Ubiquitin Thioester Research Articles

Structure and dynamics of a pentameric KCTD5/CUL3/Gβγ E3 ubiquitin ligase complex

Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3KCTD5) that ubiquitylates Gβγ and reduces Gβγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/Gβ1γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB/CUL3NTD and KCTD5CTD/Gβγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gβγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gβ and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/Gβγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex.

Unraveling the Mechanism of Action of Myricetin in the Inhibition of hUba1∼Ubiquitin Thioester Bond Formation via In Silico Molecular Modeling Techniques.

Ubiquitination is a crucial type of protein modification which helps to control substrate degradation and maintain cell homeostasis. Recent studies suggest that ubiquitination and deubiquitination are involved in regulating metabolic reprogramming in cancer cells and maintaining cancer stem cells. Uba1, a crucial protein in the ubiquitination cascade, can be targeted to develop effective inhibitors for cancer treatment. In previous work, we showed that myricetin (Myr) acts as a potential human Uba1 (hUba1) inhibitor. In this study, we have utilized computational modeling techniques to attempt to illustrate the mechanism of action of Myr. Through extra-precision docking, we confirmed that Myr binds to the adenosine triphosphate (ATP)-binding site of hUba1 (referred to as hotspot 1) with the highest binding affinity. The dynamics of this interaction revealed that hUba1 undergoes a conformational shift from open to closed upon binding of Myr. Myr also migrates outward to interact with the crossover loop simultaneously as the rotational shift of the ubiquitin fold domain (UFD) takes place, thereby blocking access to the ubiquitin binding interface of hUba1 and the crossover loop. The outward migration also explains the reversible nature of Myr binding to hUba1 in previous experiments. We hypothesize that Myr acts as an inhibitor of Uba1∼Ub thioester bond formation by causing a large domain shift toward a closed conformation. Few other analogues of Myr containing the same flavone skeleton showed promising docking scores against hUba1 and could be considered for further validation. We propose that Myr and some of its analogues reported in this study may be promising candidates for developing effective Uba1 inhibitors for cancer treatment.

Novel Disease-Causing Mutations in UBA1 Reveal Disease Mechanisms in Bone Marrow Failure and Inflammation

Background: Clonally acquired UBA1 mutations have recently been recognized as the cause of an inflammatory-hematologic overlap disease called VEXAS (vacuoles, E1, X-linked autoinflammatory, somatic) syndrome. Patients with VEXAS syndrome display bone marrow failure similar to myelodysplastic syndrome (MDS), and in addition have severe systemic inflammation. UBA1 is the E1 enzyme required for initiating most ubiquitylation in the cell, and the majority of disease-causing mutations identified occur at p.Met41, the start codon for the cytoplasmic isoform of UBA1 (UBA1b). These mutations lead to an isoform swap with loss of active UBA1b and emergence of a new catalytically impaired isoform, UBA1c, initiated from p.Met67. Despite extensive investigation into the clinical manifestations of UBA1, little is known about the mechanism of disease. Methods: We performed exome sequencing on 183 patients with clinical manifestations similar to VEXAS syndrome, but without an identified pathogenic mutation on targeted testing. Catalytic activity profiling was performed in vitro and in cells on candidate disease-causing variants identified, assessing for UBA1 thioester formation, E2 transthiolation, and overall cellular ubiquitylation. UBA1 mutations from lung cancer and SMA were also assessed. Results: Here we report the identification and molecular dissection of six novel somatic mutations in UBA1 (p.His55Tyr, p.Gly477Ala, p.Ala478Ser, p. Asp506Gly, p.Asp506Asn, and p.Ser621Cys) across nine individuals that lead to VEXAS syndrome. The majority of these mutations were identified through unbiased screening of exome sequencing data of patients with clinical manifestations similar to VEXAS syndrome but without a known pathogenic mutation. Patients with these mutations have highly overlapping symptoms with patients carrying canonical mutations. Specifically, all patients were male with a median age of disease onset of 75 years of age, had macrocytic anemia, elevated inflammatory markers along with rheumatic diagnoses, were steroid dependent and 3/5 with available bone marrow biopsies had MDS diagnoses. We systematically characterized these UBA1 mutations, along with two other previously reported pathogenic mutations (p.Met41Val, p.Ser56Phe), both in vitro and in cells. We found that only p.Met41 mutations led to a loss of UBA1b levels and production of UBA1c, while all other tested variants did not alter isoform expression or subcellular localization. Instead, these mutations reduce catalytic function of both cytoplasmic and nuclear isoforms by 40 to 90 % and can be subdivided into classes according to their impact on catalysis. Three mutations affect UBA1 function by interfering with ATP binding, one mutation renders UBA1 activity thermolabile, and three mutations specifically impair the transfer of ubiquitin from UBA1 to the E2 enzyme, while not affecting UBA1 ubiquitin thioester formation. This bottle neck in E2 charging is also present in UBA1 mutations driving lung cancer in never smokers (LCINS), but not in UBA1 mutations causing spinal muscular atrophy (SMA). Additionally, we identify E2-specific defects with distinct UBA1 mutations. Conclusions: Thus, somatic UBA1 mutations leading to cancer or inflammatory-hematologic phenotypes have a similar molecular mechanism of disease, which is different from germline SMA-causing UBA1 mutations. Together, we have expanded the genetic causes of VEXAS syndrome, identified shared properties of pathogenesis, and identified methods to screen variants of uncertain significance in disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A series of xanthenes inhibiting Rad6 function and Rad6-Rad18 interaction in the PCNA ubiquitination cascade

SummaryUbiquitination of proliferating cell nuclear antigen (PCNA) triggers pathways of DNA damage tolerance, including mutagenic translesion DNA synthesis, and comprises a cascade of reactions involving the E1 ubiquitin-activating enzyme Uba1, the E2 ubiquitin-conjugating enzyme Rad6, and the E3 ubiquitin ligase Rad18. We report here the discovery of a series of xanthenes that inhibit PCNA ubiquitination, Rad6∼ubiquitin thioester formation, and the Rad6–Rad18 interaction. Structure-activity relationship experiments across multiple assays reveal chemical and structural features important for different activities along the pathway to PCNA ubiquitination. The compounds that inhibit these processes are all a subset of the xanthen-3-ones we tested. These small molecules thus represent first-in-class probes of Rad6 function and the association of Rad6 and Rad18, the latter being a new inhibitory activity discovered for a small molecule, in the PCNA ubiquitination cascade and potential therapeutic agents to contain cancer progression.

Site-Specific Protein Ubiquitylation Using an Engineered, Chimeric E1 Activating Enzyme and E2 SUMO Conjugating Enzyme Ubc9.

Ubiquitylation—the attachment of ubiquitin (Ub) to proteins in eukaryotic cells—involves a vast number of enzymes from three different classes, resulting in heterogeneous attachment sites and ubiquitin chains. Recently, we introduced lysine acylation using conjugating enzymes (LACE) in which ubiquitin or peptide thioester is site-specifically transferred to a short peptide tag by the SUMO E2 conjugating enzyme Ubc9. This process, however, suffers from slow kinetics—due to a rate-limiting thioester loading step—and the requirement for thioesters restricts its use to in vitro reactions. To overcome these challenges, we devised a chimeric E1 containing the Ub fold domain of the SUMO E1 and the remaining domains of the Ub E1, which activates and loads native Ub onto Ubc9 and obviates the need for Ub thioester in LACE. The chimeric E1 was subjected to directed evolution to improve its apparent second-order rate constant (kcat/KM) 400-fold. We demonstrate the utility of the chimeric E1 by site-specific transfer of mono- and oligo-Ub to various target proteins in vitro. Additionally, the chimeric E1, Ubc9, Ub, and the target protein can be coexpressed in Escherichia coli for the facile preparation of monoubiquitylated proteins.

Crystal structures of an E1\u2013E2\u2013ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification

E1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1–E2–Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1–E2–ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1–E2–Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.

Mode of inhibitory binding of epigallocatechin gallate to the ubiquitin-activating enzyme Uba1 via accelerated molecular dynamics.

The green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) and some of its analogs potently inhibit the ubiquitin-activating enzyme Uba1. In an effort to understand the possible molecular basis of inhibitory activity of EGCG, we conducted a molecular docking and molecular dynamics simulation study. We found that EGCG and its two selected analogs, (−)-epicatechin-3-gallate (ECG) and (−)-epigallocatechin (EGC), bind favorably at two likely hot spots for small-molecule ligand binding on human Uba1. The compounds bind with energetics that mirror their experimental potency for inhibition of Uba1∼ubiquitin thioester formation. The binding of EGCG, ECG, and EGC at one of the hot spots, in particular, recapitulates the rank order of potency determined experimentally and suggests a possible mechanism for inhibition. A hinge-like conformational change of the second catalytic cysteine domain and the opposing ubiquitin-fold domain observed during accelerated molecular dynamics simulations of the EGCG-bound Uba1 complex that results in disruption of the ubiquitin-binding interfaces could explain the compounds' inhibitory activity. These results shed light on the possible molecular mechanism of EGCG and related catechins in the inhibition of Uba1.

Robust high-throughput assays to assess discrete steps in ubiquitination and related cascades

BackgroundUbiquitination and ubiquitin-like protein post-translational modifications play an enormous number of roles in cellular processes. These modifications are constituted of multistep reaction cascades. Readily implementable and robust methods to evaluate each step of the overall process, while presently limited, are critical to the understanding and modulation of the reaction sequence at any desired level, both in terms of basic research and potential therapeutic drug discovery and development.ResultsWe developed multiple robust and reliable high-throughput assays to interrogate each of the sequential discrete steps in the reaction cascade leading to protein ubiquitination. As models for the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, the E3 ubiquitin ligase, and their ultimate substrate of ubiquitination in a cascade, we examined Uba1, Rad6, Rad18, and proliferating cell nuclear antigen (PCNA), respectively, in reconstituted systems. Identification of inhibitors of this pathway holds promise in cancer therapy since PCNA ubiquitination plays a central role in DNA damage tolerance and resulting mutagenesis. The luminescence-based assays we developed allow for the quantitative determination of the degree of formation of ubiquitin thioester conjugate intermediates with both E1 and E2 proteins, autoubiquitination of the E3 protein involved, and ubiquitination of the final substrate. Thus, all covalent adducts along the cascade can be individually probed. We tested previously identified inhibitors of this ubiquitination cascade, finding generally good correspondence between compound potency trends determined by more traditional low-throughput methods and the present high-throughput ones.ConclusionsThese approaches are readily adaptable to other E1, E2, and E3 systems, and their substrates in both ubiquitination and ubiquitin-like post-translational modification cascades.

Multilevel structure\u2013activity profiling reveals multiple green tea compound families that each modulate ubiquitin-activating enzyme and ubiquitination by a distinct mechanism

We developed and implemented a reconstituted system to screen for modulators of the ubiquitination of proliferating cell nuclear antigen, a process that activates pathways of DNA damage tolerance and drug resistance. We identified the primary putatively health-beneficial green tea polyphenol epigallocatechin gallate (EGCG) and certain related small molecules as potent inhibitors of ubiquitination. EGCG directly and reversibly targets the ubiquitin-activating enzyme Uba1, blocking formation of the Uba1~ubiquitin thioester conjugate and thus ubiquitination and in the cell. Structure–activity relationship profiles across multiple biochemical and cellular assays for a battery of EGCG analogues revealed distinct chemical and mechanism-of-action clusters of molecules, with catechin gallates, alkyl gallates, and myricetin potently inhibiting ubiquitination. This study defines a number of related though distinct first-in-class inhibitors of ubiquitination, each series with its own unique activity pattern and mechanistic signature.

An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules

Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to many biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we present an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2-conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracts. This enables purification and mass spectrometry-based identification of modified proteins under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of APC/C in human cells. Finally, we perform E2~dID with SUMO in S. cerevisiae, showing that this approach can be easily adapted to other ubiquitin-like modifiers and experimental models.

OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly

The NEDD4-2 (neural precursor cell-expressed developmentally down-regulated 4-2) HECT ligase catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two functionally distinct E2∼ubiquitin-binding sites, analogous to the trimeric E6AP/UBE3A HECT ligase. This conserved catalytic mechanism suggests that NEDD4-2, and presumably all HECT ligases, requires oligomerization to catalyze polyubiquitin chain assembly. To explore this hypothesis, we examined the catalytic mechanism of NEDD4-2 through the use of biochemically defined kinetic assays examining rates of 125I-labeled polyubiquitin chain assembly and biophysical techniques. The results from gel filtration chromatography and dynamic light-scattering analyses demonstrate for the first time that active NEDD4-2 is a trimer. Homology modeling to E6AP revealed that the predicted intersubunit interface has an absolutely conserved Phe-823, substitution of which destabilized the trimer and resulted in a ≥104-fold decrease in kcat for polyubiquitin chain assembly. The small-molecule Phe-823 mimic, N-acetylphenylalanyl-amide, acted as a noncompetitive inhibitor (Ki = 8 ± 1.2 mm) of polyubiquitin chain elongation by destabilizing the active trimer, suggesting a mechanism for therapeutically targeting HECT ligases. Additional kinetic experiments indicated that monomeric NEDD4-2 catalyzes only HECT∼ubiquitin thioester formation and monoubiquitination, whereas polyubiquitin chain assembly requires NEDD4-2 oligomerization. These results provide evidence that the previously identified sites 1 and 2 of NEDD4-2 function in trans to support chain elongation, explicating the requirement for oligomerization. Finally, we identified a conserved catalytic ensemble comprising Glu-646 and Arg-604 that supports HECT-ubiquitin thioester exchange and isopeptide bond formation at the active-site Cys-922 of NEDD4-2.

In silico modeling of the cryptic E2∼ubiquitin–binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly

To understand the mechanism for assembly of Lys48-linked polyubiquitin degradation signals, we previously demonstrated that the E6AP/UBE3A ligase harbors two functionally distinct E2∼ubiquitin-binding sites: a high-affinity Site 1 required for E6AP Cys820∼ubiquitin thioester formation and a canonical Site 2 responsible for subsequent chain elongation. Ordered binding to Sites 1 and 2 is here revealed by observation of UbcH7∼ubiquitin-dependent substrate inhibition of chain formation at micromolar concentrations. To understand substrate inhibition, we exploited the PatchDock algorithm to model in silico UbcH7∼ubiquitin bound to Site 1, validated by chain assembly kinetics of selected point mutants. The predicted structure buries an extensive solvent-excluded surface bringing the UbcH7∼ubiquitin thioester bond within 6 Å of the Cys820 nucleophile. Modeling onto the active E6AP trimer suggests that substrate inhibition arises from steric hindrance between Sites 1 and 2 of adjacent subunits. Confirmation that Sites 1 and 2 function in trans was demonstrated by examining the effect of E6APC820A on wild-type activity and single-turnover pulse-chase kinetics. A cyclic proximal indexation model proposes that Sites 1 and 2 function in tandem to assemble thioester-linked polyubiquitin chains from the proximal end attached to Cys820 before stochastic en bloc transfer to the target protein. Non-reducing SDS-PAGE confirms assembly of the predicted Cys820-linked 125I-polyubiquitin thioester intermediate. Other studies suggest that Glu550 serves as a general base to generate the Cys820 thiolate within the low dielectric binding interface and Arg506 functions to orient Glu550 and to stabilize the incipient anionic transition state during thioester exchange.

The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly

The mechanism of Nedd4-2 has been quantitatively explored for the first time using biochemically defined kinetic assays examining rates of 125I-polyubiquitin chain assembly as a functional readout. We demonstrate that Nedd4-2 exhibits broad specificity for E2 paralogs of the Ubc4/5 clade to assemble Lys63-linked polyubiquitin chains. Full-length Nedd4-2 catalyzes free 125I-polyubiquitin chain assembly by hyperbolic Michaelis-Menten kinetics with respect to Ubc5B∼ubiquitin thioester concentration (Km = 44 ± 6 nm; kcat = 0.020 ± 0.007 s-1) and substrate inhibition above 0.5 μm (Ki = 2.5 ± 1.3 μm) that tends to zero velocity, requiring ordered binding at two functionally distinct E2∼ubiquitin-binding sites. The Ubc5BC85A product analog non-competitively inhibits Nedd4-2 (Ki = 2.0 ± 0.5 μm), consistent with the presence of the second E2-binding site. In contrast, the isosteric Ubc5BC85S-ubiquitin oxyester substrate analog exhibits competitive inhibition at the high-affinity Site 1 (Ki = 720 ± 340 nm) and non-essential activation at the lower-affinity Site 2 (Kact = 750 ± 260 nm). Additional studies utilizing Ubc5BF62A, defective in binding the canonical E2 site, demonstrate that the cryptic Site 1 is associated with thioester formation, whereas binding at the canonical site (Site 2) is associated with polyubiquitin chain elongation. Finally, previously described Ca2+-dependent C2 domain-mediated autoinhibition of Nedd4-2 is not observed under our reported experimental conditions. These studies collectively demonstrate that Nedd4-2 catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two dynamically linked E2∼ubiquitin-binding sites analogous to that recently reported for E6AP, the founding member of the Hect ligase family.

Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy

Background: X-linked spinal muscular atrophy (XL-SMA) results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 ( UBA1). Previously, four novel closely clustered mutations have been shown to cause this fatal infantile disorder affecting only males. These mutations, three missense and one synonymous, all lie within Exon15 of the UBA1 gene, which contains the active adenylation domain (AAD). Methods: In this study, our group characterized the three known missense variants in vitro. Using a novel Uba1 assay and other methods, we investigated Uba1 adenylation, thioester, and transthioesterification reactions in vitro to determine possible biochemical effects of the missense variants. Results: Our data revealed that only one of the three XL-SMA missense variants impairs the Ubiquitin-adenylating ability of Uba1. Additionally, these missense variants retained Ubiquitin thioester bond formation and transthioesterification rates equal to that found in the wild type. Conclusions: Our results demonstrate a surprising shift from the likelihood of these XL-SMA mutations playing a damaging role in Uba1’s enzymatic activity with Ubiquitin, to other roles such as altering UBA1 mRNA splicing via the disruption of splicing factor binding sites, similar to a mechanism in traditional SMA, or disrupting binding to other important in vivo binding partners. These findings help to narrow the search for the areas of possible dysfunction in the Ubiquitin-proteasome pathway that ultimately result in XL-SMA. Moreover, this investigation provides additional critical understanding of the mutations’ biochemical mechanisms, vital for the development of future effective diagnostic assays and therapeutics.

Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy.

Background: X-linked spinal muscular atrophy (XL-SMA) results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 ( UBA1). Previously, four novel closely clustered mutations have been shown to cause this fatal infantile disorder affecting only males. These mutations, three missense and one synonymous, all lie within Exon15 of the UBA1 gene, which contains the active adenylation domain (AAD). Methods: In this study, our group characterized the three known missense variants in vitro. Using a novel Uba1 assay and other methods, we investigated Uba1 adenylation, thioester, and transthioesterification reactions in vitro to determine possible biochemical effects of the missense variants. Results: Our data revealed that only one of the three XL-SMA missense variants impairs the Ubiquitin-adenylating ability of Uba1. Additionally, these missense variants retained Ubiquitin thioester bond formation and transthioesterification rates equal to that found in the wild type. Conclusions: Our results demonstrate a surprising shift from the likelihood of these XL-SMA mutations playing a damaging role in Uba1's enzymatic activity with Ubiquitin, to other roles such as altering UBA1mRNA splicing via the disruption of splicing factor binding sites, similar to a mechanism in traditional SMA, or disrupting binding to other important in vivo binding partners. These findings help to narrow the search for the areas of possible dysfunction in the Ubiquitin-proteasome pathway that ultimately result in XL-SMA. Moreover, this investigation provides additional critical understanding of the mutations' biochemical mechanisms, vital for the development of future effective diagnostic assays and therapeutics.

Nedd4-2 is a Functional Oligomer Exhibiting Cooperative Allosteric Kinetics

The Hect ligase Standard Model posits binding of the E2∼ubiquitin co-substrate to a single site within a monomeric Hect domain followed by transthiolation to form a Hect∼ubiquitin thioester intermediate. Transfer of the Hect-bound ubiquitin to lysyl residues on target proteins is then catalyzed by the ligase, processesively assembling polyubiquitin chains by distal addition. Recently, we demonstrated that the Hect ligase Nedd4-2 catalyzes polyubiquitin chain assembly by cooperative allosteric kinetics ([S]1/2=93±20 nM; kcat=0.067±0.008 s−1; nH=1.9±0.6) and exhibits substrate inhibition above 0.5 µM Ubc5B∼125I-ubiquitin, requiring the enzyme to function as an oligomer with two ordered E2∼ubiquitin binding sites of different affinities. These data support an alternative model for Hect ligase conjugation and are in agreement with our previous observations for E6AP. The current studies employ kinetic and biophysical methods to further explore the mechanism of Nedd4-2. Gel filtration chromatography and dynamic light scattering are consistent with trimerization. Truncation of the N-terminus yielding GST-Nedd4-2HECT (residues 597-955) reveals cooperative kinetics for polyubiquitin chain assembly (nH=2.6±1) with a ca. 104-fold reduction in kcat (1.9±0.2 x 10−6 s−1), demonstrating allostery intrinsic to the Hect domain and N-terminal contributions supporting oligomerization. Processing of GST yields monomeric Nedd4-2HECT resulting in monoubiquitination with hyperbolic kinetics (KM=39±7 nM; kcat=6.0±0.2 x 10−6 s−1) and loss of polyubiquitin chain assembly and substrate inhibition. Addition of Nedd4-2ΔHECT (residues 1-596) to wild type Nedd4-2 quantitatively inhibits activity; however, addition of the dominant negative Nedd4-2C922A mutant inhibits Nedd4-2 to 30% limiting activity, requiring the two sites to function in trans. These data support a model requiring trimerization and intersubunit interactions for polyubiquitin chain assembly consistent with our model of Proximal Indexation. [Supported by GM34009 to A.L.H.]

High‐Throughput Screening of HECT E3 Ubiquitin Ligases Using UbFluor

HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin thioesters and a complex mixture of polyubiquitin products provide further opportunities for off-target inhibition and increase the complexity of the assay. UbFluor is a novel ubiquitin thioester that bypasses the E1 and E2 enzymes and undergoes direct transthiolation with HECT E3 ligases. The release of fluorophore upon transthiolation allows fluorescence polarization detection of HECT E3 activity. In the presence of inhibitors, HECT E3 activity is ablated, and thus no reaction and no change in FP are observed. This assay has been adapted for high-throughput screening of small molecules against HECT E3 ligases, and its utility has been proven in the discovery of HECT E3 ligase inhibitors. © 2017 by John Wiley & Sons, Inc.

Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.

BackgroundProtein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3’s gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, connot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised.ResultsUsing the RIKEN Arabidopsis full-length cDNA library (RAFL) with the ‘split-primer’ PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time.ConclusionThe wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0660-9) contains supplementary material, which is available to authorized users.

Catalysis of ubiquitin transfer by the RBR ubiquitin ligase parkin

Mutations in the Parkin gene are responsible for an autosomal recessive form of Parkinson's disease. The parkin protein is a RING1-In-Between-RING2 (RBR) E3 ubiquitin ligase, which functions through a two-step mechanism involving a parkin~ubiquitin thioester intermediate [1]. However, compared to other ubiquitin ligases, parkin exhibits low basal activity and requires activation both in vitro and in cells. As parkin is neuroprotective in various models of Parkinson's disease, understanding how it catalyses ubiquitin transfer will be critical. We previously reported the crystal structure of full-length parkin [2]. The structure shows parkin in an auto-inhibited state and provides insight into how it is activated. The RING0 domain occludes the ubiquitin acceptor site Cys431 in RING2 whereas a novel Repressor Element of Parkin (REP) binds RING1 and blocks its E2-binding site. Remarkably, mutations that disrupt these inhibitory interactions activate parkin both in vitro and in cells. The structure also reveals that His433 and Glu444 form a catalytic dyad adjacent to Cys431. Here, we show that His433 catalyses the acyl transfer of ubiquitin carboxy terminus from Cys431 to a target lysine side-chain amino group. Mutation of His433 does not affect UbcH7~ubiquitin discharging or thioester intermediate formation, but prevents formation of polyubiquitin chains on parkin. However, mutation of His433 does not affect significantly parkin's mitochondrial recruitment and substrate ubiquitination, suggesting that other factors might be at play in vivo. We also investigate the catalytic role of other residues located around the Cys431, such as Trp462. The work provides insight into the mechanism of ubiquitination by RBR E3 ligases with important implications for Parkinson's disease.