Proteinski inženiring oligomernih oblik katepsina K

Obaha, Ana (avtor), Novinec, Marko (mentor)

Proteinski inženiring vključuje preoblikovanje proteinov z namenom pridobitve proteina, ki bo v primerjavi z nemodificiranim izvirnikom bolj primeren za specifično aplikacijo. Na področju proteinskega inženiringa proteaz so se do sedaj ukvarjali predvsem s spreminjanjem njihove specifičnosti in izboljšanjem encimske aktivnosti, področje proteinskega inženiringa proteaz z namenom oligomerizacije pa je ostalo neraziskano. Namen magistrske naloge je bil identificirati potencialne interakcijske površine monomerne papainu podobne cisteinske peptidaze katepsina K in pripraviti stabilni homodimer. Encim se izraža predvsem v osteoklastih kostnega tkiva, kjer je vključen v proces preoblikovanja kosti. Napake v regulaciji encimske aktivnosti katepsina K so povezane z različnimi bolezenskimi stanji, zaradi česar so struktura, funkcija in aktivnost encima podrobno raziskane. Zato je katepsin K primerna tarča za proteinski inženiring oligomernih encimskih struktur, ki bi bile napram monomerni obliki katepsina K bolj stabilne in aktivne. V prvem koraku raziskovalnega dela smo z uporabo bioinformatskih orodij za napovedovanje interakcijskih površin in molekulsko umeščanje izračunali tri možne načine homodimerizacije katepsina K. Največjo zakopano površino je tvoril dimer, izračunan s programom SymmDock, v katerem nastane interakcija preko izolognih površin, ki vsebujeta ostanke Lys9, Pro15, Gly168 in Ile179. Nastanek in stabilnost izračunanega dimera bi bilo v prihodnosti potrebno eksperimentalno ovrednotiti. V nadaljevanju smo v obliki rekombinantnih encimov izrazili mutanta katepsina K z vstavljenima zaporedjema, ki v katepsinu X tvorita zanki, odgovorni za dimerizacijo in stabilizacijo dimera. Mutantni obliki katepsina K nista bili encimsko aktivni, na podlagi česar smo zaključili, da vstavljeni zaporedji bistveno vplivata na zvitje proteina in/ali interakcije med prodomeno in katalitično domeno ter posledično aktivacijo cimogena. S kromatografijo z ločevanjem po velikosti smo potrdili, da eden od mutantov kljub uvedbi dimerizacijskih zank katepsina X v raztopini obstaja le v monomerni obliki. Drugi mutant, ki vsebuje le eno od dimerizacijskih zank, bi potencialno lahko tvoril dimer, vendar bi bilo njegovo strukturo potrebno podrobneje eksperimentalno ovrednotiti.

• Jezik: Slovenski jezik
• Ključne besede: dimerizacija, interakcijska površina, molekulsko umeščanje
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-127241
• COBISS.SI-ID: 66402307
• Datum objave v RUL: 27.05.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Protein engineering of oligomeric states of cathepsin K

Izvleček

Protein engineering is the process of modifying proteins with the goal of obtaining a protein that is more suitable for a particular application than the unmodified version. Protein engineering of proteases has mainly focused on modifying their specificity and improving enzyme activity, while engineering of proteases for the purpose of oligomerization has remained unexplored. The aim of this master thesis was to identify potential protein-protein interface residues and to design a novel homodimeric state of the papain-like cysteine peptidase cathepsin K. It is highly expressed in osteoclasts as the major collagenolytic protease in bone turnover. Since its excessive activity is associated with various pathological conditions, its structure, function, and enzyme activity have been studied in detail. Therefore, cathepsin K is a suitable target for protein engineering of oligomeric states of the enzyme that would be more stable and active in comparison to monomeric state of cathepsin K. To this end, using bioinformatics tools to predict protein-protein interface residues and molecular docking, we predicted three different possibilities for homodimerization of cathepsin K. The best computer-evaluated structure is a symmetric homodimer calculated with the program SymmDock with surface interaction residues Lys9, Pro15, Gly168 and Ile179. Its in vitro formation and stability would need to be evaluated experimentally. Furthermore, we produced two recombinant mutant variants of cathepsin K with insertion sequences responsible for the dimerization of cathepsin X. The mutant versions of cathepsin K were inactive, from which we concluded that the insertion sequences significantly affect protein folding or prodomain - catalytic domain interactions and consequent zymogen activation. By size-exclusion chromatography, we confirmed that one of the mutants exists only in the monomeric state despite the introduction of dimerization loops. The mutant with only one dimerization loop could potentially form a dimer, but its structure would need to be further investigated.

• Ključne besede: dimerization, protein-protein interface, molecular docking