Ubiquitin binding to A20 ZnF4 is required for modulation of NF-κB signaling

Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger?4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.     

Atypical binding of the Swa2p UBA domain to ubiquitin

Swa2p is an auxilin-like yeast protein that is involved in vesicular transport and required for uncoating of clathrin-coated vesicles. Swa2p contains a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin (Ub)-mediated processes. We have determined a structural model of the Swa2p UBA domain in complex with Ub using NMR spectroscopy and molecular docking. Ub recognition occurs predominantly through an atypical interaction in which UBA helix α1 and the N-terminal part of helix α2 bind to Ub. Mutation of Ala148, a key residue in helix α1, to polar residues greatly reduced the affinity of the UBA domain for Ub and revealed a second low-affinity Ub-binding site located on the surface formed by helices α1 and α3. Surface plasmon resonance showed that the Swa2p UBA domain binds K48- and K63-linked di-Ub in a non-linkage-specific manner. These results reveal convergent evolution of a Ub-binding site on helix α1 of UBA domains involved in membrane protein trafficking.     

A Ubiquitin Shuttle DC-UbP/UBTD2 Reconciles Protein Ubiquitination and Deubiquitination via Linking UbE1 and USP5 Enzymes

The ubiquitination levels of protein substrates in eukaryotic cells are delicately orchestrated by various protein cofactors and enzymes. Dendritic cell-derived ubiquitin (Ub)-like protein (DC-UbP), also named as Ub domain-containing protein 2 (UBTD2), is a potential Ub shuttle protein comprised of a Ub-like (UbL) domain and a Ub-binding domain (UBD), but its biological function remains largely unknown. We identified two Ub-related enzymes, the deubiquitinating enzyme USP5 and the Ub-activating enzyme UbE1, as interacting partners of DC-UbP from HEK 293T cells. Biochemical studies revealed that the tandem UBA domains of USP5 and the C-terminal Ub-fold domain (UFD) of UbE1 directly interacted with the C-terminal UbL domain of DC-UbP but on the distinct surfaces. Overexpression of DC-UbP in HEK 293T cells enhanced the association of these two enzymes and thus prompted cellular ubiquitination, whereas knockdown of the protein reduced the cellular ubiquitination level. Together, DC-UbP may integrate the functions of USP5 and UbE1 through interacting with them, and thus reconcile the cellular ubiquitination and deubiquitination processes.     

USP3 inhibits type I interferon signaling by deubiquitinating RIG-I-like receptors

Lysine 63 (K63)-linked ubiquitination of RIG-I plays a critical role in the activation of type I interferon pathway, yet the molecular mechanism responsible for its deubiquitination is still poorly understood. Here we report that the deubiquitination enzyme ubiquitin-specific protease 3 (USP3) negatively regulates the activation of type I interferon signaling by targeting RIG-I. Knockdown of USP3 specifically enhanced K63-linked ubiquitination of RIG-I, upregulated the phosphorylation of IRF3 and augmented the production of type I interferon cytokines and antiviral immunity. We further show that there is no interaction between USP3 and RIG-I-like receptors (RLRs) in unstimulated or uninfected cells, but upon viral infection or ligand stimulation, USP3 binds to the caspase activation recruitment domain of RLRs and then cleaves polyubiquitin chains through cooperation of its zinc-finger Ub-binding domain and USP catalytic domains. Mutation analysis reveals that binding of USP3 to polyubiquitin chains on RIG-I is a prerequisite step for its cleavage of polyubiquitin chains. Our findings identify a previously unrecognized role of USP3 in RIG-I activation and provide insights into the mechanisms by which USP3 inhibits RIG-I signaling and antiviral immunity.     

Recognition of polyubiquitin isoforms by the multiple ubiquitin binding modules of isopeptidase T

The conjugation of polyubiquitin to target proteins acts as a signal that regulates target stability, localization, and function. Several ubiquitin binding domains have been described, and while much is known about ubiquitin binding to the isolated domains, little is known with regard to how the domains interact with polyubiquitin in the context of full-length proteins. Isopeptidase T (IsoT/USP5) is a deubiquitinating enzyme that is largely responsible for the disassembly of unanchored polyubiquitin in the cell. IsoT has four ubiquitin binding domains: a zinc finger domain (ZnF UBP), which binds the proximal ubiquitin, a UBP domain that forms the active site, and two ubiquitin-associated (UBA) domains whose roles are unknown. Here, we show that the UBA domains are involved in binding two different polyubiquitin isoforms, linear and K48-linked. Using isothermal titration calorimetry, we show that IsoT has at least four ubiquitin binding sites for both polyubiquitin isoforms. The thermodynamics of the interactions reveal that the binding is enthalpy-driven. Mutation of the UBA domains suggests that UBA1 and UBA2 domains of IsoT interact with the third and fourth ubiquitins in both polyubiquitin isoforms, respectively. These data suggest that recognition of the polyubiquitin isoforms by IsoT involves considerable conformational mobility in the polyubiquitin ligand, in the enzyme, or in both.     

The ubiquitin-specific protease USP8 directly deubiquitinates SQSTM1/p62 to suppress its autophagic activity

ABSTRACT

SQSTM1/p62 (sequestosome 1) is a critical macroautophagy/autophagy receptor that promotes the formation and degradation of ubiquitinated aggregates. SQSTM1 can be modified by ubiquitination, and this modification modulates its autophagic activity. However, the molecular mechanisms underpinning its reversible deubiquitination have never been described. Here we report that USP8 (ubiquitin specific peptidase 8) directly interacted with and deubiquitinated SQSTM1. USP8 preferentially removed the lysine 11 (K11)-linked ubiquitin chains from SQSTM1. Moreover, USP8 deubiquitinated SQSTM1 principally at K420 within its ubiquitin-association (UBA) domain. Finally, USP8 inhibited SQSTM1 degradation and autophagic influx in cells with wild-type SQSTM1, but not its mutant with substitution of K420 with an arginine. Taken together, USP8 acts as a negative regulator of autophagy by deubiquitinating SQSTM1 at K420.

Abbreviations: BafA₁: bafilomycin A₁; BAP1: BRCA1 associated protein 1; DUB: deubiquitinating enzyme; ESCRT: endosomal sorting complex required for transport; HTT: huntingtin; K: lysine; KEAP1: kelch like ECH associated protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; shRNA: short hairpin RNA; SQSTM1: sequestosome 1; Ub: ubiquitin; UBA: ubiquitin-association; UBE2D2: ubiquitin conjugating enzyme E2 D2; UBE2D3: ubiquitin conjugating enzyme E2 D3; USP: ubiquitin specific peptidase; WT: wild-type     

Identification of a novel 29-linked polyubiquitin binding protein, Ufd3, using polyubiquitin chain analogues

Lysine 48-linked polyubiquitin chains are the best understood form of polyubiquitin and are necessary for the function of the ubiquitin-proteasome system. However, other forms of polyubiquitin (e.g., K29- and K63-linked chains) are also present in vivo. Less is known about the functional roles of these linkages or the proteins specifically interacting with these forms of polyubiquitin. Use of native polyubiquitin chains to identify binding proteins is complicated by the difficulties of synthesis and stability. Here, we report the synthesis of a nonhydrolyzable analogue of 29-linked polyubiquitin chains on an affinity support and its use in identifying proteins that bind 29-linked polyubiquitin chains. The 29-linked Ub4 resin was stable and tightly bound recombinant human Isopeptidase T (USP5), a deubiquitinating enzyme known to bind the 29-linked polyubiquitin chains. Two high affinity interactors of the 29-linked polyubiquitin analogues were identified from Saccharomyces cerevisiae lysates. They were identified as Ubp14, the yeast ortholog of Isopeptidase T, and Ufd3, a member of the ubiquitin-fusion degradation pathway with unknown function. Purified recombinant Ufd3 bound to the resin as well, confirming that Ufd3 is a novel binding partner of polyubiquitin. These results demonstrate the efficacy of using polyubiquitin analogue affinity supports to identify novel binding partners of specifically linked polyubiquitin chains. Identification of these proteins will lead to a greater understanding of the physiological relevance of different polyubiquitin linkages.     

USP8 and PARK2/parkin-mediated mitophagy

The Parkinson disease (PD)-associated E3-ubiquitin (Ub) ligase PARK2/parkin plays a central role in many stress response pathways, and in particular, in mitochondrial quality control. Within this pathway, PARK2 activation is accompanied by a robust increase in its autoubiquitination, followed by clearance of the damaged mitochondria by selective autophagy (mitophagy). Yet, little is known about how this auto-ubiquitination is regulated during mitophagy. In our study, we demonstrate that PARK2 forms predominantly K6-linked Ub conjugates on itself. Moreover, PARK2 interacts with the deubiquitinating enzyme USP8 that preferentially removes these K6-linked conjugates, thereby regulating the activity and function of PARK2 in the pathway. When USP8 is silenced, a persistence of K6-linked Ub conjugates on PARK2 delays both its translocation to damaged mitochondria and successful completion of mitophagy. Taken together, these findings implicate a novel role for K6-linked Ub conjugates and USP8-mediated deubiquitination in the regulation of PARK2 in mitochondrial quality control.     

Structural and biochemical studies of the open state of Lys48-linked diubiquitin

Ubiquitin (Ub) is a small protein highly conserved among eukaryotes and involved in practically all aspects of eukaryotic cell biology. Polymeric chains assembled from covalently-linked Ub monomers function as molecular signals in the regulation of a host of cellular processes. Our previous studies have shown that the predominant state of Lys48-linked di- and tetra-Ub chains at near-physiological conditions is a closed conformation, in which the Ub-Ub interface is formed by the hydrophobic surface residues of the adjacent Ub units. Because these very residues are involved in (poly)Ub interactions with the majority of Ub-binding proteins, their sequestration at the Ub-Ub interface renders the closed conformation of polyUb binding incompetent. Thus the existence of open conformation(s) and the interdomain motions opening and closing the Ub-Ub interface is critical for the recognition of Lys48-linked polyUb by its receptors. Knowledge of the conformational properties of a polyUb signal is essential for our understanding of its specific recognition by various Ub-receptors. Despite their functional importance, open states of Lys48-linked chains are poorly characterized. Here we report a crystal structure of the open state of Lys48-linked di-Ub. Moreover, using NMR, we examined interactions of the open state of this chain (at pH4.5) with a Lys48-linkage-selective receptor, the UBA2 domain of a shuttle protein hHR23a. Our results show that di-Ub binds UBA2 in the same mode and with comparable affinity as the closed state. Our data suggest a mechanism for polyUb signal recognition, whereby Ub-binding proteins select specific conformations out of the available ensemble of polyUb chain conformations. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway

A growing number of yeast and mammalian plasma membrane proteins are reported to be modified with K63-linked ubiquitin (Ub) chains. However, the relative importance of this modification versus monoubiquitylation in endocytosis, Golgi to endosome traffic, and sorting into the multivesicular body (MVB) pathway remains unclear. In this study, we show that K63-linked ubiquitylation of the Gap1 permease is essential for its entry into the MVB pathway. Carboxypeptidase S also requires modification with a K63-Ub chain for correct MVB sorting. In contrast, monoubiquitylation of a single target lysine of Gap1 is a sufficient signal for its internalization from the cell surface, and Golgi to endosome transport of the permease requires neither its ubiquitylation nor the Ub-binding GAT (Gga and Tom1) domain of Gga (Golgi localizing, gamma-ear containing, ARF binding) adapter proteins, the latter being crucial for subsequent MVB sorting of the permease. Our data reveal that K63-linked Ub chains act as a specific signal for MVB sorting, providing further insight into the Ub code of membrane protein trafficking.     

Molecular Recognition of M1-Linked Ubiquitin Chains by Native and Phosphorylated UBAN Domains

Although the Ub-binding domain in ABIN proteins and NEMO (UBAN) is highly conserved, UBAN-containing proteins exhibit different Ub-binding properties, resulting in their diverse biological roles. Post-translational modifications further control UBAN domain specificity for poly-Ub chains. However, precisely, how the UBAN domain structurally confers such functional diversity remains poorly understood. Here we report crystal structures of ABIN-1 alone and in complex with one or two M1-linked di-Ub chains. ABIN-1 UBAN forms a homo-dimer that provides two symmetrical Ub-binding sites on either side of the coiled-coil structure. Moreover, crystal structures of ABIN1 UBAN in complex with di-Ub chains reveal a concentration-dependency of UBAN/di-Ub binding stoichiometry. Analysis of UBAN/M1-linked di-Ub binding characteristics indicates that phosphorylated S473 in OPTN and its corresponding phospho-mimetic residue in ABIN-1 (E484) are essential for high affinity interactions with M1-linked Ub chains. Also, a phospho-mimetic mutation of A303 in NEMO, corresponding to S473 of OPTN, increases binding affinity for M1-linked Ub chains. These findings are in line with the diverse physiological roles of UBAN domains, as phosphorylation of OPTN UBAN is required to enhance its binding to Ub during mitophagy.     

TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains

The activation of NF-kappaB and IKK requires an upstream kinase complex consisting of TAK1 and adaptor proteins such as TAB1, TAB2, or TAB3. TAK1 is in turn activated by TRAF6, a RING domain ubiquitin ligase that facilitates the synthesis of lysine 63-linked polyubiquitin chains. Here we present evidence that TAB2 and TAB3 are receptors that bind preferentially to lysine 63-linked polyubiquitin chains through a highly conserved zinc finger (ZnF) domain. Mutations of the ZnF domain abolish the ability of TAB2 and TAB3 to bind polyubiquitin chains, as well as their ability to activate TAK1 and IKK. Significantly, replacement of the ZnF domain with a heterologous ubiquitin binding domain restored the ability of TAB2 and TAB3 to activate TAK1 and IKK. We also show that TAB2 binds to polyubiquitinated RIP following TNFalpha stimulation. These results indicate that polyubiquitin binding domains represent a new class of signaling domains that regulate protein kinase activity through a nonproteolytic mechanism.     

USP33 deubiquitinates PRKN/parkin and antagonizes its role in mitophagy

ABSTRACT

PRKN/parkin activation through phosphorylation of its ubiquitin and ubiquitin-like domain by PINK1 is critical in mitophagy induction for eliminating the damaged mitochondria. Deubiquitinating enzymes (DUBs) functionally reversing PRKN ubiquitination are critical in controlling the magnitude of PRKN-mediated mitophagy process. However, potential DUBs that directly target PRKN and antagonize its pro-mitophagy effect remains to be identified and characterized. Here, we demonstrated that USP33/VDU1 is localized at the outer membrane of mitochondria and serves as a PRKN DUB through their interaction. Cellular and in vitro assays illustrated that USP33 deubiquitinates PRKN in a DUB activity-dependent manner. USP33 prefers to remove K6, K11, K48 and K63-linked ubiquitin conjugates from PRKN, and deubiquitinates PRKN mainly at Lys435. Mutation of this site leads to a significantly decreased level of K63-, but not K48-linked PRKN ubiquitination. USP33 deficiency enhanced both K48- and K63-linked PRKN ubiquitination, but only K63-linked PRKN ubiquitination was significantly increased under mitochondrial depolarization. Further, USP33 knockdown increased both PRKN protein stabilization and its translocation to depolarized mitochondria leading to the enhancement of mitophagy. Moreover, USP33 silencing protects SH-SY5Y human neuroblastoma cells from the neurotoxin MPTP-induced apoptotic cell death. Our findings convincingly demonstrate that USP33 is a novel PRKN deubiquitinase antagonizing its regulatory roles in mitophagy and SH-SY5Y neuron-like cell survival. Thus, USP33 inhibition may represents an attractive new therapeutic strategy for PD patients.

Abbreviations: CCCP: carbonyl cyanide 3-chlorophenylhydrazone; DUB: deubiquitinating enzymes; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; OMM: outer mitochondrial membrane; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; TM: transmembrane; Ub: ubiquitin; UBA1: ubiquitin like modifier activating enzyme 1; UBE2L3/UbcH7: ubiquitin conjugating enzyme E2 L3; USP33: ubiquitin specific peptidase 33; WT: wild type.     

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.     

The Ubiquitin-associated (UBA) 1 Domain of Schizosaccharomyces pombe Rhp23 Is Essential for the Recognition of Ubiquitin-proteasome System Substrates Both in Vitro and in Vivo

The ubiquitin-proteasome system is essential for maintaining a functional cell. Not only does it remove incorrectly folded proteins, it also regulates protein levels to ensure their appropriate spatial and temporal distribution. Proteins marked for degradation by the addition of Lys⁴⁸-linked ubiquitin (Ub) chains are recognized by shuttle factors and transported to the 26 S proteasome. One of these shuttle factors, Schizosaccharomyces pombe Rhp23, has an unusual domain architecture. It comprises an N-terminal ubiquitin-like domain that can recognize the proteasome followed by two ubiquitin-associated (UBA) domains, termed UBA1 and UBA2, which can bind Ub. This architecture is conserved up to humans, suggesting that both domains are important for Rhp23 function. Such an extent of conservation raises the question as to why, in contrast to all other shuttle proteins, does Rhp23 require two UBA domains? We performed in vitro Ub binding assays using domain swap chimeric proteins and mutated domains in isolation as well as in the context of the full-length protein to reveal that the Ub binding properties of the UBA domains are context-dependent. In vivo, the internal Rhp23 UBA1 domain provides sufficient Ub recognition for the protein to function without UBA2.     

Structural and Biochemical Basis for Higher-Order Assembly between A20-Binding Inhibitor of NF-κB 1 (ABIN1) and M1-Linked Ubiquitins

Polyubiquitination is important in controlling NF-κB signaling. Excessive NF-κB activity has been linked to inflammatory disorders and autoimmune diseases, while ABIN1 could attenuate NF-κB activation to maintain immune homeostasis by utilizing UBAN to recognize linear (M1)-linked polyubiquitinated NF-κB activation mediators, including NEMO, IRAK1 and RIP1. PolyUb-mediated UBAN recruitment remains undetermined, since the recognition studies focused mostly on di-ubiquitin (diUb). Here we report three crystal structures of human ABIN1 UBAN (hABIN1^UBAN) in complex with M1-linked diUb, triUb, and tetraUb, respectively. Notably, the hABIN1^UBAN:diUb structure reveals that a diUb randomly binds one of the Ub-binding sites of the hABIN1^UBAN dimer and leaves the other site vacant. Together with the ITC and gel-filtration analyses, we found that M1-triUb and M1-tetraUb adopt two unique conformations, instead of an elongated one, and they preferentially use the N-terminal two-Ub unit to bind the primary Ub-binding site of a hABIN1^UBAN dimer and the C-terminal two-Ub unit to bind the secondary Ub-binding site of another hABIN1^UBAN dimer. Especially, our results suggest that two ABIN1^UBAN dimers cooperatively bind two UBAN-binding units of a tetraUb or vice versa. Since the UBAN family members share a conserved diUb-binding mode, our results suggest that M1-polyUb modification allows multiple copies of the two-tandem Ub unit to simultaneously coordinate multiple and/or different binding partners to increase their local concentrations and to facilitate the formation of a large signaling complex. Our study provides a structural-functional glimpse of M1-polyUb as a multiple-molecule binding platform to exert its intrinsic structural plasticity in mediating cellular signaling.     

A dual role for K63-linked ubiquitin chains in multivesicular body biogenesis and cargo sorting

In yeast, the sorting of transmembrane proteins into the multivesicular body (MVB) internal vesicles requires their ubiquitylation by the ubiquitin ligase Rsp5. This allows their recognition by the ubiquitin-binding domains (UBDs) of several endosomal sorting complex required for transport (ESCRT) subunits. K63-linked ubiquitin (K63Ub) chains decorate several MVB cargoes, and accordingly we show that they localize prominently to the class E compartment, which accumulates ubiquitylated cargoes in cells lacking ESCRT components. Conversely, yeast cells unable to generate K63Ub chains displayed MVB sorting defects. These properties are conserved among eukaryotes, as the mammalian melanosomal MVB cargo MART-1 is modified by K63Ub chains and partly missorted when the genesis of these chains is inhibited. We show that all yeast UBD-containing ESCRT proteins undergo ubiquitylation and deubiquitylation, some being modified through the opposing activities of Rsp5 and the ubiquitin isopeptidase Ubp2, which are known to assemble and disassemble preferentially K63Ub chains, respectively. A failure to generate K63Ub chains in yeast leads to an MVB ultrastructure alteration. Our work thus unravels a double function of K63Ub chains in cargo sorting and MVB biogenesis.     

The solution structure of the ZnF UBP domain of USP33/VDU1

USP33/VDU1 is a deubiquitinating enzyme that binds to the von Hippel-Lindau tumor suppressor protein. It also regulates thyroid hormone activation by deubiquitinating type 2 iodothyronine deiodinase. USP33/VDU1 contains a ZF UBP domain, a protein module found in many proteins in the ubiquitin-proteasome system. Several ZF UBP domains have been shown to bind ubiquitin, and a structure of a complex of the ZF UBP domain of isoT/USP5 and ubiquitin is available. In the present work, the solution structure of the ZF UBP domain of USP33/VDU1 has been determined by NMR spectroscopy. The structure differs from that of the USP5 domain, which contains only one of the three Zn ions present in the USP33/VDU1 structure. The USP33/VDU1 ZnF UBP domain does not bind to ubiquitin.