Izražanje in karakterizacija nekaterih mutantnih oblik človeškega katepsina S

magistrsko delo

Ana Cirnski (Author), Marko Novinec (Mentor), Marina Klemenčič (Thesis defence commission member), Vera Župunski (Thesis defence commission member)

Abstract

Katepsin S je papainu podobna cisteinska proteaza. Najbolj se izraža v poklicnih antigen predstavitvenih celicah, kot so dendritične celice, makrofagi in limfociti B, kjer ima pomembno vlogo pri procesiranju in predstavitvi antigenov. Tako kot ostali cisteinski katepsini se nahaja v lizosomu, kjer je reducirajoče okolje in nizek pH, ki je potreben za njihovo delovanje. Za razliko od ostalih je katepsin S stabilen tudi pri višjem pH, zato je lahko dlje časa aktiven tudi v izvenceličnem prostoru. Evolucijsko najbolj soroden mu je katepsin K, ki se izraža v različnih tkivih, največ pa v osteoklastih, kjer ima pomembno vlogo pri preoblikovanju kosti. Izloča se v kislo okolje resorpcijske lakune, kjer cepi trojno vijačnico kolagena I. Prekomerna aktivnost je povezana s številnimi boleznimi, med drugim z osteoporozo. V zadnjih letih se čedalje več pozornosti namenja razvoju alosteričnih inhibitorjev proteaz, saj v nasprotju z ortosteričnimi inhibitorji, ki se vežejo v aktivno mesto, le delno inhibirajo aktivnost proteaz in so zato bolj primerni za daljše terapije. Namen magistrskega dela je bil določiti mehanizme delovanja nekaterih že znanih (alosteričnih) inhibitorjev katepsina K na izbrane mutante katepsina S in s tem preveriti njihovo vezavo na izbrano alosterično moesto ter pomen posameznega zamenjanega aminokislinskega ostanka za vezavo teh inhibitorjev. Na podlagi strukturne primerjave obeh encimov smo z mestno specifično mutagenezo pripravili izbrane mutantne oblike človeškega katepsina S, kjer so bili zamenjani aminokislinski ostanki identični ujemajočim se ostankom v alosteričnem mestu katepsina K. S testiranjem kinetičnih parametrov potencialnih alosteričnih inhibitorjev smo določili mehanizme njihovega delovanja na posamezne mutante. Pokazali smo, da spojine 2-[2-(dietilamino)acetamido]benzojska kislina, metil (R)-(2,5-dioksopirolidin-3-il)glicinat in 2-benzilmalonska kislina delujejo na izbrane mutantne oblike katepsina S kot alosterični efektorji in da se najverjetneje vežejo v preučevano mesto. Pri vseh mutantah pride v prisotnosti teh spojin do bolj učinkovite inhibicije encimske aktivnosti v primerjavi z divjim tipom katepsina S, vendar ne moremo z gotovostjo trditi, kakšno vlogo imajo posamezni aminokislinski ostanki za vezavo alosteričnih modifikatorjev na testirane encime.

Keywords

encimi;cisteinske proteaze;cisteinski katepsini;katepsin S;katepsin K;mutante;alosterija;inhibitorji;magistrska dela;

Data

Language:	Slovenian
Year of publishing:	2019
Typology:	2.09 - Master's Thesis
Organization:	UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher:	[A. Cirnski]
UDC:	577.15(043.2)
COBISS:	1538445251
Views:	624
Downloads:	174
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Expression and characterization of selected mutant forms of human cathepsin S
Secondary abstract:	Cathepsin S is a papain-like cysteine protease. It is expressed primarily in professional antigen presenting cells (e.g. dendritic cells, macrophages and lymphocytes B) where it has an important role in processing and presenting antigens. Like other cysteine cathepsins, it is found in lysosomes. With its low pH and reducing environment, lysosome is the perfect milieu for cysteine cathepsin activity. Unlike other cysteine cathepsins, cathepsin S can also function in extracellular milieu for prolonged periods as it remains stable at neutral pH. It is evolutionarily related to cathepsin K, which is expressed in various tissues. Most abundantly, however, is expressed in osteoclasts, where it has an important role in bone remodelling. It is secreted into resorption pit where it cleaves the triple helix of type I collagen. Elevated activity is connected with numerous diseases including osteoporosis. In recent years, there has been an increase in development of allosteric inhibitors of different proteases. In contrast to orthosteric inhibitors, they only partially inhibit protease activity, which makes them more suitable for prolonged therapy. The aim of this thesis was to investigate effects of known allosteric inhibitors of cathepsin K on selected mutant forms of cathepsin S in order to verify their binding to the chosen allosteric site and the involvement of selected amino acid residues in binding of potential allosteric inhibitors. Based on the structure comparison of the two enzymes we prepared selected mutant forms of human cathepsin S by means of site-specific mutagenesis, where substituted residues were identical to matching residues in allosteric site of cathepsin K. By testing the kinetic parameters of potential allosteric inhibitors, we defined their reaction mechanisms on selected mutant forms. We showed that compounds 2- [2- (Diethylamino) acetamido] benzoic acid, methyl (R)-(2,5-dioxopyrrolidin-3-yl)glycinate and 2-benzylmalonic acid act on selected mutant forms of cathepsin S as allosteric effectors and that they most likely bind to the examined site. All tested mutant forms are inhibited more efficiently than wild type cathepsin S in the presence of these compounds. However, we cannot determine the involvement of individual residues in specificity of binding between allosteric inhibitors and tested enzymes.
Secondary keywords:	cathepsin S;cathepsin K;mutant;allostery;inhibitor;
Type (COBISS):	Master's thesis/paper
Study programme:	1000377
Embargo end date (OpenAIRE):	1970-01-01
Thesis comment:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages:	59 str.
ID:	11233014