Structures and interactions of amyloidogenic polypeptides in biomimetic environments.

This thesis aims to provide structural and biophysical insights on the molecular mechanism of amyloid aggregation. Three different molecular models were chosen for these studies; all of them being able to form fibrillar aggregates, even though starting from different conformation, therefore likely following different mechanisms. The main model is represented by amyloid beta peptides, in particular the 40 and the 42 residues peptides (Abeta40 and Abeta42), which are related to Alzheimer Disease. The ability to switch from alfa-helical conformation in membrane and apolar environment to beta-sheet-based fibril structure in aqueous solution characterizes these peptides. The second model is hemopressin, a short peptide that shows very promising pharmacological application, hampered by its aggregation propensity; this peptide is unstructured in aqueous buffers, but it forms amyloid fibrils in some pH conditions. The third model is an engineered protein, called Y65R-MNEI, derived from monellin, very interesting for its potential biotechnological application as ipocaloric sweetener. Its structures is very close to that of the parent protein MNEI which, at native state, is a soluble globular protein rich in beta-sheet. Like MNEI, it is able to form fibrils, and may represent a useful model for protein folding and aggregation. Still no aggregation study was performed on Y65R-MNEI. These molecules were studied at different levels of deepness in literature, with several different biophysical techniques. Therefore, the choice of these molecules allowed me to study the amyloid aggregation process at different stages. In particular, the effect of different physico-chemical parameters (temperature, ionic strength, pH and solvent polarity), as well as the influence of lipid interaction and of a non-enzymatic post-translational modification on the aggregation properties of these models was investigated. An integrated experimental approach was used, including biophysical techniques such as Fluorescence, CD, EPR, NMR spectroscopies and AFM, in order to fill some gaps present in literature on the aggregation process of these molecules

Striking Dependence of Protein Sweetness on Water Quality: The Role of the Ionic Strength

Sweet proteins are the sweetest natural molecules. This aspect prompted several proposals for their use as food additives, mainly because the amounts to be added to food would be very small and safe for people suffering from sucrose-linked diseases. During studies of sweet proteins as food additives we found that their sweetness is affected by water salinity, while there is no influence on protein’s structure. Parallel tasting of small size sweeteners revealed no influence of the water quality. This result is explained by the interference of ionic strength with the mechanism of action of sweet proteins and provides an experimental validation of the wedge model for the interaction of proteins with the sweet receptor

A Super Stable Mutant of the Plant Protein Monellin Endowed with Enhanced Sweetness

Sweet proteins are a class of proteins with the ability to elicit a sweet sensation in humans upon interaction with sweet taste receptor T1R2/T1R3. Single-chain Monellin, MNEI, is among the sweetest proteins known and it could replace sugar in many food and beverage recipes. Nonetheless, its use is limited by low stability and high aggregation propensity at neutral pH. To solve this inconvenience, we designed a new construct of MNEI, dubbed Mut9, which led to gains in both sweetness and stability. Mut9 showed an extraordinary stability in acidic and neutral environments, where we observed a melting temperature over 20 C higher than that of MNEI. In addition, Mut9 resulted twice as sweet than MNEI. Both proteins were extensively characterized by biophysical and sensory analyses. Notably, Mut9 preserved its structure and function even after 10 min boiling, with the greatest differences being observed at pH 6.8, where it remained folded and sweet, whereas MNEI lost its structure and function. Finally, we performed a 6-month shelf-life assessment, and the data confirmed the greater stability of the new construct in a wide range of conditions. These data prove that Mut9 has an even greater potential for food and beverage applications than MNEI

Preferential interaction of the Alzheimer peptide Aβ-(1-42) with Omega-3-containing lipid bilayers: Structure and interaction studies

Many age-related neurodegenerative diseases, including Alzheimer Disease (AD), are elicited by an interplay of genetic, environmental, and dietary factors. Food rich in Omega-3 phospholipids seems to reduce the AD incidence. To investigate the molecular basis of this beneficial effect, we have investigated by CD and ESR studies the interaction between the Alzheimer peptide Ab-(1–42) and biomimetic lipid bilayers. The inclusion of 1,2-didocosahexaenoyl- sn-glycero-3-phosphocholine does not change significantly the bilayers organization, but favors its Ab-(1–42) interaction. The Omega-3 lipid amount modulates the effect intensity, suggesting a peptide selectivity for membranes containing polyunsatured fatty acids (PUFA) and providing hints for the mechanism and therapy of AD.Many age-related neurodegenerative diseases, including Alzheimer Disease (AD), are elicited by an interplay of genetic, environmental, and dietary factors. Food rich in Omega-3 phospholipids seems to reduce the AD incidence. To investigate the molecular basis of this beneficial effect, we have investigated by CD and ESR studies the interaction between the Alzheimer peptide Aβ-(1-42) and biomimetic lipid bilayers. The inclusion of 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine does not change significantly the bilayers organization, but favors its Aβ-(1-42) interaction. The Omega-3 lipid amount modulates the effect intensity, suggesting a peptide selectivity for membranes containing polyunsatured fatty acids (PUFA) and providing hints for the mechanism and therapy of AD

Disordered Peptides Looking for Their Native Environment: Structural Basis of CB1 Endocannabinoid Receptor Binding to Pepcans

Endocannabinoid peptides, or "pepcans," are endogenous ligands of the CB1 cannabinoid receptor. Depending on their length, they display diverse activity: For instance, the nona-peptide Pepcan-9, also known as hemopressin, is a powerful inhibitor of CB1, whereas the longer variant Pepcan-12, which extends by only three amino acid residues at the N-terminus, acts on both CB1 and CB2 as an allosteric modulator, although with diverse effects. Despite active research on their pharmacological applications, very little is known about structure-activity relationships of pepcans. Different structures have been proposed for the nona-peptide, which has also been reported to form fibrillar aggregates. This might have affected the outcome and reproducibility of bioactivity studies. In an attempt of elucidating the determinants of both biological activity and aggregation propensity of Pepcan-9 and Pepcan-12, we have performed their structure characterization in solvent systems characterized by different polarity and pH. We have found that, while disordered in aqueous environment, both peptides display helical structure in less polar environment, mimicking the proteic receptor milieu. In the case of Pepcan-9, this structure is fully consistent with the observed modulation of the CB1. For Pepcan-12, whose allosteric binding site is still unknown, the presented structure is compatible with the binding at one of the previously proposed allosteric sites on CB1. These findings open the way to structure-driven design of selective peptide modulators of CB1

Disordered Peptides Looking for Their Native Environment: Structural Basis of CB1 Endocannabinoid Receptor Binding to Pepcans

Endocannabinoid peptides, or "pepcans," are endogenous ligands of the CB1 cannabinoid receptor. Depending on their length, they display diverse activity: For instance, the nona-peptide Pepcan-9, also known as hemopressin, is a powerful inhibitor of CB1, whereas the longer variant Pepcan-12, which extends by only three amino acid residues at the N-terminus, acts on both CB1 and CB2 as an allosteric modulator, although with diverse effects. Despite active research on their pharmacological applications, very little is known about structure-activity relationships of pepcans. Different structures have been proposed for the nona-peptide, which has also been reported to form fibrillar aggregates. This might have affected the outcome and reproducibility of bioactivity studies. In an attempt of elucidating the determinants of both biological activity and aggregation propensity of Pepcan-9 and Pepcan-12, we have performed their structure characterization in solvent systems characterized by different polarity and pH. We have found that, while disordered in aqueous environment, both peptides display helical structure in less polar environment, mimicking the proteic receptor milieu. In the case of Pepcan-9, this structure is fully consistent with the observed modulation of the CB1. For Pepcan-12, whose allosteric binding site is still unknown, the presented structure is compatible with the binding at one of the previously proposed allosteric sites on CB1. These findings open the way to structure-driven design of selective peptide modulators of CB1

Glycation affects fibril formation of a peptides

Increasing evidence shows that -amyloid (A) peptides, which are associated with Alzheimer disease (AD), are heavily glycated in patients, suggesting a role of this irreversible nonenzymatic post-translational modification in pathology. Previous reports have shown that glycation increases the toxicity of the A peptides, although little is known about the mechanism. Here, we used the natural metabolic by-product methylglyoxal as a glycating agent and exploited various spectroscopic methods and atomic force microscopy to study how glycation affects the structures of the A40 and A42 peptides, the aggregation pathway, and the morphologies of the resulting aggregates. We found that glycation significantly slows down but does not prevent -conversion to mature fibers. We propose that the previously reported higher toxicity of the glycated A peptides could be explained by a longer persistence in an oligomeric form, usually believed to be the toxic species

Tailored inclusion of semiconductor nanoparticles in nanoporous polystyrene-block-polymethyl methacrylate thin films

Nanoporous/nanocomposite thin films with controlled morphology at nanoscale were prepared onto transparent and conductive indium tin oxide (ITO) supports by exploiting the self-assembly of a lamellar polystyrene-b-poly(methyl methacrylate) (PS-b-PMMA) block copolymer (BCP). A perpendicular orientation of PS and PMMA lamellar nanodomains was achieved by grafting a random PS-r-PMMA copolymer to the ITO supports and successive thermal annealing. Stable and reproducible nanoporous morphologies, characterized by PS lamellar nanodomains of width equal to ≈20 nm alternating to nanochannels of width equal to ≈8 nm, were obtained by irradiating the samples with an appropriate UV-C dose able to fix the relative arrangement of PS domains through activation of cross-linking reactions and selective removal of PMMA blocks. Nanoporous hybrid composites with a stable morphology were obtained either by applying the UV irradiation protocol to BCP nanocomposites characterized by selective inclusion of zinc oxide (ZnO) nanoparticles (NPs) in the PS domains (nanocomposite first/nanopores after) or by selective infiltration of cadmium selenide (CdSe) NPs in the nanochannels left free by PMMA removal through UV irradiation (nanopores first/nanocomposite after), demonstrating the strenght of the approach