Regulation of the proteasome by Fbxo7

Living cells are largely constructed from a multitude of different proteins, each with their own individual functions and properties. Just like the components of any man-made machine, these proteins are susceptible to "wear and tear" and can become damaged over time. The health of a cell, and consequently the whole organism to which it belongs depends on the timely and specific removal of damaged proteins to prevent their accumulation in toxic aggregates that will eventually poison the cell. Cells have evolved a mechanism to identify and then destroy unwanted proteins, called the "ubiquitin-proteasome system". Damaged proteins are marked with chains of small ubiquitin molecules, targeting them to the proteasomes, the cell's "recycling bins". The failure of this system to clear protein aggregates has been linked to multiple human diseases, including Parkinson's disease, Alzheimer's disease, type II diabetes and is also implicated in those involving prion proteins which cause Creutzfeldt-Jakob disease in humans, BSE in cattle and scrapie in sheep and goats. Conversely, over-activity of the ubiquitin-proteasome system has been linked with various muscle atrophy diseases, it is therefore clear that proper regulation of this system is required for the maintenance of health.

Recently, a study detailing the properties of a controller of the rate at which the proteasomes operate has been published. This protein, called PI31, has been shown to both increase and decrease proteasome activity under different circumstances. However, PI31 does not act alone. We have identified a protein called Fbxo7 that is not only capable of marking proteins for destruction with ubiquitin but which also binds to PI31. Studies in fruit flies have shown that this interaction enhances the ability of PI31 to regulate the proteasomes, and flies lacking their analgous Fbxo7 protein, which is called nutcracker, have reduced proteasome activity, and causes sterility in male flies. We have found that loss of the Fbxo7 in mice also affects their fertility. Furthermore, mutations in the human Fbxo7 gene have also been linked to Parkinson's disease, suggesting that Fbxo7 may also cooperate with PI31 to regulate proteasome activity in mammalian systems.

We want to understand in detail how Fbxo7 participates in the regulation of proteasome activity in mammalian cells. We will use a variety of methods to investigate the relationship between PI31 and Fbxo7, determining whether the interaction between these two proteins affects their ability to regulate proteasome activity and mark proteins for degradation, respectively. We will also determine how the loss or over-representation of Fbxo7 in cells affects proteasome activity in cells using a range of assays both in cell lines and in an mouse model. By doing experiments to broaden our understanding of the protein degradation machinery, we hope to be able to affect and ultimately direct these processes in clinically relevant settings.

The ability to remove damaged proteins to prevent the formation of toxic protein aggregates is essential for cell survival. This function, performed by the ubiquitin-proteasome system (UPS), uses poly-ubiquitin tagging of defective proteins to direct them to the proteasome, a multi-subunit protease. Defects in the UPS are linked to multiple diseases of humans and animals.
Originally identified as a proteasome inhibitor, PI31 has now been shown to enhance proteasome activity in Drosophila when stabilised by the F-box protein nutcracker. Loss of nutcracker expression reduces proteasome activity and causes male sterility. The human orthologue of nutcracker, Fbxo7, also binds PI31. It also acts as a substrate-recruiting subunit for an SCF-type ubiquitin ligases and as an assembly factor for cyclin D/Cdk6 complexes.
The work proposed here will provide insight into the fundamental regulation of the proteasome by determining the consequences of the Fbxo7 and PI31 interaction on its activity in mammalian cells.
The questions we will address include:
-Does Fbxo7 regulate proteasome activity and is this dependent on PI31? We will utilise MEFs established from Fbxo7::LacZ embryos and siRNA-mediated reduction of Fbxo7 or PI31 expression in proteasome-reporter cell lines to determine their effects on proteasome activity.
-Which proteins affect the Fbxo7-PI31 interaction? We will disrupt the ability of Fbxo7 and PI31 to form dimers using targeted mutations. We will also test whether known Fbxo7-interacting proteins can disrupt their interaction.
-Does Fbxo7 regulate PI31 in vivo? We will use a range of techniques on Fbxo7LacZ mouse-derived tissues to determine whether loss of Fbxo7 causes loss or change of localization of PI31 or a change to proteasome activity.

We anticipate that our research will have a broad impact across academic, public and private arenas over both short and long time scales. The immediate beneficiaries of our work will include the other scientists in ubiquitin-proteasome research community who will receive new information on the role of Fbxo7 and PI31 in the control of proteasome function as we take the first steps into investigating their relationship in a mammalian setting. Our findings will be disseminated to the research community through the timely publication of our work and presentations at national and international conferences.

Throughout the course of the project, we expect to host under-graduate and post-graduate students who will receive training in a range of molecular, biochemical, and cell biology techniques (live cell fluorescence imaging, FACS etc), equipping them with the skills required for modern bioscience research within academic or industrial research environments. We will also regularly communicate our research and experiences within academic science with the general public at a range of different events including secondary school visits in the Cambridgeshire area and in the summer school programmes for secondary school and mature students through the Widening Participation Initiatives run by the University of Cambridge. During our current BBSRC program grant we participated in the annual Cambridge Science Festival as a forum to present our work to the general public and introduce them to some of the technologies, such as fluorescence microscopy and gene cloning using easy to understand visual and interactive aids. We intend to continue to present our work and engage the public at future science festival, and we will also take any available opportunities to communicate our findings to broader public audiences through the news media. In anticipation of these opportunities, Dr. David Nelson has already received training through the BBSRC Media Course which provided instruction and practise in the preparation of press-releases and participating in radio/TV interviews for a general audience.

Dysfunction of the proteasome, whether due to an inappropriate increase or to loss of activity has the potential to impact on many aspects of human and animal health, from age-related muscle atrophy, cancer, neurodegeneration, type II diabetes and the pathogenesis of prion-related diseases. Therefore new therapeutic drugs targeting modulators of proteasome activity have the potential to be of use to combat many diseases, especially those associated with our ageing national population. We believe that PI31 may present such a target. Only by understanding the relationship between PI31 and Fbxo7 and how it impacts on the activity of the mammalian proteasome, may we be able to best determine how to target this protein for therapeutic effect. Our discoveries may ultimately be exploited by the private sector, pharmaceutical companies which, over longer time scales (10-20 years), may be of benefit to the public and the UK healthcare system through the production of new therapeutic drugs.