BackgroundInsulin is a hormone that regulates blood glucose homeostasis and is a central protein in a medical condition termed insulin injection amyloidosis. It is intimately associated with glycaemia and is vulnerable to glycation by glucose and other highly reactive carbonyls like methylglyoxal, especially in diabetic conditions. Protein glycation is involved in structure and stability changes that impair protein functionality, and is associated with several human diseases, such as diabetes and neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Familiar Amyloidotic Polyneuropathy. In the present work, methylglyoxal was investigated for their effects on the structure, stability and fibril formation of insulin.ResultsMethylglyoxal was found to induce the formation of insulin native-like aggregates and reduce protein fibrillation by blocking the formation of the seeding nuclei. Equilibrium-unfolding experiments using chaotropic agents showed that glycated insulin has a small conformational stability and a weaker dependence on denaturant concentration (smaller m-value). Our observations suggest that methylglyoxal modification of insulin leads to a less compact and less stable structure that may be associated to an increased protein dynamics.ConclusionsWe propose that higher dynamics in glycated insulin could prevent the formation of the rigid cross-β core structure found in amyloid fibrils, thereby contributing to the reduction in the ability to form fibrils and to the population of different aggregation pathways like the formation of native-like aggregates.

Understanding which peptides and proteins have the potential to undergo amyloid formation and what driving forces are responsible for amyloid-like fiber formation and stabilization remains limited. This is mainly because proteins that can undergo structural changes, which lead to amyloid formation, are quite diverse and share no obvious sequence or structural homology, despite the structural similarity found in the fibrils. To address these issues, a novel approach based on recursive feature selection and feed-forward neural networks was undertaken to identify key features highly correlated with the self-assembly problem. This approach allowed the identification of seven physicochemical and biochemical properties of the amino acids highly associated with the self-assembly of peptides and proteins into amyloid-like fibrils (normalized frequency of β-sheet, normalized frequency of β-sheet from LG, weights for β-sheet at the window position of 1, isoelectric point, atom-based hydrophobic moment, helix termination parameter at position j+1 and ΔG° values for peptides extrapolated in 0 M urea). Moreover, these features enabled the development of a new predictor (available at http://cran.r-project.org/web/packages/appnn/index.html) capable of accurately and reliably predicting the amyloidogenic propensity from the polypeptide sequence alone with a prediction accuracy of 84.9 % against an external validation dataset of sequences with experimental in vitro, evidence of amyloid formation.

BackgroundUnlike in HIV-1 infection, the majority of HIV-2 patients produce broadly reactive neutralizing antibodies, control viral replication and survive as elite controllers. The identification of the molecular, structural and evolutionary footprints underlying these very distinct immunological and clinical outcomes may lead to the development of new strategies for the prevention and treatment of HIV infection.Methodology/Principal FindingsWe performed a side-by-side molecular, evolutionary and structural comparison of the C2, V3 and C3 envelope regions from HIV-1 and HIV-2. These regions contain major antigenic targets and are important for receptor binding. In HIV-2, these regions also have immune modulatory properties. We found that these regions are significantly more variable in HIV-1 than in HIV-2. Within each virus, C3 is the most entropic region followed by either C2 (HIV-2) or V3 (HIV-1). The C3 region is well exposed in the HIV-2 envelope and is under strong diversifying selection suggesting that, like in HIV-1, it may harbour neutralizing epitopes. Notably, however, extreme diversification of C2 and C3 seems to be deleterious for HIV-2 and prevent its transmission. Computer modelling simulations showed that in HIV-2 the V3 loop is much less exposed than C2 and C3 and has a retractile conformation due to a physical interaction with both C2 and C3. The concealed and conserved nature of V3 in the HIV-2 is consistent with its lack of immunodominancy in vivo and with its role in preventing immune activation. In contrast, HIV-1 had an extended and accessible V3 loop that is consistent with its immunodominant and neutralizing nature.Conclusions/SignificanceWe identify significant structural and functional constrains to the diversification and evolution of C2, V3 and C3 in the HIV-2 envelope but not in HIV-1. These studies highlight fundamental differences in the biology and infection of HIV-1 and HIV-2 and in their mode of interaction with the human immune system and may inform new vaccine and therapeutic interventions against these viruses.

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