-Keggin/HEWL mixture. The calculated association constant amounted to 2.2×10(2) M(-1) for the Eu-Keggin/HEWL complex. Tryptophan fluorescence quenching studies were performed and the quenching constants were calculated to be 9.1×10(4) M(-1), 4×10(4) M(-1) and 4.1×10(5) M(-1) for the lacunary Keggin/HEWL, the Eu-Keggin/HEWL and the Ce-Keggin/HEWL complexes, respectively. The number of bound POM molecules to HEWL was 1.04 for the lacunary Keggin POM, and 1.0 for Eu-Keggin, indicating the formation of a 1:1 POM/HEWL complex. The value of 1.38 for Ce-Keggin might indicate a transition from 1:1 to 1:2 interaction.

Selective hydrolysis of hen egg white lysozyme at Asp-X peptide bonds promoted by oxomolybdate.

The activity of oxomolybdate(VI) towards hen egg white lysozyme (HEWL) was examined under physiological and slightly acidic pH conditions. Purely hydrolytic cleavage of HEWL in the presence of 10 to 100 mM of oxomolybdate(VI) after incubation at pH 5.0 and 60 °C for 2 to 7 days was observed in SDS-PAGE experiments. Four cleavage sites, which all occurred at Asp-X sequences and included the Asp18-Asn19, Asp48-Gly49, Asp52-Trp53 and Asp101-Gly102 peptide bonds, were identified with Edman degradation. The molecular interaction between [MoO4](2-) and HEWL was studied by circular dichroism (CD) and (1)H-(15)N heteronuclear single quantum correlation (HSQC) NMR spectroscopy. CD spectroscopy revealed a significant decrease in the α-helical content of HEWL upon addition of oxomolybdate, while (1)H-(15)N HSQC NMR spectroscopy identified the residues which were most affected upon interaction with [MoO4](2-). (95)Mo NMR measurements, performed on oxomolybdate solutions containing HEWL, identified the monomeric [MoO4](2-) form as active species in the hydrolytic reaction. The hydrolysis of the Asp-Gly model peptide in the presence of oxomolybdate(VI) was studied by (1)H NMR, further supporting a hydrolytic mechanism where polarisation of the carbonyl is followed by internal nucleophilic attack on the Asp residue.