Ubiquitin and ubiquitin-like (UBL) protein conjugation systems control a vast array of cellular processes, and impact virtually every biological system. In this process, UBLs are activated through an ATP-driven activation and conjugation cascade before attachment to target proteins. Conjugation of ubiquitin itself is best known for its role in protein turnover via the proteasome, but ubiquitin conjugation can also provide regulatory functions in solid-state signaling networks.
Our work seeks to employ systematic genetic and proteomic approaches to elucidate the mechanisms and biology of ubiquitin and UBL protein conjugation systems, including the autophagy system. Much of our efforts have been devoted to elucidating the components and targets of a superfamily of E3 ubiquitin ligases referred to as cullin-RING ubiquitin ligases. We have explored the roles of these E3s in cell cycle and DNA damage checkpoint control and are currently employing systematic proteomic approaches to identify substrates and biological processes of many poorly understood ubiquitination pathways. We have recently elucidated the network organization of human deubiquitinating enzymes, the human autophagy system, the ERAD system, the ubiquitin modified proteome, and the mechanism of activation and action of the PARKIN ubiquitin ligase, a protein that is mutated in familial forms of Parkinson’s Disease. An additional area of interest concerns the use of proteomic methods to dissect protein networks involved in assembly and regulation of mitochondria in mammalian cells.
A major emphasis is placed on the development of proteomic tools, methods, and software for quantitative analysis of signaling pathways and ubiquitination. This includes the use of AQUA proteomics, Parallel Reaction Monitoring (PRM) and Tandem Mass Tagging (TMT) based methods. We are using these approaches to examine the dynamics of multiple signaling pathways.
Behrends C, Sowa ME, Gygi SP, and Harper, JW. (2010) Network organization of the human autophagy system. Nature 466, 68-76.
Sarraf S, Raman M, Guarani-Pereria V, Sowa ME, Huttlin EL, Gygi SP, and Harper, JW. (2013) Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496, 372-376.
Ordureau A, Sarraf SA, Duda DM, Heo J-M, Jedrychowski MP, Sviderskiy V, Olszewski JL, Koerber JT, Xie T, Beausoleil SA, Wells JA, Gygi SP, Schulman BA, and Harper, JW (2014) Quantitative proteomics reveal a feed-forward model for mitochondrial PARKIN translocation and UB chain synthesis. Molecular Cell 56:360-375.
Mancias JD, Wang X, Gygi SP, Harper, JW, Kimmelman AC. (2014) Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature 509:105-109.