| Summary: | Genotoxic stress induces significant changes in expression rates of regulatory proteins inside transformed cells. Since some of these proteins are short-lived ones and they undergo rapid proteasomal degradation, combined treatment with genotoxic agent (doxorubicin) and proteasome inhibitor (bortezomib) should launch accumulation of these proteins in cancer cells. This accumulation is even more characteristic for the multiple myeloma cells due to their high synthetic potential.
We investigated the pattern of regulatory proteins associated with PSMA3 subunit of 20S proteasome core. PSMA3 is engaged into ubiquitin-independent degradation of several proteins known to contribute in cancer development (IκBα, p21WAF1/CIP1). We suppose, that the proteins revealed may either be directed to undergo ubiquitin-independent proteolysis, or possess proteasome-regulating activity, and changes in their abundance may not only serve as an evidence of some treatment-induced changes, but also may be considered as biomarkers of the therapies efficiency.
RPMI 8226 multiple myeloma cell line was used as a model object. Before the actual treatment the proper apoptosis-inducing concentrations of doxorubicin and bortezomib were determined in order to avoid necrosis. PSMA3-interacting proteins were isolated by incubation with GST-PSMA3 fusion protein attached to glutathione sepharose beads, 2D-electrophoretically separated and identified by MALDI TOF/TOF mass-spectrometry.
As a result we obtained heterogeneous list of proteins that can be functionally divided into 12 subgroups: RNA turnover regulators, chaperones and chaperonines, metabolic regulators, translation factors, cytoskeleton proteins, mitochondrial proteins, transcriptional regulators, subunits of proteasomal regulatory complexes, and several minor groups housing single members. Due to their function and metabolism inside the cell some of the identified proteins seem to be promising for the further investigation; among them are HuR, TUSC1 (Tumour Suppressor Candidate), several hnRNP representatives, numerous chaperones (GRP78 in particular). Presence of these proteins was additionally confirmed with immunoblotting assay, which pattern also gave several promising guidelines for further elaboration of the data. To exemplify, ELAVL1 (HuR) protein is known to be a protector of mRNAs for c-myc, c-fos and some other proteins stabilising them via binding AU-rich sequences in 3′-UTR. As far as proteasomes are also capable to recognise these motifs and moreover possess RNAse activity, these two processes might compete within the cell. According to immunoblotting patterns after drug treatment of the cells HuR is significantly accumulated; this supports our assumption of it being degraded via proteasome-independent proteolysis. Relying on the obtained data this hypothesis and several others are being tested.
Present work was supported by RFBR (Projects 12-04-01397 and 15-04-04541).
|
|---|
