20.500.12556/RUL-95634 Priprava mutant amoditoksina A za ultrastrukturno lokalizacijo v mitohondriju Preparation of ammodytoxin A mutants for ultrastructural localisation in mitochondria Amoditoksin A (AtxA) je sekretorna fosfolipaza A2 (sPLA2) iz modrasovega strupa, ki ima močan presinaptično nevrotoksični ali β-nevrotoksični učinek. Delovanje AtxA na molekulskem nivoju še ni v celoti pojasnjeno. Učinki tega toksina, med drugimi, vključujejo tudi nabrekanje nevronskih mitohondrijev in porušenje njihove notranje strukture. Ob tem ostaja nejasno, na kakšen način je v molekulski mehanizem nevrotoksičnosti AtxA vpletena encimska aktivnost toksina in ali je le-ta potrebna za njegov vstop v mitohondrij. V magistrskem delu smo uspešno pripravili molekularno-biološka orodja za preučevanje vloge encimske aktivnosti AtxA v molekulskem mehanizmu nevrotoksičnosti. S pomočjo mestno-specifične mutageneze, izražanja v E. coli in in vitro renaturacije smo pripravili rekombinantne proteine, AtxA(dt) (AtxA(dt)), AtxA(D49S) in AtxA(D49S/N79C), jih temeljito okarakterizirali in potrdili, da imajo pravilno molekulsko maso in aminokislinsko zaporedje na N-koncu ter so pravilno zviti. Preverili smo jim tudi encimsko aktivnost in, v skladu s predvidevanji, ugotovili, da mutanta AtxA(D49S) in AtxA(D49S/N79C) nista bila encimsko aktivna, AtxA(dt) pa je imel fosfolipazno aktivnost. Prav tako smo potrdili predvidevanje, da mutaciji ne bosta bistveno vplivali na interakcijo rekombinantnih proteinov z Atx-vezavnim proteinom kalmodulinom (CaM). Ammodytoxin A (AtxA) is a presynaptically neurotoxic (β-neurotoxic) secreted phospholipase A2 (sPLA2) from the venom of the nose-horned viper (Vipera ammodytes ammodytes). The molecular mechanism of action of β-neurotoxins is not yet fully understood. Effects of this toxin include swelling of mitochondria and loss of mitochondrial structural integrity. It remains unclear, however, what is the role of enzymatic activity of AtxA in the neurotoxic process and how AtxA translocates into the cell and mitochondria. To study these processes we prepared in this work several molecular-biological tools. Using the methods of site-directed mutagenesis, protein expression in E. coli and in vitro renaturation we have succesfully isolated three recombinant proteins: AtxA wild type, AtxA(D49S) and AtxA(D49S/N79C). We have demonstrated that their molecular masses, N-terminal amino acid sequences and enzymatic activity were exactly as expected. By the means of heterologous competition of 125I-AtxC binding to calmodulin (CaM) we have additionally confirmed that the recombinant proteins were correctly refolded and, that the mutations D49S and N79C did not significantly affect the interaction between recombinant proteins and Atx-binding protein CaM. fosfolipaze sPLA2 amoditoksin &#946 -nevrotoksičnost proteinsko inženirstvo phospholipases sPLA2 ammodytoxin &#946 -neurotoxicity protein engineering true false false Slovenski jezik Neznan jezik Magistrsko delo/naloga 2017-09-21 07:53:10 2017-09-21 07:53:11 2022-08-10 06:39:15 0000-00-00 00:00:00 2017 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 143403 1070.pdf 1070.pdf 1 60F5D86268498FA180D30D30BF6183C7 d85031861736bf1a620a8257b49b55cbcc3cd7597c277cf2f2bda8c3df38045f ce9bc621-a1b3-11eb-a523-00155dcfd717 20.500.12556/rul/13f5f6d0-a924-4c6c-bc62-7615d3b299cd https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=104259 Biotehniška fakulteta 0 0 0