Danilo Milardi

Cations as switches of amyloid-mediated membrane disruption mechanisms: calcium and IAPP.

Disruption of the integrity of the plasma membrane by amyloidogenic proteins is linked to the pathogenesis of a number of common age-related diseases. Although accumulating evidence suggests that adverse environmental stressors such as unbalanced levels of metal ions may trigger amyloid-mediated membrane damage, many features of the molecular mechanisms underlying these events are unknown. Using human islet amyloid polypeptide (hIAPP, aka amylin), an amyloidogenic peptide associated with β-cell death in type 2 diabetes, we demonstrate that the presence of Ca(2+) ions inhibits membrane damage occurring immediately after the interaction of freshly dissolved hIAPP with the membrane, but significantly enhances fiber-dependent membrane disruption. In particular, dye leakage, quartz crystal microbalance, atomic force microscopy, and NMR experiments show that Ca(2+) ions promote a shallow membrane insertion of hIAPP, which leads to the removal of lipids from the bilayer through a detergent-like mechanism triggered by fiber growth. Because both types of membrane-damage mechanisms are common to amyloid toxicity by most amyloidogenic proteins, it is likely that unregulated ion homeostasis, amyloid aggregation, and membrane disruption are all parts of a self-perpetuating cycle that fuels amyloid cytotoxicity.

Determination of the Conformation of the Human VDAC1 N‐Terminal Peptide, a Protein Moiety Essential for the Functional Properties of the Pore

Mitochondrial porin or VDAC (voltage‐dependent anion‐selective channel) is the most abundant protein in the mitochondrial outer membrane. The structure of VDAC has been predicted to be a transmembrane β‐barrel with an α‐helix at the N terminus. It is a matter of debate as to whether this putative α‐helix plays a structural role as a component of the pore walls or a function in the pore activity. We have synthesised the human VDAC1 (HVDAC1) N‐terminal peptide Ac‐AVPPTYADLGKSARDVFTK‐NH2 (Prn2–20) and determined its structure by CD and NMR spectroscopy. CD studies show that the Prn2–20 peptide exists in aqueous solvent as an unstructured peptide with no stable secondary structure. In membrane‐mimetic SDS micelles or water/trifluoroethanol, however, it assumes an amphipathic α‐helix conformation between Tyr5 and Val16, as deduced from NMR. No ordered structure was observed in dodecyl β‐maltoside. Differential scanning calorimetric measurements were carried out in order to examine the membrane affinity of the peptide. Upon interaction with the negatively charged 1,2 dipalmitoyl‐sn‐glycero‐3‐phosphoserine membrane, Prn2–20 exhibited distinctive behaviour, suggesting that electrostatics play an important role. Interaction between the peptide and artificial bilayers indicates that the peptide lies on the membrane surface. Recombinant HVDAC1 deletion mutants, devoid of seven or 19 N‐terminal amino acids, were used for transfection of eukaryotic cells. Over‐expression of HVDAC1 increases the number of Cos cells with depolarised mitochondria, and this effect is progressively reduced in cells transfected with HVDAC1 lacking those seven or 19 amino acids. The mitochondrial targeting of the deletion mutants is unaffected. The overall picture emerging from our experiments is that the VDAC N‐terminal peptide plays a role in the proper function of this protein during apoptotic events.

α-Helical Structures Drive Early Stages of Self-Assembly of Amyloidogenic Amyloid Polypeptide Aggregate Formation in Membranes

The human islet amyloid polypeptide (hIAPP) is the primary component in the toxic islet amyloid deposits in type-2 diabetes. hIAPP self-assembles to aggregates that permeabilize membranes and constitutes amyloid plaques. Uncovering the mechanisms of amyloid self-assembly is the key to understanding amyloid toxicity and treatment. Although structurally similar, hIAPPs rat counterpart, the rat islet amyloid polypeptide (rIAPP), is non-toxic. It has been a puzzle why these peptides behave so differently. We combined multiscale modelling and theory to explain the drastically different dynamics of hIAPP and rIAPP: The differences stem from electrostatic dipolar interactions. hIAPP forms pentameric aggregates with the hydrophobic residues facing the membrane core and stabilizing water-conducting pores. We give predictions for pore sizes, the number of hIAPP peptides, and aggregate morphology. We show the importance of curvature-induced stress at the early stages of hIAPP assembly and the α-helical structures over β-sheets. This agrees with recent fluorescence spectroscopy experiments.

Extended theoretical analysis of irreversible protein thermal unfolding

The theoretical analysis of the protein denaturation model which includes an irreversible, exothermic and rate-limited step has been improved and applied to the DSC profile of Azurin. The two-step nature of the irreversible denaturation of globular proteins is usually depicted in the following simplified scheme: N <--> U <--> F, which is known as the Lumry and Eyring model. In most of the works concerning the thermal unfolding of proteins, it is usually assumed that the irreversible step of the process does not take place significantly during the short time the protein spends in the temperature range of the DSC transition, or if this is not the case, that this irreversible step occurs with a negligible thermal effect. As we will show, this last assumption cannot be accepted acritically; in fact we have found that in the case of Azurin an evident exothermic effect occurs at the end of the transition. In order to fit the experimental Cp(exc) profile of Azurin, we have analyzed a model in which the exothermic effects of the irreversible step and the variations of DeltaH with temperature are taken into account. Our model was first tested simulating a series of profiles and considering the effects of the variation of the parameters on the shape of the curves, and successfully used to fit the experimental calorimetric profile of Azurin.

A Spectroscopic and Calorimetric Investigation on the Thermal Stability of the Cys3Ala/Cys26Ala Azurin Mutant

The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the proteins overall structure or the assembly of the metal-binding site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been followed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitation wavelength. The analysis of the data shows that the thermal transition from the native to the denaturated state of the modified azurin follows the same multistep unfolding pathway as observed in wild type azurin. However, the removal of the disulfide bridge results in a dramatic reduction of the thermodynamic stability of the protein. In fact, the transition temperatures registered by the different techniques are down-shifted by about 20 degrees C with respect to wild type azurin. Moreover, the Gibbs free energy value is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contributes to the high stability of wild type azurin.

The role of aromatic side-chains in amyloid growth and membrane interaction of the islet amyloid polypeptide fragment LANFLVH

Human islet amyloid polypeptide (hIAPP) is known to misfold and aggregate into amyloid deposits that may be found in pancreatic tissues of patients affected by type 2 diabetes. Recent studies have shown that the highly amyloidogenic peptide LANFLVH, corresponding the N-terminal 12–18 region of IAPP, does not induce membrane damage. Here we assess the role played by the aromatic residue Phe in driving both amyloid formation and membrane interaction of LANFLVH. To this aim, a set of variant heptapeptides in which the aromatic residue Phe has been substituted with a Leu and Ala is studied. Differential scanning calorimetry (DSC) and membrane-leakage experiments demonstrated that Phe substitution noticeably affects the peptide-induced changes in the thermotropic properties of the lipid bilayer but not its membrane damaging potential. Atomic force microscopy (AFM), ThT fluorescence and Congo red birefringence assays evidenced that the Phe residue is not required for fibrillogenesis, but it can influence the self-assembling kinetics. Molecular dynamics simulations have paralleled the outcome of the experimental trials also providing informative details about the structure of the different peptide assemblies. These results support a general theory suggesting that aromatic residues, although capable of affecting the self-assembly kinetics of small peptides and peptide-membrane interactions, are not essential either for amyloid formation or membrane leakage, and indicate that other factors such as β-sheet propensity, size and hydrophobicity of the side chain act synergistically to determine peptide properties.

Carnosine Inhibits Aβ42 Aggregation by Perturbing the H‐Bond Network in and around the Central Hydrophobic Cluster

Aggregation of the amyloid‐β peptide (Aβ) into fibrillar structures is a hallmark of Alzheimers disease. Thus, preventing self‐assembly of the Aβ peptide is an attractive therapeutic strategy. Here, we used experimental techniques and atomistic simulations to investigate the influence of carnosine, a dipeptide naturally occurring in the brain, on Aβ aggregation. Scanning force microscopy, circular dichroism and thioflavin T fluorescence experiments showed that carnosine does not modify the conformational features of Aβ42 but nonetheless inhibits amyloid growth. Molecular dynamics (MD) simulations indicated that carnosine interacts transiently with monomeric Aβ42 by salt bridges with charged side chains, and van der Waals contacts with residues in and around the central hydrophobic cluster (17LVFFA21). NMR experiments on the nonaggregative fragment Aβ12–28 did not evidence specific intermolecular interactions between the peptide and carnosine, in agreement with MD simulations. However, a close inspection of the spectra revealed that carnosine interferes with the local propensity of the peptide to form backbone hydrogen bonds close to the central hydrophobic cluster (residues E22, S26 and N27). Finally, MD simulations of aggregation‐prone Aβ heptapeptide segments show that carnosine reduces the propensity to form intermolecular backbone hydrogen bonds in the region 18–24. Taken together, the experimental and simulation results (cumulative MD sampling of 0.2 ms) suggest that, despite the inability of carnosine to form stable contacts with Aβ, it might block the pathway toward toxic aggregates by perturbing the hydrogen bond network near residues with key roles in fibrillogenesis.

Calcium-activated membrane interaction of the islet amyloid polypeptide : Implications in the pathogenesis of type II diabetes mellitus

The role played by Ca(2+) ions in the interaction of the human islet amyloid polypeptide (hIAPP) with model membranes has been investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) experiments. In particular, the interaction of hIAPP and its rat isoform (rIAPP) with zwitterionic dipalmitoyl-phosphatidylcholine (DPPC), negatively charged dipalmitoyl-phosphatidylserine (DPPS) vesicles and with a 3:1 mixtures of them, has been studied in the presence of Ca(2+) ions. The experiments have evidenced that amorphous, soluble hIAPP assemblies interact with the hydrophobic core of DPPC bilayers. Conversely, the presence of Ca(2+) ions is necessary to activate a preferential interaction of hIAPP with the hydrophobic core of DPPS membranes. These findings support the hypothesis that an impaired cellular homeostasis of Ca(2+) ions may promote the insertion of hIAPP into the hydrophobic core of carrier vesicles which is thought to contribute to an eventual intracellular accumulation of beta-sheet rich hIAPP aggregates.

Cationic Porphyrins Are Reversible Proteasome Inhibitors

The aim of this study is to verify if water-soluble porphyrins can be used as proteasome inhibitors. We have found that cationic porphyrins inhibit proteasome peptidase activities much more effectively than the corresponding anionic derivatives. The relevance of electrostatics in driving porphyin-proteasome interactions has been confirmed by the observation that the inhibitory efficiency of the cationic macrocycles decreases with the number of positive substituents. We have also investigated various metalloporphyrins, which differ due to the different propension of the central metal ion toward axial coordination. Our experimental results indicate that the naked cationic porphyrins are the most active in reversibly inhibiting the three main protease activities of the proteasome in the micromolar range. A spectroscopic characterization of porphyrin-proteasome interactions by UV-vis spectra parallels the results of inhibition assays: the higher the inhibitory effect the stronger the spectroscopic variations are. To interpret the action of porphyrins at a molecular level, we have performed calculations evidencing that cationic porphyrins may hinder the access to the canonical proteolytic site on the proteasome β5 subunit. In particular, an inspection of the top-scoring docking modes shows that the tetracationic porphyrin blocks the catalytic pocket, close to the N termini of the β5 proteasome subunit, more efficiently than its anionic counterpart. Proteasome inhibition activity of porphyrins unites their known anticancer properties making them suitable as a scaffold for the design of novel multitargeted molecules.

DSC study of the interaction of the prion peptide PrP106–126 with artificial membranes

The incorporation of the human prion peptide PrP106–126 into 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) host model membranes has been investigated by differential scanning calorimetry. Two different types of peptide–membrane interactions have been studied. In one case, the peptide is obliged to be included into the hydrocarbon region of the lipid bilayer, in the other case, it is allowed to interact with the external surface of the membrane. The PrP106–126 incorporated into the DPPC membrane shows an increase in the gel–liquid crystal transition temperature of the bilayer with a decreased enthalpy change. The “oriented” insertion of the hydrophobic part of the fragment into the bilayer, with a consequent increase in the order of the lipidic hydrocarbon chains in the gel state, is responsible for this behavior. Independently from the procedure adopted for the preparation of the sample, the PrP106–126 fragment interacts strongly and irreversibly with the host membrane. In contrast, when PrP106–126 is incorporated in the DPPE model membrane, the gel–liquid crystal transition temperature of the bilayer and the associated enthalpy change decrease. When added externally to the DPPE model membrane, the PrP106–126 fragment has no effect on the phase behavior of the bilayer. These findings suggest that PrP106–126 has a specific affinity for DPPC membranes that might correspond to the external surface of cells.