diplomsko delo
Doroteja Armič (Author), Marina Klemenčič (Mentor)

Abstract

Legumaini ali vakuolni procesivni encimi (VPE) so cisteinske proteaze, ki jih najdemo pri praktično vseh organizmih. Imenujemo jih tudi asparaginilendopeptidaze, saj cepijo peptidno vez na C-koncu asparaginskega ali aspartatnega aminokislinskega ostanka. Pri rastlinah so prisotni v vakuoli, pri živalih pa v lizosomu. Njihova glavna vloga je procesiranje založnih proteinov, hidrolitičnih encimov in stresnih proteinov. Imajo pa tudi pomembno vlogo pri programirani celični smrti rastlin, saj sodelujejo pri kolapsu vakuole in s tem povzročijo sprostitev hidrolitičnih encimov v citosol. Glede tega so enakovredni kaspazam, ki povzročijo programirano celično smrt pri živalih. Pri rastlinah obstajajo štiri izooblike VPE, in sicer αVPE, βVPE, γVPE in δVPE. Funkcije teh izooblik so že znane. VPE iz alg pa so slabo raziskani. Vemo, da je v genomu modelne zelene alge Chlamydomonas reinhardtii prisoten en gen za vakuolni procesivni encim (CrVPE), vendar funkcija tega proteina še ni znana. S poravnavo zaporedij in ustvarjanjem filogenetskih dreves smo določili sorodnost CrVPE z izooblikami VPE iz višjih rastlin. Ugotovili smo, da je CrVPE najbolj soroden izoobliki βVPE. Med rastlinskimi VPE je znana samo struktura γVPE iz modelne rastline Arabidopsis thaliana, zato smo model strukture CrVPE primerjali s strukturo tega proteina. Glede na znana aktivna mesta in vezavne žepe VPE iz višjih rastlin smo določili aminokislinske ostanke, ki tvorijo vezavni žep in aktivno mesto pri CrVPE. Poiskali smo tudi podatke o izražanju gena za VPE pri algi Chlamydomonas reinhardtii pod različnimi pogoji in ugotovili, da se njegovo izražanje večinoma poveča ob stresnih pogojih. Izvedli smo tudi prve stopnje kloniranja CrVPE v bakteriji Escherichia coli. Ker je sintezni gen, ki smo ga naročili, vseboval neželeno insercijo, smo zaporedje spremenili z zaporednimi reakcijami PCR.

Keywords

encimi;vakuolni procesivni encimi;VPE;legumain;programirana celična smrt;CrVPE;Chlamydomonas reinhardtii;bioinformatske metode;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [D. Armič]
UDC: 577.15:575.112(043.2)
COBISS: 27913219 Link will open in a new window
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Downloads: 105
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Other data

Secondary language: English
Secondary title: Bioinformatic analysis and molecular cloning of legumain from the green alga Chlamydomonas reinhardtii
Secondary abstract: Legumains or vacuolar processing enzymes (VPE) are cysteine proteases that are present in all organisms. They are also called asparaginyl endopeptidases, because they cleave peptide bonds at the C-terminal side of asparagine or aspartic acid residues. In plant cells, they are located in the vacuole, and in animal cells, they are located in the lysosome. Their main function is the processing of storage proteins, hydrolytic enzymes and stress proteins. However, they also have an important role in plant programmed cell death, because they are involved in the vacuolar collapse and the release of hydrolytic enzymes into the cytosol. In this respect, their function equals that of caspases, which cause programmed cell death in animal cells. There are four VPE isoforms in plants: αVPE, βVPE, γVPE in δVPE. The roles of these isoforms are already known. On the other hand, algal VPEs are poorly understood. There is one gene coding for a vacuolar processing enzyme in the genome of the model green alga Chlamydomonas reinhardtii, but its function remains unknown. By performing pairwise alignments and constructing phylogenetic trees we determined the evolutionary relationship of VPE from Chlamydomonas reinhardtii (CrVPE) with VPE isoforms from higher plants. Based on these we concluded that CrVPE is most closely related to the βVPE isoform. We compared the structure model of CrVPE to the known structure of γVPE from Arabidopsis thaliana, up to now the only determined structure of a plant VPE. By comparing known active sites and substrate-binding pockets of VPEs from higher plants we determined the residues that form the substrate-binding pocket and active site in CrVPE. We examined the VPE gene expression patterns in Chlamydomonas reinhardtii under different conditions and found its upregulation during stress conditions. We also carried out the first steps of cloning CrVPE in the bacterium Escherichia coli. Due to the presence of an unwanted insertion in the coding sequence, we removed it by several consecutive PCR reactions.
Secondary keywords: VPE;legumain;Chlamydomonas reinhardtii;CrVPE;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 55 str.
ID: 12027640