Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology

Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.     

VHS domains of ESCRT‐0 cooperate in high‐avidity binding to polyubiquitinated cargo

VHS (Vps27, Hrs, and STAM) domains occur in ESCRT‐0 subunits Hrs and STAM, GGA adapters, and other trafficking proteins. The structure of the STAM VHS domain–ubiquitin complex was solved at 2.6 Å resolution, revealing that determinants for ubiquitin recognition are conserved in nearly all VHS domains. VHS domains from all classes of VHS‐domain containing proteins in yeast and humans, including both subunits of ESCRT‐0, bound ubiquitin in vitro. ESCRTs have been implicated in the sorting of Lys63‐linked polyubiquitinated cargo. Intact human ESCRT‐0 binds Lys63‐linked tetraubiquitin 50‐fold more tightly than monoubiquitin, though only 2‐fold more tightly than Lys48‐linked tetraubiquitin. The gain in affinity is attributed to the cooperation of flexibly connected VHS and UIM motifs of ESCRT‐0 in avid binding to the polyubiquitin chain. Mutational analysis of all the five ubiquitin‐binding sites in yeast ESCRT‐0 shows that cooperation between them is required for the sorting of the Lys63‐linked polyubiquitinated cargo Cps1 to the vacuole.     

Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by sequestering lysine 48-linked polyubiquitin chains

Most substrates of the 26 S proteasome are recognized only following conjugation to a Lys48-linked polyubiquitin chain. Rad23 is one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA). Recent studies have shown that UbLs interact with 26 S proteasomes, whereas UBAs bind polyubiquitin chains. These biochemical properties suggest that UbL-UBA proteins may shuttle polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this model, the effect of human Rad23A on the degradation of polyubiquitinated substrates catalyzed by purified proteasomes is exclusively inhibitory. Strong inhibition is dependent on the presence of both UBAs, independent of the UbL, and can be explained by competition between the UBA domains and the proteasome for binding to substrate-linked polyubiquitin chains. The UBA domains bind Lys48-linked polyubiquitin chains in strong preference to Lys63 or Lys29-linked chains, leading to selective inhibition of the assembly and disassembly of Lys48-linked chains. These results place constraints on the mechanism(s) by which UbL-UBA proteins promote proteasome-catalyzed proteolysis and reveal new properties of UBA domains.     

Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein.

We have recently reported that the yeast plasma membrane uracil permease undergoes cell-surface ubiquitination, which is dependent on the Npi1/Rsp5 ubiquitin-protein ligase. Ubiquitination of this permease, like that of some other transporters and receptors, signals endocytosis of the protein, leading to its subsequent vacuolar degradation. This process does not involve the proteasome, which binds and degrades ubiquitin-protein conjugates carrying Lys48-linked ubiquitin chains. The data presented here show that ubiquitination and endocytosis of uracil permease are impaired in yeast cells lacking the Doa4p ubiquitin-isopeptidase. Both processes were rescued by overexpression of wild-type ubiquitin. Mutant ubiquitins carrying Lys-->Arg mutations at Lys29 and Lys48 restored normal permease ubiquitination. In contrast, a ubiquitin mutated at Lys63 did not restore permease polyubiquitination. Ubiquitin-permease conjugates are therefore extended through the Lys63 of ubiquitin. When polyubiquitination through Lys63 is blocked, the permease still undergoes endocytosis, but at a reduced rate. We have thus identified a natural target of Lys63-linked ubiquitin chains. We have also shown that monoubiquitination is sufficient to induce permease endocytosis, but that Lys63-linked ubiquitin chains appear to stimulate this process.     

Non-Covalent Interaction between Polyubiquitin and GTP Cyclohydrolase 1 Dictates Its Degradation

GTP cyclohydrolase 1 (GTPCH1) is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4). GTPCH1 protein degradation has been reported in animal models of several diseases, including diabetes mellitus and hypertension. However, the molecular mechanisms by which GTPCH1 is degraded remain uncharacterized. Here we report a novel non-covalent interaction between polyubiquitin and GTPCH1 in vitro and in vivo. The non-covalent binding of GTPCH1 to polyubiquitin via an ubiquitin-binding domain (UBD) results in ubiquitination and degradation. Ectopic expression of ubiquitin in cultured cells accelerated GTPCH1 degradation. In cultured cells and in vitro assays, Lys48-linked ubiquitin chains, but not Lys63-linked chains, interacted with GTPCH1 and targeted it for degradation. Consistently, proteasome inhibition attenuated GTPCH1 degradation. Finally, direct mutagenesis of an isoleucine (Ile131) in the hydrophobic patch of the GTPCH1 UBD affected its ubiquitin binding and the enzyme stability. Taken together, we conclude that GTPCH1 non-covalently interacts with polyubiquitin via an ubiquitin-binding domain. The polyubiquitin binding directs GTPCH1 ubiquitination and proteasome degradation.     

A proof‐of‐concept study in engineering synthetic protein for selective recognition of substrate‐free polyubiquitin

Similar to substrate‐conjugated polyubiquitin, unanchored polyubiquitin chains are emerging as important regulators for diverse biological processes. The affinity purification of unanchored polyubiquitin from various organisms has been reported, however, tools able to distinguish unanchored polyubiquitin chains with different isopeptide linkages have not yet been described. Toward the goal of selectively identifying and purifying unanchored polyubiquitin chains linked through different Lysines, Scott et al. developed a novel strategy in their study [Proteomics 2016, 16, 1961–1969]. They designed a linker‐optimized ubiquitin‐binding domain hybrid (t‐UBD) containing two UBDs, a ZnFCUBP domain, and a linkage‐selective UBA domain, to specifically recognize unanchored Lys48‐linked polyubiquitin chains. Subsequently, a series of assays has proved the feasibility of this novel strategy for the purification of endogenous substrate‐free Lys48‐linked polyubiquitin chains from mammalian cell extracts. Their research not only provides a tool for purifying unanchored polyubiquitin with different isopeptide linkages, but also paves the way for generating reagents to study the function of unanchored polyubiquitin chains of different linkages in the future. The design of UBD hybrids for defined unanchored polyubiquitin (Lys48‐polyubiquitin) in this study also set an excellent example for future methodology studies regarding monitoring in vivo dynamic changes in the patterns of ubiquitination.     

Mapping the interactions between Lys48 and Lys63-linked di-ubiquitins and a ubiquitin-interacting motif of S5a

Numerous cellular processes are regulated by (poly)ubiquitin-mediated signaling events, which involve a covalent modification of the substrate protein by a single ubiquitin or a chain of ubiquitin molecules linked via a specific lysine. Remarkably, the outcome of polyubiquitination is linkage-dependent. For example, Lys48-linked chains are the principal signal for proteasomal degradation, while Lys63-linked chains act as nonproteolytic signals. Despite significant progress in characterization of various cellular pathways involving ubiquitin, understanding of the structural details of polyubiquitin chain recognition by downstream cellular effectors is missing. Here we use NMR to study the interaction of a ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a with di-ubiquitin, the simplest model for polyubiquitin chain, to gain insights into the mechanism of polyubiquitin recognition by the proteasome. We have mapped the binding interface and characterized the stoichiometry and the process of UIM binding to Lys48- and Lys63-linked di-ubiquitin chains. Our data provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interdomain interface. This allows UIM to enter the interface and bind directly to the same ubiquitin hydrophobic-patch surface as utilized in UIM:monoubiquitin complexes. The results indicate that up to two UIM molecules can bind di-ubiquitin, and the binding interface between UIM and ubiquitin units in di-ubiquitin is essentially the same for both Lys48- and Lys63-linked chains. Our data suggest possible structural models for the binding of UIM and of full-length S5a to di-ubiquitin.     

COP9 Signalosome Interacts ATP-dependently with p97/Valosin-containing Protein (VCP) and Controls the Ubiquitination Status of Proteins Bound to p97/VCP

Ubiquitinated proteins can alternatively be delivered directly to the proteasome or via p97/VCP (valosin-containing protein). Whereas the proteasome degrades ubiquitinated proteins, the homohexameric ATPase p97/VCP seems to control the ubiquitination status of recruited substrates. The COP9 signalosome (CSN) is also involved in the ubiquitin/proteasome system (UPS) as exemplified by regulating the neddylation of ubiquitin E3 ligases. Here, we show that p97/VCP colocalizes and directly interacts with subunit 5 of the CSN (CSN5) in vivo and is associated with the entire CSN complex in an ATP-dependent manner. Furthermore, we provide evidence that the CSN and in particular the isopeptidase activity of its subunit CSN5 as well as the associated deubiquitinase USP15 are required for proper processing of polyubiquitinated substrates bound to p97/VCP. Moreover, we show that in addition to NEDD8, CSN5 binds to oligoubiquitin chains in vitro. Therefore, CSN and p97/VCP could form an ATP-dependent complex that resembles the 19 S proteasome regulatory particle and serves as a key mediator between ubiquitination and degradation pathways.     

K63‐specific deubiquitination by two JAMM/MPN+ complexes: BRISC‐associated Brcc36 and proteasomal Poh1

An unusual deubiquitinating (DUB) activity exists in HeLa cell extracts that is highly specific for cleaving K63‐linked but not K48‐linked polyubiquitin chains. The activity is insensitive to both N‐ethyl‐maleimide and ubiquitin aldehyde, indicating that it lacks an active site cysteine residue, and gel filtration experiments show that it resides in a high molecular weight (～600 kDa) complex. Using a biochemical approach, we found that the K63‐specific DUB activity co‐fractionated through seven chromatographic steps with three multisubunit complexes: the 19S (PA700) portion of the 26S proteasome, the COP9 signalosome (CSN) and a novel complex that includes the JAMM/MPN+ domain‐containing protein Brcc36. When we analysed the individual complexes, we found that the activity was intrinsic to PA700 and the Br cc36 is opeptidase c omplex (BRISC), but that the CSN‐associated activity was due entirely to an interaction with Brcc36. None of the complexes cleave K6, K11, K29, K48 or α‐linked polyubiquitin, but they do cleave K63 linkages within mixed‐linkage chains. Our results suggest that specificity for K63‐linked polyubiquitin is a common property of the JAMM/MPN+ family of DUBs.     

Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.     

S5a promotes protein degradation by blocking synthesis of nondegradable forked ubiquitin chains

Ubiquitin (Ub)–protein conjugates formed by purified ring‐finger or U‐box E3s with the E2, UbcH5, resist degradation and disassembly by 26S proteasomes. These chains contain multiple types of Ub forks in which two Ub's are linked to adjacent lysines on the proximal Ub. We tested whether cells contain factors that prevent formation of nondegradable conjugates and whether the forked chains prevent proteasomal degradation. S5a is a ubiquitin interacting motif (UIM) protein present in the cytosol and in the 26S proteasome. Addition of S5a or a GST‐fusion of S5a's UIM domains to a ubiquitination reaction containing 26S proteasomes, UbcH5, an E3 (MuRF1 or CHIP), and a protein substrate, dramatically stimulated its degradation, provided S5a was present during ubiquitination. Mass spectrometry showed that S5a and GST–UIM prevented the formation of Ub forks without affecting synthesis of standard isopeptide linkages. The forked Ub chains bind poorly to 26S proteasomes unlike those synthesized with S5a present or linked to Lys63 or Lys48 chains. Thus, S5a (and presumably certain other UIM proteins) function with certain E3/E2 pairs to ensure synthesis of efficiently degraded non‐forked Ub conjugates.     

Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling

Diverse cellular events are regulated by post-translational modification of substrate proteins via covalent attachment of one or a chain of ubiquitin molecules. The outcome of (poly)ubiquitination depends upon the specific lysine residues involved in the formation of polyubiquitin chains. Lys48-linked chains act as a universal signal for proteasomal degradation, whereas Lys63-linked chains act as a specific signal in several non-degradative processes. Although it has been anticipated that functional diversity between alternatively linked polyubiquitin chains relies on linkage-dependent differences in chain conformation/topology, direct structural evidence in support of this model has been lacking. Here we use NMR methods to determine the structure of a Lys63-linked di-ubiquitin chain. The structure is characterized by an extended conformation, with no direct contact between the hydrophobic residues Leu8, Ile44, and Val70 on the ubiquitin units. This structure contrasts with the closed conformation observed for Lys48-linked di-ubiquitin wherein these residues form the interdomain interface (Cook, W. J., Jeffrey, L. C., Carson, M., Zhijian, C., and Pickart, C. M. (1992) J. Biol. Chem. 267, 16467-16471; Varadan, R., Walker, O., Pickart, C., and Fushman, D. (2002) J. Mol. Biol. 324, 637-647). Consistent with the open conformation of the Lys(63)-linked di-ubiquitin, our binding studies show that both ubiquitin domains in this chain can bind a ubiquitin-associated domain from HHR23A independently and in a mode similar to that for mono-ubiquitin. In contrast, Lys48-linked di-ubiquitin binds in a different, higher affinity mode that has yet to be determined. This is the first experimental evidence that alternatively linked polyubiquitin chains adopt distinct conformations.     

Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-linked polyubiquitin chains

AMPK (AMP-activated protein kinase)-related kinases regulate cell polarity as well as proliferation and are activated by the LKB1-tumour suppressor kinase. In the present study we demonstrate that the AMPK-related kinases, NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4), are polyubiquitinated in vivo and interact with the deubiquitinating enzyme USP9X (ubiquitin specific protease-9). Knockdown of USP9X increased polyubiquitination of NUAK1 and MARK4, whereas overexpression of USP9X inhibited ubiquitination. USP9X, catalysed the removal of polyubiquitin chains from wild-type NUAK1, but not from a non-USP9X-binding mutant. Topological analysis revealed that ubiquitin monomers attached to NUAK1 and MARK4 are linked by Lys(29) and/or Lys(33) rather than the more common Lys(48)/Lys(63). We find that AMPK and other AMPK-related kinases are also polyubiquitinated in cells. We identified non-USP9X-binding mutants of NUAK1 and MARK4 and find that these are hyper-ubiquitinated and not phosphorylated at their T-loop residue targeted by LKB1 when expressed in cells, suggesting that polyubiquitination may inhibit these enzymes. The results of the present study demonstrate that NUAK1 and MARK4 are substrates of USP9X and provide the first evidence that AMPK family kinases are regulated by unusual Lys(29)/Lys(33)-linked polyubiquitin chains.     

The deubiquitinating enzyme Ubp2 modulates Rsp5-dependent Lys63-linked polyubiquitin conjugates in Saccharomyces cerevisiae

The functions of Lys(63)-linked polyubiquitin chains are poorly understood, as are the enzymes that specifically generate Lys(63)-linked conjugates. Rsp5 is a HECT (homologous to E6AP C terminus) ubiquitin ligase involved in numerous processes, and an associated deubiquitinating enzyme, Ubp2, modulates its activity. A dramatic increase in Lys(63)-linked conjugates was observed in ubp2Delta cells. The formation of these was Rsp5-dependent, and ubp2Delta phenotypes could be suppressed by prevention of formation of Lys(63) conjugates. Cell wall integrity was impaired in rsp5-1 cells and in cells defective in Lys(63)-polyubiquitination, as assayed by calcofluor white sensitivity, and ubp2Delta and rup1Delta mutants suppressed the calcofluor white sensitivity of rsp5-1. A large fraction of the Lys(63) conjugates in ubp2Delta cells bound to Rsp5, and a proteomics approach was used to identify Rsp5 substrates subject to Ubp2 regulation. Two closely related proteins, Csr2 and Ecm21, were among the identified proteins. Both were efficiently Lys(63)-polyubiquitinated by Rsp5 and deubiquitinated by Ubp2. Together, these results indicate that Ubp2 modulates Lys(63)-polyubiquitination of Rsp5 substrates in vivo, including ubiquitination of two newly identified Rsp5 substrates.     

Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes?

Although cellular proteins conjugated to K48‐linked Ub chains are targeted to proteasomes, proteins conjugated to K63‐ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48‐ and K63‐ubiquitinated substrates similarly. Therefore, we investigated why K63‐ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63‐ubiquitinated molecules to the proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48‐ubiquitinated proteins and to stimulate proteasome binding. The specificities of these proteins for K48‐ or K63‐ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal‐lysosomal pathway.     

Structural basis for specific recognition of Lys 63‐linked polyubiquitin chains by NZF domains of TAB2 and TAB3

TAB2 and TAB3 activate the Jun N‐terminal kinase and nuclear factor‐κB pathways through the specific recognition of Lys 63‐linked polyubiquitin chains by its Npl4 zinc‐finger (NZF) domain. Here we report crystal structures of the TAB2 and TAB3 NZF domains in complex with Lys 63‐linked diubiquitin at 1.18 and 1.40 Å resolutions, respectively. Both NZF domains bind to the distal ubiquitin through a conserved Thr‐Phe dipeptide that has been shown to be important for the interaction of the NZF domain of Npl4 with monoubiquitin. In contrast, a surface specific to TAB2 and TAB3 binds the proximal ubiquitin. Both the distal and proximal binding sites of the TAB2 and TAB3 NZF domains recognize the Ile 44‐centred hydrophobic patch on ubiquitin but do not interact with the Lys 63‐linked isopeptide bond. Mutagenesis experiments show that both binding sites are required to enable binding of Lys 63‐linked diubiquitin. We therefore propose a mechanism for the recognition of Lys 63‐linked polyubiquitin chains by TAB2 and TAB3 NZF domains in which diubiquitin units are specifically recognized by a single NZF domain.     

Autoregulation of the 26S proteasome by in situ ubiquitination

The 26S proteasome degrades ubiquitinated proteins, and proteasomal degradation controls various cellular events. Here we report that the human 26S proteasome is ubiquitinated, by which the ubiquitin receptors Adrm1 and S5a, the ATPase subunit Rpt5, and the deubiquitinating enzyme Uch37 are ubiquitinated in situ by proteasome-associating ubiquitination enzymes. Ubiquitination of these subunits significantly impairs the 26S proteasome''s ability to bind, deubiquitinate, and degrade ubiquitinated proteins. Moreover, ubiquitination of the 26S proteasome can be antagonized by proteasome-residing deubiquitinating enzymes, by the binding of polyubiquitin chains, and by certain cellular stress, indicating that proteasome ubiquitination is dynamic and regulated in cells. We propose that in situ ubiquitination of the 26S proteasome regulates its activity, which could function to adjust proteasomal activity in response to the alteration of cellular ubiquitination levels.