Določanje funkcije proteaze CrMCA-I iz organizma Chlamydomonas reinhardtii

Peric, Tanja (avtor), Klemenčič, Marina (mentor)

Kaspaze so živalskecisteinske proteaze, ki igrajo odločilno vlogo pri regulaciji in izvedbi programirane celične smrti. Kaspaz pri neživalskih evkariontih ni, lahko pa tam najdemo njihove strukturne homologe, metakaspaze. Te ločimo na tri tipe, ki se med seboj razlikujejo po strukturi. Njihova natančna funkcija ni znana, je pa verjetno med drugim povezana tudi z odzivom na oksidativni stres. V sklopu magistrskega dela smo se osredotočili na metakaspazo tipa I iz enocelične zelene alge Chlamydomonas reinhardtii, CrMCA-I. Piprava divjega tipa te proteaze v E. coli ni bila uspešna, saj se je ta proteaza sprva agregirala v netopni frakciji, dokler ji nismo odstranili hidrofobne N-končne regije in zanke 360, ki je značilna za metakaspaze tipa I iz organizmov iz klada Viridiplantae. Tako modificiran CrMCA-I (CrMCA-I_CL) smo izrazili v E. coli in ga izolirali. Podobno smo modificirali tudi zapisa za dve metakaspazi tipa I iz modelne rastline Arabidopsis thaliana, AtMCA-Ia in AtMCA Ib, vendar nam jih ni uspelo pridobiti v topni frakciji. Izoliranemu CrMCA-I_CL smo določili aktivnost pri različnih koncentracijah kalcijevih ionov in pri različnih pH. Ugotovili smo, da je CrMCA I_CL neaktiven v odsotnosti Ca2+, največjo aktivnost pa doseže v nizkih milimolarnih koncentracijah Ca2+. Njegov pH optimum je v nevtralnem. Poleg tega smo poskusili nemodificirano obliko CrMCA-I izraziti tudi v C. reinhardtii. Pri tem smo naleteli na težave z vzpostavljanjem stabilnih kolonij po transformaciji celic. Uspelo nam je pridobiti kolonije na trdnem gojišču z antibiotikom, vendar te kolonije po inokulaciji v tekoče gojišče niso zrasle. Da bi ugotovili, ali lahko s protitelesi, ki so bila ustvarjena proti metakaspazi GtMCA-III, spremljamo prisotnost metakaspaz pri odzivu na oksidativni stres, smo pri divjem tipu C. reinhardtii in pri insercijski mutanti C. reinhardtii CrMCA I::AphVII inducirali oksidativni stres. Glede na to, da metakaspaz s protitelesi v vzorcih nismo zaznali, bomo v prihodnje morali za njihovo prisotnost in aktivnost pri oksidativnem stresu uporabiti drugačne metode.

• Jezik: Slovenski jezik
• Ključne besede: metakaspaze, CrMCA-I, Chlamydomonas reinhardtii, oksidativni stres
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-134306
• COBISS.SI-ID: 93415171
• Datum objave v RUL: 05.01.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Determining the function of protease CrMCA-I from Chlamydomonas reinhardtii

Izvleček

Caspases are cysteine proteases in animals that play an important role in programmed cell death regulation and execution. Caspases are absent in non animal eucaryotes; however, they contain structural homologues of caspases, metacaspases. These can be divided into three types that can be differentiated based on their domain architectures. Their function is yet not known, although it is likely linked to oxidative stress responses. In this work, we focused on CrMCA-I, the only type I metacaspase from the unicellular green alga Chlamydomonas reinhardtii. When expressed in E. coli, it aggregated in the insoluble fraction. However, removal of the N-terminal region and the 360 loop (CrMCA-I_CL) resulted in expression of soluble and proteolytically active protease. Since this loop is characteristic of type I metacaspases from organisms of the Viridiplantae clade, we also tried to express two sequences encoding metacaspases from Arabidopsis thaliana, AtMCA-Ia and AtMCA-Ib. However, we were not successful in their expression. We measured the activity of the isolated CrMCA-I_CL at different concentrations of calcium ions and different pH values. CrMCA-I_CL was shown to be inactive in the absence of Ca2+ ions and reached its maximum activity at low millimolar Ca2+ concentrations. Its pH optimum was at neutral values. We also tried to express non-modified CrMCA-I homologously in C. reinhardtii, but we encountered problems with establishing stable colonies after transformation. While colonies did appear on solid medium with antibiotic, no growth could be observed in liquid media. To asses if presence of metacaspases during oxidative stress response could be monitored using anti-metacaspase antibodies, we induced oxidative stress in the wild type C. reinhardtii and in CrMCA-I insertion mutant CrMCA-I::AphVII. Since no metacaspase could be detected, other methods will have to be used in the future for monitoring the presence and activity of metacaspases in this organism.

• Ključne besede: metacaspases, CrMCA-I, Chlamydomonas reinhardtii, oxidative stress