RÉSUMÉ
High levels of human group IIA secreted phospholipase A2 (hGIIA) have been associated with various inflammatory disease conditions. We have recently shown that hGIIA activity and concentration are increased in the plasma of patients with hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) and negatively correlate with C1-INH plasma activity. In this study, we analyzed whether the presence of both hGIIA and C1-INH impairs their respective function on immune cells. hGIIA, but not recombinant and plasma-derived C1-INH, stimulates the production of IL-6, CXCL8, and TNF-α from peripheral blood mononuclear cells (PBMCs). PBMC activation mediated by hGIIA is blocked by RO032107A, a specific hGIIA inhibitor. Interestingly, C1-INH inhibits the hGIIA-induced production of IL-6, TNF-α, and CXCL8, while it does not affect hGIIA enzymatic activity. On the other hand, hGIIA reduces the capacity of C1-INH at inhibiting C1-esterase activity. Spectroscopic and molecular docking studies suggest a possible interaction between hGIIA and C1-INH but further experiments are needed to confirm this hypothesis. Together, these results provide evidence for a new interplay between hGIIA and C1-INH, which may be important in the pathophysiology of hereditary angioedema.
Sujet(s)
Angio-oedèmes héréditaires , C1 Inhibiteur , Group II Phospholipases A2 , Humains , Interleukine-6 , Agranulocytes , Simulation de docking moléculaire , Facteur de nécrose tumorale alpha , C1 Inhibiteur/composition chimique , C1 Inhibiteur/métabolisme , Group II Phospholipases A2/composition chimique , Group II Phospholipases A2/métabolismeRÉSUMÉ
Phospholipases A2 (PLA2s) are found in almost every venomous snake family. In snakebites, some PLA2s can quickly cause local myonecrosis, which may lead to permanent sequelae if antivenom is administered belatedly. They hydrolyse phospholipids in membranes through a catalytic calcium ions-dependent mechanism. BthTX-II is a basic PLA2 and the second major component in the venom of Bothrops jararacussu. Herein, using the software SEQUENCE SLIDER, which integrates crystallographic, mass spectrometry and genetic data, we characterized the primary, tertiary and quaternary structure of two BthTX-II variants (called a and b), which diverge in 7 residues. Crystallographic structure BthTX-IIa is in a Tense-state with its distorted calcium binding loop buried in the dimer interface, contrarily, the novel BthTX-IIb structure is a monomer in a Relax-state with a fatty acid in the hydrophobic channel. Structural data in solution reveals that both variants are monomeric in neutral physiological conditions and mostly dimeric in an acidic environment, being catalytic active in both situations. Therefore, we propose two myotoxic mechanisms for BthTX-II, a catalytic one associated with the monomeric assembly, whereas the other has a calcium independent activity related to its C-terminal region, adopting a dimeric conformation similar to PLA2-like proteins.
Sujet(s)
Venins de crotalidé/composition chimique , Group II Phospholipases A2/composition chimique , Multimérisation de protéines , Sites de fixation , Calcium/métabolisme , Venins de crotalidé/métabolisme , Group II Phospholipases A2/métabolisme , Simulation de dynamique moléculaire , Liaison aux protéinesRÉSUMÉ
Secreted phospholipase A2s (sPLA2s) are calcium dependent enzymes involved in various biological events such as lipid metabolism and inflammation. We previously identified the second calcium (Ca2) binding site of human sPLA2 Group IIE (hGIIE) by structural study and suggested that Asn21 act as the switch of Ca2 binding to modulate the enzymatic activity, but the detailed Ca2 binding mechanism is still unclear. Combined with enzymatic assay, model analysis and calcium binding affinity data for mutated hGIIE proteins, we herein further demonstrate that the flexibly bound Ca2 is essential for the catalysis of hGIIE, unlike the stable binding of Ca2 in hGIIA that replenishes the calcium in the typical loop during the reaction. The atypical Ca2 binding feature of hGIIE will provide a better understanding on the catalytic mechanism of hGIIE.
Sujet(s)
Calcium/composition chimique , Calcium/métabolisme , Group II Phospholipases A2/composition chimique , Group II Phospholipases A2/métabolisme , Sites de fixation , Catalyse , Domaine catalytique , Group II Phospholipases A2/antagonistes et inhibiteurs , Group II Phospholipases A2/génétique , Mutation , Liaison aux protéines , Protéines recombinantesRÉSUMÉ
Bites by viperid snakes belonging to Bothrops genus produce fast and intense local edema, inflammation, bleeding and myonecrosis. In this study, we investigated the role of Myogenic Regulatory Factors (MRFs: MyoD; Myog), negatively regulated by GDF-8 (Myostatin), and ubiquitin-proteasome system pathway (UPS: MuRF-1; Fbx-32) in gastrocnemius muscle regeneration after Bothrops jararacussu snake venom (Bjussu) or its isolated phospholipase A2 myotoxins, BthTx-I (Lys-49 PLA2) and BthTx-II (Asp-49 PLA2) injection. Male Swiss mice received a single intra-gastrocnemius injection of crude Bjussu, at a dose/volume of 0.83â¯mg/kg/20⯵l, and BthTx-I or BthTx-II, at a dose/volume of 2.5â¯mg/kg/20⯵l. Control mice (Sham) received an injection of sterile saline solution (NaCl 0.9%; 20⯵l). At 24, 48, 72 and 96â¯h post injection, right gastrocnemius was collected for protein expression analyses. Based on the temporal expressional dynamics of MyoD, Myog and GDF-8/Myostatin, it was possible to propose that the myogenesis pathway was impacted most badly by BthTx-II followed by BthTx-I and lastly by B. jararacussu venom, thus suggesting that catalytic activity has likely inhibitory role on the satellite cells-mediated reparative myogenesis pathway. Inversely, the catalytic activity seems to be not a determinant for the activation of proteins ubiquitination by MuRF-1 and Fbx-32/Atrogin-1 E3 proteasome ligases, given proteolysis pathway through UPS was activated neither after Bjussu, nor after BthTx-II, but just after the catalytically-inactive BthTx-I Lys-49 PLA2-homologue exposure. The findings of this study disclose interesting perspective for further mechanistic studies about pathways that take part in the atrophy and repair after permanent damage induced by bothropic snakebites.
Sujet(s)
Venins de crotalidé/pharmacologie , Group II Phospholipases A2/pharmacologie , Protéines du muscle/métabolisme , Muscles squelettiques/effets des médicaments et des substances chimiques , Muscles squelettiques/métabolisme , Animaux , Venins de crotalidé/composition chimique , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Group II Phospholipases A2/composition chimique , Mâle , Souris , Protéines du muscle/génétique , ProtéolyseRÉSUMÉ
Secreted phospholipases A2 (sPLA2) group IIE (GIIE) is involved in several biological events, such as lipid metabolism and possibly inflammation that may mainly depend on its catalytic reaction. We previously showed that Asn21 is a critical residue that contributes to the enzymatic activity of hGIIE, but the underlying mechanism is still not clear. Here, combined with crystal structures and mutagenesis studies of the Asn21Gly mutant, we demonstrate that Asn21 acts as a switch responsible for the calcium binding and the catalytic efficiency. Our results of the atypical feature of calcium binding in hGIIE not only provide clues to understand the molecular basis of its enzymatic activity and physiological function, but also confer improved specificity for potential inhibitor design of sPLA2.
Sujet(s)
Calcium/composition chimique , Group II Phospholipases A2/composition chimique , Substitution d'acide aminé , Asparagine/composition chimique , Asparagine/génétique , Group II Phospholipases A2/génétique , Humains , Mutation faux-sens , Liaison aux protéinesRÉSUMÉ
This work shows the antitumor and antimetastatic effects of BthTX-II, an Asp-49 PLA2 from Bothrops jararacussu venom, on MDA-MB-231 human triple negative breast cancer cells. BthTX-II caused a dose-dependent cell death of MDA-MB-231 cells when compared with the non-tumorigenic breast cells by inducing apoptosis and autophagy. BthTX-II was also able to decrease the proliferation and to inhibit cell cycle progression. We also observed an upregulation of the ATM gene, which is responsible for cell-cycle arrest and DNA repair such as CCND1, CCNE1, CDC25A, E2F1, AKT1 and AKT3. Interestingly, BthTX-II inhibited invasion, migration and 3D cell growth of MDA-MB-231 cells, as well as inhibited the epithelial-mesenchymal transition (EMT) of this cell by increasing E-cadherin (CDH-1) and decreasing TWIST1, CTNNB1, vimentin and cytokeratin-5 expression. In conclusion, these results showed that BthTX-II displays antitumor and antimetastatic effects on MDA-MB-231 cells and may be useful for the development of new approaches and therapeutic strategies to manage triple negative breast cancer.
Sujet(s)
Antinéoplasiques/composition chimique , Antinéoplasiques/pharmacologie , Bothrops , Venins de crotalidé/composition chimique , Venins de crotalidé/pharmacologie , Group II Phospholipases A2/composition chimique , Group II Phospholipases A2/pharmacologie , Animaux , Antinéoplasiques/isolement et purification , Apoptose/effets des médicaments et des substances chimiques , Autophagie/effets des médicaments et des substances chimiques , Marqueurs biologiques tumoraux , Adhérence cellulaire/effets des médicaments et des substances chimiques , Points de contrôle du cycle cellulaire/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale , Mouvement cellulaire/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Venins de crotalidé/isolement et purification , Group II Phospholipases A2/isolement et purification , Humains , Venins de serpent/composition chimique , Venins de serpent/pharmacologieRÉSUMÉ
Ophidian accidents are considered an important neglected tropical disease by the World Health Organization. Particularly in Latin America, Bothrops snakes are responsible for the majority of the snakebite envenomings that are not efficiently treated by conventional serum therapy. Thus, the search for simple and efficient inhibitors to complement this therapy is a promising research area, and a combination of functional and structural assays have been used to test candidate ligands against specific ophidian venom compounds. Herein, we tested a commercial drug (acetylsalicylic acid, ASA) and a plant compound with antiophidian properties (rosmarinic acid, RA) using myographic, crystallographic and bioinformatics experiments with a phospholipase A2-like toxin, MjTX-II. MjTX-II/RA and MjTX-II/ASA crystal structures were solved at high resolution and revealed the presence of ligands bound to different regions of the toxin. However, in vitro myographic assays showed that only RA is able to prevent the myotoxic effects of MjTX-II. In agreement with functional results, molecular dynamics simulations showed that the RA molecule remains tightly bound to the toxin throughout the calculations, whereas ASA molecules tend to dissociate. This approach aids the design of effective inhibitors of PLA2-like toxins and, eventually, may complement serum therapy.
Sujet(s)
Acide acétylsalicylique , Cinnamates , Venins de crotalidé , Depsides , Group II Phospholipases A2 , Simulation de dynamique moléculaire , Animaux , Acide acétylsalicylique/composition chimique , Acide acétylsalicylique/pharmacologie , Cinnamates/composition chimique , Cinnamates/pharmacologie , Venins de crotalidé/composition chimique , Venins de crotalidé/toxicité , Cristallographie aux rayons X , Depsides/composition chimique , Depsides/pharmacologie , Group II Phospholipases A2/composition chimique , Group II Phospholipases A2/toxicité , Mâle , Souris , Structure quaternaire des protéines ,RÉSUMÉ
Inflammation-mediated disorders are on the rise and hence, there is an urgent need for the design and synthesis of new anti-inflammatory drugs with higher affinity and specificity for their potential targets. The current study presents an effective and new protocol for the synthesis of thienyl-pyrazoles through 3â¯+â¯2 annulations using a recyclable heterogeneous catalyst Amberlyst-15. Chalcones 3(a-g) prepared from 3-methylthiophene-2-carbaldehyde and acetophenones by Claisen-Schmidt approach reacted with semicarbazide hydrochloride 4 in the presence of Amberlyst-15 in acetonitrile at room temperature producing thienyl-pyrazole carboxamides 5(a-h) in good yields. Alternatively, the compounds 5(a-h) were prepared by conventional method using acetic acid (30%) medium. Structures of synthesized new pyrazoles were confirmed by spectral and crystallographic studies. All the new compounds were evaluated for their in vitro inhibition of Phospholipase A2 from Vipera russelli and preliminary studies revealed that, amongst the designed series, compounds 5b, 5c and 5h showed promising inhibition. Further, the compounds exhibited linear mixed-type inhibition behavior for the sPLA2 enzyme indicating that they bind to an allosteric site distinct from either the calcium or substrate binding site on the enzyme. These kinetic conclusions were further validated by macromolecular rigid-body docking whereby compounds 5c and 5h showed binding to distinct pockets on the protein. These findings present a promising series of lead molecules that can serve as prototypes for the treatment of inflammatory related disorders.
Sujet(s)
Site allostérique/effets des médicaments et des substances chimiques , Daboia/métabolisme , Group II Phospholipases A2/métabolisme , Inhibiteurs de la phospholipase A2/composition chimique , Inhibiteurs de la phospholipase A2/pharmacologie , Pyrazoles/composition chimique , Pyrazoles/pharmacologie , Animaux , Anti-inflammatoires non stéroïdiens/synthèse chimique , Anti-inflammatoires non stéroïdiens/composition chimique , Anti-inflammatoires non stéroïdiens/pharmacologie , Catalyse , Conception de médicament , Group II Phospholipases A2/composition chimique , Simulation de docking moléculaire , Inhibiteurs de la phospholipase A2/synthèse chimique , Pyrazoles/synthèse chimique , Styrènes/composition chimiqueRÉSUMÉ
The present study describes the purification and partial characterization of a basic anticoagulant PLA2 enzyme named as Rv(i) PLA2 from the venom of Indian Daboia russelii. The molecular mass of the protein was found to be 13,659.65 Da, and peptide mass fingerprinting revealed that it belongs to group II PLA2 family. The peptide sequence showed similarity to uncharacterized basic PLA2 enzyme having an accession no. of P86368 reported from Sri Lankan D. russelii. Rv(i) PLA2 exhibited strong phospholipase A2 and anticoagulant activity. It also induced expression of COX-2 and TNF-α mRNA in a dose-dependent manner in phorbol 12-myristate 13-acetate differentiated THP-1 cells, which play a crucial role during inflammation. Chemical modification of His residue in Rv(i) PLA2 with p-bromophenacyl bromide abolished the enzymatic, anticoagulant, and inflammatory activities. The result indicates that the catalytic site of Rv(i) PLA2 might play a vital role in inducing inflammation at the bite site during D. russelii envenomation.
Sujet(s)
Anticoagulants/toxicité , Daboia , Group II Phospholipases A2 , Médiateurs de l'inflammation/métabolisme , Venins de vipère/enzymologie , Animaux , Anticoagulants/composition chimique , Anticoagulants/isolement et purification , Lignée cellulaire tumorale , Group II Phospholipases A2/composition chimique , Group II Phospholipases A2/isolement et purification , Group II Phospholipases A2/toxicité , Inflammation/induit chimiquement , Inflammation/métabolisme , Inflammation/anatomopathologie , Venins de vipère/composition chimiqueRÉSUMÉ
Secretory phospholipase A2 group IIA (sPLA2-IIA) is an enzyme that hydrolyzes the sn-2 ester bond in glyceroacyl phospholipids present in lipoproteins and cell membranes. As many immunohistochemical studies reveal that the high expression of sPLA2-IIA in the tumorous tissue or plasma of cancer patients, though low expression in other cases, the enzyme is considered highly relevant with cancer development. Effort has been made to establish the mechanism of how sPLA2- IIA is involved in various cancers in order to understand its pathogenic role and to utilize it as a target for cancer diagnosis and therapy. This article specifically reviews recent studies regarding the relevance of sPLA2-IIA with various cancers and reported inhibitors of the protein.
Sujet(s)
Antinéoplasiques/pharmacologie , Antienzymes/pharmacologie , Group II Phospholipases A2/métabolisme , Tumeurs/enzymologie , Animaux , Femelle , Group II Phospholipases A2/antagonistes et inhibiteurs , Group II Phospholipases A2/composition chimique , Humains , Mâle , Tumeurs/traitement médicamenteuxRÉSUMÉ
Human Group IIA phospholipase A2 (hGIIA) promotes inflammation in immune-mediated pathologies by regulating the arachidonic acid pathway through both catalysis-dependent and -independent mechanisms. The hGIIA crystal structure, both alone and inhibitor-bound, together with structures of closely related snake-venom-derived secreted phospholipase enzymes has been well described. However, differentiation of biological and nonbiological contacts and the relevance of structures determined from snake venom enzymes to human enzymes are not clear. We employed molecular dynamics (MD) and docking approaches to understand the binding of inhibitors that selectively or nonselectively block the catalysis-independent mechanism of hGIIA. Our results indicate that hGIIA behaves as a monomer in the solution environment rather than a dimer arrangement that is in the asymmetric unit of some crystal structures. The binding mode of a nonselective inhibitor, KH064, was validated by a combination of the experimental electron density and MD simulations. The binding mode of the selective pentapeptide inhibitor FLSYK to hGIIA was stipulated to be different to that of the snake venom phospholipases A2 of Daboia russelli pulchella (svPLA2 ). Our data suggest that the application of MD approaches to crystal structure data is beneficial in evaluating the robustness of conclusions drawn based on crystal structure data alone. Proteins 2017; 85:827-842. © 2016 Wiley Periodicals, Inc.
Sujet(s)
Électrons , Group II Phospholipases A2/antagonistes et inhibiteurs , Simulation de dynamique moléculaire , Oligopeptides/composition chimique , Acides pentanoïques/composition chimique , Inhibiteurs de la phospholipase A2/composition chimique , Motifs d'acides aminés , Animaux , Sites de fixation , Group II Phospholipases A2/composition chimique , Humains , Simulation de docking moléculaire , Phospholipases A2/composition chimique , Liaison aux protéines , Structure en hélice alpha , Structure en brin bêta , Motifs et domaines d'intéraction protéique , Venins de vipère/composition chimique , Viperidae/métabolismeRÉSUMÉ
Secretory phospholipase A2 (sPLA2) is a key enzyme participating in the inflammatory cascade followed by the action of cyclooxygenase-2 and lipoxygenases. Therefore, inhibitors of sPLA2 could be used as potent anti-inflammatory agents to treat the early phase of inflammation. In this study, we have prepared the fenoprofen and ibuprofen analogs containing 1,3,4-oxadiazole nucleus and tested against Vipera russelli venom's basic sPLA2 (VRV-PL-VIIIa). Among the tested ligands 5(a-t),2-(2-chlorophenyl)-5-(1-(4-phenoxyphenyl) ethyl)-1,3,4-oxadiazole (5m) inhibited the catalytic activity of VRV-PL-VIIIa with an IC50 value of 11.52 µM. Biophysical studies revealed that the 5m quenches the intrinsic fluorescence of VRV-PL-VIIIa, in a concentration dependent manner. Also, the compound 5m affected VRV-PL-VIIIa conformation, which was observed by circular dichroism spectra that recorded the prominent shift in the α-helix peak and the random coil formation of VRV-PL-VIIIa. Further, molecular docking analysis revealed that the compound 5m possess strong hydrophobic interactions at catalytic triad region of the VRV-PL-VIIIa. Evident to in vitro and in silico studies, 5m strongly inhibited the hemolysis of red blood cells. Our in vivo pharmacological studies revealed that the compound 5m inhibited the edematogenic activity of VRV-PL-VIIIa in mouse foot pad. Additionally, the 5m inhibited VRV-PL-VIIIa-induced myotoxicity and lung hemorrhage in mice. Overall, our ADMET results depicted that 5m possess better druggable property. Thus, this study explored the new fenoprofen and ibuprofen analog 5m as the lead-structure that serves as an anti-inflammatory agent.
Sujet(s)
Antienzymes , Fénoprofène , Group II Phospholipases A2 , Ibuprofène , Oxadiazoles , Animaux , Antienzymes/synthèse chimique , Antienzymes/composition chimique , Antienzymes/pharmacologie , Fénoprofène/analogues et dérivés , Fénoprofène/synthèse chimique , Fénoprofène/composition chimique , Fénoprofène/pharmacologie , Group II Phospholipases A2/antagonistes et inhibiteurs , Group II Phospholipases A2/composition chimique , Group II Phospholipases A2/toxicité , Hémolyse/effets des médicaments et des substances chimiques , Ibuprofène/analogues et dérivés , Ibuprofène/synthèse chimique , Ibuprofène/composition chimique , Ibuprofène/pharmacologie , Mâle , Souris , Simulation de docking moléculaire , Oxadiazoles/synthèse chimique , Oxadiazoles/composition chimique , Oxadiazoles/pharmacologie , Structure secondaire des protéinesRÉSUMÉ
Secreted phospholipase A2 type IIA (sPLA2-IIA) is a well-established pro-inflammatory protein and has been a major target for drug discovery. However, the mechanism of its signaling action has not been fully understood. We previously found that sPLA2-IIA binds to integrins αvß3 and α4ß1 in human and that this interaction plays a role in sPLA2-IIA's signaling action. Our recent studies found that sPLA2-IIA activates integrins in an allosteric manner through direct binding to a newly identified binding site of integrins (site 2), which is distinct from the classical RGD-binding site (site 1). The sPLA2-IIA-induced integrin activation may be related to the signaling action of sPLA2-IIA. Since sPLA2-IIA is present in normal human tears in addition to rheumatoid synovial fluid at high concentrations the sPLA2-IIA-mediated integrin activation on leukocytes may be involved in immune responses in normal and pathological conditions.
Sujet(s)
Group II Phospholipases A2/composition chimique , Intégrine alpha4bêta1/composition chimique , Intégrine alphaVbêta3/composition chimique , Transduction du signal/immunologie , Régulation allostérique , Polyarthrite rhumatoïde/génétique , Polyarthrite rhumatoïde/immunologie , Polyarthrite rhumatoïde/anatomopathologie , Sites de fixation , Régulation de l'expression des gènes , Group II Phospholipases A2/génétique , Group II Phospholipases A2/immunologie , Humains , Intégrine alpha4bêta1/génétique , Intégrine alpha4bêta1/immunologie , Intégrine alphaVbêta3/génétique , Intégrine alphaVbêta3/immunologie , Simulation de docking moléculaire , Liaison aux protéines , Structure en hélice alpha , Structure en brin bêta , Motifs et domaines d'intéraction protéique , Synovie/composition chimique , Synovie/immunologie , Larmes/composition chimique , Larmes/immunologieRÉSUMÉ
Snake venoms from the Viperidae and Elapidae families often have several phospholipases A2 (PLA2s), which may display different functions despite having a similar structural scaffold. These proteins are considered an important target for the development of drugs against local myotoxic damage because they are not efficiently neutralized by conventional serum therapy. PLA2s from these venoms are generally divided into two classes: (i) catalytic PLA2s (or Asp49-PLA2s) and (ii) non-catalytic PLA2-like toxins (or Lys49-PLA2s). In many Viperidae venoms, a subset of the basic Asp49-PLA2s displays some functional and structural characteristics of PLA2-like proteins and group within the same phylogenetic clade, but their myotoxic mechanism is still largely unknown. In the present study, we have crystallized and solved the structure of myotoxin I (MT-I), a basic myotoxic Asp49-PLA2 isolated from Bothrops asper venom. The structure presents a dimeric conformation that is compatible with that of previous dimers found for basic myotoxic Asp49-PLA2s and Lys49-PLA2s and has been confirmed by other biophysical and bioinformatics techniques. This arrangement suggests a possible cooperative action between both monomers to exert myotoxicity via two different sites forming a putative membrane-docking site (MDoS) and a putative membrane disruption site (MDiS). This mechanism would resemble that proposed for Lys49-PLA2s, but the sites involved appear to be situated in a different region. Thus, as both sites are close to one another, they form a "myotoxic cluster", which is also found in two other basic myotoxic Asp49-PLA2s from Viperidae venoms. Such arrangement may represent a novel structural strategy for the mechanism of muscle damage exerted by the group of basic, Asp49-PLA2s found in viperid snake venoms.
Sujet(s)
Venins de crotalidé/enzymologie , Group II Phospholipases A2/composition chimique , Phospholipases A2/composition chimique , Protéines de reptiles/composition chimique , Séquence d'acides aminés/génétique , Animaux , Bothrops , Venins de crotalidé/composition chimique , Venins de crotalidé/toxicité , Cristallographie aux rayons X , Group II Phospholipases A2/génétique , Group II Phospholipases A2/métabolisme , Group II Phospholipases A2/toxicité , Humains , Muscles squelettiques/composition chimique , Muscles squelettiques/effets des médicaments et des substances chimiques , Phospholipases A2/génétique , Phospholipases A2/toxicité , Phylogenèse , Protéines de reptiles/génétique , Protéines de reptiles/toxicitéRÉSUMÉ
Weight loss is a well known systemic manifestation of chronic obstructive pulmonary disease (COPD). A Gly80Ser mutation on human group IID secretory phospholipase A2 (sPLA2) enhances expression of the cytokines that are responsible for weight loss. In this study, we seek to establish a structural correlation of wild type sPLA2 and the Gly80Ser mutation with function. sPLA2 with glycine and serine at the 80th positions and the M-type receptor were modelled. The enzymes were docked to the receptor and molecular dynamics was carried out to 70 ns. Structural analysis revealed the enzymes to comprise three helices (H1-H3), two short helices (SH1 and SH2), and five loops including a calcium binding loop (L1-L5), and to be stabilized by seven disulfide bonds. The overall backbone folds of the two models are very similar, with main chain RMSD of less than 1 Å. The active site within the substrate binding channel shows a catalytic triad of water-His67-Asp112, showing a hydrogen bonded network. Major structural differences between wild type and mutant enzymes were observed locally at the site of the mutation and in their global conformations. These differences include: (1) loop-L3 between H2 and H3, which bears residue Gly80 in the wild type, is in a closed conformation with respect to the channel opening, while in the mutant enzyme it adopts a relatively open conformation; (2) the mutant enzyme is less compact and has higher solvent accessible surface area; and (3) interfacial binding contact surface area is greater, and the quality of interactions with the receptor is better in the mutant enzyme as compared to the wild type. Therefore, the structural differences delineated in this study are potential biophysical factors that could determine the increased potency of the mutant enzyme with macrophage receptor for cytokine secreting function, resulting in exacerbation of cachexia in COPD.
Sujet(s)
Group II Phospholipases A2/composition chimique , Modèles moléculaires , Broncho-pneumopathie chronique obstructive/complications , Broncho-pneumopathie chronique obstructive/enzymologie , Récepteurs à la phospholipase A2/composition chimique , Perte de poids/génétique , Sites de fixation , Group II Phospholipases A2/génétique , Humains , Simulation de dynamique moléculaire , Structure secondaire des protéines , Broncho-pneumopathie chronique obstructive/génétiqueRÉSUMÉ
Maslinic acid is a natural pentacyclic triterpenoid which has anti-inflammatory properties. A recent study showed that secretory phospholipase A2 (sPLA2) may be a potential binding target of maslinic acid. The human group IIA (hGIIA)-sPLA2 is found in human sera and their levels are correlated with severity of inflammation. This study aims to determine whether maslinic acid interacts with hGIIA-sPLA2 and inhibits inflammatory response induced by this enzyme. It is shown that maslinic acid enhanced intrinsic fluorescence of hGIIA-sPLA2 and inhibited its enzyme activity in a concentration-dependent manner. Molecular docking revealed that maslinic acid binds to calcium binding and interfacial phospholipid binding site, suggesting that it inhibit access of catalytic calcium ion for enzymatic reaction and block binding of the enzyme to membrane phospholipid. The hGIIA-sPLA2 enzyme is also responsible in mediating monocyte recruitment and differentiation. Results showed that maslinic acid inhibit hGIIA-sPLA2-induced THP-1 cell differentiation and migration, and the effect observed is specific to hGIIA-sPLA2 as cells treated with maslinic acid alone did not significantly affect the number of adherent and migrated cells. Considering that hGIIA-sPLA2 enzyme is known to hydrolyze glyceroacylphospholipids present in lipoproteins and cell membranes, maslinic acid may bind and inhibit hGIIA-sPLA2 enzymatic activity, thereby reduces the release of fatty acids and lysophospholipids which stimulates monocyte migration and differentiation. This study is the first to report on the molecular interaction between maslinic acid and inflammatory target hGIIA-sPLA2 as well as its effect towards hGIIA-sPLA2-induced THP-1 monocyte adhesive and migratory capabilities, an important immune-inflammation process in atherosclerosis.
Sujet(s)
Group II Phospholipases A2/antagonistes et inhibiteurs , Monocytes/cytologie , Triterpènes/pharmacologie , Calcium/métabolisme , Domaine catalytique/effets des médicaments et des substances chimiques , Différenciation cellulaire/effets des médicaments et des substances chimiques , Lignée cellulaire , Mouvement cellulaire/effets des médicaments et des substances chimiques , Relation dose-effet des médicaments , Régulation de l'expression des gènes codant pour des enzymes , Group II Phospholipases A2/composition chimique , Humains , Simulation de docking moléculaire , Monocytes/effets des médicaments et des substances chimiques , Monocytes/enzymologieRÉSUMÉ
Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 µM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer's disease.
Sujet(s)
Enzymes de conversion de l'endothéline/métabolisme , Group II Phospholipases A2/composition chimique , Group II Phospholipases A2/pharmacologie , Néprilysine/métabolisme , Protéines de reptiles/composition chimique , Protéines de reptiles/pharmacologie , Alanine/métabolisme , Séquence d'acides aminés , Activation enzymatique/effets des médicaments et des substances chimiques , Dosages enzymatiques , Cellules HEK293 , Humains , Cinétique , Peptides/composition chimique , Peptides/métabolisme , Domaines protéiques , Relation structure-activitéRÉSUMÉ
Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.
Sujet(s)
Sérums antivenimeux , Bothrops , Venins de crotalidé , Group II Phospholipases A2 , Simulation de docking moléculaire , Anticorps à chaîne unique , Animaux , Sérums antivenimeux/composition chimique , Sérums antivenimeux/génétique , Sérums antivenimeux/immunologie , Camélidés du Nouveau Monde/génétique , Camélidés du Nouveau Monde/immunologie , Venins de crotalidé/composition chimique , Venins de crotalidé/immunologie , Venins de crotalidé/toxicité , Group II Phospholipases A2/composition chimique , Group II Phospholipases A2/immunologie , Group II Phospholipases A2/toxicité , Mâle , Souris , Anticorps à chaîne unique/composition chimique , Anticorps à chaîne unique/génétique , Anticorps à chaîne unique/immunologieRÉSUMÉ
Snake venoms are composed of approximately 90% of proteins with several pharmacological activities having high potential in research as biological tools. One of the most abundant compounds is phospholipases A2 (PLA2), which are the most studied venom protein due to their wide pharmacological activity. Using a combination of chromatographic steps, a new PLA2 K49 was isolated and purified from the whole venom of the Bothriopsis taeniata and submitted to analyses mass spectrometry. An automatic "de novo" sequencing of this new PLA2 K49 denominated Btt-TX was performed using Peaks Studio 6 for analysis of the spectra. Additionally, a triplex approach CID/HCD/ETD has been performed, to generate higher coverage of the sequence of the protein. Structural studies correlating biological activities were made associating specific Btt-TX regions and myotoxic activity. Lysine acetylation was performed to better understand the mechanism of membrane interaction, identifying the extreme importance of the highly hydrophobic amino acids L, P and F for disruption of the membrane. Our myotoxical studies show a possible membrane disruption mechanism by Creatine Kinase release without a noticeable muscle damage, that probably occurred without phospholipid hydrolyses, but with a probable penetration of the hydrophobic amino acids present in the C-terminal region of the protein.
Sujet(s)
Group II Phospholipases A2/composition chimique , Group II Phospholipases A2/toxicité , Muscles squelettiques/effets des médicaments et des substances chimiques , Analyse de séquence de protéine/méthodes , Venins de vipère/composition chimique , Viperidae/métabolisme , Séquence d'acides aminés , Animaux , Relation dose-effet des médicaments , Souris , Données de séquences moléculaires , Relation structure-activité , Venins de vipère/toxicitéRÉSUMÉ
One of the main challenges in toxicology today is to develop therapeutic alternatives for the treatment of snake venom injuries that are not efficiently neutralized by conventional serum therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a fundamental role in skeletal muscle necrosis, which can result in permanent sequelae and disability. This leads to economic and social problems, especially in developing countries. In this work, we performed structural and functional studies with Piratoxin-I, a Lys49-PLA2 from Bothropspirajai venom, complexed with two compounds present in several plants used in folk medicine against snakebites. These ligands partially neutralized the myotoxic activity of PrTX-I towards binding on the two independent sites of interaction between Lys49-PLA2 and muscle membrane. Our results corroborate the previously proposed mechanism of action of PLA2s-like and provide insights for the design of structure-based inhibitors that could prevent the permanent injuries caused by these proteins in snakebite victims.