RESUMEN
Proteasome-mediated proteolysis is critical for regulation of vast majority of cellular processes. In addition to their well-documented functions in the nucleus and cytoplasm proteasomes have also been found in extracellular space. The origin and functions of these proteasomes, dubbed as circulating/plasmatic or extracellular proteasomes, are unclear. To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ-based quantitative proteomics (iTRAQ LC/MS-MS). Our analysis of purified from K562 cells or conditioned medium intact proteasome complexes led to an identification and quantification of 114 proteins, out of which 19 were 26S proteasome proteins (all subunits of the 20S proteasome and a small number of the 19S regulatory particle proteins), and 3 belonged to the ubiquitin system. Sixty-two of proteasome interacting proteins (PIPs) were differentially represented in CP versus EP, with folds difference ranging from 1.5 to 4.8. The bioinformatics analysis revealed that functionally most of EP-PIPs were associated with protein biosynthesis and, unlike CP-PIPs, were under represented by chaperon/ATP-binding proteins. Identities of some of the proteasome proteins and PIPs were verified by Western blotting. Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes. Specifically, the EP prep contained only three 19S subunits versus at least 18 in the CP one, suggesting that the extracellular proteasomes are deficient in 19S complexes, which may imply that they have special biological functions. J. Cell. Physiol. 232: 842-851, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Espacio Extracelular/metabolismo , Marcaje Isotópico/métodos , Subunidades de Proteína/metabolismo , Proteómica/métodos , Medios de Cultivo Condicionados/farmacología , Humanos , Células K562 , Modelos Biológicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Cromatografía de Afinidad/métodos , Proteínas Fluorescentes Verdes/metabolismo , Imagen Molecular , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Complejo de la Endopetidasa Proteasomal/metabolismo , Supervivencia Celular , Células HeLa , Humanos , Subunidades de Proteína/metabolismo , Proteolisis , Proteínas Recombinantes de FusiónRESUMEN
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.