期刊
BIOCHIMIE
卷 93, 期 4, 页码 715-724出版社
ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
DOI: 10.1016/j.biochi.2010.12.014
关键词
Ubiquitination; Protein-protein interactions; Medulloblastoma; Hedgehog pathway; Structure-function relationships; Light scattering
资金
- Italian MIUR
- Pasteur Institute-Cenci Bolognetti Foundation
The family of human proteins containing a potassium channel tetramerization domain (KCTD) includes 21 members whose function is largely unknown. Recent reports have however suggested that these proteins are implicated in very important biological processes. KCTD11/REN, the best-characterized member of the family to date, plays a crucial role in the ubiquitination of HDAC1 by acting, in complex with Cullin3, as an E3 ubiquitin ligase. By combining bioinformatics and mutagenesis analyses, here we show that the protein is expressed in two alternative variants: a short previously characterized form (sKCTD11) composed by 232 amino acids and a longer variant (IKCTD11) which contains an N-terminal extension of 39 residues. Interestingly, we demonstrate that IKCTD11 starts with a non-canonical AUU codon. Although both sKCTD11 and IKCTD11 bear a POZ/BTB domain in their N-terminal region, this domain is complete only in the long form. Indeed, sKCTD11 presents an incomplete POZ/BTB domain. Nonetheless, sKCTD11 is still able to bind Cul3, although to much lesser extent than IKCTD11, and to perform its biological activity. The heterologous expression of sKCTD11 and IKCTD11 and their individual domains in Escherichia coli yielded soluble products as fusion proteins only for the longer form. In contrast to the closely related KCTD5 which is pentameric, the characterization of both IKCTD11 and its POZ/BTB domain by gel filtration and light scattering indicates that the protein likely forms stable tetramers. In line with this result, experiments conducted in cells show that the active protein is not monomeric. Based on these findings, homology-based models were built for IKCTD11 BIB and for its complex with Cul3. These analyses indicate that a stable IKCTD11 BTB-Cul3 three-dimensional model with a 4:4 stoichiometry can be generated. Moreover, these models provide insights into the determinants of the tetramer stability and into the regions involved in IKCTD11-Cul3 recognition. (C) 2011 Elsevier Masson SAS. All rights reserved.
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