Karakterizacija humanog mišićnog proteina Ankrd2

Abstract

Predmet istraživanja ovog rada je humani mišićni protein Ankrd2 koji se eksprimira u sporim mišićnim vlaknima i potencijalni je transkripcioni faktor čija je količina povećana u toku hipertrofije skeletnih mišića. To je protein od 37 kDa koji poseduje signal za lokalizaciju u jedru, četiri ankirinska ponovka i veoma je sličan jedarnom proteinu humanih endotelnih ćelija, C-193, čija je ekspresija indukovana citokinima. Pacovski i mišji ortologoni humanog proteina C-193, CARP odnosno MARP su pretežno eksprimirani u srcu i verovatno su uključeni u kontrolu hipertrofije srca. Pretpostavlja se da Ankrd2 u skeletnim mišićima ima sličnu funkciju kao CARP/MARP/C-193 u srcu. Za utvrdjivanje intraćelijske lokalizacije i ekspresije proteina Ankrd2 u različitim tkivima napravljena su tri mišja poliklonska antitela (na ceo protein, N-terminalni i C-terminalni region) i monoklonsko antitelo (na C-terminalni region proteina Ankrd2). Western blot analizom proteinskih ekstrakata različitih humanih tkiva pokazano je da se protein Ankrd2 eksprimira uglavnom u skeletnim mišićima i u manjoj meri u srcu i bubregu. U toku diferencijacije humanih (CHQ5B) i mišjih (C2C12) mišićnih ćelija dolazi do povećanja količine proteina Ankrd2. U cilju detaljne analize intraćelijske lokalizacije proteina Ankrd2 uradjeni su eksperimenti indirektne imunofluorescencije koristeći humane mioblaste i miotube. Generalno, u toku diferencijacije povećava se i broj ćelija koje fluoresciraju i intenzitet signala. Takodje, u humanim mioblastima, za razliku od miotuba, detektovana je specifična jedarna fluorescencija. Jedarni signal je u obliku tački i eksperimenti kolokalizacije su pokazali da se proteini Ankrd2 i PML nalaze u istim jedarnim strukturama nazvanim PML jedarna tela. U cilju izučavanja proteinsko-proteinskih interakcija pripremljeni su rekombinantni proteini fuzionisani sa GST markerom (F-Ankrd2 (5-333 ak), N-Ankrd2 (5-120 ak) i C-Ankrd2 (279-333 ak)) koji su korišćeni u GST „pull-down” eksperimentima. Ovom metodom je pokazano da protein Ankrd2 interaguje sa nekoliko, za sada neidentifikovanih, proteina. U aminokiselinskoj sekvenci proteina Ankrd2 nalaze se četiri potencijalna mesta za fosforilaciju kazein kinazom II (CKII). U ovom radu je pokazano da protein Ankrd2 može biti fosforilisan in vitro ovom kinazom, s tim što je samo četvrto mesto (SGRE, 318-321 ak) fosforilisano. Eksperimenima retardacije DNK na gelu utvrdjeno je da proteini Ankrd2 i IkB, inhibitor transkripcionog faktora NFkB, nemaju sličnu funkciju iako imaju strukturne sličnosti (oba proteina poseduju ankirinske ponovke, signal za lokalizaciju u jedru i mesta za fosforilaciju raznim kinazama). Za razliku od IkB, protein Ankrd2 ne sprečava formiranje kompleksa izmedju DNK i NFkB.

The object of this study was the new human muscle protein Ankrd2 found preferentially in slow muscle fibers that is possibly a transcription factor up-regulated in hypertrophy. It is a protein of 37 kDa, which has a signal for nuclear targeting, four ankyrin repeat motifs and shows significant similarity to a cytokine inducible nuclear protein C-193 from human endothelial cells. The rat and mouse orthologs of human C-193 called respectively CARP and MARP are mainly expressed in heart and probably involved in the control of cardiac hypertrophy. It is possible that Ankrd2 may play a similar role to CARP/MARP/C-193, but in skeletal muscle rather than heart. For intracellular localization and tissue distribution of Ankrd2 three mouse polyclonal antibodies (raised against the full length, N-terminal and C-terminal regions of the protein) and one monoclonal antibody (raised against C-terminal region of Ankrd2) were made. Western blot analysis of protein extracts isolated from different human tissues showed that Ankrd2 is expressed mainly in human skeletal muscle and to a lesser extent in heart and kidney. During muscle cell differentiation there is an increase of Ankrd2 signal in both mouse (C2C12) and human (CHQ5B) muscle cells that can be detected by Western blot analysis. Immunofluorescence experiments were undertaken in primary human myoblasts and myotubes with the scope of pinpointing the intracellular localization of Ankrd2 protein. In general, during differentiation both the number of fluorescing cells and the intensity of signal increase. Also, in human myoblasts but not in differentiated myotubes very specific nuclear fluorescence can be detected. The nuclear signal is in the form of speckles and co-localization experiments showed that Ankrd2 and PML co-localize in PML nuclear bodies. In order to study protein-protein interactions GST recombinant proteins F-Ankrd2 (5-333 aa), N-Ankrd2 (5-120 aa) and C-Ankrd2 (279-333 aa) were prepared and used for the GST „pull-down” experiments. Results show that Ankrd2 interacts with several proteins, but so far these proteins are not identified. The deduced amino acid sequence of Ankrd2 contains four putative casein kinase II (CKII) phosphorylation sites. Ankrd2 can be phosphorylated in vitro with CKII, but only the fourth site (318-321 aa, SGRE) is phosphorylated. In order to discover wether Ankrd2 has a similar function to IkB, the inhibitor of transcriptional factor NFkB (as they have structural similarities (ankyrin repeats, nuclear localization signal, phosphorylation sites)) electromobility shift assays (EMSA) were performed. Ankrd2 does not prevent the formation of a complex between NFkB and its DNA binding site whereas IkB in the same experiments was able to do so, thus suggesting that Ankrd2 does not function as muscle specific IkB protein.