A novel type of ubiquitination regulates apoptosis signaling by BH3-only protein Bid.
11 / 2008 - 11 / 2012
Background - Bcl-2 family member Bid is a key intermediate in apoptosis signaling by death receptors and cytolytic T cells. We have discovered that Bid function is regulated by a novel form of polyubiquitination, in which the ubiquitin acceptor sites are neither lysines, nor the amino-terminus. Upon chemical reduction, ubiquitin was partially lost from Bid, implicating thioester linkage to cysteine residues. However, mutation of all cysteines did not abrogate ubiquitination, implicating other residues. These are most likely serine and/or threonine, given that ubiquitin was lost upon mild hydrolysis. Ubiquitin ligation to cysteine or serine/threonine residues has thus far only been described for two viral ubiquitin ligases. We are the first to demonstrate that mammalian cells can carry out this process. Moreover, we have elucidated the functional significance of this unconventional ubiquitination: it selectively targets Bid?s amino-terminal fragment for degradation, thus liberating its BH3 domain for apoptosis-induction. Aim ? We will determine the exact nature of Bid ubiquitination and its mode of regulation. Thereby we aim to define this novel type of ubiquitin modification, for which the endogenous mammalian enzymatic machinery is unknown and which may apply to other signaling molecules as well. Plan of investigation ? We will use two experimental systems: expression of Bid and variants thereof in cells and introduction of these in an in vitro ubiquitination system. In these systems, we will identify the nature of this unusual ubiquitin conjugation and enzymes regulating Bid's ubiquitination status. We will use for these purposes a combination of molecular biological and innovative chemical approaches. This project is based on unique findings in our research group and provides a unique opportunity to capitalize on recent investments in (chemical) technology in our institute. Collaboration wil be carried out with dr. Huib Ovaa, organic chemist, and prof. Titia Sixma, structural biologist at the NKI and with dr. Benedikt Kessler, mass spectrometrist at Oxford University. The results will advance our basic understanding of ubiquitination in cell signaling and are expected to have a therapeutic spin-off.