Functional proteomics of Arion vulgaris and Drosophila melanogaster by mass spectrometry approach

Abstract

Functional proteomics represent an approach which could provide answers to two major questions: the revelation of biological functions of unknown proteins and the description of cellular mechanisms at the protein level. The Spanish slug, Arion vulgaris, is one of the hundred most invasive species in Central Europe. No transcriptomic or proteomic study on A. vulgaris has been carried out so far. In this thesis, the first transcriptomic database from adult specimens of A. vulgaris is reported. To facilitate and enable proteomics in this non-model organism, a mRNA-derived protein database was constructed for protein identification. A gel-based proteomic approach was used to obtain the first generation of a comprehensive slug mantle proteome. A total of 2128 proteins were unambiguously identified from which 48 proteins represent novel proteins with no significant homology in NCBI non-redundant database. Combined transcriptomic and proteomic analysis revealed an extensive repertoire of novel proteins with a role in innate immunity, including many associated pattern recognition proteins, effector proteins and cytokine-like proteins. The immense and very successful adaptation and spreading of A. vulgaris suggests that it developed highly effective mechanisms to deal with infections and natural predators. The number and diversity in gene families encoding lectins point to a complex defense system, probably as a result of adaptation to a pathogen-rich environment. These results are providing a fundamental and important resource for subsequent studies on molluscs, as well as for putative antimicrobial compounds for drug discovery and biomedical applications...

Funkcionalna proteomika danas predstavlja metodu koja može da ukaže na odgovor na dva pitanja: koja je biološka funkcija novootkrivenih proteina i da okarakteriše ćelijske procese na proteinskom nivou. Arion vulgaris predstavlja jednu od sto najinvaznivijih vrsta u Evropi. Do sada u nauĉnoj literaturi nisu zabeleženi podaci o iRNK i proteinskom profilu ove vrste. U prvom delu teze obraĊena je data problematika gde su prikazani rezultati analize iRNA na osnovu ĉega je napravljena baza podataka koja je korišćena za identifikaciju proteina. Ukupan broj identifikovanih proteina, dobijenih tripsinskom digestijom u gelu i analizom na masenom spektrometru, iznosi 2128. Od ukupnog broja indentifikovanih proteina, 48 nema homologe u nacionalnom centru za biotehnološke informacije (NCBI) i predstavljaju do sada neopisane proteine. Kombinacijom transkriptoma i proteoma opisan je ĉitav repertoar kako novih, tako i već poznatih proteina koji imaju ulogu u uroĊenom imunom sistemu, kao na primer „associated pattern recognition“, efektorski i citokinu-sliĉni proteini. Dobijeni rezultati ukazuju na mehanizam borbe protiv infekcija i prirodnih neprijatelja, koji verovatno omogućavaju uspešnu adaptaciju i široku rasprostranjenost ove vrste. Velika raznovrsnost gena koji kodiraju lektine ukazuje na odbrambeni sistem vrste Arion vulgaris u sredini koji je bogata patogenima. Rezultati koji su proizašli iz ove teze pružaju fundametalno znanje o proteomskom profilu ove vrste mekušaca, ukljuĉujući i 48 novih proteina. Drugi deo teze je fokusiran na „gel-free bottom-up“ analizu proteina iz membranskih frakcija glave Drosophila melanogaster...