Biokemijska karakterizacija in primerjava lastnosti treh izbranih rekombinantno izraženih metakaspaz iz organizmov Chlamydomonas reinhardtii in Guillardia theta

Štrancar, Vida (author), Klemenčič, Marina (mentor)

Regulirana celična smrt (RCD) je evolucijsko ohranjen proces, ki je bil opažen pri posameznih predstavnikih vseh živečih organizmov. Sprva so ta pojav pripisovali le evkariontskim večceličnim organizmom, vendar so kasneje opazili, da do tega pojava prihaja tudi pri enoceličnih organizmih kot so kvasovke, alge in bakterije. Pri izvedbi RCD pri živalskih organizmih so ključne cisteinske endopeptidaze kaspaze. Pri ostalih organizmih kaspaze niso prisotne, so pa prisotni njim homologni proteini, metakaspaze. Kljub strukturni homologiji med kaspazami in metakaspazami se ti dve skupini po funkcionalnih lastnostih bistveno razlikujeta. Metakaspaze namreč cepijo substrate za pozitivno nabitimi aminokislinskimi ostanki ter za svojo katalitično aktivnost ne potrebujejo oligomerizacije. Večina metakaspaz je katalitično aktivna le v prisotnosti kalcijevih ionov. Glavni namen tega dela je bil izraziti, očistiti in okarakterizirati predstavnice vseh treh tipov metakaspaz. Metakaspazi GtMC1 (tip I) in GtMC2 (tip III) izvirata iz sekundarnega endosimbionta, alge Guillardia theta. CrMC2 je metakaspaza tipa II iz zelene alge Chlamydomonas reinhardtii. Proteolitična aktivnost vseh treh metakaspaz je podobna, saj proteinske substrate cepijo le v prisotnosti kalcijevih ionov. Vse tri proteaze cepijo majhna sintetična substrata Z-FR-AMC in Z-RR-AMC, vendar se njihove Michaelisove konstante precej razlikujejo. V delu smo preverjali tudi inhibitorno sposobnost proteinskega inhibitorja CrSERPIN-a ob spremljanju hidrolize majhnega sintetičnega substrata. CrSERPIN najbolj inhibira CrMC2, najmanj pa GtMC2. Testirali smo tudi štiri različne tetrapeptidne sonde za detekcijo metakaspazne aktivnosti, ki se ireverzibilno vežejo v aktivno mesto metakaspaz. Sonde EKAK-AOMK, EKTK-AOMK, IRSK-AOMK in IISK-AOMK so bile načrtovane tako, da so specifične za metakaspaze. Njihov peptidni del je prilagojen tako, da je čimbolj podoben substratni specifičnosti že okarakteriziranih metakaspaz. Konstante inaktivacije sond so najvišje za metakaspazo CrMC2, iz česar sklepamo, da so sonde najbolj primerne za detekcijo metakaspaz tipa II. Sonda z najvišjimi konstantami inaktivacije za posamezno metakaspazo je IRSK-AOMK, kar kaže na to, da imajo vse tri metakaspaze podobno kemijsko okolje vezavnega žepa za substrat.

• Language: Slovenian
• Keywords: metakaspaze, cisteinske proteaze, SERPIN, Chlamydomonas reinhardtii, Guillardia theta
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-127825
• COBISS.SI-ID: 70487043
• Publication date in RUL: 24.06.2021

Secondary language

• Language: English
• Title: Biochemical characterization and feature comparison of three recombinant metacaspases from Chlamydomonas reinhardtii and Guillardia theta

Abstract

Regulated cell death (RCD) is an evolutionarily conserved process that can be observed in representatives of all living organisms. It was first described in eukaryotic multicellular organisms, but was later also observed in unicellular organisms such as algae and bacteria. Main executioners of metazoan RCD are cysteine endopeptidases called caspases. Caspases are not present in non-metazoan organisms. Instead, other cysteine proteases homologous to caspases are found in these organisms. They are termed metacaspases. Despite structural homology to caspases, metacaspases display different characteristics: they cleave their substrates after positively charged amino acids, do not require dimerization for catalytic activity, and most of them require calcium ions for their proteolytic activity. The main objective of this thesis was the expression, purification and characterization of three metacaspases, each representative of the three types. The metacaspases GtMC1 and GtMC2, a type I and type III metacaspase, respectively, originate from the algal organism Guillardia theta, which is a secondary endosymbiont. CrMC2 is a type II metacaspase from a green algal organism Chlamydomonas reinhardtii. Proteolytic activities of the three metacaspases are similar: they cleave protein substrates only in the presence of calcium ions. Cleavage of the small synthetic substrates Z-FR-AMC and Z-RR-AMC is also successful, however Michaelis constants for their hydrolysis are substantially different. The inhibitory efficiency of the protein inhibitor CrSERPIN was also tested using a small synthetic fluorogenic substrate. CrSERPIN inhibits CrMC2 most effectively and GtMC2 least effectively. We also tested four different tetrapeptide probes for the detection of metacaspases that irreversibly bind to their active sites. The probes EKAK-AOMK, EKTK-AOMK, IRSK-AOMK and IISK-AOMK were specifically designed for the detection of metacaspases and their peptide moieties were adjusted according to the substrate specificity of other characterized metacaspases. The inactivation constants of the probes are highest for the metacaspase CrMC2, indicating that these probes are most suitable for the detection of type II metacaspases. The probe with the highest inactivation constant for each metacaspase is IRSK-AOMK, indicating that all three metacaspases have a similar chemical nature of the substrate binding pocket.

• Keywords: metacaspases, cysteine proteases, SERPIN, Chlamydomonas reinhardtii, Guillardia theta