Biotin-ubiquitin tagging of proteins and ubiquitination of poly(ADP-ribose) polymerase-1.

摘要

The degradation of tightly controlled proteins involved in cell proliferation, differentiation, and gene expression is frequently regulated by the covalent modification of ubiquitin. Ubiquitination of proteins may also participate in other cellular processes such as receptor targeting, DNA repair, and translation. Poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the synthesis of poly(ADP-ribose) and is a key enzyme involved in DNA repair, apoptosis, necrosis, and transcription. Whether PARP-1 is modified or regulated by ubiquitination is not known.; A synthetic ubiquitin cDNA was first fused to a biotinylation peptide cDNA and biotin holoenzyme synthetase cDNA as a tripartite cDNA. Biotin tagged ubiquitin was successfully produced in E. coli and purified by affinity chromatography as the tripartite cDNA was inserted into a pET vector for expression. Biotin-ubiquitin provided a new non-radioactive method for monitoring protein ubiquitination. Biotin-ubiquitin facilitated expression and purification of large mammalian proteins. Several large mammalian proteins including human ubiquitin activating enzyme and SUMO (small ubiquitin-like modifier) activating enzyme were expressed and purified as functional biotin-ubiquitin fusion proteins. Additionally, the biotin-ubiquitin tag can be efficiently and specifically cleaved using a chicken ubiquitin hydrolase UBP41 to produce unmodified mammalian proteins.; The ubiquitination of full-length PARP-1 was demonstrated in vitro and in vivo. Polyubiquitination of recombinant PARP-1 was observed in HeLa cell extracts or with affinity purified ubiquitination enzymes by immunoprecipitation with monoclonal anti-PARP antibody followed by SDS gel electrophoresis and immunoblot with anti-ubiquitin or biotin ubiquitin. The PARP-1 polyubiquitination was strongly inhibited by activated DNA. The 89 kDa fragment of the caspase-3 cleaved PARP-1 exhibited dramatically reduced ubiquitination. These results are consistent with the polyubiquitination within the DNA binding domain. The polyubiquitin chain was apparently mediated through Lys48 not Lys63. Mouse fibroblasts in the presence but not in the absence of a proteasome inhibitor accumulated polyubiquitinated PARP-1 in vivo. These results suggest that the polyubiquitinated PARP-1 is destined for degradation and is most likely involved in transcription regulation and less likely in apoptosis or necrosis.