Structural vulnerability in EPCR suggests functional modulation.

The endothelial protein C receptor (EPCR) is a fundamental component of the vascular system in mammals due to its contribution in maintaining blood in a non-prothrombotic state, which is crucial for overall life development. It accomplishes this by enhancing the conversion of protein C (PC) into the anticoagulant activated protein C (APC), with this property being dependent on a known EPCR conformation that enables direct interaction with PC/APC. In this study, we report a previously unidentified conformation of EPCR whereby Tyr154, critical for PC/APC binding, shows a striking non-canonical configuration.

Self-Assembling Protein Nanoparticles in the Design of Vaccines: 2022 Update.

Vaccines constitute a pillar in the prevention of infectious diseases. The unprecedented emergence of novel immunization strategies due to the COVID-19 pandemic has again positioned vaccination as a pivotal measure to protect humankind and reduce the clinical impact and socioeconomic burden worldwide. Vaccination pursues the ultimate goal of eliciting a protective response in immunized individuals.

Identification of a broad lipid repertoire associated to the endothelial cell protein C receptor (EPCR).

Evidence is mounting that the nature of the lipid bound to the endothelial cell protein C receptor (EPCR) has an impact on its biological roles, as observed in anticoagulation and more recently, in autoimmune disease. Phosphatidylethanolamine and phosphatidylcholine species dominate the EPCR lipid cargo, yet, the extent of diversity in the EPCR-associated lipid repertoire is still unknown and remains to be uncovered. We undertook mass spectrometry analyses to decipher the EPCR lipidome, and identified species not yet described as EPCR ligands, such as phosphatidylinositols and phosphatidylserines.

Structural bases for the higher adherence to ACE2 conferred by the SARS-CoV-2 spike Q498Y substitution.

A remarkable number of SARS-CoV-2 variants and other as yet unmonitored lineages harbor amino-acid substitutions with the potential to modulate the interface between the spike receptor-binding domain (RBD) and its receptor ACE2. The naturally occurring Q498Y substitution, which is present in currently circulating SARS-CoV-2 variants, has drawn the attention of several investigations. While computational predictions and in vitro binding studies suggest that Q498Y increases the binding affinity of the spike protein for ACE2, experimental in vivo models of infection have shown that a triple mutant carrying the Q498Y replacement is fatal in mice.

Structural plasticity in I-A links autoreactivity to hybrid insulin peptides in type I diabetes.

We recently provided evidence for promiscuous recognition of several different hybrid insulin peptides (HIPs) by the highly diabetogenic, I-A-restricted 4.1-T cell receptor (TCR). To understand the structural determinants of this phenomenon, we solved the structure of an agonistic HIP/I-A complex, both in isolation as well as bound to the 4.

One evolutionarily selected amino acid variation is sufficient to provide functional specificity in the cold shock protein paralogs of Staphylococcus aureus.

Bacterial genomes encode several families of protein paralogs. Discrimination between functional divergence and redundancy among paralogs is challenging due to their sequence conservation. Here, we investigated whether the amino acid differences present in the cold shock protein (CSP) paralogs of Staphylococcus aureus were responsible for functional specificity.

NadA3 Structures Reveal Undecad Coiled Coils and LOX1 Binding Regions Competed by Meningococcus B Vaccine-Elicited Human Antibodies.

serogroup B (MenB) is a major cause of sepsis and invasive meningococcal disease. A multicomponent vaccine, 4CMenB, is approved for protection against MenB. Neisserial adhesin A (NadA) is one of the main vaccine antigens, acts in host cell adhesion, and may influence colonization and invasion.

Crystal structure reveals vaccine elicited bactericidal human antibody targeting a conserved epitope on meningococcal fHbp.

Data obtained recently in the United Kingdom following a nationwide infant immunization program against serogroup B Neisseria meningitidis (MenB) reported >80% 4CMenB vaccine-mediated protection. Factor H-binding protein (fHbp) is a meningococcal virulence factor and a component of two new MenB vaccines. Here, we investigated the structural bases underlying the fHbp-dependent protective antibody response in humans, which might inform future antigen design efforts.

Self-assembling protein nanoparticles in the design of vaccines.

For over 100 years, vaccines have been one of the most effective medical interventions for reducing infectious disease, and are estimated to save millions of lives globally each year. Nevertheless, many diseases are not yet preventable by vaccination. This large unmet medical need demands further research and the development of novel vaccines with high efficacy and safety.

MAIT recognition of a stimulatory bacterial antigen bound to MR1.

MR1-restricted mucosal-associated invariant T (MAIT) cells represent a subpopulation of αβ T cells with innate-like properties and limited TCR diversity. MAIT cells are of interest because of their reactivity against bacterial and yeast species, suggesting that they play a role in defense against pathogenic microbes. Despite the advances in understanding MAIT cell biology, the molecular and structural basis behind their ability to detect MR1-Ag complexes is unclear.

The molecular basis for Mucosal-Associated Invariant T cell recognition of MR1 proteins.

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved αβ T-cell lineage that express a semi-invariant T-cell receptor (TCR) restricted to the MHC related-1 (MR1) protein. MAIT cells are dependent upon MR1 expression and exposure to microbes for their development and stimulation, yet these cells can exhibit microbial-independent stimulation when responding to MR1 from different species. We have used this microbial-independent, cross-species reactivity of MAIT cells to define the molecular basis of MAIT-TCR/MR1 engagement and present here a 2.

The molecular basis for recognition of CD1d/α-galactosylceramide by a human non-Vα24 T cell receptor.

CD1d-mediated presentation of glycolipid antigens to T cells is capable of initiating powerful immune responses that can have a beneficial impact on many diseases. Molecular analyses have recently detailed the lipid antigen recognition strategies utilized by the invariant Vα24-Jα18 TCR rearrangements of iNKT cells, which comprise a subset of the human CD1d-restricted T cell population. In contrast, little is known about how lipid antigens are recognized by functionally distinct CD1d-restricted T cells bearing different TCRα chain rearrangements.

Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor.

Invariant Natural Killer T (iNKT) cells use highly restricted αβ T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted α1 helix resulting in an open A' pocket.

sPLA2-V inhibits EPCR anticoagulant and antiapoptotic properties by accommodating lysophosphatidylcholine or PAF in the hydrophobic groove.

The endothelial protein C receptor (EPCR) plays an important role in cardiovascular disease by binding protein C/activated protein C (APC). EPCR structure contains a hydrophobic groove filled with an unknown phospholipid needed to perform its function. It has not been established whether lipid exchange takes place in EPCR as a regulatory mechanism of its activity.

The immutable recognition of CD1d.

CD1d presents lipid antigens to natural killer T cells. In this issue of Immunity, Wun et al. (2011) and Mallevaey et al.

Improved dendritic cell-based immunization against hepatitis C virus using peptide inhibitors of interleukin 10.

Unlabelled: The high levels of interleukin 10 (IL-10) present in chronic hepatitis C virus (HCV) infection have been suggested as responsible for the poor antiviral cellular immune responses found in these patients. To overcome the immunosuppressive effect of IL-10 on antigen-presenting cells such as dendritic cells (DCs), we developed peptide inhibitors of IL-10 to restore DC functions and concomitantly induce efficient antiviral immune responses. Two IL-10-binding peptides (p9 and p13) were selected using a phage-displayed library and their capacity to inhibit IL-10 was assessed in a bioassay and in STAT-3 (signal transducer and activator of transcription 3) phosphorylation experiments in vitro.

A peptide inhibitor of FOXP3 impairs regulatory T cell activity and improves vaccine efficacy in mice.

Immunosuppressive activity of regulatory T cells (Treg) may contribute to the progression of cancer or infectious diseases by preventing the induction of specific immune responses. Using a phage-displayed random peptide library, we identified a 15-mer synthetic peptide, P60, able to bind to forkhead/winged helix transcription factor 3 (FOXP3), a factor required for development and function of Treg. P60 enters the cells, inhibits FOXP3 nuclear translocation, and reduces its ability to suppress the transcription factors NF-κB and NFAT.

Blocking endothelial protein C receptor (EPCR) accelerates thrombus development in vivo.

The endothelial protein C receptor (EPCR) plays an anticoagulant role by improving protein C activation. Although low levels of activated protein C (APC) constitute a thrombosis risk factor, the relationship between modulating EPCR function and thrombosis has not been addressed so far. Monoclonal antibodies (mAb) against murine EPCR were raised, and their ability to block protein C/APC binding was tested.

The endothelial cells downregulate the generation of factor VIIa through EPCR binding.

Traces of activated factor VII (FVIIa) are required to maintain haemostasis. Activated factor X (FXa) is the main activator of FVII in the absence of tissue factor. However, little is known about how this mechanism is regulated.

Losartan metabolite EXP3179 blocks NADPH oxidase-mediated superoxide production by inhibiting protein kinase C: potential clinical implications in hypertension.

Oxidative stress plays a critical role in the pathogenesis of hypertension. The NADPH oxidase constitutes a major source of superoxide anion in phagocytic cells, and its activation is associated with matrix metalloproteinase (MMP)-9 secretion by these cells. We investigated the effects of the angiotensin II type 1 receptor antagonist losartan and its metabolites (EXP3174 and EXP3179) on NADPH oxidase activity and MMP-9 secretion in human phagocytic cells.

A global benchmark study using affinity-based biosensors.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times.

Recombinant expression of biologically active murine soluble EPCR.

Endothelial cell protein C receptor (EPCR) downregulates the coagulation system and prevents thrombosis by binding to protein C/activated protein C (APC) and factor VII/activated factor VII (VIIa). Recombinant APC and factor VIIa have been shown to be useful in a variety of clinical conditions. Murine models could prove extremely helpful in order to study in vivo actions of these drugs.

In vitro and in vivo down-regulation of regulatory T cell activity with a peptide inhibitor of TGF-beta1.

Down-regulation of CD4+CD25+ regulatory T (Treg) cell function might be beneficial to enhance the immunogenicity of viral and tumor vaccines or to induce breakdown of immunotolerance. Although the mechanism of suppression used by Treg cells remains controversial, it has been postulated that TGF-beta1 mediates their immunosuppressive activity. In this study, we show that P17, a short synthetic peptide that inhibits TGF-beta1 and TGF-beta2 developed in our laboratory, is able to inhibit Treg activity in vitro and in vivo.

The functional properties of a truncated form of endothelial cell protein C receptor generated by alternative splicing.

Background: A soluble form of endothelial cell protein C receptor (sEPCR) is generated by shedding of the cellular form. sEPCR binds to protein C and factor VIIa and inhibits both the activation of protein C and the activity of activated protein C and factor VIIa. High sEPCR levels may increase the risk of thrombosis.

Identification of peptide inhibitors of transforming growth factor beta 1 using a phage-displayed peptide library.

Pathologies such as liver fibrosis and scleroderma are characterized by harmful levels of transforming growth factor beta 1 (TGFbeta1). These levels could be neutralized if inhibitors of this cytokine were available. With this aim we searched for peptides with binding affinity for TGFbeta1 using a phage-displayed random 15-mer peptide library.