diplomsko delo
Nika Goršek (Avtor), Marina Klemenčič (Mentor)

Povzetek

Programirana celična smrt ima pomembno vlogo pri rasti, razvoju in ohranjanju večceličnega organizma. Proces je vključen v ohranjanje velikosti tkiva, embrionalni razvoj, nastajanje okončin, odstranjevanje poškodovanih in okuženih celic ter v delovanje imunskega sistema. Apoptoza je poleg nekroze in avtofagije ena izmed glavnih tipov programirane celične smrti. Ključno vlogo pri poteku apoptoze imajo cisteinske proteaze, imenovane kaspaze. Tudi pri rastlinah je programirana celična smrt pomembna za razvoj in interakcije z okoljem. Med rastlinsko in živalsko celično smrtjo obstajajo določene razlike. Celična stena rastlin onemogoča nastajanje apoptotskih telesc, odsotnost fagocitov onemogoča odstranjevanje ostankov celic, v samih rastlinskih genomih pa ni zapisa za regulatorje apoptoze, družin Bcl-2, Bax in kaspaz. Najdemo pa lahko zapise za metakaspaze. Te proteaze so strukturni homologi kaspaz, ki prav tako vsebujejo katalitično diado sestavljeno iz cisteina in histidina. Od kaspaz se razlikujejo v substratni specifičnosti. Metakaspaze cepijo za pozitivno nabitim aminokislinskim ostankom za razliko od kaspaz, ki cepijo za negativno nabitim aminokislinskim ostankom. Razlikujejo pa se tudi v aktivaciji cimogenov. Ta je pri metakaspazah odvisna od kalcijevih ionov, pri kaspazah pa je avtokatalitično procesiranje odvisno od dimerizacije. Metakaspaze glede na razporeditev domen p20 in p10 razdelimo na tri tipe. Pri tipu I si domeni sledita ena za drugo, na N-končnem delu pa je prisotna tudi prodomena, ki se po proteolitičnem procesiranju odstrani. Za tip II je značilna dolga povezovalna regija med domenama p20 in p10. Pri tipu III pa je domena p10 pred domeno p20. Cilj diplomskega dela je bila izraziti in izolirati rekombinantne metakaspaze vseh treh tipov ter določitev njihovih osnovnih biokemijskih lastnosti. Kot predstavnika metakaspaz tipov I in III smo uporabili metakaspazi iz alge Guillardia theta, kot predstavnika tipa II pa smo uporabili metakaspazo modelne zelene alge Chlamydomonas reinhardtii. Za pripravo proteinov smo uporabili ekspresijski sistem Escherichia coli, sev BL21[DE3]. Za določevanje encimske aktivnosti proteaz smo uporabili substrata FITC-kazein in Z-FR-AMC, ki ima pred mestom cepitve pozitivni aminokislinski ostanek. Meritve encimske aktivnosti so pokazale, da vse tri metakaspaze za delovanje potrebujejo milimolarne koncentracije kalcijevih ionov. Pri optimalnem pH pa si metakaspaze niso tako enotne. Metakaspazi 1 in 2 iz organizma G. theta najbolje delujeta pri pH 6,0, metakaspaza 2 iz C. reinhardtii pa za optimalno delovanje potrebuje pH 8,5.

Ključne besede

alge;Guillardia theta;Chlamydomonas reinhardtii;programirana celična smrt;encimi;cisteinske proteaze;metakaspaze;GtMC1;CrMC2;GtMC2;diplomska dela;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.11 - Diplomsko delo
Organizacija: UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [N. Goršek]
UDK: 577.152.34(043.2)
COBISS: 1538536643 Povezava se bo odprla v novem oknu
Št. ogledov: 656
Št. prenosov: 218
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Expression and basic characterisation of the three algal metacaspase types
Sekundarni povzetek: Programmed cell death plays a vital role in growth, development and maintenance of a multicellular organism. The process is crucial in tissue size retention, embryonal development, limb formation, elimination of damaged and infected cells and in proper functioning of the immune system. Apoptosis, together with necrosis and autophagy, presents the main type of programmed cell death. A crucial role in apoptosis is played by cysteine proteases termed caspases. Programmed cell death has an important role not only in animals but also in plant development and interaction with the environment. Key features distinguish plant programmed cell death from the one in animals. Plant cell wall hinders the formation of apoptotic bodies, the absence of phagocytes prevents the elimination of cell remains. Last but not least there are no coding sequences for apoptosis regulators, Bcl-2 and Bax families and caspases. Plant genomes, however, contain genes encoding metacaspases. Metacaspases, like their structural homologues caspases, possess catalytic dyad composed of cysteine and histidine. The difference between these two members of the C14 family is the substrate specificity. Caspases cleave after negatively charged amino acid residues as opposed to metacaspases, which preferably cleave after the positively charged amino acid residues. Furthermore, metacaspases require calcium ions for their activation, whereas caspases are known for their activation through dimerisation. Regarding the organisation of two caspase-like domains, p20 and p10, we distinguish three types of metacaspases. Type I metacaspases possess the same architecture as caspases, p10 being on the C-terminus of the p20 domain. Type I metacaspases can also contain a prodomain, which can be found in front of p20 and is cleaved after proteolytic processing. Type II is known for its long linker separating the two domains. Lastly, type III metacaspases have a different arrangement of the domains, p10 being at the N-terminus of p20. The aim of this thesis was to prepare and isolate recombinant metacaspases of all three types and to determine their proteolytic properties. We used metacaspase 1 and 2 (GtMC1, GtMC2) from an algal organism Guillardia theta as representatives of type I and III, respectively, and metacaspase 2 from a model algal organism Chlamydomonas reinhardtii (CrMC2) as an example of type II metacaspases. E. coli expression system was used to express proteins. Proteolytic properties were determined using two substrates that are efficiently cleaved by metacaspases, FITC-casein and Z-FR-AMC. The data collected showed that all three metacaspases required millimolar concentrations of calcium ions for their activation. When it comes to pH dependency the metacaspases are not so unified. Two proteases from G. theta exhibited their optimal activity in acidic (pH 6,0), whereas metacaspase from C. reinhardtii prefered slightly basic conditions (pH 8,5).
Sekundarne ključne besede: cysteine proteases;metacaspases;
Vrsta dela (COBISS): Diplomsko delo/naloga
Študijski program: 1000371
Komentar na gradivo: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo
Strani: 46 str.
ID: 11415864