1H, 13C and 15N backbone and side-chain resonance assignments of the N-terminal ubiquitin-binding domains of USP25

Ubiquitin Specific Protease 25 (USP25), a member of the deubiquitinase family, is involved in several disease-related signal pathways including myogenesis, immunity and protein degradation. It specially catalyzes the hydrolysis of the K48-linked and K63-linked polyubiquitin chains. USP25 contains one ubiquitin-associated domain and two ubiquitin-interacting motifs (UIMs) in its N-terminal region, which interact with ubiquitin and play a role in substrate recognition. Besides, it has been shown that the catalysis activity of USP25 is either impaired by sumoylation or enhanced by ubiquitination within its UIM. To elucidate the structural basis of the cross-regulation of USP25 function by non-covalent binding and covalent modifications of ubiquitin and SUMO2/3, a systematic structural biology study of USP25 is required. Here, we report the ¹H, ¹³C and ¹⁵N backbone and side-chain resonance assignments of the N-terminal ubiquitin binding domains (UBDs) of USP25 with BMRB accession number of 19111, which is the first step of the systematic structural biology study of the enzyme.     

Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25

Vertebrates express two distinct families of SUMO proteins (SUMO1 and SUMO2/3) that serve distinct functions as posttranslational modifiers. Many proteins are modified specifically with SUMO1 or SUMO2/3, but the mechanisms for paralog selectivity are poorly understood. In a screen for SUMO2/3 binding proteins, we identified Ubiquitin Specific Protease 25 (USP25). USP25 turned out to also be a target for sumoylation, being more efficient with SUMO2/3. Sumoylation takes place within USP25's two ubiquitin interaction motifs (UIMs) that are required for efficient hydrolysis of ubiquitin chains. USP25 sumoylation impairs binding to and hydrolysis of ubiquitin chains. Both SUMO2/3-specific binding and sumoylation depend on a SUMO interaction motif (SIM/SBM). Seven amino acids in the SIM of USP25 are sufficient for SUMO2/3-specific binding and conjugation, even when taken out of structural context. One mechanism for paralog-specific sumoylation may, thus, involve SIM-dependent recruitment of SUMO1 or SUMO2/3 thioester-charged Ubc9 to targets.     

Regulation of USP28 Deubiquitinating Activity by SUMO Conjugation

USP28 (ubiquitin-specific protease 28) is a deubiquitinating enzyme that has been implicated in the DNA damage response, the regulation of Myc signaling, and cancer progression. The half-life stability of major regulators of critical cellular pathways depends on the activities of specific ubiquitin E3 ligases that target them for proteosomal degradation and deubiquitinating enzymes that promote their stabilization. One function of the post-translational small ubiquitin modifier (SUMO) is the regulation of enzymatic activity of protein targets. In this work, we demonstrate that the SUMO modification of the N-terminal domain of USP28 negatively regulates its deubiquitinating activity, revealing a role for the N-terminal region as a regulatory module in the control of USP28 activity. Despite the presence of ubiquitin-binding domains in the N-terminal domain, its truncation does not impair deubiquitinating activity on diubiquitin or polyubiquitin chain substrates. In contrast to other characterized USP deubiquitinases, our results indicate that USP28 has a chain preference activity for Lys¹¹, Lys⁴⁸, and Lys⁶³ diubiquitin linkages.     

Tat-SIRT1 tango

In this issue of Molecular Cell, Meulmeester et al. (2008) identify USP25 as a SUMO2/3-interacting protein and substrate. A USP25 SUMO interaction motif directs SUMO2/3 specificity, and SUMO modification diminishes USP25's ability to bind and degrade polyubiquitin chains.     

Interaction of the tail with the catalytic region of a class II E2 conjugating enzyme

Ubiquitination plays an important role in many biological processes, including DNA repair, cell cycle regulation, and protein degradation. In the latter pathway the ubiquitin-conjugating enzymes or E2 enzymes are important proteins forming a key E2-ubiquitin thiolester prior to substrate labelling. While the structure of the 150-residue catalytic domain has been well characterized, a subset of E2 enzymes (class II) carry a variable length C-terminal `tail' where structural detail is not available. The presence of this C-terminal extension plays an important role in target recognition, ubiquitin chain assembly and oligomerization. In this work NMR spectroscopy was used to determine the secondary structure of the 215-residue yeast E2 protein Ubc1 and the interactions of its C-terminus with the catalytic domain. The C-terminal tail of Ubc1 was found to contain three -helices between residues D169-S176, K183-L193 and N203-L213 providing the first evidence for a well-defined secondary structure in this region. Chemical shift mapping indicated that residues in the L2 loop of the catalytic domain were most affected indicating the C-terminus of Ubc1 likely interacts with this region. This site of interaction is distinct from that observed in the E2-ubiquitin thiolester and may act to protect the catalytic C88 residue and direct the interaction of ubiquitin in the thiolester intermediate.     

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.     

Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein-protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain-HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.     

Analysis of the domain structure of elongation factor-2 kinase by mutagenesis.

A number of elongation factor-2 kinase (eEF-2K) mutants were constructed to investigate features of this kinase that may be important in its activity. Typical protein kinases possess a highly conserved lysine residue in subdomain II which follows the GXGXXG motif of subdomain I. Mutation of two lysine residues, K340 and K346, which follow the GXGXXG motif in eEF-2K had no effect on activity, showing that such a lysine residue is not important in eEF-2K activity. Mutation of a conserved pair of cysteine residues C-terminal to the GXGXXG sequence, however, completely inactivated eEF-2K. The eEF-2K CaM binding domain was localised to residues 77-99 which reside N-terminal to the catalytic domain. Tryptophan 84 is an important residue within this domain as mutation of this residue completely abolishes CaM binding and eEF-2K activity. Removal of approximately 130 residues from the C-terminus of eEF-2K completely abolished autokinase activity; however, removal of only 19 residues inhibited eEF-2 kinase activity but not autokinase activity, suggesting that a short region at the C-terminal end may be important in interacting with eEF-2. Likewise, removal of between 75 and 100 residues from the N-terminal end completely abolished eEF-2K activity.     

Biochemical characterization of USP7 reveals post-translational modification sites and structural requirements for substrate processing and subcellular localization

Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinating enzymes. Among other functions, USP7 is involved in the regulation of stress response pathways, epigenetic silencing and the progress of infections by DNA viruses. USP7 is a 130-kDa protein with a cysteine peptidase core, N- and C-terminal domains required for protein-protein interactions. In the present study, recombinant USP7 full length, along with several variants corresponding to domain deletions, were expressed in different hosts in order to analyze post-translational modifications, oligomerization state, enzymatic properties and subcellular localization patterns of the enzyme. USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in mammalian cells. In in vitro activity assays, N- and C-terminal truncations affected the catalytic efficiency of the enzyme different. Both the protease core alone and in combination with the N-terminal domain are over 100-fold less active than the full length enzyme, whereas a construct including the C-terminal region displays a rather small decrease in catalytic efficiency. Limited proteolysis experiments revealed that USP7 variants containing the C-terminal domain interact more tightly with ubiquitin. Besides playing an important role in substrate recognition and processing, this region might be involved in enzyme dimerization. USP7 constructs lacking the N-terminal domain failed to localize in the cell nucleus, but no nuclear localization signal could be mapped within the enzyme's first 70 amino acids. Instead, the tumor necrosis factor receptor associated factor-like region (amino acids 70-205) was sufficient to achieve the nuclear localization of the enzyme, suggesting that interaction partners might be required for USP7 nuclear import.     

Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin

The interaction between ubiquitinated proteins and intracellular proteins harboring ubiquitin binding domains (UBDs) is critical to a multitude of cellular processes. Here, we report that Rabex-5, a guanine nucleotide exchange factor for Rab5, binds to Ub through two independent UBDs. These UBDs determine a number of properties of Rabex-5, including its coupled monoubiquitination and interaction in vivo with ubiquitinated EGFRs. Structural and biochemical characterization of the UBDs of Rabex-5 revealed that one of them (MIU, motif interacting with ubiquitin) binds to Ub with modes superimposable to those of the UIM (ubiquitin-interacting motif):Ub interaction, although in the opposite orientation. The other UBD, RUZ (Rabex-5 ubiquitin binding zinc finger) binds to a surface of Ub centered on Asp58(Ub) and distinct from the "canonical" Ile44(Ub)-based surface. The two binding surfaces allow Ub to interact simultaneously with different UBDs, thus opening new perspectives in Ub-mediated signaling.     

Ubiquitin-specific protease 4 is inhibited by its ubiquitin-like domain

USP4 is a member of the ubiquitin-specific protease (USP) family of deubiquitinating enzymes that has a role in spliceosome regulation. Here, we show that the crystal structure of the minimal catalytic domain of USP4 has the conserved USP-like fold with its typical ubiquitin-binding site. A ubiquitin-like (Ubl) domain inserted into the catalytic domain has autoregulatory function. This Ubl domain can bind to the catalytic domain and compete with the ubiquitin substrate, partially inhibiting USP4 activity against different substrates. Interestingly, other USPs, such as USP39, could relieve this inhibition.     

The role of UBL domains in ubiquitin-specific proteases

Ubiquitin conjugation and deconjugation provides a powerful signalling system to change the fate of its target enzymes. Ubiquitination levels are organized through a balance between ubiquitinating E1, E2 and E3 enzymes and deubiquitination by DUBs (deubiquitinating enzymes). These enzymes are tightly regulated to control their activity. In the present article, we discuss the different ways in which DUBs of the USP (ubiquitin-specific protease) family are regulated by internal domains with a UBL (ubiquitin-like) fold. The UBL domain in USP14 is important for its localization at the proteasome, which enhances catalysis. In contrast, a UBL domain in USP4 binds to the catalytic domain and competes with ubiquitin binding. In this process, the UBL domain mimics ubiquitin and partially inhibits catalysis. In USP7, there are five consecutive UBL domains, of which the last two affect catalytic activity. Surprisingly, they do not act like ubiquitin and activate catalysis rather than inhibiting it. These C-terminal UBL domains promote a conformational change that allows ubiquitin binding and organizes the catalytic centre. Thus it seems that UBL domains have different functions in different USPs. Other proteins can modulate the roles of UBL domains in USP4 and USP7. On one hand, the inhibition of USP4 can be relieved when the UBL is sequestered by another USP. On the other, the activation of USP7 is increased, when the UBL-activated state is stabilized by allosteric binding of GMP synthetase. Altogether, UBL domains appear to be able to regulate catalytic activity in USPs, but they can use widely different mechanisms of action, in which they may, as in USP4, or may not, as in USP7, use the direct resemblance to ubiquitin.     

SUMO modification of the ubiquitin-conjugating enzyme E2-25K

Post-translational modification with small ubiquitin-related modifier (SUMO) alters the function of many proteins, but the molecular mechanisms and consequences of this modification are still poorly defined. During a screen for novel SUMO1 targets, we identified the ubiquitin-conjugating enzyme E2-25K (Hip2). SUMO attachment severely impairs E2-25K ubiquitin thioester and unanchored ubiquitin chain formation in vitro. Crystal structures of E2-25K(1-155) and of the E2-25K(1-155)-SUMO conjugate (E2-25K(*)SUMO) indicate that SUMO attachment interferes with E1 interaction through its location on the N-terminal helix. The SUMO acceptor site in E2-25K, Lys14, does not conform to the consensus site found in most SUMO targets (PsiKXE), and functions only in the context of an alpha-helix. In contrast, adjacent SUMO consensus sites are modified only when in unstructured peptides. The demonstration that secondary structure elements are part of SUMO attachment signals could contribute to a better prediction of SUMO targets.     

USP13 enzyme regulates Siah2 ligase stability and activity via noncatalytic ubiquitin-binding domains

The RING finger E3 ubiquitin ligase Siah2 is implicated in control of diverse cellular biological events, including MAPK signaling and hypoxia. Here we demonstrate that Siah2 is subject to regulation by the deubiquitinating enzyme USP13. Overexpression of USP13 increases Siah2 stability by attenuating its autodegradation. Consequently, the ability of Siah2 to target its substrates prolyl hydroxylase 3 and Spry2 (Sprouty2) for ubiquitin-mediated proteasomal degradation is attenuated. Conversely, inhibition of USP13 expression with corresponding shRNA decreases the stability of both Siah2 and its substrate Spry2. Thus, USP13 limits Siah2 autodegradation and its ubiquitin ligase activity against its target substrates. Strikingly, the effect of USP13 on Siah2 is not mediated by its isopeptidase activity: mutations in its ubiquitin-binding sequences positioned within the ubiquitin-specific processing protease and ubiquitin-binding domains, but not within putative catalytic sites, abolish USP13 binding to and effect on Siah2 autodegradation and targeted ubiquitination. Notably, USP13 expression is attenuated in melanoma cells maintained under hypoxia, thereby relieving Siah2 inhibition and increasing its activity under low oxygen levels. Significantly, on melanoma tissue microarray, high nuclear expression of USP13 coincided with high nuclear expression of Siah2. Overall, this study identifies a new layer of Siah2 regulation mediated by USP13 binding to ubiquitinated Siah2 protein with a concomitant inhibitory effect on its activity under normoxia.     

A noncanonical cysteine protease USP1 is activated through active site modulation by USP1-associated factor 1

Ubiquitin-specific proteases (USPs) constitute the largest family of the human deubiquitinating enzymes. USP1 belongs to the cysteine protease family and contains a catalytic triad comprised of C90, H593, and D751. Notably, the catalytic activity of USP1 is stimulated through the formation of a tight complex with a WD40 repeat protein UAF1 (USP1-associated factor 1). Our kinetic analyses revealed a general base catalysis in USP1/UAF1, in contrast to an ion-pair mechanism as demonstrated for papain and cathepsin. The pK(a) value of the catalytic cysteine was determined to be 8.67 ± 0.07 in a pH-dependent inactivation study of USP1/UAF1 by iodoacetamide. A normal solvent kinetic isotope effect of 2.8 for k(cat) and 3.0 for k(cat)/K(m) was observed in the USP1/UAF1-catalyzed hydrolysis of ubiquitin-AMC substrate. Moreover, proton inventory analysis supported the transfer of a single solvent-derived proton in the transition state. Our study also revealed the molecular basis for the activation of USP1 by UAF1. Although the pK(a) of the catalytic cysteine in USP1 and USP1/UAF1 was almost identical, the pK(a) of the catalytic histidine in USP1/UAF1 was 0.43 pH unit lower than that in USP1, which facilitates general base catalysis at a neutral pH and contributes to the elevated catalytic efficiency. We ruled out that the higher catalytic efficiency is due to a tighter binding of ubiquitin. Our results support a regulatory mechanism in which UAF1 activates USP1 by modulating its active site conformation. This finding has a general implication for the regulation of USPs that form complex with partner proteins.     

SUMO protein modification

SUMO (small ubiquitin-related modifier) family proteins are not only structurally but also mechanistically related to ubiquitin in that they are posttranslationally attached to other proteins. As ubiquitin, SUMO is covalently linked to its substrates via amide (isopeptide) bonds formed between its C-terminal glycine residue and the epsilon-amino group of internal lysine residues. The enzymes involved in the reversible conjugation of SUMO are similar to those mediating the ubiquitin conjugation. Since its discovery in 1996, SUMO has received a high degree of attention because of its intriguing and essential functions, and because its substrates include a variety of biomedically important proteins such as tumor suppressor p53, c-jun, PML and huntingtin. SUMO modification appears to play important roles in diverse processes such as chromosome segregation and cell division, DNA replication and repair, nuclear protein import, protein targeting to and formation of certain subnuclear structures, and the regulation of a variety of processes including the inflammatory response in mammals and the regulation of flowering time in plants.