Regulated protein degradation

Proteolysis (or protein degradation) is mediated by proteases, which vary from small proteins such as extracellular trypsin and the intracellular caspases to large, ATP-dependent, multifunctional proteases called proteasomes. Fifty years ago, at the time of the founding of the IUB(MB), it was believed that most intracellular proteins were long lived. This assumption survived nearly intact until the 1980s, when two complementary sets of discoveries were made, largely by two groups of researchers – the laboratory of Avram Hershko at Technion (http://www.technion.ac.il) and my laboratory, then at MIT (http://web.mit.edu). Through the elegant use of biochemical fractionation and enzymology, Hershko, his student Aaron Ciechanover and his collaborator Irwin Rose discovered in 1978–1983 that some proteins added to a reticulocyte extract became covalently conjugated to a protein called ubiquitin, and that ubiquitylated proteins were processively destroyed by an ATP-dependent protease in the extract [1Ciechanover A. et al.A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes.Biochem. Biophys. Res. Comm. 1978; 81: 1100-1105Crossref PubMed Scopus (446) Google Scholar, 2Hershko A. et al.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. 1980; 77: 1783-1786Crossref PubMed Scopus (530) Google Scholar]. Hershko and colleagues went on to identify the enzymes – termed E1, E2 and E3 – that carry out ubiquitin-protein conjugation [3Hershko A. et al.Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown.J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar] (Figure 1). The ATP-dependent protease that mediates the destruction of ubiquitin-protein conjugates was characterized by several laboratories in the 1990s, and is now called the 26S proteasome [4Baumeister W. et al.The proteasome: paradigm of a self-compartmentalizing protease.Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1319) Google Scholar]. During 1984–1990, my colleagues and I discovered the first biological functions of the ubiquitin system, and deciphered the source of its specificity, that is, the degradation signals in short-lived proteins that enable their conjugation to ubiquitin. From genetic, biochemical and cell biological studies with mammalian cells and the yeast Saccharomyces cerevisiae, we discovered that the ubiquitin system (until then defined in cell extracts) was essential for the bulk of protein degradation in living cells, was required for cell viability, and had major roles in the cell cycle, DNA repair, protein synthesis, transcriptional regulation and stress responses [5Finley D. et al.Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85.Cell. 1984; 37: 43-55Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 6Bachmair A. et al.In vivo half-life of a protein is a function of its amino-terminal residue.Science. 1986; 234: 179-186Crossref PubMed Scopus (1502) Google Scholar, 7Bachmair A. Varshavsky A. The degradation signal in a short-lived protein.Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (362) Google Scholar, 8Jentsch S. et al.The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme.Nature. 1987; 329: 131-134Crossref PubMed Scopus (596) Google Scholar, 9Finley D. et al.The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses.Cell. 1987; 48: 1035-1046Abstract Full Text PDF PubMed Scopus (679) Google Scholar, 10Goebl M.G. et al.The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme.Science. 1988; 241: 1331-1335Crossref PubMed Scopus (369) Google Scholar, 11Finley D. et al.The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis.Nature. 1989; 338: 394-401Crossref PubMed Scopus (598) Google Scholar, 12Chau V. et al.A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1207) Google Scholar, 13Hochstrasser M. Varshavsky A. In vivo degradation of a transcriptional regulator: the yeast Matα2 repressor.Cell. 1990; 61: 697-708Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 14Johnson E.S. et al.Cis–trans recognition and subunit-specific degradation of short-lived proteins.Nature. 1990; 346: 287-291Crossref PubMed Scopus (124) Google Scholar, 15Bartel B. et al.The recognition component of the N-end rule pathway.EMBO J. 1990; 9: 3179-3189Crossref PubMed Scopus (306) Google Scholar]. Together, these complementary advances revealed three distinct, previously unknown facts: (i) that intracellular protein degradation involves a new protein modification, ubiquitin conjugation [1Ciechanover A. et al.A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes.Biochem. Biophys. Res. Comm. 1978; 81: 1100-1105Crossref PubMed Scopus (446) Google Scholar, 2Hershko A. et al.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. 1980; 77: 1783-1786Crossref PubMed Scopus (530) Google Scholar, 3Hershko A. et al.Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown.J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar]; (ii) that the selectivity of this conjugation is determined by specific degradation signals (degrons) in short-lived proteins [6Bachmair A. et al.In vivo half-life of a protein is a function of its amino-terminal residue.Science. 1986; 234: 179-186Crossref PubMed Scopus (1502) Google Scholar, 7Bachmair A. Varshavsky A. The degradation signal in a short-lived protein.Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (362) Google Scholar, 12Chau V. et al.A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1207) Google Scholar, 14Johnson E.S. et al.Cis–trans recognition and subunit-specific degradation of short-lived proteins.Nature. 1990; 346: 287-291Crossref PubMed Scopus (124) Google Scholar, 15Bartel B. et al.The recognition component of the N-end rule pathway.EMBO J. 1990; 9: 3179-3189Crossref PubMed Scopus (306) Google Scholar]; and (iii) that ubiquitin-dependent processes play a major part in cellular physiology [5Finley D. et al.Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85.Cell. 1984; 37: 43-55Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 8Jentsch S. et al.The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme.Nature. 1987; 329: 131-134Crossref PubMed Scopus (596) Google Scholar, 9Finley D. et al.The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses.Cell. 1987; 48: 1035-1046Abstract Full Text PDF PubMed Scopus (679) Google Scholar, 10Goebl M.G. et al.The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme.Science. 1988; 241: 1331-1335Crossref PubMed Scopus (369) Google Scholar, 11Finley D. et al.The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis.Nature. 1989; 338: 394-401Crossref PubMed Scopus (598) Google Scholar, 13Hochstrasser M. Varshavsky A. In vivo degradation of a transcriptional regulator: the yeast Matα2 repressor.Cell. 1990; 61: 697-708Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 16Hochstrasser M. et al.The short-lived Matα2 transcriptional regulator is ubiquitinated in vivo.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4606-4610Crossref PubMed Scopus (209) Google Scholar], primarily by controlling the levels of intracellular proteins, a function previously thought to be mediated almost exclusively by transcription or translation. As a result, the ubiquitin field, which was small in the 1980s, has undergone an enormous expansion and is now one of the largest arenas in biomedical science. From ∼1990 to present, extensive studies by many laboratories have greatly enlarged the initially discovered biological functions and degrons of the ubiquitin system and have dissected, in mechanistic and functional detail, a multitude of ubiquitin-dependent pathways. By virtue of regulating the levels of intracellular proteins, ubiquitin-dependent proteolysis mediates a great variety of cellular and metacellular (organismal) functions, including cell growth, division, differentiation, signal transduction, stress responses, programmed cell death, embryogenesis, immunity and activities of the nervous system [17Varshavsky A. The ubiquitin system.Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (518) Google Scholar, 18Pickart C. Back to the future with ubiquitin.Cell. 2004; 116: 181-190Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar]. Until the 1980s, intracellular protein degradation was believed to be a simple, and even mundane, process that served mainly to dispose of damaged or otherwise abnormal proteins. Among the reasons for that misperception was the difficulty of connecting the long-recognized proteolytic system in the lysosomes to specific pathways of intracellular regulation. As a result, most researchers of gene expression in the 1960s and 1970s assumed that the circuits they cared about did not involve short-lived proteins. Cellular regulation was believed to be a separate affair, mediated primarily by repressors and activators of gene expression, which were assumed, often tacitly, to be long-lived. As we know now, just the opposite proved true – especially in eukaryotes, in which most transcriptional regulators are conditionally short-lived proteins whose cellular levels are determined by the rates of their ubiquitin-dependent destruction at least as much as by the rates of their synthesis. Although not everyone shared the constricting, pre-modern view of intracellular proteolysis, definitive evidence for major biological functions of intracellular protein degradation had to wait until the 1980s. The resulting fundamental shift of paradigm is now so much taken for granted that it's easy to forget its specific origins in the biological (function-based) discoveries that began in the 1980s and continue, at an ever increasing rate, to this day. The key insight that transformed our understanding of the logic of biological circuits was that control via regulated protein degradation rivals, and often surpasses in significance the classical regulation by transcription and translation. In 1990, during his postdoctoral studies in my laboratory, Mark Hochstrasser identified a regulatory protein, the transcriptional repressor Matα2, as the first physiological in vivo substrate of the ubiquitin system [13Hochstrasser M. Varshavsky A. In vivo degradation of a transcriptional regulator: the yeast Matα2 repressor.Cell. 1990; 61: 697-708Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 16Hochstrasser M. et al.The short-lived Matα2 transcriptional regulator is ubiquitinated in vivo.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4606-4610Crossref PubMed Scopus (209) Google Scholar]. This advance was followed by the demonstration in 1991 by Michael Glotzer, Andrew Murray and Marc Kirschner, and independently by Hershko and coworkers, that a ubiquitin-dependent pathway degraded another regulatory protein, a mitotic cyclin [19Glotzer M. et al.Cyclin is degraded by the ubiquitin pathway.Nature. 1991; 349: 132-138Crossref PubMed Scopus (2062) Google Scholar, 20Hershko A. et al.Methylated ubiquitin inhibits cyclin degradation in clam embryo extracts.J. Biol. Chem. 1991; 266: 16376-16379Abstract Full Text PDF PubMed Google Scholar]. Today, it is clear that nearly every protein in a cell is either an actual or potential substrate of the ubiquitin system. This is, in part, because even proteins that reside in (or travel through) compartments such as the endoplasmic reticulum (ER) can be targeted for ubiquitylation and degradation. A protein that misfolds in the ER or is 'perceived' by quality-control systems to be otherwise abnormal can be retro-transported from the ER back to the cytosol for its ubiquitin-dependent degradation [18Pickart C. Back to the future with ubiquitin.Cell. 2004; 116: 181-190Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar]. Integral membrane proteins can also be destroyed by the ubiquitin system, either directly or by their ubiquitin-mediated targeting to lysosomes. Ubiquitin-dependent proteolysis is regulated in space and time with exquisite precision, in part by activation or inactivation (via covalent modifications or sequestrations) of specific degrons, and also by regulation of the activity, expression or localization of ligases that recognize substrates of the ubiquitin system and conjugate ubiquitin to them. These ubiquitin ligases (specifically, their E3 components, the functions of which include recognition of degrons) form an exceptionally large protein family, with >500 distinct E3 ligases in a mammal [18Pickart C. Back to the future with ubiquitin.Cell. 2004; 116: 181-190Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar]. In 1989, Vincent Chau and colleagues in my laboratory discovered that ubiquitin conjugation results in a substrate-linked polyubiquitin chain of unique topology, and that polyubiquitin chains are essential for the proteasome-mediated protein degradation [12Chau V. et al.A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1207) Google Scholar]. One key feature of this degradation is its subunit selectivity, that is, the ability of the ubiquitin system to eliminate one subunit of an oligomeric protein while leaving the rest of the protein intact. This fundamental capability was discovered in 1990 by Erica Johnson, David Gonda and myself, in the context of the N-end rule pathway, the targets of which include proteins with destabilizing N-terminal residues [14Johnson E.S. et al.Cis–trans recognition and subunit-specific degradation of short-lived proteins.Nature. 1990; 346: 287-291Crossref PubMed Scopus (124) Google Scholar]. It is specifically the subunit conjugated to a polyubiquitin chain that gets destroyed [14Johnson E.S. et al.Cis–trans recognition and subunit-specific degradation of short-lived proteins.Nature. 1990; 346: 287-291Crossref PubMed Scopus (124) Google Scholar]. The subunit selectivity of the ubiquitin system is both powerful and flexible in that it enables protein degradation to be wielded as an instrument of protein remodeling for either positive or negative control. Among many examples are activation of a major transcription factor nuclear factor-κB (NF-κB) via degradation of its inhibitory ligand IF-κB, and inactivation of cyclin-dependent kinases via degradation of their regulatory cyclin subunits. To be functionally significant, proteolysis does not have to be processive. Some of the earliest examples are the RecA-mediated cleavage of the lambda repressor that induces the lambda prophage, and RecA-mediated cleavage of the LexA repressor that initiates the SOS response to DNA damage in prokaryotes. One example of the important role of limited intracellular proteolysis in eukaryotes is the 1993 discovery, by Robert Horvitz and coworkers, that a nematode gene for an essential component of the apoptotic (programmed cell death) pathway encoded a protease, later termed caspase [21Yuan J. et al.The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme.Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2301) Google Scholar]. Specific proteases of this class are activated by upstream effectors of apoptosis, and function by producing cleavages in a subset of cellular proteins, thereby both killing a cell and preparing it for engulfment by other cells. Another example of regulation by limited proteolysis is the discovery, by the laboratory of Kim Nasmyth in 1999, that the cleavage by a protease termed separase of a subunit of cohesin complexes that hold together sister chromatids is a crucial step in the pathway of chromosome segregation at mitosis [22Uhlmann F. et al.Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1.Nature. 1999; 400: 37-42Crossref PubMed Scopus (770) Google Scholar]. In 2001, Hai Rao in my laboratory, in a collaboration with Frank Uhlmann in the Nasmyth laboratory, discovered that the ubiquitin-dependent N-end rule pathway selectively destroys the C-terminal fragment of cohesin subunit cleaved by separase, and that post-cleavage degradation of this fragment is essential for high-fidelity chromosome segregation [23Rao H. et al.Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability.Nature. 2001; 410: 955-960Crossref PubMed Scopus (234) Google Scholar]. The separase itself is also regulated by the ubiquitin system. This is one of many examples in which ubiquitin-dependent circuits, controlled by different E3 ubiquitin ligases, not only drive the cell-cycle oscillator but also enable its high fidelity, in part via the N-end rule pathway [23Rao H. et al.Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability.Nature. 2001; 410: 955-960Crossref PubMed Scopus (234) Google Scholar]. The brevity of the descriptions here cannot do justice to the broad and deep understanding of intracellular proteolysis that has been gained since the 1980s. For example, regulated protein degradation has also been shown to have major roles in prokaryotes, which are sophisticated about their proteolysis. It involves ATP-dependent, proteasome-like proteases, associated 'recognition' proteins and specific degrons [24Kenniston J.A. et al.Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1390-1395Crossref PubMed Scopus (89) Google Scholar]. Remarkably, however, it does not involve ubiquitin and ubiquitin conjugation, although the characteristic ubiquitin fold (a short α helix interacting with a β sheet) is encountered in both eukaryotic and prokaryotic proteins. Moreover, a specific ubiquitin-dependent pathway of eukaryotes can have an analog in prokaryotes that is fundamentally similar but independent of ubiquitin [25Tobias J.W. et al.The N-end rule in bacteria.Science. 1991; 254: 1374-1377Crossref PubMed Scopus (448) Google Scholar]. The apparent total absence of ubiquitin conjugation in prokaryotes – in contrast to the vast scope and importance of this reaction in eukaryotes – is a striking dichotomy, the origins and causes of which are a mystery. Its resolution is made difficult by the circumstance that bedevils most other attempts to reconstruct early life: some of essential, 'constraining' pieces of information about that life have been irretrievably lost, yielding an under-determined landscape. Conjectures about that landscape, although often interesting, might also prove to be, in the end, unfalsifiable. For a realm as large and complex as the ubiquitin system, it is a given that its malfunctions are both numerous and diverse. Hence, the enormous importance of the ubiquitin field for understanding and treating human diseases, including cancer, neurodegenerative syndromes, immunological abnormalities and a myriad of other illnesses, genetic or otherwise, that can be traced to ubiquitin-dependent processes. Research on regulated protein degradation, from its mechanistic and physiological beginnings in the 1980s, has become a veritable monster of a field. Several pharmaceutical companies are developing compounds that target specific components of the ubiquitin system. The fruits of their labors have already become, or will soon become, clinically useful drugs. Efforts in this area will yield, I hope, not only 'conventional' inhibitors or activators of enzymes but also more sophisticated drugs that will direct the ubiquitin system to target, destroy and, thereby, inhibit functionally any specific protein. Studies in my laboratory are supported by grants from the NIH and the Ellison Medical Foundation. I am most grateful to past and present colleagues for their contributions, some of which are mentioned above. I also thank R.D. Magnuson for his helpful comments on the article.