Matteo Pappalardo

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.

Phenylurea herbicides induce cytogenetic effects in Chinese hamster cell lines

The intensive use of herbicides over the last few decades has caused a general increase of environmental pollution. It is thus very important to evaluate the possible genotoxic properties of these chemical compounds as well as identifying their mode of action. Phenylurea herbicides are selective agents widely used for the control of infestant plants. Of these herbicides, which are widely used in agriculture, we analysed four of the less intensively studied molecules. More precisely, we investigated the genotoxic effects of fenuron, chlorotoluron, diuron, and difenoxuron by analyses of chromosomal aberrations (CAs) and sister chromatid exchange (SCE) in exposed mammalian cells. We used the Chinese hamster ovary (CHO) and epithelial liver (CHEL) cell lines, endowed with the absence or the presence, respectively, of an enzymatic system to activate pro-mutagenic compounds. Our results show that all herbicides tested induce, at high concentrations, an increasing number of CAs in non-metabolising CHO cells. Instead, in the exposed CHEL cell line, the four herbicides induced CAs also at the lowest dose-level. In the CHEL cells, a statistically significant increase of SCE was also observed. The phenylurea herbicides showed direct genotoxic activity, but the cytogenetic effects were greatly enhanced after metabolic conversion. These data, together with other information on phenylurea herbicides, are of great interest from the environmental point of view, and for human health. In fact, intensive use of herbicides contaminates soil, surface water, groundwater and agricultural products, and thus should be taken in particular consideration not only for those initiatives to specifically protect exposed workers, but also to safeguard the health of consumers of agricultural products.

In silico bioremediation of polycyclic aromatic hydrocarbon: A frontier in environmental chemistry

In recent years, the number of studies in the field of bioremediation has been growing steadily. Although a large number of studies provide information that is highly detailed and offer great amounts of knowledge on a given subject, the downside is that the hunt for more information requires the combined efforts of researchers from many areas, which are becoming increasingly difficult to attain. In this review, we present an overview of recent work investigating enzyme degradation of polycyclic aromatic hydrocarbons. In the first part, this review examines several of the new enzymes able to degrade pollutants, with special attention being given to those with a well-resolved structure. The second part explores some of the most recent work in which computational approaches, such as molecular dynamics, docking, density functional theory and database retrieval, have been employed to study enzymes with specific bioremediation activities.

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.

Steered molecular dynamics studies reveal different unfolding pathways of prions from mammalian and non-mammalian species

Prion diseases are associated with an abnormal conformational transition involving the prion protein and are known to affect mammals. Here, the different mechanical behaviour of two mammalian, human (HuPrP) and Syrian hamster (ShaPrP), and two non-mammalian, chicken (ChPrP) and turtle (TuPrP), prions was assessed by steered molecular dynamics simulations performed on the globular domains of the four proteins. In mammalian prions a greater resistance to external stretching forces and an earlier occurrence of irreversible events were observed. The different unfolding profile of mammalian prions, ascribable to the intramolecular interactions involving helix 1 with helix 3, implicate the existence of metastable non-native states which may prompt abnormal pathways of protein misfolding and aggregation.

The Role Played by the α-Helix in the Unfolding Pathway and Stability of Azurin: Switching Between Hierarchic and Nonhierarchic Folding

The role played by the α‐helix in determining the structure, the stability and the unfolding mechanism of azurin was addressed by studying a helix‐depleted azurin variant produced by sitedirected mutagenesis. The protein structure was investigated by CD, 1D 1H NMR, fluorescence spectroscopy measurements and MD simulations, whilst EPR, UV‐visible and cyclic voltammetry experiments were carried out to investigate the geometry and the properties of the CuII site. The effects of the α‐helix depletion on the thermal stability and the unfolding pathway of the protein were determined by DSC, UV/visible and fluorescence measurements at increasing temperature. The results show that, in the absence of the α‐helix segment, the overall protein structure is maintained, and that only the Cu site is slightly modified. In contrast, the protein stability is diminished by about 60 % with respect to the wild‐type azurin. Moreover, the unfolding pathway of the mutant azurin involves the presence of detectable intermediates. In comparison with previous studies concerning other small β‐sheet cupredoxins, the results as a whole support the hypothesis that the presence of the α‐helix can switch the folding of azurin from a hierarchic to a nonhierarchic mechanism in which the highly conserved β‐sheet core provides a scaffold for cooperative folding of the wild‐type protein.

Computational Comparison of Imidazoline Association with the I2 Binding Site in Human Monoamine Oxidases

Imidazoline ligands in I2-type binding sites in the brain alter monoamine turnover and release. One example of an I2 binding site characterized by binding studies, kinetics, and crystal structure has been described in monoamine oxidase B (MAO B). MAO A also binds imidazolines but has a different active site structure. Docking and molecular dynamics were used to explore how 2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI) binds to MAO A and to explain why tranylcypromine increases tight binding to MAO B. The energy for 2-BFI binding to MAO A was comparable to that for tranylcypromine-modified MAO B, but the location of 2-BFI in the MAO A could be anywhere in the monopartite substrate cavity. Binding to the tranylcypromine-modified MAO B was with high affinity and in the entrance cavity as in the crystal structure, but the energies of interaction with the native MAO B were less favorable. Molecular dynamics revealed that the entrance cavity of MAO B after tranylcypromine modification is both smaller and less flexible. This change in the presence of tranylcypromine may be responsible for the greater affinity of tranylcypromine-modified MAO B for imidazoline ligands.

Computational study on the interaction of a ring-hydroxylating dioxygenase from Sphingomonas CHY-1 with PAHs

The massive computational resources available in the framework of a grid paradigm approach represent an emerging tool in the bioinformatics field. In this paper, we used the above approach in the rapid determination of the interactions between the ring-hydroxylating dioxygenase, comprised six enzymatic subunits, and polycyclic aromatic hydrocarbons (PAHs) in their optimal positions. The results were obtained by simulating enzyme dynamics at 300 K through molecular dynamics calculations. For the first time, the equilibrated structure of the dioxygenase revealed a network of channels throughout the enzyme that were sufficiently large to allow a flow of small ions or molecules from the inner core of the complex to its exterior surface. The ring-hydroxylating dioxygenase was able to interact with some of the studied PAHs. Additionally, not only the number of aromatic rings but also the PAH shape were critical in predicting the ability of the dioxygenase to interact with these types of molecules. Docking calculations shed light on a new possible binding site that is far from the enzymatic one, which is potentially interesting in considering the stability of the enzyme itself.

How to Choose the Suitable Template for Homology Modelling of GPCRs: 5-HT7 Receptor as a Test Case

G protein‐coupled receptors (GPCRs) are a super‐family of membrane proteins that attract great pharmaceutical interest due to their involvement in almost every physiological activity, including extracellular stimuli, neurotransmission, and hormone regulation. Currently, structural information on many GPCRs is mainly obtained by the techniques of computer modelling in general and by homology modelling in particular. Based on a quantitative analysis of eighteen antagonist‐bound, resolved structures of rhodopsin family “A” receptors – also used as templates to build 153 homology models – it was concluded that a higher sequence identity between two receptors does not guarantee a lower RMSD between their structures, especially when their pair‐wise sequence identity (within trans‐membrane domain and/or in binding pocket) lies between 25 % and 40 %. This study suggests that we should consider all template receptors having a sequence identity ≤50 % with the query receptor. In fact, most of the GPCRs, compared to the currently available resolved structures of GPCRs, fall within this range and lack a correlation between structure and sequence. When testing suitability for structure‐based drug design, it was found that choosing as a template the most similar resolved protein, based on sequence resemblance only, led to unsound results in many cases. Molecular docking analyses were carried out, and enrichment factors as well as attrition rates were utilized as criteria for assessing suitability for structure‐based drug design.