BASIC SCIENCE Basic Science for the Clinician 40 Ubiquitin, Programmed Protein Degradation, and the Proteasome Leonard H. Sigal, MD, FACP, FACR Abstract: mRNA is made from DNA. Protein is made from mRNA. Although one might say that “DNA is forever,” the same cannot be said for mRNA or protein. These molecules are made in response to the cell’s present needs; once the cell’s circumstances change, a whole new repertoire of proteins may be needed and the previous set of proteins may be unnecessary, perhaps even deleterious. So, the cell must be able to eliminate the characters in the previous act in favor of the actors needed for the current act. In addition, there is good evidence that the DNA to mRNA to protein flow may not be efficient; abnormal proteins, as well as damaged or misfolded proteins, are quite common and must also be eliminated. This process depends on the ability of the cell to tag the protein to be eliminated with a small protein (or chain of these proteins) that targets the protein to a special structure for digestion into its constituent amino acids for recycling into new proteins. This very common protein tag was identified in the 1970s and called “ubiquitin”—it truly was everyplace! In addition, ubiquitin is crucial to targeting normal proteins to their appropriate place in or on the cell and for recycling of proteins. Ubiqutination of proteins and what follows this tagging are crucial to the normal function of cells. The complexity of these processes is being used for therapy in oncology now and perhaps in immunology and rheumatology in the near future. Key Words: ubiquitin, proteasome, immunoproteasome, ubiquitin ligases, SUMO (J Clin Rheumatol 2006;12: 255–258) U biquitin was first described in the 1970s, but its central role as a cellular regulator only became apparent in 1984; it was the subject of the Nobel Prize in 2004. As its name suggests, this 76 amino acid protein is found everywhere in cells; this ubiquity speaks to the crucial role it plays in a From the Pharmaceutical Research Institute, Bristol-Myers Squibb and the Division of Rheumatology and Connective Tissue Research, Department of Medicine, Department of Pediatrics and Department of Molecular Genetics & Microbiology, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, New Brunswick, New Jersey. Reprints: Leonard H. Sigal, MD, Pharmaceutical Research Institute/BristolMyers Squibb, J.3100, P.O. Box 4000, Princeton, NJ 08543-4000. E-mail: leonard.sigal@bms.com Copyright © 2006 by Lippincott Williams & Wilkins ISSN: 1076-1608/06/1205-0255 DOI: 10.1097/01.rhu.0000239903.71688.c5 variety of cellular functions. Related proteins have been described (perhaps more are to come) and the complexity of how ubiquitin gets added to proteins, how this tag is translated into specific addressing for shipping to specific sites in the cell, and how the tag is interpreted at these sites is not yet well understood, but suggests many more therapeutic targets in oncology and immunology. Ubiquitination (also called ubiquitylation) is involved in destruction of defective protein transcripts and degradation of short-lived proteins involved in cell-cycle control, signal transduction and control of that process, and transcriptional regulation. It is involved in DNA repair, receptor downregulation, antigen presentation, apoptosis, and targeting of proteins to various intracellular locations. A useful analogy may be drawn with phosphorylation of protein tyrosine, serine, or threonine residues—posttranslational modification of these residues changes the function of the proteins. A rapid-on, rapid-off change of a variety of functions is enabled by both phosphorylation and ubiquitination. ADDITION OF UBIQUITIN: THE UBIQUITIN LIGASES Ubiquitin is added to the targeted proteins by means of a series of “ubiquitin ligases.” The C-terminal glycine of ubiquitin is attached to the ␧ amino group of a lysine residue on the target protein, forming an isopeptide or amide bond. This is accomplished by 3 sets of enzymes: E1, E2, and E3. E1 (activating enzyme) activates the ubiquitin molecule in an ATP-dependent process. E2 (ubiquitin conjugating enzyme) transfers the ubiquitin molecule to the E2 molecule itself, forming a thioester bond; thus, the E2 is a carrier of the ATP-activated ubiquitin. Each E2 enzyme then can interact with a number of E3 enzymes (protein ligase), which recruit both the E2– ubiquitin complex and the target protein, and then transfer the ubiquitin from the E2 to the target protein. A fourth set of enzymes (E4s) has recently been described; E4 allow additional ubiquitins to link to the initial ubiquitin, leading to polyubiquitination of the targeted protein. There are 2 major families of E3 ligases containing 2 different but related active domains known as HECT (homology to the E6-associated protein carboxyl terminus) and RING (really interesting new gene—I love that name!). HECT One of the HECT-containing E3 ligases is called the E6-associated protein. Papillomavirus proteins form a com- JCR: Journal of Clinical Rheumatology • Volume 12, Number 5, October 2006 255 JCR: Journal of Clinical Rheumatology • Volume 12, Number 5, October 2006 Sigal plex with the E6-associated protein that then causes degradation of the p53 tumor suppressor, thus leading to an increased risk of malignant degeneration. Defective maternal copies of the E6-associated protein has been implicated in the neurodegenerative syndrome, Angelman syndrome, and defects in a related protein, Nedd4, have been implicated in the regulation of renal epithelial sodium channels and, if Nedd4 is deleted, Liddle syndrome (hereditary hypertension) develops. Itch (one of its mutations causes mice to itch) is a member of the HECT family and is involved in T-cell differentiation; it modifies IL-7 receptor function and B-cell development and activation. RING RING is a family of E3 ligases occurring in single or multisubunits. Cbl is a single subunit RING finger protein. SCF (Skp1-Cullin1-Fbox), APC (anaphase promoting complex) and VCB (VHL-elongin C/elongin B) are multisubunits. SCF has been implicated in lymphocyte signaling. VHL associates with HIF␣ (hypoxia-induced factor ␣ noted in a previous article as being associated with the pathogenesis of rheumatoid arthritis) causing ubiqutination and degradation of HIF␣. Another Family PIAS (protein inhibitor of activated STAT—signal transducer and activator of transcription) are RING-type E3 ligases that are involved in the regulation of p53, androgen receptors, and cJun; we will discuss PIAS again in more depth in a future paper devoted to the STAT proteins and other control mechanisms. UBIQUITIN: ROLE IN CELL FUNCTION Now, just what does ubiquitin control you ask? Well, perhaps the best-known example of ubiquitination occurs after certain receptors bind their ligands and ultimately signal through NF␬B to the nucleus, leading to the transcription of many proinflammatory or proapoptotic genesis. It is by activating an ubiquitin ligase that I␬B is ubiquitinated and degraded, thereby freeing NF ␬B to be transported to the nucleus; thus, these receptor-mediated signals depend on ubiquitin. IL-1 binding to the IL-1 receptor leads to activation of IRAK (IL-1 receptor-associated kinase), MyD88, and TRAF-6 (TNF-associated factor 6). TRAF-6 is a RING finger protein and is polyubiquitinated during its activation. Other pathways may converge on polyubiquitination of TRAF-6, including RANK-L and possibly bone morphogenetic protein R1A (BMP-R1A)—another example of ubiquitin at the nexus of receptor-mediated signaling. TGF␤ signals through a number of receptors and accessory proteins known as Smads. TGF␤ plays a critical role at many stages of development and controls many functions in adults, eg, tumor suppression. There are balancing forces to TGF␤, eg, Ecto, acting in ectodermal development and present in many tumors. Ecto is a RING-type ubiquitin ligase that adds a ubiquitin to Smad4, blocking Smad-dependent TGF␤ signaling. Another is Smurf-1 (Smad ubiquitin regu- 256 latory factor-1), which is a member of the HECT family of E3 ligases and causes the degradation of Smad-1 and Smad-5. Ubiquitination of cyclin B and securin allows the enzyme separase to free sister DNA strands during mitosis. Ubiquitination of transcription factors cJun, JunC, and JunD leads to degradation of these important factors; their degradation has been implicated in control of oncogenesis. GRAIL mentioned previously in the article on tolerance is a RING finger protein whose activation inhibits IL2 and IL4 production. Ubiqutination of certain growth factors may be needed to activate transcription of many genes. Ubiqutination is crucial in the uncoiling of DNA (through ubiquitination of histones) and methylation of DNA that renders it more available for transcription. The addition of ubiquitin to DNA is required for DNA methylation, which leads to gene silencing. Ubiquitin is involved in the maintenance of the telomere through management of proteins that modify the telomere; another closely related protein, called SUMO, is also involved in the process; more on SUMO a little later. The number of ubiquitins attached to a protein helps determine targeting of the protein: does it go to the cell surface, to lysosome for degradation versus endosome for recycling. Ubiquitination of certain cell surface proteins, eg, certain receptors, major histocompatibility complex (MHC) class I, causes the proteins to be taken into the endosome– lysosomal pathway for degradation. At least 2 E3 ligases are involved in monoubiqutination of receptors targeted for endocytosis: SOCS (suppressor of cytokine signaling) and Cbl. The Cbl family recognizes phosphorylated tyrosine (through its aminoterminal Src homology, known as SH2) and has a RING motif. Increasing levels of Cbl lead to downregulation of receptor tyrosine kinases and of receptor-associated protein tyrosine kinases (PTKs). Increased levels of Cbl in mast cells leads to inhibition of IgE signaling through the Fc␧R1. Decreased levels of Cbl in T cells leads to enhanced T-cell receptor clustering, cytoskeletal reorganization, and CD28 independent assembly of the supramolecular activation complex (SMAC); thus, ubiqutination is involved in T-cell costimulation, especially in decreasing IL2 production and proliferation. Various viruses have learned how to make use of ubiquitination. The virus that causes Kaposi sarcoma encodes a protein that promotes ubiqutination of MHC class I proteins, increasing their degradation and thereby decreasing the immune response to the virus. In fact, recent studies have shown that these herpesviruses actually encode 2 E3 ligases that ubiquitinate MHC class I molecules on cysteine rather than the usual lysine; this may allow the virus to broaden its MHC class I targets and more effectively block the host’s immune response against the virus. Ubiquitination of viral proteins is required for membrane fusion and budding of HIV and the Ebola virus. DUB2 (one of the enzymes that deubiquitinates proteins) is constitutively expressed in HTLV-1infected cells, causing increased IL-2-stimulated proliferation and prolonged activity of STAT5. © 2006 Lippincott Williams & Wilkins JCR: Journal of Clinical Rheumatology • Volume 12, Number 5, October 2006 SUMO: A MEMBER OF AN EVOLVING UBIQUITIN FAMILY? SUMO (small ubiquitin-like modifier) is a 97 amino acid protein first described in 1997 that is structurally related to ubiquitin. SUMO has been implicated in viral entry into cells, neurodegenerative syndromes like Alzheimer and Huntington chorea, gene transcription, DNA repair, transport of proteins, and RNA in and out of the nucleus through pores and building the mitotic spindle apparatus. Three SUMOs have been described (very original: 1, 2, and 3); these are added by the same families of E1, E2, and E3 ligases involved in ubiquitination. Sumoylation does not cause degradation of proteins. Rather, sumoylation causes increased protein stability, enhanced protein–protein interactions, and helps to determine subcellular translocation and transcriptional control. Most sumoylated proteins are found within the nucleus and perhaps 50% are transcription factors. Sumoylation helps to direct histone deacetylase 4 traffic into the nucleus. HDAC4 is involved in removing acetyl groups from histone, resulting in a more condensed and nontranscriptionally available chromatin. HDAC4 must be sumoylated to be active (of note, histone must be ubiquinated to methylate DNA, which leads to gene silencing). There is a progressive decrease in a number of the enzymes in the HDAC family in chronic obstructive pulmonary disease, a decrease which correlates with the severity of the lung damage. A variety of heat shock factors (HSF) are sumoylated, which results in the transcription of a number of genes that then stabilize the cell against stresses, eg, heat. HSF2 (not to be confused with the hsp— heat shock proteins— described in a previous article in this series) is also involved in condensing/compacting chromatin for cell division. When topoisomerase II is sumoylated (undergoing a constant “on” and “off” cycle in the normal function of the cell), there is less chromosomal adhesion and ultimate separation of the DNA strands. Sumoylation also helps in the formation of the kinetochore, attaching chromosomes to microtubule fibers of the mitotic spindle. Addition of SUMO to several DNA repair proteins regulates their function. The list includes p53 (called “the guardian of the genome”) and PCNA (proliferating cell nuclear antigen). Of note, PCNA can be either ubiqutinated or sumoylated, at the same site; the former results in increased repair activity, the latter in decreased activity. The Huntington protein (Htt) is mutated in Huntington chorea and becomes more neurotoxic if SUMO is added. Human papillomavirus and herpesviruses use addition of SUMO to target themselves to the nucleus of the infected cell. Some viruses block sumoylation, which disinhibits gene transcription, allowing more efficient viral gene expression. THE PROTEASOME AND THE IMMUNOPROTEASOME The term UPS has been used to describe how ubiquitin targeting leads to degradation of proteins—standing for “ubiquitin proteasome system”— efficient shipping and handling of proteins. The proteasome is a multiprotein complex that recognizes posttranslational polyubiquitination of pro© 2006 Lippincott Williams & Wilkins Ubiquitin, Protein Degradation, and the Proteasome teins. Its size is measured in sedimentation units (Svedbergs) as 26S. The 26S proteasome is the main protease in the cytoplasm, ultimately degrading over 80% of all cellular proteins and generating the C-termini of most peptide ligands of MHC class I—another example of the evolving immune system making use of a perfectly good proteolytic mechanism to supply itself with targets, ie, antigens. The 26S proteasome consists of 33 proteins in 2 subcomplexes—a 20S proteolytic core and a 19S regulatory particle (also known as the PA700 –proteasome activator). The 20S proteolytic core is constructed like a cylinder with the outer 2 rings consisting of 7 different ␣ type subunits that bind to the 19S regulatory complex. The inner 2 rings of the 20S complex consist of 7 different ␤ subunits. Three of these, designated ␦ (␤1), MB1 (␤5), and MC14 (␤2), bear the active centers of the proteasome. ␥ interferon (IFN␥) causes replacement of these by LMP2 (␤1i), LMP7 (␤5i), and MECL-1 (␤2i), which causes a change in the cleavage patterns of the proteasome. IFN␥ also induces the 11S regulator (also called REG) of the proteasome PA28 (proteasome activator). PA28 consists of ␣ and ␤ subunits forming a heptameric ring that binds to the 20S or 26S proteasome, causing a change in conformation that results in enhanced proteolytic activity. With increases in PA28 levels, there is an increase in availability of some epitopes. Defects in expression in these subunits cause the lack of expression of some epitopes. PA28␣ and ␤ subunits are differentially regulated in dendritic cells: expression of ␣ is high in both immature and mature dendritic cells (DC), whereas expression of ␤ is low in immature DC and strongly increased in mature DC. NF-␬B is involved in upregulation of PA28␤. IFN␥ has no effect on the expression of the PA28 ␣ and ␤ subunits, in contrast to the upregulation of MHC molecules on the DC surface and the replacement of the 20S ␤1 (␦), ␤2 (MB1), and ␤5 (Z) subunits of the proteasome by LMP2 LMP7 and MECL-1, respectively, in the IFN-␥-mediated shift to the immunoproteasome. Thus, the DC is provided with its “00” status—a license to kill. The effects of IFN␥ is to turn the standard proteasome into the immunoproteasome, as noted, an example of how the immune system took advantage of the proteasome. In addition to increasing the expression of LMP2, MECL1, and LMP7, there is an increase in cell surface expression of MHC molecules and of the protein TAP (transporter associated with antigen presentation), which takes peptides from the immunoproteasome to the endoplasmic reticulum, where it is loaded onto MHC class I molecules. The 20S proteasome is expressed in all cells, but its subunit composition varies from organ to organ. The role of the proteasome is to eliminate many proteins from the cell. Some of these are defective ribosomal products (called DRiPs). Up to 30% of all cell proteins (with a survival half-life of only 10 minutes compared with a half-life of approximately 3000 minutes for the other 70% of proteins) go directly to the proteasome; these may be short-lived proteins or DRiPs. Most of the peptides on cell surface class I MHC molecules are derived from this pool of intracellular proteins. 257 Sigal JCR: Journal of Clinical Rheumatology • Volume 12, Number 5, October 2006 Of note, these peptides may derive from gene’s “untranslated regions” and from all 3 frames from each DNA strand. Within the 19S regulatory complex is the site recognized by the ubiquitin tag on proteins meant for degradation within the proteasome. Ubiquitin binds to the 19S complex and, in an ATP-dependent process, the ubiquitin is removed, and the protein is unfolded and inserted into the proteolytic core’s inner tunnel. The ATPase activity of the 19S complex is contained in its “cap” or “lid.” The Rpn 10 proteins within the “lid” bind to polyubiquitin chains and recruit the substrate. The Rpn 11 protein (a metalloprotease) is the deubiquitinating enzyme; it allows unfolding of the protein and translocation of the substrate into the proteolytic lumen of the 20S particle. This is a one-way trip for the protein; peptides and single amino acids are the end result. The timing and specificity of ubiquitin-mediated proteolysis is controlled at the level of recognition of the substrate protein by a specific ubiquitin ligase. Many ubiquitin ligases interact with the 26S proteasome. There are different adaptor proteins that bind to the 26S proteasome and participate in the recognition of the polyubiquitin chain; there is speculation that these may contribute to the specificity of proteolysis, although no mechanism has been identified. THERAPIES BASED ON MANIPULATION OF PROTEASOME FUNCTION All of these insights have been used to design therapies. PS341 (bortezomib) is a proteasome inhibitor showing effectiveness in multiple myeloma (Richardson et al). Multiple mechanisms have been suggested for why a proteasome inhibitor might be useful in treating malignancy, including the absence of cyclins (causing a halt in cell proliferation), a buildup of I␬B causing inhibition of NF␬B (leading to decrease angiogenesis), and the buildup of many intracellular proteins, which might overtax cancer cells’ limited ability to withstand stress. Inhibition of the proteasome results in increased levels of p53 (the tumor suppressor), p21 and p27 (cyclin-dependent kinase inhibitors that stop cell-cycle progression at the G1-S phase by inactivating cyclin/cyclin-dependent kinase complexes), and Bax (dephosphorylated Bax inhibits the antiapoptotic mitochondrial proteins BclII and Bcl-xL) and decreased levels of NF␬B (mechanism described elsewhere) and p44/42 MAPK (mitogen-activated protein kinase, involved in proliferation and suppression of apoptosis; mediates cell signaling by Her2 on breast cancer cells). Net result: decreased tumor growth. PS519 has been tested in streptococcal cell wall-induced arthritis (a model of rheumatoid arthritis), cerebral ischemia, experimental autoimmune encephalomyelitis (a model of multiple sclerosis), asthma, and psoriasis. Just as bortezomib is showing promise in multiple myeloma, preliminary results in these inflammatory and postischemia models 258 suggest that proteasome inhibition may show promise in rheumatic diseases. THE PROTEASOME IN AUTOIMMUNE DISEASE The 20S proteasome may be an autoantigen of interest. Circulating levels of the 20S proteasome are increased in myositis, systemic lupus erythematosus (SLE), primary Sjögren syndrome, rheumatoid arthritis, and autoimmune hepatitis and levels correlate with clinical activity. Antibodies to the 20S proteasome were found in approximately 60% of German patients with SLE and myositis in levels higher than in controls, in approximately 20% to 25% of Japanese patients with SLE and primary Sjögren syndrome and in 66% of patients with multiple sclerosis, 35% with SLE, and 16% with primary Sjögren in a Spanish study. It remains a matter of speculation as to whether the immunoproteasome or other modifications of normal proteasomal function under the influence of ␥ interferon or other cytokines (perhaps ␣ interferon?) contribute to presentation of autoantigens. This is an obvious question and may be answered in the coming years. Studies of ubiquitin and SUMO and their effects on many cell functions have yielded great insights into how to treat malignancies, inflammatory and postischemic phenomena, and viral infections. The modification of the “UPS” to improve immunosurveillance is another fascinating example of the evolution of the adaptive/acquired immune system. Much of interests to rheumatologists will come from our emerging better understanding of these systems. RECOMMENDED READING 1. Ben-Niriah Y. Regulatory functions of ubiquitination in the immune system. Nat Immunol. 2002;3:20 –26. 2. Dalton WS. The proteasome. Semin Oncol. 2004;31(suppl 16):3–9. 3. Elliott PJ, Zollner TM, Boehncke W-H. Proteasome inhibition: a new anti-inflammatory strategy. J Molec Med. 2003;81:1432–1440. 4. Fang S, Weissman AM. A field guide to ubiquitylation. CMLS Cell Mol Life Sci. 2004;61:1546 –1561. 5. Garber K. Taking garbage in, tossing cancer out? Science. 2002;295: 612– 613. 6. Kloetzel P-M, Ossendorp F. Proteasome and peptidase function in MHC-class-I-mediated antigen presentation. Curr Opin Immunol. 2004; 16:76 – 81. 7. Liu Y-C. Ubiquitin ligases and the immune response. Annu Rev Immunol. 2004;22:81–127. 8. Marx J. Ubiquitin lives up to its name. Science. 2002;297:1792–1794. 9. Marx J. SUMO wrestles its way to prominence in the cell. Science. 2005;307:836 – 839. 10. Nasmyth K. How do so few control so many? Cell. 2005;120:739 –746. 11. Reinstein E. Immunologic aspects of protein degradation by the ubiquitin–proteasome system. IMAJ. 2004;6:420 – 425. 12. Richardson, Sonneveld, Schuster, et al. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med. 2005;352: 2487–2498. 13. Voorhees PM, Dees EC, O’Neill B, et al. The proteasome as a target for cancer therapy. Clin Cancer Res. 2003;9:6316 – 6325. 14. Yewdell J. Hide and seek in the peptidome. Science. 2003;301:1334 – 1335. 15. Zhang J. Ubiquitin ligases in T cell activation and autoimmunity. Clin Immunol. 2004;111:234 –240. © 2006 Lippincott Williams & Wilkins