RESUMO
The largest family of transmembrane receptors are G-protein-coupled receptors (GPCRs). These receptors respond to perceived environmental signals and infect their host plants. Family A of the GPCR includes opsin. However, there is little known about the roles of GPCRs in phytopathogenic fungi. We studied opsin in Leptosphaeria maculans, an important pathogen of oilseed rape (Brassica napus) that causes blackleg disease, and compared it with six other fungal pathogens of oilseed rape. A phylogenetic tree analysis of 31 isoforms of the opsin protein showed six major groups and six subgroups. All three opsin isoforms of L. maculans are grouped in the same clade in the phylogenetic tree. Physicochemical analysis revealed that all studied opsin proteins are stable and hydrophobic. Subcellular localization revealed that most isoforms were localized in the endoplasmic reticulum membrane except for several isoforms in Verticillium species, which were localized in the mitochondrial membrane. Most isoforms comprise two conserved domains. One conserved motif was observed across all isoforms, consisting of the BACTERIAL_OPSIN_1 domain, which has been hypothesized to have an identical sensory function. Most studied isoforms showed seven transmembrane helices, except for one isoform of V. longisporum and four isoforms of Fusarium oxysporum. Tertiary structure prediction displayed a conformational change in four isoforms of F. oxysporum that presumed differences in binding to other proteins and sensing signals, thereby resulting in various pathogenicity strategies. Protein-protein interactions and binding site analyses demonstrated a variety of numbers of ligands and pockets across all isoforms, ranging between 0 and 13 ligands and 4 and 10 pockets. According to the phylogenetic analysis in this study and considerable physiochemically and structurally differences of opsin proteins among all studied fungi hypothesized that this protein acts in the pathogenicity, growth, sporulation, and mating of these fungi differently.
RESUMO
The global outbreak of SARS-CoV-2/COVID-19 provided the stage to accumulate an enormous biomedical data set and an opportunity as well as a challenge to test new concepts and strategies to combat the pandemic. New research and molecular medical protocols may be deployed in different scientific fields, e.g., glycobiology, nanopharmacology, or nanomedicine. We correlated clinical biomedical data derived from patients in intensive care units with structural biology and biophysical data from NMR and/or CAMM (computer-aided molecular modeling). Consequently, new diagnostic and therapeutic approaches against SARS-CoV-2 were evaluated. Specifically, we tested the suitability of incretin mimetics with one or two pH-sensitive amino acid residues as potential drugs to prevent or cure long-COVID symptoms. Blood pH values in correlation with temperature alterations in patient bodies were of clinical importance. The effects of biophysical parameters such as temperature and pH value variation in relation to physical-chemical membrane properties (e.g., glycosylation state, affinity of certain amino acid sequences to sialic acids as well as other carbohydrate residues and lipid structures) provided helpful hints in identifying a potential Achilles heel against long COVID. In silico CAMM methods and in vitro NMR experiments (including 31P NMR measurements) were applied to analyze the structural behavior of incretin mimetics and SARS-CoV fusion peptides interacting with dodecylphosphocholine (DPC) micelles. These supramolecular complexes were analyzed under physiological conditions by 1H and 31P NMR techniques. We were able to observe characteristic interaction states of incretin mimetics, SARS-CoV fusion peptides and DPC membranes. Novel interaction profiles (indicated, e.g., by 31P NMR signal splitting) were detected. Furthermore, we evaluated GM1 gangliosides and sialic acid-coated silica nanoparticles in complex with DPC micelles in order to create a simple virus host cell membrane model. This is a first step in exploring the structure-function relationship between the SARS-CoV-2 spike protein and incretin mimetics with conserved pH-sensitive histidine residues in their carbohydrate recognition domains as found in galectins. The applied methods were effective in identifying peptide sequences as well as certain carbohydrate moieties with the potential to protect the blood-brain barrier (BBB). These clinically relevant observations on low blood pH values in fatal COVID-19 cases open routes for new therapeutic approaches, especially against long-COVID symptoms.
RESUMO
Insulin and lysozyme share the common features of being prone to aggregate and having biomedical importance. Encapsulating lysozyme and insulin in micellar nanoparticles probably would prevent aggregation and facilitate oral drug delivery. Despite the vivid structural knowledge of lysozyme and insulin, the environment-dependent oligomerization (dimer, trimer, and multimer) and associated structural dynamics remain elusive. The knowledge of the intra- and intermolecular interaction profiles has cardinal importance for the design of encapsulation protocols. We have employed various biophysical methods such as NMR spectroscopy, X-ray crystallography, Thioflavin T fluorescence, and atomic force microscopy in conjugation with molecular modeling to improve the understanding of interaction dynamics during homo-oligomerization of lysozyme (human and hen egg) and insulin (porcine, human, and glargine). The results obtained depict the atomistic intra- and intermolecular interaction details of the homo-oligomerization and confirm the propensity to form fibrils. Taken together, the data accumulated and knowledge gained will further facilitate nanoparticle design and production with insulin or lysozyme-related protein encapsulation.