Ciljni proteini za ADP-ribozilaciju iz bakterije Streptomyces coelicolor

master's thesis

• Title (english): ADP-ribosylation target proteins from bacterium Streptomyces coelicolor
• Author: Petra Kozulić
• Mentor: Andreja Mikoč (mentor)
• Committee member: Morana Dulić (predsjednik povjerenstva)
• Committee member: Iva Juranović Cindrić (član povjerenstva)
• Committee member: Ivica Đilović (član povjerenstva)
• Committee member: Aleksandra Maršavelski (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2020-10-28, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

ADP-ribozilacija je reverzibilna posttranslacijska modifikacija u kojoj se adenozin-difosfat-riboza (ADP-riboza) iz nikotinamid-adenin-dinukleotida (NAD) prenosi na ciljni protein. Enzimi koji prenose ADP-ribozu na ciljne proteine su ADP-ribozil-transferaze, a enzimi koji ovu modifikaciju uklanjaju su ADP-ribozil-hidrolaze. ADP-ribozilacija kontrolira brojne važne stanične procese u eukariota i prokariota. Bakterije iz roda Streptomyces, a posebno S. coelicolor, dobri su modelni organizmi za proučavanje ADP-ribozilacije kod bakterija, jer posjeduju gene za nekoliko vrsta enzima koji bi mogli biti uključeni u reakcije ADP-ribozilacije i de-ADP-ribozilacije. K tome, ADP-ribozilacija regulira proizvodnju antibiotika, sekundarnih metabolita koje ove bakterije proizvode.
Kod S. coelicolor identificirano je nekoliko ciljnih proteina za ADP-ribozilaciju. U sklopu ovoga rada iz genoma bakterije S. coelicolor tehnologijom rekombinantne DNA klonirani su geni pet odabranih proteina za koje je pokazano da su mete ADP-ribozilacije. Geni su prekomjerno eksprimirani, a rekombinantni proteini pročišćeni afinitetnom kromatografijom. Provedeno je testiranje pročišćenih ciljnih proteina kao supstrata u reakciji ADP-ribozilacije in vitro pomoću enzima ADP-ribozil-transferaze SCO5461, čija je aktivnost od prije poznata, ali nisu poznati njeni proteinski supstrati. Reakcijom ADP-ribozilacije in vitro potvrđena je sposobnost enzima SCO5461 da modificira samoga sebe te je pokazano da ADP-ribozilira tri odabrana ciljna proteina.
Ovaj rad doprinosi istraživanju ADP-ribozilacije kod streptomiceta koje će omogućiti bolje razumijevanje i kontrolu metaboličkih puteva ovih važnih proizvođača velikog broja bioaktivnih spojeva.

Abstract (english)

ADP-ribosylation is a reversible post-translational modification in which adenosine-diphosphate-ribose (ADP-ribose) is transferred from nicotinamide-adenine-dinucleotide (NAD) to the target protein. The enzymes which transfer ADP-ribose onto target proteins are ADP-ribosyl-transferases, while the enzymes which reverse this modification are ADP-ribosyl hydrolases. ADP-ribosylation controls many important cellular processes in eukaryotes and prokaryotes. Bacteria of the genus Streptomyces, in particular S. coelicolor, are good model organisms for studying ADP-ribosylation in bacteria because they have genes for several enzyme types that might be included in ADP-ribosylation and de-ADP-ribosylation reactions. In addition, ADP-ribosylation regulates the production of antibiotics which are secondary metabolites produced by these bacteria.
Several target proteins for ADP-ribosylation have been identified in S. coelicolor. In this research recombinant DNA technology has been used to clone the genes from five selected proteins from genome of S. coelicolor bacterium, which have been proven to be ADP-ribosylated. Genes are overexpressed, while recombinant proteins are purified by affinity chromatography. The testing was carried out on purified target proteins as substrates in reaction of ADP-ribosylation in vitro by using the enzyme ADP-ribosyl-transferase SCO5461 of which activity has been previously known, but its protein substrates have been unknown. ADP-ribosylation reaction in vitro has confirmed the ability of SCO5461 enzyme to modify itself and also demonstrated its ability to modify three selected target proteins.
This work contributes to the research of ADP-ribosylation in streptomyces, which will provide better understanding and control of metabolic pathways of these important producers of numerous bioactive compounds.

• Keywords: ADP-ribozilacija; protein; posttranslacijska modifikacija; Streptomyces coelicolor
• Keywords (english): ADP-ribosylation; protein; post-translational modification; Streptomyces coelicolor
• Language: croatian
• URN:NBN: urn:nbn:hr:217:053774
• Study programme: Title: Graduate university studies in chemistry; specializations in: Research programme; Course: Research programme; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2024-10-28
• Created on: 2020-12-10 14:10:37