Biochimica et Biophysica Acta 1695 (2004) 73–87 http://www.elsevier.com/locate/bba Review family 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 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 , 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 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 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 , 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 of InBa [35–39].InArabidopsis, implicated in spindle pole body duplication in S. either deletion or overexpression of Nedd8 conjugation cerevisiae [44]. pathway components impairs SCF function. Therefore, The contribution of another type II ubiquitin-like regulation of neddylation is crucial for proper SCF function protein, parkin, to the pathogenesis of neurodegenerative [40,41]. disease has been better defined than that of the PLIC SUMO and Nedd8 are two of the best characterized type proteins. Mutations in parkin are linked to autosomal I ubiquitin family members. Despite their similarity to recessive inherited juvenile parkinsonism. Parkinson’s ubiquitin, they play entirely different roles in the cell. disease is characterized by the loss of dopaminergic Indeed, the posttranslational modification of proteins by neurons in the substantia nigra and by the presence of type I ubiquitin family members has been shown to have a Lewy bodies, which are cytoplasmic inclusions that contain wide variety of biological consequences, including regu- ubiquitin. Defects in the proteasome are linked to Parkin- lation of subcellular localization, regulation of protein/ son’s disease, and inhibition of the proteasome can cause protein interactions, and protection from degradation. the neuronal apoptosis and inclusion formation that are the hallmarks of this disease [57,58]. In fact, parkin is itself an 2.3. Type II ubiquitin-like domain proteins E3 ubiquitin ligase, whose substrates include the Pael receptor, CDCrel-1, O-glycosylated alpha-synuclein, par- Type II ubiquitin-like domain proteins constitute the kin-associated endothelin-like cell receptor, and synphilin. second group of ubiquitin family members. They are Accumulation of certain substrates, such as the Pael defined by the presence of ubiquitin-like domains within receptor and synuclein, is also associated with neuro- the context of a larger protein. These ubiquitin-like domains degeneration [59]. lack the C-terminal diglycine motif and are not cleaved or There are many other type II ubiquitin family members ligated to other proteins (Fig. 1B). that cannot be discussed here due to space constraints. The S. cerevisiae protein Rad23 and its two human They play essential roles in diverse functional pathways, homologs, hHR23a and hHR23b, are type II ubiquitin-like including DNA repair [42], apoptosis [60–62], protein domain proteins, with a ubiquitin-like domain at their N- folding [63–66], and signaling [67]. As described above, terminus and two ubiquitin-associated domains. Their best- however, there is increasing evidence pointing to a role for characterized function is in DNA repair, particularly in some type II ubiquitin family proteins in the ubiquitin– nucleotide excision repair [42] and in base excision repair proteasome pathway. The mechanism by which they do so [43].InS. cerevisiae, Rad23 has also been implicated in is presently the subject of intense study in several spindle pole body duplication and progression laboratories. [44–46]. Overexpression of Rad23 suppresses the toxicity caused by overexpression of N-end rule pathway compo- 2.4. Interactions between type II ubiquitin-like domain nents [47], and both the yeast and human homologs have proteins and the proteasome been shown to interact with proteasomal subunits as well as with ubiquitin itself [12,48–51]. These data suggest an Various type II ubiquitin family members have been additional mechanistic role for Rad23/hHR23 homologs in found to interact directly with the proteasome. Besides the ubiquitin–proteasome pathway. Rad23/hHR23 and Dsk2/PLIC, the list of such proteins Similar observations have been noted for the S. includes the cofactor BAG-1 and the Nedd8- cerevisiae protein Dsk2 and its human homologs interacting protein NUB1 [68]. All of these, except for hPLIC-1, hPLIC-2, and ubiquilin. They are also type II BAG-1, have been shown to bind the S5a regulatory subunit ubiquitin-like domain proteins with a ubiquitin-like of the proteasome. S5a is found free in the cell and is also domain at the N-terminus and a ubiquitin-associated part of the 19S regulatory particle, where it is located domain at the C-terminus. Like Rad23/hHR23, the three between the lid and base subcomplex. HHR23a and hPLIC- human homologs have been shown to interact with the 2 have been shown to interact with S5a via their ubiquitin- proteasome as well as with ubiquitin [48,51–53]. They like domains [68]. 76 K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87

Recent reports have identified other proteasomal subunits compares type I ubiquitin-like proteins, ubiquitin-like that can also bind ubiquitin or ubiquitin-like domains. The domains from type II ubiquitin family proteins, and AAA ATPase S6V(also known as Rpt5 in yeast) is found in ubiquitin. As seen in Fig. 3, these conserved residues are the base of the proteasome, within the 19S regulatory directed towards the protein interior and are mostly hydro- particle, and has been shown to interact with polyubiquitin phobic. A conserved salt bridge between ubiquitin residues chains only when it is within the proteasomal complex [69]. K27 and D52 (colored blue in Fig. 3) connects the long a- As an ATPase, S6Vmight provide the link between substrate helix with the loop following h4. The conserved residues recognition by polyubiquitin chain binding and the sub- and salt bridge are important for the stabilization of the sequent unfolding of the substrate that must occur in ubiquitin fold. preparation for its translocation into the proteolytic chamber of the 20S proteasome core [69]. 3.2. Ubiquitin family members have a similar protein fold Studies in yeast recently revealed that two additional but different electrostatic surface potentials components of the base subcomplex, Rpn1 and Rpn2, bind ubiquitin-like domains. These two proteins are non-ATPase The structures of several ubiquitin family members have subunits whose human homologs are S2 and S1, respec- already been elucidated and are represented in Fig. 2. The tively. One report demonstrated that Rpn1 alone is capable solution structures of SUMO-1 and of the ubiquitin-like of direct interaction with the ubiquitin-like domains of domain of hPLIC-2 have been solved by NMR spectroscopy Rad23 and Dsk2 [50]. Based on data from cross-linking [74,75]. The structure of the hPLIC-2 ubiquitin-like domain experiments, another research group proposed that these two was used to build a model of this domain in hHR23a, by ubiquitin-like domain proteins each bind an Rpn1/Rpn2 creating an alignment based on NMR data of the regular protein complex within the proteasome [70]. secondary structural elements as well as hydrogen bonding patterns between the h-strands [75]. As displayed in Fig. 2, SUMO-1 and the ubiquitin-like 3. Structural comparisons of ubiquitin family proteins domain from hPLIC-2 have similar structural topology to ubiquitin. A detailed inspection, however, reveals that the 3.1. Ubiquitin has a highly conserved structure orientation of h3 and h5 relative to a1 in the hPLIC-2 ubiquitin-like domain is more similar to that of ubiquitin The structure of ubiquitin was revealed by X-ray than to that of SUMO-1. A quantitative analysis of the crystallography [71,72]. The ubiquitin fold forms quickly backbone architecture of these three proteins supports this and cooperatively [73] around a hydrophobic core and is observation, as the root mean square deviation to the highly stable. It consists of five h-strands, an a-helix of 3.5 ubiquitin-like domain of hPLIC-2 for backbone atoms of turns and a 310-helical turn (Fig. 2A) [71,72]. The first and residues situated in helices or h-strands is 2.0 2 for last h-strands (h1 and h5) are oriented parallel to each other. ubiquitin and 2.8 2 for SUMO-1. All other neighboring h-strands are anti-parallel. Although the ubiquitin-like domains of hPLIC-2, ubiq- A stereoview of the ubiquitin structure is presented in uitin, and SUMO-1 all have similar folds, their electrostatic Fig. 3, where certain residues have been highlighted in red. surface potentials differ significantly. Fig. 5 displays the These residues correspond to those that are conserved in electrostatic potential mapped onto a surface diagram for the over 90% of ubiquitin family members. They are also ubiquitin-like domains of hHR23a (Fig. 5A) and hPLIC-2 highlighted in the alignment shown in Fig. 4,which (Fig. 5B), ubiquitin (Fig. 5C) and SUMO-1 (Fig. 5D). The

Fig. 2. A comparison of the secondary structural diagrams of ubiquitin family members reveals that they have a highly conserved fold. (A) Ubiquitin, (B) the ubiquitin-like domain of the type I ubiquitin family protein SUMO-1, (C) the ubiquitin-like domain of the type II ubiquitin family protein hPLIC-2. This figure was generated with the program MOLMOL [96]. K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87 77

Fig. 3. Stereoview of the ubiquitin structure. The conserved residues are highlighted in Fig. 4, colored red. These residues are predominantly directed towards the interior surface of the protein. K27 and D52, which form a conserved salt bridge, are colored blue. orientation displayed on the left in this figure is rotated by 4. Definition of the S5a-binding surfaces on ubiquitin 1808 relative to that on the right. Basic, acidic, and family proteins hydrophobic regions are indicated in blue, red and white, respectively. As seen in the orientation on the left, SUMO-1 4.1. Using NMR spectroscopy to map protein interaction is dramatically more acidic than both ubiquitin and type II surfaces ubiquitin-like domains. The face in question includes E67 and E89 in SUMO-1 and, as discussed below, is implicated Use of NMR spectroscopy allows structural insight into in binding S5a. the different binding properties of ubiquitin and of the

Fig. 4. Sequence alignment of ubiquitin-like proteins/domains with ubiquitin. Protein names are provided in Fig. 1. Residues that are highly conserved in all family members are colored red. 78 K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87

Fig. 5. Comparison of the electrostatic surface potential of ubiquitin family proteins. The electrostatic potential is mapped onto the surface diagrams of the ubiquitin-like domains of hHR23a (A) and hPLIC-2 (B), ubiquitin (C), and SUMO-1 (D). The orientation of the figures on the left is identical to that presented in Fig. 2 whereas that on the right is rotated by 1808. The parameters used to generate each of these surface potentials are 42 to 21 and 42 to 21 kT. This figure was generated with GRASP [97]. K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87 79 ubiquitin-like domains with S5a, and enables the S5a- We have used the [1H,15N]-HSQC spectra of 15N-labeled binding surface of the ubiquitin-fold to be characterized. It ubiquitin-like domains of hPLIC-2 or hHR23a alone and is a powerful tool for the mapping of protein interaction with equimolar ratios of S5a to study their interaction surfaces. Two-dimensional [1H,15N]-HSQC spectra surfaces with S5a (Fig. 6). Comparison of the [1H,15N]- acquired on 15N-labeled proteins display resonance cross- HSQC spectra acquired either alone or in complex with S5a peaks for all amide nitrogen and proton atoms and therefore reveals the disappearance of signal that originates from allow all residues (excluding proline) to be simultaneously resonance broadening, which is in turn due to the increased observed. The chemical shift at which atoms appear in rotational correlation time of the complex and chemical NMR spectra is sensitive to their local chemical environ- exchange from switching between the free and bound states. ment. Atoms at interaction surfaces thus tend to experience The increased rotational correlation time is caused by the the greatest changes in their environment upon ligand increased molecular weight of the complex [77].For binding, assuming that there are no structural changes example, in the case of the hPLIC-2 ubiquitin-like domain, resulting from the interaction. The chemical shift perturba- the molecular weight is increased from 14 kDa in the free tions that these atoms undergo can be monitored by form to 55 kDa when complexed with S5a. [1H,15N]-HSQC spectra and can thus be used to map In the [1H,15N]-HSQC spectrum acquired on hPLIC-2 binding surfaces [75,76]. ubiquitin-like domain in complex with S5a, some strong

Fig. 6. [1H,15N]-HSQC spectra of the ubiquitin-like domains of hPLIC-2 (A) and hHR23a. Spectra of hPLIC-2 (A) and hHR23a (B) alone (left) and with equimolar unlabeled S5a (right) are shown. 80 K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87 crosspeaks are observable. These originate from the N- absence or presence of an equimolar concentration of S5a, terminal and C-terminal regions of our hPLIC-2 construct we were able to measure the change in the amide nitrogen and include the first 30 residues of hPLIC-2 protein (Fig. 1) and proton chemical shift. and 22 residues following the ubiquitin-like domain that The chemical shift changes were then plotted as shown in were added to facilitate purification. All of these residues Fig. 7 according to Eq. (1), are randomly coiled, whether the hPLIC-2 construct was qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiÀÁ T 2 2 alone or complexed with S5a. 0:2 dN þ dH ð1Þ Although residues in the ubiquitin-like domains are severely broadened, a detailed comparison of the where dN and dH represent the changes in nitrogen and [1H,15N]-HSQC spectra of the ubiquitin-like domains of proton chemical shifts (in ppm), respectively, upon S5a hPLIC-2 and of hHR23a reveals that some resonances addition. These data are much easier to interpret in the experienced chemical shift changes upon addition of S5a. context of the three-dimensional structure and were At equimolar concentration with S5a, all of the crosspeaks therefore mapped onto the surface diagrams of the could be observed in 1D traces along the nitrogen and respective proteins as illustrated in Fig. 8. In this figure, proton dimensions. By comparing the 1D traces in both residues experiencing greatest perturbation in their amide dimensions of [1H,15N]-HSQC spectra acquired in the nitrogen and proton chemical shifts, indicating the highest

Fig. 7. NMR chemical shift perturbation data for the ubiquitin-like domains of hPLIC-2 ubiquitin-like domain andqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi hHR23a. Data for the ubiquitin like domains T 2 2 of hPLIC-2 (A) and hHR23a (B) are shown. These data are displayed for each residue according to Eq. (1), 0:2 dN þ dH, where dN and dH represent the change in nitrogen and proton chemical shifts (in ppm) upon S5a addition, respectively. Residues that were excluded from this analysis include P40, P49, N51, and F57 of the hPLIC-2 ubiquitin-like domain and P21, P42, and P60 of the hHR23a ubiquitin-like domain. K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87 81

Fig. 8. Identifying the S5a contact surface of the ubiquitin-like domains of hPLIC-2 (A) and of hHR23a (B) as well as of ubiquitin (C). The orientation of these figures is identical to that shown in Fig. 5. A gradient based on the data displayed in Fig. 7 was built and plotted onto each surface diagram, such that the residues colored darkest blue experience greatest chemical shift perturbation upon S5a addition and residues displayed in white are not perturbed by S5a addition. Residues that could not be measured, including prolines, are indicated in red. This figure was produced with the program GRASP [97].

involvement in the interaction, are colored dark blue. The The S5a contact surface revealed by this technique is residues in white are those that do not experience localized to an analogous surface on the hHR23a and changes in the resonance frequencies of their amide hPLIC-2 ubiquitin-like domains, and predominantly nitrogen and proton atoms, and which are thus not at the involves residues located in the third, fourth, and fifth h- S5a contact surface. Residues, including proline, whose strands (Fig. 8). Reference to Fig. 5 reveals that the residues chemical shift perturbation could not be measured, are at the S5a contact surface of each of these ubiquitin-like indicated in red. domains are hydrophobic with peripheral basic residues. 82 K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87

Furthermore, titration experiments indicate that the ubiq- other proteasomal subunits bind this ubiquitin-like domain, uitin-like domains of hPLIC-2 and hHR23a each bind S5a they do so at the same surface as S5a. with a 1:1 stoichoimetry [75]. We used the above technique to study the interaction 4.3. Using structure-based homology of protein surfaces to between mono-ubiquitin and S5a. Competition experiments predict proteasome subunit interaction between mono-ubiquitin and polyubiquitin chains suggest that polyubiquitin chains preferentially bind the 26S Subtle differences in electrostatic charge or hydropho- proteasome [22]. From our NMR studies, we found that bicity may underlie significant alterations in function, as mono-ubiquitin binds S5a and that the contact surface on exemplified by the decreased binding affinity of ubiquitin ubiquitin is analogous to those of the ubiquitin-like domains for S5a that arises from the presence of the charged and of the type II ubiquitin family proteins, hHR23a and hPLIC- polar residues on its S5a-binding surface. 2 [75] (Fig. 8C). This surface includes three residues (L8, The type I ubiquitin-like protein SUMO-1 has a very I44, and V70) that had been previously identified by different electrostatic surface from ubiquitin as well as from mutagenesis studies as important for proteasome-binding the ubiquitin-like domains of either hPLIC-2 or hHR23a [22,78,79]. Contact with I44 and V70 would be structurally (Fig. 5). The surface in SUMO-1 that is analogous to the difficult without contact with R42. Accordingly, we found S5a-binding surfaces of the hPLIC-2 and hHR23a ubiquitin- that the positively charged residues R42 and Q49 are also like domains and of ubiquitin is substantially more within the S5a contact surface. negatively charged. Therefore, we expected that SUMO-1 would not bind S5a. This prediction was confirmed by 4.2. Comparison of S5a-binding surfaces on ubiquitin titration experiments on 15N-labeled SUMO-1 with unla- family proteins beled S5a, where no perturbations of the SUMO-1 spectra were observed in the presence of S5a [75]. Although We were also able to compare the relative S5a-binding SUMO-1 shares a fold very similar to that of the other affinity of ubiquitin to the ubiquitin-like domains of hPLIC- proteins in this study, it has evolved very different surface 2 and hHR23a. This analysis was done by comparing the properties, presumably to perform different functions not spectral changes of each of these proteins at identical molar directly involving proteolysis or binding to the proteasome. ratios with S5a. Ubiquitin can bind S5a at two different We have extended our structural analysis of type II segments, only one of which can bind the ubiquitin-like ubiquitin family proteins to the chaperone cofactor BAG-1, domains of hPLIC-2 and hHR23a [80–82]. Therefore, the which has also been reported to bind the 26S proteasome ubiquitin-like domains should be saturated with S5a when [83]. BAG-1 binds the E3 ubiquitin ligase CHIP, and the they are at equimolar concentration whereas ubiquitin BAG-1/CHIP complex accepts protein substrates from the binding is expected to be complete when ubiquitin is in 70-kDa heat shock molecular chaperones Hsc/ for twofold excess over S5a. ubiquitin-mediated protein degradation [84]. We generated We indeed observed complete binding at equimolar a model structure of the BAG-1 ubiquitin-like domain from concentration with S5a for the ubiquitin-like domains, the experimentally determined structure of the ubiquitin- which indicates that the complexes formed by these proteins like domain of hPLIC-2, based on the alignment shown in are specific. In contrast, saturation of ubiquitin required a Fig. 4. The secondary structural diagrams of both threefold molar excess of S5a, indicating that ubiquitin ubiquitin-like domains are thus the same. The BAG-1 complexes weakly with S5a and dissociates easily [75]. ubiquitin-like domain surface potential at the h-sheet face Examination of the electrostatic surface potential of is similar to that in the ubiquitin-like domains of both ubiquitin (Fig. 5C) indicates that the weaker binding hPLIC-2 and hHR23a (Fig. 9), suggesting that this surface between ubiquitin and S5a is probably due to the charged of BAG-1 will also be used to contact the S5a subunit of and polar residues on the relevant surface of ubiquitin, the proteasome. including R42, Q49 and the nearby D52. To test whether there are other binding sites for the proteasome on the hPLIC-2 ubiquitin-like domain, we 5. Functional implications of the interactions between performed site-directed mutagenesis of a few residues in type II ubiquitin family proteins and the proteasome the hPLIC-2 ubiquitin-like domain that had been identified as important for S5a-binding, namely, H99A, I75A and 5.1. The role of S5a in ubiquitin-mediated protein A77S [75]. The resulting mutant hPLIC-2 ubiquitin-like degradation domain could bind neither S5a nor the 26S proteasome. Therefore, the surface of the hPLIC-2 ubiquitin-like domain S5a binds polyubiquitin chains [85,86] as well as a that is involved in binding S5a is also required for its subset of type II ubiquitin family members, including interaction with the proteasome. It is possible that the ability hHR23, PLIC, and NUB1. HHR23 and PLIC additionally of the hPLIC-2 ubiquitin-like domain to bind S5a is crucial bind ubiquitin [53,87] and NUB1 binds NEDD8 [87]. It has for its interaction with the proteasome. Alternatively, if therefore been proposed that these type II ubiquitin family K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87 83

Fig. 9. Electrostatic surface potential mapped onto the surface diagram of BAG-1. This structure was generated by using the homology module of the InsightII software package (Molecular Simulations). The orientation and parameters of this figure are identical to that presented in Fig 5. proteins could serve as linkers between ubiquitin/NEDD8- with polyubiquitin may serve other purposes. Furthermore, conjugated proteins and S5a. This hypothesis is not relevant the ability of free S5a to bind polyubiquitin indicates that to S. cerevisiae, where the yeast homolog Rpn10 lacks the it may play an additional role in ubiquitin-mediated second ubiquitin-binding site that is required for interaction degradation, albeit one that is still not mechanistically with type II ubiquitin family members [50]. Instead, the defined. Rpn1 and Pre2 proteasomal subunits have been found to recognize Dsk2 and Rad23 in yeast [50,53]. 5.2. The role of Rad23/hHR23 proteins in the In fact, deletion of the Rpn10 from S. cerevisiae does not ubiquitin–proteasome pathway result in any significant defect in ubiquitin-mediated proteolysis, suggesting that it is not essential for recognition Various type II ubiquitin family proteins are believed to of polyubiquitinated substrates [88]. Other proteasomal play a role in the ubiquitin–proteasome pathway. They subunits may also bind polyubiquitin chains, and indeed, include parkin, BAG-1, NUB1, Rad23/hHR23, and Dsk2/ the S6VATPase in the proteasomal base has recently been PLIC. Such connections have caused new scientific ques- identified as a ubiquitin receptor within the proteasome [69]. tions to emerge as they implicate proteasome activity in The ability of the proteasome to bind polyubiquitin chains, novel functional pathways. This discussion will focus on however, is decreased in the absence of Rpn10/S5a [50], Rad23/hHR23, since it is one of the more extensively indicating that this subunit may be important for some studied of these proteins. ubiquitin-binding function of the proteasome. Rad23/hHR23 has a well-characterized function in Most of the Rpn10/S5a within a cell is free of the pro- nucleotide excision repair but much data currently support teasome, and only a small proportion is found associated an additional role for it in the ubiquitin–proteasome with the proteasome at steady-state levels. Interestingly, pathway. However, this functional role of Rad23/hHR23 free S5a, but not proteasome-associated S5a, has been remains highly controversial. Early evidence suggested that shown to bind polyubiquitin strongly [69]. Also, deletion Rad23/hHR23 interaction with the proteasome was essen- of both the RAD23 and RPN10 in S. cerevisiae tial for nucleotide excision repair as deletion of the results in a synthetic phenotype, with pleiotropic defects ubiquitin-like domain, which is required for proteasome that include increased levels of polyubiquitinated proteins binding, causes increased UV-sensitivity in cell lines [12]. [89]. This accumulation is also observed in a mutant strain Later work provided evidence that the 19S regulatory with a double deletion of RAD23 and DSK2, as well as in particle of the proteasome interacts with Rad23 to play a a triple deletion mutant of RAD23, DSK2 and RPN10 nonproteolytic role in nucleotide excision repair [91]. This [90]. Therefore, Rad23, Dsk2 and Rpn10 may have yet hypothesis is based in part on the observation that defects in undefined but overlapping functions in ubiquitin-mediated the 19S particle cause defective nucleotide excision repair, protein degradation. whereas those in the 20S core, where proteolysis occurs, do Given the biological necessity of targeted protein not [91]. degradation, it would not be surprising if there were Other data indicate that Rad23/hHR23 may function in redundancy in polyubiquitin chain recognition by the ubiquitin-mediated protein degradation independent of proteasome. It is also clear from the studies mentioned nucleotide excision repair. In fact, there is evidence that it above that S5a plays an important role in this process. can both inhibit and promote ubiquitin-mediated protein Nonetheless, S5a may not be the major polyubiquitin degradation. The inhibitory role is noticeable in studies chain receptor within the proteasome and its interaction where the protein expression level is high. More specifi- 84 K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87 cally, high levels of Rad23 have been shown to inhibit the formation of polyubiquitin chains in vitro and to stabilize proteolytic substrates in vivo [49,92]. This inhibition was found to be dependent on the presence of the ubiquitin- associated domains of Rad23/hHR23 and their ability to interact with ubiquitin [49]. It has therefore been suggested that Rad23 is important for the translocation of ubiquiti- nated proteins to the proteasome [49]. Notably, the yeast homolog Rpn10 also appears to play a role in this process. In the presence of high levels of Rad23, the interaction between ubiquitinated proteins and the proteasome is detectable and Rpn10-dependent [93]. It is possible that this role of Rad23 has implications for nucleotide excision repair as its interaction with ubiquitinated proteins increases after DNA damage [93]. In addition to its inhibitory role described above, Rad23/ hHR23 has also been shown to promote the degradation of certain proteins. These two seemingly contradictory roles might be substrate-dependent. This hypothesis is exempli- fied by comparing the effects of Rad23/hHR23 over- expression on p53 and Rad4. Such overexpression results in decreased steady-state levels and transcriptional activity of p53 [94]. In contrast, Rad23 overexpression inhibits the ubiquitination of the nucleotide excision repair factor Rad4, which forms a complex with Rad23 to recognize damaged DNA [95]. This inhibition is associated with an increased rate of DNA repair, suggesting that regulation of Rad4 levels by Rad23 is important for proper nucleotide excision repair function [95].

5.3. A model of how hHR23 may function in the recruitment Fig. 10. A model of how hHR23 may function to recruit certain proteins to of ubiquitinated proteins to the proteasome the proteasome for protein degradation. We confirmed by NMR spectro- scopy that the ubiquitin-like domain of hHR23a binds S5a and mapped the Proper Rad23/hHR23 function requires both the N- S5a-binding region. We have also confirmed that ubiquitin binds the second ubiquitin-associated domain and have also mapped the interaction regions terminal ubiquitin-like domain and the ubiquitin-associated by NMR spectroscopy. These results combined with published work from domains [93]. As described above, the ubiquitin-like other groups suggest that S5a and hHR23 could function together for domain mediates binding to S5a [68] whereas the ubiq- proteasome recruitment of certain proteins. uitin-associated domains interact with polyubiquitin chains [49,51,87]. Therefore, Rad23/hHR23 can and does facilitate could thus be transferred from hHR23a to S5a, enabling the formation of ternary complexes with the proteasome them to bind S5a with little entropic penalty. This and ubiquitinated substrates [93]. Also, deletion experi- mechanism may represent one of multiple strategies for ments in yeast have shown that Rad23/hHR23 and Rpn10/ the regulation of ubiquitin-mediated protein degradation. S5a play overlapping roles in ubiquitin-mediated protein degradation [89,90]. Data from the genetic and biochemical experiments Acknowledgements described above, together with biophysical data from NMR spectroscopy experiments, suggest a model of how We are grateful to Dr. Patton Fast and University of hHR23a and S5a function together to recruit ubiquitinated Minnesota Supercomputing Institute for providing resources proteins to the proteasome. S5a has higher affinity for the necessary for making figures in this review. ubiquitin-like domain of hHR23a than for ubiquitin, which readily binds the C-terminal ubiquitin-associated domain of hHR23a [98]. As illustrated in Fig. 10, in the ternary References complex, the polyubiquitin chain bound to the ubiquitin- associated domain of hHR23a could be in close proximity to [1] W. Baumeister, J. Walz, F. Zuhl, E. Seemuller, The proteasome: the ubiquitin-binding segments of S5a, which interacts with paradigm of a self-compartmentalizing protease, Cell 92 (1998) the ubiquitin-like domain of hHR23a. Polyubiquitin chains 367–380. K.J. Walters et al. / Biochimica et Biophysica Acta 1695 (2004) 73–87 85

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