Title Stanični mehanizmi uklanjanja mRNA i proteina SAHH kod pacijenata s nedostatnom aktivnosti S-adenozilhomocistein hidrolaze Title (english) Cellular mechanisms involved in turnover of pathogenic ahcy transcripts and protein identified in ahcy deficiency patients Author Filip Rokić Mentor Oliver Vugrek (mentor)Mentor Igor Jurak (komentor)Committee member Ivan Gudelj (predsjednik povjerenstva)Committee member Tihomir Balog (član povjerenstva)Committee member Neda Slade (član povjerenstva)Granter University of Rijeka Defense date and country 2024-06-19, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Universal decimal classificationUDC ) 577 - Biochemistry. Molecular biology. Biophysics Abstract Nedostatna aktivnost S-adenozilhomocistein hidrolaze (SAHH) je rijetka nasljedna
bolest ciklusa metionina uzrokovana točkastim mutacijama u genu SAHH. SAHH služi za
uklanjanje S-adenozilhomocisteina (SAH), nusprodukta i snažnog inhibitora brojnih staničnih
metiltransferaza, čime SAHH igra ključnu ulogu u održavanju staničnog metilacijskog
potencijala. Smanjena aktivnost ovog esencijalnog enzima rezultira značajnim biokemijskim
poremećajima kod ljudi, uzrokujući multisistemsku bolest karakteriziranu neurorazvojnim
zastojem, miopatijom i disfunkcijom jetre. Do danas je opisano svega 16 pacijenata s različitim
težinama bolesti, od kasne kliničke manifestacije do smrtnih ishoda tijekom dojenačke dobi.
Trenutni terapijski pristup uključuje dijetalnu restrikciju unosa metionina koja pokazuje
kliničku korist kod nekih bolesnika, iako varirajuće učinkovitosti. Prethodne studije pokazale
su da je razina inaktivacije enzima povezana s težinom bolesti i potencijalno, s ishodom
terapije. Cilj ovog doktorskog rada je istražiti funkcionalne posljedice tri varijante gena SAHH
(p.W112*, p.Y143C, p.Y328D) koje su otkrivene kod dva pacijenta s izrazito teškom
biokemijskom i kliničkom prezentacijom nedostatne aktivnosti SAHH-a do danas opisane u
literaturi. Naglasak ove studije je na učincima mutacija na gensku ekspresiju i staničnim
mehanizmima u pozadini značajnog nedostatka proteina SAHH, pružajući molekularne uvide
koji mogu pomoći u razvoju i promjeni novih terapijskih strategija u budućnosti.
Materijali i metode
U sklopu ove studije proveli smo sveobuhvatne analize razina mutiranih transkripata i
proteina SAHH u fibroblastima pacijenata i ekspresijskim sustavima in vitro. Rekombinantni
plazmidi koji sadrže mutirane cDNA pripremljeni su u svrhu izvanstanične ekspresije proteina
i pripreme ljudskih staničnih modela (Hek293, HepG2, SW480) putem lentivirusne
transdukcije. Biblioteke cDNA konstruirane su iz ukupne RNA izolirane iz stanica pacijenata
i staničnih modela HepG2 za interno sekvenciranje transkriptoma (mRNAseq).
Bioinformatička analiza korištena je za kvantificiranju ekspresije pojedinih alela SAHH u
fibroblastima pacijenata. Diferencijalna analiza ekspresije, funkcionalna anotacija i analiza
genske ontologije (GO) provedene su kako bi se identificirali geni s promijenjenom
ekspresijom u modelima HepG2. Razine mutiranih proteina analizirane su nakon tretmana
ljudskih staničnih modela inhibitorom proteasoma MG-132. Na kraju, provedena je računalna
analiza in silico kako bi se predvidjeli učinci pogrešnih mutacija na stabilnost proteina.
Rezultati
Analiza ekspresije proteina kombiniranih heterozigotnih fibroblasta (W112*/Y143C i
Y328D/Y143C) otkrila je smanjene razine SAHH-a kod oba pacijenta. Uz to, stanice koje
sadrže preuranjeni stop-kodon (PTC) pokazale su i smanjene razine transkripata.
Rekombinantni mutanti SAHH uspješno su sintetizirani korištenjem izvanstaničnog
ekspresijskog sustava in vitro, bez razlika u prinosu između varijanti. Analiza staničnih modela
sa stabilnom ekspresijom proteina pokazala je izrazito nisku ekspresiju SAHH-a s mutacijama
W112* i Y328D, za razliku od normalnih razina varijante SAHH-Y143C. Sekvenciranje
transkriptoma otkrilo je neuravnoteženu ekspresiju alela kod jednog pacijenta zbog potpunog
nedostatka transkripata koji sadrže PTC. U staničnim modelima HepG2, identificirano je 58
diferencijalno eksprimiranih gena koji su prvenstveno povezani s regulacijom ekspresije gena
i biosintezom staničnih makromolekula. Detaljna analiza istaknula je E3-ubikvitinsku ligazu
TRIM33 kao potencijalnog regulatornog faktora uključenog u nedostatak proteina SAHHY328D.
Inhibicija proteasoma povećala je ekspresiju varijante SAHH-Y328D u svim
pripremljenim staničnim modelima. Dodatno, konfokalna mikroskopija otkrila je izraženu
tendenciju ovog mutanta za nakupljanje unutar stanice. Konačno, analiza in silico potvrdila je
da aminokiselinska supstitucija Y328D značajno remeti stabilnost SAHH-a zbog značajnih
strukturnih promjena u hidrofobnoj jezgri proteina.
Zaključci
Značajno smanjene razina mRNA i proteina SAHH primijećene u stanicama pacijenata
mogu pridonositi težini fenotipa nedostatne aktivnosti SAHH-a. Prema dobivenim rezultatima,
mutacije Y328D i W112* primarni su čimbenici koji pridonose nedostatku ekspresije proteina
uočene kod pacijenata. Značajni nedostatak SAHH-a rezultat je stanične regulacije genske
ekspresije mehanizmima NMD i UPS. TRIM33 je predložen kao potencijalni regulator
pojačane proteasomske degradacije mutanta SAHH-Y328D.
Abstract (english) Introduction and aims
S-adenosylhomocysteine hydrolase (AHCY) deficiency is a rare hereditary disease of
methionine cycle caused by point mutations in the AHCY gene. AHCY functions as a disposal
route for S-adenosylhomocysteine (SAH), a byproduct and strong inhibitor of numerous
cellular methyltransferases, thus AHCY plays a pivotal role in maintaining cellular methylation
potential. Reduced activity of this essential enzyme results in significant biochemical
aberrations in humans, leading to a multisystemic disorder characterized by
neurodevelopmental delay, myopathy and liver dysfunction. To date, only 16 patients have
been reported with diverse disease severity, ranging from late clinical onset to fatal outcomes
during infancy. Current therapeutic approach involves dietary methionine restriction which
demonstrates clinical benefits in some patients, albeit with varying efficacy. Previous studies
have shown that the magnitude of mutational impact is associated with clinical presentation of
the disease and possibly therapy outcome. The aim of this doctoral thesis is to investigate
functional consequences of three AHCY variants (p.W112*, p.Y143C, p.Y328D) discovered in
two patients with extremely severe biochemical and clinical manifestations of AHCY
deficiency reported so far. This study focuses on differential expression effects of these
mutations and their underlying mechanisms involved in aberrant AHCY turnover, providing
molecular insights that can help guide the development and application of novel therapeutic
strategies in the future.
Materials and methods
In this study, we performed comprehensive analyses of mutant AHCY transcript and
protein levels in patient fibroblasts and in vitro expression systems. Recombinant plasmids
harboring mutant cDNAs were prepared for cell-free protein expression and the preparation of
human cell models (Hek293T, HepG2, SW480) via lentiviral transduction. cDNA libraries
were constructed from total RNA isolated from patient-derived cells and HepG2 models for inhouse
transcriptome sequencing (mRNAseq). Bioinformatic analysis was employed to quantify
the expression of individual AHCY alleles in patient fibroblasts. Differential expression
analysis, functional annotation and gene ontology (GO) analysis were performed to identify
genes with altered expression in HepG2 models. Mutant protein levels were analyzed following
treatment of human cell models with proteasome inhibitor MG-132. Lastly, in silico
computational analysis was performed to predict the effects of missense mutations on protein
stability.
Results
Protein expression analysis of compound heterozygote fibroblasts (W112*/Y143C and
Y328D/Y143C) revealed reduced AHCY levels in both patients. Additionally, cells harboring
premature termination codon (PTC) also displayed reduced transcript levels. Recombinant
AHCY mutants were successfully synthesized using in vitro cell free expression system,
without any discrepancies in yield across all variants. Analysis of stably expressing human cell
models showed markedly lower expression of AHCY harboring W112* and Y328D mutation,
contrasting normal levels of Y143C variant. Transcriptome sequencing revealed an allelic
imbalance in one patient due to complete absence of transcripts containing PTC. In HepG2 cell
models, 58 differentially expressed genes were identified that are primarily associated with
gene expression regulation and biosynthesis of cellular macromolecules. Detailed analysis
highlighted ubiquitin ligase TRIM33 as a potential regulator involved in aberrant turnover of
AHCY-Y328D mutant. Proteasome inhibition increased expression of Y328D variant across
all prepared human cell models. Moreover, microscopy reveled pronounced tendency of this
mutant protein to aggregate within the cell. Finally, in silico analysis confirmed that Y328D
substitution significantly disrupts AHCY stability due to structural changes in protein’s
hydrophobic core.
Conclusion
Considerable decrease of mRNA and protein levels observed in patient-derived cells
may contribute to the severity of AHCY deficiency phenotype. According to the results
obtained, Y328D and W112* mutations are primary factors contributing to the loss of protein
expression observed in the respective patients. Aberrant AHCY turnover is the result of cellular
regulation of gene expression through NMD and UPS mechanisms. TRIM33 is suggested to
have a regulatory role in proteasomal degradation of AHCY-Y328D mutant.
Keywords
nedostatna aktivnost SAHH-a
mutacije
genska ekspresija
NMD
UPS
Keywords (english)
AHCY deficiency
mutations
gene expression
NMD
UPS
Language croatian URN:NBN urn:nbn:hr:193:246003 Promotion 2024 Study programme Title: Medicinal chemistry Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2024-06-26 10:43:02
