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Sekretorne fosfolipaze A2 (sPLA2) so ena od družin fosfolipaz A2, encimov, ki katalizirajo hidrolizo estrske vezi v glicerofosfolipidu na mestu sn-2, pri čemer se sprostita prosta maščobna kislina in lizofosfolipid. Vendar pa sPLA2 lahko delujejo tudi kot ligandi za raznolike proteinske receptorje, zato je njihova (pato)fiziologija kompleksna in kljub intenzivnim raziskavam še vedno pogosto nepojasnjena na molekulskem nivoju. Med boljše preučenimi je molekulski mehanizem delovanja ß-nevrotoksičnih sPLA2 iz kačjih strupov (ß-ntx), ki poškodujejo živčne končiče, kar povzroči blokado živčno-mišičnega prenosa in posledično flacidno paralizo. Pri tem je ključna interakcija ß-ntx z mitohondriji. Toksične učinke na živčne celice ima v patoloških okoliščinah tudi sesalska sPLA2 skupine IIA (GIIA), npr. pri nekaterih nevrodegenerativnih boleznih. Ker so sesalske GIIA in viperidne ß-ntx ortologne molekule, bi lahko imeli opaženi učinki enako molekulsko osnovo. Da bi pridobili bolj jasen vpogled v (pato)fiziološko delovanje sesalskih in ß-nevrotoksičnih GIIA na molekulskem nivoju, smo raziskali procese, ki jih amoditoksin (Atx) in njegov podganji ortolog (rGIIA) prožita v mitohondrijih živčnih celic, ter raziskali njun vnos in razmeščanje v teh celicah. Pripravili smo rekombinantna proteina Atx in rGIIA ter njuna encimsko neaktivna mutanta Atx(D49S) in rGIIA(D49S). Pokazali smo, da vsi štirje produkti interagirajo s podenoto II citokrom c oksidaze (CCOX-II). rGIIA in rGIIA(D49S) sta se na ta ključni kompleks dihalne verige vezala s približno 100-krat nižjo afiniteto kot Atx. Kljub temu pa sta obe molekuli rGIIA močno zavirali encimsko aktivnost CCOX v mitohondrijih, izoliranih iz celic PC12. rGIIA se je vezala tudi na mitohondrijski protein z navidezno molekulsko maso 20 kDa, katerega identiteta pa zaenkrat ostaja neznana. Z elektronsko in konfokalno mikroskopijo smo pokazali, da se Atx in rGIIA vnašata v celice PC12 in kolokalizirata z mitohondriji neodvisno od njune fosfolipazne aktivnosti. To nakazuje, da obe sPLA2 za celični vnos uporabljata enako pot. Atx(D49S) in rGIIA(D49S) sta imela tudi podoben učinek v celicah PC12, kjer sta znižala mitohondrijski membranski potencial, ter tkivnih rezinah podganje možganske skorje, kjer sta zavirala encimsko aktivnost CCOX. Rezultati torej kažejo, da ß-nevrotoksične GIIA iz kačjih strupov in sesalske GIIA učinkujejo na nevronske mitohondrije po podobnem molekulskem scenariju, ter nakazujejo verjeten mehanizem, po katerem je GIIA vpletena v Alzheimerjevo bolezen. V prizadevanju za razvoj novih postopkov za diagnostiko, preventivo in zdravljenje Alzheimerjeve ter sorodnih nevrodegenerativnih bolezni bodo naši rezultati dobro izhodišče za testiranje GIIA kot novega zgodnjega označevalca ter potencialne terapevtske tarče pri teh boleznih. Secretory phospholipases A2 (sPLA2) are a family of phospholipase A2 enzymes that catalyze the hydrolysis of the ester bond in glycerophospholipids at the sn-2 position, releasing free fatty acids and lysophospholipids. However, sPLA2s can also act as ligands for diverse protein receptors, making their (patho)physiology complex and, despite intensive research, often unexplained at the molecular level. Among the best studied is the molecular mechanism of action of β-neurotoxic sPLA2s from snake venoms (β-ntx). β-ntx damage nerve endings, resulting in the blockade of neuromuscular transmission and subsequent flaccid paralysis. In this process, interaction of β-ntx with mitochondria plays a key role. Mammalian group IIA sPLA2 (GIIA) can also have toxic effects on nerve cells under pathological conditions, e.g. in some neurodegenerative diseases. Since mammalian GIIA and viperid β-ntx are orthologous molecules, the observed effects could have the same molecular basis. To gain a better insight into the (patho)physiological action of mammalian and β-neurotoxic GIIA sPLA2s at the molecular level, we investigated the processes triggered by ammodytoxin (Atx) and its rat ortholog (rGIIA) in the mitochondria of nerve cells and investigated their uptake and localization in these cells. We prepared recombinant Atx and rGIIA and their enzymatically inactive mutants, Atx(D49S) and rGIIA(D49S). We showed that all four products interact with cytochrome c oxidase subunit II (CCOX-II). rGIIA and rGIIA(D49S) bound to this essential complex of the respiratory chain with approximately 100-fold lower affinity than Atx. Nevertheless, both rGIIA molecules strongly inhibited CCOX enzyme activity in isolated mitochondria from PC12 cells. rGIIA also bound to a mitochondrial protein with an apparent molecular mass of 20 kDa, however, the identity of this receptor remains unknown. With transmission electron and confocal microscopy, we showed that Atx and rGIIA internalize into PC12 cells and colocalize with mitochondria independently of their phospholipase activity. This suggests that both sPLA2s use the same pathway for cellular uptake. Atx(D49S) and rGIIA(D49S) also had a similar inhibitory effect on mitochondrial membrane potential and CCOX activitiy in PC12 cells and rat brain tissue sections, respectively. The results therefore show that β-neurotoxic GIIA from snake venoms and mammalian GIIA share a similar molecular background in their effects on neuronal mitochondria, suggesting a possible mechanism by which mammalian GIIA is involved in Alzheimer’s disease. In an effort to develop new procedures for diagnosis, prevention and treatment of Alzheimer's and related neurodegenerative diseases, the results of this work will be a good starting point for testing GIIA as a new early marker and potential therapeutic target in these diseases.

Amoditoksin A (AtxA) je sekretorna fosfolipaza A2 (sPLA2) iz modrasovega strupa, ki ima močan presinaptično nevrotoksični ali &#946 -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 (&#946 -neurotoxic) secreted phospholipase A2 (sPLA2) from the venom of the nose-horned viper (Vipera ammodytes ammodytes). The molecular mechanism of action of &#946 -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.