Biochimica et Biophysica Acta 1695 (2004) 73–87 http://www.elsevier.com/locate/bba Review Ubiquitin family proteins and their relationship to the proteasome: a structural perspective Kylie J. Waltersa, Amanda M. Gohb, Qinghua Wanga, Gerhard Wagnerc, Peter M. Howleyb,* aDepartment of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States bDepartment of Pathology, Harvard Medical School, Boston, MA 02115, United States cDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, United States Available online 26 October 2004 Abstract Many biological processes rely on targeted protein degradation, the dysregulation of which contributes to the pathogenesis of various diseases. Ubiquitin plays a well-established role in this process, in which the covalent attachment of polyubiquitin chains to protein substrates culminates in their degradation via the proteasome. The three-dimensional structural topology of ubiquitin is highly conserved as a domain found in a variety of proteins of diverse biological function. Some of these so-called bubiquitin family proteinsQ have recently been shown to bind components of the 26S proteasome via their ubiquitin-like domains, thus implicating proteasome activity in pathways other than protein degradation. In this chapter, we provide a structural perspective of how the ubiquitin family of proteins interacts with the proteasome. D 2004 Elsevier B.V. All rights reserved. Keywords: Protein degradation; Ubiquitin; Ubiquitin-like domain; Proteasome 1. Introduction A highly conserved ubiquitin structural fold is present in numerous eukaryotic proteins and has ancestral ties to the Ubiquitin-mediated protein degradation is one of the prokaryotic protein ThiS [18]. Ubiquitin family proteins have major mechanisms for controlled proteolysis, which is crucial been defined by their structural homology with ubiquitin and for many cellular events, including the control of life span of are divided into two families: type I ubiquitin-like proteins regulatory proteins, the removal of improperly folded and type II ubiquitin-like domain proteins. Like ubiquitin, proteins that would otherwise aggregate, and the production these proteins are involved in a variety of different biological of immunocompetent peptides [1–6]. The role of ubiquitin in pathways. Some ubiquitin-like domains have been found to degradation is its most extensively studied function: poly- bind proteasomal subunits, although the functional signifi- ubiquitin chains are covalently attached to proteins and serve cance of these interactions remains the subject of much as a signal for their recognition and degradation by the 26S speculation. In this chapter, we illustrate how the interactions proteasome [2]. Ubiquitin has also been found to be involved between ubiquitin family members and proteasomal subunits in protein trafficking [7–11], DNA repair [12–14], signaling may be characterized by NMR spectroscopy, and we discuss pathways [15], and transcription [16,17]. the implications of such interactions for proteasome activity. 2. The ubiquitin family of proteins Abbreviations: E6AP, E6-associated protein; hHR23a, human homolog A of Rad23; hPLIC-2, human proteasome ligase interaction component-2; 2.1. A prominent role for ubiquitin in targeted protein HSQC, heteronuclear single quantum coherence; NMR, nuclear magnetic degradation resonance; ppm, parts per million; S5a, subunit 5a; SUMO-1, small ubiquitin-related modifier-1 * Corresponding author. The best-characterized role of ubiquitin is in targeted E-mail address: [email protected] (P.M. Howley). protein degradation by the 26S proteasome, which consists 0167-4889/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2004.10.005 74 K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87 of two 19S regulatory particles and a 20S core. Proteins be expressed during each transcription/translation cycle. The are posttranslationally modified by ubiquitin via an fusion proteins are subsequently separated into functional enzymatic cascade, which begins with the formation of a molecules by isopeptidase cleavage at the C-terminus, thioester bond between a ubiquitin-activating enzyme (E1) where there is usually a diglycine motif [23–26]. and the C-terminal glycine of ubiquitin. This activated SUMO-1 (Small Ubiquitin-related MOdifier) is a much ubiquitin is subsequently transferred to a ubiquitin- studied type I ubiquitin family protein. It is conserved conjugating enzyme (E2) [19–21]. An E3 ubiquitin ligase from yeast to mammals and its sequence is 18% identical then catalyzes the formation of an isopeptide bond to that of ubiquitin. The list of substrates for SUMO-1 is between ubiquitin and lysine residues of a specific target growing steadily and the effects of SUMO-1 conjugation protein, either by directly accepting the ubiquitin and are diverse. Sumoylation may affect the intracellular transferring it to the substrate, or by bringing the substrate localization of the proteins to which they are conjugated. into proximity with the E2–ubiquitin complex. This For example, the sumoylation of RanGAP1 targets it to process is repeated, with more ubiquitin moieties being the nuclear pore complex [27] and the attachment of covalently conjugated to those already in the nascent SUMO-1 to the PML protein (identified as a fusion polyubiquitin chain. These polyubiquitin chains are protein in promyelocytic leukemia patients) is crucial for recognized by the 19S regulatory complex of the 26S its localization to nuclear bodies [28]. The conjugation of proteasome and the proteins to which they are conjugated SUMO-1 to transcription factors may regulate their are degraded [22]. activity, as has been shown for c-Jun, p53, and c-Myb [29–31]. Sumoylation may also protect proteins from 2.2. Type I ubiquitin-like proteins degradation, as it does for InBa [32]. There is evidence for cross-talk between the ubiquitination and sumoylation The first class of ubiquitin family proteins, the type I machinery [14], the mechanism and functional signifi- ubiquitin-like proteins, are similar to ubiquitin: they can cance of which are still unclear. Interestingly, increased either exist freely or be covalently attached to other proteins levels of sumoylated proteins have been found to be via an enzymatic cascade (Fig. 1A). Like ubiquitin, they are associated with polyglutamine diseases such as Hunting- expressed as fusion products, allowing several moieties to ton’s disease [33]. Fig. 1. Schematic diagram illustrating the structural domains within several type I (A) and type II (B) ubiquitin family members. Proteins included are NEDD8; SUMO1, small ubiquitin-related modifier-1; FUBI, failure of ureteric bud invasion protein; UCRP, ubiquitin cross-reactive protein precursor; hHR23a, human homolog of Rad23-a; hHR23b, human homolog of Rad23-b; hPLIC-1, human proteasome ligase interaction component-1; hPLIC-2, human proteasome ligase interaction component-2; ubiquilin-3; A1Up, ataxin-1 ubiquitin-like interacting protein; NUB1, NEDD8 ultimate buster-1; BAG1, BCL-2 binding athanogene- 1 protein; Parkin; ElonginB; HERP, homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 protein; OASL, 59-kDa2V-5V- oligoadenylate synthetase like protein; GdX, ubiquitin-like protein from chromosome Xq28; U7I3, ubiquitin conjugating enzyme 7 interacting protein 3; S3A1, splicing factor 3 subunit 1; BAT3, HLA-B-associated transcript 3; Uch14, ubiquitin carboxyl-terminal hydrolase 14. Ubiquitin-like and ubiquitin-associated domains are represented as UBL and UBA, respectively. Other identified functional domains within these proteins are also included as BAG, bcl-2-associated athanogene domain; COL, collagen-like domain; IBR, in between ring finger domains; PAP, polyA-related domain; ProR, proline-rich region; SYNTH, 2V-5V- oligoadenylate synthetase N-terminal region; SWAP, suppressor-of-white-apricot splicing regulator domain; UCH-1, ubiquitin carboxyl-terminal hydrolase family 1; UCH-2, ubiquitin carboxyl-terminal hydrolase family 2; XPC, XPC-binding domain; ZnF, zinc finger domain. K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87 75 Nedd8, also known as RUB (Related to Ubiquitin) in have also been shown to bind the E3 ubiquitin ligase yeast, is a type I ubiquitin family member that functions in E6AP [52,54]. These interactions suggest that the PLIC targeted protein degradation. The Nedd8 conjugation path- proteins are functionally linked to the ubiquitin–protea- way is conserved and essential in many eukaryotes, some pathway, which is further supported by reports that including Schizosaccharomyces cerevisiae, Arabidopsis, they regulate the stability of the GABAA receptor and of and humans, but notably not in Saccharomyces cerevisiae presenilin [55,56]. Regulation of the GABAA receptor is (S. cerevisiae) [34]. The only known substrates for Nedd8 important for inhibitory neurotransmission and mutations are cullins, which are a key component of the SCF in presenilin are associated with early-onset Alzheimer’s ubiquitin ligase complexes. Neddylation of the cullins is disease, thereby implicating the PLIC proteins in proper important in the regulation of SCF function. For example, nervous system function. In addition to its role in Nedd8 conjugation to Cul-1 activates ubiquitin-mediated ubiquitin-mediated protein degradation, Dsk2 has been proteolysis of p27 and