Molecular mechanisms of stress-associated cysteine proteases of green alga Chlamydomonas reinhardtii

Van Midden, Katarina Petra (author), Klemenčič, Marina (mentor)

Proteases are essential regulators of plant stress responses, yet the mechanisms of their functions remain poorly understood. Cysteine proteases, including metacaspases, vacuolar processing enzymes, and papain-like cysteine proteases, have been implicated in regulating plant cell death triggered by biotic and abiotic stress. The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) with its simpler genome compared to higher plants serves as a good model for the study of these enzymes. However, the lack of molecular characterisation of these proteases limits further functional studies. In this work, we investigated the molecular mechanisms of selected stress-associated cysteine proteases of Chlamydomonas. We performed a biochemical characterisation of both Chlamydomonas metacaspases, CrMCA-I and CrMCA-II, which revealed information about their cleavage preferences, selectivity, and activation, providing knowledge for further investigation of their cellular functions. As part of this work, the first successful recombinant production and characterisation of a plant type I metacaspase in soluble form was achieved in E. coli by the removal of a short, hydrophobic sequence, unique to plant type I metacaspases. Additionally, we evaluated novel metacaspase-targeting activity-based probes, which selectively detect active metacaspase forms, enabling us to study their maturation mechanisms in vitro. Furthermore, we investigated the presence and activity of cysteine proteases during cell death induced by 5 mM hydrogen peroxide (H$_2$O$_2$) in Chlamydomonas using fluorogenic peptides, activity-based probes and proteomics. Experiments aimed at detecting active metacaspases using activity-based probes in Chlamydomonas extracts revealed an unexpected activity of a previously uncharacterised papain-like cysteine protease, CrCEP1. The protease was found to ligate peptides to the N-termini of the photosystem-II proteins PsbO, PsbP and PsbQ, revealing its dual protease and transpeptidase activity, with potential physiological implications. The results presented in this work enhance our understanding of the two Chlamydomonas metacaspases and the papain-like cysteine protease CrCEP1 and pave the way for further research into the roles of proteases regulating stress response in Chlamydomonas.

• Language: English
• Keywords: Chlamydomonas reinhardtii, stress-induced cell death, metacaspases, papain-like cysteine proteases, activity-based probes, transpeptidation
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2024
• PID: 20.500.12556/RUL-165005
• COBISS.SI-ID: 219280643
• Publication date in RUL: 20.11.2024

Secondary language

• Language: Slovenian
• Title: Molekularni mehanizmi s stresom povezanih cisteinskih proteaz zelene alge Chlamydomonas reinhardtii

Abstract

Proteaze so pomembni regulatorji odziva na stres pri rastlinah, vendar točnih mehanizmov njihovega delovanja še ne poznamo. Pokazano je bilo, da so cisteinske proteaze, med katere spadajo metakaspaze, vakuolarni procesivni encimi in papainu podobne cisteinske proteaze, povezane z regulacijo celične smrti pri rastlinah, ki jo lahko sprožijo biotski ali okoljski stresi. Enocelična zelena alga Chlamydomonas reinhardtii (C. reinhardtii) je zaradi svojega enostavnega genoma v primerjavi z višjimi rastlinami odličen modelni organizem za proučevanje teh encimov, vendar slabo poznavanje molekularnih lastnosti njenih proteaz zavira nadaljnje raziskave njihovih funkcij. V tem delu smo proučevali molekularne mehanizme delovanja izbranih s stresom povezanih cisteinskih proteaz alge C. reinhardtii. Z biokemijsko karakterizacijo obeh metakaspaz, CrMCA-I in CrMCA-II, smo raziskali optimalne pogoje za cepitev, njuno substratno specifičnost in mehanizem aktivacije, kar bo omogočilo nadaljnje proučevanje njunih fizioloških funkcij. V sklopu tega dela smo prvi uspešno rekombinantno pripravili in okarakterizirali rastlinsko metakaspazo tipa I. Encim smo pripravili v topni obliki v E. coli z odstranitvijo kratke hidrofobne regije, ki jo vsebujejo le rastlinske metakaspaze tipa I. Poleg tega smo testirali novo orodje – sonde za detekcijo aktivnih metakaspaz. Te so selektivno zaznale le metakaspaze v njihovi aktivni obliki, kar nam je omogočilo proučevanje mehanizmov aktivacije in proteoliznega delovanja teh encimov in vitro. Dodatno smo s fluorogenimi substrati, sondami za detekcijo aktivnosti proteaz in proteomskimi analizami preverili, katere cisteinske proteaze so prisotne in aktivne med celično smrtjo, ki se sproži ob dodatku 5 mM vodikovega peroksida (H$_2$O$_2$) v C. reinhardtii. Uporaba sond za detekcijo metakaspaz je vodila do odkritja nepričakovane aktivnosti prej neopisane papainu podobne proteaze CrCEP1. Ugotovili smo, da lahko proteaza ligira peptide na N-konce proteinov PsbO, PsbP in PsbQ, ki so komponente fotosistema II. S tem smo odkrili dvojno proteazno-transpeptidazno aktivnost CrCEP1 s potencialno fiziološko vlogo. Predstavljeni rezultati izboljšujejo naše razumevanje obeh metakaspaz C. reinhardtii in papainu podobne cisteinske proteaze CrCEP1 ter obenem utirajo pot nadaljnjim raziskavam vloge proteaz pri odzivu C. reinhardtii na stres.

• Keywords: Chlamydomonas reinhardtii, s stresom povezana celična smrt, metakaspaze, papainu podobne cisteinske proteaze, sonde za detekcijo aktivnih proteaz, transpeptidacija