The impact of nitric oxide toxicity on the evolution of the glutathione transferase superfamily: A proposal for an evolutionary driving force

Background: Why do ancestral GSTs utilize cysteine/serine as catalytic residues, whereas more recently evolved GSTs utilize tyrosine? Results: Only the more recently evolved GSTs display enough affinity to bind and make harmless the toxic DNDGIC (a natur

Molecular dynamics approaches in the study of biomolecular systems of increasing complexity: peptides, proteins and membranes

Molecular dynamics (MD) simulations are a powerful tool to study biochemical processes at the atomic level and to complement experiments by providing otherwise unattainable structural and dynamic information. In this thesis, different simulative approaches were applied to two classes of problems. The first regards the mechanism of lipid bilayer perturbation by antimicrobial peptides (AMPs), which kill bacteria by disrupting their membranes. Our computational results on peptides temporin L, LAH4, trichogin GAIV and PMAP-23 clarified several aspects of the mechanism of action of AMPs, illustrating how peptide sequence modulates aggregation and insertion in the lipid bilayer, and showing several facets of membrane disruption by AMPs, such as formation of bilayer defects, membrane thinning and perturbation of lipid dynamics. Overall, these data indicate that AMPs activity is regulated by several complex equilibria that should be taken into account in the rational design of new antibiotic drugs. Other studies focused on RRAS and SHP-2, two proteins involved in the MAPK pathway and in a family of disorders called RASopathies. We analyzed an RRAS mutation, isolated in a patient affected by Noonan syndrome, causing an enhancement in the rate of GDP dissociation. MD simulations revealed that this effect is related to the perturbation of the conformational transitions of the RRAS molecular switch. Similarly, simulations showed that the motions of an α-helix resulted to be essential in the function of SHP-2, thus providing new indications on the possible molecular effects of several pathogenic mutations. These studies underline once more the importance of conformational fluctuations in the physiological and aberrant function of proteins. All MD simulations reported in this thesis were consistent with the available experimental data, thus confirming the reliability of in silico approaches in obtaining novel insights in the characterization of complex biomolecular systems

Molecular Dynamics Simulations of the Host Defense Peptide Temporin L and Its Q3K Derivative: An Atomic Level View from Aggregation in Water to Bilayer Perturbation

Temporin L (TempL) is a 13 residue Host Defense Peptide (HDP) isolated from the skin of frogs. It has a strong affinity for lipopolysaccharides (LPS), which is related to its high activity against Gram-negative bacteria and also to its strong tendency to neutralize the pro-inflammatory response caused by LPS release from inactivated bacteria. A designed analog with the Q3K substitution shows an enhancement in both these activities. In the present paper, Molecular Dynamics (MD) simulations have been used to investigate the origin of these improved properties. To this end, we have studied the behavior of the peptides both in water solution and in the presence of LPS lipid-A bilayers, demonstrating that the main effect through which the Q3K substitution improves the peptide activities is the destabilization of peptide aggregates in water

Co-occurring WARS2 and CHRNA6 mutations in a child with a severe form of infantile parkinsonism

Objective: To investigate the molecular cause(s) underlying a severe form of infantile-onset parkinsonism and characterize functionally the identified variants. Methods: A trio-based whole exome sequencing (WES) approach was used to identify the candidate variants underlying the disorder. In silico modeling, and in vitro and in vivo studies were performed to explore the impact of these variants on protein function and relevant cellular processes. Results: WES analysis identified biallelic variants in WARS2, encoding the mitochondrial tryptophanyl tRNA synthetase (mtTrpRS), a gene whose mutations have recently been associated with multiple neurological phe-notypes, including childhood-onset, levodopa-responsive or unresponsive parkinsonism in a few patients. A substantial reduction of mtTrpRS levels in mitochondria and reduced OXPHOS function was demonstrated, supporting their pathogenicity. Based on the infantile-onset and severity of the phenotype, additional variants were considered as possible genetic modifiers. Functional assessment of a selected panel of candidates pointed to a de novo missense mutation in CHRNA6, encoding the α6 subunit of neuronal nicotinic receptors, which are involved in the cholinergic modulation of dopamine release in the striatum, as a second event likely contributing to the phenotype. In silico, in vitro (Xenopus oocytes and GH4C1 cells) and in vivo (C. elegans) analyses demon-strated the disruptive effects of the mutation on acetylcholine receptor structure and function