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

Oncotarget (2017-12-20)
Valentina A Kulichkova, Tatiana O Artamonova, Olga G Lyublinskaya, Mikhail A Khodorkovskii, Alexey N Tomilin, Anna S Tsimokha
RESUMEN

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.

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Sigma-Aldrich
Monoclonal Anti-α-Tubulin antibody produced in mouse, clone B-5-1-2, purified immunoglobulin, buffered aqueous solution
Sigma-Aldrich
Monoclonal Anti-β-Tubulin I+II antibody produced in mouse, clone JDR.3B8, ascites fluid

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