Valentina Brandi

Huntington’s disease is a rare neurodegenerative and autosomal dominant disorder. HD is caused by a mutation in the gene coding for huntingtin (Htt). The result is the production of a mutant Htt with an abnormally long polyglutamine repeat that leads to pathological Htt aggregates. Although the structure of human Htt has been determined, albeit at low resolution, its functions and how they are performed are largely unknown. Moreover, there is little information on the structure and function of Htt in other organisms. The comparison of Htt homologs can help to understand if there is a functional conservation of domains in the evolution of Htt in eukaryotes. In this work, through a computational approach, Htt homologs from lower eukaryotes have been analysed, identifying ordered domains and modelling their structure. Based on the structural models, a putative function for most of the domains has been predicted. A putative C. elegans Htt-like protein has also been analysed following the same approach. The results obtained support the notion that this protein is a orthologue of human Htt.

Butylated hydroxytoluene (BHT) is one of the most commonly used synthetic antioxidants in food, cosmetic, pharmaceutical and petrochemical products. BHT is considered safe for human health; however, its widespread use together with the potential toxicological effects have increased consumers concern about the use of this synthetic food additive. In addition, the estimated daily intake of BHT has been demonstrated to exceed the recommended acceptable threshold. In the present work, using BHT as a case study, the usefulness of computational techniques, such as reverse screening and molecular docking, in identifying protein–ligand interactions of food additives at the bases of their toxicological effects has been probed. The computational methods here employed have been useful for the identification of several potential unknown targets of BHT, suggesting a possible explanation for its toxic effects. In silico analyses can be employed to identify new macromolecular targets of synthetic food additives and to explore their functional mechanisms or side effects. Noteworthy, this could be important for the cases in which there is an evident lack of experimental studies, as is the case for BHT.

Pseudomonas aeruginosa is an opportunistic human pathogen highly relevant from a biomedical viewpoint. It is one of the main causes of infection in hospitalized patients and a major cause of mortality of cystic fibrosis patients. This is also due to its ability to develop resistance to antibiotics by various mechanisms. Therefore, it is urgent and desirable to identify novel targets for the development of new antibacterial drugs against Pseudomonas aeruginosa. In this work this problem was tackled by an in silico approach aimed at providing a reliable structural model and functional annotation for the Pseudomonas aeruginosa periplasmic proteins for which these data are not available yet. A total of 83 protein sequences were analyzed, and the corresponding structural models were built, leading to the identification of 32 periplasmic “substrate-binding proteins”, 14 enzymes and 4 proteins with different functions, including lipids and metals binding. The most interesting cases were found within the “enzymes” group with the identification of a lipase, which can be regarded as a virulence factor, a protease involved in the assembly of β-barrel membrane proteins and a l,d-transpeptidase, which could contribute to confer resistance to β-lactam antibiotics to the bacterium.

A polyglutamine expansion of the N-terminal region of huntingtin (Htt) causes Huntington's disease, a severe neurodegenerative disorder. Htt huge multidomain structure, the presence of disordered regions, and the lack of sequence homologs of known structure, so far prevented structural studies of Htt, making the study of its structure-function relationships very difficult. In this work, the presence and location of five Htt ordered domains (named from Hunt1 to Hunt5) has been detected and the structure of these domains has been predicted for the first time using a combined threading/ab initio modeling approach. This work has led to the identification of a previously undetected HEAT repeats region in the Hunt3 domain. Furthermore, a putative function has been assigned to four out of the five domains. Hunt1 and Hunt5, displaying structural similarity with the regulatory subunit A of protein phosphatase 2A, are predicted to play a role in regulating the phosphorylation status of cellular proteins. Hunt2 and Hunt3 are predicted to be homologs of two yeast importins and to mediate vescicles transport and protein trafficking. Finally, a comprehensive analysis of the Htt interactome has been carried out and is discussed to provide a global picture of the Htt's structure-function relationships.

The opportunistic pathogen Staphylococcus aureus (S. aureus) is a major cause of nosocomial- and community-acquired infections. In addition, many antibiotic-resistant strains are emerging worldwide, thus, there is an urgent unmet need to pinpoint novel therapeutic and prophylactic strategies. In the present study, we characterized the impact of infection with the pandemic methicillin-resistant USA300 S. aureus strain on human primary dendritic cells (DC), key initiators and regulators of immune responses. In particular, among staphylococcal virulence factors, the function of EsxA and EsxB, two small acidic dimeric proteins secreted by the type VII-like secretion system Ess (ESAT-6-like secretion system), was investigated in human DC setting. A comparative analysis of bacterial entry, replication rate as well as DC maturation, apoptosis, signaling pathway activation and cytokine production was performed by using wild type (wt) USA300 and three isogenic mutants carrying the deletion of esxA (ΔesxA), esxB (ΔesxB) or both genes (ΔesxAB). The S. aureus mutant lacking only the EsxA protein (ΔesxA) stimulated a stronger pro-apoptotic phenotype in infected DC as compared to wt USA300, ΔesxAB and ΔesxB strains. When the mutant carrying the esxB deletion (ΔesxB) was analyzed, a higher production of both regulatory and pro-inflammatory mediators was found in the infected DC with respect to those challenged with the wt counterpart and the other esx mutants. In accordance with these data, supernatant derived from ΔesxB-infected DC promoted a stronger release of both IFN-γ and IL-17 from CD4+ T cells as compared with those conditioned with supernatants derived from wild type USA300-, ΔesxAB- and ΔesxA-infected cultures. Although the interaction of S. aureus with human DC is not yet fully understood, our data suggest that both cytokine production and apoptotic process are modulated by Esx factors, thus indicating a possible role of these proteins in the modulation of DC-mediated immunity to S. aureus.

In the year 2000, the third member of the globin family was discovered in human and mouse brain and named neuroglobin (Ngb). Neuroglobin overexpression significantly protects both heart and brain from hypoxic/ischemic and oxidative stress‐related insults, whereas decreased Ngb levels lead to an exacerbation of tissue injuries. Moreover, Ngb overexpression protects neurons from mitochondrial dysfunctions and neurodegenerative disorders such as Alzheimer disease; however, it facilitates the survival of cancer cells. Neuroglobin, representing a switch point for cell death and survival, has been reported to recognize a number of proteins involved in several metabolic pathways including ionic homeostasis maintenance, energy metabolism, mitochondrial function, and cell signaling. Here, the recognition properties of Ngb are reviewed to highlight its roles in health and disease.

The Gram-positive bacterium Streptococcus mutans is the principal causative agent of human tooth decay, an oral disease that affects the majority of the world’s population. Although the complete S. mutans genome is known, approximately 700 proteins are still annotated as hypothetical proteins, as no three-dimensional structure or homology with known proteins exists for them. Thus, the significant portion of genomic sequences coding for unknown-function proteins makes the knowledge of pathogenicity and survival mechanisms of S. mutans still incomplete. Plasmids are found in virtually every species of Streptococcus, and some of these mediate resistance to antibiotics and pathogenesis. However, there are strains of S. mutans that contain plasmids, such as LM7 and UA140, to which no function has been assigned yet. In this work, we describe an in silico study of the structure and function of all the S. mutans proteins encoded by pLM7 and pUA140 plasmids to gain insight into their biological function. A combination of different structural bioinformatics methodologies led to the identification of plasmidic proteins potentially required for the bacterial survival and pathogenicity. The structural information obtained on these proteins can be used to select novel targets for the design of innovative therapeutic agents towards S. mutans.

Alternative oxidases (AOXs) are mitochondrial cyanide-resistant membrane-bound metallo-proteins catalyzing the oxidation of ubiquinol and the reduction of oxygen to water bypassing two sites of proton pumping, thus dissipating a major part of redox energy into heat. Here, the structure of Arabidopsis thaliana AOX 1A has been modeled using the crystal structure of Trypanosoma brucei AOX as a template. Analysis of this model and multiple sequence alignment of members of the AOX family from all kingdoms of Life indicate that AOXs display a high degree of conservation of the catalytic core, which is formed by a four-α-helix bundle, hosting the di-iron catalytic site, and is flanked by two additional α-helices anchoring the protein to the membrane. Plant AOXs display a peculiar covalent dimerization mode due to the conservation in the N-terminal region of a Cys residue forming the inter-monomer disulfide bond. The multiple sequence alignment has also been used to infer a phylogenetic tree of AOXs whose analysis shows a polyphyletic origin for the AOXs found in Fungi and a monophyletic origin of the AOXs of Eubacteria, Mycetozoa, Euglenozoa, Metazoa, and Land Plants. This suggests that AOXs evolved from a common ancestral protein in each of these kingdoms. Within the Plant AOX clade, the AOXs of monocotyledon plants form two distinct clades which have unresolved relationships relative to the monophyletic clade of the AOXs of dicotyledonous plants. This reflects the sequence divergence of the N-terminal region, probably due to a low selective pressure for sequence conservation linked to the covalent homo-dimerization mode.