The serine-rich repeat glycoprotein Srr2 mediates Streptococcus agalactiae interaction with host fibronectin.

BACKGROUND: Group B Streptococcus (GBS) is a commensal of healthy adults and an important pathogen in newborns, the elderly and immunocompromised individuals. GBS displays several virulence factors that promote colonisation and host infection, including the ST-17 strain-specific adhesin Srr2, previously characterised for its binding to fibrinogen. Another common target for bacterial adhesins and for host colonization is fibronectin, a multi-domain glycoprotein found ubiquitously in body fluids, in the extracellular matrix and on the surface of cells. RESULTS: In this study, fibronectin was identified as a novel ligand for the Srr2 adhesin of GBS. A derivative of the ST-17 strain BM110 overexpressing the srr2 gene showed an increased ability to bind fibrinogen and fibronectin, compared to the isogenic wild-type strain. Conversely, the deletion of srr2 impaired bacterial adhesion to both ligands. ELISA assays and surface plasmon resonance studies using the recombinant binding region (BR) form of Srr2 confirmed a direct interaction with fibronectin with an estimated Kd of 92 nM. Srr2-BR variants defective in fibrinogen binding also exhibited no interaction with fibronectin, suggesting that Srr2 binds this ligand through the dock-lock-latch mechanism, previously described for fibrinogen binding. The fibronectin site responsible for recombinant Srr2-BR binding was identified and localised in the central cell-binding domain of the protein. Finally, in the presence of fibronectin, the ability of a Δsrr2 mutant to adhere to human cervico-vaginal epithelial cells was significantly lower than that of the wild-type strain. CONCLUSION: By combining genetic and biochemical approaches, we demonstrate a new role for Srr2, namely interacting with fibronectin. We characterised the molecular mechanism of this interaction and demonstrated that it plays a role in promoting the adhesion of GBS to human cervico-vaginal epithelial cells, further substantiating the role of Srr2 as a factor responsible for the hypervirulence of GBS ST-17 strains. The discovery of the previously undescribed interaction between Srr2 and fibronectin establishes this adhesin as a key factor for GBS colonisation of host tissues.

From haemadin to haemanorm: Synthesis and characterization of full-length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm).

Hirudin from Hirudo medicinalis is a bivalent α-Thrombin (αT) inhibitor, targeting the enzyme active site and exosite-I, and is currently used in anticoagulant therapy along with its simplified analogue hirulog. Haemadin, a small protein (57 amino acids) isolated from the land-living leech Haemadipsa sylvestris, selectively inhibits αT with a potency identical to that of recombinant hirudin (KI = 0.2 pM), with which it shares a common disulfide topology and overall fold. At variance with hirudin, haemadin targets exosite-II and therefore (besides the free protease) it also blocks thrombomodulin-bound αT without inhibiting the active intermediate meizothrombin, thus offering potential advantages over hirudin. Here, we produced in reasonably high yields and pharmaceutical purity (>98%) wild-type haemadin and the oxidation resistant Met5 → nor-Leucine analogue, both inhibiting αT with a KI of 0.2 pM. Thereafter, we used site-directed mutagenesis, spectroscopic, ligand-displacement, and Hydrogen/Deuterium Exchange-Mass Spectrometry techniques to map the αT regions relevant for the interaction with full-length haemadin and with the synthetic N- and C-terminal peptides Haem(1-10) and Haem(45-57). Haem(1-10) competitively binds to/inhibits αT active site (KI = 1.9 µM) and its potency was enhanced by 10-fold after Phe3 → ß-Naphthylalanine exchange. Conversely to full-length haemadin, haem(45-57) displays intrinsic affinity for exosite-I (KD = 1.6 µM). Hence, we synthesized a peptide in which the sequences 1-9 and 45-57 were joined together through a 3-Glycine spacer to yield haemanorm, a highly potent (KI = 0.8 nM) inhibitor targeting αT active site and exosite-I. Haemanorm can be regarded as a novel class of hirulog-like αT inhibitors with potential pharmacological applications.

A novel genetically-encoded bicyclic peptide inhibitor of human urokinase-type plasminogen activator with better cross-reactivity toward the murine orthologue.

The inhibition of human urokinase-type plasminogen activator (huPA), a serine protease that plays an important role in pericellular proteolysis, is a promising strategy to decrease the invasive and metastatic activity of tumour cells. However, the generation of selective small molecule huPA inhibitors has proven to be challenging due to the high structural similarity of huPA to other paralogue serine proteases. Efforts to generate more specific therapies have led to the development of cyclic peptide-based inhibitors with much higher selectivity against huPA. While this latter property is desired, the sparing of the orthologue murine poses difficulties for the testing of the inhibitor in preclinical mouse model. In this work, we have applied a Darwinian evolution-based approach to identify phage-encoded bicyclic peptide inhibitors of huPA with better cross-reactivity towards murine uPA (muPA). The best selected bicyclic peptide (UK132) inhibited huPA and muPA with Ki values of 0.33 and 12.58 µM, respectively. The inhibition appears to be specific for uPA, as UK132 only weakly inhibits a panel of structurally similar serine proteases. Removal or substitution of the second loop with one not evolved in vitro led to monocyclic and bicyclic peptide analogues with lower potency than UK132. Moreover, swapping of 1,3,5-tris-(bromomethyl)-benzene with different small molecules not used in the phage selection, resulted in an 80-fold reduction of potency, revealing the important structural role of the branched cyclization linker. Further substitution of an arginine in UK132 to a lysine resulted in a bicyclic peptide UK140 with enhanced inhibitory potency against both huPA (Ki = 0.20 µM) and murine orthologue (Ki = 2.79 µM). By combining good specificity, nanomolar affinity and a low molecular mass, the bicyclic peptide inhibitor developed in this work may provide a novel human and murine cross-reactive lead for the development of a potent and selective anti-metastatic therapy.

Exogenous human α-Synuclein acts in vitro as a mild platelet antiaggregant inhibiting α-thrombin-induced platelet activation.

α-Synuclein (αSyn) is a small disordered protein, highly conserved in vertebrates and involved in the pathogenesis of Parkinson's disease (PD). Indeed, αSyn amyloid aggregates are present in the brain of patients with PD. Although the pathogenic role of αSyn is widely accepted, the physiological function of this protein remains elusive. Beyond the central nervous system, αSyn is expressed in hematopoietic tissue and blood, where platelets are a major cellular host of αSyn. Platelets play a key role in hemostasis and are potently activated by thrombin (αT) through the cleavage of protease-activated receptors. Furthermore, both αT and αSyn could be found in the same spatial environment, i.e. the platelet membrane, as αT binds to and activates platelets that can release αSyn from α-granules and microvesicles. Here, we investigated the possibility that exogenous αSyn could interfere with platelet activation induced by different agonists in vitro. Data obtained from distinct experimental techniques (i.e. multiple electrode aggregometry, rotational thromboelastometry, immunofluorescence microscopy, surface plasmon resonance, and steady-state fluorescence spectroscopy) on whole blood and platelet-rich plasma indicate that exogenous αSyn has mild platelet antiaggregating properties in vitro, acting as a negative regulator of αT-mediated platelet activation by preferentially inhibiting P-selectin expression on platelet surface. We have also shown that both exogenous and endogenous (i.e. cytoplasmic) αSyn preferentially bind to the outer surface of activated platelets. Starting from these findings, a coherent model of the antiplatelet function of αSyn is proposed.

NOG-Derived Peptides Can Restore Neuritogenesis on a CRASH Syndrome Cell Model.

Homo- and heterophilic binding mediated by the immunoglobulin (Ig)-like repeats of cell adhesion molecules play a pivotal role in cell-cell and cell-extracellular matrix interactions. L1CAM is crucial to neuronal differentiation, in both mature and developing nervous systems, and several studies suggest that its functional interactions are mainly mediated by Ig2-Ig2 binding. X-linked mutations in the human L1CAM gene are summarized as L1 diseases, including the most diagnosed CRASH neurodevelopmental syndrome. In silico simulations provided a molecular rationale for CRASH phenotypes resulting from mutations I179S and R184Q in the homophilic binding region of Ig2. A synthetic peptide reproducing such region could both mimic the neuritogenic capacity of L1CAM and rescue neuritogenesis in a cellular model of the CRASH syndrome, where the full L1CAM ectodomain proved ineffective. Presented functional evidence opens the route to the use of L1CAM-derived peptides as biotechnological and therapeutic tools.

The Role of Proteolysis in Amyloidosis.

Amyloidoses are a group of diseases associated with deposits of amyloid fibrils in different tissues. So far, 36 different types of amyloidosis are known, each due to the misfolding and accumulation of a specific protein. Amyloid deposits can be found in several organs, including the heart, brain, kidneys, and spleen, and can affect single or multiple organs. Generally, amyloid-forming proteins become prone to aggregate due to genetic mutations, acquired environmental factors, excessive concentration, or post-translational modifications. Interestingly, amyloid aggregates are often composed of proteolytic fragments, derived from the degradation of precursor proteins by yet unidentified proteases, which display higher amyloidogenic tendency compared to precursor proteins, thus representing an important mechanism in the onset of amyloid-based diseases. In the present review, we summarize the current knowledge on the proteolytic susceptibility of three of the main human amyloidogenic proteins, i.e., transthyretin, ß-amyloid precursor protein, and α-synuclein, in the onset of amyloidosis. We also highlight the role that proteolytic enzymes can play in the crosstalk between intestinal inflammation and amyloid-based diseases.

Staphylococcus aureus iron-regulated surface determinant B (IsdB) protein interacts with von Willebrand factor and promotes adherence to endothelial cells.

Staphylococcus aureus is the cause of a spectrum of diseases in humans and animals. The molecular basis of this pathogenicity lies in the expression of a variety of virulence factors, including proteins that mediate adherence to the host plasma and extracellular matrix proteins. In this study, we discovered that the iron-regulated surface determinant B (IsdB) protein, besides being involved in iron transport and vitronectin binding, interacts with von Willebrand Factor (vWF). IsdB-expressing bacteria bound to both soluble and immobilized vWF. The binding of recombinant IsdB to vWF was blocked by heparin and reduced at high ionic strength. Furthermore, treatment with ristocetin, an allosteric agent that promotes the exposure of the A1 domain of vWF, potentiates the binding of IsdB to vWF. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound recombinant A1 domain with KD values in the micromolar range. The binding of IsdB and adhesion of S. aureus expressing IsdB to monolayers of activated endothelial cells was significantly inhibited by a monoclonal antibody against the A1 domain and by IsdB reactive IgG from patients with staphylococcal endocarditis. This suggests the importance of IsdB in adherence of S. aureus to the endothelium colonization and as potential therapeutic target.

A conserved Neurite Outgrowth and Guidance motif with biomimetic potential in neuronal Cell Adhesion Molecules.

The discovery of conserved protein motifs can, in turn, unveil important regulatory signals, and when properly designed, synthetic peptides derived from such motifs can be used as biomimetics for biotechnological and therapeutic purposes. We report here that specific Ig-like repeats from the extracellular domains of neuronal Cell Adhesion Molecules share a highly conserved Neurite Outgrowth and Guidance (NOG) motif, which mediates homo- and heterophilic interactions crucial in neural development and repair. Synthetic peptides derived from the NOG motif of such proteins can boost neuritogenesis, and this potential is also retained by peptides with recombinant sequences, when fitting the NOG sequence pattern. The NOG motif discovery not only provides one more tile to the complex puzzle of neuritogenesis, but also opens the route to new neural regeneration strategies via a tunable biomimetic toolbox.

Sperm Cholesterol Content Modifies Sperm Function and TRPV1-Mediated Sperm Migration.

Transient receptor potential channels-vanilloid receptor 1 (TRPV1) regulates thermotaxis in sperm-oriented motility. We investigated the role of membrane cholesterol (Chol) on TRPV1-mediated human sperm migration. Semen samples were obtained from five normozoospemic healthy volunteers. Sperm membrane Chol content, quantified by liquid chromatography-mass spectrometry, was modified by incubating cells with 2-hydroxypropyl-ß-cyclodextrin (CD) or the complex between CD and Chol (CD:Chol). The effect on sperm migration on a 10 µM capsaicin gradient (CPS), a TRPV1 agonist, was then investigated. Motility parameters were evaluated by Sperm Class Analyser. Intracellular calcium concentration and acrosome reaction were measured by staining with calcium orange and FITC-conjugated anti-CD46 antibody, respectively. TRPV1-Chol interaction was modelled by computational molecular-modelling (MM). CD and CD:Chol, respectively, reduced and increased membrane Chol content in a dose-dependent manner, resulting in a dose-dependent increase and reduction of sperm migration in a CPS gradient. MM confirmed a specific interaction of Chol with a TRPV1 domain that appeared precluded to the Chol epimer epicholesterol (Epi-Chol). Accordingly, CD:Epi-Chol was significantly less efficient than CD:Chol, in reducing sperm migration under CPS gradient. Chol inhibits TRPV1-mediated sperm function by directly interacting with a consensus sequence of the receptor.

Interaction of Lactoferrin with Unsaturated Fatty Acids: In Vitro and In Vivo Study of Human Lactoferrin/Oleic Acid Complex Cytotoxicity.

As shown recently, oleic acid (OA) in complex with lactoferrin (LF) causes the death of cancer cells, but no mechanism(s) of that toxicity have been disclosed. In this study, constitutive parameters of the antitumor effect of LF/OA complex were explored. Complex LF/OA was prepared by titrating recombinant human LF with OA. Spectral analysis was used to assess possible structural changes of LF within its complex with OA. Structural features of apo-LF did not change within the complex LF:OA = 1:8, which was toxic for hepatoma 22a cells. Cytotoxicity of the complex LF:OA = 1:8 was tested in cultured hepatoma 22a cells and in fresh erythrocytes. Its anticancer activity was tested in mice carrying hepatoma 22a. In mice injected daily with LF-8OA, the same tumor grew significantly slower. In 20% of animals, the tumors completely resolved. LF alone was less efficient, i.e., the tumor growth index was 0.14 for LF-8OA and 0.63 for LF as compared with 1.0 in the control animals. The results of testing from 48 days after the tumor inoculation showed that the survival rate among LF-8OA-treated animals was 70%, contrary to 0% rate in the control group and among the LF-treated mice. Our data allow us to regard the complex of LF and OA as a promising tool for cancer treatment.

Von Willebrand Factor Multimers and the Relaxation Response: A One-Year Study.

BACKGROUND AND AIM: Mental stress represents a pivotal factor in cardiovascular diseases. The mechanism by which stress produces its deleterious ischemic effects is still under study but some of the most explored pathways are inflammation, endothelial function and balancing of the thrombotic state. In this scenario, von Willebrand factor (vWF) is a plasma glycoprotein best known for its crucial hemostatic role, also acting as key regulatory element of inflammation, being released by the activated vascular endothelium. Antistress techniques seem to be able to slow down inflammation. As we have recently verified how the practice of the Relaxation Response (RR), which counteracts psychological stress, causes favorable changes in some inflammatory genes' expressions, neurotransmitters, hormones, cytokines and inflammatory circulating microRNAs with coronary endothelial function improvement, we aimed to verify a possible change even in serum levels of vWF. Experimental procedure: We measured vWF multimers and the total protein carbonyl contents in the sera of 90 patients with ischemic heart disease (and 30 healthy controls) immediately before and after an RR session, three times (baseline, 6 months, 12 months), during a one-year follow-up study. RESULTS: According to our data, large vWF multimers decrease during the RR, as does the plasma total carbonyl content. CONCLUSION: vWF levels seem to vary rapidly between anti-inflammatory and antithrombotic behaviors dependent on psychological activity, leading to relaxation and also possibly changes in its quaternary structure.

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen.

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.

A Novel Aza-Derivative Inhibits agr Quorum Sensing Signaling and Synergizes Methicillin-Resistant Staphylococcus aureus to Clindamycin.

Increasing antibiotic resistance and diminishing pharmaceutical industry investments have increased the need for molecules that can treat infections caused by dangerous pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Quorum Sensing (QS) is a signaling mechanism that regulates bacterial virulence in pathogens. A report demonstrating that the anti-inflammatory drug Diflunisal reduces MRSA virulence factors' expression prompted us to design, synthesize and test 16 aza-analogs as inhibitors of S. aureus virulence factors controlled by the accessory gene regulator (agr) QS system. At first, we evaluated by qRT-PCR the activity of compounds on rnaIII expression, a QS related gene. Azan-7 was the most active molecule tested and it did not show cytotoxic activity in human cell lines. Moreover, we demonstrated that it did not affect bacterial proliferation. Regulation of MRSA virulence genes by Azan-7 was investigated using qRT-PCR and RNAseq. Azan-7 significantly reduced hla, psmα, hysA, agrA, cap1A, and cap1C gene expression. In silico docking demonstrated that Azan-7 binds the response regulator AgrA. This data was confirmed by electrophoretic mobility shift assay (EMSA) reporting that Azan-7 binding to AgrA protein strongly reduced the AgrA-DNA complex formation at the P3 promoter region involved in the regulation of rnaIII transcription. Azan-7 inhibited MRSA-mediated haemolysis, reduced survival of the pathogen at low pH levels, and increased macrophage killing. In addition, Azan-7 enhanced MRSA susceptibility to clindamycin both in planktonic growth and biofilm. Azan-7 did not induce resistance over 10 days in culture. It was equally active against all the AgrA MRSA subtypes encountered among clinical isolates, but it was not active against Staphylococcus epidermidis, although the AgrA proteins show an approximate 80% homology. These results demonstrate that Azan-7 inhibits the expression of MRSA virulence factors by interfering in the QS and synergizes MRSA biofilm with clindamycin, indicating the compound as a promising candidate for the treatment of MRSA infections.

A serine protease secreted from Bacillus subtilis cleaves human plasma transthyretin to generate an amyloidogenic fragment.

Aggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1-3% older adults. Fragmented hTTR is the standard composition of amyloid deposits in SSA, but the protease(s) responsible for amyloidogenic fragments generation in vivo is(are) still elusive. Here, we show that subtilisin secreted from Bacillus subtilis, a gut microbiota commensal bacterium, translocates across a simulated intestinal epithelium and cleaves hTTR both in solution and human plasma, generating the amyloidogenic fragment hTTR(59-127), which is also found in SSA amyloids in vivo. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart.

Endocrine disruption of vitamin D activity by perfluoro-octanoic acid (PFOA).

Perfluoroalkyl substances (PFAS) are a class of compounds used in industry and consumer products. Perfluorooctanoic acid (PFOA) is the predominant form in human samples and has been shown to induce severe health consequences, such as neonatal mortality, neurotoxicity, and immunotoxicity. Toxicological studies indicate that PFAS accumulate in bone tissues and cause altered bone development. Epidemiological studies have reported an inverse relationship between PFAS and bone health, however the associated mechanisms are still unexplored. Here, we present computational, in silico and in vitro evidence supporting the interference of PFOA on vitamin D (VD). First, PFOA competes with calcitriol on the same binding site of the VD receptor, leading to an alteration of the structural flexibility and a 10% reduction by surface plasmon resonance analysis. Second, this interference leads to an altered response of VD-responsive genes in two cellular targets of this hormone, osteoblasts and epithelial cells of the colorectal tract. Third, mineralization in human osteoblasts is reduced upon coincubation of PFOA with VD. Finally, in a small cohort of young healthy men, PTH levels were higher in the exposed group, but VD levels were comparable. Altogether these results provide the first evidence of endocrine disruption by PFOA on VD pathway by competition on its receptor and subsequent inhibition of VD-responsive genes in target cells.

The J-elongated conformation of ß2-glycoprotein I predominates in solution: implications for our understanding of antiphospholipid syndrome.

ß2-Glycoprotein I (ß2GPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a life-threatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound ß2GPI to that in solution. ß2GPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, ß2GPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and O-circular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant ß2GPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular or twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound ß2GPI arises from the ability of its preexisting J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of ß2GPI provides a strategy to block pathogenic aPLs in APS.

Application of Circular Dichroism and Fluorescence Spectroscopies To Assess Photostability of Water-Soluble Porcine Lens Proteins.

The eye lens is mainly composed of the highly ordered water-soluble (WS) proteins named crystallins. The aggregation and insolubilization of these proteins lead to progressive lens opacification until cataract onset. Although this is a well-known disease, the mechanism of eye lens protein aggregation is not well understood; however, one of the recognized causes of proteins modification is related to the exposure to UV light. For this reason, the spectroscopic properties of WS lens proteins and their stability to UV irradiation have been evaluated by different biophysical methods including synchrotron radiation circular dichroism, fluorescence, and circular dichroism spectroscopies. Moreover, dynamic light scattering, gel electrophoresis, transmission electron microscopy, and protein digestion followed by tandem LC-MS/MS analysis were used to study the morphological and structural changes in protein aggregates induced by exposure to UV light. Our results clearly indicated that the exposure to UV radiation modified the protein conformation, inducing a loss of ordered structure and aggregation. Furthermore, we confirmed that these changes were attributable to the generation of reactive oxygen species due to the irradiation of the protein sample. This approach, involving the photodenaturation of proteins, provides a benchmark in high-throughput screening of small molecules suitable to prevent protein denaturation and aggregation.

SHBG141-161 Domain-Peptide Stimulates GPRC6A-Mediated Response in Leydig and ß-Langerhans cell lines.

GPRC6A is acknowledged as a major regulator of energy metabolism and male fertility through the action of undercarboxylated osteocalcin (ucOCN), representing a possible therapeutic target. We recently showed that the sex hormone-binding globulin (SHBG) binds to GPRC6A through the likely involvement of the 141-161 domain. To confirm this model, here we investigated the possible binding and agonist activity of SHBG(141-161) domain-peptide (SHBG141-161) on GPRC6A. The binding of SHBG141-161 to GPRC6A and downstream dissociation from Gαi(GDP) protein was computationally modelled. SHBG141-161 was obtained by solid-phase synthesis, characterized by circular dichroism (CD) and the receptor binding was assessed by displacement of ucOCN on HEK-293 cells transfected with GPRC6A gene. Agonist activity of SHBG141-161 was assessed on Leydig MA-10 and Langerhans ß-TC6 cell lines through the GPRC6A-mediated release of testosterone (T) and insulin. SHBG141-161 was predicted to bind to GPRC6A and to reduce the affinity for Gαi(GDP) at computational level. Conformational properties and binding to GPRC6A of the synthetic SHBG141-161 were confirmed by CD and displacement experiments. SHBG141-161 stimulated cell secretion of T and insulin, with dose dependency from 10-13 to 10-11M for T release (respectively P = 0,041 10-13M; P = 0,032 10-12M; P = 0,008 10-11M vs basal) and for 10-12 to 10-10M for insulin (respectively P = 0,041 10-12M; P = 0,007 10-11M; P = 0,047 10-10M; P = 0,045 vs basal). Blockade with anti GPRC6A IgG abolished the response to SHBG141-161, suggesting agonist specificity. SHBG141-161 showed stimulating activity on GPRC6A, representing a template peptide with possible therapeutic use for metabolic and endocrine disorders.

Fibronectin-binding protein B (FnBPB) from Staphylococcus aureus protects against the antimicrobial activity of histones.

Staphylococcus aureus is a Gram-positive bacterium that can cause both superficial and deep-seated infections. Histones released by neutrophils kill bacteria by binding to the bacterial cell surface and causing membrane damage. We postulated that cell wall-anchored proteins protect S. aureus from the bactericidal effects of histones by binding to and sequestering histones away from the cell envelope. Here, we focused on S. aureus strain LAC and by using an array of biochemical assays, including surface plasmon resonance and ELISA, discovered that fibronectin-binding protein B (FnBPB) is the main histone receptor. FnBPB bound all types of histones, but histone H3 displayed the highest affinity and bactericidal activity and was therefore investigated further. H3 bound specifically to the A domain of recombinant FnBPB with a KD of 86 nm, ∼20-fold lower than that for fibrinogen. Binding apparently occurred by the same mechanism by which FnBPB binds to fibrinogen, because FnBPB variants defective in fibrinogen binding also did not bind H3. An FnBPB-deletion mutant of S. aureus LAC bound less H3 and was more susceptible to its bactericidal activity and to neutrophil extracellular traps, whereas an FnBPB-overexpressing mutant bound more H3 and was more resistant than the WT. FnBPB bound simultaneously to H3 and plasminogen, which after activation by tissue plasminogen activator cleaved the bound histone. We conclude that FnBPB provides a dual immune-evasion function that captures histones and prevents them from reaching the bacterial membrane and simultaneously binds plasminogen, thereby promoting its conversion to plasmin to destroy the bound histone.

Effects of point mutations in the binding pocket of the mouse major urinary protein MUP20 on ligand affinity and specificity.

The mouse Major Urinary Proteins (MUPs) contain a conserved ß-barrel structure with a characteristic central hydrophobic pocket that binds a variety of volatile compounds. After release of urine, these molecules are slowly emitted in the environment where they play an important role in chemical communication. MUPs are highly polymorphic and conformationally stable. They may be of interest in the construction of biosensor arrays capable of detection of a broad range of analytes. In this work, 14 critical amino acids in the binding pocket involved in ligand interactions were identified in MUP20 using in silico techniques and 7 MUP20 mutants were synthesised and characterised to produce a set of proteins with diverse ligand binding profiles to structurally different ligands. A single amino acid substitution in the binding pocket can dramatically change the MUPs binding affinity and ligand specificity. These results have great potential for the design of new biosensor and gas-sensor recognition elements.