Ubiquitin-specific cysteine protease 2a (USP2a) regulates the stability of Aurora-A

The ubiquitin/proteasome pathway plays critical roles in virtually all aspects of cell biology. Enzymes of the ubiquitin pathway add (ligases) or remove (deubiquitinases) ubiquitin tags to or from their target proteins in a selective fashion. USP2a is a member of a subfamily of deubiquitinases, called ubiquitin-specific cysteine proteases (USPs). Although USP2a has been reported to be a bona fide oncogene that regulates the stability of MDM2, MDMX, and FAS, it is likely that there are other unidentified substrates for USP2a. In this study, we show that USP2a mediates mitotic progression by regulating the stability of Aurora-A. Through cell-based screening of a USP siRNA library, we discovered that knockdown of USP2a reduced the protein levels of Aurora-A. USP2a interacts with Aurora-A directly in vitro and in vivo. In addition, Aurora-A is a substrate for USP2a in vitro and in vivo. Our study provides a novel mechanism for the role of USP2a in mediating the stability of Aurora-A.     

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.     

Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2

Deubiquitinating enzymes (DUBs) catalyze the removal of attached ubiquitin molecules from amino groups of target proteins. The large family of DUBs plays an important role in the regulation of the intracellular homeostasis of different proteins and influences therefore key events such as cell division, apoptosis, etc. The DUB family members UCH-L3 and USP2 are believed to inhibit the degradation of various tumor-growth-promoting proteins by removing the trigger for degradation. Inhibitors of these enzymes should therefore lead to enhanced degradation of oncoproteins and may thus stop tumor growth. To develop an enzymatic assay for the search of UCH-L3 and USP2 inhibitors, C-terminally labeled ubiquitin substrates were enzymatically synthesized. We have used the ubiquitin-activating enzyme E1 and one of the ubiquitin-conjugating enzymes E2 to attach a fluorescent lysine derivative to the C terminus of ubiquitin. Since only the epsilon-NH(2) group of the lysine derivatives was free and reactive, the conjugates closely mimic the isopeptide bond between the ubiquitin and the lysine side chains of the targeted proteins. Various substrates were synthesized by this approach and characterized enzymatically with the two DUBs. The variant consisting of the fusion protein between the large N-terminal NusA tag and the ubiquitin which was modified with alpha-NH(2)-tetramethylrhodamin-lysine, was found to give the highest dynamic range in a fluorescence polarization readout. Therefore we have chosen this substrate for the development of a miniaturized, fluorescence-polarization-based high-throughput screening assay.     

UBL/UBA ubiquitin receptor proteins bind a common tetraubiquitin chain

The ubiquitin-proteasome pathway is essential throughout the life cycle of a cell. This system employs an astounding number of proteins to ubiquitylate and to deliver protein substrates to the proteasome for their degradation. At the heart of this process is the large and growing family of ubiquitin receptor proteins. Within this family is an intensely studied group that contains both ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains: Rad23, Ddi1 and Dsk2. Although UBL/UBA family members are reported to regulate the degradation of other proteins, their individual roles in ubiquitin-mediated protein degradation has proven difficult to resolve due to their overlapping functional roles and interaction with each other and other ubiquitin family members. Here, we use a combination of NMR spectroscopy and molecular biology to reveal that Rad23 and Ddi1 interact with each other by using UBL/UBA domain interactions in a manner that does not preclude their interaction with ubiquitin. We demonstrate that UBL/UBA proteins can bind a common tetraubiquitin molecule and thereby provide strong evidence for a model in which chains adopt an opened structure to bind multiple receptor proteins. Altogether our results suggest a mechanism through which UBL/UBA proteins could protect chains from premature de-ubiquitylation and unnecessary elongation during their transit to the proteasome.     

Ubiquitin-interacting Motifs Confer Full Catalytic Activity,but Not Ubiquitin Chain Substrate Specificity,to Deubiquitinating Enzyme USP37

Ubiquitin-specific proteases (USPs) consist of a family of deubiquitinating enzymes with more than 50 members in humans. Three of them, including USP37, contain ubiquitin-interacting motifs (UIMs), an ∼20-amino acid α-helical stretch that binds to ubiquitin. However, the roles of the UIMs in these USP enzymes remain unknown. USP37 has three UIMs, designated here as UIMs 1, 2, and 3 from the N-terminal side, between the Cys and His boxes comprising the catalytic core. Here, we examined the role of the UIMs in USP37 using its mutants that harbor mutations in the UIMs. The nuclear localization of USP37 was not affected by the UIM mutations. However, mutations in UIM2 or UIM3, but not UIM1, resulted in a significant decrease in USP37 binding to ubiquitinated proteins in the cell. In vitro, a region of USP37 harboring the three UIMs also bound to both Lys⁴⁸-linked and Lys⁶³-linked ubiquitin chains in a UIM2- and UIM3-dependent manner. The level of USP37 ubiquitination was also reduced by mutations in UIM2 or UIM3, suggesting their role in ubiquitination of USP37 itself. Finally, mutants lacking functional UIM2 or UIM3 exhibited a reduced isopeptidase activity toward ubiquitinated proteins in the cell and both Lys⁴⁸-linked and Lys⁶³-linked ubiquitin chains. These results suggested that the UIMs in USP37 contribute to the full enzymatic activity, but not ubiquitin chain substrate specificity, of USP37 possibly by holding the ubiquitin chain substrate in the proximity of the catalytic core.     

Differential ubiquitin binding of the UBA domains from human c-Cbl and Cbl-b: NMR structural and biochemical insights

The Cbl proteins, RING-type E3 ubiquitin ligases, are responsible for ubiquitinating the activated tyrosine kinases and targeting them for degradation. Both c-Cbl and Cbl-b have a UBA (ubiquitin-associated) domain at their C-terminal ends, and these two UBA domains share a high sequence similarity (75%). However, only the UBA from Cbl-b, but not from c-Cbl, can bind ubiquitin (Ub). To understand the mechanism by which the UBA domains specifically interact with Ub with different affinities, we determined the solution NMR structures of these two UBA domains, cUBA from human c-Cbl and UBAb from Cbl-b. Their structures show that these two UBA domains share the same fold, a compact three-helix bundle, highly resembling the typical UBA fold. Chemical shift perturbation experiments reveal that the helix-1 and loop-1 of UBAb form a predominately hydrophobic surface for Ub binding. By comparing the Ub-interacting surface on UBAb and its counterpart on cUBA, we find that the hydrophobic patch on cUBA is interrupted by a negatively charged residue Glu12. Fluorescence titration data show that the Ala12Glu mutant of UBAb completely loses the ability to bind Ub, whereas the mutation disrupting the dimerization has no significant effect on Ub binding. This study provides structural and biochemical insights into the Ub binding specificities of the Cbl UBA domains, in which the hydrophobic surface distribution on the first helix plays crucial roles in their differential affinities for Ub binding. That is, the amino acid residue diversity in the helix-1 region, but not the dimerization, determines the abilities of various UBA domains binding with Ub.     

The E2-25K ubiquitin-associated (UBA) domain aids in polyubiquitin chain synthesis and linkage specificity

E2-25K is an ubiquitin-conjugating enzyme with the ability to synthesize Lys48-linked polyubiquitin chains. E2-25K and its homologs represent the only known E2 enzymes which contain a C-terminal ubiquitin-associated (UBA) domain as well as the conserved catalytic ubiquitin-conjugating (UBC) domain. As an additional non-covalent binding surface for ubiquitin, the UBA domain must provide some functional specialization. We mapped the protein–protein interface involved in the E2-25K UBA/ubiquitin complex by solution nuclear magnetic resonance (NMR) spectroscopy and subsequently modeled the structure of the complex. Domain–domain interactions between the E2-25K catalytic UBC domain and the UBA domain do not induce significant structural changes in the UBA domain or alter the affinity of the UBA domain for ubiquitin. We determined that one of the roles of the C-terminal UBA domain, in the context of E2-25K, is to increase processivity in Lys48-linked polyubiquitin chain synthesis, possibly through increased binding to the ubiquitinated substrate. Additionally, we see evidence that the UBA domain directs specificity in polyubiquitin chain linkage.     

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     

Recruitment of Ubiquitin within an E2 Chain Elongation Complex

The ubiquitin (Ub) proteolysis pathway uses an E1, E2, and E3 enzyme cascade to label substrate proteins with ubiquitin and target them for degradation. The mechanisms of ubiquitin chain formation remain unclear and include a sequential addition model, in which polyubiquitin chains are built unit by unit on the substrate, or a preassembly model, in which polyubiquitin chains are preformed on the E2 or E3 enzyme and then transferred in one step to the substrate. The E2 conjugating enzyme UBE2K has a 150-residue catalytic core domain and a C-terminal ubiquitin-associated (UBA) domain. Polyubiquitin chains anchored to the catalytic cysteine and free in solution are formed by UBE2K supporting a preassembly model. To study how UBE2K might assemble polyubiquitin chains, we synthesized UBE2K-Ub and UBE2K-Ub₂ covalent complexes and analyzed E2 interactions with the covalently attached Ub and Ub₂ moieties using NMR spectroscopy. The UBE2K-Ub complex exists in multiple conformations, including the catalytically competent closed state independent of the UBA domain. In contrast, the UBE2K-Ub₂ complex takes on a more extended conformation directed by interactions between the classic I44 hydrophobic face of the distal Ub and the conserved MGF hydrophobic patch of the UBA domain. Our results indicate there are distinct differences between the UBE2K-Ub and UBE2K-Ub₂ complexes and show how the UBA domain can alter the position of a polyubiquitin chain attached to the UBE2K active site. These observations provide structural insights into the unique Ub chain-building capacity for UBE2K.     

Ubiquitin manipulation by an E2 conjugating enzyme using a novel covalent intermediate

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.     

Isolation and Characterization of Ubiquitin-activating Enzyme E1-domain Containing 1, UBE1DC1

Ubiquitin and other ubiquitin-like proteins play important roles in post-translational modification. They are phylogenetically well-conserved in eukaryotes. Activated by other proteins, ubiquitin and ubiquitin-like proteins can covalently modify target proteins. The enzymes responsible for the activation of this modification have been known to include UBA1, SAE2, UBA3, SAE1 and ULA1. Here we report a new ubiquitin activating enzyme like cDNA, named ubiquitin activating enzyme E1-domain containing 1 (UBE1DC1), whose cDNA is 2654 base pairs in length and contains an open reading frame encoding 404 amino acids. The UBE1DC1 gene consists of 12 exons and is located at human chromosome 3q22. The result of RT-PCR showed that UBE1DC1 is expressed in most of human tissues. These two authors contributed equally to this paper. The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY253672.     

A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between terbium and yellow fluorescent protein

Deubiquitinating enzymes (DUBs) proteolytically cleave ubiquitin from ubiquitinated proteins, and inhibition of DUBs that rescue oncogenic proteins from proteasomal degradation is of emerging therapeutic interest. Recently, USP2 and UCH37 have been shown to deubiquitinate tumor-growth-promoting proteins, and other DUBs have been shown to be overexpressed in cancer cells. Therefore inhibition of DUBs is of interest as a potential therapeutic strategy for treating cancer. DUBs require the presence of properly folded ubiquitin protein in the substrate for efficient proteolysis, which precludes the use of synthetic peptide substrates in DUB activity assays. Because of the requirement for full-length ubiquitin, substrates suitable for use in fluorescent assays to identify or study DUB inhibitors have been difficult to prepare. We describe the development of a time-resolved fluorescence resonance energy transfer (FRET)-based DUB substrate that incorporates full-length ubiquitin that is site-specifically labeled using genetically encoded yellow fluorescent protein (YFP) and a chemically attached terbium donor. The intact substrate shows a high degree of FRET between terbium and YFP, whereas DUB-dependent cleavage leads to a decrease in FRET.     

Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in Escherichia coli

The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by ubiquitin activating enzyme, E1. Previously, we purified and characterized multiple species of E1 from wheat germ. We now describe the isolation and characterization of a cDNA clone encoding E1 from wheat. This clone (UBA1) was isolated from a cDNA expression library with anti-wheat E1 antibodies. It contained an open reading frame coding for 1051 amino acids and directed the synthesis of a protein that comigrated with a wheat germ E1 of 117 kDa. UBA1 was confirmed as encoding E1 by (i) comparison of the peptide map of the protein product of UBA1 synthesized in Escherichia coli with that of purified E1 from wheat, and (ii) amino acid sequence identity of peptides generated from purified E1 with regions of the derived amino acid sequence of UBA1. The isolation of two additional cDNAs closely related to UBA1 indicated that E1 was encoded by a small gene family in wheat. Nonetheless, a single poly(A+) mRNA size class of 4 kilobases hybridized with UBA1. When expressed in E. coli, the product of UBA1 catalyzed the formation of a thiol ester linkage between ubiquitin and an ubiquitin carrier protein. The ability of E. coli containing UBA1 to synthesize an active protein will allow us to identify domains important for E1 function using in vitro mutagenesis.     

The UBA-UIM Domains of the USP25 Regulate the Enzyme Ubiquitination State and Modulate Substrate Recognition

USP25m is the muscle isoform of the deubiquitinating (DUB) enzyme USP25. Similarly to most DUBs, data on USP25 regulation and substrate recognition is scarce. In silico analysis predicted three ubiquitin binding domains (UBDs) at the N-terminus: one ubiquitin-associated domain (UBA) and two ubiquitin-interacting motifs (UIMs), whereas no clear structural homology at the extended C-terminal region outside the catalytic domains were detected. In order to asses the contribution of the UBDs and the C-terminus to the regulation of USP25m catalytic activity, ubiquitination state and substrate interaction, serial and combinatorial deletions were generated. Our results showed that USP25m catalytic activity did not strictly depend on the UBDs, but required a coiled-coil stretch between amino acids 679 to 769. USP25 oligomerized but this interaction did not require either the UBDs or the C-terminus. Besides, USP25 was monoubiquitinated and able to autodeubiquitinate in a possible loop of autoregulation. UBDs favored the monoubiquitination of USP25m at the preferential site lysine 99 (K99). This residue had been previously shown to be a target for SUMO and this modification inhibited USP25 activity. We showed that mutation of K99 clearly diminished USP25-dependent rescue of the specific substrate MyBPC1 from proteasome degradation, thereby supporting a new mechanistic model, in which USP25m is regulated through alternative conjugation of ubiquitin (activating) or SUMO (inhibiting) to the same lysine residue (K99), which may promote the interaction with distinct intramolecular regulatory domains.     

Structural and functional studies of USP20 ZnF‐UBP domain by NMR

Deubiquitinase USP20/VDU2 has been demonstrated to play important roles in multiple cellular processes by controlling the life span of substrate proteins including hypoxia‐inducible factor HIF1α, and so forth. USP20 contains four distinct structural domains including the N‐terminal zinc‐finger ubiquitin binding domain (ZnF‐UBP), the catalytic domain (USP domain), and two tandem DUSP domains, and none of the structures for these four domains has been solved. Meanwhile, except for the ZnF‐UBP domain, the biological functions for USP20's catalytic domain and tandem DUSP domains have been at least partially clarified. Here in this study, we determined the solution structure of USP20 ZnF‐UBP domain and investigated its binding properties with mono‐ubiquitin and poly‐ubiquitin (K48‐linked di‐ubiquitin) by using NMR and molecular modeling techniques. USP20's ZnF‐UBP domain forms a spherically shaped fold consisting of a central β‐sheet with either one α‐helix or two α‐helices packed on each side of the sheet. However, although having formed a canonical core structure essential for ubiquitin recognition, USP20 ZnF‐UBP presents weak ubiquitin binding capacity. The structural basis for understanding USP20 ZnF‐UBP's ubiquitin binding capacity was revealed by NMR data‐driven docking. Although the electrostatic interactions between D264 of USP5 (E87 in USP20 ZnF‐UBP) and R74 of ubiquitin are kept, the loss of the extensive interactions formed between ubiquitin's di‐glycine motif and the conserved and non‐conserved residues of USP20 ZnF‐UBP domain (W41, E55, and Y84) causes a significant decrease in its binding affinity to ubiquitin. Our findings indicate that USP20 ZnF‐UBP domain might have a physiological role unrelated to its ubiquitin binding capacity.     

Solution structure of the flexible class II ubiquitin-conjugating enzyme Ubc1 provides insights for polyubiquitin chain assembly

E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation. While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation. Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae. The structure shows the N-terminal catalytic domain adopts an alpha/beta fold typical of other E2 proteins. This domain is physically separated from its C-terminal domain by a 22-residue flexible tether. The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA). NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin. This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation.     

Lysine 92 amino acid residue of USP46, a gene associated with 'behavioral despair' in mice, influences the deubiquitinating enzyme activity

Deubiquitinating enzymes (DUBs) regulate diverse cellular functions by their activity of cleaving ubiquitin from specific protein substrates. Ubiquitin-Specific Protease 46 (USP46) has recently been identified as a quantitative trait gene responsible for immobility in the tail suspension test and forced swimming test in mice. Mice with a lysine codon (Lys 92) deletion in USP46 exhibited loss of 'behavioral despair' under inescapable stresses in addition to abnormalities in circadian behavioral rhythms and the GABAergic system. However, whether this deletion affects enzyme activity is unknown. Here we show that USP46 has deubiquitinating enzyme activity detected by USP cleavage assay using GST-Ub52 as a model substrate. Interestingly, compared to wild type, the Lys 92 deletion mutant resulted in a decreased deubiquitinating enzyme activity of 27.04%. We also determined the relative expression levels of Usp46 in rat tissues using real-time RT-PCR. Usp46 mRNA was expressed in various tissues examined including brain, with the highest expression in spleen. In addition, like rat USP46, both human and mouse USP46 are active toward to the model substrate, indicating the USP cleavage assay is a simple method for testing the deubiquitinating enzyme activity of USP46. These results suggest that the Lys 92 deletion of USP46 could influence enzyme activity and thereby provide a molecular clue how the enzyme regulating the pathogenesis of mental illnesses.     

Crystal structure of the ubiquitin-associated (UBA) domain of p62 and its interaction with ubiquitin

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.     

Identification of a novel isopeptidase with dual specificity for ubiquitin- and NEDD8-conjugated proteins

Covalent conjugation of proteins by ubiquitin or ubiquitin-like molecules is an important form of post-translational modification and plays a critical role in many cellular processes. Similar to the concept of phosphorylation and dephosphorylation, these conjugates are regulated by a large number of deconjugating enzymes. Here, we report the cloning of a 2,141-base pair DNA fragment from human placenta cDNA library by a strategy that involves expressed sequence tag data base searching, polymerase chain reaction, and rapid amplification of cDNA ends. Nucleotide sequence analysis revealed that the cloned cDNA contains an open reading frame of 1,143 base pairs encoding a novel protease, USP21, which is composed of 381 residues with a calculated molecular mass of 43 kDa. The human USP21 gene is located on chromosome 1q21 and encodes a member of the ubiquitin-specific protease family with highly conserved Cys and His domains. The activity and specificity of USP21 were determined by using a COS cell expression system in vivo. We showed that USP21 is capable of removing ubiquitin from ubiquitinated proteins as expected. Furthermore, USP21 is capable of removing NEDD8 from NEDD8 conjugates but has no effect on Sentrin-1 conjugates. As expected from its biochemical activity, overexpression of USP21 has a profound growth inhibitory effect on U2OS cells. Thus, USP21 is the first ubiquitin-specific protease shown to have dual specificity for both ubiquitin and NEDD8 and may play an important role in the regulation of cell growth.     

Multiple interactions of rad23 suggest a mechanism for ubiquitylated substrate delivery important in proteolysis

The mechanism underlying the delivery of ubiquitylated substrates to the proteasome is poorly understood. Rad23 is a putative adaptor molecule for this process because it interacts with ubiquitin chains through its ubiquitin-associated motifs (UBA) and with the proteasome through a ubiquitin-like element (UBL). Here, we demonstrate that the UBL motif of Rad23 also binds Ufd2, an E4 enzyme essential for ubiquitin chain assembly onto its substrates. Mutations in the UBL of Rad23 alter its interactions with Ufd2 and the proteasome, and impair its function in the UFD proteolytic pathway. Furthermore, Ufd2 and the proteasome subunit Rpn1 compete for the binding of Rad23, suggesting that Rad23 forms separate complexes with them. Importantly, we also find that the ability of other UBL/UBA proteins to associate with Ufd2 correlates with their differential involvement in the UFD pathway, suggesting that UBL-mediated interactions may contribute to the substrate specificity of these adaptors. We propose that the UBL motif, a protein-protein interaction module, may be used to facilitate coupling between substrate ubiquitylation and delivery, and to ensure the orderly handoff of the substrate from the ubiquitylation machinery to the proteasome.