20.500.12556/RUL-126159 Študije strukturnih lastnosti in interakcij človeških proteoglikanov testikanov Structural and interactions studies of human proteoglycans testicans Testikani so modularni proteoglikani zunajceličnega matriksa pri vretenčarjih s širokim tkivnim spektrom izražanja. Proteinsko družino sestavljajo trije predstavniki, testikan-1, -2 in -3, ki so visoko ohranjeni. V njihovi polipeptidni verigi so tri regije, homologne drugim proteinom, ustrezajo pa folistatinu podobni domeni (FS), zunajcelični domeni, ki veže kalcijeve ione (EC), ter tiroglobulinski domeni (TY), medtem ko sta N- in C-končni regiji unikatni. Med drugim sodelujejo pri pritrditvi celic, njihovi migraciji in izraščanju nevritov, raziskave pa kažejo na vsaj delno funkcijsko zamenljivost znotraj proteinske družine. Do sedaj opisane specifične funkcije in lastnosti (vezava kalcijevih ionov, inhibicija cisteinskih proteaz ter metaloproteaz matriksa membranskega tipa) so povezane s posameznimi domenami in regijami, a njihova prostorska organizacija ni znana. Z namenom njene osvetlitve smo podrobneje okarakterizirali testikan-2 kot predstavnika proteinske družine. Proučili smo vezavo kalcijevih ionov in njen vpliv na strukturo, ki smo jo povezali z učinkovanjem testikanov na migracijo celic. Najprej smo z uporabo izotermne titracijske kalorimetrije in strukturnih modelov kalcij-vezavne domene (EC) pokazali, da je aktivno le eno od obeh potencialnih kalcij-vezavnih mest in da je le-to pri zunajceličnih koncentracijah kalcija vedno zasedeno. Vezava na drug potencialni motiv EF2 je onemogočena zaradi substitucije na enem od kanoničnih koordinacijskih mest, kjer je aminokislinski ostanek z manjšo polarno stransko skupino v primerjavi z EF1 zamenjan z aminokislinskim ostankom z veliko hidrofobno stransko skupino, to pa velja za vse tri člane proteinske družine. V nadaljevanju smo z bioinformatsko analizo aminokislinskih zaporedij pokazali, da sta N- in C-končni regiji vse treh testikanov najverjetneje strukturno neurejeni. To je skladno z rezultati analize z uporabo sipanja X-žarkov pod majhnim kotom (SAXS), ki kažejo na strukturno neurejenost N-končne unikatne regije, medtem ko je regija domen FS–EC–TY strukturno bistveno bolj urejena, na področju domene EC prevladujejo α-vijačnice, v domenah FS in TY pa so poleg α-vijačnic prisotne tudi ploskve beta. To ilustrira tudi družina modelov, pripravljenih na osnovi eksperimentalnih podatkov SAXS ter modelov posameznih domen, ki kaže na meddomenske interakcije znotraj tripleta FS–EC–TY. Vezava kalcijevih ionov prispeva k strukturni stabilizaciji in se odraža v nekoliko večji kompaktnosti molekule testikana-2. Z uporabo testov zapiranja celične vrzeli smo tudi pokazali, da je ravno jedro FS–EC–TY tisto, ki je povezano s povečano celično migracijo. Enako smo pokazali tudi za testikan-1, kar nakazuje na funkcionalno redundančnost v okviru te fiziološke vloge. Naši rezultati nudijo prvi vpogled v strukturno organizacijo testikanov ter s tem predstavljajo dobre temelje za nadaljnje raziskave na nivoju struktura–funkcija. Testicans are modular proteoglycans of the vertebrate extracellular matrix with a broad tissue spectrum of expression. The protein family consists of three representatives, testican-1, -2, and -3, which are highly conserved. In their polypeptide chain, there are three regions homologous to other proteins, corresponding to follistatin-like domain (FS), extracellular calcium-binding domain (EC), and thyroglobulin domains (TY), while the N- and C-terminal regions are unique. They are involved in cell attachment, migration, and neurite outgrowth, among other functions, and studies suggests at least partial functional redundancy within the protein family. Although the specific functions and properties described so far (binding of calcium ions, inhibition of cysteine proteases,and membrane-type matrix metalloproteases) have been assigned to individual domains and regions, their spatial organization is unknown. To shed light on this, we further characterized testican-2 as a representative of the protein family, in terms of calcium ion binding and structure, and was related this to its influence on cell migration. First, using isothermal titration calorimetry and structural models of the calcium-binding domain (EC), we showed that only one of the two potential calcium binding sites is active and that it is always occupied at extracellular calcium concentrations. Binding to another potential EF2 motif is deactivated by substitution at one of the canonical coordination sites, where an amino acid residue with a smaller polar side group compared to EF1 is replaced by a residue with a large hydrophobic side group, and this applies to all three members of the protein family. Subsequently, we used disorder analysis to show that the N- and C-terminal regions of all three testicans are most likely structurally disordered. This is consistent with the results of low angle X-ray scattering (SAXS) analysis showing the structural disorder of the N-terminal unique region, while the FS–EC–TY domain region is structurally more ordered. This is also illustrated by a family of models generated based on experimental SAXS data and individual domain models, which indicate interdomain interactions within the FS–EC–TY triplet. Calcium binding contributes to structural stabilization and is reflected in the slightly larger compactness of the testican-2 molecule. Subsequently, using cell gap closure tests, we showed that the core domain triplet FS–EC–TY is associated with increased cell migration. The same was shown for testican-1, suggesting functional redundancy within this physiological role. Our results provide a first insight into the structural organization of testicans and thus provide a good basis for further research at the level of structure-function. testikan SPOCK vezava kalcija SAXS celična migracija testican SPOCK calcium-binding SAXS cell migration true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2021-04-13 11:55:00 2021-04-13 11:55:01 2022-09-05 03:54:23 0000-00-00 00:00:00 2021 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1313 62643971 Krajnc_Anja_-_Studije_strukturnih_lastnosti_in_interakcij_cloveskih_proteoglikanov_testikanov.pdf Krajnc_Anja_-_Studije_strukturnih_lastnosti_in_interakcij_cloveskih_proteoglikanov_testikanov.pdf 1 644D82DC9397B96000F29EC5EC2F9A3C cb70d38c95b1ad208e8f6776935f989111745ca9f14da0ea9feb9a9e824b44f1 de587f34-a1bb-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=142048 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0