The influence of selected peptide and protein inhibitors on the activity of some algal type I, II and III metacaspases

Proteaze so hidrolaze, ki jih najdemo pri rastlinah, živalih in mikroorganizmih in omogočajo znotrajcelično in zunajcelično razgradnjo proteinov. V splošnem jih delimo v štiri večje skupine, in sicer metaloproteaze, aspartatne, serinske in cisteinske proteaze. Aktivnost slednjih kontrolirajo njihovi endogeni inhibitorji, najbolj pogosti so cistatini, tiropini in α2-makroglobulin. Vrsta cisteinskih proteaz so tudi kaspaze. V celicah so prisotne kot prokaspaze v neaktivni obliki. Za njihovo aktivacijo je nujna proteolitična cepitev na dveh mestih. Metakaspaze so edini kaspazni homologi, ki poleg domene p20 vsebujejo tudi domeno p10. Od kaspaz se razlikujejo po tem, da za svojo katalitično aktivnost ne potrebujejo oligomerizacije. Metakaspaze najdemo pri glivah, praživalih in rastlinah. Sodelujejo v procesu programirane celične smrti in uravnavajo celično homeostazo. Glede na položaj domen p20 in p10 v polipeptidni verigi poznamo metakaspaze tipa I, tipa II in zaenkrat še najmanj raziskan, tip III. Namen moje diplomske naloge je bil ugotoviti, kako intenzivno različni inhibitorji zavirajo aktivnost posameznega tipa metakaspaze. Uporabili smo metakaspazo tipa II, CrMC2, iz alge Chlamydomonas reinhardtii ter metakaspazo tipa I, GtMC1, in metakaspazo tipa III, GtMC2, iz organizma Guillardia theta. Preizkusili smo sedem različnih inhibitorjev: antipain, levpeptin, E-64, FFR-CMK, Z-VAD, PMSF in serpin. Teste encimskih aktivnosti smo izvedli pri pH 8,0 v prisotnosti kalcijevih ionov. Aktivnost encima smo spremljali z merjenjem fluorescence, pri čemer smo kot substrat uporabili FR-AMC. Rezultati so pokazali, da so vsi inhibitorji razen serpina podobno učinkovito zavirali delovanje posameznega tipa metakaspaze. Serpin je učinkovito zaviral le delovanje GtMC1. Kot najmočnejši inhibitor vseh treh tipov metakaspaz se je izkazal FFR-CMK, sledita pa mu antipain in levpeptin. Na podlagi naše raziskave lahko trdimo, da GtMC1, CrMC2 in GtMC2 najmanj zavirajo inhibitorji PMSF, E-64 in Z-VAD.Proteases are hydrolases found in plants, animals and microorganisms. They enable intracellular and extracellular degradation of proteins. In general, proteases divided into four major groups: metalloproteases, aspartatic, serine and cysteine proteases. The activity of the latter is controlled by their endogenous inhibitors, the most common being cystatins, thyropins, and α2-macroglobulin. Among the best characterized cysteine proteases are caspases. They are present in cells as procaspases in an inactive form and depend on autoproteolytic cleavage and dimerization for their activity. Metacaspases are the only caspase homologues that besides the p20 domain also contain the p10 domain. Unlike caspases, metacaspases do not require oligomerization for their catalytic activity. These proteases are found in fungi, protozoa and plants. They participate in the process of programmed cell death and regulate cellular homeostasis. Depending on the position of the p20 and p10 domains in the polypeptide chain, type I, type II and, the least studied, type III metacaspases are known. The aim of this study was to determine to what extent various inhibitors inhibit the activity of each of the three metacaspase types. For this we used a type II metacaspase, CrMC2, from the algae Chlamydomonas reinhardtii and type I metacaspase, GtMC1, and type III metacaspase, GtMC2, from the algae Guillardia theta. As inhibitors antipain, leupeptin, E-64, FFR-CMK, Z-VAD-FMK, PMSF and serpin were tested. Enzyme activity measurements were performed at pH 8.0 in the presence of calcium ions. The activity of enzymes was monitored by fluorescence measurement using FR-AMC as the substrate. The results showed that all inhibitors but serpin exhibited similar inhibition towards each of the three metacaspase types. Serpin, on the other hand, effectively inhibited only the activity of GtMC1. FFR-CMK was the most potent inhibitor for all three metacaspase types, followed by antipain and leuepeptin. PMSF, E-64 and Z-VAD exhibited little inhibitory effect towards any of the three metacaspase types