Glycosaminoglycans (GAGs) are linear polysaccharides. In proteoglycans (PGs), they are attached to a core protein. GAGs and PGs can be found as free molecules, associated with the extracellular matrix or expressed on the cell membrane. They play a role in the regulation of a wide array of physiological and pathological processes by binding to different proteins, thus modulating their structure and function, and their concentration and availability in the microenvironment. Unfortunately, the enormous structural diversity of GAGs/PGs has hampered the development of dedicated analytical technologies and experimental models. Similarly, computational approaches (in particular, molecular modeling, docking and dynamics simulations) have not been fully exploited in glycobiology, despite their potential to demystify the complexity of GAGs/PGs at a structural and functional level. Here, we review the state-of-the art of computational approaches to studying GAGs/PGs with the aim of pointing out the “bitter” and “sweet” aspects of this field of research. Furthermore, we attempt to bridge the gap between bioinformatics and glycobiology, which have so far been kept apart by conceptual and technical differences. For this purpose, we provide computational scientists and glycobiologists with the fundamentals of these two fields of research, with the aim of creating opportunities for their combined exploitation, and thereby contributing to a substantial improvement in scientific knowledge.

Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants’ evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was predicted to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation could confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we have shown that Q675H spike mutation is documented in all the VOCs. This finding highlights that VOCs are still evolving to enhance viral fitness and to adapt to the human host. At the same time, it may suggest Q675H spike mutation involvement in SARS-CoV-2 evolution.

Several human disorders, including Alzheimer’s disease (AD), are characterized by the aberrant formation of amyloid fibrils. In many cases, the amyloid core is flanked by disordered regions, known as fuzzy coat. The structural properties of fuzzy coats, and their interactions with their environments, however, have not been fully described to date. Here, we generate conformational ensembles of two brain-derived amyloid filaments of Aβ42, corresponding respectively to the familial and sporadic forms of AD. Our approach, called metadynamic electron microscopy metainference (MEMMI), provides a characterization of the transient interactions between the fuzzy coat and the cross-β core of the filaments. These calculations indicate that the familial AD filaments are less soluble than the sporadic AD filaments, and that the fuzzy coat contributes to solubilizing both types of filament. These results illustrate how the metainference approach can help analyze cryo-EM maps for the characterization of the properties of amyloid fibrils.

HIV-1 transactivating factor Tat is released by infected cells. Extracellular Tat homodimerizes and engages several receptors, including integrins, vascular endothelial growth factor receptor 2 (VEGFR2) and heparan sulfate proteoglycan (HSPG) syndecan-1 expressed on various cells. By means of experimental cell models recapitulating the processes of lymphocyte trans-endothelial migration, here, we demonstrate that upon association with syndecan-1 expressed on lymphocytes, Tat triggers simultaneously the in cis activation of lymphocytes themselves and the in trans activation of endothelial cells (ECs). This “two-way” activation eventually induces lymphocyte adhesion and spreading onto the substrate and vascular endothelial (VE)-cadherin reorganization at the EC junctions, with consequent endothelial permeabilization, leading to an increased extravasation of Tat-presenting lymphocytes. By means of a panel of biochemical activation assays and specific synthetic inhibitors, we demonstrate that during the above-mentioned processes, syndecan-1, integrins, FAK, src and ERK_1/2 engagement and activation are needed in the lymphocytes, while VEGFR2, integrin, src and ERK_1/2 are needed in the endothelium. In conclusion, the Tat/syndecan-1 complex plays a central role in orchestrating the setup of the various in cis and in trans multimeric complexes at the EC/lymphocyte interface. Thus, by means of computational molecular modelling, docking and dynamics, we also provide a characterization at an atomic level of the binding modes of the Tat/heparin interaction, with heparin herein used as a structural analogue of the heparan sulfate chains of syndecan-1.

Mutations in the Myelin Protein Zero gene (MPZ), encoding P0, the major structural glycoprotein of peripheral nerve myelin, are the cause of Charcot–Marie-Tooth (CMT) type 1B neuropathy, and most P0 mutations appear to act through gain-of-function mechanisms. Here, we investigated how misglycosylation, a pathomechanism encompassing several genetic disorders, may affect P0 function. Using in vitro assays, we showed that gain of glycosylation is more damaging for P0 trafficking and functionality as compared with a loss of glycosylation. Hence, we generated, via CRISPR/Cas9, a mouse model carrying the MPZ^D61N mutation, predicted to generate a new N-glycosylation site in P0. In humans, MPZ^D61N causes a severe early-onset form of CMT1B, suggesting that hyperglycosylation may interfere with myelin formation, leading to pathology. We show here that MPZ^D61N/+ mice develop a tremor as early as P15 which worsens with age and correlates with a significant motor impairment, reduced muscular strength and substantial alterations in neurophysiology. The pathological analysis confirmed a dysmyelinating phenotype characterized by diffuse hypomyelination and focal hypermyelination. We find that the mutant P0D61N does not cause significant endoplasmic reticulum stress, a common pathomechanism in CMT1B, but is properly trafficked to myelin where it causes myelin uncompaction. Finally, we show that myelinating dorsal root ganglia cultures from MPZ^D61N mice replicate some of the abnormalities seen in vivo, suggesting that they may represent a valuable tool to investigate therapeutic approaches. Collectively, our data indicate that the MPZ^D61N/+ mouse represents an authentic model of severe CMT1B affirming gain-of-glycosylation in P0 as a novel pathomechanism of disease.

Cystic fibrosis is a hereditary disease mainly caused by the deletion of the Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. Cystic fibrosis remains a potentially fatal disease, but it has become treatable as a chronic condition due to some CFTR-rescuing drugs that, when used in combination, increase in their therapeutic effect due to a synergic action. Also, dietary supplementation of natural compounds in combination with approved drugs could represent a promising strategy to further alleviate cystic fibrosis symptoms. On these bases, we screened by in silico drug repositioning 846 small synthetic or natural compounds from the AIFA database to evaluate their capacity to interact with the highly druggable lumacaftor binding site of F508del-CFTR. Among the identified hits, nicotinamide (NAM) was predicted to accommodate into the lumacaftor binding region of F508del-CFTR without competing against the drug but rather stabilizing its binding. The effective capacity of NAM to bind F508del-CFTR in a lumacaftor-uncompetitive manner was then validated experimentally by surface plasmon resonance analysis. Finally, the capacity of NAM to synergize with lumacaftor increasing its CFTR-rescuing activity was demonstrated in cell-based assays. This study suggests the possible identification of natural small molecules devoid of side effects and endowed with the capacity to synergize with drugs currently employed for the treatment of cystic fibrosis, which hopefully will increase the therapeutic efficacy with lower doses.