Search

Subversion of the host immune system by viruses is often mediated by molecular decoys that sequester host proteins pivotal to mounting effective immune responses. The widespread mammalian pathogen parapox Orf virus deploys GIF, a member of the poxvirus immune evasion superfamily, to antagonize GM-CSF (granulocyte macrophage colony-stimulating factor) and IL-2 (interleukin-2), two pleiotropic cytokines of the mammalian immune system. However, structural and mechanistic insights into the unprecedented functional duality of GIF have remained elusive. Here we reveal that GIF employs a dimeric binding platform that sequesters two copies of its target cytokines with high affinity and slow dissociation kinetics to yield distinct complexes featuring mutually exclusive interaction footprints. We illustrate how GIF serves as a competitive decoy receptor by leveraging binding hotspots underlying the cognate receptor interactions of GM-CSF and IL-2, without sharing any structural similarity with the cytokine receptors. Our findings contribute to the tracing of novel molecular mimicry mechanisms employed by pathogenic viruses., Viruses often subvert the host immune system using molecular decoys to prevent an effective immune response. Here, the authors examine the structural details of the viral decoy receptor GIF and its antagnosim of GM-CSF and IL-2.

Protein glycation (PG) usually induces the loss of protein function and consequently, the development of diabetes-related diseases1. It has been assumed that this process occurs as a result of a structural modification induced by PG, which is also able to trigger protein aggregation. Therefore, neurodegenerative disorders such as Parkinson or Alzheimer diseases increase their prevalence under diabetes mellitus. Trying to describe in detail the molecular mechanism that links PG with protein aggregation we have studied the glycation of lysozyme (HEWL) with ribose2. HEWL has been used as a model since it holds a reduced aggregation tendency under its native state and possesses putative glycation targets (lysine and arginine)3. On the other hand, ribose induces protein aggregation4 and it physiological concentration rapidly rises in diabetic patients5. Through the combination of different biophysical techniques (NMR, CD, SAXS, etc.), we have proved that HEWL glycation mediated by ribose mainly occurs on lysine without altering the protein structure. However, glycation induces a change on the overall protein charge generating different hydrophobic patches which are responsible for the native aggregation of glycated HEWL. This aggregation process initiates with the formation of soluble oligomers which finally assemble to form insoluble aggregates., Journal of International Society of Antioxidants in Nutrition & Health, Vol 3, No 2 (2016): Journal of ISANH Volume 3: Special Issue for Maillard Reaction & Glycation

Non-enzymatic glycation of proteins is a post-translational modification process which plays an important role in diabetes mellitus [1]. It involves covalent bond formation between free amino groups of proteins and reducing sugars that affects the physical and functional properties of proteins [2]. The present study was aimed at the investigation of the effects of glycation of hen egg white lysozyme (HEWL) by methylglyoxal and glycolaldehyde. The objectives of this study were to evaluate the enzyme activity during the glycation process, to identify the structures of advanced glycation end products (AGEs) formed on glycated HEWL, to investigate the effect of glycation on the secondary structure of glycated HEWL, and to analyse the morphology of the resulting aggregates. By using interdisciplinary techniques, we have shown that the kinetic rate of enzymatic activity loss of HEWL by methylglyoxal is two times higher than that for glycolaldehyde. Nevertheless, no changes in the secondary structure of HEWL were detected by CD. Furthermore, AGEs formed in the glycated HEWL by methylglyoxal promotes cross-linking in the glycated enzyme. Finally, the resulting aggregates exhibit the same spherical morphology but with different size (glycolaldehyde induces formation of bigger aggregates)., Journal of International Society of Antioxidants in Nutrition & Health, Vol 3, No 2 (2016)

Calpain is an essential and ubiquitous Ca2+ dependent protease that acts as a bio-modulator by modifying the functionality of substrate proteins, rather than digesting them. It plays an important role in processes such as apoptosis, cell cycle regulation, signal transduction and cell migration. Hyperactivation of calpain by calcium has severe pathological implications in e.g. Alzheimer's disease and cancer. Therefore calpain is an interesting therapeutic target for specific inhibition. Calpastatin is a dedicated endogenous inhibitor that binds to the catalytic cleft of calpain in a Ca2+ dependent way. Our project aims at developing an in silico methodology for de novo synthetic protein design using the available structural data of the calpain-calpastatin system as reference. In-depth profiling of known calpastatins and calpain substrates was carried out in silico using a diverse set of bioinformatics methods. Calpain's substrate and inhibitor preferences were extensively evaluated incorporating various features, including but not limited to amino acid composition, biochemical properties, secondary structure, conformational stability and binding energy. Based on the relevant parameters of the system we designed an initial library of potential binders and carried out high-throughput docking using HADDOCK followed by ranking of candidates based on their binding energies according to FoldX, thus effectively narrowing down the pool of structures. The best scoring candidates among these in silico designed peptides are currently being evaluated for interaction with calpain by Bio-Layer Interferometry. Upon finalizing the in vitro phase of the project, our design will be evaluated in vivo.

Calpain is an essential and ubiquitously expressed Ca2+ dependent protease that acts as a bio-modulator, modifying the functionality of substrate proteins rather than digesting them. It plays an important role in processes such as apoptosis, cell cycle regulation, signal transduction and cell migration. Pathological implications of disorders in the calpain system range from Alzheimer's disease and type II diabetes to cancer and cerebral/cardiac ischemia. These conditions are often typified by altered calcium homeostasis leading to hyperactivation of calpain. Therefore calpain is an important therapeutic target for specific inhibition. Calpastatin is such an endogenous inhibitor that binds to the catalytic chasm of calpain in a Ca2+ dependent way. Our project aims at evaluating critical parameters of the calpain-calpastatin system in silico and to design a synthetic construct that will specifically inhibit calpain. In-depth profiling of known calpastatins and calpain substrates was carried out in silico using various bioinformatics screening methods. Calpain-substrate and inhibitor preferences and tendencies were evaluated in regard to various traits, including but not limited to amino acid composition, biochemical properties, secondary structure, stability and docking to known interacting sites of calpains. This allowed us to define a structure-based and rational design of synthetic constructs that are currently being evaluated in vitro in regard to efficiency, examining properties such as calpain-binding and inhibition capabilities, stability and specificity. Upon finalizing the in vitro phase of the project, our design will be evaluated in in vivo experiments using a cell biology model.

Subversion of the host immune system by viruses is often mediated by molecular decoys that sequester host proteins pivotal to mounting effective immune responses. The widespread mammalian pathogen parapox Orf virus deploys GIF, a member of the poxvirus immune evasion superfamily, to antagonize GM-CSF (granulocyte macrophage colony-stimulating factor) and IL-2 (interleukin-2), two pleiotropic cytokines of the mammalian immune system. However, structural and mechanistic insights into the unprecedented functional duality of GIF have remained elusive. Here we reveal that GIF employs a dimeric binding platform that sequesters two copies of its target cytokines with high affinity and slow dissociation kinetics to yield distinct complexes featuring mutually exclusive interaction footprints. We illustrate how GIF serves as a competitive decoy receptor by leveraging binding hotspots underlying the cognate receptor interactions of GM-CSF and IL-2, without sharing any structural similarity with the cytokine receptors. Our findings contribute to the tracing of novel molecular mimicry mechanisms employed by pathogenic viruses.