Effects of allergic diseases and age on the composition of serum IgG glycome in children

Acknowledgements Glycan analysis was partly supported by European Commission GlycoBioM (contract #259869), IBD-BIOM (contract #305479), HighGlycan (contract #278535), MIMOmics (contract #305280), HTP-GlycoMet (contract #324400) and IntegraLife (contract #315997) grants. The SEATON cohort was partly funded by the UK Medical Research Council (contract #80219) and Asthma UK (contract #00/011 and 02/017) grants.Peer reviewedPublisher PD

Inactivation of the glutathione peroxidase GPx4 by the ferroptosis-inducing molecule RSL3 requires the adaptor protein 14-3-3 epsilon

RSL3, a drug candidate prototype for cancer chemotherapy, triggers ferroptosis by inactivating GPx4. Here we report the purification of the protein indispensable for GPx4 inactivation by RSL3. MS analysis reveals 14-3-3 isoforms as candidates and recombinant human 14-3-3epsilon confirms the identification. The function of 14-3-3\uf065 is redox-regulated. Moreover, overexpression and silencing of the gene coding for 14-3-3\uf065 consistently control the inactivation of GPx4 by RSL3. The interaction of GPx4 with a redox-regulated adaptor protein, operating in cell signalling, further contributes to frame it within redox-regulated pathways of cell survival and death and opens new therapeutic perspectives

Defining the genetic control of human blood plasma N-glycome using genome-wide association study

Glycosylation is a common post-translational modification of proteins. Glycosylation is associated with a number of human diseases. Defining genetic factors altering glycosylation may provide a basis for novel approaches to diagnostic and pharmaceutical applications. Here we report a genome-wide association study of the human blood plasma N-glycome composition in up to 3811 people measured by Ultra Performance Liquid Chromatography (UPLC) technology. Starting with the 36 original traits measured by UPLC, we computed an additional 77 derived traits leading to a total of 113 glycan traits. We studied associations between these traits and genetic polymorphisms located on human autosomes. We discovered and replicated 12 loci. This allowed us to demonstrate an overlap in genetic control between total plasma protein and IgG glycosylation. The majority of revealed loci contained genes that encode enzymes directly involved in glycosylation (FUT3/FUT6, FUT8, B3GAT1, ST6GAL1, B4GALT1, ST3GAL4, MGAT3 and MGAT5) and a known regulator of plasma protein fucosylation (HNF1A). However, we also found loci that could possibly reflect other more complex aspects of glycosylation process. Functional genomic annotation suggested the role of several genes including DERL3, CHCHD10, TMEM121, IGH and IKZF1. The hypotheses we generated may serve as a starting point for further functional studies in this research area

Unraveling the mechanism of ferroptosis

Ferroptosis, a form of regulated cell death (RCD), has been recently reported to be primed by missing activity of the ubiquitous selenoperoxidase glutathione peroxidase 4 (GPx4). GPx4 catalyzes a glutathione (GSH)-dependent reduction of membrane hydroperoxides to corresponding alcohols. Since ferroptosis is executed by lipid peroxidation, oxygen, polyunsaturated fatty acids (PUFA), and iron are necessary constraints. Furthermore, since phospholipid hydroperoxides (PL-OOH) have a key role in membrane lipid peroxidation –i.e. their decomposition generates radicals in the membranes- GPx4-mediated regulation of ferroptosis is consistent with both, the mechanisms of lipid peroxidation and protection which have been finely elucidated in vitro in the last forty years of the XX century. In this study, we used as a suitable model of ferroptotic cell death, that primed by erastin in HT1080 cells. Erastin, by preventing cystine import, in these cells decreases the cellular level of GSH and, consequently, GPx4 activity. On this model, we disclosed a critical constraint permitting membrane lipid oxidation and therefore ferroptosis execution. Unexpectedly, we observed that modulating the electron flow through the mitochondria respiratory chain, is indeed unsuccessful in modulating ferroptosis sensitivity. For this indispensable function we identified, instead, the activity of mitochondrial alpha-keto acid dehydrogenases. The deduced mechanism we propose is the formation of O2•- during re-oxidation of dihydrolipoate in the last step of the oxidative decarboxylation, a reaction, already observed in isolated mitochondria. In relation to ferroptosis, we propose that it is the HO2• that generates a carbon centered radical in a PUFA, which, following oxygen addiction and stabilization by hydrogen transfer, forms a PL-OOH. We also studied the mechanisms underlying the inhibition of GPx4 by a prototype of electrophile: (1S,3R) RSL3. This study allowed to disclose the protein 14-3-3 epsilon as an adaptor protein necessary to achieve GPx4 inactivation by (1S,3R) RSL3. By these observations, we clarified the requirements for GPx4 inactivation by electrophiles, which, in vivo is supposed to have a regulatory role. Further, we provided evidence that the ‘inactivation-permitting activity’ of 14-3-3 epsilon on GPx4 is redox regulated by thiol-disulphide transition. This links inhibition by electrophiles to intracellular redox status. This set of new information contributes to the view of cell death by ferroptosis as intrinsically connected to aerobic life through a fine modulation of specific metabolic events. It is an amazing thought that electron transitions between oxygen and iron permits in aerobic organism both, life and death. The latter, therefore, comes up as a fundamental component of aerobic life

Lack of glutathione peroxidase-8 in the ER impacts on lipid composition of HeLa cells microsomal membranes

GPx8 is a glu\u200btathione per\u200box\u200bi\u200bdase ho\u200bmolog in\u200bserted in the mem\u200bbranes of en\u200bdo\u200bplas\u200bmic retic\u200bu\u200blum (ER), where it seem\u200bingly plays a role in con\u200btrol\u200bling re\u200bdox sta\u200btus by pre\u200bvent\u200bing the spill of H2O2. We ad\u200bdressed the im\u200bpact of GPx8 si\u200blenc\u200bing on the lipidome of mi\u200bcro\u200bso\u200bmal mem\u200bbranes, us\u200bing sta\u200bbly GPx8-si\u200blenced HeLa cells. The two cell lines were clearly sep\u200ba\u200brated by Prin\u200bci\u200bpal Com\u200bpo\u200bnent Analy\u200bsis (PCA) and Par\u200btial Least Square Dis\u200bcrim\u200bi\u200bnant analy\u200bsis (PLS-DA) of lipidome. Con\u200bsid\u200ber\u200bing in de\u200btail the in\u200bdi\u200bvid\u200bual lipid classes, we ob\u200bserved that un\u200bsat\u200bu\u200brated glyc\u200berophos\u200bpho\u200blipids (GPL) de\u200bcreased, while only in phos\u200bphatidyli\u200bnos\u200bi\u200btols (PI) a sub\u200bsti\u200btu\u200btion of mo\u200bnoun\u200bsat\u200bu\u200brated fatty acids (MUFA) for polyun\u200bsat\u200bu\u200brated fatty acids (PUFA) was ob\u200bserved. Among sphin\u200bgolipids (SL), ce\u200bramides (CER) de\u200bcreased while sphin\u200bgomyelins (SM) and neu\u200btral gly\u200bcoph\u200bin\u200b! golipids (nGSL) in\u200bcreased. Here, in ad\u200bdi\u200btion, longer chains than in con\u200btrols in the amide fatty acid were pre\u200bsent. The in\u200bcrease up to four folds of the CER (d18:1; c24:0) con\u200btain\u200bing three hex\u200bose units, was the most re\u200bmark\u200bable species in\u200bcreas\u200bing in the dif\u200bfer\u200ben\u200btial lipidome of siG\u200bPx8 cells. Quan\u200bti\u200bta\u200btive RT-PCR com\u200bplied with lipidomic analy\u200bsis specif\u200bi\u200bcally show\u200bing an in\u200bcreased ex\u200bpres\u200bsion of: i) acyl-CoA syn\u200bthetase 5 (AC\u200bSL5); ii) CER syn\u200bthase 2 and 4; iii) CER trans\u200bporter (CERT); iv) UDP-glu\u200bco\u200bsyl trans\u200bferase (UDP-GlcT), as\u200bso\u200bci\u200bated to a de\u200bcreased ex\u200bpres\u200bsion of UDP-galac\u200bto\u200bsyl trans\u200bferase (UDP-GalT). A role of the un\u200bfolded pro\u200btein re\u200b! sponse (UPR) and the spliced form of the tran\u200bscrip\u200btion fac\u200btor XBP1 on the tran\u200bscrip\u200btional changes of GPx8 si\u200blenced cells was ruled-out. Sim\u200bi\u200blarly, also the in\u200bvolve\u200bment of Nrf2 and NF-!B. Al\u200bto\u200bgether our re\u200bsults in\u200bdi\u200b! cate that GPx8-si\u200blenc\u200bing of HeLa yields a mem\u200bbrane de\u200bpleted by about 24% of polyun\u200bsat\u200bu\u200brated GPL and a cor\u200bre\u200bspond\u200bing in\u200bcrease of sat\u200bu\u200brated or mo\u200bnoun\u200bsat\u200bu\u200brated SM and spe\u200bci$c nGSL. This is ten\u200bta\u200btively in\u200bter\u200b! preted as an adap\u200btive mech\u200ba\u200bnism lead\u200bing to an in\u200bcreased re\u200bsis\u200btance to rad\u200bi\u200bcal ox\u200bi\u200bda\u200btions. More\u200bover, the marked shift of fatty acid com\u200bpo\u200bsi\u200btion of PI emerges as a pos\u200bsi\u200bbly rel\u200be\u200bvant is\u200bsue in re\u200bspect to the im\u200bpact of GPx8 on sig\u200bnal\u200bing path\u200bways