Final Report Summary - UB (Construction of Well- defined Ubiquitin Conjugates)
The modification of proteins with ubiquitin and Ub-like proteins (Ubls) regulates a multitude of biological processes. The introduction of this very complex interface (which in effect creates a new protein with additional options for further (protein-protein) interactions, may explain the vast variety of biological processes regulated by ubiquitination. In general, the C-terminal carboxylate of Ub (ls) is linked to a target protein by an isopeptide bond with the e-amine of lysine (Lys) or by a peptide bond with an N-terminal amine. This ATP-dependent conjugation involves a complex enzymatic cascade consisting of E1, E2 and E3 enzymes. For any biological or structural study of Ub (l) structures, access to (sufficient amounts of) well-defined Ub (l) structures is of prime importance. Although some well-defined Ub conjugates can be synthesized by biochemical techniques, these techniques have severe drawbacks: one needs to identify and isolate E2/3 ligases which can selectively construct the desired linkage; this strategy is not necessary compatible with other post-translational modifications. The full extent of this dynamicity is still not fully understood and it seems that variety in linkage-sites and structure is greater than anticipated. In the case of Ub for example, its 7 lysines can by self-conjugation create diverse polyUb chains with their own signalling function.
Then there is the removal of Ub by deubiquitinating enzymes (DUBs). As these proteases process inactive Ub precursors, proofread ubiquitinated substrates and remove Ub from cellular adducts, they are of considerable importance. Their involvement in various diseases and that eukaryotic genomes encode many potential DUBs, has intensified the need for well-defined and specific DUB targeting probes, facilitating development of DUB-targeted pharmacological agents. Overall, with current research into Ub (l) processing progressing rapidly, the need for (chemical) tools has become of great importance. In contrast to the currently used biochemical techniques, chemical strategies can be targeted at a wider range of structures, i. e. natural or non-natural.
Our aim was to develop novel chemical strategies towards well-defined Ub modified conjugates. These can be used to investigate DUBs on several levels:
monitor DUB activity (assay development)
use as antigens by creating a non-hydrolysable linkage between UB and the polypeptide
use as activity based DUB probes, in a target sequence specifc context.
Our group has succeeded in developing chemical ubiquitination strategies that allow us to synthesize native and non-native linked Ub-conjugates. Importantly, our approaches also allow us to construct Ub-like polypeptide conjugates. We have used all these types of conjugates for not only answering fundamental quuestions (e. g. polyUb chain preference of DUBs, binding studies) but also to develop assay reagents for drug discovery efforts.
Our results and collaborative projects based on our conjugates have been published in first-class research journals. Our ubiquitin-system centered chemical technologies have also resulted in three patent applications and form the basis of a spin-off (http://www.ubiqbio.com) that was founded end of 2010.
Contact details:
Dr Farid El Oualid
Tel: + 31 (0) 205122012
e-mail: f. eloualid@nki. nl
Project website address: http://research. nki. nl/Ovaalab/
Then there is the removal of Ub by deubiquitinating enzymes (DUBs). As these proteases process inactive Ub precursors, proofread ubiquitinated substrates and remove Ub from cellular adducts, they are of considerable importance. Their involvement in various diseases and that eukaryotic genomes encode many potential DUBs, has intensified the need for well-defined and specific DUB targeting probes, facilitating development of DUB-targeted pharmacological agents. Overall, with current research into Ub (l) processing progressing rapidly, the need for (chemical) tools has become of great importance. In contrast to the currently used biochemical techniques, chemical strategies can be targeted at a wider range of structures, i. e. natural or non-natural.
Our aim was to develop novel chemical strategies towards well-defined Ub modified conjugates. These can be used to investigate DUBs on several levels:
monitor DUB activity (assay development)
use as antigens by creating a non-hydrolysable linkage between UB and the polypeptide
use as activity based DUB probes, in a target sequence specifc context.
Our group has succeeded in developing chemical ubiquitination strategies that allow us to synthesize native and non-native linked Ub-conjugates. Importantly, our approaches also allow us to construct Ub-like polypeptide conjugates. We have used all these types of conjugates for not only answering fundamental quuestions (e. g. polyUb chain preference of DUBs, binding studies) but also to develop assay reagents for drug discovery efforts.
Our results and collaborative projects based on our conjugates have been published in first-class research journals. Our ubiquitin-system centered chemical technologies have also resulted in three patent applications and form the basis of a spin-off (http://www.ubiqbio.com) that was founded end of 2010.
Contact details:
Dr Farid El Oualid
Tel: + 31 (0) 205122012
e-mail: f. eloualid@nki. nl
Project website address: http://research. nki. nl/Ovaalab/