20.500.12556/RUL-109428 Vezava kalcijevih ionov in njihova potencialna vloga pri regulaciji aktivnosti alfa-aktinina 4 Binding of calcium ions and their potential role in regulation of alpha-actinin-4 activity α-aktinin-4 je citoskeletni protein iz družine spektrinov. Sestavljajo ga tri domene in sicer domena za vezavo aktina ter paličasta in kalmodulinu podobna domena. Aktivno obliko α-aktinina-4 predstavlja antiparalelni homodimer, katerega osrednja vloga je prečno povezovanje aktinskih filamentov, preko tega pa posredno sodeluje pri tvorbi fokalnih stikov in adherentnih pasov. Za razliko od sorodnega α-aktinina-1 je prisoten tudi v nukleoplazmi, kjer naj bi deloval kot transkripcijski faktor številnih genov, vpletenih v tumorske spremembe celic. Sklepamo, da je izooblika 4, podobno kot izooblika 1, regulirana preko vezave kalcijevih ionov. Za α-aktinin-1 je bilo pokazano, da vezava Ca2+-iona povzroči konformacijske spremembe v domeni CaMD, kar povzroči spremembo afinitete do aktina in posledično reorganizacijo aktinskega citoskeleta. Da bi določili afiniteto vezave ionov Ca2+ in podrobneje sturukturno okarakterizirali protein smo pripravili dva proteinska konstrukta: kalmodulinu podobna domena in konstrukt, skrajšan na N-koncu za 45 aminokislinskih ostankov, ki so glede na napoved strukturno neurejeni in lahko kot taki negativno vplivajo na kristalizacijo. Z izotermno titracijsko kalorimetrijo (ITC) smo določili število vezavnih mest za ione Ca2+ na eno domeno (n = 1,19) ter afiniteto vezave (Kd = 474 µM). Za namen kristalizacije smo pripravili tudi metilirano (metilacija lizinskih aminokislinskih ostankov) obliko α-aktinina-4 in preko velikostne izključitvene kromatografije, sklopljene s sipanjem svetlobe, pokazali, da še vedno tvori stabilen homodimer. Naši rezultati predstavljajo dobro osnovo za podrobnejše strukturne in funkcijske raziskave α-aktinina-4. α-actinin-4 is a cytoskeletal protein from the spectrin family. It consists of three domains, an actin-binding domain, a rod and calmodulin-like domain. The active form of α-actinin-4 is represented by antiparallel homodimer, whose central role is the cross-linking of actin filaments, through which it indirectly participates in the formation of focal contacts and adherent bands. Unlike related α-actinin-1, it is also present in the nucleoplasm, where it is thought to act as a transcription factor for many genes implicated in tumor cell changes. We conclude that isoform 4, like isoform 1, is regulated via calcium ion binding. For α-actinin-1, Ca2+-ion binding has been shown to induce conformational changes in the CaMD domain, resulting in a change in affinity for actin and subsequent reorganization of the actin cytoskeleton. In order to determine the binding affinity of Ca2+-ions and to characterize the protein in more detail, two protein constructs were prepared: a calmodulin-like domain and a construct shortened at the N-terminus by 45 amino acid residues that are structurally disordered according to the forecast and, as such, have a negative effect on crystallization. The number of binding sites for Ca2+-ions per domain (n = 1.19) and the binding affinity (Kd = 474 µM) were determined by isothermal titration calorimetry (ITC). For the purpose of crystallization, a methylated (methylation of lysine amino acid residues) form of α-actinin-4 was also prepared and, via size exclusion chromatography coupled with light scattering, revealed that it still forms a stable homodimer. Our results provide a good basis for more detailed structural and functional studies of α-actinin-4. α-aktinin-4 regulacija kalcij ITC metilacija kristalizacija α-actinin-4 regulation calcium ITC methylation crystallization true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2019-09-02 11:00:09 2019-09-02 11:00:16 2022-08-21 03:42:04 0000-00-00 00:00:00 2019 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 6382 1538305475 Ivanovski_Andrej_-_Vezava_kalcijevih_ionov_in_njihova_potencialna_vloga_pri_regulaciji_aktivnost.pdf Ivanovski_Andrej_-_Vezava_kalcijevih_ionov_in_njihova_potencialna_vloga_pri_regulaciji_aktivnost.pdf 1 1E85D6D7EBD74BDEB2DB5003761C0CBC 3fa057351def748b32b31267bcd5c2dcde0d7fd65e06676ec1178d6979c1e734 dd3e8e24-a1b6-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=120992 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0