Immunoassay for the Quantification of Intracellular Multi‐Ubiquitin Chains

Abstract

A sandwich ELISA has been developed to measure intracellular levels of multi-ubiquitin chains. The mixture of multi-ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus "'I-ubiquitin) and lysozyme with ubiquitin-ligating enzymes and ATP, was partially purified and established as a standard named the multi-ubiquitin-chain reference preparation 1 (MUCRPI). The concentration of MUCRPI was calculated from the recovered radioactivity of "'I-ubiquitin. All measurements by the ELISA were expressed in terms of MUCRPI. The ELISA showed good sensitivity (98 pg/ml), precision (intra-assays < 6%) and reproducibility (interassay < 9%). In addition, there was no substantial cross-reaction with mono-, di-and tri-ubiquitin, or mono-ubiquitinated and di-ubiquitinated lysozyme in the ELISA, and large multi-ubiquitin chains ( n > approximately 6 ) may be fully reactive. These results combined with excellent results in the recovery and dilution tests guarantee accurate measurement of multi-ubiquitin chains in cell extracts prepared with a lysis buffer (water soluble) or the buffer supplemented 8 M urea (urea soluble). The level of the water-soluble multi-ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea-soluble chain level was higher in the reticulocytes. Heat-shock treatment of HeLa cells increased the urea-soluble multi-ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi-ubiquitin-conjugate turnover.Keywords: ELISA ; heat shock ; multi-ubiquitin chain; reticulocyte; ubiquitin.Ubiquitin is found in either a free form or a form bound covalently to intracellular proteins in vivo (Hershko, 1988;Finley and Chau, 1991). In the latter form, the ubiquitin C-terminus is ligated to &-amino groups of appropriate Lys residues in target proteins by a family of ubiquitin-conjugating enzymes: a ubiquitin-activating enzyme (El), a ubiquitin-carrier protein (E2), and a ubiquitin-protein ligase (E3) (Hershko et al., 1983;Hershko and Ciechanover, 1992). In this process, several ubiquitin moieties are usually ligated sequentially to the initial acceptor protein, forming multi-ubiquitin chains in which the ubiquitin Cterminus is conjugated to the &-amino group of Lys48 of another ubiquitin moiety (Chau et al., 1989). The multi-ubiquitin chain acts as a signal to induce degradation of target proteins by 2 6 s proteasome (Chau et al., 1989;Hershko and Ciechanover, 1992). Therefore, the chain structure is essential for ubiquitin-dependent proteolysis.The ubiquitin-dependent proteolysis has been implicated in a variety of cellular processes, including stress response (Finley et al., 1987;Carlson et a]., 1987), cell-cycle control (Glotzer et al., 1991;Nishizawa et al., 1993) and apoptosis (Delic et al., 1993). Intracellular levels of ubiquitin and ubiquitin-protein conjugates are changed in response to heat shock (Parag et al., Abbreviations. MUCRPI , multi-ubiquitin chain reference preparation 1 ; E l , ubiquitin-activating enzy...

Proteomic techniques to probe the ubiquitin landscape

“…The development of monoclonal antibodies recognizing polyubiquitin but not monoubiquitin facilitated the targeted analysis of polyubiquitinated material [69]. One of these monoclonal antibodies (FK2) was selected because of its high specificity toward polyubiquitinated proteins [70] and was first introduced in proteomic studies of the ubiquitinome [71] ( Figure 4C). This study identified 670 proteins that were enriched under highly denaturing conditions, but lacked the detection of GG-peptides…”

Purification and identification of monoubiquitin-phosphoglycerate mutase B complex from human colorectal cancer tissues

“…The electrophoretic technique employed in our study, however, is less accurate for such applications. Based on our ELISA system for multiubiquitin chains, 20 development of an asymmetric ELISA for the ubiquitinated PGM-B, in which 2 antibodies to ubiquitin and the PGM-B are used for capture and detection of the antigen, respectively, is planned and it will be useful in evaluating the clinical significance.…”

“…The levels of multi-ubiquitin chains were expressed in terms of the multi-ubiquitin chain reference preparation 1 (MUCRP1). 20 …”

“…The monoclonal antibody FK2 against conjugated ubiquitin, 19 which recognizes not only conjugated ubiquitin but also free ubiquitin in solution, 18 was purified as described previously 20 and covalently coupled to Sepharose using NHS-activated Sepharose 4FF (Amersham Pharmacia Biotech, Piscataway, NJ) in accordance with the manufacturer's instructions. The affinity-purified antibody specific to human PGM type B was kindly provided by Oriental Yeast Co., Ltd. (Tokyo, Japan).…”

“…Free ubiquitin and multi-ubiquitin chain were quantified using a competitive radioimmunoassay 22 and a symmetric sandwich ELISA, 20 respectively. The levels of multi-ubiquitin chains were expressed in terms of the multi-ubiquitin chain reference preparation 1 (MUCRP1).…”