Optimizacija aktivacije prokatepsina L in vitro

Hauptman, Nastja (author), Pečar Fonović, Urša (mentor)

Peptidaze so encimi, ki katalizirajo cepitev peptidne vezi v polipeptidni verigi in tako omogočajo razgradnjo proteinov do manjših peptidov ali do aminokislinskih preostankov. Prisotne so v vseh živih organizmih in so vključene tako v fiziološke kot v patološke procese. V medicini predstavljajo zaradi svoje regulatorne vloge ter udeleženosti pri patoloških procesih potencialne tarče za zdravljenje. Med najbolj poznane človeške cisteinske peptidaze spadajo katepsini, ki se v aktivni obliki največkrat nahajajo v lizosomih. Katepsin L je široko izražena lizosomska peptidaza, ki je vključena v mnogo različnih celičnih funkcij. Pomembno vlogo ima pri znotrajceličnem obnavljanju proteinov, epidermalni homeostazi, pri predstavitvi antigenov ter tudi pri razvoju las. Prav tako pa je katepsin L nadpovprečno izražen v maligno spremenjenjih celicah. Povečano je izražen pri raku jajčnikov, dojk, prostate, pljuč, želodca, trebušne slinavke in debelega črevesa. Motnje v njegovi aktivnosti vodijo tudi do hudih nepravilnosti v razvoju kože, las in kosti. Tako ima kontrola izražanja katepsina L in uravnavanje njegove aktivnosti zdravilen učinek na mnoga bolezenska stanja. Tekom diplomskega dela smo optimizirali pogoje za avtoaktivacijo prokatepsina L s spreminjanjem temperature, različnih načinov inkubacije (inkubator, vodna kopel) in časa aktivacije. Izvedli smo štiri avtoaktivacije in rezultate pridobili z elektroforezo, v prisotnosti natrijevega dodecil sulfata in prenosom western, s katerim smo razlikovali med neaktivno in aktivno obliko katepsina L. Aktivnost katepsina L po avtoaktivaciji smo merili preko razgradnje fluorogenega substrata Z-FR-AMC. Meritve so pokazale, da je do največje aktivacije prišlo pri vzorcih, ki so bili inkubirani na 37 °C, v vodni kopeli 60 in 90 minut. Do nizke izmerjene aktivnosti je prišlo pri tretji aktivaciji, kjer smo aktivirali večji volumen. Pri ponovni aktivaciji manjšega volumna tretje aktivacije, je prišlo do popolne razgradnje encima.

• Language: Slovenian
• Keywords: Katepsin L, aktivacija
• Work type: Bachelor thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-111439
• Publication date in RUL: 01.10.2019

Secondary language

• Language: English
• Title: Optimisation of procathepsin L activation in vitro

Abstract

Peptidases are enzymes that catalyse the cleavage of the peptide bond in the polypeptide chain, thus enabling the degradation of proteins to smaller peptides or to amino acid residues. They are present in all living organisms and are included both in physiological and pathological processes. In medicine, they represent a potential target for therapy, because of their regulatory role and their involvement in pathological processes. Among the most well-known human cysteine peptidases are cathepsins, which are most commonly found in lysosomes in the active form. Cathepsin L is a widely expressed lysosomal protease, which is involved in many different cellular functions. It plays an important role in intracellular protein renewal, epidermal homeostasis, antigen presentation, and hair development. Likewise, cathepsin L is above-average expressed in malignantly transformed cells. It is increased in prostate, ovarian, lung, breast, colon, pancreatic and gastric cancer. The disorders in his activity also lead to severe abnormalities in the development of skin, hair and bones. Thus, the control of the cathepsin L expression and regulation of its activity has a therapeutic effect for the treatment of a broad range of disease states. During the graduation work, we optimized the conditions for autoactivation of procathepsin L by varying the temperature, various incubation modes (incubator, water bath) and activation time. Four autoactivations were performed and the results obtained by electrophoresis in the presence of sodium dodecyl sulphate and western blot with which we distinguished between the inactive and active form of cathepsin L. The activity of cathepsin L after autoactivation was measured by degradation of the fluorogenic substrate Z-FR-AMC. Measurements showed that the maximum activation occurred in samples that were incubated at 37 °C in water bath for 60 and 90 minutes. A low activity occurred in the third activation, where a larger volume was activated. After the reactivation of a smaller volume of third activation, a complete degradation of the enzyme occurred.

• Keywords: Cathepsin L, activation