Francesco Attanasio

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.

Protective Effects of L- and D-Carnosine on α-Crystallin Amyloid Fibril Formation: Implications for Cataract Disease

Mildly denaturing conditions induce bovine alpha-crystallin, the major structural lens protein, to self-assemble into fibrillar structures in vitro. The natural dipeptide l-carnosine has been shown to have potential protective and therapeutic significance in many diseases. Carnosine derivatives have been proposed as potent agents for ophthalmic therapies of senile cataracts and diabetic ocular complications. Here we report the inhibitory effect induced by the peptide (l- and d-enantiomeric form) on alpha-crystallin fibrillation and the almost complete restoration of the chaperone activity lost after denaturant and/or heat stress. Scanning force microscopy (SFM), thioflavin T, and a turbidimetry assay have been used to determine the morphology of alpha-crystallin aggregates in the presence and absence of carnosine. DSC and a near-UV CD assay evidenced that the structural precursors of amyloid fibrils are polypeptide chain segments that lack stable structural elements. Moreover, we have found a disassembling effect of carnosine on alpha-crystallin amyloid fibrils. Finally, we show the ability of carnosine to restore most of the lens transparency in organ-cultured rat lenses exposed to similar denaturing conditions that were used for the in vitro experiments.

Design and synthesis of new trehalose‐conjugated pentapeptides as inhibitors of Aβ(1–42) fibrillogenesis and toxicity

Aggregation of the amyloid Aβ peptide and its accumulation into insoluble deposits (plaques) are believed to be the main cause of neuronal dysfunction associated with Alzheimers disease (AD); small molecules that can interfere with the Aβ amyloid fibril formation are therefore of interest for a potential therapeutic strategy. Three new trehalose‐conjugated peptides of the well known β‐sheet breaker peptide iAβ5p, were synthesized. The disaccharide was covalently attached to different sites of the LPFFD peptide chain, i.e. at the N‐terminus, C‐terminus or at the Asp side chain. CD spectroscopy in different solvents was used to assess changes in the peptide conformation of these compounds. The effects of these glycopeptides on the self‐assembly and morphology of Aβ aggregates were investigated by ThT fluorescence assay and dynamic Scanning Force Microscopy, respectively. All the synthesized compounds were tested as inhibitors of Aβ toxicity toward pure cultures of rat cortical neurons. Copyright

Soluble Sugar-Based Quinoline Derivatives as New Antioxidant Modulators of Metal-Induced Amyloid Aggregation

Oxidative stress and protein aggregation have been demonstrated to be the major factors involved in neurodegenerative diseases. Metal ions play a pivotal role, acting as mediators of neurotoxicity either by favoring or redox cycling. Thus, they represent a promising and suitable therapeutic target for the treatment of neurodegenerative disorders. In particular, the development of bifunctional or multifunctional molecules, which have antiaggregant and metal-chelating/antioxidant properties, may be considered as a valuable strategy for the treatment of neurodegeneration considering its multifactorial nature. Herein, we report the design and the characterization of four new multifunctional sugar-appended 8-hydroxyquinolines focusing on the effects of the conjugation with trehalose, a nonreducing disaccharide involved in the protection of proteins and cells against environmental stresses. These glycoconjugates do not exhibit any antiproliferative activity against three human cell lines of different histological origin, unlike 8-hydroxyquinolines. The multiple properties of the new derivatives are highlighted, reporting their Cu(2+) and Zn(2+) binding ability, and antioxidant and antiaggregant capacities. In particular, these latter were determined by different assays, including the evaluation of their ability to modulate or even suppress the aggregation of Aβ1-40 and Aβ1-42 peptides induced by copper or zinc ions.

Multifunctional 8‐Hydroxyquinoline‐Appended Cyclodextrins as New Inhibitors of Metal‐Induced Protein Aggregation

Mounting evidence suggests a pivotal role of metal imbalances in protein misfolding and amyloid diseases. As such, metal ions represent a promising therapeutic target. In this context, the synthesis of chelators that also contain complementary functionalities to combat the multifactorial nature of neurodegenerative diseases is a highly topical issue. We report two new 8-hydroxyquinoline-appended cyclodextrins and highlight their multifunctional properties, including their Cu(II) and Zn(II) binding abilities, and capacity to act as antioxidants and metal-induced antiaggregants. In particular, the latter property has been applied in the development of an effective assay that exploits the formation of amyloid fibrils when β-lactoglobulin A is heated in the presence of metal ions.

Membrane interactions and conformational preferences of human and avian prion N-terminal tandem repeats: the role of copper(II) ions, pH, and membrane mimicking environments.

The flexible N-terminal domain of the prion protein (PrP(c)) is believed to play a pivotal role in both trafficking of the protein through the cell membrane and its pathogenic conversion into the β sheet-rich scrapie isoform (PrP(sc)). Unlike mammalian PrP(c), avian prion proteins are not known to undergo any pathogenic conformational conversions. Consequently, some critical advances in our understanding of the molecular mechanisms underlying prion pathogenesis are expected from comparative studies of the biophysical properties of the N-terminal domains of the two proteins. The present study addresses the role played by different environmental factors, i.e., copper(II), pH, and membrane-mimicking environments, in assisting the conformational preferences of huPrP60-91 and chPrP53-76, two soluble peptides encompassing the N-terminal copper(II) binding domains of the human and chicken prion proteins, respectively. Moreover, the membrane interactions of huPrP60-91, chPrP53-76, and their copper(II) complexes were evaluated by Trp fluorescence in conjunction with measurements of the variation in thermotropic properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) unilamellar vesicles. Circular dichroism experiments revealed that huPrP60-91 adopts a predominant polyproline II conformation in aqueous solution that is destabilized at basic pH or in the presence of trifluoroethanol (TFE). Unlike anionic sodium dodecyl sulfate (SDS), which seems to stabilize the polyproline II conformation further, zwitterionic dodecylphosphocholine (DPC) micelles do not affect the peptide structure. On the contrary, copper(II) promptly promotes an increase in β-turn-rich structures. Differential scanning calorimetry (DSC) and Trp fluorescence assays carried out on DPPC model membranes after incubation with huPrP60-91 showed a marked tendency of the peptide to slowly penetrate the lipid bilayer with a concomitant conformational transition toward an extended β-sheet-like structure. Such an event, which was ascribed to the hydrophobic Trp side chain residues, was shown to also depend on the level of copper(II) occupancy along the peptide. Conversely, the CD spectra of chPrP53-76 aqueous solutions indicated the presence of a mixture of random-coil/β-turn-like structures whose resulting equilibrium was influenced by SDS and copper(II) addition. Furthermore, chPrP53-76 did not exhibit any tendency to interact with model membranes in either the presence or absence of copper(II). The results reported here provide evidence of the different roles played by environmental factors in affecting the conformation and membrane activity of human and avian prion N-terminal domains.

Structural determinants in prion protein folding and stability.

Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases, involving post-translational modifications of the cellular prion protein. Epidemiological studies on Creutzfeldt-Jakob disease, a prototype prion disorder, show a majority of cases being sporadic, while the remaining occurrences are either genetic or iatrogenic. The molecular mechanisms by which PrP(C) is converted into its pathological isoform have not yet been established. While point mutations and seeds trigger the protein to cross the energy barriers, thus causing genetic and infectious transmissible spongiform encephalopathies, respectively, the mechanism responsible for sporadic forms remains unclear. Since prion diseases are protein-misfolding disorders, we investigated prion protein folding and stability as functions of different milieus. Using spectroscopic techniques and atomistic simulations, we dissected the contribution of major structural determinants, also defining the energy landscape of prion protein. In particular, we elucidated (i) the essential role of the octapeptide region in prion protein folding and stability, (ii) the presence of a very enthalpically stable intermediate in prion-susceptible species, and (iii) the role of the disulfide bridge in prion protein folding.

Aggregation properties of the peptide fragments derived from the 17-29 region of the human and rat IAPP: a comparative study with two PEG-conjugated variants of the human sequence.

The amyloidogenic amino acid sequence Ac-VHSSNNFGAILSS-NH(2), corresponding to the 17-29 peptide region of human amylin (hIAPP17-29), was modified by grafting a hydrophilic PEG chain in order to obtain a novel class of peptides to be used as models to study the aggregation process of the full-length IAPP. The amphiphilic feature of the pegylated peptide fragment at the N-terminus (PEG-N-hIAPP17-29) drives the aggregation process toward stable micellar clusters without fibrillogenesis, despite the presence of beta-sheet interaction between peptides at pH values higher than 4.0. The hIAPP17-29-C-PEG, in which the PEG moiety is linked to the C-terminus, does not possess analogous amphiphilic character and the ability of PEG in forming H-bonds with the solvent overcomes that of the peptide chain, thereby causing peptide flocculation. The comparison with the unmodified hIAPP17-29 and the rats peptide sequence Ac-VRSSNNLGPGLPP-NH(2)(rIAPP17-29) revealed the crucial role of hydrogen bonding between peptide and solvent in determining the aggregate structure and preventing fibril formation, as well as the non-negligible effect of a small amount of organic solvent in the aqueous solution which affects the aggregation process and rate.

Are fibril growth and membrane damage linked processes? An experimental and computational study of IAPP12–18 and IAPP21–27 peptides

Islet amyloid polypeptide (IAPP) is a 37-residue hormone known to deposit as fibrillar aggregates in pancreatic β-cells of patients affected by T2DM. Although it has been proposed that both the fibrillogenic potential and membrane-activity may play a key role in IAPP cytotoxicity, a direct causative relationship between these two properties has not yet been firmly established. More recently, it has been observed that membrane damage may occur independently from fiber formation of IAPP and that these properties may be encoded by different sequences of IAPP. To further check this hypothesis, the membrane-activity and aggregation properties of the two neutral segments LANFLVH (IAPP12–18) and NNFGAIL (IAPP21–27), that recent theoretical studies have reported to possess the highest and the lowest fibrillogenic potential respectively, have been studied by means of a combined experimental and computational approach. The whole of the results demonstrate that if neutral peptides and lipids are employed, the most fibrillogenic peptide has the lowest membrane damaging effect and vice versa. These findings are expected to contribute to our rational understanding of the factors involved in the formation of amyloidosis and in the mechanisms of peptide-induced membrane damage.

Monomeric ß-amyloid interacts with type-1 insulin-like growth factor receptors to provide energy supply to neurons

ß-amyloid (Aß1−42) is produced by proteolytic cleavage of the transmembrane type-1 protein, amyloid precursor protein. Under pathological conditions, Aß1−42self-aggregates into oligomers, which cause synaptic dysfunction and neuronal loss, and are considered the culprit of Alzheimers disease (AD). However, Aß1−42 is mainly monomeric at physiological concentrations, and the precise role of monomeric Aß1−42 in neuronal function is largely unknown. We report that the monomer of Aß1−42 activates type-1 insulin-like growth factor receptors and enhances glucose uptake in neurons and peripheral cells by promoting the translocation of the Glut3 glucose transporter from the cytosol to the plasma membrane. In neurons, activity-dependent glucose uptake was blunted after blocking endogenous Aß production, and re-established in the presence of cerebrospinal fluid Aß. APP-null neurons failed to enhance depolarization-stimulated glucose uptake unless exogenous monomeric Aß1−42 was added. These data suggest that Aß1−42 monomers were critical for maintaining neuronal glucose homeostasis. Accordingly, exogenous Aß1−42 monomers were able to rescue the low levels of glucose consumption observed in brain slices from AD mutant mice.