Human proteasomes analysed with monoclonal antibodies

Abstract

The proteasome or multicatalytic endopeptidase from eukaryotic cells consists of at least 14 subunits that fall into two families, alpha and beta. Subunit-specific monoclonal antibodies against ten different subunits of human proteasomes have been produced, together with an antibody that reacts with a motif (prosbox 1), common to alpha-type subunits. Four of the subunit-specific antibodies were able to precipitate proteasomes. The subunit composition of HeLa-cell proteasomes precipitated with these four different antibodies were identical, as judged from two-dimensional electrophoresis. One of the four antibodies was used to obtain proteasomes from cell lines (HeLa, Daudi, IMR90 and BSC-1) and human tissues (placenta, kidney, and liver). Electrophoretic analysis of these proteasomes, combined with peptide mapping of some subunits, suggests that they all contain 14 types of subunits as their major constituents. However, one subunit was present in two isoelectric isoforms in all cells examined. Two other subunits occurred in two or three isoelectric isoforms in placenta, liver and kidney, but not in the cell cultures. Extracts of human cells (HeLa, IMR90, Daudi and erythrocytes) were analysed by non-denaturing electrophoresis and immunoblotting. All of the 11 subunits detected by antibodies were present in a pair of ATP-stabilized protein complexes, presumed to be the 26 S proteinase, and in a doublet of complexes which migrated more slowly than purified proteasomes. Besides being present in proteasomes, one subunit was also found to occur in the free state in cell extracts.

Central Role of the Proteasome in Senescence and Survival of Human Fibroblasts

“…, and S14 (p31) (PW8835) subunits were purchased from Affiniti Research Products Ltd. Antibodies against ␤ 7 (N3), ␣ 6 (C2), ␣ 7 (C8), S4, S5a, S6a (TBP1), S6b (TBP7), S8 (p45), ␤ 1i (LMP2), and ␤ 5i (LMP7) were a generous gift from Dr. K. Hendil (27,28). Primary antibodies against ubiquitinated proteins (sc8017), ApoJ (sc6419), p16 (sc467), p21 (sc817), and ␤-actin (sc1616) as well as secondary antibodies were purchased from Santa Cruz Biotechnology Inc. Oxidized proteins were detected with anti-2,4-dinitrophenol antibody from the OxyBlot TM protein oxidation detection kit (Intergen).…”

Novel Propeptide Function in 20 S Proteasome Assembly Influences β Subunit Composition

“…MCP21 is a mouse monoclonal antibody that recognizes human C3 and immunoprecipitates both 20 S proteasomes and 13-16 S preproteasomes (34). The hybridoma was obtained from the European Collection of Animal Cell Cultures (Salisbury, Wiltshire, UK).…”

Effects of Major‐Histocompatibility‐Complex‐Encoded Subunits on the Peptidase and Proteolytic Activities of Human 20S Proteasomes

“…The hybridoma mAb MCP21 specific for the atype subunit HC 3 was kindly provided and described in detail by Klavs Hendil (Hendil and Uerkvitz, 1991 ;Kaltoft et al, 1992;Hendil et al, 1995). To produce polyclonal antibodies against LMP2 and LMP7 rabbits were immunized with peptides derived from the C-termini of LMP2 (CHRVILGNELPKFYDE) and LMP7 (CTDVSDLLHQYREANQ).…”

“…Method 2. Proteasomes were isolated by affinity chromatography as described (Hendil and Uerkvitz, 1991). 1.5 mg purified mAb MCP21 was coupled to a 1 ml N-hydroxysuccinimide-activated HiTrap column (Pharmacia) according to the instructions of the manufacturer.…”

“…The 20s proteasome plays a major role in non-lysosomal degradative processes (Fujiwara et al, 1990;Heinemeyer et al, 1991;Mason and Rivett, 1994; and as the proteolytic core of the 26s complex in the ubiquitin-dependent pathway Hershko and Ciechanover, 1992;Rechsteiner et al, 1993). 20S/26S complexes are postulated to be important for the generation of antigenic peptides (Goldberg and Rock, 1992;Monaco, 1992;Michalek et al, 1993;.…”