Proteomic analysis of affinity-purified extracellular proteasomes reveals exclusively 20S complexes.

Proteasome-mediated proteolysis is important for many basic cellular processes. In addition to their functions in the cell, proteasomes have been found in physiological fluids of both healthy and diseased humans including cancer patients. Higher levels of these proteasomes are associated with higher cancer burden and stage. The etiology and functions of these proteasomes, referred to as circulating, plasmatic, or extracellular proteasomes (ex-PSs), are unclear. Here we show that human cancer cell lines, as well as human endometrium-derived mesenchymal stem cells (hMESCs), release proteasome complexes into culture medium (CM). To define ex-PS composition, we have affinity purified them from CM conditioned by human leukemia cell line K562. Using matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS), we have identified core 20S proteasome subunits and a set of 15 proteasome-interacting proteins (PIPs), all previously described as exosome cargo proteins. Three of them, PPIase A, aldolase A, and transferrin, have never been reported as PIPs. The study provides compelling arguments that ex-PSs do not contain 19S or PA200 regulatory particles and are represented exclusively by the 20S complex.

Simultaneous EGFP and tag labeling of the ß7 subunit for live imaging and affinity purification of functional human proteasomes.

The proteasome is a multi-subunit protein complex that serves as a major pathway for intracellular protein degradation, playing important functions in various biological processes. The C-terminus of the ß7 (PSMB4) proteasome subunit was tagged with EGFP and with a composite element for affinity purification and TEV cleavage elution (HTBH). When the construct was retrovirally delivered into HeLa cells, virtually all of the ß7-EGFP-HTBH fusion protein was found to be incorporated into fully functional proteasomes. This ensured that subcellular localization of the EGFP signal in living HeLa cells could be attributed to ß7-EGFP-HTBH within the proteasome complex rather than to free protein. The ß7-EGFP-HTBH fusion can, therefore, serve as a valuable tool for in vivo imaging of proteasomes as well as for high-affinity purification of these complexes and associated molecules for subsequent analyses.

DNA damage modulates interactions between microRNAs and the 26S proteasome.

26S proteasomes are known as major non-lysosomal cellular machines for coordinated and specific destruction of ubiquitinylated proteins. The proteolytic activities of proteasomes are controlled by various post-translational modifications in response to environmental cues, including DNA damage. Besides proteolysis, proteasomes also associate with RNA hydrolysis and splicing. Here, we extend the functional diversity of proteasomes by showing that they also dynamically associate with microRNAs (miRNAs) both in the nucleus and cytoplasm of cells. Moreover, DNA damage induced by an anti-cancer drug, doxorubicin, alters the repertoire of proteasome-associated miRNAs, enriching the population of miRNAs that target cell cycle checkpoint regulators and DNA repair proteins. Collectively, these data uncover yet another potential mode of action for proteasomes in the cell via their dynamic association with microRNAs.

26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli.

26S proteasome is a large multi-subunit protein complex involved in proteolytic degradation of proteins. In addition to its canonical proteolytic activity, the proteasome is also associated with recently characterized endoribonuclease (endo- RNAse) activity. However, neither functional significance, nor the mechanisms of its regulation are currently known. In this report, we show that 26S proteasome is able to hydrolyze various cellular RNAs, including AU-rich mRNA of c-myc and c-fos. The endonucleolytic degradation of these mRNAs is exerted by one of the 26S proteasome subunits, PSMA5 (alpha5). The RNAse activity of 26S proteasome is differentially affected by various extra-cellular signals. Moreover, this activity contributes to the process of degradation of c-myc mRNA during induced differentiation of K562 cells, and may be controlled by phosphorylation of the adjacent subunits, PSMA1 (alpha6) and PSMA3 (alpha7). Collectively, the data presented in this report suggest a causal link between cell signalling pathways, endo-RNAse activity of the 26S proteasome complex and metabolism of cellular RNAs.

Changes in composition and activities of 26S proteasomes under the action of doxorubicin--apoptosis inductor of erythroleukemic K562 cells.

Changes in the subunit composition, phosphorylation of the subunits, and regulation of the activities of 26S proteasomes in proliferating cells undergoing programmed cell death have not been studied so far. Moreover, there are no reports on phosphorylation of proteasome subunits both in normal and in neoplastic cells during apoptosis. The data of the present study show for the first time that apoptosis inductor doxorubicin regulates subunit composition, enzymatic activities, and phosphorylation state of 26S proteasomes in neoplastic (proerythroleukemic K562) cells or, in other words, induces reprogramming of proteasome population. Furthermore, the phosphorylation state of proteasomes is found to be the mechanism controlling specificity of proteasomal proteolytic and endoribonuclease activities.

U87 RNA, a novel C/D box small nucleolar RNA from mammalian cells.

A novel 72 nt small nucleolar RNA (snoRNA) called U87 was found in rat liver cells. This RNA possesses the features of C/D box snoRNA family: boxes C, D', C', D, and 11 nt antisense element complementary to 28S ribosomal RNA (rRNA). The vast majority of C/D box snoRNAs direct site-specific 2'-O-ribose methylation of rRNAs. U87 RNA is suggested to be involved in 2'-O-methylation of a G(3468) residue in 28S rRNA. U87 RNA was detected in different mammalian species with slight length variability. Rat and mouse U87 RNA gene was characterized. Unlike the majority of C/D box snoRNAs U87 RNA lacks the terminal stem required for snoRNA processing. However, U87 gene is flanked by 7 bp inverted repeats potentially able to form a terminal stem in U87 RNA precursor.