Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1

Uch37 is one of the three principal deubiquitinating enzymes (DUBs), and the only ubiquitin carboxy-terminal hydrolase (UCH)-family protease, that is associated with mammalian proteasomes. We show that Uch37 is responsible for the ubiquitin isopeptidase activity in the PA700 (19S) proteasome regulatory complex. PA700 isopeptidase disassembles Lys 48-linked polyubiquitin specifically from the distal end of the chain, a property that may be used to clear poorly ubiquitinated or unproductively bound substrates from the proteasome. To better understand Uch37 function and the mechanism responsible for its specificity, we investigated how Uch37 is recruited to proteasomes. Uch37 binds through Adrm1, a previously unrecognized orthologue of Saccharomyces cerevisiae Rpn13p, which in turn is bound to the S1 (also known as Rpn2) subunit of the 19S complex. Adrm1 (human Rpn13, hRpn13) binds the carboxy-terminal tail of Uch37, a region that is distinct from the UCH catalytic domain, which we show inhibits Uch37 activity. Following binding, Adrm1 relieves Uch37 autoinhibition, accelerating the hydrolysis of ubiquitin-7-amido-4-methylcoumarin (ubiquitin-AMC). However, neither Uch37 alone nor the Uch37-Adrm1 or Uch37-Adrm1-S1 complexes can hydrolyse di-ubiquitin efficiently; rather, incorporation into the 19S complex is required to enable processing of polyubiquitin chains.     

Regulators of the proteasome pathway, Uch37 and Rpn13, play distinct roles in mouse development

Rpn13 is a novel mammalian proteasomal receptor that has recently been identified as an amplification target in ovarian cancer. It can interact with ubiquitin and activate the deubiquitinating enzyme Uch37 at the 26S proteasome. Since neither Rpn13 nor Uch37 is an integral proteasomal subunit, we explored whether either protein is essential for mammalian development and survival. Deletion of Uch37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development. In contrast, the majority of Rpn13-deficient mice survived to adulthood, although they were smaller at birth and fewer in number than wild-type littermates. Absence of Rpn13 produced tissue-specific effects on proteasomal function: increased proteasome activity in adrenal gland and lymphoid organs, and decreased activity in testes and brain. Adult Rpn13(-/-) mice reached normal body weight but had increased body fat content and were infertile due to defective gametogenesis. Additionally, Rpn13(-/-) mice showed increased T-cell numbers, resembling growth hormone-mediated effects. Indeed, serum growth hormone and follicular stimulating hormone levels were significantly increased in Rpn13(-/-) mice, while growth hormone receptor expression was reduced in the testes. In conclusion, this is the first report characterizing the physiological roles of Uch37 and Rpn13 in murine development and implicating a non-ATPase proteasomal protein, Rpn13, in the process of gametogenesis.     

Relative structural and functional roles of multiple deubiquitylating proteins associated with mammalian 26S proteasome

We determined composition and relative roles of deubiquitylating proteins associated with the 26S proteasome in mammalian cells. Three deubiquitylating activities were associated with the 26S proteasome: two from constituent subunits, Rpn11/S13 and Uch37, and one from a reversibly associated protein, Usp14. RNA interference (RNAi) of Rpn11/S13 inhibited cell growth, decreased cellular proteasome activity via disrupted 26S proteasome assembly, and inhibited cellular protein degradation. In contrast, RNAi of Uch37 or Usp14 had no detectable effect on cell growth, proteasome structure or proteolytic capacity, but accelerated cellular protein degradation. RNAi of both Uch37 and Usp14 also had no effect on proteasome structure or proteolytic capacity, but inhibited cellular protein degradation. Thus, proper proteasomal processing of ubiquitylated substrates requires Rpn11 plus either Uch37 or Usp14. Although the latter proteins feature redundant deubiquitylation functions, they also appear to exert noncatalyic effects on proteasome activity that are similar to but independent of one another. These results reveal unexpected functional relationships among multiple deubiquitylating proteins and suggest a model for mammalian 26S proteasome function whereby their concerted action governs proteasome function by linking deubiquitylation to substrate hydrolysis.     

Uch2/Uch37 is the major deubiquitinating enzyme associated with the 26S proteasome in fission yeast

Conjugation of proteins to ubiquitin plays a central role for a number of cellular processes including endocytosis, DNA repair and degradation by the 26S proteasome. However, ubiquitination is reversible as a number of deubiquitinating enzymes mediate the disassembly of ubiquitin-protein conjugates. Some deubiquitinating enzymes are associated with the 26S proteasome contributing to and regulating the particle's activity. Here, we characterise fission yeast Uch2 and Ubp6, two proteasome associated deubiquitinating enzymes. The human orthologues of these enzymes are known as Uch37 and Usp14, respectively. We report that the subunit Uch2/Uch37 is the major deubiquitinating enzyme associated with the fission yeast 26S proteasome. In contrast, the activity of Ubp6 appears to play a more regulatory and/or structural role involving the proteasome subunits Mts1/Rpn9, Mts2/Rpt2 and Mts3/Rpn12, as Ubp6 becomes essential when activity of these subunits is compromised by conditional mutations. Finally, when the genes encoding Uch2/Uch37 and Ubp6 are disrupted, the cells are viable without showing obvious signs of impaired ubiquitin-dependent proteolysis, indicating that other deubiquitinating enzymes may remedy for the redundancy of these enzymes.     

Solution Structure of Yeast Rpn9: INSIGHTS INTO PROTEASOME LID ASSEMBLY*

The regulatory particle (RP) of the 26 S proteasome functions in preparing polyubiquitinated substrates for degradation. The lid complex of the RP contains an Rpn8-Rpn11 heterodimer surrounded by a horseshoe-shaped scaffold formed by six proteasome-COP9/CSN-initiation factor (PCI)-containing subunits. The PCI domains are essential for lid assembly, whereas the detailed molecular mechanisms remain elusive. Recent cryo-EM studies at near-atomic resolution provided invaluable information on the RP architecture in different functional states. Nevertheless, atomic resolution structural information on the RP is still limited, and deeper understanding of RP assembly mechanism requires further studies on the structures and interactions of individual subunits or subcomplexes. Herein we report the high-resolution NMR structures of the PCI-containing subunit Rpn9 from Saccharomyces cerevisiae. The 45-kDa protein contains an all-helical N-terminal domain and a C-terminal PCI domain linked via a semiflexible hinge. The N-terminal domain mediates interaction with the ubiquitin receptor Rpn10, whereas the PCI domain mediates interaction with the neighboring PCI subunit Rpn5. The Rpn9-Rpn5 interface highlights two structural motifs on the winged helix module forming a hydrophobic center surrounded by ionic pairs, which is a common pattern for all PCI-PCI interactions in the lid. The results suggest that divergence in surface composition among different PCI pairs may contribute to the modulation of lid assembly.     

Consequences of COP9 signalosome and 26S proteasome interaction

The COP9 signalosome (CSN) occurs in all eukaryotic cells. It is a regulatory particle of the ubiquitin (Ub)/26S proteasome system. The eight subunits of the CSN possess sequence homologies with the polypeptides of the 26S proteasome lid complex and just like the lid, the CSN consists of six subunits with PCI (proteasome, COP9 signalosome, initiation factor 3) domains and two components with MPN (Mpr-Pad1-N-terminal) domains. Here we show that the CSN directly interacts with the 26S proteasome and competes with the lid, which has consequences for the peptidase activity of the 26S proteasome in vitro. Flag-CSN2 was permanently expressed in mouse B8 fibroblasts and Flag pull-down experiments revealed the formation of an intact Flag-CSN complex, which is associated with the 26S proteasome. In addition, the Flag pull-downs also precipitated cullins indicating the existence of super-complexes consisting of the CSN, the 26S proteasome and cullin-based Ub ligases. Permanent expression of a chimerical subunit (Flag-CSN2-Rpn6) consisting of the N-terminal 343 amino acids of CSN2 and of the PCI domain of S9/Rpn6, the paralog of CSN2 in the lid complex, did not lead to the assembly of an intact complex showing that the PCI domain of CSN2 is important for complex formation. The consequence of permanent Flag-CSN2 overexpression was de-novo assembly of the CSN complex connected with an accelerated degradation of p53 and stabilization of c-Jun in B8 cells. The possible role of super-complexes composed of the CSN, the 26S proteasome and of Ub ligases in the regulation of protein stability is discussed.     

A High Affinity hRpn2-Derived Peptide That Displaces Human Rpn13 from Proteasome in 293T Cells

Rpn13 is a proteasome ubiquitin receptor that has emerged as a therapeutic target for human cancers. Its ubiquitin-binding activity is confined to an N-terminal Pru (pleckstrin-like receptor for ubiquitin) domain that also docks it into the proteasome, while its C-terminal DEUBAD (DEUBiquitinase ADaptor) domain recruits deubiquitinating enzyme Uch37 to the proteasome. Bis-benzylidine piperidone derivatives that were found to bind covalently to Rpn13 C88 caused the accumulation of polyubiquitinated proteins as well as ER stress-related apoptosis in various cancer cell lines, including bortezomib-resistant multiple myeloma lines. We find that a 38-amino acid peptide derived from the C-terminus of proteasome PC repeat protein hRpn2/PSMD1 binds to hRpn13 Pru domain with 12 nM affinity. By using NMR, we identify the hRpn13-interacting amino acids in this hRpn2 fragment, some of which are conserved among eukaryotes. Importantly, we find the hRpn2-derived peptide to immunoprecipitate endogenous Rpn13 from 293T cells, and to displace it from the proteasome. These findings indicate that this region of hRpn2 is the primary binding site for hRpn13 in the proteasome. Moreover, the hRpn2-derived peptide was no longer able to interact with endogenous hRpn13 when a strictly conserved phenylalanine (F948 in humans) was replaced with arginine or a stop codon, or when Y950 and I951 were substituted with aspartic acid. Finally, over-expression of the hRpn2-derived peptide leads to an increased presence of ubiquitinated proteins in 293T cells. We propose that this hRpn2-derived peptide could be used to develop peptide-based strategies that specifically target hRpn13 function in the proteasome.     

Structural insights into endosomal sorting complex required for transport (ESCRT-I) recognition of ubiquitinated proteins

The endosomal sorting complex required for transport (ESCRT-I) is a 350-kDa complex of three proteins, Vps23, Vps28, and Vps37. The N-terminal ubiquitin-conjugating enzyme E2 variant (UEV) domain of Vps23 is required for sorting ubiquitinated proteins into the internal vesicles of multivesicular bodies. UEVs are homologous to E2 ubiquitin ligases but lack the conserved cysteine residue required for catalytic activity. The crystal structure of the yeast Vps23 UEV in a complex with ubiquitin (Ub) shows the detailed interactions made with the bound Ub. Compared with the solution structure of the Tsg101 UEV (the human homologue of Vps23) in the absence of Ub, two loops that are conserved among the ESCRT-I UEVs move toward each other to grip the Ub in a pincer-like grasp. The contacts with the UEV encompass two adjacent patches on the surface of the Ub, one containing several hydrophobic residues, including Ile-8(Ub), Ile-44(Ub), and Val-70(Ub), and the second containing a hydrophilic patch including residues Asn-60(Ub), Gln-62(Ub), Glu-64(Ub). The hydrophobic Ub patch interacting with the Vps23 UEV overlaps the surface of Ub interacting with the Vps27 ubiquitin-interacting motif, suggesting a sequential model for ubiquitinated cargo binding by these proteins. In contrast, the hydrophilic patch encompasses residues uniquely interacting with the ESCRT-I UEV. The structure provides a detailed framework for design of mutants that can specifically affect ESCRT-I-dependent sorting of ubiquitinated cargo without affecting Vps27-mediated delivery of cargo to endosomes.     

Modulation of K11-linkage formation by variable loop residues within UbcH5A

Ubiquitination refers to the covalent addition of ubiquitin (Ub) to substrate proteins or other Ub molecules via the sequential action of three enzymes (E1, E2, and E3). Recent advances in mass spectrometry proteomics have made it possible to identify and quantify Ub linkages in biochemical and cellular systems. We used these tools to probe the mechanisms controlling linkage specificity for UbcH5A. UbcH5A is a promiscuous E2 enzyme with an innate preference for forming polyubiquitin chains through lysine 11 (K11), lysine 48 (K48), and lysine 63 (K63) of Ub. We present the crystal structure of a noncovalent complex between Ub and UbcH5A. This structure reveals an interaction between the Ub surface flanking K11 and residues adjacent to the E2 catalytic cysteine and suggests a possible role for this surface in formation of K11 linkages. Structure-guided mutagenesis, in vitro ubiquitination and quantitative mass spectrometry have been used to characterize the ability of residues in the vicinity of the E2 active site to direct synthesis of K11- and K63-linked polyubiquitin. Mutation of critical residues in the interface modulated the linkage specificity of UbcH5A, resulting in generation of more K63-linked chains at the expense of K11-linkage synthesis. This study provides direct evidence that the linkage specificity of E2 enzymes may be altered through active-site mutagenesis.     

RING‐between‐RINGs—keeping the safety on loaded guns

EMBO J (2012) 31 19, 3833–3844 doi:10.1038/emboj.2012.217; published online September072012EMBO Rep (2012) 13 9, 840–846 doi:10.1038/embor.2012.105; published online September072012The ‘RING-between-RING''-type E3 ubiquitin ligase HOIP acts via a novel RING/HECT-hybrid ubiquitin transfer mechanism and catalyses the formation of linear ubiquitin chains by non-covalently binding the acceptor ubiquitin. But in the absence of a binding partner, HOIP is auto-inhibited. This explains why assembly of either HOIP/HOIL-1L or HOIP/SHARPIN is required to catalyse linear chain formation.Post-translational modification of a protein with Ubiquitin (Ub) requires the activity of three enzymes: a Ub activating enzyme (E1), a Ub conjugating enzyme (E2), and a Ub ligase (E3). Final Ub transfer is performed by an E3 enzyme, which mediates the ligation of Ub from an E2∼Ub conjugate (‘∼'' denotes a thioester) onto a substrate. E3s are commonly divided into two mechanistic classes: RING/U-box E3s and HECT E3s. RING/U-box E3s facilitate the transfer of Ub from the E2∼Ub directly onto a substrate amino group. In contrast, HECTs transfer Ub from the E2∼Ub to the substrate via a HECT∼Ub intermediate. This mechanistic difference leads to an important distinction regarding what determines the type of Ub product (i.e., the specific Ub-chain linkage) formed: in ubiquitination pathways involving RING-type E3 ligases, the E2 determines the product formed, whereas for HECT-catalysed pathways, the E3 governs product formation (Christensen et al, 2007; Kim and Huibregtse, 2009).RING-between-RING (RBR) E3s comprise a class of E3s that appear to have special properties. Although RBR E3s have been considered as a subfamily of RING E3s, the RBR E3 HHARI (Human Homologue of ARIadne) was recently shown to form a HECT-like E3∼Ub intermediate (Wenzel et al, 2011). Two other members of the RBR family, HOIL-1 and HOIP, form the Linear Ub Chain Assembly Complex (LUBAC), the only E3 ligase known to catalyse the synthesis of linear Ub chains (Kirisako et al, 2006). Linear Ub chains are produced by head-to-tail conjugation of Ub molecules through their N- and C-termini and have been shown to activate the canonical NF-κB pathway (Tokunaga et al, 2009).Two studies by the Rittinger and Sixma groups now reveal important insights regarding the formation of linear Ub chains by the dimeric RBR E3 complex HOIP/HOIL-1L (Smit et al, 2012; Stieglitz et al, 2012). Results from these studies highlight three emerging themes among RBR ligases: a RING/HECT-hybrid Ub transfer mechanism; auto-inhibition of RBR E3 activity, and a role for E3:Ub interactions.The RBR E3 ligase domain consists of two distinct RING domains, called RING1 and RING2, connected by an IBR (In-Between-Ring) domain. Despite its name, RING2 is not a canonical RING domain as it contains an active site Cysteine (Cys), which has recently been shown to form a thioester E3∼Ub intermediate, as directly detected for the RBR E3 HHARI. Although the Ub-loaded species could not be detected for the RBR E3 parkin, mutation of the analogous cysteine residue abrogated parkin''s ligase activity implying that it works via the same mechanism. On the basis of these observations, Wenzel et al (2011) proposed that the RBR E3s are a family of RING/HECT hybrids that use RING1 to bind an E2 (RING-like) and RING2 to present the active site Cys (HECT-like) as shown schematically in Figure 1. Both Smit et al (2012) and Stieglitz et al (2012) observed a HOIP∼Ub thioester, confirming that HOIP also acts via a RING/HECT-hybrid mechanism. Furthermore, Smit et al (2012) used a clever strategy to uncouple the first transfer event (E2∼Ub to E3) from the final transfer event (E3∼Ub to substrate Ub) to verify that the E3∼Ub intermediate is a prerequisite for Ub transfer onto a substrate and not just a serendipitous side product. The results extend the number of RBR E3s for which a thioester intermediate has been observed and support the notion that RBR E3s are indeed RING/HECT hybrids.Open in a separate windowFigure 1Three common themes are emerging among RBR ligases: a RING/HECT-hybrid Ub transfer mechanism; auto-inhibition of RBR E3 activity, and a role for E3:Ub interactions. RBR E3s are characterized by their RBR domain that consists of two distinct RING domains, RING1 that binds the E2, and RING2 that harbours the active site Cys. Two new studies on the RBR E3 HOIP show that (a) domain(s) in HOIP''s N-terminal region inhibits its ligase activity and (b) a domain C-terminal to HOIP''s RBR binds and orients an acceptor Ub to direct linear Ub-chain formation (‘Linear Ub chain Determining Domain'' or LDD). (A)Three ways in which auto-inhibition might occur are illustrated: (1) inhibition of E2∼Ub binding by RING1, (2) obstruction of the active site cysteine on RING2, and/or (3) occlusion of acceptor Ub binding on the LDD. (B) A possible flow of events that occur once auto-inhibition released is shown. Details of each step and how specifically auto-inhibition is released are still unknown.Previous studies have established that HOIP Ub ligase activity and subsequent activation of NF-κB require either the RBR-containing protein, HOIL-1L, or SHARPIN, an adaptor protein associated with LUBAC (Ikeda et al, 2011; Tokunaga et al, 2011). The two current studies now show that although full-length HOIP exhibits very low activity on its own, removal of the N-terminal ∼700 residues results in robust ligase activity. Thus, HOIP appears to be auto-inhibited in the absence of a binding partner. Further analysis revealed that HOIP''s UBA (Ub-Associated) domain is partly responsible for auto-inhibition, although additional N-terminal domains appear to have auto-inhibitory effects as well. SHARPIN, which contains a UBL (Ub-Like) domain, can relieve auto-inhibition of HOIP. Similarly, the addition of the HOIL-1L UBL domain, previously shown to interact with the HOIP UBA domain (Yagi et al, 2012), relieves inhibition. Interestingly, the addition of full-length HOIL-1L results in even greater ubiquitination activity.Stieglitz et al (2012) show that the RBR E3 HOIL-1L has very low E3 activity on its own. Intriguingly, they found that mutation of the HOIL-1L RING2 active site Cys (C460A) reduced activity of the HOIP/HOIL-1L complex back to levels comparable to HOIP activity in presence of HOIL UBL alone. This suggests a more active, catalytic role for HOIL-1L in linear Ub-chain formation than previously appreciated. The details regarding this role must await further studies, but involvement of an active site Cys residue on a second RING2 domain suggests a possible reciprocal transfer mechanism. Perhaps linear chains can be pre-built via such a mechanism and passed en bloc to substrate, similarly to mechanisms used by some HECT-type bacterial E3 ligases (Levin et al, 2010).Parkin, another RBR E3, also exhibits auto-inhibition (Chaugule et al, 2011), but the auto-inhibitory mechanism and the release thereof differ from HOIP. Unlike parkin''s N-terminal UBL, which is thought to interact within the RBR domain at RING2, HOIP''s UBA does not bind detectably in trans to any region in the RBR domain (Stieglitz et al, 2012). Furthermore, addition of its UBA in trans does not inhibit the activity of HOIP RBR E3 as was seen with parkin and its UBL domain. The auto-inhibition of parkin is likely released by substrate binding, because addition of either the UIM of Eps15 or the SH3 domain of endophilin-A, both known to bind the parkin UBL, can restore the activity of parkin (Chaugule et al, 2011). In addition, phosphorylation of Ser65 within the UBL of parkin by PINK-1 activates parkin, presumably by releasing the UBL from RING2 (Kondapalli et al, 2012). In contrast, HOIP overcomes its auto-inhibition through binding either HOIL-1L or SHARPIN. There is no additive effect when both binding partners are present, consistent with the notion that both proteins act via their UBL domains, although this remains to be demonstrated for SHARPIN. The activity of either SHARPIN/HOIP or HOIL-1L/HOIP can activate NF-κB (Ikeda et al, 2011; Tokunaga et al, 2011), but how the protein complexes differ in their cellular roles remains to be further analysed.The finding that HOIP and parkin exhibit auto-inhibition raises the question whether there is something special about the RBR E3s that require auto-inhibition. In this regard, we note that RBR E3s bind the E2 UbcH7 with significantly tighter affinity than canonical RING E3s bind their E2s (Dove and Klevit, unpublished). In the absence of a substrate, RING1 loaded with UbcH7∼Ub would lead to non-productive transfer of Ub from UbcH7∼Ub to the active site of RING2. Occlusion of the active site by auto-inhibition may therefore act as a safety check until its activity is required for transfer of Ub to a substrate. As yet, there is no evidence to indicate whether substrate binding will release HOIP auto-inhibition, as it does for parkin, but this remains a possibility.The revelation that removal of all domains N-terminal to the HOIP RING1 domain yields a highly active ligase allowed both groups to explore questions pertaining to how linear chains are built. Remarkably, constructs comprised of only the RBR domain through the C-terminus of HOIP are sufficient to specify linear Ub chains. (The two groups use HOIP constructs that differ by only two N-terminal residues (697/699–1072) but Stieglitz et al call their construct RBR whereas Smit et al call it RBR-LDD.) (Smit et al, 2012; Stieglitz et al, 2012). Smit et al (2012) demonstrate that the region immediately C-terminal to RING2 is required for linear chain building activity and name the region the ‘LDD'' (Linear Ub chain Determining Domain). Their results indicate that the LDD binds and orients the acceptor Ub to promote transfer of the donor Ub from the RING2 active site to the N-terminus of the acceptor Ub (Figure 1). Parkin has also been suggested to bind free Ub. Details about whether parkin binds acceptor or donor Ub and whether Ub binding determines Ub-chain specificity are still unknown.There is precedence for acceptor Ub binding by HECT E3s and this interaction is essential for chain formation by NEDD4 and its yeast orthologue Rsp5 (Kim et al, 2011; Maspero et al, 2011). In another example, the inactive E2 variant MMS2 binds an acceptor Ub and orients the Ub-Lys63 into the active site of Ubc13 thereby guaranteeing K63-linked chain formation by the E2 (Eddins et al, 2006). Besides proper orientation of the acceptor Ub, chemical differences between α- and ɛ-amino groups likely contribute to linear Ub-chain specificity. For example, E2s known to be active with RING-type E3s can transfer Ub onto the amino acid lysine, but not the other amino acids containing α-amino groups indicating specificity towards the ɛ-amino of lysine (Wenzel et al, 2011).Catalysed by the unexpected discovery that HHARI is a HECT/RING hybrid E3, details about how the RBR class of E3s function are beginning to emerge. We now know, either directly or indirectly, that at least 4 RBR E3s of the 13 identified in humans (HHARI, HOIL, HOIP, and parkin) require a trans-thiolation event using an active site cysteine within RING2. Conservation of this cysteine among all RBR E3s strongly suggests that the RING/HECT-hybrid mechanism is conserved and therefore defines the class. The hybrid mechanism also offers an explanation for the heretofore puzzling observation that, despite being categorized as a RING E3, HOIP determines the type of Ub chain formed. The ability to bind an acceptor Ub close to the RING2 active site likely contributes to how the RBR E3s dictate the type of product they produce. Finally, both HOIP and parkin are auto-inhibited. It remains to be seen whether HOIP''s auto-inhibitory domains work via inhibition of E2∼Ub binding by RING1, obstruction of the active site cysteine on RING2, and/or occlusion of acceptor Ub binding on the LDD (Figure 1). Regardless of the mechanistic details, the ability to modulate their activity may be a common trait of the RBR E3s. Given recent rapid progress, our understanding of this special class of E3s will continue to grow apace.     

Ubiquitin interactions of NZF zinc fingers

Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains. One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes. We now report the solution structure of the NZF domain from Npl4 in complex with Ub. The structure reveals that three key NZF residues (13TF14/M25) surrounding the zinc coordination site bind the hydrophobic 'Ile44' surface of Ub. Mutations in the 13TF14/M25 motif inhibit Ub binding, and naturally occurring NZF domains that lack the motif do not bind Ub. However, substitution of the 13TF14/M25 motif into the nonbinding NZF domain from RanBP2 creates Ub-binding activity, demonstrating the versatility of the NZF scaffold. Finally, NZF mutations that inhibit Ub binding by the NZF domain of Vps36/ESCRT-II also inhibit sorting of ubiquitylated proteins into the yeast vacuole. Thus, the NZF is a versatile protein recognition domain that is used to bind ubiquitylated proteins during vacuolar protein sorting, and probably many other biological processes.     

A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination

Protein ubiquitination is a powerful regulatory modification that influences nearly every aspect of eukaryotic cell biology. The general pathway for ubiquitin (Ub) modification requires the sequential activities of a Ub-activating enzyme (E1), a Ub transfer enzyme (E2), and a Ub ligase (E3). The E2 must recognize both the E1 and a cognate E3 in addition to carrying activated Ub. These central functions are performed by a topologically conserved alpha/beta-fold core domain of approximately 150 residues shared by all E2s. However, as presented herein, the UbcH5 family of E2s can also bind Ub noncovalently on a surface well removed from the E2 active site. We present the solution structure of the UbcH5c/Ub noncovalent complex and demonstrate that this noncovalent interaction permits self-assembly of activated UbcH5c approximately Ub molecules. Self-assembly has profound consequences for the processive formation of polyubiquitin (poly-Ub) chains in ubiquitination reactions directed by the breast and ovarian cancer tumor susceptibility protein BRCA1.     

A novel recognition site for polyubiquitin and ubiquitin-like signals in an unexpected region of proteasomal subunit Rpn1

The ubiquitin (Ub)–proteasome system is the primary mechanism for maintaining protein homeostasis in eukaryotes, yet the underlying signaling events and specificities of its components are poorly understood. Proteins destined for degradation are tagged with covalently linked polymeric Ub chains and subsequently delivered to the proteasome, often with the assistance of shuttle proteins that contain Ub-like domains. This degradation pathway is riddled with apparent redundancy—in the form of numerous polyubiquitin chains of various lengths and distinct architectures, multiple shuttle proteins, and at least three proteasomal receptors. Moreover, the largest proteasomal receptor, Rpn1, contains one known binding site for polyubiquitin and shuttle proteins, although several studies have recently proposed the existence of an additional uncharacterized site. Here, using a combination of NMR spectroscopy, photocrosslinking, mass spectrometry, and mutagenesis, we show that Rpn1 does indeed contain another recognition site that exhibits affinities and binding preferences for polyubiquitin and Ub-like signals comparable to those of the known binding site in Rpn1. Surprisingly, this novel site is situated in the N-terminal section of Rpn1, a region previously surmised to be devoid of functionality. We identified a stretch of adjacent helices as the location of this previously uncharacterized binding site, whose spatial proximity and similar properties to the known binding site in Rpn1 suggest the possibility of multivalent signal recognition across the solvent-exposed surface of Rpn1. These findings offer new mechanistic insights into signal recognition processes that are at the core of the Ub–proteasome system.     

Domain analysis reveals that a deubiquitinating enzyme USP13 performs non-activating catalysis for Lys63-linked polyubiquitin

Deubiquitination is a reverse process of cellular ubiquitination important for many biological events. Ubiquitin (Ub)-specific protease 13 (USP13) is an ortholog of USP5 implicated in catalyzing hydrolysis of various Ub chains, but its enzymatic properties and catalytic regulation remain to be explored. Here we report studies of the roles of the Ub-binding domains of USP13 in regulatory catalysis by biochemical and NMR structural approaches. Our data demonstrate that USP13, distinct from USP5, exhibits a weak deubiquitinating activity preferring to Lys63-linked polyubiquitin (K63-polyUb) in a non-activation manner. The zinc finger (ZnF) domain of USP13 shares a similar fold with that of USP5, but it cannot bind with Ub, so that USP13 has lost its ability to be activated by free Ub. Substitution of the ZnF domain with that of USP5 confers USP13 the property of catalytic activation. The tandem Ub-associated (UBA) domains of USP13 can bind with different types of diUb but preferentially with K63-linked, providing a possible explanation for the weak activity preferring to K63-polyUb. USP13 can also regulate the protein level of CD3δ in cells, probably depending on its weak deubiquitinating activity and the Ub-binding properties of the UBA domains. Thus, the non-activating catalysis of USP13 for K63-polyUb chains implies that it may function differently from USP5 in cellular deubiquitination processes.     

The N-terminal domain of Rpn4 serves as a portable ubiquitin-independent degron and is recognized by specific 19S RP subunits

The number of proteasomal substrates that are degraded without prior ubiquitylation continues to grow. However, it remains poorly understood how the proteasome recognizes substrates lacking a ubiquitin (Ub) signal. Here we demonstrated that the Ub-independent degradation of Rpn4 requires the 19S regulatory particle (RP). The Ub-independent degron of Rpn4 was mapped to an N-terminal region including the first 80 residues. Inspection of its amino acid sequence revealed that the Ub-independent degron of Rpn4 consists of an intrinsically disordered domain followed by a folded segment. Using a photo-crosslinking-label transfer method, we captured three 19S RP subunits (Rpt1, Rpn2 and Rpn5) that bind the Ub-independent degron of Rpn4. This is the first time that specific 19S RP subunits have been identified interacting with a Ub-independent degron. This study provides insight into the mechanism by which Ub-independent substrates are recruited to the 26S proteasome.     

Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria

Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co‐expression of affinity‐tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in‐vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non‐ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9‐GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.     

Bidirectional remodeling of β1-integrin adhesions during chemotropic regulation of nerve growth

Background

The proteasome is a multi-subunit protein machine that is the final destination for cellular proteins that have been marked for degradation via an ubiquitin (Ub) chain appendage. These ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors Rpn10, Rpn13, or Rpt5, or are shuttled to the proteasome by Rad23, Dsk2, or Ddi1. The latter proteins share an Ub association domain (UBA) for binding poly-Ub chains and an Ub-like-domain (UBL) for binding to the proteasome. It has been proposed that shuttling receptors dock on the proteasome via Rpn1, but the precise nature of the docking site remains poorly defined.

Results

To shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in Rpn1 that disrupt its binding to UBA-UBL proteins. Here we demonstrate that delivery of Ub conjugates and docking of Ddi1 (and to a lesser extent Dsk2) to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved D517 residue of Rpn1. Moreover, degradation of the Ddi1-dependent proteasome substrate, Ufo1, is blocked in rpn1-D517A yeast cells. By contrast, Rad23 recruitment to the proteasome is not affected by rpn1-D517A.

Conclusions

These studies provide insight into the mechanism by which the UBA-UBL protein Ddi1 is recruited to the proteasome to enable Ub-dependent degradation of its ligands. Our studies suggest that different UBA-UBL proteins are recruited to the proteasome by distinct mechanisms.     

Structural insights into specificity and diversity in mechanisms of ubiquitin recognition by ubiquitin-binding domains

UBDs [Ub (ubiquitin)-binding domains], which are typically small protein motifs of <50 residues, are used by receptor proteins to transduce post-translational Ub modifications in a wide range of biological processes, including NF-κB (nuclear factor κB) signalling and proteasomal degradation pathways. More than 20 families of UBDs have now been characterized in structural detail and, although many recognize the canonical Ile44/Val70-binding patch on Ub, a smaller number have alternative Ub-recognition sites. The A20 Znf (A20-like zinc finger) of the ZNF216 protein is one of the latter and binds with high affinity to a polar site on Ub centred around Asp58/Gln62. ZNF216 shares some biological function with p62, with both linked to NF-κB signal activation and as shuttle proteins in proteasomal degradation pathways. The UBA domain (Ub-associated domain) of p62, although binding to Ub through the Ile44/Val70 patch, is unique in forming a stable dimer that negatively regulates Ub recognition. We show that the A20 Znf and UBA domain are able to form a ternary complex through independent interactions with a single Ub molecule, supporting functional models for Ub as a 'hub' for mediating multi-protein complex assembly and for enhancing signalling specificity.     

Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase

Ubiquitin (Ub)-protein ligases (E3s) frequently modify their substrates with multiple Ub molecules in the form of a polyubiquitin (poly-Ub) chain. Although structurally distinct poly-Ub chains (linked through different Ub lysine (Lys) residues) can confer different fates on target proteins, little is known about how E3s select the Lys residue to be used in chain synthesis. Here, we used a combination of mutagenesis, biochemistry, and mass spectrometry to map determinants of linkage choice in chain assembly catalyzed by KIAA10, an HECT (Homologous to E6AP C-Terminus) domain E3 that synthesizes K29- and K48-linked chains. Focusing on the Ub molecule that contributes the Lys residue for chain formation, we found that specific surface residues adjacent to K48 and K29 are critical for the usage of the respective Lys residues in chain synthesis. This direct mechanism of linkage choice bears similarities to the mechanism of substrate site selection in sumoylation catalyzed by Ubc9, but is distinct from the mechanism of chain linkage selection used by the Mms2/Ubc13 (Ub E2 variant (UEV)/E2) complex.