Priprava in biokemijska karakterizacija rekombinantnih egerolizinov s podaljšanim N-koncem

Uzar, Aleksandra (author), Anderluh, Gregor (mentor), Pavšič, Miha (comentor)

Egerolizini so relativno majhni proteini (15–20 kDa) z značilnim zvitjem s prevladujočimi β-strukturami. Predstavniki družine so prisotni v mnogih taksonomskih skupinah, največ pa jih najdemo pri glivah. Kljub relativno široki taksonomski porazdelitvi je le nekaj egerolizinov natančno raziskanih. Biološka vloga egerolizinov pri glivah še vedno ni popolnoma razjasnjena. Trenutno obstaja le hipotetičen nabor možnih bioloških vlog. Glede na znane podatke verjetno delujejo med rastjo in razvojem organizma ali igrajo pomembno vlogo pri obrambi in preživetju organizmov. Značilnost nekaterih egerolizinov je, da delujejo hemolitično in citolitično, a v nekaterih primerih do aktivnosti pride šele ob vezavi partnerskega proteina MACPF. Opažena je bila še sposobnost vezave lipidov, pri tem pa se specifičnost vezave na določeno vrsto lipida med različnimi egerolizini razlikuje. V sklopu tega dela smo obravnavali štiri egerolizine z neobičajnim N-končnim podaljškom iz različnih glivnih organizmov. Zanimivi so predvsem zato, ker so na podlagi zaporedja drugačni kot večina ostalih egerolizinov. Raziskovali smo, kako Nkončni podaljšek vpliva na strukturo in funkcijo egerolizinov. Rezultate smo primerjali z znanim egerolizinom OlyA (ostreolizin A) in z aktinoporinom EqtII (ekvinatoksin II), katerega N-končno zaporedje z motivom α-vijačnice pomembno vpliva na njegovo citolitično aktivnost. Z bioinformacijsko analizo smo napovedali in preučili strukturne modele tarčnih egerolizinov ter nekatere druge biokemijske lastnosti. Predvideli smo, da imajo štirje egerolizini: fusver, fusgra, phiatt in traver podaljšan N-konec z napovedano strukturo αvijačnice. Ta je pri egerolizinih fusver, fusgra, phiatt amfipatična in bi lahko služila vezavi v membrano. Pripravili smo vse štiri egerolizine in pokazali, da noben od njih ni hemolitičen, niti se ne veže na različne liposome. Za namen določitve strukture, smo egerolizine poskusili kristalizirati, kar nam v izbranem naboru kristalizacijskih pogojev ni uspelo.

• Language: Slovenian
• Keywords: egerolizin, podaljšan egerolizin, lipidne membrane, bioinformacijska analiza, ostreolizin A, ekvinatoksin II
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-134910
• COBISS.SI-ID: 97991683
• Publication date in RUL: 11.02.2022

Secondary language

• Language: English
• Title: Preparation and biochemical characterization of recombinant aegerolysins with an N-terminal extension

Abstract

Aegerolysins are relatively small proteins (15–20 kDa) with characteristic β-structural fold. Representatives of the aegerolysin protein family are found in many taxonomic groups; however, they are most commonly found in fungi. Despite a relatively broad taxonomic distribution, only a few aegerolysin-like proteins have been thoroughly researched. The biological role of aegerolysins in fungi is still not fully elucidated. Currently, there is only a hypothetical set of possible biological roles. According to known data, they are likely to act during the growth and development of the organism and/or they play an important role in the defence and survival of the organisms. Some aegerolysins are known to have hemolytic and cytolytic activity, but in most cases that activity occurs only upon interaction with a partner MACPF-domain protein. The ability to bind lipids has also been observed, but the specificity of binding to a particular type of lipid varies between representative proteins. As part of this work, we considered four fungal aegerolysins with a specific N-terminal extension. They are interesting mainly because of their difference from most aegerolysins in terms of the sequence. We investigated how the N-terminal extension affects the structure and function of aegerolysins. The results were compared to the known aegerolysin OlyA (ostreolysin A) and to actinoporin EqtII (equinatoxin II), whose Nterminal α-helix structure significantly affects its cytolytic activity. Using bioinformaticc analysis, we predicted and studied structural models of target aegerolysins and some other biochemical properties. We predicted that four aegerolysins: fusver, fusgra, phiatt, and traver have an elongated N-terminus with a predicted α-helix structure. This is amphipathic in the case of aegerolysin fusver, fusgra, phiatt and could potentially bind to the membrane. We prepared and purified all four aegerolysins and showed that none of them were hemolytic, nor did they bind to different liposomes. For the purposes of determining the structure, we tried to crystallize aegerolysins. We were not able to achieve the aegerolysins crystallization in the selected set of crystallization conditions.

• Keywords: aegerolysin, extended aegerolysin, lipid membranes, bioinformatic analysis, ostreolysin A, equinatoxin II