20.500.12556/RUL-184337 Molekularni mehanizem citotoksičnosti proteinov NLP Molecular mechanism of cytotoxicity of NLP proteins NLP-ji so apoplastni efektorji z dvojno vlogo, saj delujejo kot imunogeni molekulski vzorci in kot porotvorni citotoksini, vendar mehanizem njihovega toksičnega delovanja ostaja slabo pojasnjen. Izločajo jih mikrobni patogeni, kot so glive, bakterije in oomicete. Za oomicetne NLP-je je bilo pokazano, da se vežejo na glikozilinozitol fosfoceramid (GIPC), sfingolipid plazemske membrane rastlin. V tej raziskavi smo z biokemijskimi in biofizikalnimi pristopi proučevali nekrotično aktivnost ter vezavo na GIPC proteinov MpNEP2 iz glive Moniliophthora perniciosa ter NLPPp iz oomicete Phytophthora parasitica in NLPPya iz Phytium aphanidermatum. Pripravili smo modelne membranske sisteme, ki so vsebovali GIPC, izolirane iz tobaka (dvokaličnica) in pora (enokaličnica). Pokazali smo, da se MpNEP2 in NLPPya vežeta na liposome z GIPC iz obeh rastlinskih virov ter izkazujeta porotvorno aktivnost, s čimer prvič dokazujemo, da NLP permeabilizirajo membrane tako dvokaličnic kot enokaličnic. NLPPp se prav tako veže na liposome z GIPC, vendar šibkeje, kar se odraža v manj vezanega proteina na sedimentacijskem testu ter v nižjem odzivu in hitrejši disociaciji pri meritvah s površinsko plazmonsko resonanco. Kristalna zgradba NLPPp je razkrila ohranjeno NLP-zvitje, vendar je primerjalna analiza pokazala ožji in manj dostopen vezavni žleb ter bolj negativno nabito površino v primerjavi z NLPPya in MpNEP2, kar je skladno z njegovo šibkejšo vezavo in nižjo aktivnostjo. Točkovne mutacije načrtovane na podlagi primerjave z NLPPya, usmerjene v vezavni žleb, niso bistveno vplivale na aktivnost, kar nakazuje, da je funkcija NLP odvisna od kombinacije strukturne organizacije, konformacijske dinamike in elektrostatskih lastnosti vezavnega mesta. NLPs are apoplastic effectors with both immunogenic and pore-forming modes of action, yet their toxic mechanism remains poorly understood. They are secreted by microbial pathogens, such as fungi, bacteria and oomycetes. Oomycetal NLPs were reported to bind to GIPC, a sphingolipid of the plant plasma membrane. Here, we used biophysical and biochemical approaches to investigate the necrotic activity and GIPC binding of fungal MpNEP2 from Moniliophthora perniciosa, and oomycetal NLPPp from Phytophthora parasitica and NLPPya from Phytium aphanidermatum. Model membrane systems containing GIPCs extracted from tobacco (dicot) and leek (monocot) were prepared. MpNEP2 and NLPPya bound to GIPC-containing liposomes from both plant sources and exhibited pore-forming activity, demonstrating for the first time that NLPs permeabilize not only dicot but also monocot membranes. NLPPp also bound GIPC-containing liposomes, albeit more weakly, as indicated by sedimentation assay and lower response units and faster dissociation in surface plasmon resonance measurements. The crystal structure of NLPPp revealed a conserved NLP fold, but structural comparison showed a narrower, less accessible binding crevice and a more negatively charged surface relative to NLPPya and MpNEP2, consistent with its reduced binding and activity. Site-directed mutagenesis designed based on the comparison with NLPPya, targeting the binding crevice, did not significantly enhance activity, indicating that NLP function depends on the combined effects of structural organization, conformational dynamics, and electrostatic properties. rastlinski toksini NLP rastlinska plazemska membrana glikozilinozitol fosfoceramidi makromolekularna kristalografija interakcije med proteini in lipidnimi membranami umetni lipidni membranski sistemi plant toxins NLP plant plasma membrane glycosylinositol phosphoceramides macromolecular crystalography interactions between proteins and lipid membranes artificial membrane systems true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2026-07-05 07:15:28 2026-07-05 07:15:43 2026-07-06 10:21:19 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 280497 283705859 57743.pdf 57743.pdf 1 582C4BF3AEFB7A62B4ACFF922A686337 f523eea4d87c7528335cbb2c22c358c53474f798106f34c7f978401fd1127072 7bf7a97e-7830-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=237017 Biotehniška fakulteta 0 0 0