UC Irvine UC Irvine Previously Published Works Title Global approaches to understanding ubiquitination

Ubiquitination the linkage of one or more molecules of the protein ubiquitin to another protein regulates a wide range of biological processes in all eukaryotes. We review the proteome-wide strategies that are being used to study aspects of ubiquitin biology, including substrates, components of the proteasome and ubiquitin ligases, and deubiquitination. Published: 29 September 2005 Genome Biology 2005, 6:233 (doi:10.1186/gb-2005-6-10-233) The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2005/6/10/233 © 2005 BioMed Central Ltd Ubiquitin, a small protein of 76 amino acids, is highly conserved in all eukaryotes. In a multi-step process, ubiquitin is covalently linked to lysine residues of substrate proteins. If a single molecule of ubiquitin is linked to a protein, this is referred to as mono-ubiquitination, a process that is of particular importance for protein trafficking but has also been shown to regulate retrovirus budding and to modulate protein function directly [1]. A lysine residue of a ubiquitin molecule attached to a substrate can itself serve as an acceptor for an additional ubiquitin molecule, and this process can be repeated so that poly-ubiquitinated proteins form. Polyubiquitin chains serve as recognition signals for the 26S proteasome, the major regulator of protein abundance in cells, and poly-ubiquitination thus often initiates proteolysis of the substrate. But poly-ubiquitination can also regulate protein function directly without affecting stability, in ways similar to mono-ubiquitination and other post-translational modifications. The mechanisms underlying proteolysis-independent regulation by poly-ubiquitination are only poorly understood but might function by changing conformation or adding or obscuring a binding site (Figure 1; for reviews see [1-3]). The transfer of ubiquitin is a multi-step process that involves at least three classes of enzymes: ubiquitin-activating enzymes, generally called E1 enzymes; ubiquitin-conjugating enzymes or E2s; and ubiquitin ligases, E3s (Figure 1). E3 ubiquitin ligases are of particular importance because they confer substrate specificity to the system by interacting directly with substrate proteins and thereby directing the transfer of ubiquitin. The human genome encodes an estimated 500-600 ubiquitin ligases, a number comparable to the 518 predicted kinases [4,5]. If you consider that each ubiquitin ligase is active on several substrates, you can get some impression of the complexity and importance of the ubiquitin system. Ubiquitination is a highly dynamic process and is balanced by deconjugation of ubiquitin by deubiquitinating enzymes (DUBs). The more than 70 DUBs that are estimated to be encoded in the human genome are responsible for the reversible nature of ubiquitin modifications and have important roles in recycling ubiquitin from proteasome substrates, in stabilizing proteins by counteracting their poly-ubiquitination, and in opposing the proteolysis-independent regulatory roles of ubiquitin modifications (for reviews see [6,7]). DUBs together with E1, E2 and E3 enzymes and the proteasome make up the ubiquitin-proteasome system. The large number of proteins that constitute the ubiquitinproteasome system and the enormous number of ubiquitination substrates mean that global approaches are required if we are to understand fully the role of ubiquitination in cell biology, development, and disease. Large-scale studies of the entire system are still in their early stages, but they have already made important contributions to the field. Here, we review the approaches taken in some of these studies and their findings. Proteomic approaches to characterizing the ubiquitin-proteasome system Multi-protein complexes and protein-protein interactions have important roles in the ubiquitin-proteasome system. Both the 26S proteasome (see Figure 1c) and E3 ubiquitin ligases have been studied extensively using protein-complex purification coupled with mass-spectrometric protein identification [8-11]. Studies of the subunit composition of proteasomes from various organisms have revealed that the 26S proteasome complex consists of the 20S complex (made up of seven and seven subunits) and the 19S complex (made up of six ATPase and twelve non-ATPase subunits) [12]. The 20S complex is well characterized as forming the catalytic core; the 19S regulatory complex is believed to be responsible for substrate recognition and unfolding (Figure 1c), but the specific functions of most of the 19S subunit components are still not well understood. There is accumulating evidence for a non-proteolytic role for the proteasome in processes such as transcription, chromatin packaging, and DNA repair [13,14]. Additional subunits found in the majority of proteasome complexes have been identified following the development of new protein-purification and protein-identification techniques [8,15]. Recently, hybrid 26S proteasome complexes have been characterized, in which one copy of the 19S is present at one end of the 20S core and the other end is capped by Bml10, a newly characterized HEAT repeat protein in yeast. Blm10 and its mammalian ortholog PA28 function as 20S activators [16,17]. These complexes seem to have labile structures that cannot be preserved during the purification steps. Although the hybrid complexes reconstituted in vitro have higher peptidase activity than the classic versions, their roles in the degradation of ubiquitinated substrates in vivo are unclear [16,17]. Given the heterogeneous population and functional diversity of the various proteasome complexes, it remains a challenging task to purify and identify the subpopulations of proteasome complexes and to correlate the differences in their composition with their distinct functions in vivo. A diverse groups of proteasome-interacting proteins, including ubiquitin ligases, DUBs, heat-shock proteins and many other proteins have been identified by affinity purification and mass spectrometry as well as from genome-wide two-hybrid 233.2 Genome Biology 2005, Volume 6, Issue 10, Article 233 Kaiser and Huang http://genomebiology.com/2005/6/10/233 Genome Biology 2005, 6:233 Figure 1 The ubiquitin proteasome system. (a) Ubiquitin is activated by a ubiquitin-activating enzyme (E1) and transferred onto substrate proteins by ubiquitinconjugating enzymes (E2) and ubiquitin ligases (E3), resulting in (b) either attachment of a single ubiquitin molecule (mono-ubiquitination), attachment of multiple ubiquitin units to several substrate lysine residues on the same protein (multi-ubiquitination) or synthesis of ubiquitin chains (poly-ubiquitination). (c) Many poly-ubiquitinated proteins are subsequently degraded by the 26S proteasome, which consists of the catalytic 20S complex and the regulatory 19S particles. Degradation substrates are either delivered to the proteasome by soluble ubiquitin receptors or recognized by the intrinsic ubiquitin-binding activity of the 19S particle. At the 19S proteasome the ubiquitin chain is disassembled, and the substrate is unfolded before it can enter the cavity of the 20S subunit where proteolysis takes place. Finally, proteolytic fragments exit the proteasome in a poorly understood way. (d) Ubiquitination can also directly regulate protein function in a proteolysis-independent manner, via mono-, multior poly-ubiquitinated proteins. 20S