MASS-SPECTROMETRIC ANALYSIS OF PROTEASOMAL SUBUNITS POSSESSING ENDORIBONUCLEASE ACTIVITY
A.G. Mittenberg,1,* T.N. Moiseeva,1 V.O. Kuzyk,1 E.P. Podolskaya,2
I.N. Evteeva,1 N.A. Barlev 1,3
1 Institute of Cytology RAS, 2 Institute for Analytical Instrumentation RAS, St. Petersburg, Russia, and
3 Department of Biochemistry, Leicester University, United Kingdom;
* e-mail: a.mittenberg@gmail.com
Proteasomes act as the main apparatus of non-lysosomal intracellular proteolysis and participate in the regulation of most important cellular processes. Despite considerable
progress in the understanding of proteasome's functioning, some issues, in particular, RNase activity of these ribonucleoprotein complexes and its regulation remain scarcely
explored. In this paper we found several proteins corresponding by electrophoretic mobility to subunits of the complex 20S proteasome to possess endoribonuclease activity
with respect to both sense and antisense sequences of the c-myc mRNA 3R-UTR. Mass-spectrometric analysis of tryptic hydrolysates of these proteins revealed in the
samples the presence of 20S proteasome subunits - α1 (PSMA6), α5 (PSMA5), α6 (PSMA1) and α7 (PSMA3). A number of novel phosphorylation sites
in subunits α1 (PSMA6) and α7 (PSMA3), and the form of subunit α5 (PSMA5) with a deletion of N-terminal 20 amino acid residues detected. The observed
differences of individual subunits in the possession endonuclease activity could be apparently ex-plained by postranslational modifications of these proteins, in particular - by
phosphorylation. It is shown that the specificity of the proteasomal RNase activity varies after dephosphorylation and also influenced by Ca and Mg cations. The conclusions made
about the impact of the PTM status of proteasome subunits on the specificity of their RNase activity.
Key words: proteasomes, ribonucleases, mRNA stability, posttranslational modifications, phosphorylation,
mass-spectrometry
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