Function and regulation of ABC transport proteins in rainbow trout (Oncorhynchus mykiss) tissues and cell lines

doctoral thesis

• Title (croatian): Funkcija i regulacija abc transportnih proteina u tkivima i staničnim linijama kalifornijske pastrve (Oncorhynchus mykiss)
• Author: Jovica Lončar
• Mentor: Tvrtko Smital (mentor)
• Committee member: Vladimir Mrša (predsjednik povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; Zagreb
• Defense date and country: 2015-07-16, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology
• Universal decimal classification (UDC): 60 - Biotechnology. Genetic engineering. Products of cloning

Abstract

We studied expression, regulation and function of the ABC transport proteins in ecotoxicologically relevant rainbow trout (Oncorhynchus mykiss) tissues and respective cell lines. We have done a thorough database search and found 49 ABC genes in rainbow trout. We did phylogenetic and syntenic analysis of the ABCB, C, G and H subfamilies, annotated new genes and confirmed conserved syntenic relationship for most of the genes of interest. We then evaluated gene expression of toxicologically relevant ABC transporter proteins in 7 rainbow trout tissues and cell lines. In order to test protein function we utilized the multixenobiotic resistance (MXR) assay that evaluates transport activity of target ABC proteins in relation to accumulation or efflux of specific protein substrates in inhibited versus uninhibited protein state. Finally, we obtained insight into transcriptional regulation of ABC transporters in response to model ligands/activators of toxicologically relevant transcription factors. To summarize, we offer new evidence that shows that although ABC transporters are expressed in all tested trout tissues and permanent cell lines, there are notable differences in expression levels. In addition, functional activity of most assayed transporters and transcription factors’ network implicated in transporter regulation was confirmed in studied trout cell lines. The obtained data should be taken in account when using these cell lines, especially in studies directed to extrapolation of the obtained data to mammalian systems. Stable trout cell lines appear to be a suitable model for basic screening of potential interactors, but not an optimal research model for detailed, single protein oriented studies for which heterologous overexpressing cell lines should rather be developed/used.

Abstract (croatian)

Proučavali smo ekspresiju, regulaciju i funkciju ABC transportnih proteina u ekotoksikološki važnim tkivima i staničnim linijama pastrve (Oncorhynchus mykiss). Detaljnom pretragom genomskih baza pronašli smo 49 gena kod pastrve. Proveli smo filogenetsku i sintenijsku analizu ABCB, C, G i H obitelji, imenovali nove gene te potvrdili sintenijsku očuvanost za najveći broj izučavanih gena. Potom smo izmjerili gensku ekspresiju toksikološki značajnih ABC transportnih proteina iz 7 tkiva i staničnih linija pastrve. Kako bi utvrdili funkciju proteina koristili smo test multiksenobiotičke otpornosti (MXR) koji procjenjuje transportnu aktivnost ciljanog proteina kroz nakupljanje ili izbacivanje određenog supstrata u neinhibiranom nasuprot inhibiranog stanja. Konačno, stekli smo uvide u transkripcijsku regulaciju ABC transportera u ovisnosti o modelnim ligandima/aktivatorima toksikološki značajnih transkripcijskih faktora. Sažeto, naša studija donosi nove dokaze koji pokazuju da ABC transporteri, iako eksprimirani u svim testiranim tkivima i staničnim linijama, pokazuju značajne razlike u razini ekspresije. Nadalje, na staničnim linijama smo potvrdili funkcionalnu aktivnost većine testiranih transportera kao i postojanje mreže transkripcijskih faktora uključenih u regulaciju transportera. Dobivene rezultate bi trebalo uzimati u obzir pri radu s navedenim staničnim linijama, posebno u studijama koje ciljaju na ekstrapolaciju rezultata na sisavce. Stabilne pastrvine stanične linije se čine kao pogodan model za temeljnu provjeru mogućih interaktora, ali ne i optimalan istraživački model za detaljno istraživanje pojedinačnog proteina za što bi trebalo koristiti/razviti heterologni stanični sustav s pojačano eksprimiranim pojedinačnim proteinom.

• Keywords: ABC transporters; rainbow trout; trout cell lines; qPCR; MXR
• Keywords (croatian): ABC transporteri; pastrva; pastrvine stanične linije; qPCR; MXR
• Language: english
• URN:NBN: urn:nbn:hr:159:347060
• Promotion: 2016-04-17
• Study programme: Title: Postgraduate PhD Study in Biotechnology and Bioprocess Engineering; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti (doktor/doktorica znanosti)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2018-05-17 11:40:54