Iskanje novih membranskih tarč proteinov iz družine egerolizinov

• Main Author: Krpan, Teja
• Other Authors: Sepčić, Kristina
• Format: Master Thesis
• Language: Slovenian
• Published: 2018
• Subjects: membranske tarče; egerolizini; ceramid fosfoetanolamin; ostreolizin A; nigerolizin A2; RahU; membrane targets; aegerolysins; ceramide phosphoetanolamine; ostreolysin A; nigerolysin A2; Domen; sami
• Online Access: https://repozitorij.uni-lj.si/IzpisGradiva.php?id=105136; https://repozitorij.uni-lj.si/Dokument.php?id=115732&dn=; https://plus.si.cobiss.net/opac7/bib/4894799?lang=sl

V družino egerolizinov uvrščamo čez 350 proteinov. Njihova biološka vloga ni povsem razjasnjena, najverjetneje pa sodelujejo pri razvoju in obrambi organizmov, ki jih proizvajajo. Egerolizini sami ali pa v kombinaciji s partnerskim proteinom delujejo porotvorno. Ker se vežejo le na določene lipide, se njihova potencialna uporaba kaže v zatiranju škodljivcev ali označevanju določenih domen v membrani, t. i. membranskih raftov. Nekatere od znanih membranskih tarč egerolizinov so ceramid fosfoetanolamin (CPE), CPE v kombinaciji s holesterolom (Hol) in kombinacija sfingomielina (SM) in Hol. Z namenom boljše opredelitve njihove biološke vloge smo želeli poiskati morebitne nove membranske tarče rekombinantnih egerolizinov, ostreolizina A (OlyA) iz gobe Pleurotus ostreautus, nigerolizina A2 (NigA2) iz nitaste glive Aspergillus niger in RahU iz bakterije Pseudomonas aeruginosa. Na različne načine smo testirali vezavo na lipide, izolirane iz žuželčjih celic Sf9 (iz molja Spodoptera frugiperda), ki so bili ločeni na polarno in nepolarno fazo. Ugotovili smo, da se egerolizini vežejo na lipide iz nepolarne faze. Tekočinska kromatografija v povezavi s tandemsko masno spektrometrijo z elektrorazpršilno ionizacijo (LC-ESI-MS/MS) je pokazala, da sta najbolj zastopana lipida v tej fazi fosfoetanolamin (PE) in fosfatidilholin (PC). Med lipidi nepolarne faze pa so tudi različne zvrsti CPE. Izkazalo se je, da trije testirani egerolizini vežejo različne zvrsti CPE. Z metodo točkovnega nanosa lipidov smo izvedli širše testiranje lipidnih ekstraktov različnih organizmov. Ugotovili smo, da so se izbrani rekombinantni egerolizini vezali na lipide nepolarne faze kvasovk in nitastih gliv in na lipide polarne faze bakterij, s čimer smo potrdili našo hipotezo o obstoju še nepoznanih membranskih tarč egerolizinov. The biological role of proteins of aegerolysin family is not defined but most probably they participate in development and defence of the organisms that produce them. Since their targets are specific lipids, aegerolysins show potential to be used as insecticides or as markers of specific membrane domains, so called membrane rafts. Some of the known lipid targets of aegerolysins are ceramide phosphoetanolamine (CPE), CPE combined with cholesterol (Chol) and the combination of sphingomyelin with Chol. With the purpose of better understanding their biological role, we tried to find new membrane targets of three recombinant aegerolysins, ostreolysin A (OlyA) from Pleurotus ostreatus, nigerolysin A2 (NigA2) from Aspergillus niger and RahU from Pseudomonas aeruginosa. During the extraction, lipids of Sf9 insect cells (derived from the moth Spodoptera frugiperda) were separated into two distinct phases: lipids of polar phase and lipids of nonpolar phase. We performed several binding tests and discovered that the three aegerolysins bind to the lipids of the nonpolar phase. Liquid chromatography- electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) showed that phosphoetanolamine (PE) and phosphocholine (PC) are the two most abundant lipids in nonpolar phase.However lipids of this phase also contain several CPE species. and the three aegerolysins bound to different ones. We performed a lipid dot blot assay on a broad range of lipid extracts from different organisms, i. e. yeasts, filamentous fungi and bacteria. While the binding of aegerolysins was observed to lipids of nonpolar phases of yeast and filamentous fungi, we also discover that the chosen aegerolysins bind to the lipids of the polar phase of bacterial lipids. Those may represent new potential targets, but further investigations are required.