Utjecaj nedostatne aktivnosti S-adenozilhomocistein hidrolaze na metilaciju proteina [Impact of S-Adenosylhomocysteine hydrolase deficiency on protein methylation]

Ćuk, Mario (2013) Utjecaj nedostatne aktivnosti S-adenozilhomocistein hidrolaze na metilaciju proteina [Impact of S-Adenosylhomocysteine hydrolase deficiency on protein methylation]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

S-Adenosylhomocysteine hydrolase (SAHH) deficiency (Y143C SAHH W112X) is a recently discovered inherited multiple organ disease characterized by elevated plasma Sadenozilmethionine (AdoMet) and S-Adenosylhomocysteine (AdoHcy) concentrations, and pathologically low methylation index (MI). Both, an unclear pathogenesis and limited treatment options were major reasons for studying posttranslational protein methylation in terms of SAHH deficiency. This was analyzed both indirectly, by measuring asymmetric dimethylarginine (aDMA) concentration in both, plasma and fibroblasts, (aDMA), before the treatment and during the treatment, and directly, using specific antibodies against methylated target proteins. The obtained results showed that an abnormally low methylation index, associated with SAHH deficiency, relates to disturbances of aDMA metabolism and protein hypomethylation. In Y143C SAHH W112X patients, aDMA concentrations were statistically significantly increased, but normalized following low methionine diet. In Y143C SAHH W112X fibroblast, aDMA concentrations were statistically significantly reduced after addition of AdoHcy. In control fibroblasts, addition of both AdoMet and AdoHcy have led to statistically significant reduction of aDMA concentrations, reaching the value similar to those in Y143C SAHH W112X fibroblast, in native conditions. The reproducabilty of these results has been confirmed in several independent experiments. Exploring deficiency of SAHH, a multiple functioning enzyme involved in numerous transmethylation reactions, we discovered hypomethylation of another multifunctional protein, heterogeneous nuclear ribonucleoprotein type A1 (hnRNP-A1), a substantial regulator of mRNA processing and splicing. The results showed arginine hypomethylation of hnRNP-A1. This may result hnRNP-A1 dysfunction , which could be manifested as abnormal alternative splicing of its own mRNA and unbalanced synthesis and/or degradation of both constitutive (KoB) and alternative (AiA) protein isoform. Any kind of dysfunction including these proteins may lead to a series of secondary consequences, in particular those affecting protein interactions and networks. All these additionally complicate the pathogenesis of SAHH deficiency. The level of posttranslational arginine methylation of selected proteins in terms of SAHH deficiency was further investigated and hnRNP-A1 was found to be hypomethylated. This change surely influnce the hnRNP-A1 secondary and tertiary structure and, consequentially, its interaction with another bio-macromolecules, contributing to development of disease that we now assume to be a "global dismethylopathy or epigenopathy". Reduced level of hnRNP-A1 arginine methylation additionaly contributes to this dysfuncionality which reflects to the processes of mRNA splicing and processing, in various tissues and organs. This ultimately leads to a multiple organ disease, present in patients with SAHH deficiency.

Abstract in Croatian

Manjak aktivnosti S-adenozilhomocistein hidrolaze (SAHH) je novootkrivena nasljedna višeorganska bolest koju biokemijski karakteriziraju povišene koncentracije S-adenozilmetionina (AdoMet) i Sadenozilhomocisteina (AdoHcy) te patološki nizak metilacijski indeks (MI). Nedovoljno jasna patogeneza i nemogućnost izlječenja povod su za istraživanje posttranslacijske metilacije proteina. Razinu metilacije smo određivali neizravno, mjerenjem koncentracije asimetričnog dimetilarginina (aDMA) u plazmi i modelu fibroblasta in vitro, u različitim uvjetima, prije i nakom liječenja, te izravno, primjenom protutijela usmjerenih na ciljne proteine i metilne skupine. Rezultati istraživanja su pokazali da patološki snižen metilacijski indeks, koji se javlja u okviru nedostatne aktivnosti SAHH, dovodi do poremećaja metabolizma aDMA zbog dismetilacije proteina. U plazmi pacijenata Y143C SAHH W112X, koncentracija aDMA je statistički značajno povišena, a liječenje dijetom siromašnom metioninom je normalizira. U modelu in vitro, koncentracija aDMA u mediju i fibroblastima Y143C SAHH W112X je značajno niža u uvjetima dodatno sniženog MI, uz dodatak AdoHcy. Dodavanje AdoMet i AdoHcy kontrolnim fibroblastima, dovelo je do sniženja koncentracije aDMA, slično kao što je to u fibroblastima Y143C SAHH W112X, u nativnim uvjetima. Na većem broju eksperimenata pokazali smo reproducibilnost rezultata. Istražujući multifunkcionalan protein SAHH koji je, uključen u brojne transmetilacijske reakcije, otkrili smo poremetnju u procesiranju (količina izoformi proteina i njihov omjer, razina posttranslacijske metilacije) drugog multifunkcionalan proteina, heterogenog ribonuklearnog proteina tipa A1 (hnRNP-A1), uključenog u procesiranje mRNA i kôd izrezivanja. Ovo za posljedicu može imati disfunkciju proteina hnRNP-A1, koja se može manifestirati poremećenim alternativnim izrezivanjem vlastite mRNA i neuravnoteženom sintezom i/ili razgradnjom izoformi KoB i AiA. Poremećaj na razini ova dva proteina dovodi do niza posljedica na razini funkcioniranja proteinskih mreža te time ionako kompleksnu patogenezu bolesti zbog manjka SAHH dodatno kompliciraju. Istražili smo posttranslacijsku razinu metilacije arginina odabranih proteina. Manjak SAHH dovodi do promjena na razini posttranslacijske metilacije proteina, s vrlo vjerojatno, raznim dodatnim sekundarnim, tercijarnim i brojnim drugim međudjelovanjima koja vode stvaranju "dismetilacijskog kruga". Stoga bismo lako mogli pretpostaviti da je bolest zbog manjka SAHH „globalna dismetilopatija ili epigenopatija“. Poseban patogenetski aspekt bolesti zbog manjka SAHH koji smo pokazali je hipometilacija arginina hnRNP-A1 koja remeti njegovu funkciju prilikom izrezivanja i procesiranja mRNA, a time i integraciju u složenu proteinsku mrežu što u konačnici vodi višeorganskoj bolesti kakvu viđamo u pacijenata s nedostatnom aktivnošću S-adenozilhomocistein hidrolaze.

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