Our Science – Weissman Website
Allan M. Weissman, M.D.
The components of the ubiquitin system comprise a highly complex and finely tuned set of mechanisms whereby the fate and function of specific proteins are regulated. Modification of proteins with ubiquitin impacts on almost all cellular processes through a variety of different mechanisms. The most well-known of these is through targeting of proteins for degradation in the 26S proteasome. Additionally, ubiquitination can affect the trafficking of proteins within the cell in a variety of different ways. This modification can also activate signal transduction pathways, modulate gene expression and plays critical roles in both double and single stranded DNA repair. Ubiquitination is a hierarchical pathway with one predominant ubiquitin-activating enzyme (E1), over 30 different ubiquitin conjugating enzymes (E2s) and more than 500 different potential ubiquitin ligases (E3s). Ubiquitin ligases interact with E2s that have been loaded with ubiquitin, recognize specific substrates and mediate the transfer of ubiquitin to substrates where stable isopeptide linkages are formed and in many cases chain of ubiquitin are generated. While we are interested in all aspects of the ubiquitin system, work in our laboratory is primarily focused on ubiquitin ligases, their interactions with specific E2s, structure-function relationship, and the roles of these proteins in modulating critical cellular processes, particularly those that are associated with cancer and other human diseases. We also have an interest in the discovery of inhibitors of specific components of the ubiquitin system that may serve as the basis for novel therapeutics.
Structure-Function Relations and Substrate Identification for Ubiquitin Ligases
There are only a handful of different ubiquitin ligase domains. Most prominent among these are the RING and RING-like family of E3s. Within this family of proteins there are a variety of other regions that appear to play roles in modulating ligase activity. Ongoing studies in the laboratory are oriented towards determining the roles that various domains and other structural features play in the function of ubiquitin ligases and in identifying specific substrates for these ligases. Ubiquitin ligases currently under study include AO7 - the first RING E3 that we discovered, Hdm2/Mdm2 - the primary ubiquitin ligase for p53 and gp78 [also known as the human tumor autocrine motility factor (AMFR) or RNF45]. Our research has uncovered a complex domain structure for gp78, which includes a RING finger, a CUE domain that binds ubiquitin, and a novel and highly specific binding site for gp78's cognate E2, which we refer to as the Ube2g2 binding region (G2BR) (Chen et al., 2006). A major area of interest is to understand how these domains within gp78 function together to mediate ubiquitination. In collaboration with our colleagues at CCR, Drs. R. Andrew Byrd and Xinhua Ji, we have determined the structure of Ube2g2 in complex with the G2BR. The binding of the G2BR to Ube2g2, at a site removed from the RING finger (‘backside binding’), results in allosteric changes in Ube2g2 such that the capacity of the E2 to be loaded with ubiquitin is altered. However, most striking is that this binding translates into a marked increase in the affinity of Ube2g2 for the gp78 RING finger resulting in an enhancement of ubiquitination (Das et al., 2009), this work was the subject of an NCI press release and a Preview in Structure (Wang and Schulman, 2009). We now understand that this allostery is manifest in interactions between Ube2g2 and the gp78 RING finger. Moreover, we have determined that there is additionally reverse allostery that facilitates E2 release, presumably after ubiquitin transfer, thereby promoting a 'ubiquitination machine' (Das et al., 2013).
In yeast, both major RING finger ERAD E3s, HRD1 and DOA10 share a common component known as Cue1p, which is required for their function. Analogous to gp78, we have now determined a specific binding site within the yeast Cue1p protein for the yeast ERAD E2, Ubc7p. This Ubc7p binding region (U7BR) has the capacity to stimulate RING finger-dependent ubiquitination (Kostova et al., 2009). Again, in collaboration with Drs. Byrd and Ji, we have structurally and functionally characterized the U7BR in complex with Ubc7p and determined that the U7BR has multiple allosteric effects on Ubc7p, all of which enhance ubiquitination (Metzger et al., 2013).
Determining the fate of transmembrane proteins in the secretory pathway
A major area of interest is in understanding the mechanisms responsible for degradation of proteins by ERAD (endoplasmic reticulum-associated degradation). This set of process represents a major mechanism whereby misfolded and unassembled proteins are targeted for degradation and also plays important roles in responses to ER stress and in regulating critical regulatory proteins. We have characterized the role of Ube2g2 and gp78 in ERAD and determined, in collaboration with Dr. Chand Khanna, that in sarcoma, gp78 plays a causal role in survival of metastatic cancer cells. A striking finding is that gp78 targets a known metastasis suppressor protein, known as KAI1 or CD82, for ubiquitin-mediated proteasomal degradation (Tsai et al., 2007, NCI press release). This finding is important not only because of its potential therapeutic implications but also as it establishes a new level of regulation of metastasis suppressors, through control of protein stability. We are continuing to study the relationship between gp78 and metastasis through analysis of different tumor types in mouse models that we have established. Related studies are oriented towards determining the range of substrates for gp78 and functional and physical interactions between the multiple mammalian ERAD E3s.
Inhibitors of the ubiquitin conjugating system
The discovery that the RING fingers in general are ubiquitin ligase domains (Lorick et al., 1999) has led us to become involved in the search for inhibitors of specific RING finger ubiquitin ligases whose activity may play an important role in cancer. This work, in collaboration with Dr. Karen Vousden and IGEN/MSD has led to the discovery of a family of inhibitors of Hdm2, an E3 that plays a critical role in regulating levels of p53, a critical tumor suppressor and the 'guardian of the genome' (Yang et al., 2005). We have subsequently discovered a highly soluble and more potent member of this family that is currently being evaluated by NCI for its potential as a lead for therapeutic development (Kitagaki et al., 2008). A side benefit of our screening efforts has been the discovery of a family of small molecules that exhibit relative specificity towards inhibiting E1 and which specifically kill transformed cells (Yang et al.,2007). The lead compound from this screen, Pyr-41, has now been commercialized as the first cell permeable inhibitor of the ubiquitin E1. In addition, together with CCR collaborators we are exploring the potential to inhibit ERAD in general or gp78 in specific as a therapeutic modality.
Ubiquitin and Mitochondria
Our studies on ubiquitination at membrane-bound organelles have now expanded to mitochondria. We provided the first unequivocal evidence for a crucial role for ubiquitination in regulating a critical component of the mitochondrial fusion machinery, the yeast mitofusin Fzo1p, and determined that regulated degradation of Fzo1p is integral to fusion in yeast (Cohen et al., 2008, Cohen et al., 2011 ). We have also expanded our studies to mammalian mitochondria where we have determined a new pathway linking genotoxic stress to phosphorylation, ubiquitination and proteasomal degradation of human mitofusin 2. This in turn leads to mitochondrial fragmentation and apoptosis (Leboucher, Tsai et al., 2012) . Current and ongoing studies are oriented towards exploring other E3s involved in regulation of mitofusin 2, exploring mitochondrial quality control pathways and the role of the ubiquitin-proteasome system in the degradation of mitochondrial proteins that are localized to compartments other than the outer mitochondrial membrane.
This page was last updated on 12/9/2013.