Proteasome (Prosome) Subunit Variations during the Differentiation of Myeloid U937 Cells

Abstract

20S proteasomes (prosomes/multicatalytic proteinase) are protein particles built of 28 subunits in variable composition. We studied the changes in proteasome subunit composition during the differentiation of U937 cells induced by phorbol‐myristate‐acetate or retinoic acid plus 1,25‐dihydroxy‐cholecalciferol by western blot, flow cytometry and immuno‐fluorescence. p25K (C3), p27K (IOTA) and p30/33K (C2) subunits were detected in both the nucleus and cytoplasm of undifferentiated cells. Flow cytometry demonstrated a biphasic decrease in proteasome subunits detection during differentiation induced by RA+VD. PMA caused an early transient decrease in these subunits followed by a return to their control level, except for p30/33K, which remained low. Immuno‐fluorescence also showed differences in the cytolocalization of the subunits, with a particular decrease in antigen labeling in the nucleus of RA+VD‐induced cells, and a scattering in the cytoplasm and a reorganization in the nucleus of PMA‐induced cells. Small amounts of proteasomal proteins were seen on the outer membrane of non‐induced cells; these membrane proteins disappeared when treated with RA+VD, whereas some increased on PMA‐induced cells. The differential changes in the distribution and type of proteasomes in RA+VD and PMA‐induced cells indicate that, possibly, 20S proteasomes may play a role in relation to the mechanisms of differentiation and the inducer used.

The Ubiquitin-associated Domain of hPLIC-2 Interacts with the Proteasome

“…By comparing ␣-p25 with ␣-p27 and a third proteasomal core antibody against the ␣6 subunit, 62A32, we have previously shown that both bands are proteasome related and that these antibodies can detect proteasomal core antigens in Western blotting (Kleijnen et al, 2000;Walters et al, 2002). Most likely, the two ␣-p25 bands represent different splice variants that may depend on the particular cell line one uses, because a similar situation has been reported for the 62A32 antibody (Silva Pereira et al, 1992;Henry et al, 1997). It was discovered that the proteasomal core antigens could be preferentially eluted from the beads with a wash in NP-40 lysis buffer supplemented with 150 mM NaCl (the GST proteins remain on the beads), allowing for clean Western blotting.…”

Plasma proteasome level is a potential marker in patients with solid tumors and hemopoietic malignancies

“…Anomalies in the ubiquitin‐proteasome pathway have been described in various pathologic settings, including malignant disease,18 and proteasome inhibitors can induce tumor regression in animal models 19. Along with others, we have published modifications in the intracellular expression and subunit composition of proteasome in leukemic cells4–8 as well as abnormal proteasome levels in tumor and stromal cells in some patients of breast carcinoma 20. Moreover, gene expression of the proteasome subunit iota is increased in patients with ALL and decreased in patients with AML 21…”

“…In 1990, Kumatori et al4 first demonstrated an abnormally high expression level of proteasomes in human leukemic cells. We subsequently published findings that, in some examples of neoplastic differentiation, the amount and subunit composition of cell proteasomes indeed varied 5–8. Proteasomes can be detected and measured on the cell surface of lymphocytes9 and in cell culture supernatants or patient serum 10.…”

Chronically active: activation of microglial proteolysis in ageing and neurodegeneration

“…13 PMA was shown to diminish the inclusion of the p30/33K proteasome subunit and the amount of p25K and p27K, 35 but whether this indeed increases proteasome activity is not yet known. There are conflicting data regarding the effect of PMA activation on lysosomal enzyme activity.…”