Karakterizacija interakcije katepsina B s heparinom z mestno-specifično mutagenezo

Lebar, Blaž (avtor), Novinec, Marko (mentor)

Katepsin B pripada družini papainu podobnih cisteinskih proteaz. Sodeluje pri vrsti celičnih procesov, kjer je odgovoren za cepitev proteinov. Posebna zaporna zanka mu omogoča tako endo- kot eksopeptidazno aktivnost. Pretežno se nahaja v lizosomu. Mnoge študije so ga identificirale kot pomembnega pri različnih patoloških stanjih, med drugim tudi pri artritisu, Alzheimerjevi bolezni in raku. Pri slednjem odigra pomembno vlogo pri migraciji tumorskih celic s cepitvijo komponent zunajceličnega matriksa. Zaradi tega se je pojavila potreba po raziskovanju inhibitorjev tega encima. Eno izmed obetavnih področij je alosterična regulacija, kjer se molekule vežejo v mesto oddaljeno od aktivnega mesta. S tem sprožijo konformacijsko spremembo v encimu, ki privede do njegove spremenjene aktivnosti. Znano je, da je heparin alosterični inhibitor katepsina B. Njegovo vezavno mesto je bilo računalniško umeščeno na območja s presežkom pozitivnega naboja na površini, kar se sklada s pretežno negativnim nabojem na heparinu. V sklopu naše naloge smo nadaljevali raziskave na področju identifikacije vezavnega mesta za heparin. Pripravili smo kombinacije treh mutantov, ki so bile v dosedanjih študijah najbolj obetavne: R85A, K130A in K141A. S tremi dvojnimi ter enim trojnim mutantom smo izvedli kinetične meritve v prisotnosti heparina in določili vrednosti ravnotežne konstante disociacije KD ter jih primerjali z divjim tipom. Pokazali smo, da so ta mesta sicer odgovorna za vezavo heparina, vendar niso edina. Heparin se je še vedno vezal, afiniteta se je znižala v podobnem redu velikosti kot pri enojnih mutantih. Vezavnega mesta nismo uspeli nedvoumno pokazati. Prav tako smo poskušali identificirati male organske molekule, ki bi lahko na divji tip katepsina B delovale podobno kot heparin. Izmed vseh preiskanih smo identificarali le eno: kavno kislino. Vezavo smo okarakterizirali – gre za linearni akompetitivni inhibitor z vrednostjo K_iu približno 110 µM.

• Jezik: Slovenski jezik
• Ključne besede: katepsin B, heparin, alosterična regulacija, inhibitor
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2020
• PID: 20.500.12556/RUL-119693
• COBISS.SI-ID: 31305731
• Datum objave v RUL: 10.09.2020

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Characterization of interaction of cathepsin B with heparin by site-specific mutagenesis

Izvleček

Cathepsin B belongs to the family of papaine-like cysteine proteases. It is involved in numerous cellular processes and can cleave a variety of proteins. A unique structure called the occluding loop enables it to exhibit endo- and exopeptidase activity. It is usually located within the lysosome. Many studies have identified cathepsin B in pathological processes, including arthritis, Alzheimer's disease and cancer. In the latter it plays an important role during migration of tumor cells by cleaving different proteins of the extracellular matrix. These findings have stimulated research of different inhibitors of cathepsin B. One such possibilitya is by allosteric regulation, where the regulatory molecule binds to the enzyme at a site distant from the active site. Its binding triggers conformational changes which affects enzyme activity. It is known that heparin is an allosteric inhibitor of cathepsin B. Computational simulations proposed that two clusters of positively charged residues on the surface of cathepsin B are directly responsible for the binding of negatively charged heparin. The aim of our study was to extend current studies of the location of the binding site and to unambiguously determine its location. We prepared mutant variants of recombinant human cathepsin B carrying two or three mutations of residues R85A, K130A and K141A, which were previously indicated to be involved in the binding of heparin. Kinetic studies were performed to determine the equilibrium dissociation constants KD for heparin. Comparison with the wild type showed that the investigated residues contribute to the binding of heparin, but there are also other residues involved. Heparin still bound to cathepsin B and the affinity of binding was comparable to single mutants. The binding site for heparin is thus still not unambiguously determined. In addition, a small compound library was screened in search of molecules, which act similarly on cathepsin B as heparin. Only one was identified: caffeic acid. We showd that it acts as a linear uncompetitive inhibitor with an K_iu value about 110 µM.

• Ključne besede: cathepsin B, heparin, allosteric regulation, inhibitor