Allan M. Weissman, M.D.
Our lab studies regulated protein post-translational modifications. We’re particularly interested in the ubiquitin-proteasome system as it relates to normal physiology, cancer, cardiovascular disease, and other disorders. Research is focused on three major areas: 1) structure-function relationships of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s); 2) ubiquitin-mediated endoplasmic reticulum (ER)-associated degradation (ERAD) and its relationship to stress, the unfolded protein response and regulating the survival and death of cancer cells; and 3) the roles of the ubiquitin system at mitochondria and its disease implications. We utilize a wide variety of approaches and employ systems that include yeast, cancer cell lines, mice, and human tissue samples.
1) ubiquitin, 2) ubiquitin ligases, 3) ubiquitin-conjugating enzymes, 4) endoplasmic reticulum-associated degradation, 5) mitochondria, 6) unfolded protein response, 7) cell biology of cancer
The ubiquitin-proteasome system (UPS) comprises a highly complex, finely tuned, set of mechanisms that determines the fate and function of proteins, and which regulates essentially all cellular processes. The most well-known function of ubiquitin is to target proteins to the 26S proteasome. Ubiquitination also affects the trafficking of proteins within the cell, activates signal transduction pathways, modulates gene expression, and plays critical roles in DNA repair.
This hierarchical system includes a predominant ubiquitin-activating enzyme (E1), over 30 different ubiquitin conjugating enzymes (E2s) and more than 500 substrate-specific ubiquitin ligases (E3s). Each E3 interacts with one or more E2 that has been ‘loaded’ with ubiquitin, and mediates the transfer of ubiquitin to substrates where stable isopeptide linkages are formed and, in many cases, ubiquitin chains are generated. Ubiquitination is potentially reversible through the activity of ~100 cellular deubiquitinating enzymes (DUBs).
Our lab is interested in E3s and interacting E2s, their substrates and their cognate DUBs. As a mechanism-oriented lab, we explore structure-function relations and relate these back to physiology and disease. Particular areas of cellular focus pertain to the roles of this system in the secretory pathway and at mitochondria, where there are clear associations of the UPS with physiology and disease. Integrated into this is an interest in applying our knowledge to establishing proof of principle for new therapeutic modalities for cancer and other diseases.
Structure-Function Relations for RING-type Ubiquitin Ligases
Most prominent among E3s are the RING-type family of E3s, which recognize E2s through their RING or RING-like domains. Many members of this family have a variety of other regions that modulate activity. Ongoing studies are oriented towards determining the roles played by these regions in E3 function and in identifying specific substrates.
One RING E3 that has served as the basis for much study is gp78 [also known as the human tumor autocrine motility factor (AMFR) or RNF45]. We have uncovered a complex domain structure for this polytopic ER (endoplasmic reticulum) resident E3, which includes 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 in understanding how these domains 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 the E2, Ube2g2, in complex with the G2BR and the gp78 RING. The binding of the G2BR to Ube2g2, at a site removed from the RING (backside binding), markedly enhances ubiquitination. We now understand that this is governed by complex allosteric changes involving the G2BR, Ube2g2 and the gp78 RING finger (Das et al., 2009; Das et al., 2013). We have discovered E2-specific backside binding domains in other E3s. These carry out a variety of functions and have the potential to either enhance or decrease ubiquitination (Kostova et al., 2009; Metzger et al., 2013, Li et al., 2015).
Ongoing studies are oriented towards further understanding these domains through structure-activity studies and in vivo studies, characterizing other related, domains and potentially exploiting these unique regions to modulate E3 function and to use this information as the basis for therapeutics.
The Ubiquitin System in the Secretory Pathway and Mitochondria and its role in Oncogenesis and Metastasis
A major area of interest is in understanding the mechanisms responsible for degradation of proteins by ERAD (ER-associated degradation). This set of processes 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 xenografts, gp78 plays a causal role in survival of metastatic cancer cells, in part by targeting a metastasis suppressor protein for proteasomal degradation (Tsai et al., 2007). This 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 continue to study the relationship between gp78 and metastasis through analysis of different tumor types in mouse models, which we have established. Related studies are oriented towards determining the range of substrates for gp78 and assessing these for roles in malignancy as well as determining the functional and physical interactions between the multiple mammalian ERAD E3s.
Our studies on ubiquitination at membrane-bound organelles have 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 ). This led to an expansion of studies to mammalian mitochondria. Here we have determined a new pathway linking genotoxic stress to phosphorylation, ubiquitination and proteasomal degradation of human mitofusin 2, which leads to mitochondrial fragmentation and apoptosis (Leboucher, Tsai et al., 2012) .
Ongoing studies in mammals and yeast are oriented towards exploring other E3s involved in regulation of mitofusin 2, elucidating mitochondrial quality control pathways and understanding the role of the ubiquitin-proteasome system in the degradation of mitochondrial proteins that are localized to compartments other than the outer mitochondrial membrane. Studies in mice are focused on the role of proteins that control mitochondrial fusion and fission in cancer and metastasis.
Selected Recent Publications
Deubiquitinating enzyme VCIP135 dictates the duration of the botulinum neurotoxin type A intoxication.Proc Natl Acad Sci U S A. 114(26): E5158-E5166, 2017. [ Journal Article ]
The Ubiquitin Ligase (E3) Psh1p is required for proper segregation of both Centromeric and Two-Micron Plasmids in Saccharomyces cerevisiae.G3 (Bethesda). 7(11): 3731-3743, 2017. [ Journal Article ]
An ubiquitin-dependent balance between mitofusion turnover and fatty acids desaturation regulates mitochondrial fusion.Nat Commun. 8: 15832, 2017. [ Journal Article ]
Insights into Ubiquitination from the Unique Clamp-like binding of the Ring E3 A07 to the E2 UbcH5B.J Biol Chem. 290(51): 30225-39, 2015. [ Journal Article ]
A structually unique E2-binding domain activates ubiquitination by the ERAD E2, Ubc7p, through multiple mechanisms.Mol Cell. 50(4) : 516-27, 2013. [ Journal Article ]
Dr. Allan Weissman received his B.S. from Stony Brook University and his M.D. from Albert Einstein College of Medicine in 1981. After a residency in Internal Medicine at Washington University, he came to NIH where he was a post-doctoral fellow in NICHD. In 1989, he joined the NCI as an independent investigator. In 2001 he was appointed Laboratory Chief and is currently the Chief of the Laboratory of Protein Dynamics and Signaling.
|M. Iveth Garcia Ph.D.||Postdoctoral Fellow (CRTA)|
|Loren J. Kozar B.S.||Guest Researcher|
|Jennifer Mariano M.S.||Research Biologist|
|Meredith Metzger Ph.D.||Research Fellow|
|Claudia I. Meyer B.S.||Postbaccalaureate Fellow (CRTA)|
|Christopher Smith Ph.D.||Postdoctoral Fellow (CRTA)|
|Yien Che Tsai Ph.D.||Staff Scientist|
|Mei Yang M.D.||Research Biologist|