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A5. Oxidativ modifikation af lipider - Biologi

A5. Oxidativ modifikation af lipider - Biologi


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Figur: Oxidativ modifikation af lipider

De indledende stadier af kardiovaskulær sygdom synes at involvere udviklingen af ​​fedtsyrestriber under arterievæggene. Makrofager, en immuncelle, har receptorer, som ser ud til at genkende oxiderede lipoproteiner i blodet, som de optager. Cellerne bliver så til fedtholdige skumceller, som danner striberne. Oxidation af fedtsyrer i lipoproteiner (muligvis af ozon) kan producere lipidperoxider og til proteinoxidation i lipoproteiner. Kortikale neuroner fra føtale Downs Syndrom-patienter viser 3-4 gange niveauer af intracellulære reaktive O2-arter og øgede niveauer af lipidperoxidation sammenlignet med kontrolneuroner. Denne skade forhindres ved behandling af neuronerne i kultur med frie radikaler eller katalase.

Figur: oxideret LDL-optagelse

En nylig undersøgelse af peroxiredoxiner af Neumann et al. viste vigtigheden af ​​disse genprodukter i mus. Peroxidredoxiner (som katalyserer omdannelsen af ​​peroxider og thioredoxin til vand og oxideret thioredoxin) er små proteiner med et aktivt sted cystein og findes i de fleste organismer. Transskription af pattedyrperoxiredoxin 1-genet aktiveres af oxidativt stress. De inaktiverede genet, som producerede en mus, der kunne formere sig og virkede vital, men som havde en forkortet levetid. Disse mus udviklede svær hæmolytisk anæmi og flere typer kræft. Høje niveauer af reaktive oxygenarter og deraf følgende øgede niveauer af oxiderede proteiner blev fundet i røde blodlegemer hos knockout-musene med anæmi. Høje niveauer af 8-oxoguanin, som følge af oxidativ beskadigelse af DNA, blev fundet i tumorceller.


OP44 - Systembiologi af oxidativ stress: første indsigt i lipidoxidation og proteinmodifikation krydstaler

Nyere forskning viser, at oxidativt stress (OS) påvirker alle niveauer af cellulær organisation og inducerer forstyrrelse i genekspression, epigenetisk regulering, mRNA- og proteinniveauer og cellulær metabolisme. Oxidative modifikationer af proteiner og lipider repræsenterer et andet niveau af kompleksitet i cellulær regulering understøttet af talrige undersøgelser, der viser, hvordan disse modifikationer regulerer genekspression og cellulære signalveje. Det er dog næsten umuligt at specificere input og (pato)fysiologiske konsekvenser af OS ved hjælp af data fra et enkelt modifikationsniveau. I dag er det klart, at kun en holistisk tilgang, som foreslået af systembiologi, vil give et integreret syn på OS-relateret cellulær regulering og patologier ved at overveje alle kendte OS-determinanter, identificere nye og etablere funktionelle forbindelser mellem dem.

Som et første forsøg på at studere OS via redox-systembiologi anvendte vi en multi-omics-tilgang til at karakterisere virkningen af ​​OS på kardiomyocytter (CM) ved hjælp af en dynamisk model for nitrosativ stress. Reaktive carbonylerede lipider, hydroxylerede og trunkerede phospholipider, nitrerede fedtsyrer og oxysteroler blev kvantificeret i forhold til en kontrol i CM-lipidekstrakter efter 15, 30, 70 min og 16 timers OS. Samtidig blev proteinfraktionen analyseret for en lang række post-translationelle modifikationer, såsom phosphorylering, carbonylering, cysteinoxidation og nitrosylering. Omics-data blev kombineret og understøttet af biokemiske og mikroskopiske undersøgelser af oxidationsdynamik, rumlig fordeling og funktionelle effekter. Kombinationen af ​​lipidomik, datadrevet proteomik og systembiologi integration tillod således identifikation af mere end 200 proteiner modificeret af reaktive lipidperoxidationsprodukter, hvoraf mange var involveret i calciumsignalveje, regulering af actincytoskelet, fokal adhæsion og phosphatidylinositol signalsystem. Biokemiske og mikroskopiske undersøgelser bekræftede yderligere OS-afledt svækkelse af Ca-signalering og cytoskeletproteinfordeling.


Lipidperoxidation

Måling af lipidperoxidation

Lipidperoxidation kan bestemmes kvantitativt eller kvalitativt ved en række forskellige metoder. Det kan måles ved tab af fedtsyrer mængder af primære peroxidationsprodukter mængder af sekundære produkter såsom carbonyler og kulbrintegasser og reduktion i antioxidantaktivitet. Nogle af de almindeligt anvendte metoder er beskrevet nedenfor. Analyse af fedtsyrer ved gasvæskekromatografi (GLC) eller højtydende væskekromatografi (HPLC) bruges til at måle tabet af umættede fedtsyrer, en konsekvens af lipidperoxidation. Lipidhydroperoxider, det primære produkt af peroxidation, kan måles direkte ved HPLC med kemiluminescensdetektorer. Jodfrigørelse og glutathionperoxidasemetoder bruges ofte til måling af lipidperoxider. Lipidperoxider oxiderer I - til I2 for titrering med thiosulfat og dermed forbrug af thiosulfat indikerer indirekte mængden af ​​lipidperoxider. Hydrogenperoxider og hydroperoxider oxiderer reduceret GSH til GSSG. Tilsætningen af ​​glutathionreduktase og NADPH reducerer GSSG tilbage til GSH, hvilket kræver indtagelse af NADPH, som kan relateres til peroxidindhold. Spin-fælder (phenyl-t-butylnitron) bruges ofte til at fange mellemradikaler. Produkter af lipidperoxidnedbrydning, såsom kulbrintegasser og cytotoksiske aldehyder, kan måles ved GLC eller HPLC. Lipidperoxidationsprodukter kan forårsage skade på DNA, og dannelse af 8-oxo-2'-deoxyguanosin (8oxodG) er en markør for oxidativ skade på DNA. Indhold af 8oxodG i DNA kan kvantificeres ved HPLC med en EC-detektor og ved en immunokemisk metode (ELISA).

De mest populære assays til måling af lipidperoxidation er thiobarbitursyre (TBA)-testen og dienkonjugationsbestemmelse. I TBA-testen opvarmes lipidholdige prøver med TBA og et LP-produkt, malondialdehyd (MDA) ved lav pH for at tillade dannelsen af ​​et lyserødt kompleks (se reaktion nedenfor). Densiteten af ​​farven er relateret til omfanget af lipidperoxidation. På grund af sin enkelhed og økonomi bruges denne metode i vid udstrækning i in vivo og in vitro indstillinger. Under processen med lipidperoxidation dannes dienkonjugationer (en dobbeltbinding-enkeltbinding-dobbeltbindingsstruktur) (se enzymkatalyseret peroxidation), som absorberer ultraviolet (UV) lys i bølgelængdeområdet 230-235 nm. Absorptionen af ​​UV-lys ved denne bølgelængde kan relateres til indholdet af dienkonjugater i lipidekstrakter af væv og dermed graden af ​​lipidperoxidation.

Andre nyudviklede, rutinemæssigt anvendte metoder er 4-hydroxynonenal assay og 8-iso-prostaglandin F2a assay. Elisa-kits bruges nu mest til at detektere forskellige lipidperoxidationsaddukter. Disse sæt er let tilgængelige på markedet, er ret specifikke og giver nøjagtige resultater. Da både MDA og hydroxynonenal (HNE) har vist sig at være i stand til at binde til proteiner og danne stabile addukter, betragtes de også som avancerede lipidperoxidationsslutprodukter (ALE'er). Ethanal, propanal, hexanal, glyoxal, methylglyoxal, 4-hydroxy-2-hexenal, acrolein, formaldehyd og acetaldehyd betragtes som ALE'er. Disse er reaktive carbonylforbindelser (RCC'er) og sukkerglycoxidationsprodukter (kaldet avancerede glycation-slutprodukter), der normalt akkumuleres med aldring og oxidativ stress-relaterede sygdomme såsom åreforkalkning, diabetes eller neurodegenerative sygdomme. RCC'er inducerer 'carbonylstress' karakteriseret ved dannelsen af ​​addukter og tværbindinger på proteiner, hvilket gradvist fører til proteinfejl og skader i alle væv og patologiske konsekvenser, herunder cytotoksicitet, celledysfunktion, inflammation og apoptotisk celledød. Yderligere interaktion med proteiner af disse ALE'er kan forårsage både strukturelle og funktionelle ændringer af oxiderede proteiner. Specifikt kan 4-HNE reagere med lysin-, histidin- eller cysteinrester i protein for at danne addukter. Den enzymimmunoassay, der er udviklet, er til hurtig påvisning og kvantificering af disse addukter. I disse assays bestemmes mængden af ​​addukt i proteinprøver ved at sammenligne dets absorbans med absorbansen for en kendt addukt - BSA-standardkurve.

LDL-kolesterol, generelt omtalt som dårligt kolesterol, er endnu farligere, når det bliver oxideret. Oxideret LDL (OxLDL) er mere reaktivt med omgivende væv og kan samle sig i arteriernes indre beklædning. Makrofager, kolesterol og andre lipider kan ophobes på stedet (aterosklerose), og i sidste ende danner en plak, der kan føre til hjerteanfald, slagtilfælde eller død. LDL-oxidation påvirker både lipid- og proteinkomponenterne i LDL. Reaktive aldehydprodukter dannet under oxidationen af ​​PFA'er, såsom MDA og 4-HNE, er i stand til at binde sig kovalent til e-aminogrupperne af lysinrester i ApoB-100 for at danne MDA-Lys og HNE-Lys addukter (MDA-LDL) og HNE-LDL). Avanceret glykosylering såsom dannelsen af ​​CML-LDL og CEL-LDL er også involveret i LDL-oxidation. Enzymimmunoassayet er udviklet til påvisning og kvantificering af humant OxLDL i plasma-, serum- eller andre biologiske væskeprøver. Sættet, som er let tilgængeligt, indeholder en kobberoxideret LDL-standard.

Historisk er måling af MDA ved TBARS (thiobarbitursyrereaktive stoffer) assay blevet brugt hyppigt til at bestemme omfanget af LP. Det betragtes som uspecifikt og er generelt dårligt, når det anvendes på biologiske prøver. Nylige assays er baseret på måling af MDA- eller HNE-lysinaddukter. Disse vil sandsynligvis være mere anvendelige til biologiske prøver, da addukter af disse reaktive aldehyder er relativt stabile. Opdagelsen af ​​isoprostanerne som lipidperoxidationsprodukter, målt ved GCMS eller immunoassay, har åbnet en ny vej for indirekte kvantificering af lipidperoxidation in vivo.


Korrelation mellem lipoprotein(a) og lipidperoxidation ved psoriasis: enzymets paraoxonase-1 rolle

Baggrund: Psoriasis er en kronisk, inflammatorisk hudsygdom forbundet med unormal plasmalipidmetabolisme og med en høj frekvens af kardiovaskulære hændelser. Ændringer af plasmalipider og en stigning i niveauerne af biokemiske markører for lipidperoxidation er blevet rapporteret hos personer med psoriasis, hvilket tyder på en sammenhæng mellem psoriasis, lipoproteiner og oxidativ skade.

Mål: For yderligere at undersøge forholdet mellem lipoproteiner og oxidativt stress ved psoriasis.

Metode: Niveauerne af plasmalipider, lipoprotein(a) [Lp(a)] og markører for lipidperoxidation blev evalueret i forsøgspersoner med psoriasis (n=23) og i kontroller (n=25). Hos de samme forsøgspersoner blev aktiviteten af ​​paraoxonase-1 (PON1), en antioxidant og et antiinflammatorisk enzym forbundet med high-density lipoproteiner, undersøgt.

Resultater: Resultaterne viste højere niveauer af Lp(a) i serum fra patienter med psoriasis sammenlignet med kontroller (P<0·001). Højere niveauer af lipidhydroperoxider (P<0·001) og lavere PON1-aktivitet blev observeret i serum fra patienter sammenlignet med raske forsøgspersoner, hvilket bekræfter, at psoriasis er forbundet med oxidativt stress. Ubalancen mellem oxidativt stress og antioxidantenzymer og stigningen i Lp(a)-serumniveauer var relateret til omfanget og sværhedsgraden af ​​psoriasis. Endelig viste vores resultater, at Lp(a)-niveauer var positivt korreleret med markører for lipidperoxidation og negativt relateret til PON1-aktivitet, hvilket tyder på, at forsøgspersoner med højere niveauer af Lp(a) er mere udsat for oxidativ skade.

Konklusioner: Vores resultater giver yderligere bevis for, at oxidativt stress og svækkelse af antioxidantsystemet i plasma hos patienter kan spille en rolle i patogenese og progression af psoriasis og relaterede komplikationer.


In vivo oxidativ modifikation af erytrocytmembranproteiner i kobbermangel

Oxidativt stress er blevet postuleret at bidrage til patologien forbundet med kobbermangel i kosten. In vivo er erytrocytter sandsynlige mål for oxidativ skade, fordi de udsættes for høje koncentrationer af ilt og indeholder hæmjern, der kan autooxidere, hvilket resulterer i dannelsen af ​​superoxidanioner. Aktiviteten af ​​det vigtige antioxidantenzym, kobber, zinksuperoxiddismutase, falder markant i erytrocytter under kobbermangel. Effekten af ​​kobbermangel i kosten på indikatorer for oxidativt stress blev undersøgt i erytrocytmembraner hos rotter, der blev holdt på en oprenset kobber-mangel kost i 35 dage efter fravænning. Erytrocytter blev adskilt i unge og gamle populationer på en Percoll-gradient før membranisolering og kvantificering af lipidperoxider og proteincarbonyler. Proteincarbonyler, bestemt ved Western blot-immunoassay, blev overvejende påvist i både alfa- og beta-kæderne af spektrin. Alfa- og beta-underenheder af spektrin i erytrocytmembraner fra kobber-mangelfulde rotter indeholdt højere mængder af carbonyler end kontroller, uanset populationen af ​​erytrocytter, der blev undersøgt. Denne undersøgelse tyder på, at spektrin kan være et specifikt mål for oxidativ skade, når erytrocytkobber, zinksuperoxiddismutaseaktivitet reduceres af kobbermangel.


Modifikationer og oxidation af lipider og proteiner i humant serum påvist ved termokemiluminescens

Påvisning af elektronisk exciterede arter (EES) i kropsvæsker kan udgøre et vigtigt diagnostisk værktøj i forskellige patologier. Eksempler på sådanne produkter er triplet exciterede carbonyler (TEC), som kan være en kilde til fotonemission i området 400-550 nm. Formålet med denne undersøgelse var at bestemme det faktiske bidrag fra lipid- og proteinkomponenter (proteincarbonyler) til fotonemission genereret af termokemiluminescens (TCL) under opvarmning af biologiske væsker. I denne undersøgelse blev en ny TCL Photometer-enhed, designet af Lumitest Ltd, Israel, brugt. Prøver blev opvarmet til en konstant temperatur på 80 ± 0,5°C i 280 s, og fotonemission blev målt på flere tidspunkter. For at sammenligne resultaterne af TCL-målinger med konventionelle metoder til påvisning af lipid- og proteinoxidation blev hver undersøgte prøve også opvarmet i et vandbad ved 80°C i 10-280 s. Lipid- og proteinoxidation blev efterfølgende målt ved anvendelse af konventionelle metoder. TCL af fire flerumættede fedtsyrer (PUFA) med tre til seks dobbeltbindinger blev målt. Forhøjelsen af ​​PUFA TCL-amplituden korrelerede med stigningen i antallet af dobbeltbindinger af PUFA. En korrelation mellem stigningen i TCL-intensitet og proteincarbonyldannelse i bovint serumalbumin (BSA) blev også observeret. I det venøse blodserum viste vores undersøgelse, at en stigning i TCL-intensiteten under opvarmning afspejlede spaltningen af ​​TEC af lipid-oprindelse. Vores undersøgelse tyder på, at biologiske molekyler såsom proteiner, lipider og andre molekyler, som kan blive ustabile under opvarmning, er i stand til at generere EES. Vi demonstrerede, at en TCL-kurve kan bruges som en kinetisk model til måling af oxidative processer, som afspejler modifikationer af forskellige molekyler involveret i de oxidative stress-fænomener. Copyright © 2003 John Wiley & Sons, Ltd.


1. Introduktion

Af de mange COVID-19-vacciner under udvikling repræsenterer de to vacciner, der har vist de mest lovende resultater i at forhindre COVID-19-infektion, en ny klasse af vaccineprodukter: de er sammensat af messenger-ribonukleinsyre (mRNA)-strenge indkapslet i lipid-nanopartikler ( LNP'er). Effektiviteten af ​​disse mRNA-vacciner udviklet af BioNTech/Pfizer og Moderna er omkring 95 % (Baden et al., 2021 Polack et al., 2020), og de var de første mRNA-vacciner, der modtog ‘nødbrugstilladelse’ (af FDA) ) og �tinget godkendelse’ af EMA. Disse mRNA COVID-19-vacciner koder for det virale Spike (S)-glycoprotein af SARS-CoV-2, der inkluderer to prolinsubstitutioner (K986P- og V987P-mutationer), for at stabilisere præfusionskonformationen af ​​glycoproteinet (Wrapp et al., 2020) . Ved intramuskulær (IM) administration muliggør LNP-systemet optagelse af værtsceller og levering af mRNA inde i cytosolen, hvor translationen af ​​mRNA-sekvensen til S-proteinet sker i ribosomerne. Efter post-translationsbehandling af værtscellerne præsenteres S-proteinet som et membranbundet antigen i dets præfusionskonformation på den cellulære overflade, hvilket tilvejebringer antigenmålet for B-celler. Derudover går en del af de tidsmæssigt producerede Spike-proteiner ind i antigenpræsentationsveje, hvilket giver antigengenkendelse af T-celler via MHC-præsentation af T-celleepitoper (Verbeke et al., 2021). EMA-vurderingsrapporten formulerer virkningsmekanismen for mRNA-vacciner på injektionsstedet som følger: �ministration af LNP-formulerede RNA-vacciner IM resulterer i forbigående lokal inflammation, der driver rekruttering af neutrofiler og antigenpræsenterende celler (APC'er) til stedet for levering. Rekrutterede APC'er er i stand til LNP-optagelse og proteinekspression og kan efterfølgende migrere til de lokale drænende lymfeknuder, hvor T-celle-priming forekommer (EMA, 2020a).’ På grund af denne iboende medfødte immunaktivitet er det ikke nødvendigt at formulere mRNA'et vacciner med yderligere adjuvanser. Interessant nok bruger Pfizer/BioNTech og Moderna specifikt nukleosid-modificeret mRNA, der reducerer (i stedet for at øge) den iboende mRNA-immunogenicitet, hvilket understreger behovet for korrekt at balancere den medfødte immunaktivitet af mRNA-vacciner (se nedenfor). Det in vivo antigenproduktion efter administration, der kan opnås med mRNA-vacciner, sammen med de selvadjuvante egenskaber af mRNA-LNP-vacciner, fører i sidste ende til den effektive generering af neutraliserende antistofresponser og cellulær immunitet, hvilket mindsker risikoen for at udvikle COVID-19 for vaccinemodtagere.

mRNA-vacciner har flere fordele i forhold til andre typer vacciner. En generel fordel ved mRNA-vacciner er, at deres udvikling er relativt hurtig, da mRNA-LNP'er er en ægte platformsteknologi. Efter identifikation af det eller de beskyttende proteinantigen(er) og sekventering af det eller de tilsvarende gener, kan mRNA'et laves inden for uger (Jackson et al., 2020). Da de mRNA'er, der koder for forskellige antigener, er kemisk og fysisk meget ens, følger formuleringsdesign og fremstillingsprocesser af nye mRNA-vacciner de samme trin (Petsch et al., 2012). Sammenlignet med replikationsmangelfulde virale vektorer kan mRNA-vacciner være mere effektive til COVID-19-forebyggelse. I modsætning til virale vektorbaserede vacciner genererer de ikke immunitet mod bæreren. I denne henseende ligner mRNA-vacciner desoxyribonukleinsyre (DNA)-baserede vacciner. DNA-vacciner har dog stadig en lille chance for potentiel genomintegration. I modsætning til mRNA-vacciner har DNA-vacciner desuden vist ret lav immunogenicitet i tidlige kliniske forsøg, muligvis fordi DNA-baserede vacciner skal have adgang til kernen for at udøve deres virkning, hvilket komplicerer effektiv levering. Samlet set gør fleksibelt design, standardiserede produktionsprocesser og relativt kortvarig cytoplasmatisk tilstedeværelse mRNA-vacciner meget kraftfulde, især i en pandemisk situation med hurtigt muterende vira.

En af de største udfordringer, man støder på, når man udvikler mRNA-vacciner, er deres dårlige stabilitet. I øjeblikket administreres de fleste mRNA-vacciner IM, hvor det mRNA, der optages af værtsceller, fører til antigenekspression (Hasssett et al., 2019). Tidlig forskning på mRNA-vacciner har vist, at nøgent mRNA hurtigt nedbrydes efter administration (Pardi et al., 2015, Wayment-Steele et al., 2020). Derfor er der i løbet af de sidste par år blevet gjort en indsats for at forbedre in vivo stabilitet af mRNA efter administration. Dette førte til måder at optimere mRNA-strukturen ved at bremse dens nedbrydning (se under afsnittet ‘mRNA-stabilitet’). En anden succesfuld og i øjeblikket meget brugt tilgang er at indkapsle og beskytte mRNA'et i LNP'er (Pardi et al., 2015). Dette reducerer for tidlig mRNA-nedbrydning efter administration og øger levering til cytosolen af ​​antigen-præsenterende celler (Liang et al., 2017, Lindsay et al., 2019).

Selvom der er gjort fremskridt for at øge stabiliteten in vivo og effektiviteten af ​​mRNA-LNP-vacciner, er der blevet lagt meget mindre opmærksomhed på deres stabilitet under opbevaring (Crommelin et al., 2021). For effektivt at kunne distribuere en vaccine over hele verden, bør den have en tilstrækkelig lang holdbarhed, helst ved køleskabstemperaturer (2𠄸 ଌ) eller derover. I øjeblikket er der næppe nogen data tilgængelige i det offentlige domæne om, hvad der sker, når mRNA-LNP-formuleringer opbevares i lange perioder. Desuden er det uklart, i hvilket omfang indfangning af mRNA i LNP'er påvirker opbevaringsstabiliteten af ​​mRNA-vaccinen. Derudover er meget lidt kendt om strukturen og morfologien af ​​LNP'er formuleret med mRNA, den kemiske stabilitet af LNP-komponenterne og den kolloide stabilitet af mRNA-LNP-systemet. Hvad man nu ved, er, at for at kunne opbevare de nuværende mRNA COVID-19-vacciner i længere tid, skal de nedfryses. De nuværende mRNA COVID-19-vacciner fra Moderna og BioNTech/Pfizer skal opbevares mellem � og � ଌ og mellem � og �° , EMA, EMA, EMA. , 2021). Til dato er nedbrydningsprocesserne og årsagerne til, at kravene til opbevaringstemperatur er forskellige, ikke fuldt ud forstået.

Kravet om at opbevare mRNA-LNP'erne i en frossen tilstand hæmmer vaccinedistributionen. Især den meget lave temperatur på � til � ଌ er en stor hindring, når det kommer til vaccinetransport, opbevaring og distribution blandt slutbrugere verden over. De fleste andre vacciner kan opbevares ved 2𠄸 ଌ. Det er klart, at der er behov for og mulighed for at finde måder at stabilisere mRNA-LNP-vacciner på for at tillade ikke-frossen opbevaring. Denne gennemgang giver et overblik over tilgange til at gøre mRNA-vacciner mere stabile, så de kan opbevares længere ved mindre ekstreme temperaturer. For at udforske emnet diskuteres mRNA-LNP-vaccinernes egenskaber og deres indflydelse på opbevaringsstabilitet. Disse oplysninger bruges til at identificere årsagerne til mRNA-vaccine-ustabilitet og til at udforske teknologiske muligheder for stabilitetsforbedring.


Introduktion

Reaktive oxygenarter (ROS) er obligatoriske metaboliske produkter af aerobe celler. De holdes på lave niveauer af et antioxidantsystem, der inkluderer enzymer såsom superoxiddismutase (SOD), katalase (CAT), glutathionperoxidaser (GPX'er), thioredoxiner (TRX'er) og peroxiredoxiner (PRDX'er) og andre molekyler med rensende egenskaber som f.eks. som glutathion (GSH), ubiquinol, vitamin C og E, og så videre [1]. Men når antioxidantsystemet er dysreguleret, og produktionen af ​​ROS forværres, bliver disse aktive molekyler skadelige biprodukter af cellulær metabolisme [1].

Pattedyrsspermatozoer er følsomme over for ROS, såsom superoxidanionen (O2 •− ), hydrogenperoxid (H2O2), nitrogenoxid (NO • ), hydroxyl (HO • ), og peroxynitritanion (ONOO − ). Når ROS-niveauer øges, påvirkes sædfunktionen, hvilket fører til infertilitet [2-6]. Denne stigning i ROS-niveauer betegnes oxidativt stress og er resultatet af en overdreven produktion af ROS og/eller et fald i antioxidantforsvarssystemet [7, 8]. Den oxidative skade retter sig mod alle cellekomponenter, hvilket reducerer sædmotilitet og mitokondriel aktivitet [5, 9, 10]. Det første bevis på en sammenhæng mellem oxidativ skade og mandlig infertilitet blev demonstreret af Thaddeus Manns og Bayard Storeys banebrydende arbejde [11, 12].

Den infertile befolkning har været stigende i løbet af de sidste par årtier [13]. Behandlingseffektiviteten er dog dårlig, fordi årsagen er ukendt i 40%-50% af tilfældene [14]. Flere faktorer er relateret til infertilitet, såsom eksponering for miljøforurenende stoffer, kemikalier, medicin, røg, toksiner, stråling og endda sygdomme [15-18]. Et fællestræk ved ovenstående er produktionen af ​​oxidativ stress. Under sådanne forhold oxideres vitale cellekomponenter (proteiner, lipider og DNA), hvilket kompromitterer cellefunktion og overlevelse [8, 19]. I mindst 25 % af tilfældene påvises forhøjede niveauer af ROS i både sæd og spermatozoer fra infertile patienter [2-5]. I nogle tilfælde af mandlig infertilitet er antioxidantsystemet til stede i sæden [20, 21] ikke tilstrækkeligt til at beskytte spermatozoer mod ROS-afhængige skader. Spermatozoer fra infertile patienter har peroxidation af membranlipider [22], DNA-fragmentering og oxidation af baser [23, 24], lavt mitokondrielt membranpotentiale [25, 26] og inaktivering af enzymer forbundet med motilitet [27, 28].

Proteinmodifikationer i spermatozoer på grund af reaktive oxygenarter

Spermproteiner er målet for redoxafhængige modifikationer, der vil føre, afhængigt af niveauerne af ROS, til enten aktivering/inaktivering af signalveje, der er vigtige for sædfysiologi, eller til oxidativ skade og svækkelse af vitale funktioner (tabel 1 og 2). Nogle af disse modifikationer er reversible, hvilket muliggør en stram regulering af cellulære processer involveret i redox-signalering [29, 30]. En skematisk repræsentation af de vigtigste redox-proteinmodifikationer er vist i figur 1.

Redox-afhængige proteinmodifikationer. Afhængigt af typen af ​​produceret ROS produceres forskellige proteinmodifikationer for enten at kontrollere cellulære processer eller, når disse ROS er på høje niveauer, fremme skade ved inaktiveringsproteinfunktion (dvs. enzymatisk aktivitet, ændring af konformationel struktur osv.). Hydrogenperoxid (H2O2) og andre peroxider fremmer forskellige proteinmodifikationer afhængigt af de niveauer, som de er til stede i cellen, vil mildt oxidativt stress for det meste fremme thioloxidation og S-glutathionylering. Disse redoxafhængige modifikationer vendes let tilbage af antioxidanter (ikke- og enzymatiske forbindelser). Et stærkt oxidativt stress fører til sulfonering, en modifikation, der er sværere at vende tilbage (det kræver en enzymatisk reaktion med energiforbrug). 4-HNE danner proteinaddukter, der irreversibelt inaktiverer enzymer såsom succinatdehydrogenase i spermatozoer. Superoxid (O2 •− ) spontant eller ved superoxid dismutase aktivitet dismuteres til H2O2 eller det kunne kombineres med nitrogenoxid (NO • ) for at give ONOO − . Nitrogenoxid og ONOO - fremmer S-nitrosylering og tyrosinnitrering, der kan deltage i fysiologiske og patologiske processer.

Redox-afhængige proteinmodifikationer. Afhængigt af typen af ​​produceret ROS produceres forskellige proteinmodifikationer for enten at kontrollere cellulære processer eller, når disse ROS er på høje niveauer, fremme skade ved inaktiveringsproteinfunktion (dvs. enzymatisk aktivitet, ændring af konformationel struktur osv.). Hydrogenperoxid (H2O2) og andre peroxider fremmer forskellige proteinmodifikationer afhængigt af de niveauer, som de er til stede i cellen, vil mildt oxidativt stress for det meste fremme thioloxidation og S-glutathionylering. Disse redoxafhængige modifikationer vendes let tilbage af antioxidanter (ikke- og enzymatiske forbindelser). Et stærkt oxidativt stress fører til sulfonering, en modifikation, der er sværere at vende tilbage (det kræver en enzymatisk reaktion med energiforbrug). 4-HNE danner proteinaddukter, der irreversibelt inaktiverer enzymer såsom succinatdehydrogenase i spermatozoer. Superoxid (O2 •− ) spontant eller ved superoxid dismutaseaktivitet dismuteres til H2O2 eller det kunne kombineres med nitrogenoxid (NO • ) for at give ONOO − . Nitrogenoxid og ONOO - fremmer S-nitrosylering og tyrosinnitrering, der kan deltage i fysiologiske og patologiske processer.

Redox-afhængige proteinmodifikationer forbundet med fysiologiske processer i spermatozoer.

Ændring . Fysiologiske processer. Arter . Reference .
Thiol oxidation Binding af sædceller med oviduktalt epitel Kvæg [ 142, 143]
Spermmotilitet Hamster, menneske, rotte [ 144– 147]
Spermkapacitet Menneske, kvæg [ 120, 123, 143, 148]
Sperm kromatin ombygning Menneske, mus, hest, kanin, rotte [ 36, 37, 39, 149– 151]
S-nitrosylering Spermmotilitet Human [ 76, 152]
Tyrosin nitrering Spermkapacitet Human [ 83, 153]
Ændring . Fysiologiske processer. Arter . Reference .
Thiol oxidation Binding af spermatozoon med oviductalt epitel Kvæg [ 142, 143]
Spermmotilitet Hamster, menneske, rotte [ 144– 147]
Spermkapacitet Menneske, kvæg [ 120, 123, 143, 148]
Sperm kromatin ombygning Menneske, mus, hest, kanin, rotte [ 36, 37, 39, 149– 151]
S-nitrosylering Spermmotilitet Human [ 76, 152]
Tyrosin nitrering Spermkapacitet Human [ 83, 153]

Redox-afhængige proteinmodifikationer forbundet med fysiologiske processer i spermatozoer.

Ændring . Fysiologiske processer. Arter . Reference .
Thiol oxidation Binding af spermatozoon med oviductalt epitel Kvæg [ 142, 143]
Spermmotilitet Hamster, menneske, rotte [ 144– 147]
Spermkapacitet Menneske, kvæg [ 120, 123, 143, 148]
Sperm kromatin ombygning Menneske, mus, hest, kanin, rotte [ 36, 37, 39, 149– 151]
S-nitrosylering Spermmotilitet Human [ 76, 152]
Tyrosin nitrering Spermkapacitet Human [ 83, 153]
Ændring . Fysiologiske processer. Arter . Reference .
Thiol oxidation Binding af spermatozoon med oviductalt epitel Kvæg [ 142, 143]
Spermmotilitet Hamster, menneske, rotte [ 144– 147]
Spermkapacitet Menneske, kvæg [ 120, 123, 143, 148]
Sperm kromatin ombygning Menneske, mus, hest, kanin, rotte [ 36, 37, 39, 149– 151]
S-nitrosylering Spermmotilitet Human [ 76, 152]
Tyrosin nitrering Spermkapacitet Human [ 83, 153]

Redox-afhængige proteinmodifikationer forbundet med skadelige virkninger i spermatozoer.

Ændring . Tilknyttet resultat. Arter . Reference .
Thiol oxidation Mandlig infertilitet Human [ 38, 39, 42]
Nedsat sædmotilitet Hamster, menneske, rotte [ 42, 154, 155]
Blokering af sæd-æg-fusion Mus [ 156, 157]
4-HNE proteinaddukter Nedsat sædmotilitet Menneske, hest [ 62, 158, 159]
S-glutathionylering Nedsat sædmotilitet Human [ 34]
Forringelse af sædkapacitet Human [ 34]
Tyrosin nitrering Nedsat sædmotilitet Human [ 34, 80]
Forringelse af sædkapacitet Human [ 34]
Sulfonering Nedsat sædmotilitet Menneske, rotte [ 42, 60, 154]
Forringelse af epididymal modning Rotte [ 154]
Ændring . Tilknyttet resultat. Arter . Reference .
Thiol oxidation Mandlig infertilitet Human [ 38, 39, 42]
Nedsat sædmotilitet Hamster, menneske, rotte [ 42, 154, 155]
Blokering af sæd-æg-fusion Mus [ 156, 157]
4-HNE proteinaddukter Nedsat sædmotilitet Menneske, hest [ 62, 158, 159]
S-glutathionylering Nedsat sædmotilitet Human [ 34]
Forringelse af sædkapacitet Human [ 34]
Tyrosin nitrering Nedsat sædmotilitet Human [ 34, 80]
Forringelse af sædkapacitet Human [ 34]
Sulfonering Nedsat sædmotilitet Menneske, rotte [ 42, 60, 154]
Forringelse af epididymal modning Rotte [ 154]

Redox-afhængige proteinmodifikationer forbundet med skadelige virkninger i spermatozoer.

Ændring . Tilknyttet resultat. Arter . Reference .
Thiol oxidation Mandlig infertilitet Human [ 38, 39, 42]
Nedsat sædmotilitet Hamster, menneske, rotte [ 42, 154, 155]
Blokering af sæd-æg-fusion Mus [ 156, 157]
4-HNE proteinaddukter Nedsat sædmotilitet Menneske, hest [ 62, 158, 159]
S-glutathionylering Nedsat sædmotilitet Human [ 34]
Forringelse af sædkapacitet Human [ 34]
Tyrosin nitrering Nedsat sædmotilitet Human [ 34, 80]
Forringelse af sædkapacitet Human [ 34]
Sulfonering Nedsat sædmotilitet Menneske, rotte [ 42, 60, 154]
Forringelse af epididymal modning Rotte [ 154]
Ændring . Tilknyttet resultat. Arter . Reference .
Thiol oxidation Mandlig infertilitet Human [ 38, 39, 42]
Nedsat sædmotilitet Hamster, menneske, rotte [ 42, 154, 155]
Blokering af sæd-æg-fusion Mus [ 156, 157]
4-HNE proteinaddukter Nedsat sædmotilitet Menneske, hest [ 62, 158, 159]
S-glutathionylering Nedsat sædmotilitet Human [ 34]
Nedsættelse af sædkapacitet Human [ 34]
Tyrosin nitrering Nedsat sædmotilitet Human [ 34, 80]
Nedsættelse af sædkapacitet Human [ 34]
Sulfonering Impairment of sperm motility Human, rat [ 42, 60, 154]
Impairment of epididymal maturation Rotte [ 154]

Thiol oxidation

Cysteine, a sulfur-containing amino acid, is a potent nucleophile under physiological conditions. This remarkable reactivity is due to the thiol (-SH) group. The formation of disulfide bridges (-SS-) by thiol oxidation is a common strategy to fold a protein generating a structure to assure, for instance, enzymatic activity or interaction with receptors, plasma membrane components, etc. A specific ratio -SS-/–SH within a protein molecule is essential to assure its function, and this rate can be affected by ROS. An oxidative stress will oxidize free -SH, thus preventing the formation of -SS- where and when it is needed during a physiological process and will translate in the impairment of protein function. A good example of this situation is the effects of high levels of ROS on the ATP production by human spermatozoa. It was observed that elevated levels of ROS, generated by direct addition of H2O2 or by adding xanthine-xanthine oxidase system (generator of O2 •− and of H2O2) to the incubation medium, reduce human sperm motility in a dose- and time-dependent manner [ 31]. This reduction was correlated with a decrease in the ATP production by the spermatozoon [ 32], thus suggesting that the impairment of sperm motility was a depletion of energy. Human spermatozoa depend on aerobic oxidation of glucose accomplish by the conjunction of glycolysis, Krebs cycle, and the oxidative phosphorylation by the electron transport chain. In 1992, de Lamirande and Gagnon [ 32] suggested that one possible target of ROS could be the glyceraldehyde 3-phosphate dehydrogenase, which is linked to the fiber sheath explaining the drop in ATP production observed in ROS-treated spermatozoa. Recently, it was demonstrated that the glyceraldehyde 3-phosphate dehydrogenase, a key enzyme of the glycolytic pathway, can be inactivated by oxidation of the –SH in its active site by exogenous H2O2 [ 33].

An appropriate level of thiol oxidation in proteins is necessary for sperm motility [ 144– 147]. However, the machinery that makes the spermatozoon to move is very sensitive to ROS [ 42, 154, 155] levels that decrease motility significantly do not impair the viability of these spermatozoa [ 34]. But most importantly, ROS alter motility differently for instance, 100 μM H2O2 promotes a significant decrease in motility and inhibits capacitation of human spermatozoa compared to nontreated controls. However, 100 μM DA-NONOate, a NO • donor, do not affect sperm motility and even stimulate capacitation [ 34]. It is also important to highlight that 200–500 μM DA-NONOate inhibits sperm capacitation without impairing motility or viability [ 34]. Altogether, these findings indicate that there is a need to identify which specifics ROS are at high levels in infertile men to better find treatment strategies to avoid their toxic effects.

Another possible target of ROS is tubulin, a structural protein of the sperm flagellum. We found that increasing concentrations of H2O2 promoted an increase in thiol oxidation levels of α-tubulin in human spermatozoa [ 1]. Thiol oxidation of α-tubulin impaired microtubule polymerization and thus affecting the appropriate functioning of the flagellum [ 35].

Sperm chromatin remodeling is completed during epididymal maturation [ 36, 37]. During this process, an appropriate balance of thiol oxidation in protamines assures a healthy sperm chromatin structure. Both reduction and over oxidation of protamine thiols are associated with male infertility [ 38, 39].

Peroxiredoxins are selenium-free enzymes with one (PRDX6) or two cysteines (PRDX1 to 5) in their active site that are highly reactive with H2O2 and other peroxides. They compose the primary antioxidant system in ejaculated human spermatozoa since the amount of reduced GSH is insufficient (∼0.1 mM), the absence of CAT, and inactivation of mitochondrIal GPX4 as ROS scavenger [ 40, 41]. They are considered essential elements of the antioxidant defense of spermatozoa since infertile men have low amounts of PRDXs with high levels of thiol oxidation. This modification promotes the inactivation of their enzymatic activity and thus generating an increase of oxidative stress and DNA damage [ 42]. Mouse spermatozoa lacking PRDX6 display low motility and high levels of lipid peroxidation (a marker of oxidative stress) and poor sperm quality. This poor sperm quality is associated with subfertility which is exacerbated with age [ 2, 3]. The absence of PRDX4 also leads to loss of spermatogenic cells and increase of apoptosis in the testis without a significant reduction of fertility of the knockout males [ 43]. Although not measured, the authors concluded that the spermatogenic cell loss and the increase in apoptosis are due to an oxidative stress generated by the absence of PRDX4.

Peroxiredoxins are highly sensitive to ROS, and an active reductant system must be taken in place to maintain active their peroxidase activity [ 44]. In the case of 2-Cys PRDXs, this re-activation is done by the TRX/TRX reductase/NADPH system. The functional deletion of thioredoxin domain-containing proteins Txndc2 and Txndc3 in mouse spermatozoa correlated with an increase of age-related oxidative stress [ 45]. The thiol oxidized PRDX6 cannot be reduced by the TRXs and requires the presence of reduced GSH and glutathione-S-transferase pi (GSTpi) [ 46]. Ascorbate can also reduce PRDX6 as it was reported in yeast [ 47]. However, later studies using yeast and mammalian somatic cells revealed that the GSH/GSTpi system is the physiological mechanism to reduce PRDX6 [ 48– 50]. Since the GSH content is very low in spermatozoa [ 51], it is possible that ascorbate may be necessary to maintain the peroxidase activity of PRDX6. In mouse, we demonstrated that PRDX6 is important to protect the paternal genome from oxidative damage [ 52, 53]. The fact ascorbic acid protected human sperm DNA against oxidative damage [ 54] supports the hypothesis yet to be proven that PRDX6 may be reduced by ascorbate rather than by the GSH/GST system in spermatozoa.

Rat epididymal spermatozoa, collected after 24 h of the end of treatment for 2 weeks with tert-BHP, a compound that generates an oxidative stress in vivo, showed increasing levels of thiol oxidation of PRDX1 and PRDX6, the most abundant PRDXs in the rat spermatozoa [ 4]. This treatment was meant to generate the oxidative stress during the epididymal maturation, and this PRDXs oxidation reflects the scavenging activity of these enzymes in an attempt to fight against the oxidative stress caused. Moreover, the total amount of PRDX1, PRDX4, and PRDX6 increased in the treated spermatozoa, suggesting an active transfer of these enzymes from the epididymal epithelium to the maturing spermatozoa [ 4]. This active transfer of antioxidant enzymes has also been reported for other proteins including GPX5 and TRX [ 55–59].

Infertile men have a lower quantity of PRDXs in seminal plasma and spermatozoa than healthy donors [ 42]. Sperm PRDX6 was low in 67% and 39% varicocele and idiopathic infertile patients, respectively. Sperm PRDX1 was only low in 42% of varicocele patients [ 42]. In most of the cases of infertility, higher levels of thiol oxidation of PRDX1 and PRDX6 were observed in sperm from these infertile men. The thiol oxidation ratio (thiol oxidized PRDX/reduced PRDX) negatively and positively correlated with sperm motility and DNA damage and lipid peroxidation, respectively [ 42]. Interestingly, sperm levels of high molecular mass complexes of hyperoxidized PRDX6 were greater in both infertile men groups than in donors and the PRDX6 thiol oxidation ratio correlated positively with lipid peroxidation in spermatozoa [ 42]. These higher molecular mass complexes contain the sulfonated form of PRDXs (PRDX-SO2) a kind of redox-dependent modification that occurs when a strong oxidative stress is established [ 60].

Another significant modification of thiol groups by ROS is the formation of protein adducts with electrophiles such as aldehyde 4-hydroxynonenal (4-HNE) and acrolein, products of lipid peroxidation [ 61]. Both electrophiles promoted an increase in mitochondrial ROS production and reduction, DNA damage and reduction of motility in spermatozoa [ 62, 63]. The 4-HNE-dependent protein modification promotes the inactivation of succinate dehydrogenase and dynein heavy chain, both important proteins of the motility machinery of human spermatozoa. The modification of heat shock protein A2 by 4-HNE promoted its degradation by the proteasome system in male germ cells [ 64]. The recent evidence that the inhibition of arachidonate 15-lipoxygenase prevented the 4-HNE-induced protein damage in male germ cells [ 65] supports the potential advantage of pharmacological inhibition of this enzyme to protect germ cells from oxidative damage. Acrolein is capable of forming adducts with proteins and DNA and thus promoting its deleterious effects observed in spermatozoa [ 62, 63] that may include enzyme inactivation and mutagenesis [ 66, 67]. It has been reported that acrolein impairs the activity of the TRX/TRD system and PRDX [ 68].

S-Glutathionylation

Glutathione is a water-soluble tripeptide ubiquitously distributed in tissues. It is the predominant nonprotein source of intracellular SH groups (∼1–10 mM in most of the mammalian cells) present in the cytosol (∼90% of the total GSH), mitochondria, endoplasmic reticulum, and possibly in the nucleus [ 69, 70]. Protein S-glutathionylation occurs when GSH reacts with protein SH groups, often resulting in enzyme inactivation [ 71–74]. This mechanism may appear detrimental for the cell, but it is protective because it prevents further protein oxidation and it is reversible [ 8, 75].

Mammalian spermatozoa have a little amount of GSH (1–13 nmoles GSH/10 9 spermatozoa) and particularly in humans is approximately 0.3 mM [ 51] compared to the 10 mM found in most somatic cells [ 5]. Thus, the contribution of this antioxidant compound in the defense against oxidative stress is limited. This restriction will impact on antioxidant enzymes, for instance, PRDXs that require GSH for reduction of their thiol groups after ROS oxidized them. This particular topic will be discussed later in this review.

Human and mouse sperm proteins are subjected to S-glutathionylation we observed that mouse spermatozoa, lacking PRDX6, show higher levels of this modification compared to wild-type controls [ 34]. Human spermatozoa treated with H2O2 or tet-butyl hydroperoxide (tert-BHP) showed higher levels of glutathionylated proteins than nontreated controls [ 34]. Although the increase of S-glutathionylation was an antioxidant response, it was not sufficient as the oxidative stress generated either by lacking PRDX6 or by exogenously increasing the levels of ROS severely affected sperm motility.

The response of human spermatozoa to high levels of H2O2 or tert-BHP depended on the concentration of each ROS H2O2 generates S-glutathionylation at lower concentrations than tert-BHP, indicating a more reactive molecule capable of damaging the spermatozoon. Interestingly, this significant reduction in total and progressive motility was observed in viable spermatozoa [ 34]. This observation highlights the differential effects of ROS on sperm functions.

S-Glutathionylation was observed in the cytosolic and Triton X-100-insoluble fractions in human spermatozoa treated with high concentration of H2O2 [ 34]. Members of the human sperm antioxidant system may be inactivated by S-glutathionylation and therefore trigger a strong oxidative stress associated with the impairment of sperm motility and capacitation. PRDX1, PRDX5, and PRDX6 are found in the Triton X-100-soluble fraction, and PRDX6 is also found in the cytosolic and Triton X-100 soluble fractions [ 60]. We then can hypothesize that S-glutathionylation also contributes with the inactivation of these PRDXs, explaining the high oxidative stress associated with poor sperm quality observed in infertile men [ 42].

S-Nitrosylation and tyrosine nitration

Nitric oxide (NO • ) or its derivatives (e.g. ONOO − ) generate S-nitrosylation in proteins. High levels of NO • and ONOO − can modify structural proteins and enzymes, thus altering cellular function. However, low levels of these ROS are involved in redox signaling [ 6, 7]. About 240 s-nitrosylated proteins were identified in human spermatozoa treated with the NO • donor nitrosocysteine [ 76]. Enzymes involved in energy production, motility, ion channels, and in antioxidant defense were identified as modified by s-nitrosylation, indicating that this modification may be involved in redox regulation of sperm physiological processes. Of interest, PRDX1, GST, and thioredoxin domain-containing protein 3 and 11 carried this modification (for a complete list of s-nitrosylated proteins, see Lefièvre et al. [ 76]).

Peroxiredoxins are susceptible to NO • or ONOO − attack for instance, S-nitrosylation impairs the ability of PRDX2 to reduce peroxide, thus promoting neuronal cell oxidative stress [ 77] and, as mentioned above, PRDX1 was identified as one of the S-nitrosylated proteins due to nitroso-cysteine treatment in human spermatozoa [ 76].

Tyrosine (Tyr) nitration is also a protein modification promoted by NO • and ONOO − . A Tyr residue reacts with these ROS producing a nitro (-NO2) group. This modification will alter protein function leading to either a physiological or pathological effect, depending on the protein target and the level of ROS generated [ 78, 79]. It has been reported that spermatozoa from asthenozoospermic patients had high levels of Tyr nitration determined by immunocytochemistry [ 80]. Human spermatozoa treated with DA-NONOate show increased levels of Tyr nitration in a dose-dependent manner, a modification associated with the impairment of sperm motility [ 34]. Tyr-nitrated proteins were located in the Triton X-100-insoluble fraction of human spermatozoa. Immunocytochemistry studies showed that these modified proteins were mainly found in the flagellum in nonpermeabilized spermatozoa but also in the head when the sperm cells were permeabilized with methanol [ 34, 81].

Protein targets for NO • and ONOO − that display Tyr nitration and may account for impairment of sperm motility are enzymes belong to glycolysis (glyceraldehyde 3-P- dehydrogenase and enolase) and the Krebs cycle (aconitase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and dihydro lipoamide dehydrogenase) (see references in Morielli and O’Flaherty [ 34]). By inactivating these enzymes by Tyr nitration, the production of ATP is severely diminished leading to an impairment of sperm motility. α-tubulin also can be modified by Tyr nitration [ 82], interfering with appropriate microtubule polymerization in the sperm flagellum.

Tyrosine nitration was found mostly in the Triton X-100-insoluble fraction [ 34], where PRDX1, PRDX5, and PRDX6 were found [ 60]. It is possible then that PRDXs are the target of NO • or ONOO − and be inactivated by Tyr nitration, leading to an increase in ROS levels that will impair sperm motility and capacitation.

Tyrosine nitration is also associated with sperm capacitation. Levels of Tyr nitration increased in spermatozoa exposed to ONOO − in a dose-dependent manner but at concentrations that do not affect sperm motility [ 34, 83]. The prevention of the time-dependent Tyr nitration by SOD (O2 •− scavenger) or L-NMMA (inhibitor of nitric oxide synthase (NOS)) that prevent capacitation in spermatozoa under capacitating conditions strongly suggests that ONOO − is endogenously produced during human sperm capacitation [ 84].

Redox signaling during sperm capacitation

Phosphorylation of proteins (e.g. enzymes, receptors and transcription factors) [ 85–92] and the redox regulation (which involves the oxidation of a signaling molecule) [ 93–96] represent two primary mechanisms regulating cell function. In essence, they resemble on–off switches for proteins [ 93, 96]. Under physiological conditions, the reversibility of these reactions is spontaneous or assured by reductive pathways or are catalyzed by enzymes [ 93, 97].

Growing evidence highlights the importance of H2O2 signaling in cell physiology [ 98– 100].

The redox regulation is essential for sperm capacitation [ 101–104]. Low levels of ROS trigger early, intermediate, and late phosphorylation events [ 105–109] that culminate with the increased capacitation-associated Tyr phosphorylation [ 101, 102]. Catalase, GPXs, and PRDXs are recognized scavengers of H2O2, but PRDXs are more versatile with a dual action as antioxidant and as modulators of H2O2-dependent signaling [ 44, 98, 100, 110–114]. This dual action of PRDXs is necessary for mammalian spermatozoa because these cells lack CAT (peroxisomes, containing the enzyme, are eliminated from germ cells during spermatogenesis [ 115, 116], and GPX4 is a structural protein involved in the mitochondrial sheath without antioxidant capacity in the ejaculated spermatozoa [ 117]. Other GPXs such as GPX2, 3, and 5 are not present in human spermatozoa [ 118, 119], and inhibition of either CAT or the GPX system did not increase the levels of lipid peroxidation in human spermatozoa [ 40]. Thus, the highly abundant and reactive PRDXs with different ROS become major players not only in the protection against oxidative damage but the regulation of the redox signaling in human spermatozoa [ 40–42, 60, 101–103].

We observed differential subcellular localization of the six PRDX isoforms in human spermatozoa and that PRDX1, PRDX4, PRDX5, and PRDX6 react with different concentrations of H2O2 [ 60]. Interestingly, PRDX6 is highly abundant and the only member of the family present in all the subcellular compartments of human spermatozoa and to react with H2O2 at levels (50 μM) that promote CAP, as well as with other ROS [ 60].

The thiol oxidation is also associated with physiological functions in human spermatozoa, a temporal inhibition of PRDXs by thiol oxidation allows the increase of ROS at levels that will not promote damage but will trigger the capacitation-associated phosphorylation events such as phosphorylation of PKA substrates and Tyr residues [ 120]. The inhibition of 2-Cys PRDXs with thiostrepton promoted the prevention of sperm capacitation and the establishment of oxidative stress. Thus, PRDX1–5 are important to assure the acquisition of fertilizing ability by the human spermatozoon [ 120].

PRDX6 is the only member of the family with phospholipase A2 activity which is important to remove lipid peroxides from the membranes [ 121, 122]. When 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phospho-methanol lithium (MJ33) was present in the capacitation medium, spermatozoa not only were unable to undergo capacitation but also they displayed higher levels of lipid peroxidation compared to those capacitated without the inhibitor. These results indicate that PRDX6 is dominant in the protection of human spermatozoa against lipid peroxidation to assure normal function as the other 2-Cys PRDXs were not inhibited by MJ33 [ 120].

Human sperm capacitation is associated with rapid and reversible changes in protein -SH groups that appear to be redox regulated [ 123, 124]. This shift in the redox status of thiol groups is a very dynamic mechanism of switching on or off protein activity. The increases or decreases in -SH content were prevented by exogenous addition of SOD or CAT to the capacitating medium [ 124]. Some sperm proteins, with molecular mass and isoelectric point similar to those of PRDX1, PRDX4, and PRDX6 [ 125, 126], are oxidized by H2O2 during capacitation. This evidence supports the findings that thiol oxidation of PRDXs allows the redox signaling necessary to trigger phosphorylations events occurring during sperm capacitation [ 105, 107–109, 127].


INTESTINAL Lipid Metabolism and Chylomicron Assembly

Intestinal Lipid Absorption

Through absorption of dietary lipids, the intestine is a key regulator of stored and circulating lipids. Primarily it is enterocytes in the small intestine that actively regulate the release of dietary lipids into circulation (503-505). The predominant lipids derived from diet are triglycerides, phospholipids and cholesteryl esters. In the intestinal lumen, ingested lipids are emulsified by bile salts to enhance their hydrolysis by lipases (Figure 9) (506-509). Triglycerides make up the largest percentage of the intestinal lipids. Lipolysis of triglycerides releases free fatty acids (non-esterified fatty acids) and monoacylglycerides (Figure 9). These are absorbed on the luminal surface of the enterocytes both by free diffusion and actively by protein-mediated transport into the enterocyte cytosol (Figure 9) (508-510). The principal transporters identified to date are CD36 (now known as SR-B2 (511)) and several fatty acid binding and transport proteins (512-514).

Figur 9.

Intestinal Triglyceride and Cholesterol Metabolism. In the intestinal lumen, dietary triglyceride (TG) and cholesterol are emulsified by bile salts which enhance their uptake. Lipases in the intestinal lumen digest triglycerides to free fatty acids (FFA) and monoacylglycerides (MAG). These are absorbed into the enterocyte where they are used in the synthesis of TG, phospholipid and cholesteryl ester (CE). Much of the synthesized TG in enterocytes is packaged, along with phospholipids, cholesterol and proteins into chylomicrons, which are secreted at the basolateral surface of the enterocyte and enter the lymphatic system. The assembly of chylomicrons begins in the endoplasmic reticulum. During the synthesis of apolipoprotein B48 (apoB48), the protein acquires phospholipid from the endoplasmic reticulum membrane and also cholesterol and TG to form a primordial chylomicron. Continued acquisition of TG and CE and smaller, exchangeable proteins (e.g. apolipoprotein A-IV and apolipoprotein C-III) in the endoplasmic reticulum enlarges the particle to form a prechylomicron. Prechylomcirons are transported to the Golgi apparatus in specialized COPII vesicles. In the Golgi apparatus, the prechylomicron matures into a chylomicron. The maturation process includes the glycosylation of apoB48, the acquisition of additional proteins (e.g. apolipoprotein A-I) and lipid. Secretory vesicles formed from the Golgi carry the mature chylomicrons to the basolateral surface of the enterocyte. Fusion of the secretory vesicle membrane with the plasma membrane releases the chylomicron into the extracellular space where it is taken up into lacteals near the enterocyte and, thus, enters the lymphatic circulation. Dietary cholesterol in the intestinal lumen is taken into the enterocyte by a process involving Niemann-Pick C1-like protein 1 (NPC1L1). Enterocyte cholesterol and CE can be incorporated into chylomicrons and secreted with TG. In addition, enterocyte cholesterol can be directly excreted into the intestinal lumen using the heterodimer ATP-binding cassette transporter G5 and G8 (ABCG5/G8). Enterocyte cholesterol can also be transported to and incorporated into the basolateral membrane for efflux into the circulation.

Chylomicron Assembly and Secretion

In the enterocyte, the free fatty acids and monoacylglycerides are used to synthesize triglycerides, phospholipids, and cholesteryl esters (Figure 9) (508,509,513,515-517). The majority of the triglycerides formed in the enterocytes are repackaged into large, buoyant lipoproteins, called chylomicrons, and secreted from the basolateral surface of the cell (Figure 9). These particles play a central role in the transport of triglycerides and fat-soluble vitamins to the rest of the body (518).

The assembly of the chylomicron particle from precursors is a complex process. Each particle contains a single copy of apolipoprotein B48 and assembly begins with the synthesis of this protein in the rough endoplasmic reticulum. Apolipoprotein B48 is a truncated form of apolipoprotein B100 that is formed by posttranscriptional editing (519,520). As apolipoprotein B48 is synthesized and translocated across the endoplasmic reticulum membrane, it becomes lipidated to form a phospholipid-rich, dense primordial chylomicron in the lumen of the endoplasmic reticulum (Figure 9). The primordial chylomicron contains apolipoprotein B48, phospholipid, cholesterol and minor amounts of cholesteryl ester and triglyceride (513,521,522). The assembly process requires microsomal triglyceride transfer protein (523). In the absence of sufficient lipid, or if microsomal triglyceride transfer protein function is impaired, apolipoprotein B48 is ubiquitinated and targeted for proteasome degradation (524). The importance of this initiating assembly step is seen in patients with a defect in the MTP gene leading to the rare recessive disorder abetalipoproteinemia. Individuals with abetalipoproteinemia have almost undetectable levels of apoB or and very low total cholesterol levels in their plasma because of the inability to assemble apoB-containing lipoproteins in their enterocytes or hepatocytes. Among the sequelae experienced by these patients are accumulation of triglycerides in their intestines and livers and a deficiency of lipid-soluble vitamins in their plasma (525,526). If untreated, these patients develop severe neurological problems mostly related to vitamin E and A deficiency.

After formation, the initial primordial particle expands by the acquisition of additional triglyceride and cholesteryl ester (Figure 9). The additional lipid is acquired by fusion with non-apolipoprotein B48 containing particles that are rich in triglyceride and cholesteryl ester. The exact origin of these lipid particles and their precise composition is currently actively debated (504,505,513,527,528), but the fusion of the primordial chylomicron with the apolipoprotein B48-free particles occurs in the endoplasmic reticulum (513). The resulting particle is a prechylomicron (Figure 9). In addition to apolipoprotein B48, the prechylomicron surface can contain multiple copies of other small, exchangeable apoproteins including apolipoprotein A-IV and apolipoprotein C-III. Exchangeable apoproteins are soluble proteins that are not as tightly adherent to the particle surface and so can be exchanged between lipid particles.

Prechylomicrons are transported out of the endoplasmic reticulum and delivered to the Golgi apparatus for further processing (Figure 9). Transport occurs in specialized vesicles that can accommodate their large size. The unique vesicles contain a number of specific proteins necessary for the transport and docking process. Vesicle-associated membrane protein-7, coatomer protein II and Sar1b, a small GTPase component of the coatomer protein II vesicle assembly machinery (Figure 9) are among the specialized proteins on the lipid transport vesicles (505,529-531). The maturation of the particle in the Golgi apparatus includes further glycosylation of apolipoprotein B48 and the addition of apolipoprotein A-I to the surface (505,532,533). After processing, the mature chylomicron is packaged into Golgi-derived secretory vesicles and transported to the basolateral surface and exocytosed into the lymph (Figure 9) (527,534,535).

The assembly of chylomicrons in enterocytes is a complex process requiring a number of coordinated steps and specific factors to work in unison. A failure in any of these can lead to lipid-related disease states. For instance, mutations in the SAR1B gene lead to retention of prechylomicrons within membrane-bound structures in the enterocytes (529). The condition is marked in childhood by decreased blood cholesterol levels, lipid accumulation in the enterocytes, chronic fat malabsorption with steatorrhea, and deficiencies in fat-soluble vitamin and essential fatty acids.

Chylomicron Cholesterol

Although chylomicrons are triglyceride-rich, they also carry substantial amounts of cholesterol (536,537). The cholesterol in chylomicrons comes from the general pool of enterocyte cholesterol. Enterocytes acquire cholesterol by uptake at the luminal surface, acquisition from lipoproteins at the basal lateral surface, and by de novo synthesis within the enterocyte. Niemann-Pick C1-Like 1 protein is a key component of the luminal acquisition machinery (Figure 9) (538), while the low density lipoprotein receptor appears to be a major mediator of cholesterol acquisition at the basolateral surface (539,540). The incorporation of cholesterol into chylomicrons contributes to the circulating levels of cholesterol, and increases in intestinal synthesis of chylomicrons due to increased dietary lipids contributes to cardiovascular risk and atherosclerosis, albeit by complex mechanisms (516,541,542).

Non-Chylomicron Intestinal Lipid Metabolism

Enterocytes can also regulate circulating lipids by means other than chylomicron secretion. In the presence of excess fatty acids or cholesterol, the enterocyte can store excess lipid in their esterified forms (triglycerides and cholesteryl esters, respectively) within cytoplasmic lipid droplets (543-545). The neutral lipids in the droplets can subsequently be mobilized by hydrolysis as needed by the cell. The free fatty acids liberated from storage droplets can be incorporated into the chylomicron production pathway to become part of secreted chylomicrons.

Finally, the intestine also regulates circulating cholesterol levels by taking up excess circulating cholesterol and excreting it into the intestinal lumen for clearance in the feces. This process is known as trans-intestinal cholesterol excretion. It acts as an adjunct to liver biliary secretion and can account for as much as 30% of neutral sterol excretion (546). Trans-intestinal cholesterol excretion occurs at the luminal surface of the enterocytes by a process that primarily utilizes the ATP-binding cassette transporter pair ABCG5/G8 (Figure 9) but can use other pathways as well (547).

Resumé

It is clear that intestinal lipid processing is a key contributor to the circulating levels of both triglyceride and cholesterol. Dietary, genetic and metabolic factors that disrupt the process of enterocyte lipid metabolism potentially can alter lipid homeostasis and produce disease states.


Abstrakt

Oxidative stress is known to play an important role in the pathogenesis of a number of diseases. In particular, it is linked to the etiology of Alzheimer’s disease (AD), an age-related neurodegenerative disease and the most common cause of dementia in the elderly. Histopathological hallmarks of AD are intracellular neurofibrillary tangles and extracellular formation of senile plaques composed of the amyloid-beta peptide (Aβ) in aggregated form along with metal-ions such as copper, iron or zinc. Redox active metal ions, as for example copper, can catalyze the production of Reactive Oxygen Species (ROS) when bound to the amyloid-β (Aβ). The ROS thus produced, in particular the hydroxyl radical which is the most reactive one, may contribute to oxidative damage on both the Aβ peptide itself and on surrounding molecule (proteins, lipids, …). This review highlights the existing link between oxidative stress and AD, and the consequences towards the Aβ peptide and surrounding molecules in terms of oxidative damage. In addition, the implication of metal ions in AD, their interaction with the Aβ peptide and redox properties leading to ROS production are discussed, along with both in vitro og in vivo oxidation of the Aβ peptide, at the molecular level.


Se videoen: Farmakologie signálních molekul (Juli 2022).


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