3 The Ubiquitin-Proteasome System: Death of Proteins is Required for Life of Cells Aaron Ciechanover etween the 1960s and 1980s, much of life science substrate that is specifically bound to a member of the B research was focused on the study of nucleic acids and ubiquitin-protein ligase family, E3, of which a number of the translation of their encoded information into the different classes are known to exist. For the HECT synthesis of new proteins. In comparison, protein degrada- (Homologous to the E6-AP C-Terminus) domain E3s, the tion was a neglected area, considered to be a non-specific, ubiquitin is transferred once again from the E2 enzyme to dead-end process. While it was known that proteins turn an active site cysteine residue on the E3 to generate a over, the large extent and high specificity of the process, third high-energy thiol ester intermediate, E3-S~ubiquitin, whereby distinct proteins can have half-lives that range prior to its transfer to the ligase-bound substrate. RING from a few minutes to several days, was not appreciated. finger-containing E3s catalyze direct transfer of the However, beginning in 1978 and throughout the next activated ubiquitin moiety to the E3-bound substrate. E3s decade, the discovery of the complex cascade of the ubiq- catalyze the last step in the conjugation process, namely uitin proteasome system (UPS) dramatically changed the the covalent attachment of ubiquitin to the substrate. The ε field. As a result, it is now clear that the degradation of ubiquitin molecule is generally transferred to an -NH2 cellular proteins is a highly complex, temporally controlled group of an internal lysine residue in the substrate to and tightly regulated process that plays major roles in a generate a covalent isopeptide bond. In some cases, variety of basic cellular pathways during both cell life and however, ubiquitin is conjugated to the N-terminal amino cell death, and hence in both health and disease. With the group of the substrate. By successively adding activated multitude of protein substrates targeted and the myriad of ubiquitin moieties to internal lysine residues on the previ- processes involved, it is not surprising that aberrations in ously conjugated ubiquitin molecule (mostly to Lys48), a the UPS are implicated in the pathogenesis of several polyubiquitin chain is synthesized that is subsequently diseases, including neurodegeneration and various malig- recognized by the downstream 26S proteasome complex. nancies. However, despite intensive research in this area, Thus, E3s play a key role in the ubiquitin-mediated prote- key questions remain unanswered concerning intracellular olytic cascade by serving as the specific recognition factors protein degradation. Among these are the modes of of the system. In certain cases, the first ubiquitin moiety is specific and timed recognition for the degradation of the conjugated to the substrate by one E3, while chain elon- many substrates, and the mechanisms that underlie aberra- gation is catalyzed by a different ligase often termed E4. tions in the system that lead to pathogenesis of diseases. Certain substrates are conjugated by a single ubiquitin moiety in a three-step mechanism similar to the attach- ment of ubiquitin that occurs during polyubiquitination. Mechanisms of Ubiquitination and Monoubiquitinated substrates are not degraded as the Degradation single ubiquitin moiety is not recognized by the 26S Ubiquitination proteasome complex. Rather, the single ubiquitin moiety Degradation of a protein via the UPS involves two discrete serves as a routing signal that, in most cases, targets and successive steps (see Figure 1 for a general scheme of membrane proteins to the lysosome/vacuole. For recent the UPS). The protein substrate is first tagged by covalent reviews on E3s and ubiquitination, see [4-7] and on the attachment of multiple ubiquitin molecules to generate the role of monoubiquitination, see [8]. polyubiquitin chain that serves as a recognition marker to the downstream 26S proteasome complex. The tagged About the Author protein is then degraded by the 26S proteasome com- plex (Prod. No. P 3863) leading to the subsequent release Dr. Aaron Ciechanover received his M.D. from "Hadassah" of free and reusable ubiquitin [1-3]. This last process is Medical School of the Hebrew University in Jerusalem and a mediated by ubiquitin recycling enzymes such as isopepti- D.Sc. in Biochemistry from the Technion in Haifa, Israel. While a dases (Prod. No. I 1154), also known as ubiquitin-specific Ph.D. student with Dr. Avram Hershko, the two discovered that proteases (UBPs) and deubiquitinating enzymes (DUBs). covalent attachment of ubiquitin to the target substrate signals it for degradation. They also discovered the three enzymes, E1, E2, and E3, that catalyze conjugation, and elucidated their mode Conjugation of ubiquitin (Prod. No. U 6253), a highly of action. As a postdoctoral fellow in the laboratory of Dr. evolutionarily conserved 76 amino acid residue poly- Harvey Lodish at the Massachusetts Institute of Technology, peptide, to the protein substrate proceeds via a three-step Cambridge, MA, USA, he also worked with Drs. Alexander cascade mechanism. Initially, the ubiquitin-activating Varshavsky and Daniel Finley. The three have elucidated the enzyme, E1, activates ubiquitin in an ATP-requiring enzymatic aberration in a cell cycle arrested mutant as a defect in E1, and used this model to demonstrate that the ubiquitin reaction to generate a high-energy thiol ester inter- system is involved in targeting short-lived proteins in cells. He is mediate, E1-S~ubiquitin. One of several E2 enzymes currently a distinguished professor in the Department of ® known as ubiquitin-carrier proteins or ubiquitin-conju- Biochemistry and the Center of Tumor and Vascular Biology in gating enzymes (UBCs, Prod. No. U 8382) then transfer the Bruce Rappaport Faculty of Medicine and the Rappaport the activated ubiquitin from E1 via an additional high- Institute at the Technion, Haifa Israel. His research focuses on the ® energy thiol ester intermediate, E2-S~ubiquitin, to the regulation of transcriptional factors by the ubiquitin system. Order: 1.800.325.3010 (US) Technical Service: 1.800.325.5832 (US) Outside US:Order: 1.800.325.3010 (US) Technical Contact local Sigma-Aldrich Office 4 The Ubiquitin-Proteasome System...(continued) B. RING- E1 E2 E3 finger E2 E3 Ubiquitin-carrier protein; Ubiquitin- Ubiquitin-conjugating enzyme, activating UBC Proteolytic substrates enzyme A. SS SS Ubiquitin S HECT E2 E3 Domain E3 E3 S S E3 S S 26S Proteasome Peptides Peptides Figure 1. The ubiquitin-proteasome system. Ubiquitin is first activated by the ubiquitin-activating enzyme, E1, toS a high energy thiol ester inter- mediate. It is then transferred, still as a high energy intermediate, to a member of the ubiquitin-carrier proteins family of enzymes, E2 (also known as a ubiquitin-conjugating enzyme - UBC). From E2 it can be transferred directly to the substrate, S, that is bound specifically to a member of the ubiq- uitin ligase family of proteins, E3. This occurs when the E3 belongs to the RING finger family of ligases (A). In the case of an HECT (Homologous to the E6-AP C-Terminal domain) domain-containing E3 ligase (B), the activated ubiquitin moiety is transferred first to the E3, to generate yet another high-energy thiol ester intermediate, before it is transferred to the E3-bound target substrate. Additional ubiquitin moieties are added successively to the previously conjugated one in a similar mechanism to generate a poly-ubiquitin chain. The poly-ubiquitinated substrate is bound specifically to the 26S proteasome complex. The substrate is degraded to short peptides, and free and reusable ubiquitin is released via the activity of de-ubiquitinating enzymes (DUBS). Vol. 19 No. 3 Vol. Degradation the 20S complex, is made up of six homologous ATPases Degradation of polyubiquitinated substrates is carried out (Rpt1-6) together with three non-ATPase subunits (Rpn1, by a large, ~2 mega dalton protease complex, referred to 2, and 10). The lid of the RP is made of eight non-ATPase as the 26S proteasome, that does not recognize non- subunits (Rpn 3-9), which can be released from the pro- modified substrates. In one established case, that of the teasome or can rebind under certain conditions. polyamine-synthesizing enzyme ornithine decarboxylase Altogether, the 19S RP comprises 17 subunits. One impor- (ODC), the proteasome recognizes and degrades the sub- tant function of the 19S RP is to recognize ubiquitinated strate without prior ubiquitination. The proteasome is a proteins and other potential substrates of the proteasome. Celltransmissions multicatalytic protease that degrades polyubiquitinated Two ubiquitin-binding subunits of the 19S RP have been • proteins to short peptides. It is composed of two sub- identified, referred to as Rpn10 (S5a in mammalian cells) complexes: a 20S core particle (CP, Prod. No. P 3988) and Rpt5 (S6'). However, their importance and mode of that carries the catalytic activity, and a regulatory 19S action have yet to be determined. A second function of regulatory particle (RP). The 20S CP is a barrel-shaped the 19S RP is to open an orifice in the α ring that will structure composed of four stacked rings; two identical allow entry of the substrate into the proteolytic chamber. outer α rings and two identical inner β rings. The eukary- Also, since a folded protein would not be able to enter otic α and β rings are each composed of seven distinct through the narrow proteasomal channel, it is assumed subunits, giving the 20S complex the general structure of that the 19S particle unfolds substrates and inserts them α β β α into the 20S CP. Both the channel opening function and 1-7 1-7 1-7 1-7. The catalytic sites are localized to some of the β subunits. Most of the 20S complexes are capped by the unfolding of the substrate require metabolic energy, two 19S complexes, one on each extremity.