439 Melanoma cells export functional proteasomes by way of microvesiculation: a possible mechanism for generation of plasmatic proteasomes

Abstract

Plasmatic proteasomes (p-proteasomes) are a reliable marker for metastatic dissemination in melanoma patients. Recent works have suggested that p-proteasome partially originated from tumour cell secretion via exosomes, which are also considered as a new biomarker for metastatic melanoma. Our objectives were to investigate the origin, mechanism of secretion and functionality of the melanoma p-proteasomes. We show by proteasome purification either from culture supernatant of melanoma cell lines (A-375, SK-MEL-28) or from plasma of melanoma patients, that the extracellular proteasome is released essentially as an assembled 20S core particle, contrarily to proteasome analyzed from cell lysate, which presents additional 26S capped proteasome. Using specific fluorogenic substrates we demonstrate that these extracellular proteasomes are functional and possess all the peptidase activities associated with b1, b2 and b5 subunits. There is no correlation between exosome and proteasome concentrations determined by ELISA in the plasma of melanoma patients. Immunoisolation of secreted proteasome using a7 antibody adsorbed on magnetic beads and pull-down experiments show that secreted proteasomes are not enwrapped in exosomes. To our point of view, previous reports of 20S proteasome subunits in exosomes are rather due to a co-purification along the exosome preparation. Extracellular proteasome concentration is increased in the secretome of melanoma cell lines after stimulation of membrane blebbing with the ionophore A23187. Moreover, proteasome is associated with microvesicles as shown by gel filtration experiments and immobilization of microvesicles on lectin-sepharose. Altogether our results show that melanoma cells secrete functional 20S proteasomes at least in part by membrane blebbing and microvesiculation and not through exosomal pathway.