Carmelo La Rosa

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.

Zinc stabilization of prefibrillar oligomers of human islet amyloid polypeptide

The aggregation of human islet amyloid polypeptide (hIAPP) has been linked to beta-cell death in type II diabetes. Zinc present in secretory granules has been shown to affect this aggregation. A combination of EXAFS, NMR, and AFM experiments shows that the influence of zinc is most likely due to the stabilization of prefibrillar aggregates of hIAPP.

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.

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.

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.

Probing the Sources of the Apparent Irreproducibility of Amyloid Formation: Drastic Changes in Kinetics and a Switch in Mechanism Due to Micellelike Oligomer Formation at Critical Concentrations of IAPP

The aggregation of amyloidogenic proteins is infamous for being highly chaotic, with small variations in conditions sometimes leading to large changes in aggregation rates. Using the amyloidogenic protein IAPP (islet amyloid polypeptide protein, also known as amylin) as an example, we show that a part of this phenomenon may be related to the formation of micellelike oligomers at specific critical concentrations and temperatures. We show that pyrene fluorescence can sensitively detect micellelike oligomer formation by IAPP and discriminate between micellelike oligomers from fibers and monomers, making pyrene one of the few chemical probes specific to a prefibrillar oligomer. We further show that oligomers of this type reversibly form at critical concentrations in the low micromolar range and at specific critical temperatures. Micellelike oligomer formation has several consequences for amyloid formation by IAPP. First, the kinetics of fiber formation increase substantially as the critical concentration is approached but are nearly independent of concentration below it, suggesting a direct role for the oligomers in fiber formation. Second, the critical concentration is strongly correlated with the propensity to form amyloid: higher critical concentrations are observed for both IAPP variants with lower amyloidogenicity and for native IAPP at acidic pH in which aggregation is greatly slowed. Furthermore, using the DEST NMR technique, we show that the pathway of amyloid formation switches as the critical point is approached, with self-interactions primarily near the N-terminus below the critical temperature and near the central region above the critical temperature, reconciling two apparently conflicting views of the initiation of IAPP aggregation.

Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β

The aggregation of amyloid-β (Aβ) on lipid bilayers has been implicated as a mechanism by which Aβ exerts its toxicity in Alzheimers disease (AD). Lipid bilayer thinning has been observed during both oxidative stress and protein aggregation in AD, but whether these pathological modifications of the bilayer correlate with Aβ misfolding is unclear. Here, we studied peptide-lipid interactions in synthetic bilayers of the short-chain lipid dilauroyl phosphatidylcholine (DLPC) as a simplified model for diseased bilayers to determine their impact on Aβ aggregate, protofibril, and fibril formation. Aβ aggregation and fibril formation in membranes composed of dioleoyl phosphatidylcholine (DOPC) or 1- palmitoyl-2-oleoyl phosphatidylcholine mimicking normal bilayers served as controls. Differences in aggregate formation and stability were monitored by a combination of thioflavin-T fluorescence, circular dichroism, atomic force microscopy, transmission electron microscopy, and NMR. Despite the ability of all three lipid bilayers to catalyze aggregation, DLPC accelerates aggregation at much lower concentrations and prevents the fibrillation of Aβ at low micromolar concentrations. DLPC stabilized globular, membrane-associated oligomers, which could disrupt the bilayer integrity. DLPC bilayers also remodeled preformed amyloid fibrils into a pseudo-unfolded, molten globule state, which resembled on-pathway, protofibrillar aggregates. Whereas the stabilized, membrane-associated oligomers were found to be nontoxic, the remodeled species displayed toxicity similar to that of conventionally prepared aggregates. These results provide mechanistic insights into the roles that pathologically thin bilayers may play in Aβ aggregation on neuronal bilayers, and pathological lipid oxidation may contribute to Aβ misfolding.

Thermodynamics and kinetics of the thermal unfolding of plastocyanin

Abstract The thermal denaturation of plastocyanin in aqueous solution was investigated by means of DSC, ESR and absorbance techniques, with the aim of determining the thermodynamic stability of the protein and of characterizing the thermally induced conformational changes of its active site. The DSC and absorbance experiments indicated an irreversible and kinetically controlled denaturation path. The extrapolation of the heat capacity and optical data at infinite scan rate made it possible to calculate the kinetic and thermodynamic parameters associated with the denaturation steps. The denaturation pathway proposed, and the parameters found from the calorimetric data, were checked by computer simulation using an equation containing the information necessary to describe the denaturation process in detail. ESR and absorbance measurements have shown that structural changes of the copper environment occur during the protein denaturation. In particular, the geometry of the copper-ligand atoms changes from being tetrahedral to square planar and the disruption of the active site precedes the global protein denaturation. The thermodynamic enthalpic change, the half-width transition temperature, and the value of ΔCp, were used to calculate the thermodynamic stability, ΔG, of the reversible process over the entire temperature range of denaturation. The low thermal stability found for plastocyanin, is discussed in connection with structural factors stabilizing the native state of a protein.

Self-Assembling Pathway of HiApp Fibrils within Lipid Bilayers

The presence of insoluble fibrillar deposits in the pancreas is one of the most common pathological features in patients affected by diabetes mellitus type II. The constituent of these deposits is the hormone known as islet amyloid polypeptide (IAPP) or amylin, a 37-amino-acid polypeptide cosecreted and costored with insulin by Langherans b-cells. This hormone plays a central role in the regulation of glucose’s homeostasis. Human amylin (hIAPP) forms amyloid fibrils in vitro; on the other hand, rat amylin (rIAPP), which differs from the human variant by only six amino acid residues, does not form fibrils and is not toxic. The dysfunction and death of Langherans b-cells has been attributed to the conversion from soluble monomers of hIAPP to insoluble b-sheet fibrils, which lead to extracellular buildup. However, recent studies have refuted this hypothesis, and it is unclear whether Langherans cell death is due to the fibrils or to simple structures. In any case, there is experimental evidence, recently reviewed by Jayasinghe and Langen, of the crucial involvement of lipid membranes in the fibrillogenic process. The fibrillogenesis process of hIAPP follows a nucleation-dependent mechanism that comprise a first phase (lag time), in which monomers form small, soluble, unstructured aggregates, 16] followed by a second phase, in which unstructured aggregates transform into b-sheet-rich structures that form the mature fibrils through a self-assembling process. Moreover, it has been suggested that the main mechanism for hIAPP bcell cytotoxicity can be ascribed to membrane permeabilization, through the formation of membrane channels by soluble hIAPP or by hIAPP-induced bilayer disruption (detergent like). In this work, by using hIAPP in the presence of 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes, we provide evidence that the process of membrane destruction is related to the formation of fibrils. POPC lipid membranes were used both as vesicles (LUVs) and supported lipid bilayers (SLBs) in order to simulate the extracellular membrane. Fluorescence spectroscopy, atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) have been employed to characterize the membranes and the interacting hIAPP-membrane systems. In particular, fluorescence versus time measurements have been used to investigate the leakage of a fluorescent probe from LUVs as a test for aspecific membrane pore formations, the thioflavin T (ThT) assay has been used to test the fibril formation, and AFM has been used to obtain morphological information on the studied systems. Finally, the kinetics of hIAPP adsorption onto SLBs have been studied by using QCMD (see the Supporting Information). In Figure 1, the results of fluorescence versus time experiments of hIAPP and 6-carboxyfluorescein-filled LUVs, are shown (red dots). It can be seen that an increase of fluores-

Testing a fluorinated compound as a protective material for calcarenite

Abstract A polyfluorinated compound was studied as a material for the specific protection of calcarenite. Water capillary absorption and vapour permeability measurements were carried out in order to evaluate efficiency as a protective agent. Particular attention was given to characterising its resistance against bio-deterioration induced by microorganisms such as blue and green algae. Chemical surface modifications were induced by UV-irradiation in a specially designed climatic chamber and were investigated through comparative tests on untreated and artificially weathered samples using X-ray photoelectron spectroscopy (XPS). This paper discusses the potential use of the compound studied as a specific coating material for the protection of calcarenite.