magistrsko delo
Doroteja Armič (Author), Marina Klemenčič (Mentor), Marko Novinec (Thesis defence commission member), Marko Dolinar (Thesis defence commission member)

Abstract

Vakuolni procesivni encimi (VPE) so cisteinske proteaze, ki jih najdemo v rastlinah. Lokalizirani so v vakuolah, kjer cepijo peptidne vezi za asparaginskimi in aspartatnimi aminokislinskimi ostanki. VPE se sintetizirajo v obliki neaktivnih prekurzorjev, ki se avtokatalitsko procesirajo in aktivirajo pri kislem pH. Takšni pogoji so v vakuoli, kjer so VPE tudi aktivni. Po avtoaktivaciji lahko VPE opravljajo svojo funkcijo procesiranja drugih vakuolnih proteinov, ki se sintetizirajo v obliki neaktivnih prekurzorjev. Med drugim je VPE pomembna komponenta rastlinske programirane celične smrti, saj ima zaradi zmožnosti avtokatalitskega procesiranja in aktivacije prekurzorjev hidrolitičnih encimov funkcijo iniciatorja tega procesa. Cilj magistrskega dela je bil v celicah E. coli izraziti vakuolni procesivni encim iz alge Chlamydomonas reinhardtii (CrVPE) ter okarakterizirati njegovo avtokatalitsko procesiranje in aktivnost. Pričakovali smo, da se bo pri kislem pH (pod 5) CrVPE avtoaktiviral ter cepil peptidna substrata Ac-ESEN-AMC in Z-YVAD-AFC, kar je značilno za vakuolne procesivne encime. Pripravili smo pet konstruktov CrVPE. Protein CrVPE divjega tipa in njegov neaktivni mutant nam je uspelo izraziti v topni frakciji, vendar nam ju ni uspelo izolirati, saj sta se tekom izolacije razgradila. V celotni topni frakciji lizata celic E. coli po izražanju proteina CrVPE divjega tipa nismo izmerili relevantne aktivnosti na substrata Ac-ESEN-AMC in Z-YVAD-AFC; prav tako nismo opazili avtokataliskega procesiranja. Protein CrVPE, ki smo ga izrazili, torej ni bil aktiven. Poleg proteina CrVPE divjega tipa in njegovega neaktivnega mutanta smo pripravili še tri skrajšane variante proteina CrVPE, ki niso imele N- in/ali C-končne prodomene. Ugotovili smo, da se zrel protein CrVPE brez N- in C-končne prodomene ter protein CrVPE brez N-končne prodomene akumulirata v inkluzijskih telesih. Na podlagi tega predvidevamo, da je N-končna prodomena pomembna za pravilno zvitje proteina CrVPE in deluje kot šaperon.

Keywords

cisteinske proteaze;legumain;vakuolni procesni encim;Chlamydomonas reinhardtii;CrVPE;rastlinska programirana celična smrt;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [D. Armič]
UDC: 577.15(043.2)
COBISS: 138564355 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Expression and biochemical characterization of a vacuolar processing enzyme from the unicellular alga Chlamydomonas reinhardtii
Secondary abstract: Vacuolar processing enzymes (VPEs) are cysteine proteases found in plants. They are localized in the vacuole, where they cleave peptide bonds after asparagine and aspartate amino acid residues. VPEs are synthesized as inactive precursors and are autocatalytically processed and activated at acidic pH. Such conditions are found in the vacuole, where VPEs are active. After autoactivation, VPEs can perform its function of processing other vacuolar proteins that are synthesized as inactive precursors. VPE is an important component of plant programmed cell death, because it has the ability of autocatalytic processing and activation of precursors of hydrolytic enzymes. VPE therefore acts as an initiator of this process. The goal of this thesis was the expression of the vacuolar processing enzyme from the alga Chlamydomonas reinhardtii (CrVPE) in E. coli and characterization of its autocatalytic processing and activity. We hypothesized that CrVPE would autoactivate at acidic pH (below 5) and cleave peptide substrates Ac-ESEN-AMC and Z-YVAD-AFC, which are two of the characteristics of vacuolar processing enzymes. We prepared five CrVPE constructs. Wild type CrVPE and its inactive mutant were expressed in the soluble fraction, but we were not able to isolate them, because they degraded during the process of isolation. We did not detect any relevant activity on substrates Ac-ESEN-AMC and Z-YVAD-AFC in the soluble fraction of E. coli cell lysate after expression of CrVPE; we also did not observe any autocatalytic processing. We therefore concluded that the expressed CrVPE was not active. In addition to the wild type CrVPE and its inactive mutant, we also prepared three shortened variants of CrVPE lacking the N- and/or C-terminal prodomain. We found that the mature CrVPE without the N- and C-terminal prodomain and CrVPE without the N-terminal prodomain are accumulated in inclusion bodies. We speculate that the N-terminal prodomain is important for the correct folding of CrVPE and thus functions as a chaperone.
Secondary keywords: legumain;vacuolar processing enzyme;Chlamydomonas reinhardtii;CrVPE;Encimi;Univerzitetna in visokošolska dela;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 106 str.
ID: 17623725