Secreted phospholipases A2 in hereditary angioedema with C1-inhibitor deficiency

BackgroundHereditary angioedema (HAE) caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein (C1-INH-HAE) is a disabling, potentially fatal condition characterized by recurrent episodes of swelling. We have recently found that patients with C1-INH-HAE have increased plasma levels of vascular endothelial growth factors and angiopoietins (Angs), which have been associated with vascular permeability in several diseases. Among these and other factors, blood endothelial cells and vascular permeability can be modulated by extracellular or secreted phospholipases A2 (sPLA2s).ObjectiveWe sought to investigate the enzymatic activity and biological functions of sPLA2 in patients with C1-INH-HAE.MethodssPLA2s enzymatic activity was evaluated in the plasma from 109 adult patients with C1-INH-HAE and 68 healthy donors in symptom-free period and attacks. Plasma level of group IIA sPLA2 (hGIIA) protein was measured in selected samples. The effect of C1-INH-HAE plasma on endothelial permeability was examined in vitro using a vascular permeability assay. The role of hGIIA was determined using highly specific sPLA2 indole inhibitors. The effect of recombinant hGIIA on C1-INH activity was examined in vitro by functional assay.ResultsPlasma sPLA2 activity and hGIIA levels are increased in symptom-free C1-INH-HAE patients compared with controls. sPLA2 activity negatively correlates with C1-INH protein level and function. C1-INH-HAE plasma increases endothelial permeability in vitro, and this effect is partially reverted by a specific hGIIA enzymatic inhibitor. Finally, recombinant hGIIA inhibits C1-INH activity in vitro.ConclusionsPLA2 enzymatic activity (likely attributable to hGIIA), which is increased in C1-INH-HAE patients, can promote vascular permeability and impairs C1-INH activity. Our results may pave the way for investigating the functions of sPLA2s (in particular, hGIIA) in the pathophysiology of C1-INH-HAE and may inform the development of new therapeutic targets

PplD Is a De-N-Acetylase of the Cell Wall Linkage Unit of Streptococcal Rhamnopolysaccharides

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins

Lack of group X secreted phospholipase A₂ increases survival following pandemic H1N1 influenza infection

The role of Group X secreted phospholipase A2 (GX-sPLA2) during influenza infection has not been previously investigated. We examined the role of GX-sPLA2 during H1N1 pandemic influenza infection in a GX-sPLA2 gene targeted mouse (GX−/−) model and found that survival after infection was significantly greater in GX−/− mice than in GX+/+ mice. Downstream products of GX-sPLA2 activity, PGD2, PGE2, LTB4, cysteinyl leukotrienes and Lipoxin A4 were significantly lower in GX−/− mice BAL fluid. Lung microarray analysis identified an earlier and more robust induction of T and B cell associated genes in GX−/− mice. Based on the central role of sPLA2 enzymes as key initiators of inflammatory processes, we propose that activation of GX-sPLA2 during H1N1pdm infection is an early step of pulmonary inflammation and its inhibition increases adaptive immunity and improves survival. Our findings suggest that GX-sPLA2 may be a potential therapeutic target during influenza

Repression of PLA2R1 by c-MYC and HIF-2alpha promotes cancer growth

Loss of secreted phospholipase A2 receptor (PLA2R1) has recently been found to render human primary cells more resistant to senescence whereas increased PLA2R1 expression is able to induce cell cycle arrest, cancer cell death or blockage of cancer cell transformation in vitro, suggesting that PLA2R1 displays tumor suppressive activities. Here we report that PLA2R1 expression strongly decreases in samples of human renal cell carcinoma (RCC). Knockdown of PLA2R1 increases renal cancer cell tumorigenicity supporting a role of PLA2R1 loss to promote in vivo RCC growth. Most RCC result from Von Hippel-Lindau (VHL) tumor suppressor loss-of-function and subsequent gain-of-function of the oncogenic HIF-2alpha/c-MYC pathway. Here, by genetically manipulating VHL, HIF-2alpha and c-MYC, we demonstrate that loss of VHL, stabilization of HIF-2alpha and subsequent increased c-MYC activity, binding and transcriptional repression, through induction of PLA2R1 DNA methylation closed to PLA2R1 transcriptional start site, results in decreased PLA2R1 transcription. Our results describe for the first time an oncogenic pathway leading to PLA2R1 transcriptional repression and the importance of this repression for tumor growth

The Free Fatty Acid-Binding Pocket is a Conserved Hallmark in Pathogenic β-Coronavirus Spike Proteins from SARS-CoV to Omicron

As coronavirus disease 2019 (COVID-19) persists, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) emerge, accumulating spike (S) glycoprotein mutations. S receptor binding domain (RBD) comprises a free fatty acid (FFA)–binding pocket. FFA binding stabilizes a locked S conformation, interfering with virus infectivity. We provide evidence that the pocket is conserved in pathogenic β-coronaviruses (β-CoVs) infecting humans. SARS-CoV, MERS-CoV, SARS-CoV-2, and VOCs bind the essential FFA linoleic acid (LA), while binding is abolished by one mutation in common cold–causing HCoV-HKU1. In the SARS-CoV S structure, LA stabilizes the locked conformation, while the open, infectious conformation is devoid of LA. Electron tomography of SARS-CoV-2–infected cells reveals that LA treatment inhibits viral replication, resulting in fewer deformed virions. Our results establish FFA binding as a hallmark of pathogenic β-CoV infection and replication, setting the stage for FFA-based antiviral strategies to overcome COVID-19

Molecular and functional characterization of polymorphisms in the secreted phospholipase A2 group X gene: relevance to coronary artery disease

Among secreted phospholipases A2 (sPLA2s), human group X sPLA2 (hGX sPLA2) is emerging as a novel attractive therapeutic target due to its implication in inflammatory diseases. To elucidate whether hGX sPLA2 plays a causative role in coronary artery disease (CAD), we screened the human PLA2G10 gene to identify polymorphisms and possible associations with CAD end-points in a prospective study, AtheroGene. We identified eight polymorphisms, among which, one non-synonymous polymorphism R38C in the propeptide region of the sPLA2. The T-512C polymorphism located in the 5′ untranslated region was associated with a decreased risk of recurrent cardiovascular events during follow-up. The functional analysis of the R38C polymorphism showed that it leads to a profound change in expression and activity of hGX sPLA2, although there was no detectable impact on CAD risk. Due to the potential role of hGX sPLA2 in inflammatory processes, these polymorphisms should be investigated in other inflammatory diseases

La phospholipase A2 sécrétée de groupe X (maturation protéolytique et rôles fonctionnels)

Les phospholipases A2 constituent une superfamille de protéines comprenant au moins onze phospholipases A2 sécrétées (sPLA2) et douze phospholipases A2 intracellulaires. Ces protéines catalysent l hydrolyse des phospholipides en position sn-2, libérant un acide gras et un lysophospholipide. Elles contrôlent ainsi la production d une variété de médiateurs lipidiques qui sont importants pour de multiples fonctions cellulaires dans différents contextes physiologiques ou physiopathologiques (maladies inflammatoires et cancer). La sPLA2 de groupe X a été clonée au laboratoire en 1997 et possède des propriétés moléculaires uniques. Son ARN messager est présent dans différents tissus, mais semble peu régulé par des stimuli proinflammatoires. L enzyme est unique dans sa capacité à libérer des médiateurs lipidiques à partir des phopholipides cellulaires ou des lipoprotéines et possède aussi des propriétés antimicrobiennes variées. Un élément clé de la régulation fonctionnelle de la sPLA2 de groupe X est vraisemblablement lié à la présence d un propeptide dans sa partie N-terminale. Le lieu de la maturation protéolytique de sPLA2 de groupe X (dans la cellule avant sécrétion ou à l extérieur après sécrétion du proenzyme), les protéases impliquées et la régulation de cette maturation dans des conditions physiologiques ou physiopathologiques sont inconnus. Le travail de cette thèse a permis de mieux comprendre comment peut s effectuer la maturation de la sPLA2 de groupe X et quels sont ses rôles physiologiques et physiopathologiques. Concernant la maturation, nos études in vitro sur protéines purifiées et en cellules transfectées (HEK293) ont permis de montrer qu une protéase de type furine contribue de façon majeure à l activation de l enzyme, vraisemblablement au cours de sa sécrétion. Nos travaux suggèrent aussi qu une maturation est possible par d autres protéases et dans le milieu extracellulaire comme par exemple dans les cellules LOVO. Nous avons aussi tenté de mettre en évidence cette maturation dans des tissus murins dans certaines conditions physiologiques et physiopathologiques. Nous avons notamment trouvé que la sPLA2-X était la sPLA2 majeure présente dans l acrosome des spermatozoïdes. Enfin nous avons observé un polymorphisme présent dans le propeptide de la sPLA2 humaine qui conduit à la formation d une protéine inactive et rapidement dégradée. Dans la deuxième partie de cette thèse, nous avons montré que la forme active de la sPLA2 de groupe X, mais pas son proenzyme était capable i) de stimuler la prolifération cellulaire dans un contexte de cancer colorectal, ii) d exercer une action toxique contre le parasite de la malaria P. falciparum lors de l infection de globules rouges humains, et iii) de contrôler la réaction acrosomique des spermatozoïdes de souris, avec un impact important sur le taux de fécondité dans des tests de fécondation in vitro.NICE-BU Sciences (060882101) / SudocSudocFranceF

Lipids play music at the cellular membrane: From membranes dynamics to signaling via lipid mediators, vesicles and lipid droplets

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