D r. 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. Dr. Weissman’s main interests are in the function and regulation of transmembrane receptors, the role of the ubiquitin system in protein regulation and in development of approaches to manipulate specific components of the ubiquitin system as a basis for therapeutics.
I would like to express my gratitude for the honor of being asked by Dean Raghuram and Professor Kannan to deliver the 11th Yellapragada SubbaRow Memorial Lecture at GGSIU.
It is highly appropriate that a lecture on the ubiquitin-proteasome system be part of this series dedicated to this outstanding scientist.
Dr. SubbaRow played a critical role in developing our understanding of cellular energy stored in phosphate-containing molecules (phosphocreatine and ATP) and was also responsible for synthesizing one of our most important anti-cancer cytotoxic pharmaceuticals, methotrexate. The area that I study grew out of the search for the mechanism of ATP-dependent intracellular protein degradation and has enormous practical and theoretical implications for cancer. This cancer relevance is too broad a topic for this narrative. However please see the referenced review article (1).
Until the late 1970’s, it was not uniformly accepted that ATP-dependent degradation took place within the cell. The reason for this rested, in part, on the fact that breaking of peptide bonds is exergonic in nature. Although there were previous studies, convincing evidence for an ATP-dependent degradative pathway came from a study by Eltinger and Goldberg (2) and a body of work from Hershko, Ciechanover, Rose and colleagues in the late 1970s into the early 1980s. This latter series of publications characterized the process of ubiquitination-mediated protein degradation (3-9). The discovery of this system was recognized by the awarding of the Nobel Prize in Chemistry in 2004 to Hershko, Ciechanover and Rose (http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2004/).
It subsequently became clear that the degradation of these “doomed proteins” was the result of being fed into the proteasome, a multicatalytic 26S structure. The roles of ATP hydrolysis in this process are in the energy-dependent attachment of ubiquitin to proteins, and threading of these proteasome-targeted proteins into the central core of this machine with the assistance of AAA ATPases, which are integral to the proteasome.
We now understand that ubiquitin-dependent proteolysis is key to essentially all regulated cellular processes and is critical to the capacity of the cell to destroy misfolded proteins. Moreover, we appreciate that ubiquitination (also referred to as ubiquitylation) plays other non-proteasomal roles in the cell, such as in endocytosis, kinase activation and DNA repair. Ubiquitination is a hierarchical process with one major and another ‘minor’ cellular ubiquitin-activating enzymes (E1), which activate the C terminus of ubiquitin in an ATP-dependent manner; ~40 ubiquitin-carrier enzymes (or ubiquitin conjugating enzymes; E2s), which accept ubiquitin onto their active site cysteines from that of E1 via a transthiolation reaction involving the C terminus of ubiquitin; and over 500 ubiquitin ligases (E3s), which function together with E2s and provide the exquisite substrate specificity that characterizes this system. In humans ~100 deubiquitinating enzymes serve to remove ubiquitin from substrates or play other roles in disassembling ubiquitin chains. Substrate proteins can be modified by single ubiquitin moieties, generally though isopeptide (involving internal lysine residues) or N-terminal peptide linkages. In all cases this involves the C-terminus of ubiquitin. Alternatively, proteins can be modified with chains of ubiquitin linked together as the result of a processive process in which the next ubiquitin in the chain is linked to any of seven internal lysines or the N terminus of a ubiquitin already attached, directly or indirectly, to a target substrate. An important aspect of ubiquitination is that it is the nature of the ubiquitin modification on the substrate protein that determines its fate. So, while targeting to the proteasome is a major outcome, there are some ubiquitin modifications (such as linkages through lysine 63 or the N-terminus of ubiquitin) that specify different results, such as stimulation of lysosomal targeting, DNA repair or kinase activation, and are not known to target to the proteasome.
I was fortunate to enter this field relatively early, when only a few naturally-occurring cellular substrates were known. This occurred when we found, in 1992, that the T cell antigen receptor, when stimulated, was modified with ubiquitin (10). This is a modification that, in retrospect, likely leads to its endocytosis and lysosomal rather than proteasomal degradation. Nonetheless, this finding introduced me to this nascent field and the highly-talented and interactive scientists that populated it. I immediately changed the focus of my group at the NCI to studying ubiquitination.
We went on to characterize this modification of stimulated T cell receptors and then to focus on the enzymes and substrates of this system in mammalian cells. We also moved our work from assembled T cell receptors at the cell surface to examining the fate of unassembled subunits from the endoplasmic reticulum – an issue that had intrigued many for years. It became clear from our work that these proteins were being moved back out into the cytoplasm, ubiquitinated and targeted to the proteasome (11) by a process now known as endoplasmic reticulum-associated degradation (ERAD). At this point our work was stimulated to move forward by three discoveries in our lab.
The first was that the RING finger is a ubiquitin ligase domain. This discovery exponentially expanded the number of potential substrate-specific ligases in the cell and led many others to enter the field (12). We now understand that RING fingers interact with E2 enzymes through well-specified, low-affinity (>100 µM) interactions. The interaction between E2s and RINGs leads to the direct transfer of ubiquitin from E2 to substrates for the specific RING E3. The second discovery was the characterization of a key E2 enzyme involved in ERAD, Ube2G2 (originally coined MmUbc7), by Swati Tiwari, formerly faculty at GGSIU and now a Professor at JNU (13). The third discovery was that a RING finger protein known as gp78 is an ubiquitin ligase resident to the endoplasmic reticulum that functions with Ube2G2 as its E2 (14). gp78 has subsequently been found, through the work of our group and a number of other labs, to be involved in the degradation of many unassembled, misfolded and highly regulated proteins from the endoplasmic reticulum.
Since these findings, our laboratory has had a focus in understanding ERAD and also in understanding the function of gp78. The reason why gp78 became of such interest to us was two-fold. First, we established it as the first known pro-metastatic ubiquitin ligase, with this function, at least in part, being due to targeting a metastasis suppressor for degradation (15). The second reason was that gp78 has a region, distinct from the RING finger (the G2BR), which interacts with Ube2G2 in a high-affinity manner (16). This binding led us and our collaborators at NCI, R. Andrew Byrd (and particularly Ranabir Das – now a Professor ay NCBS) and Xinhua Ji, to explore this high affinity-binding site. Through structural analysis we determined that this binding site neither overlapped the region of Ube2G2:RING finger binding nor encroached on the active site cysteine of the E2. Instead the G2B2 binds to the ‘backside’ of Ube2G2 and allosterically results in an increase in the affinity of the E2 for the gp78 RING, which facilitates ubiquitination (17, 18). Stimulated by our findings with gp78, together with the Byrd and Ji labs, we went on to determine that additional RING E3s and associated proteins, under study in the laboratory, also have backside binding sites on their specific E2s. Interestingly, despite the similar binding region on each of the different E2s, the effects of binding on E2-RING E3 activity were distinct. This concept of backside binding of E3 components to E2s is a previously unappreciated mechanism by which RING finger E3 function can be either positively or negatively regulated (19, 20). Work from other groups has also contributed to this concept in very important ways (21-24). It now becomes an important question in the field, and indeed in the regulation of cell biological processes in general, to understand the prevalence of such binding sites among ubiquitin E3s and proteins associated with E3s. The details of these findings and their functional consequences in each case are quite complex. For this reason I have referred the reader to the primary literature as well as to a review that addresses this particular topic (25).
Moving beyond the particulars, what I hope to have conveyed in my lecture, is that we are just beginning to scratch the surface in terms of understanding how the activities of ubiquitin ligases are regulated. We carry out studies in test tubes, as we should, analyzing this process, but it is clear that we often don’t know what yet unknown factors, which either fail to make it into ‘the mix’ or are there but uncharacterized, may either positively or negatively effect the robustness and the nature of this regulated modification. Thus, there is much more to be learned about the complex factors that influence the exquisite regulatory role of ubiquitination in cellular protein homeostasis. With a molecular understanding of this complexity, comes enormous opportunities for intervention in cancer and other diseases.
(1) Lipkowitz, S. and Weissman, A. M. (2011) RINGs of good and evil: RING finger ubiquitin ligases at the crossroads of tumour suppression and oncogenesis. Nat. Rev. Cancer 11, 629-643. (2) Etlinger, J. D. and Goldberg, A. L. (1977) A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Proc. Natl. Acad. Sci. U S A 74, 54-58. (3) Hershko, A., Heller, H., Elias, S. and Ciechanover, A. (1983) Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258, 8206-8214. (4) Hershko, A., Ciechanover, A., Heller, H., Haas, A. L. and Rose, I. A. (1980) Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis. Proc. Natl. Acad. Sci. U S A 77, 1783-1786. (5) Hershko, A., Ciechanover, A. and Rose, I. A. (1979) Resolution of the ATP-dependent proteolytic system from reticulocytes: a component that interacts with ATP. Proc. Natl. Acad. Sci. U S A 76, 3107-3110. (6) Ciehanover, A., Hod, Y. and Hershko, A. (1978) A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. Biochem. Biophys. Res. Commun. 81, 1100-1105. (7) Ciechanover, A., Heller, H., Katz-Etzion, R. and Hershko, A. (1981) Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system. Proc. Natl. Acad. Sci. U S A 78, 761-765. (8) Ciechanover, A., Elias, S., Heller, H., Ferber, S. and Hershko, A. (1980) Characterization of the heat-stable polypeptide of the ATP-dependent proteolytic system from reticulocytes. J. Biol. Chem. 255, 7525-7528. (9) Ciechanover, A., Heller, H., Elias, S., Haas, A. L. and Hershko, A. (1980) ATP-dependent conjugation of reticulocyte proteins with the polypeptide required for protein degradation. Proc. Natl. Acad. Sci. U S A 77, 1365-1368. (10) Cenciarelli, C., Hou, D., Hsu, K. C., Rellahan, B. L., Wiest, D. L., Smith, H. T., Fried, V. A. and Weissman, A. M. (1992) Activation-induced ubiquitination of the T cell antigen receptor. Science 257, 795-797. (11) Yang, M., Omura, S., Bonifacino, J. S. and Weissman, A. M. (1998) Novel aspects of degradation of T cell receptor subunits from the endoplasmic reticulum (ER) in T cells: importance of oligosaccharide processing, ubiquitination, and proteasome-dependent removal from ER membranes. J. Exp. Med. 187, 835-846. (12) Lorick, K. L., Jensen, J. P., Fang, S., Ong, A. M., Hatakeyama, S. and Weissman, A. M. (1999) RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc. Natl. Acad. Sci. U S A 96, 11364-11369. (13) Tiwari, S. and Weissman, A. M. (2001) Endoplasmic reticulum (ER)-associated degradation of T cell receptor subunits. Involvement of ER-associated ubiquitin-conjugating enzymes (E2s). J. Biol. Chem. 276, 16193-16200. (14) Fang, S., Ferrone, M., Yang, C., Jensen, J. P., Tiwari, S. and Weissman, A. M. (2001) The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum. Proc. Natl. Acad. Sci. U S A 98, 14422-14427. (15) Tsai, Y. C., Mendoza, A., Mariano, J. M., Zhou, M., Kostova, Z., Chen, B., Veenstra, T., Hewitt, S. M., Helman, L. J., Khanna, C. and Weissman, A. M. (2007) The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation. Nat. Med. 13, 1504-1509. (16) Chen, B., Mariano, J., Tsai, Y. C., Chan, A. H., Cohen, M. and Weissman, A. M. (2006) The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site. Proc. Natl. Acad. Sci. U S A 103, 341-346. (17) Das, R., Mariano, J., Tsai, Y. C., Kalathur, R. C., Kostova, Z., Li, J., Tarasov, S. G., McFeeters, R. L., Altieri, A. S., Ji, X., Byrd, R. A. and Weissman, A. M. (2009) Allosteric activation of E2-RING finger-mediated ubiquitylation by a structurally defined specific E2-binding region of gp78. Mol. Cell 34, 674-685. (18) Das, R., Liang, Y. H., Mariano, J., Li, J., Huang, T., King, A., Tarasov, S. G., Weissman, A. M., Ji, X. and Byrd, R. A. (2013) Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine. EMBO J. 32, 2504-2516. (19) Metzger, M. B., Liang, Y. H., Das, R., Mariano, J., Li, S., Li, J., Kostova, Z., Byrd, R. A., Ji, X. and Weissman, A. M. (2013) A structurally unique E2-binding domain activates ubiquitination by the ERAD E2, Ubc7p, through multiple mechanisms. Mol. Cell 50, 516-527. (20) Li, S., Liang, Y. H., Mariano, J., Metzger, M. B., Stringer, D. K., Hristova, V. A., Li, J., Randazzo, P. A., Tsai, Y. C., Ji, X. and Weissman, A. M. (2015) Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B. J. Biol. Chem. 290, 30225-30239. (21) Brzovic, P. S., Lissounov, A., Christensen, D. E., Hoyt, D. W. and Klevit, R. E. (2006) A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination. Mol. Cell 21, 873-880. (22) Hibbert, R. G., Huang, A., Boelens, R. and Sixma, T. K. (2011) E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6. Proc. Natl. Acad. Sci. U S A 108, 5590-5595. (23) Williams, C., van den Berg, M., Panjikar, S., Stanley, W. A., Distel, B. and Wilmanns, M. (2012) Insights into ubiquitin-conjugating enzyme/ co-activator interactions from the structure of the Pex4p:Pex22p complex. EMBO J. 31, 391-402. (24) Buetow, L., Gabrielsen, M., Anthony, N. G., Dou, H., Patel, A., Aitkenhead, H., Sibbet, G. J., Smith, B. O. and Huang, D. T. (2015) Activation of a primed RING E3-E2-ubiquitin complex by non-covalent ubiquitin. Mol. Cell 58, 297-310. (25) Metzger, M. B., Pruneda, J. N., Klevit, R. E. and Weissman, A. M. (2014) RING-type E3 ligases: master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination. Biochim. Biophys. Acta 1843, 47-60.
Allan M. Weissman, M.D., Center for Cancer Research, National Cancer Institute
Building 560, Room 22-103, Frederick, MD 21702-1201. Ph: 301-846-7540 email@example.com