magistrsko delo
Andrej Ivanovski (Author), Miha Pavšič (Mentor), Aljaž Gaber (Thesis defence commission member), Gregor Gunčar (Thesis defence commission member)

Abstract

Aktivnost mnogih citosolnih proteinov je odvisna od koncentracije kalcija. Eden izmed pomembnih znotrajceličnih proteinov, ki se odziva na kalcijeve ione, je α-aktinin-4. α aktinini so citoskeletni proteini iz družine spektrinov, ki igrajo povezovalno vlogo med različnimi celičnimi strukturami. Sestavljajo jih štiri regije in sicer aktin vezavna domena, paličasta regija s štirimi spektrinskimi ponovitvami, povezovalna regija “vrat” in kalmodulinu podobna domena. α aktinine delimo na dve skupini glede na njihovo tkivno specifičnost. Mišični izoobliki, α-aktinina 2 in 3, ne vežeta Ca2+-ionov. Po drugi strani ne-mišični izoobliki, α aktinina-1 in -4, vežeta Ca2+-ione, kar vpliva na njuno strukturo in posledično na prostorsko ureditev z njima povezanih aktinskih filamentov. Aktivno obliko α-aktinina-4 predstavlja antiparalelen homodimer z aktin-vezavno domeno na obeh koncih, katerega osrednja vloga je prečno povezovanje aktinskih filamentov v snope in mreže. Za razliko od sorodnega α-aktinina-1 je prisoten tudi v nukleoplazmi, kjer naj bi deloval kot transkripcijski ko-aktivator številnih genov, vpletenih v tumorske spremembe celic. Dosedanje raziskave so pokazale, da vezava Ca2+-iona v kalmodulinu podobno domeno α aktinina-1 sproži konformacijske spremembe v tem delu molekule, ki se po predvidevanjih prenesejo na aktin-vezavo domeno in posledično reorganizacijo aktinskega citoskeleta. Z namenom primerjati Ca2+-vezavne lastnosti ne-mišičnih α aktininov smo se osredotočili na še neokarakterizirano izoobliko 4. V okviru magistrskega dela smo pripravili proteinske kristale dimerne oblike α-aktinina-4, ki vsebuje vse domene udeležene v zgoraj opisani interakciji, ter posneli set difrakcijskih podatkov do ločljivosti 2,9 Å. Z bioinformatsko analizo, kjer smo primerjali zaporedja kalmodulinu podobnih domen različnih izooblik, smo določili tiste aminokislinske ostanke v kalmodulinu podobni domeni α aktinina-4, ki so najverjetneje ključni pri koordinaciji Ca2+ iona. Podobno kot pri α aktininu-1 je tudi pri tej izoobliki pri vezavi Ca2+-ionov aktivna le ena izmed štirih t. i. EF-dlani. Da bi to hipotezo preverili smo v kalmodulinu podobno domeno α aktinina-4 uvedli različne točkovne mutacije, mutantne oblike pa nato okarakterizirali z uporabo izotermne titracijske kalorimetrije. Potrdili smo, da je res aktivno le eno od potencialnih vezavnih mest za Ca2+, afiniteta za vezavo pa je 4-krat nižja kot pri α aktininu-1. Predvidevamo, da je opažena razlika eden izmed faktorjev, ki vplivajo na različno obnašanje teh dveh izooblik ter posledično na njuno različno preferenčno vključenost v citoskeletne strukture kot so fokalni stiki in ukrivljene membranske strukture.

Keywords

aktinski citoskelet;alfa-aktinin-4;regulacija;kalcij;kristalizacija;struktura;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [A. Ivanovski]
UDC: 577.112(043.2)
COBISS: 138548995 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Characterization of calcium ion binding in calmodulin-like α-actinin-4 domain and preparation of crystals for structural research
Secondary abstract: The activity of many cytosolic proteins depends on calcium concentration. One of the important intracellular proteins that respond to calcium ions is α-actinin-4. α actinins are cytoskeletal proteins of the spectrin family that play a crosslinking role between different cellular structures. They consist of four regions, namely an actin binding domain, a rod region with four spectrin repeats, linker region “neck” and a calmodulin-like domain. α actinins are divided into two groups according to their tissue specificity. The muscle isoforms, α-actinin-2 and -3, do not bind Ca2+-ions. On the other hand, the non-muscle isoforms, α-actinin-1 and -4, bind Ca2+-ions, which affects their structure and consequently the spatial arrangement of the actin filaments connected to them. The active form of α-actinin-4 is an antiparallel homodimer with an actin-binding domain at both ends, the central role of which is the cross-linking of actin filaments into bundles and networks. Unlike the related α-actinin-1, it is also present in the nucleoplasm, where it is thought to act as a transcription co-activator for many genes involved in tumorigenic cell changes. Research so far has shown that the binding of Ca2+-ion in the calmodulin-like domain of α-actinin-1 triggers conformational changes in this part of the molecule, which are predicted to be transferred to the actin-binding domain and the resulting reorganization of the actin cytoskeleton. To compare the Ca2+-binding properties of non-muscle α actinins, we focused on the yet uncharacterized isoform 4. As part of the master's thesis, we prepared protein crystals of the dimeric form of α-actinin-4, which contains all the domains involved in the interaction described above, and recorded a set of diffraction data up to a resolution of 2.9 Å. By bioinformatic analysis, where we compared the sequences of the calmodulin-like domains of different isoforms, we determined those amino acid residues in the calmodulin-like domain of α-actinin-4, which are most likely to be crucial in the coordination of the Ca2+-ion. Like α-actinin-1, only one of the four EF1-hands is active in binding Ca2+-ions. In order to test this hypothesis, we introduced various point mutations in the calmodulin-like domain of α-actinin-4, and the mutant forms were then characterized using isothermal titration calorimetry. We confirmed that only one of the potential binding sites for Ca2+ is indeed active, and the binding affinity is 4 times lower than that of α-actinin-1. We assume that the observed difference is one of the factors influencing the different behavior of these two isoforms and consequently, to their different preferential involvement in cytoskeletal structures such as focal junctions and curved membrane structures.
Secondary keywords: alpha-actinin-4;regulazion;calcium;crystallization;structure;Univerzitetna in visokošolska dela;
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: XVI, 63 str.
ID: 17389061