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Effect of selected heavy metal ions on activity of cathepsins B, L and S
No full textEncimi predstavljajo pomembno skupino makromolekul, ki v telesu sodelujejo pri veliki večini procesov. Zaradi povezave s fiziološkimi in patološkimi procesi je regulacija njihovega delovanja izjemno pomembna. Pri patoloških stanjih so encimi pogosto prekomerno aktivni zato predstavljajo inhibitorji encimske aktivnosti pomembno terapevtsko sredstvo za nadzorovanje in zdravljenje bolezni. Kot regulatorji encimske aktivnosti lahko delujejo proteini, peptidi, organske molekule ali pa kovine oz. kovinski kompleksi.
Med ene izmed bolj raziskanih encimov spadajo katepsini, lizosomske, cisteinske proteaze, ki se izražajo v vseh tipih celic, kjer nespecifično razgrajujejo različne proteinske komponente celic. Njihova patološka vloga je običajno povezana z zunajcelično lokalizacijo ter razgradnjo komponent zunajceličnega matriksa. Povezani so s kardiovaskularnimi, avtoimunskimi ter nevrološkimi obolenji, osteoporozo, aterosklerozo ter nenazadnje z napredovanjem in metastaziranjem malignih tumorjev.
V predhodnih študijah so preučevali vpliv izbranih ionov težkih kovin (Cd(II), Ce(III), Ce(IV), Ga(III), La(III), Pb(II) ter Zn(II)) na delovanje katepsina K, ki ima pomembno vlogo pri razgradnji in obnavljanju kostnine. Kovinski ioni predstavljajo dobro izhodišče za pripravo kovinskih kompleksov, ki lahko selektivno delujejo samo na izbran encim ter delno ali v celoti inhibirajo njegovo delovanje.
V okviru te študije smo preučili vpliv izbranih kovinskih ionov na aktivnost treh katepsinov (B, L ter S). Vpliv kovinskih ionov smo spremljali s pomočjo kinetičnih meritev encimske aktivnosti, s pomočjo katerih smo nato določili vrednosti Ki oz. EC50 ter mehanizme delovanja za posamezen kovinski kation. Pri uporabi sintetičnih substratov, se je pri vseh encimih izkazalo, da Cd(II), Pb(II) ter Zn(II) ioni delujejo kot linearni kompetitivni inhibitorji, kot najbolj učinkovit inhibitor so delovali Zn(II), kot najšibkejši pa Pb(II) ioni. Preostali kationi (Ce(III), Ce(IV), Ga(III) ter La(III)) so pri različnih katepsinih delovali na različne načine. Pri katepsinu B so vsi kationi razen Ce(III) ionov delovali kot delni inhibitorji, v primeru Ce(III) ionov pa je do inhibicije prišlo šele pri visokih koncentracijah kationa. Vsi štirje kationi so prav tako delovali kot delni inhibitorji katepsina S, pri katepsinu L pa so delovali po mehanizmu linearne kompetitivne inhibicije. V vseh primerih so se kot boljši inhibitorji izkazali Ga(III) ter Ce(IV) ioni, kar je najverjetneje povezano z manjšim ionskim polmerom teh kationov. Pri razgradnji makromolekulskih substratov pa so bili rezultati nekoliko drugačni, inhibicija je bila v vseh primerih nižja. Pri razgradnji elastina so kovinski ioni nekoliko bolje inhibirali delovanje katepsina S kakor katepsina L, pri razgradnji azokazeina pa so se kot učinkovit inhibitor izkazali samo Zn(II) ioni. Ostali kovinski ioni bodisi niso vplivali na aktivnost encima, ali pa je bil njihov vpliv zanemarljivo majhen.Enzymes, an important group of macromolecules, are involved in most processes in the human body. Because of their involvement in both physiological and pathological processes, regulation of enzyme activity is of the utmost importance. Enzymes are often overly active in various pathological conditions. Inhibitors of enzyme activity therefore represent an important therapeutical implement for both managing and treating the disease. Different types of molecules, such as proteins, peptides, organic molecules, metals, or complex compounds can regulate enzyme activity. The lysosomal, cysteine proteases called cathepsins, which are expressed in all cell types, where they non-specifically degrade various protein components of cells are among the most often studied enzymes. Their pathological role is usually associated with their extracellular localization and degradation of the components of extracellular matrix. They are involved in different diseases, such as cardiovascular, autoimmune, and neurological diseases as well as osteoporosis, atherosclerosis and last but not least, in the progression and metastasis of malignant tumours.
Previous studies have already examined the effect of selected heavy metal ions (Cd(II), Ce(III), Ce(IV), Ga(III), La(III), Pb(II) and Zn(II)) on the activity of cathepsin K, an important enzyme in bone degradation and repair. Metal ions offer a good basis for the development of complex compounds that can selectively act on a specific enzyme and partially or fully inhibit its activity.
The aim of this study was to examine the effect of these heavy metal ions on activity of other cathepsins, namely cathepsins B, L and S. We tested the effects of selected metal ions by means of kinetic measurement of enzyme activity, which in turn enabled us to determine the Ki or EC50 values and mechanism of action for each metal cation. When using a synthetic substrate Cd(II), Zn(II) and Pb(II) ions act as linear competitive inhibitors of all three enzymes, of these the Zn(II) ions were the most effective inhibitor, while Pb(II) ions were the least effective inhibitor. The remaining cations (Ce(III), Ce(IV), Ga(III) ter La(III)) acted differently on different cathepsins. All cations except Ce(III) ions acted as partial inhibitors of cathepsin B, whilst low inhibition occurred only at very high concentrations of Ce(III) ions. All 4 cations acted as partial inhibitors of cathepsin S activity and as full inhibitors of cathepsin L activity. In all cases Ga(III) and Ce(IV) ions proved to be better inhibitors, which is most likely due to their smaller ionic radii. The results of macromolecular degradation assay the results were slightly different, with inhibition being lower in all cases. In the elastin degradation assay, metal ions proved to inhibit the activity of cathepsin S slightly better than that of cathepsin L. On the other hand, in the azocasein assay, only Zn(II) ions proved to be an effective inhibitor of both cathepsin L and S, while the effect of other metal ions was either non-existent or negligible
Identification of human cytoplasmic proteins that interact with two pyrazole derivatives
Get PDFIdentifikacija malih molekul, ki interagirajo z biološkimi makromolekulami, je eden temeljnih pristopov pri iskanju novih zdravilnih učinkovin. Pri tem ponavadi izhajamo iz znane makromolekule in odkrivamo male molekule, ki z njo interagirajo. Alternativno pa lahko iščemo tudi makromolekule, ki interagirajo z neko izbrano malo molekulo, katere biološka (fenotipska) aktivnost je lahko znana ali pa ne. Najbolj razširjena tehnika, ki jo lahko uporabimo v ta namen, je afinitetna kromatografija. Cilj magistrske naloge je bil identificirati proteine, ki specifično interagirajo z dvema podobnima malima molekulama, 4-(2-aminoetil)-1-cikloheksil pirazol-5-olom (ligand 1) in 4-(2-aminoetil)-1-fenil pirazol-5-olom (ligand 2), ki sta bili sintetizirani na Katedri za organsko kemijo FKKT UL. Mali molekuli smo imobilizirali na afinitetni nosilec NHS-activated Sepharose™ 4 Fast flow in pripravljeni koloni uporabili za izolacijo tarčnih proteinov. Preiskovani vzorec je bil lizat celic človeških monocitov U937, gojenih v suspenzijski kulturi. Analize NaDS-PAGE so pokazale, da se liganda med seboj razlikujeta v naboru interagirajočih proteinov. Eluate s kolone z ligandom 1 smo analizirali z masno spektrometrijo in na ta način identificirali enega od interagirajočih proteinov kot ISOC2 – slabo poznan človeški protein, ki vsebuje izohorizmatazno domeno in interagira z zaviralcem tumorjev p16INK4a. Če je ISOC2 regulator p16INK4a, je potencialna nova tarča za razvoj nove strategije za zdravljenje raka in staranja, ligand 1 pa prva znana mala molekula, ki se nanj specifično veže. Za namen podrobnejše karakterizacije interakcije in vitro smo v okviru tega dela pripravili tudi sistem za izražanje človeškega ISOC2 v bakteriji E. coli. Analiza rekombinantnega proteina s kromatografijo z ločevanjem po velikosti je pokazala, da je rekombinanten protein najverjetneje homodimer, po čemer se razlikuje od svojih homologov, ki so večinoma homotetrameri.Understanding the interactions between small molecules and macromolecules from different perspectives is important for the advancement of basic science and drug development. Towards this goal, we usually begin with a known macromolecule and discover small molecules that interact with it. Alternatively, looking for macromolecules that interact with a selected small molecule, whose biological (phenotypic) activity may or may not be known. Affinity chromatography is the most widely used technique for isolating specific target proteins from a complex proteome. The aim of the master\u27s thesis was to identify proteins that specifically interact with two similar small molecules, 4- (2-aminoethyl) -1-cyclohexyl pyrazol-5-ol (ligand 1) and 4- (2-aminoethyl)-1-phenyl pyrazole-5-ol (ligand 2), which were synthesized at the Department of Organic Chemistry UL FCCT. Both molecules were individually immobilized on NHS-activated Sepharose™ 4 Fast flow agarose and the prepared columns were used to isolate target proteins. The test sample was a lysate of U937 human monocyte cells cultured in suspension. SDS-PAGE analyzes showed that the ligands differed from each other in the set of interacting proteins. Eluates from the ligand 1 column were sent for mass spectrometry analysis, thus identifying one of the interacting proteins as ISOC2, a little-known human protein that has an isochorimatase domain and interacts with the tumor inhibitor p16INK4a. If ISOC2 were a regulator of p16INK4a, it might be a new target for development of a novel strategy for the treatment of cancer and aging. For the purpose of more detailed characterization of the in vitro interaction, we developed a system for expressing the human ISOC2 form in E. coli. Analysis of the recombinant protein by size separation chromatography showed that it was most likely a homodimer, distinguishing it from its homologues, which are mostly homotetramers
Characterisation of interactions between a pyrazole derivative and L-threonine dehydrogenase and succinate dehydrogenase of Escherichia coli
No full textZaradi naraščajoče odpornosti mikroorganizmov proti antibiotikom se mnoge raziskovalne skupine trudijo identificirati biološke makromolekule in metabolne poti znotraj celic mikroorganizmov, ki bi lahko delovale kot tarče za novo odkrite antibiotike. Predhodno smo pokazali, da derivat pirazola 4-(2-aminoetil)-1-(piridin-2-il)-1H-pirazol-5-ol zavira rast bakterije E. coli in se veže na proteina L-treonin dehidrogenaza in sukcinat dehidrogenaza v celičnem lizatu. L-treonin dehidrogenaza, ki nima človeškega homologa, sodeluje pri metabolizmu aminokislin in medcelični komunikaciji, celičnih procesih, ki pri prokariontih in evkariontih potekata zelo različno. Katalizira začetni korak pretvorbe aminokisline L-treonin v L-glicin, prav tako pa L-treonin lahko služi kot izhodna spojina za sintezo analogov avtoinduktorja-3, pomembnega za zaznavanje kvoruma. Sukcinat dehidrogenaza je encim citratnega cikla in je za razliko od L-treonin dehidrogenaze prisotna pri E. coli in pri človeku, njena inhibicija lahko vpliva torej tudi na celice gostiteljskega organizma.
Namen magistrske naloge je bil podrobneje okarakterizirati interakcije derivata pirazola z obema encimoma. V ta namen smo pripravili rekombinantno L-treonin dehidrogenazo in topni podenoti A in B sukcinat dehidrogenaze v E. coli. Naš primarni cilj je bil ugotoviti, ali spojina deluje inhibitorno na encim L-treonin dehidrogenaza. Aktivnost L-treonin dehidrogenaze v prisotnosti derivata pirazola smo spremljali z merjenjem fluorescence nastajanja produkta NADH. Vezavo male molekule na proteine L-treonin dehidrogenazo ter podenoti A in B sukcinat dehidrogenaze pa smo preverjali z uporabo izotermne titracijske kalorimetrije (ITC).
Z uporabo ITC smo ugotovili, da se derivat pirazola veže na obe citosolni podenoti sukcinat dehidrogenaze in na L-treonin dehidrogenazo z afinitetami v mikromolarnem območju. Prav tako je derivat pirazola inhibiral delovanje L-treonin dehidrogenaze predvsem pri koncentracijah substrata, nižjih od Km. Iz tega lahko sklepamo, da gre za linearni kompetitivni mehanizem inhibicije L-treonin dehidrogenaze.
Uporabljen derivat pirazola glede na naše rezultate ne deluje tako, kot bi moral delovati antibiotik. Zaradi vezave na sukcinat dehidrogenazo lahko deluje citotoksično za celice gostitelja. Spojina prav tako verjetno deluje kot kelator kovinskih ionov, kar pomeni, da lahko posredno inhibira tudi mnoge druge bakterijske in gostiteljske encime. S testiranjem delovanja analogov spojine bi postopno lahko odkrili molekule z ožjim naborom bakterijskih proteinskih tarč in močnejšo inhibicijo L-treonin dehidrogenaze.Due to the increasing resistance of microorganisms against antibiotics, many research groups are trying to identify novel biological macromolecules and metabolic pathways that could serve as targets for newly discovered antibiotics.
Two of the cellular processes, that proceed differently in prokaryotes than in eukaryotes, are amino acid metabolism and cellular communication. The protein L-threonine dehydrogenase is involved in bothit starts the conversion of the amino acid L-threonine to L-glycine, but L-threonine may also lead to the synthesis of autoinducer-3 analogues, important for quorum sensing. Unlike L-threonine dehydrogenase, succinate dehydrogenase is present in E. coli and humans, so its inhibition might also affect the host cell. We have previously shown that a pyrazole derivative 4-(2-aminoethyl)-1-(pyridin-2-yl)-1H-pyrazole-5-ol inhibits the growth of bacterium Escherichia coli and binds to the proteins L-threonine dehydrogenase and succinate dehydrogenase. The enzyme L-threonine dehydrogenase has no human counterpart and is involved in amino acid metabolism and cellular communication. Both cellular processes operate differently in prokaryotes than in eukaryotes. L-threonine dehydrogenase initiates the conversion of the amino acid L-threonine to L-glycine or its conversion to autoinducer-3 analogues, which are important for quorum sensing. In contrast, succinate dehydrogenase, an enzyme of the citric acid cycle. Is found in E. coli and in humans, so its inhibition could also affect the host cell.
In this master’s’ thesis, our aim was to better characterize interactions between the pyrazole derivative and the two dehydrogenases. To this end, we overexpressed and purified recombinant L-threonine dehydrogenase and soluble subunits A and B of succinate dehydrogenase in E. coli. Our primary goal was to determine whether the compound had an inhibitory effect on L-threonine dehydrogenase. Inhibition of L-threonine dehydrogenase in the presence of the pyrazole derivative was monitored by measuring the fluorescence of NADH formed during the reaction. Binding of the compound to L-threonine dehydrogenase and to subunits A and B of succinate dehydrogenase was detected by isothermal titration calorimetry (ITC).
We found that the pyrazole derivative weakly binds both domains of succinate dehydrogenase and L-threonine dehydrogenase. It also inhibits the activity of L-threonine dehydrogenase, especially when the substrate concentration was lower than Km. This suggests a linear competitive mechanism of inhibition.
According to our results, the pyrazole derivative we used does not act as an antibiotic compound. Due to its binding to succinate dehydrogenase, it may have a cytotoxic effect on the host cell. The compound also likely acts as a metal ion chelator, meaning that it could potentially inhibit many other enzymes in bacterial and host cells. Nevertheless, by developing novel analogues of the compound, it may be possible to discover molecules with a narrower range of bacterial protein targets and stronger inhibition of L-threonine dehydrogenase
Inhibition of homodimerization of the Mpro protease and bacterial histidine kinase EnvZ by small molecule inhibitors in the bacterium Escherichia coli
No full textHomodimerizacija proteinov je ključna za uravnavanje številnih procesov v celicah. Encimi morajo za katalitično aktivnost pogosto dimerizirati, na ta način je torej opravljanje morfološke funkcije uravnavano z oligomernim stanjem. S porastom pojava odpornosti na antibiotike in pojavom novih virusov, se vse več raziskav ukvarja tudi s preučevanjem dimerizacijske površine kot potencialne tarče v razvoju novih zdravil, Iskanje novih inhibitorjev dimerizacije pa terja učinkovite metode presejanja knjižnic malih molekul, ki omogočajo identifikacijo oligomernega stanja. Tekom diplomskega dela smo skušali preveriti uporabnost sistemov LEXGFP in TOXGFP za detekcijo homodimerizacije v bakteriji Escherichia coli pri presejanju knjižnic malomolekulskih inhibitorjev dimerizacije v celicah. Analizirali smo oligomerno stanje štirih ključnih bakterijskih oz. virusnih encimov, za katere so poznani inhibitorji dimerizacije – proteaze Mpro iz SARS-CoV-2, bakterijske histidin kinaze EnvZ iz E. coli, proteaze citomegalovirusa in proteaze herpes virusa povezanega s Kaposijevim sarkomom. V bakteriji Escherichia coli smo uspeli potrdili le homodimerizacijo prvih dveh, nato pa smo njuno oligomerno stanje analizirali še po uporabi malomolekulskih inhibitorjev homodimerizacije. Statistično značilne rezultate smo uspeli pridobiti le s sistemom TOXGFP, za potrditev uporabnosti tega sistema pri presejanju knjižnic inhibitorjev dimerizacije pa so potrebne dodatne raziskave.Homodimerization is crucial for the regulation of numerous cellular processes. Enzymes often require dimerization for catalytic activity, thus fulfilling morphological functions is regulated by their oligomeric state. With the rise of antibiotic resistance and the emergence of new viruses, more research is focused on utilizing the dimerization surface as a potential target in drug development. Identifying novel dimerization inhibitors requires effective methods for screening libraries of small molecules, that allow the identification of the oligomeric state. With this thesis, we attempted to verify the potential of two systems for detecting homodimerization in Escherichia coli – namely LEXGFP and TOXGFP – as tolls for screening libraries of small molecule dimerization inhibitors in bacterial cells. We analysed the oligomeric state of four key bacterial or viral enzymes, for which dimerization inhibitors are known – Mpro protease from SARS-CoV-2, bacterial histidine kinase EnvZ from Escherichia coli, the cytomegalovirus protease, and the protease of the herpesvirus associated with Kaposi\u27s sarcoma. We managed to confirm only the homodimerization of the first two. We then analysed their oligomeric state after the use of dimerization inhibitors. Statistically significant results were obtained only with the TOXGFP system, and further research is needed to confirm the usability of this system for screening libraries of dimerization inhibitors
Protein engineering of oligomeric states of cathepsin K
Get PDFProteinski 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.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
Enzyme-catalyzed formation of covalently cross-linked dimers of the cysteine peptidase xylellain
No full textOligomerni proteini so proteini, sestavljeni iz več polipeptidnih verig. Med encimi prevladujejo homodimeri, tj. dimeri, kjer sta obe podenoti enaki. S prečnim kovalentnim povezovanjem proteina lahko izboljšamo njegovo stabilnost, povečamo specifičnost in kompleksnost reakcij, dosežemo pa lahko tudi alosterične in kooperativne učinke, ki povečajo hitrost reakcije. Nespecifično oligomerizacijo proteinov lahko dosežemo na več različnih načinov, pri čemer se najpogosteje proteine kovalentno poveže s prečnimi povezovalci.
Cilj diplomske naloge je bil prečno kovalentno povezati ksilelain, ki je ekstracelularna cisteinska proteaza, ki izhaja iz bakterije vrste Xylella fastidiosa.
Rekombinantna ksilelaina z dodanim tirozinskim aminokislinskim ostankom in z dodanim cisteinskim aminokislinskim ostankom, izražena in izolirana iz E. coli, smo kovalentno povezali z uporabo tirozinaze. To je encim, ki oksidira tirozin v o-kinon, slednji pa nato reagira s tiolno skupino cisteinskega aminokislinskega ostanka. Kovalentno povezovanje ksilelaina s pomočjo tirozinaze se kljub našim poskusom optimizacije ni izkazala za učinkovito, poleg tega pa nam ni uspelo izolirati dimera ksilelaina, zato smo ksilelain kovalentno povezali še z uporabo SnoopLigaze, kjer smo s tem encimom povezali ksilelaina s SnoopTagJr in z DogTag oznako.
Da bi preverili, ali ima približanje obeh podenot ksilelaina alosterične učinke, smo pri obeh pridobljenih dimerih ksilelaina izmerili še učinek heparina na aktivnost dimera. Dimer ksilelaina, kovalentno povezan s SnoopLigazo, ni bil aktiven, zato smo odvisnost aktivnosti dimera ksilelaina od koncentracije heparina merili v mešanici dimera, kovalentno povezanega s tirozinazo, in nezreagiranega ksilelaina. Izmerili smo, da je K heparina 0,337 µg/mL, F mešanice pa je 36,9 %.Oligomeric proteins are proteins composed of multiple polypeptide chains. Among enzymes, homodimers are predominant, i.e., dimers where both subunits are identical. By cross-linking the protein covalently, we can improve its stability, increase specificity and complexity of reactions, and achieve allosteric and cooperative effects that enhance reaction rates. Non-specific oligomerization of proteins can be achieved in various ways, most commonly by covalently linking proteins with cross-linkers.
The goal of this thesis was to covalently link xylelain, an extracellular cysteine protease originating from the bacterium Xylella fastidiosa.
Recombinant xylelain, with an added tyrosine amino acid residue and an added cysteine amino acid residue, expressed and isolated from E. coli, was covalently linked using tyrosinase. It is an enzyme that oxidizes tyrosine to o-quinone, which then reacts with the thiol group of the cysteine amino acid residue. Despite our optimization efforts, covalent linking of xylelain using tyrosinase was not effective, and we were also unable to isolate the xylelain dimer. Therefore, we additionally covalently linked xylelain using SnoopLigase, where we used this enzyme to link xylelain with the SnoopTagJr and DogTag labels.
To determine whether the proximity of the two xylelain subunits has allosteric effects, we measured the effect of heparin on the activity of the obtained xylelain dimers. The xylelain dimer covalently linked with SnoopLigase was not active, so we measured the dependence of xylelain dimer activity on heparin concentration in a mixture of the dimer covalently linked with tyrosinase and unreacted xylelain. We measured that the K of heparin is 0.337 µg/mL, and the F of the mixture is 36.9%
Karakterizacija vpliva aminokislinskih derivatov sukcinimida na aktivnost katepsina L
Get PDFČloveški katepsin L je papainu podobna cisteinska endopeptidaza izražena v večini celic. Ima pomembno vlogo pri razgradnji proteinov, predstavitvi antigenov in regulaciji celičnega cikla. Namen naše raziskave je bil identificirati alosterične inhibitorje katepsina L. Slednji imajo prednost pred ortosteričnimi, saj zagotavljajo večjo specifičnost vezave. Kot tarčno alosterično mesto smo si izbrali tisto, kamor se dokazano vežejo alosterični efektorji na katepsinu K, ki sodi v družino katepsinu L podobnih peptidaz. Kot potencialne inhibitorje smo testirali spojine, ki so bile sintetizirane kot možni inhibitorji katepsina K in S. Testiranja smo izvedli z meritvijo aktivnosti encima s fluorogenim substratom Z-LR-AMC. Identificirali smo 12 linearnih in 5 hiperboličnih inhibitorjev. Da pa bi preverili, ali se spojina res veže v alosterično mesto, smo naredili mutanto, ki ima pet aminokislinskih ostankov v alosteričnem mestu zamenjanih s tistimi iz sorodnega katepsina V. Kot najboljši inhibitor s hiperboličnim mehanizmom delovanja se je izkazala spojina 2. Z logistično enačbo s štirimi parametri smo določili faktor EC50 670 ± 300 µM. Spojina zmanjša aktivnost encima za 30 %. Afiniteta vezave se pri mutiranih oblikah encima spremeni, zato lahko trdimo, da se veže v tarčno alosterično mesto.Human cathepsin L is a papain-like cysteine endopeptidase expressed in most cells. It plays crucial roles in protein degradation, antigen presentation and regulation of the cell cycle. Our research is focused on identifying allosteric inhibitors of cathepsin L. As our target allosteric site we choose a site where allosteric effectors bind on the related cathepsin K. We characterized the effects of potential allosteric inhibitors from a library of amino acid derivatives of succinimide that were synthesized as potential inhibitors of cathepsins K and S. By measuring the activity of cathepsin L with the synthetic fluorogenic substrate Z-LR-AMC we identified 12 linear inhibitors and 5 hyperbolic inhibitors. To test whether the inhibitors bind to the allosteric site we produced a mutant variant of cathepsin L with five amino acids in target allosteric site substituted with those from the closely related cathepsin V. The best hyperbolic inhibitor was compound 2 which lowered the activity of the enzyme by 30 % and had an EC50 value of 670 ± 300 µM for wild-type cathepsin L. The affinity of binding on mutant enzyme of chatepsin L is different so compound 2 most likely binds to allosteric site
Characterization of S. aureus menaquinone biosynthesis pathway enzyme MenD as a basis for developing new antibiotics
No full textZaradi tvorbe biofilma, ki ji omogoča preživetje tudi v nelagodnih razmerah, bakterija S. aureus predstavlja veliko težavo pri zdravljenju z antibiotiki. Odkrili so, da je za anaerobno tvorbo biofilma potreben menakinon. Molekula DHNA, ki je »downstream« metabolit v biosintezi menakinona, inhibira drugi encim klasične biosintezne poti MenD. V diplomskem delu smo pripravili rekombinantna proteina MenF in MenD, ki sta prva encima v klasični poti. MenD je prvi specifičen encim za to pot, ki pretvarja izokorizmat v 2-sukcinil-5-enolpiruvil-6-hidroksi-3-cikloheksen-1-karboksilat (SEPHCHC). Naš cilj je bil z encimom MenF iz korizmata pripraviti izokorizmat, nato pa tega uporabiti za merjenje aktivnosti MenD ter testirati potencialen inhibitorni vpliv derivatov hidroksinaftojske kisline, podobnih DHNA, na encim MenD. Pri eksperimentalnem delu smo uspešno pripravili oba rekombinantna encima ter uporabili MenF za pretvorbo korizmata v izokorizmat, vpliva derivatov na MenD pa žal nismo uspeli testirati, saj je bil encim zaradi nezmožnosti tetramerizacije neaktiven. Predvidevamo, da je razlog za to v podaljšanem N-koncu, ki smo ga uvedli pri molekulskem kloniranju. V prihodnje bi bilo potrebno optimizirati odcep N-končne oznake oz. namesto tega uvesti C-končno oznako. Inhibicija encima MenD predstavlja dobro iztočnico za razvoj novih antibiotikov, ki se osredotočajo na inhibicijo sinteze menakinona, zato bi bilo področje smiselno še dodatno raziskati.Due to its biofilm forming ability, which allows it to survive in adverse conditions, S. aureus is a major problem for antibiotic treatment. It was recently discovered that anaerobic biofilm formation requires menaquione. DHNA, which is a downstream metabolite in menaquinone biosynthesis, inhibits the second enzyme of the classical menaquinone biosnynthesis pathway MenD. In this thesis, we prepared recombinant proteins MenF and MenD, which are the first two enzymes in the classical pathway. MenD is the first committed enzyme of the pathway, which converts isochorismate to 2 succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC). Our aim was to prepare isochorismate from chorismate using the enzyme MenF and then use isochorismate to observe MenD activity and test the potential inhibitory effect of DHNA like hydroxynaphthoic acid derivatives on the MenD enzyme. In our experiments, we successfully prepared both recombinant proteins and used MenF to convert chorismate to isochorismate, but were unable to test the inhibitory effects of the derivatives on MenD because the enzyme was inactive due to its inability to tetramerise. We presume that this is due to an extended N-terminus introduced in molecular cloning. In future work, the N-terminal tag cleavage should be optimized or a C-terminal tag introduced instead. Inhibition of MenD represents a good starting point for developing new antibiotics focusing on menaquinone biosynthesis inhibition, so further research in this area would be beneficial
Karakterizacija vpliva aminokislinskih derivatov sukcinimida na aktivnost katepsina B
Get PDFKatepsin B je predstavnik družine papainu podobnih cisteinskih peptidaz. Po svoji zgradbi se precej razlikuje od ostalih predstavnikov te družine, saj ima dodaten strukturni element, imenovan zaporna zanka. Zaporna zanka mu omogoča, da lahko deluje kot endo- ali eksopeptidaza. Endopeptidazna aktivnost katepsina B je povezana z različnimi patološkimi stanji, kot so revmatične bolezni.
V okviru diplomskega dela smo preverjali vpliv nekaterih aminokislinskih derivatov sukcinimida na aktivnost človeškega katepsina B. Določili smo delež preostale encimske aktivnosti pri nasičenju z inhibitorjem ter potentnost inhibitorja v obliki vrednosti EC50. Iz nabora spojin smo identificirali spojine 6, 8 in 15, ki so delovale kot hiperbolični inhibitorji katepsina B ter 4, 5, 11 in 12, ki so delovale kot popolni inhibitorji. Izbranim inhibitorjem smo preverili tudi inhibitorni vpliv hidrolize makromolekulskega substrata azokazeina. Rezultati so pokazali, da so hiperbolični inhibitorji v večini obdržali svoje inhibitorne sposobnosti, linearni pa niso bistveno vplivali na razgradnjo azokazeina s katepsinom B.Cathepsin B is a representative of the papain-like cysteine peptidase family. Its structure is different from the other representatives of this family, in that it has an additional structural element called the occluding loop. This element allows it to act as an endo- or exopeptidase. Endopeptidase activity of cathepsin B is involved in various pathological conditions, such as rheumatic diseases.
In thesis, we tested the effects of selected amino acid derivatives of succinimide on the activity of human cathepsin B. We determined residual enzyme activity at saturation with inhibitors and the potency of the inhibitors expressed as EC50 were determined. From our in-house compound library, we identified compounds 6, 8 and 15, as partial inhibitors of cathepsin B and compounds 4, 5, 11 and 12 as linear inhibitors, respectively. Selected inhibitors were also assayed for their inhibitory effect on the hydrolysis of the macromolecular substrate azocaseins. Partial inhibitors, for the most part, retained their inhibitory activity, whereas linear inhibitors did not significantly affect the caseinolytic activity of cathepsin B
Effect of caffeic acid and iron(III) ions and their complexes on cathepsin B activity
No full textZa pravilno delovanje živih organizmov je bistvenega pomena stroga regulacija encimske aktivnosti, saj nezadostno ali prekomerno delovanje encimov povezujemo z razvojem številnih patoloških stanj. Kot ena izmed za raziskave zanimivih skupin encimov se je izkazala skupina papainu podobnih proteaz in sicer so to cisteinski katepsini. Katepsin B je cisteinska peptidaza s katalitično diado sestavljeno iz cisteinskega in histidinskega ostanka. Izraža se v vseh tipih celic in večinoma nespecifično razgrajuje proteine in peptide. Njegova aktivnost je povezana z dolgim seznamom bolezni kot so nevrološka, avtoimunska, skeletna, kardiovaskularna obolenja ter nastanek in metastaziranje različnih malignih tumorjev. Zaradi tega so katepsini zelo priljubljena tema številnih raziskav na temo popolne ali delne inhibicije encimske aktivnosti. Kot inhibitorji aktivnosti katepsina B se dinamično preučujejo različne proteinske molekule, organske molekule, kovinski ioni in kovinski kompleksi.
V okviru magistrske naloge smo preučevali inhibitorni vpliv kavne kisline v kombinaciji s kovinskimi ioni na aktivnost katepsina B. V prejšnjih raziskavah so sicer ugotovili inihbitorne učinke tako kavne kisline kot nekaterih kovinskih ionov na aktivnost katepsina B, vendar pa smo v tej študiji preverili vpliv kompleksov kavne kisline in izbranih kovinskih ionov na aktivnost encima ter ugotovili, ali ima kompleks močnejši inhibitorni učinek na aktivnost katepsina B kot posamezni komponenti. V začetnih poskusih sem preverila učinek kavne kisline v prisotnosti nekaterih kovinskih ionov, da bi določila, kateri ion najmočneje inhibira aktivnosti katepsina B v kombinaciji s kavno kislino. Kot najboljši so se izkazali železovi(III) ioni. Vpliv kavne kisline in železovih ionov na encimsko aktivnost pri treh različnih pH vrednostih sem spremljala s fluorometričnim zasledovanjem hidrolize sintetičnega substrata Z-FR-AMC, rezultate analizirala s programom GraphPad Prism in določila vrednosti konstant inhibicije in EC50 ter mehanizem delovanja in situ pripravljenega kompleksa. Ugotovili smo, da so inhibitorni učinki kavne kisline izboljšani, ko v reakcijske mešanice dodamo ustrezno količino železovih(III) ionov. Določene vrednosti EC50 za inhibicijo katepsina B z in situ pripravljenim kompleksom pri različnih pH so 6,9 ± 5,3 µM (pH 4,5), 7,1 ± 2,3 µM (pH 5,5), 24,9 ± 5,4 µM (pH 7,4) in so nižje v primerjavi z EC50 vrednostmi pri inhibiciji s posameznimi komponentami. Kompleks se je pokazal kot inhibitor encimske aktivnosti, ki deluje po mehanizmu linearne akompetitivne inhibicije s konstanto akompetitivne inhibicije Kiu= 4,27± 0,29 µM.
Poskusila sem tudi sintetizirati in okarakterizirati kompleks kavne kisline in železovega(III) iona v množinskem razmerju 3:1, vendar je sinteza in posledično tudi karakterizacija kompleksa pokazala le delni uspeh. Problemi so bili bodisi v čistosti ali pa v topnosti nastalih produktov. Zagotovo pa je nadaljevanje raziskav v smeri sinteze kompleksa kavne kisline in kovinskih ionov in priprave kristalne strukture kompleksa smiselno, saj bi tako dobili boljši vpogled in bolj jedrnato razlago o načinu delovanja kompleksa kavne kisline in železovih ionov pri inhibiciji katepsina B.The strict regulation of the enzyme activity is essential for the proper functioning of all living organisms, due to the known connection of the extreme, either minimum or maximum, activity of the enzymes to the numerous pathological conditions. A group of papain-like proteases, namely cysteine cathepsins, has awoken a special interest amongst researchers. Cathepsin B is a cysteine peptidase with a catalytic dyad consisting of a cysteine and a histidine residue. It is expressed in all types of cells and it commonly degrades proteins and peptides non-specifically. The activity of the cathepsin B is closely associated with a long list of conditions such as neurological, autoimmune, skeletal, cardiovascular diseases and the formation and metastasis of various malignant tumors. For this reason, cathepsins are a very popular subject of many studies of complete or partial inhibition of enzyme activity. Different protein molecules, organic molecules, metal ions and metal complexes are dynamically studied as inhibitors of the activity of the cathepsin B.
During the research for the Master thesis, we have studied the inhibitory effect of the caffeic acid in combination with iron(III) ions on the activity of cathepsin B. In previous studies, the inhibitory effects of both these elements separately have been proven. However, our research focuses on understanding of the effect of the complex of caffeic acid and the selected metal ion on the activity of enzyme, and determing whether the complex, rather then it\u27s separated components, presents a more powerful inhibitory effect on cathepsin B\u27s activity. In the initial experiments, I have checked the effect of caffeic acid in the presence of different metal ions, in order to determine which ion most strongly inhibits the activity of cathepsin B when combined with caffeic acid. Iron(III) ions were proven to be the best choice. I have measured the effect of the caffeic acid and iron(III) ions on the activity of the enzyme in three different pH values by flourometric tracking of the hydrolysis of the synthetic substrate Z-FR-AMC. Further on I analized the data with GraphPad Prism program and determined the values of the inhibition constants and the EC50 and the mechanism of action of the in situ prepared complex. We found that the inhibitory effects of caffeic acid are improved when an appropriate amount of iron(III) ions is added to the reaction mixtures. Determined values of the EC50 for the inhibition of cathepsine B with the in situ prepared complex in different pH are 6,9 ± 5,3 µM (pH 4,5), 7,1 ± 2,3 µM (pH 5,5), 24,9 ± 5,4 µM (pH 7,4), and are lower when compared to the EC50 values of the inhibition by individual components of the complex. The complex was proven to be an enzyme activity inhibitor acting by a uncompetitivne inhibition mechanism, with the constant of uncompetitive inhibition Kiu= 4,27± 0,29 µM.
I have also attempted to synthesize and characterize the complex of caffeic acid and iron(III) ion in the ratio 3:1, however the synthesis and, consequently, the characterization of the complex showed only partial success. The problems appeared either in the purity or in the solubility of the formed products. Certainly, continuing research in the direction of the synthesis of the complex of caffeic acid and metal ions and the preparation of the crystal structure of the complex makes sense, because this may provide a better insight and a more concise explanation of the mode of action of the complex of caffeic acid and iron(III) ions in the inhibition of cathepsin B
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