CHARACTERIZATION OF THE EXTRACELLULAR PROTEASOMES AND ITS INTERACTING PROTEINS BY iTRAQ MASS SPECTROMETRY
Iu.Ia. Zaykova,1,2 V.A. Kulichkova,1 Iu.B. Ermolaeva,1 A. Bottrill,3
N.A. Barlev,1,3,4 A.S. Tsimokha 1,*
1 Institute of Cytology, RAS, St. Petersburg, 2 Saint-Petersburg State Polytechnical University, Saint-Petersburg,
31 University of Leicester, Leicester, UK, 4 Saint-Petersburg State Institute of Technology, Saint-Petersburg;
*e-mail: atsimokha@mail.cytspb.rssi.ru
The analysis of the extracellular proteasomes by isobaric tagging for relative and absolute quantifications (iTRAQ) mass spectrometry was carried out. Here we have shown a
standard set of 26S proteasomal subunits in the composition of the extracellular proteasomes. Moreover, extracellular proteasomes have a number of PA200 activators, which, as
previously thought, are localized in the cell's nucleus. Posstanslational modifications (PTMs) of subunits of the extracellular proteasomes were revealed by iTRAQ mass spectrometry.
We for the first time identified several ubiquitination and acetylation sites on subunits α2 (K196), α4 (K189 è K234), α6 (K217), and Rpn6 (A2). It was obtained a
large number of proteasome-interacting proteins that are involved in various cell processes, such as transcription, DNA repair, translation, cytoskeletal proteins and the proteins of the
ubiquitin-proteasome system (UPS). Immunoblot analysis confirmed the interactions between purified extracellular proteasomes and nine proteins which were randomly selected from
the set of interacting proteins.
Key words: extracellular proteasomes, iTRAQ mass spectrometry, posttranslational modifications, ubiquitination
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