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Karakterizacija vezave izbranih egerolizinov na lipidne vezikle z različno sestavo.
Mordej, Margareta (Author), Sepčić, Kristina (Mentor) More about this mentor... This link opens in a new window, Novak, Maruša (Co-mentor)

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Abstract
Pomembna lastnost egerolizinov je vezava na različne lipide, lipidne vezikle ali lipidne dvosloje. Egerolizini lahko oblikujejo pore sami ali v kombinaciji s partnerskim proteinom z domeno MACPF (angl. membrane attack complex/perforin). Zaradi tega so uporabni kot označevalci lipidov in primerni za zatiranje škodljivih organizmov. Eden izmed pomembnih vezavnih partnerjev egerolizinov je ceramid fosfoetanolamin (CPE), sfingolipid, ki je najbolj zastopan v membranah nevretenčarjev (predvsem žuželk) in praživali. V sklopu magistrskega dela smo se osredotočili na tri egerolizine: nigerolizin A2 (NigA2) iz nitaste glive Aspergillus niger, pleurotolizin A2 (PlyA2) iz gobe Pleurotus eryngii in RahU iz bakterije Pseudomonas aeruginosa z namenom bolje opredeliti njihove vezavne lastnosti na lipidne vezikle, sestavljene iz različnih deležev CPE in holesterola (Hol) ali drugih sterolov. To smo preverili s testom sedimentacije in s površinsko plazmonsko resonanco. S testom sproščanja kalceina iz različnih lipidnih veziklov, smo spremljali permeabilizacijsko aktivnost PlyA2 v kombinaciji s partnerskim proteinom pleurotolizinom B (PlyB). Odkrili smo, da je PlyA2 najprimernejši marker za označevanje membran, ki vsebujejo CPE, medtem ko sta NigA2 in RahU za to manj primerna. Za uspešno vezavo PlyA2 na lipidne vezikle je potrebnih vsaj 5 molskih % CPE in 30 molskih % Hol. Prav tako se dani egerolizin veže na lipidne vezikle sestavljene iz CPE in različnih sterolov (najmočnejša vezava je bila zaznana v primeru 7-dehidroholesterola in β-sitosterola). Pokazali smo tudi, da PlyA2 v kombinaciji s PlyB permeabilizira membrane lipidnih veziklov, ki vsebujejo CPE in različne sterole, kar nakazuje na možnost uporabe teh dveh proteinov za zatiranje škodljivih organizmov.

Language:Slovenian
Keywords:egerolizini, nigerolizin A2, RahU, pleurotolizin A2, ceramid fosfoetanolamin, različni steroli
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2018
Publisher:[M. Mordej]
UDC:606:63:577.115(043.2)
COBISS.SI-ID:9009273 Link is opened in a new window
Views:301
Downloads:182
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Secondary language

Language:English
Title:Binding characterization of selected aegerolysins to lipid vesicles with different composition.
Abstract:
Important feature of aegerolysins is binding to various lipids, lipid vesicles or lipid bilayers. Aegerolysins can form pores directly or in combination with a partnering protein containing a MACPF (membrane attack complex/perforin) domain. They can therefore be used as specific lipid markers and for the pest control. One of the important binding partners of aegerolysins is ceramide phosphoethanolamine (CPE), a sphingolipid that is very abundant in the membranes of the invertebrates (particularly insects) and protozoa. Within M. Sc. Thesis we focused on three aegerolysins: nigerolysin A2 (NigA2) from the filamentous fungus Aspergillus niger, pleurotolysin A2 (PlyA2) from the mushroom Pleurotus eryngii and RahU from the bacterium Pseudomonas aeruginosa. Our aim was to determine their binding characteristics to the lipid vesicles composed of different molar ratios of CPE and cholesterol (Chol) or other sterols using sedimentation assay and surface plasmon resonance. Monitoring the calcein release from various lipid vesicles, the permeabilization activity of aegerolysin PlyA2 in combination with its partnering protein, pleurotolysin B (PlyB), was tested. We have discovered that PlyA2 is the most appropriate marker for labeling membranes containg CPE, while NigA2 and RahU are less suitable. For the successful binding of PlyA2 to lipid vesicles, at least 5 mol % of CPE and 30 mol % of Chol are required. In addition, PlyA2 binds to lipid vesicles composed of CPE and various sterols (the strongest binding was detected in the case of 7-dehydrocholesterol and β-sitosterol). We have also shown that PlyA2 in combination with PlyB permeabilizes membranes of lipid vesicles containing CPE and various sterols, indicating their potential to be used as pest control agents.

Keywords:aegerolysins, nigerolysin A2, RahU, pleurotolysin A2, ceramide phosphoethanolamine, different sterols

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