A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes

Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub(F45W)-Xaa) and di-ubiquitin chains (Ub(F45W)-diUb). After removal of the intact substrate by Ni(2+)-NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub(F45W) product. The results show that three UCHs, i.e. UCH-L1, UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs.     

Ubiquitin metabolism in HeLa cells starved of amino acids.

Radio-iodinated ubiquitin (Ub) was introduced into HeLa cells by red blood cell-mediated microinjection. The half-life and solubility of Ub, as well as the molecular weight distributions of Ub conjugates, were then measured in HeLa cells grown in complete medium or in medium lacking amino acids and fetal calf serum. Ub metabolism was similar in the two sets of cells. Thus, the dramatic changes in Ub metabolism induced by thermal stress are not observed upon amino acid deprivation.     

Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation

A series of nonhydrolyzable ubiquitin dimer analogues has been synthesized and evaluated as inhibitors of ubiquitin-dependent processes. Dimer analogues were synthesized by cross-linking ubiquitin containing a terminal cysteine (G76C) to ubiquitin containing cysteine at position 11 ((76-11)Ub(2)), 29 ((76-29)Ub(2)), 48 ((76-48)Ub(2)), or 63 ((76-63)Ub(2)). A head-to-head dimer of cysteine G76C ((76-76)Ub(2)) served as a control. These analogues are mimics of the different chain linkages observed in natural polyubiquitin chains. All analogues showed weak inhibition toward the catalytic domain of UCH-L3 and a UBP pseudogene. In the absence of ubiquitin, isopeptidase T was inhibited only by the dimer linked through residue 29. In the presence of 0.5 microM ubiquitin, isopeptidase T was inhibited by several of the dimer analogues, with the (76-29)Ub(2) dimer exhibiting a K(i) of 1.8 nM. However, USP14, the human homologue of yeast Ubp6, was not inhibited at the concentrations tested. Some analogues of ubiquitin dimer also acted as selective inhibitors of conjugation and deconjugation of ubiquitin catalyzed by reticulocyte fraction II. (76-76)Ub(2) and (76-11)Ub(2) did not inhibit the conjugation of ubiquitin, while (76-29)Ub(2), (76-48)Ub(2), and (76-63)Ub(2) were potent inhibitors of conjugation. This specificity is consistent with the known ability of cells to form K29-, K48-, and K63-linked polyubiquitin chains. While (76-11)Ub(2), (76-29)Ub(2), and (76-63)Ub(2) inhibited release of ubiquitin from a pool of total conjugates, (76-48)Ub(2) and (76-76)Ub(2) showed no significant inhibition. Isopeptidase T was shown to specifically disassemble two conjugates (assumed to be di- and triubiquitin with masses of 26 and 17 kDa) formed in the reticulocyte lysate system. This activity was inhibited differentially by all dimer analogues. The inhibitor selectivity for deconjugation of the 26 and 17 kDa conjugates was similar to that observed for isopeptidase T. The observations suggest that these two conjugated proteins of the reticulocyte lysate are specific substrates for isopeptidase T in lysates.     

Structural properties of polyubiquitin chains in solution

Because polyubiquitin chain structure modulates Ub-mediated signaling, knowledge of the physiological conformations of chain signals should provide insights into specific recognition. Here, we characterized the solution conformations of K48-linked Ub(2) and Ub(4) using a combination of NMR techniques, including chemical shift mapping of the interdomain interface, domain orientation measurements on the basis of 15N relaxation and residual dipolar couplings, and the solvent accessibility studies. Our data indicate a switch in the conformation of Ub(2), from open to closed, with increasing pH. The closed conformation features a well-defined interface that is related to, but distinguishable from, that observed in the Ub(2) crystal structure. This interface is dynamic in solution, such that important hydrophobic residues (L8, I44, V70) that are sequestered at the interface in the closed conformation may be accessible for direct interactions with recognition factors. Our results suggest that the distal two units of Ub(4), which is the minimum signal for efficient proteasomal degradation, may adopt the closed Ub(2) conformation.     

Ubiquitin-Mediated Stress Response in a Rat Model of Brain Transient Ischemia/Hypoxia

Ubiquitin (Ub) is a small 76-residue protein, involved in intracellular protein degradation through a specific ATP-dependent system, which uses Ub as a tag to label proteins committed to be hydrolyzed by a specific 26 S protease. PGP-9.5 is another important component of the Ub system, i.e. a neuron-specific carboxyl-terminal hydrolase, which recycles Ub from Ub-polypeptide complexes. We have investigated the expression of Ub and PGP-9.5 in rat hippocampal neurons in an early phase of reperfusion in a model of transient global brain ischemia/hypoxia (bilateral occlusion of common carotid arteries for 10 min accompanied by mild hypoxia—15% O₂—for 20 min), by means of immunohistochemical methods using light and electron microscopy. The intensity of Ub and PGP-9.5 immunoreactivity was evaluated by image analysis. We have detected a marked increase of Ub immunoreactivity (UIR) in neurons of CA1, CA2, CA3, CA4, and dentate gyrus subfields 1 hr after ischemia/hypoxia (but not after hypoxia only), statistically significant as confirmed by image analysis. Such increase in immunoreactivity in ischemic/hypoxic rats was localized essentially in the nuclei of hippocampal neurons. There were no changes in PGP-9.5 immunoreactivity. The data suggest that in the present model of rat brain ischemia/hypoxia Ub is involved in the neuronal stress response.     

Detection and characterization of the in vitro e3 ligase activity of the human MID1 protein

Human MID1 (midline-1) is a microtubule-associated protein that is postulated to target the catalytic subunit of protein phosphatase 2A for degradation. It binds alpha4 that then recruits the catalytic subunit of protein phosphatase 2A. As a member of the TRIM (tripartite motif) family, MID1 has three consecutive zinc-binding domains—RING (really interesting new gene), Bbox1, and Bbox2—that have similar ββα-folds. Here, we describe the in vitro characterization of these domains individually and in tandem. We observed that the RING domain exhibited greater ubiquitin (Ub) E3 ligase activity compared to the Bbox domains. The amount of autopolyubiquitinated products with RING-Bbox1 and RING-Bbox1-Bbox2 domains in tandem was significantly greater than those of the individual domains. However, no polyubiquitinated products were observed for the Bbox1-Bbox domains in tandem. Using mutants of Ub, we observed that these MID1 domain constructs facilitate Ub chain elongation via Lys63 of Ub. In addition, we observed that the high-molecular-weight protein products were primarily due to polyubiquitination at one site (Lys154) on the Bbox1 domain of the RING-Bbox1 and RING-Bbox1-Bbox2 constructs. We observed that MID1 E3 domains could interact with multiple E2-conjugating enzymes. Lastly, a 45-amino-acid peptide derived from the C-terminus of alpha4 that binds tightly to Bbox1 was observed to be monoubiquitinated in the assay and appears to down-regulate the amount of polyubiquitinated products formed. These studies shed light on MID1 E3 ligase activity and show how its three zinc-binding domains can contribute to MID1's overall function.     

Functional heterogeneity of ubiquitin carrier proteins

In the formation of covalent ubiquitin-protein conjugates that occurs during ATP- and ubiquitin-dependent proteolysis in reticulocyte extracts, ubiquitin (Ub) is activated to a thiol ester of the activating enzyme E1 (via the Ub carboxyl terminus), transferred to low-molecular weight "carrier proteins" (E2s) to form E2-Ub thiol esters, and then transferred by a third enzyme (E3) to amino groups on target proteins (Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983) J. Biol. Chem. 258, 8206-8214). We report here the fractionation of Ub carrier proteins by molecular weight, and their characterization with respect to several activities. The Ub thiol ester forms of at least four of the five E2s catalyze Ub transfer to a number of small amines, in a reaction that does not require E3; only primary amines on primary carbons can serve as Ub acceptors. E3-independent Ub transfer to the small, basic proteins histones H2A and H2B, and cytochrome c, is also observed. The Ub thiol ester forms of two of the E2s were found to catalyze Ub transfer to cytochrome c. Only a single E2 functions in E3-dependent conjugate formation (with the substrates creatine phosphokinase, reduced/carboxymethylated serum albumin, and oxidized RNase) and in E3-dependent protein breakdown (with the substrate serum albumin). This E2 has a subunit molecular weight of 14,000 and migrates as a dimer on Sephacryl 200.     

Immunochemical Properties of Ubiquitin Conjugates in the Paired Helical Filaments of Alzheimer Disease

Immunocytochemical and peptide sequencing studies indicate that the regulatory protein ubiquitin (Ub) is incorporated into the paired helical filaments (PHF) of Alzheimer disease. In this study, we showed that some antibodies raised to PHF recognize epitopes of Ub. Analysis of the Ub sequences recognized by the antibodies raised to PHF, along with the known specificity of several monoclonal antibodies raised to artificial Ub conjugates, indicates the immunochemical representation of Ub residues 34-76 in PHF. The Ub epitopes recognized by antibodies raised to PHF are distinct from those recognized by antibodies raised to artificial Ub conjugates in two respects. First, antibodies that are raised to PHF and that recognize Ub react with PHF equally, whether denatured or not, whereas those raised to artificial Ub conjugates show greater reaction after denaturation. Second, mapping of the epitopes recognized by two monoclonal antibodies to PHF onto Ub indicates a distinction in the Ub residues recognized, compared with monoclonal antibodies raised to artificial Ub conjugates. The proximity of their epitopes to the site of conjugation, as well as their affinity for PHF polypeptides, suggests that the PHF antibodies that recognize Ub may be directed specifically to Ub epitopes defined by the protein conjugated to Ub.     

Ubiquitin is a novel substrate for human insulin-degrading enzyme

Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β, peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (k_cat = 2 s^− 1) followed by a slow cleavage between residues 72 and 73 (k_cat = 0.07 s^−  1), thereby producing the inactive 1-74 fragment of Ub (Ub1-74) and 1-72 fragment of Ub (Ub1-72). IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼ 90-fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG <  0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that the interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE.     

Main chain and side chain dynamics of the ubiquitin conjugating enzyme variant human Mms2 in the free and ubiquitin-bound States

Protein ubiquitination involves a cascade of enzymatic steps where ubiquitin (Ub) is sequentially transferred as a thiolester intermediate from an E1 enzyme to an E2 enzyme and finally to the protein target with the help of a Ub-protein ligase. Protein ubiquitination brought about by the Ubc13-Mms2 (E2-E2) complex has a unique role in the cell, unrelated to protein degradation. The Mms2-Ubc13 heterodimer links Ub molecules to one another through an isopeptide bond between its own C-terminus and Lys-63 on another Ub. The role of Mms2 is to orient a target-bound Ub molecule such that its Lys-63 is proximal to the C-terminus of the Ub molecule that is covalently linked to the active site of Ubc13. To gain insight into the influence of protein dynamics on the affinity of Ub for Mms2, we have determined pico- to nanosecond time scale fluctuations of the main chain and methyl side chains of human Mms2 in the free and Ub-bound states using solution state (15)N and (2)H nuclear magnetic resonance relaxation measurements. Analysis of the relaxation data allows for a semiquantitative estimation of the conformational entropy change (TDeltaS(NMR)) for the main chain and side chain methyl groups of Mms2 upon binding Ub. The value of TDeltaS(NMR) for the main chain and side chain methyl groups of Mms2 is -8 +/- 2 and -2 +/- 2 kcal mol(-)(1), respectively. The experimental DeltaG(binding) for the Mms2.Ub complex is -6 kcal mol(-)(1). Estimation of DeltaG(binding) using an empirical structure-based approach that does not account for changes in main chain entropy yields a value of -17 +/- 2 kcal mol(-)(1). However, inclusion of TDeltaS(NMR) for the main chain of Mms2 increases the estimated DeltaG(binding) to -9 +/- 3 kcal mol(-)(1). Assuming that changes in Ub main chain dynamics contribute to TDeltaS(NMR) to the same extent as Mms2, the estimated DeltaG(binding) is further reduced to -1 +/- 4 kcal mol(-)(1), a value close to the experimental DeltaG(binding).     

Conformational dynamics of wild-type Lys-48-linked diubiquitin in solution

Proteasomal degradation is mediated through modification of target proteins by Lys-48-linked polyubiquitin (polyUb) chain, which interacts with several binding partners in this pathway through hydrophobic surfaces on individual Ub units. However, the previously reported crystal structures of Lys-48-linked diUb exhibit a closed conformation with sequestered hydrophobic surfaces. NMR studies on mutated Lys-48-linked diUb indicated a pH-dependent conformational equilibrium between closed and open states with the predominance of the former under neutral conditions (90% at pH 6.8). To address the question of how Ub-binding proteins can efficiently access the sequestered hydrophobic surfaces of Ub chains, we revisited the conformational dynamics of Lys-48-linked diUb in solution using wild-type diUb and cyclic forms of diUb in which the Ub units are connected through two Lys-48-mediated isopeptide bonds. Our newly determined crystal structure of wild-type diUb showed an open conformation, whereas NMR analyses of cyclic Lys-48-linked diUb in solution revealed that its structure resembled the closed conformation observed in previous crystal structures. Comparison of a chemical shift of wild-type diUb with that of monomeric Ub and cyclic diUb, which mimic the open and closed states, respectively, with regard to the exposure of hydrophobic surfaces to the solvent indicates that wild-type Lys-48-linked diUb in solution predominantly exhibits the open conformation (75% at pH 7.0), which becomes more populated upon lowering pH. The intrinsic properties of Lys-48-linked Ub chains to adopt the open conformation may be advantageous for interacting with Ub-binding proteins.     

Structural basis of ubiquitin recognition by mammalian Eap45 GLUE domain

ESCRT-II, a complex that sorts ubiquitinated membrane proteins to lysosomes, localizes to endosomes through interaction between the Vps36 subunit's GLUE domain and phosphatidylinositides (PIs). In yeast, a ubiquitin (Ub)-interacting NZF domain is inserted in Vps36 GLUE, whereas its mammalian counterpart, Eap45 GLUE, lacks the NZF domain. In the Eap45 GLUE-Ub complex structure, Ub binds far from the proposed PI-binding site of Eap45 GLUE, suggesting their independent binding.     

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

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

Plasticity of polyubiquitin recognition as lysosomal targeting signals by the endosomal sorting machinery

Lysosomal targeting is fundamental for the regulated disposal of ubiquitinated membrane proteins from the cell surface. To elucidate ubiquitin (Ub) configurations that are necessary and sufficient as multivesicular body (MVB)/lysosomal-sorting motifs, the intraendosomal destination and transport kinetics of model transmembrane cargo molecules bearing monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated cytoplasmic tails were determined. Monomeric CD4 chimeras with K63-linked poly-Ub chains and tetrameric CD4-mono-Ub chimeras were rapidly targeted to the lysosome. In contrast, lysosomal delivery of CD4 chimeras exposing K48-linked Ub chains was delayed, whereas delivery of monoubiquitinated CD4 chimeras was undetectable. Similar difference was observed in the lysosomal targeting of mono- versus polyubiquitinated invariant chain and CD4 ubiquitinated by the MARCH (membrane-associated RING-CH) IV Ub ligase. Consistent with this, Hrs (hepatocyte growth factor regulated tyrosine kinase phosphorylated substrate), an endosomal sorting adaptor, binds preferentially to K63-Ub chain and negligibly to mono-Ub. These results highlight the plasticity of Ub as a sorting signal and its recognition by the endosomal sorting machinery, and together with previous data, suggest a regulatory role for assembly and disassembly of Ub chains of specific topology in lysosomal cargo sorting.     

Multiple tryptophan probes reveal that ubiquitin folds via a late misfolded intermediate

Much of our understanding of protein folding mechanisms is derived from experiments using intrinsic fluorescence of natural or genetically inserted tryptophan (Trp) residues to monitor protein refolding and site-directed mutagenesis to determine the energetic role of amino acids in the native (N), intermediate (I) or transition (T) states. However, this strategy has limited use to study complex folding reactions because a single fluorescence probe may not detect all low-energy folding intermediates. To overcome this limitation, we suggest that protein refolding should be monitored with different solvent-exposed Trp probes. Here, we demonstrate the utility of this approach by investigating the controversial folding mechanism of ubiquitin (Ub) using Trp probes located at residue positions 1, 28, 45, 57, and 66. We first show that these Trp are structurally sensitive and minimally perturbing fluorescent probes for monitoring folding/unfolding of the protein. Using a conventional stopped-flow instrument, we show that ANS and Trp fluorescence detect two distinct transitions during the refolding of all five Trp mutants at low concentrations of denaturant: T₁, a denaturant-dependent transition and T₂, a slower transition, largely denaturant-independent. Surprisingly, some Trp mutants (Ub^M1W, Ub^S57W) display Trp fluorescence changes during T₁ that are distinct from the expected U → N transition suggesting that the denaturant-dependent refolding transition of Ub is not a U → N transition but represents the formation of a structurally distinct I-state (U → I). Alternatively, this U → I transition could be also clearly distinguished by using a combination of two Trp mutations Ub^F45W-T66W for which the two Trp probes that display fluorescence changes of opposite sign during T₁ and T₂ (Ub^F45W-T66W). Global fitting of the folding/unfolding kinetic parameters and additional folding-unfolding double-jump experiments performed on Ub^M1W, a mutant with enhanced fluorescence in the I-state, demonstrate that the I-state is stable, compact, misfolded, and on-pathway. These results illustrate how transient low-energy I-states can be characterized efficiently in complex refolding reactions using multiple Trp probes.     

Binding surface mapping of intra- and interdomain interactions among hHR23B,ubiquitin, and polyubiquitin binding site 2 of S5a

hHR23B is the human homologue of the yeast protein RAD23 and is known to participate in DNA repair by stabilizing xeroderma pigmentosum group C protein. However, hHR23B and RAD23 also have many important functions related to general proteolysis. hHR23B consists of N-terminal ubiquitin-like (UbL), ubiquitin association 1 (UBA1), xeroderma pigmentosum group C binding, and UBA2 domains. The UBA domains interact with ubiquitin (Ub) and inhibit the assembly of polyubiquitin. On the other hand, the UbL domain interacts with the poly-Ub binding site 2 (PUbS2) domain of the S5a protein, which can carry polyubiquitinated substrates into the proteasome. We calculated the NMR structure of the UbL domain of hHR23B and determined binding surfaces of UbL and Ub to UBA1, UBA2, of hHR23B and PUbS2 of S5a by using chemical shift perturbation. Interestingly, the surfaces of UbL and Ub that bind to UBA1, UBA2, and PUbS2 are similar, consisting of five beta-strands and their connecting loops. This is the first report that an intramolecular interaction between UbL and UBA domains is possible, and this interaction could be important for the control of proteolysis by hHR23B. The binding specificities of UbL and Ub for PUbS1, PUbS2, and general ubiquitin-interacting motifs, which share the LALA motif, were evaluated. The UBA domains bind to the surface of Ub including Lys-48, which is required for multiubiquitin assembly, possibly explaining the observed inhibition of multiubiquitination by hHR23B. The UBA domains bind to UbL through electrostatic interactions supported by hydrophobic interactions and to Ub mainly through hydrophobic interactions supported by electrostatic interactions.     

Cyclization of polyubiquitin by the E2-25K ubiquitin conjugating enzyme

For most substrates of ubiquitin (Ub)-dependent degradation, recognition by the proteasome is mediated by a covalently attached signal assembled from multiple ubiquitins linked to each other via the C terminus of one Ub and the epsilon-amine of Lys(48) of another Ub. Among Ub-conjugating enzymes, E2-25K is unique in its ability to synthesize in vitro unanchored Lys(48)-linked poly-Ub chains from mono- or poly-Ub, E1, and ATP; thus, E2-25K has distinct binding sites for donor and acceptor (poly)Ub. During studies of chain assembly by E2-25K, we observed that Lys(48)-linked tri-Ub was efficiently converted to a new species that upon SDS-polyacrylamide gel electrophoresis migrated between linear di-Ub and tri-Ub. Analysis of this product by mass spectrometry and tryptic digestion showed that it was a cyclic form of tri-Ub. Cyclization of tri-Ub requires E1, E2-25K, ATP, and that the linear substrate has a free Gly(76) C terminus on the proximal end Ub and a Lys(48) side chain available on the distal end Ub. E2-25K similarly can catalyze the cyclization of longer poly-Ub chains, including tetra- and penta-Ub. Although cyclic tri-Ub resists hydrolysis by the PA700 or isopeptidase T deubiquitinating enzymes, it can be disassembled to Ub monomers by isopeptidase(s) in a red blood cell extract. Thus, if cyclic poly-Ub forms in vivo, it will not accumulate as a dead-end product.     

Ubiquitin Ser65 phosphorylation affects ubiquitin structure,chain assembly and hydrolysis

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.     

Taking it step by step: mechanistic insights from structural studies of ubiquitin/ubiquitin-like protein modification pathways

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a diverse array of cellular pathways through post-translational attachment to protein substrates. Ub/Ubl-mediated signaling is initiated through E1, E2, and E3-mediated conjugation, transduced by proteins that recognize Ub/Ubl-modified substrates, and terminated by proteases which remove the Ub/Ubl from the substrate. Recent structural studies have elucidated mechanisms pertinent to Ub/Ubl conjugation, recognition, and deconjugation, highlighting essential steps during Ub/Ubl modification that illustrate common and divergent mechanistic themes within this important process.