Uloga aminoacil-tRNA-sintetaza u staničnom odgovoru na oksidacijski stres

doctoral thesis

• Title (english): The role of aminoacyl-tRNA synthetases in cellular oxidative stress response
• Author: Valentina Ević
• Mentor: Jasmina Rokov Plavec (mentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Ivana Leščić Ašler (član povjerenstva)
• Committee member: Morana Dulić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2024-03-07, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry
• Universal decimal classification (UDC): 54 - Chemistry. Crystallography. Mineralogy

Abstract

Aminoacil-tRNA-sintetaze (AARS) su enzimi koji kataliziraju nastanak kovalentne veze između pripadne aminokiseline i tRNA pri čemu nastaje aminoacil-tRNA, supstrat u procesu translacije. Citosolne AARS zbog redukcijskog okoliša inače nemaju disulfidnu vezu, no pronađena je u kristalnoj strukturi citosolne seril-tRNA-sintetaze (SerRS) iz biljke Arabidopsis thaliana. U ovom radu dizajnirane su jednostruko (SerRS_C213S, SerRS_C244S) i dvostruko mutirane inačice (SerRS_C213S/C244S) i pokazano je da dolazi do reverzibilnog nastajanja disulfidne veze što omogućuje enzimu nepromijenjenu aktivnost u oksidacijskom stresu. Također je istražen utjecaj mistranslacije uzrokovane ugradnjom valina (Val) ili norvalina (Nva) umjesto izoleucina na odgovor na oksidacijski stres. U tu svrhu korišteni su sojevi bakterije Escherichia coli koji eksprimiraju izoleucil-tRNA-sintetazu s inaktiviranom domenom za popravak pogreške (PS7066 i ED-). Rezultati su pokazali da prethodni uzgoj stanica u mistranslatirajućim uvjetima do stacionarne faze uzrokuje bolje preživljenje i rast u oksidacijskom stresu u odnosu na stanice koje nisu rasle u mistranslatirajućim uvjetima. Nadalje, uočeno je da uzgoj stanica s inaktiviranim RpoS-om u mediju s višim koncentracijama valina ili norvalina izaziva adaptaciju na oksidacijski stres koja je neovisna o RpoS-u, dok stanice uzgojene pri nižim koncentracijama valina ili norvalina ne preživljavaju u uvjetima oksidacijskog stresa.

Abstract (english)

Aminoacyl-tRNA synthetases (AARS) are enzymes that catalyze covalent bond formation between cognate amino acid and tRNA to produce aminoacyl-tRNA - substrate in the translation process. Due to the reductive environment, cytosolic AARSs do not have disulfide bonds, however, one was found in the crystal structure of cytosolic seryl-tRNA synthetase (SerRS) from the plant Arabidopsis thaliana. In this study, point (SerRS_C213S, SerRS_C244S) and double mutants (SerRS_C213S/C244S) were designed and it was shown that the reversible disulfide bond formation allows unchanged enzyme activity under oxidative stress. Also, the effect of isoleucine-to-valine (Val) or norvaline (Nva) mistranslation on oxidative stress response was investigated. For this purpose, Escherichia coli strains expressing isoleucyl-tRNA synthetase with a mutation in the editing domain were used (PS7066 and ED-). The results show that prior cell growth in mistranslating conditions to stationary phase leads to higher survival and growth rates under oxidative stress compared to cells that were not grown in mistranslating conditions. Furthermore, it was shown that prior growth of cells with inactivated RpoS in media with higher concentrations of Val or Nva leads to adaptation to oxidative stress that is RpoS- independent, while the cells grown with lower concentrations of Val or Nva do not survive in oxidative stress conditions.

• Keywords: aminoacil-tRNA-sintetaza; disulfidna veza; izoleucil-tRNA-sintetaza; mistranslacija; odgovor na stres; oksidacijski stres; seril-tRNA-sintetaza
• Keywords (english): minoacyl-tRNA synthetase; disulfide bond; isoleucyl-tRNA synthetase; mistranslation; stress response; oxidative stress; seryl-tRNA synthetase
• Language: croatian
• URN:NBN: urn:nbn:hr:217:345036
• Promotion: 2024
• Project: Number: IP-2016-06-6272; Title: Aminoacil-tRNA-sintetaze kao čuvari standardnog genetičkog koda; Title: Aminoacyl-tRNA synthetases as gatekeepers of the standard genetic code; Acronym: AARSCODE; Leader: Ita Gruić-Sovulj; Jurisdiction: Croatia; Funder: Hrvatska zaklada za znanost; Funding stream: Research Projects
• Project: Number: DOK-2018-09-1578; Title: Projekt razvoja karijera mladih istraživača - izobrazba novih doktora znanosti, mentor: J. Rokov Plavec; Title: Young Researchers’ Career Development Project - training new doctoral students, mentor: J. Rokov Plavec; Acronym: DOK-2018-09-Rokov Plavec; Leader: Jasmina Rokov Plavec; Jurisdiction: Croatia; Funder: Hrvatska zaklada za znanost; Funding stream: Young Researchers’ Career Development Project - Training New Doctoral Students
• Study programme: Title: Doctoral study; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica u području prirodnih znanosti (doktor/doktorica u području prirodnih znanosti)
• Type of resource: Text
• Extent: 130, XXXIII str. ; 30 cm
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2026-03-07
• Created on: 2024-03-11 12:23:39