Biology subjects

Petrlova, J.

Publications and source records attributed to Petrlova, J..

5 recordsLinked to original sources

bioRxiv2023

Community-based peptidomic clustering reveals pathogen-specific proteolytic signatures in wounds

Recent advances in mass spectrometry-based peptidomics has catalyzed the identification and quantification of thousands endogenous peptides across diverse biological systems. However, the large theoretical peptidomic landscape and high proportion of missing values poses several challenges for downstream analyses and limits the comparability of clinical samples. Here, we present a generalizable computational workflow that clusters peptides with overlapping sequences to reduce the dimensionality of peptidomic data, improve the definition of protease cut-sites, enhance inter-sample comparability, and enable the implementation of reliable and large-scale data analysis methods akin to those employed in other omics fields. We showcase the algorithm by performing large-scale quantitative analysis of wound fluid peptidomes of highly defined porcine wounds and human clinical non-healing wounds. The analysis revealed signature phenotype-specific peptide regions reflecting pathogen-specific proteolytic activity at the earliest stages of colonization, resulting in novel class of potential peptide cluster-based biomarkers.

bioinformatics↗

bioRxiv2023

Protein aggregation in wound fluid confines bacterial lipopolysaccharide and reduces inflammation

Bacterial lipopolysaccharide (LPS) induces the rapid formation of protein aggregates in human wound fluid. We aimed to define such LPS-induced aggregates and the functional consequences of protein aggregation using a combination of mass spectrometry analyses, biochemical imaging, and experimental animal models. We show that such wound-fluid aggregates contain a multitude of protein classes, including sequences from coagulation factors, annexins, histones, antimicrobial proteins/peptides, and apolipoproteins. Proteins and peptides with a high aggregation propensity were identified, and selected components were verified biochemically by western blot analysis. Staining by thioflavin T and the Amytracker probe demonstrated the presence of amyloid-like aggregates formed after exposure to LPS in vitro in human wound fluid and in vivo in porcine wound models. Using NF-{kappa}B-reporter mice and IVIS bioimaging, we show that such wound-fluid LPS aggregates induce a significant reduction in local inflammation compared with LPS in plasma. The results show that protein/peptide aggregation is a mechanism for confining LPS and reducing inflammation and further underscore the connection between host defense and amyloidogenesis.

immunology↗

bioRxiv2021

SARS-CoV-2 spike protein as a bacterial lipopolysaccharide delivery system in an overzealous inflammatory cascade

Accumulating evidence indicates a potential role for bacterial lipopolysaccharide (LPS) in the overactivation of the immune response during SARS-CoV-2 infection. LPS is recognised by Toll-like receptor 4 (TLR4) in innate immunity. Here, we showed that LPS binds to multiple hydrophobic pockets spanning both the S1 and S2 subunits of the SARS-CoV-2 spike (S) protein. LPS binds to the S2 pocket with a lower affinity compared to S1, suggesting its possible role as an intermediate in the TLR4 cascade. Congruently, nuclear factor-kappa B (NF-{kappa}B) activation in vitro is strongly boosted by S2. In vivo, however, a boosting effect is observed for both S1 and S2, with the former potentially facilitated by proteolysis. Collectively, our study suggests the S protein may act as a delivery system for LPS in host innate immune pathways. The LPS binding pockets are highly conserved across different SARS-CoV-2 variants and therefore represent potential therapeutic targets.

microbiology↗

bioRxiv2020

SARS-CoV-2 Spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity

ABSTRACTThere is a well-known and established link between high lipopolysaccharide (LPS) levels in blood and the metabolic syndrome (MS). MS is a risk factor for developing severe COVID-19 and acute respiratory distress syndrome (ARDS). Here we define an interaction between SARS-CoV-2 Spike (S) protein and LPS and its link to aggravated inflammation in vitro and in vivo. Electrophoresis under native conditions demonstrated that SARS-CoV-2 S protein binds to Escherichia coli LPS, forming high molecular weight aggregates. Microscale thermophoresis analysis further defined the interaction, having a KD of ~47 nM, similar to the observed affinity between LPS and the human receptor CD14. Moreover, S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-κB) and cytokine responses in monocytic THP-1 cells and human blood, respectively. In an experimental model of localized inflammation, employing NF-κB reporter mice and in vivo bioimaging, S protein in conjunction with LPS significantly increased the inflammatory response when compared with S protein and LPS alone. Apart from providing information on LPS as a ligand for S protein, our results are of relevance for studies on comorbidities involving bacterial endotoxins, such as the MS, or co-existing acute and chronic infections in COVID-19 patients.Competing Interest StatementA.S is a founder and shareholder of in2cure AB, a company developing therapies based on host defense peptides. A patent application related to the present work, with A.S. and G.P. listed as inventors, has been filed.AbbreviationsARDSacute respiratory distress syndromeCOVID-19coronavirus disease 2019MSmetabolic syndromeLBPLPS-binding proteinLPSlipopolysaccharideNF-κBnuclear factor-kappa BSARS-CoV-2 Spike proteinS proteinTLR4Toll-like receptor 4View Full Text

immunology↗

bioRxiv2017

Structural basis for endotoxin neutralization and anti-inflammatory activity of thrombin-derived C-terminal peptides

Thrombin-derived C-terminal peptides (TCP) of about 2 kDa are present in wounds, where they exert anti-endotoxic functions. In an effort to elucidate the structural and molecular aspects of these functions, we here employ a combination of nuclear magnetic resonance spectroscopy (NMR), ellipsometry, fluorescence spectroscopy, circular dichroism (CD) measurements, and in silico multiscale modeling to define interactions and the bound conformation of a TCP generated by neutrophil elastase, HVF18 (HVFRLKKWIQKVIDQFGE) in complex with bacterial lipopolysaccharide (LPS). In contrast to the disordered state of HVF18 in aqueous solution, its binding to LPS leads to a structural transition, wherein the N- terminus of the peptide forms a unique {beta}-turn whilst the C-terminus becomes helical. In silico modelling and simulations demonstrated that HVF18, as well as related peptides, target the LPS-binding site of CD14, and this interaction was experimentally supported using microscale thermophoresis. Collectively, the results demonstrate the role of structural transitions in LPS complex formation as well as in CD 14 interaction, and provide a molecular explanation for the previously observed therapeutic effects of TCPs in experimental models of bacterial sepsis and endotoxin shock.\n\nSignificanceThrombin-derived C-terminal peptides (TCPs) of various sizes are present in human wounds, where they bind bacteria as well as \"free\" lipopolysaccharide (LPS), and thereby reduce inflammation. In this work, employing a combination of cellular, biophysical and structural studies, combined with in silico multiscale modeling, we present the molecular structure of a TCP in association with LPS, and define a previously undisclosed interaction between TCPs and CD14. Further, we show that TCPs exhibit relatively weak but specific affinities, all in the M range, to both LPS and CD14. These novel structural insights into the function of this class of host-defense molecules will facilitate rational design of novel \"dual function\" anti-infectives, which target both bacteria and inflammatory signaling.

biophysics↗