Regulation of protein networks occurs at all levels, from transcription to protein degradation and modification. A key question in mammalian biology has been how the proteome is regulated independently of transcription. With over 600 E3 ubiquitin ligases in human cells, compared to over 1,000 transcription factors, regulation of protein stability is likely on the same scale of significance and complexity as transcriptional regulation. However, protein stability remains an uncharacterized feature for most proteins and this deficiency has arisen because development of tools for a proteome-wide study of protein turnover is technologically challenging.
Our research aims at developing and employing systematic high throughput genomic and proteomic approaches to the general problem of protein degradation regulation and ubiquitin ligase target identification. Ubiquitin-mediated proteolysis (UPS) is the major proteolytic pathway in the cell and the substrate specificity in UPS is largely conferred by E3 ligases. Many E3s are potential drug targets, but linking an E3 with its substrates is currently a laborious endeavor with few general solutions. We, together with Dr. Stephen Elledge's lab, developed a method called GPS, for Global Protein Stability, which allows us to simultaneously monitor the stability of ~8,000 proteins in live cells. The method uses a highly parallel multiplex analysis of single cells by FACS coupled with a microarray readout. Many novel findings concerning the mammalian proteome were revealed, such as a strong correlation between protein size and protein stability, and the fact that there are distinct amino acid distributions and cellular functions associated with stable versus unstable proteins. In addition, we applied the GPS approach to identify the substrates of SCF, a member of cullin-RING E3 ligases (CRL). We recovered 75% of the previously known SCF targets and identified several hundred novel substrates. These results demonstrate the potential of GPS as a general platform for the global discovery of E3-substrate networks.
GPS approach opened many adventures for protein turnover studies. GPS profiling could be used to identify proteins whose stabilities change in response to stimuli, genetic perturbations, or during developmental transitions. GPS could also be used to generate disease-specific protein stability signatures that may be useful for diagnoses and elucidating disease mechanisms. GPS could be coupled with loss of function (RNAi), gain of function or chemical screens to discover proteins/compounds that regulate the stability of a protein of interest. Furthermore, the integration of global protein stability information from GPS with other datasets, such as the transcriptome, interactome and phosphoproteome, will provide a global view of regulatory networks and identify crosstalk between protein turnover and other forms of biological regulation.
Our current projects focus on developing the next generation of GPS with improved sensitivity, fidelity, applicability and throughput. We also continue to characterize the function of CRL family E3 ligases and seek to elucidate the mechanism and biology of CRL-mediated protein degradation. In the long term, we are interested in investigating how protein turnover regulation coordinates with other levels of regulation in biological networks, and in developing other high throughput technologies for global analysis of additional forms of protein regulation.