Ubiquitin Specific Protease 11 (USP11): structure and enzymology

The units that make up an organism, the cells, rely on the presence of a large number of proteins that are required at different times. In order to quickly respond to environmental changes or to dispose of damaged proteins, cells harbour an effective system for protein destruction that is tightly regulated as any mistakes can have serious effects for the cell. Protein destruction consists of a series of events. At first a small protein termed ubiquitin is attached to a protein molecule that serves as a signal to direct these molecules into a certain pathway. The formation of a particular type of chain of several ubiquitin molecules on a protein results in them being recognised by a large molecular machine, the proteasome that ultimately digests the protein into small pieces. The attachment of only one ubiquitin moiety or other types of ubiquitin chain to a protein can also lead to other outcomes unrelated to protein destruction. Because the correct timing is essential, there are mechanisms to remove or alter these signals. Check point molecules called deubiquitinating enzymes can remove ubiquitin molecules and consequently alter the fate of a target protein. The most abundant class of these deubiquitinating enzymes in humans are the ubiquitin specific proteases (USPs). They regulate ubiquitin-dependent metabolic pathways by cleaving ubiquitin-protein bonds. Each USP specifically recognizes a limited number of ubiquitinated proteins that they can salvage from destruction or channel into different pathways within cells. The structure of these molecules is complicated in that they need to be able to provide binding sites for the protein, in order to be specific, as well as the attached ubiquitin chain and be highly selective in their action. Removing the ubiquitin signal from the wrong protein could have disastrous effects for the cell. This research aims at gaining insights into how one ubiquitin specific protease, USP11, achieves this specificity, whether it is able to distinguish between different ubiquitin chains and how its molecular make-up influences its function. To this end we will determine structures of discrete parts of USP11 and identify binding surfaces for the protein and the ubiquitin chain. As USP11 also interacts with other proteins we will look for additional binding sites. A second attempt to gain a better understanding of these important regulatory proteins will consist of looking at the shape and flexibility of the whole molecule in solution. Ultimately we will establish which parts of USP11 are involved in the recognition of the viral protein HPV-16E7 that once infection has occurred exploits the function of USP11 in order to extend its own life-span. Together, this will provide unique novel insights into how these proteases work, which will help us to better understand this check-point system in regulating normal protein destruction pathways, signalling events and in viral infection.

Individual cellular proteins are degraded and re-synthesized at different rates in order to ensure the proper functioning of the cell. Protein degradation is therefore essential as a means to reduce the steady state level of a particular protein and needs to be tightly regulated. The covalent attachment of one or several moieties of the small protein ubiquitin to target proteins influences virtually all cellular events, but is most well known as signal for selective protein degradation by the proteasome. Similar to phosphorylation, ubiquitination is reversible, and is catalysed by deubiquitinating enzymes (DUBs). As such, DUBs play an important regulatory role in controlling a protein's life span and the propagation of signalling cascades. In the human genome, ubiquitin-specific proteases (USPs) constitute the largest family of DUBs and are potential drug targets. Despite their fundamental importance for the life cycle of a protein and cellular signalling, our molecular understanding of how they work is surprisingly poor. The project will investigate the structure, specificity and mechanism of USP11, a paradigm for a sub-class of USPs that are characterised by the predicted presence of an N-terminal DUSP (domain in ubiquitin specific proteases) and two ubiquitin-like (UBL) domains. We will use structural biology techniques such as X-ray crystallography and Small-angle Scattering, in conjunction with biochemical studies. We want to identify interaction surfaces and understand how ubiquitin chains and target proteins such as the viral oncoprotein HPV-16E7 are recognized and specificity is achieved. These structural insights will reveal fundamental principles of ubiquitin deconjugation.

Short-term, research outcomes will be communicated and disseminated through publications in high impact international journals, talks and poster presentations at national and international conferences, university research and open days, and on departmental web-sites. Medium-term, the structures will provide a template for structure-guided drug design targeting not only the active site of USP11 (or the related USP4 and USP15 for which the USP11 structure will provide the basis for the construction of homology models) but also newly identified interaction surfaces (e.g. the USP11-HPV-16E7 interaction) to design inhibitors of protein-protein interactions. Long-term it can be envisaged that this will lead to new therapeutic strategies and as such will spur the interest of pharmaceutical companies. The University of Nottingham has as strong reputation as an entrepreneurial University ('Entrepreneurial University of the Year' at the prestigious Times Higher Education Awards 2008) and there is support provided for translational activities and patent application processes through dedicated administrative staff. The applicant will also be able to draw on previous experience in translational research (see case for support). This ultimately could impact on improved health of the UK society (examples listed below) 1. Private sector: Pharmaceutical companies Ubiquitin proteasomal system as a target for drug discovery in cancer The observation that proteasome inhibitors are able to induce apoptosis preferentially in tumor cells opened the way to their use as potential drugs (Orlowski RZ, Kuhn DJ. Clin Cancer Res. 2008 ;14(6):1649-57. Proteasome inhibitors in cancer therapy: lessons from the first decade). One of these drugs, bortezomib (Velcade), was introduced in cancer therapy and its use was approved for the treatment of multiple myeloma and mantle cell lymphoma. Bortezomib is the first drug targeting the Ubiquitin Proteaseome System, it was introduced by Millennium in 2007 and generated $1B revenue in 2008. The drug bortezomib affects many cellular pathways and more selective targeting of the UPS via inhibition of Ubiquitin ligases or proteases may have increased benefit and reduced side effect. This industry will benefit from this work by reading publications from the grant or downloading structures depositied in the PDB database. 2. Charities. Cancer charities (CRUK,) Human papillomaviruses and cervical cancer Infection with high-risk human papillomaviruses (HR-HPVs) is the cause of most cervical cancers, as well as a number of other malignancies and researchers working in this area (Cancer Research UK) would benefit from greater understanding of the USP11 removal of ubiquitin from the E7 oncoprotein. HR-HPVs express two oncogenes, E6 and E7, whose products disable the two predominant tumour suppressor. The stability of E7 is thus important to ensure its fully functional status. USP11 can greatly increase the steady state level of HPV-16E7 by reducing ubiquitination and attenuating E7 degradation. Cervical cancer is a leading cause of cancer mortality and the second most common malignancy in women worldwide and research groups internationally (US, Germany, Korea, UK) will be interested in this research to develop novel therapeutics. Such therapeutics would lower disease burden (worldwide annually 500,000 new cases of invasive cervical cancer) and medical care expenses. These groups will become aware of the data from this grant by searching publication archives or will be contacted directly. 3. Design of new drugs using crystal structures as templates The USP11 crystal structures determined from this project will be depositied in the PDB database upon publication. These structures can be used by the research community as templates to design specific inhibitors using structure based drug design methodology (pharaceutical industry, charities and academic groups are currenltly engaged in using this methodology