Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases.

E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains to control an enormous swath of eukaryotic biology. Yet the molecular mechanisms underlying this exceptional linkage specificity and millisecond kinetics of poly-ubiquitylation remain unclear. Here we obtain cryogenic-electron microscopy (cryo-EM) structures that provide pertinent insight into how such poly-ubiquitin chains are forged. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation. NEDD8 releases the RING domain from the CRL, but unlike previous CRL-E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s.

A Novel Pathway for Biosynthesis of the Herbicidal Phosphonate Natural Product Phosphonothrixin Is Widespread in Actinobacteria.

Phosphonothrixin is an herbicidal phosphonate natural product with an unusual, branched carbon skeleton. Bioinformatic analyses of the ftx gene cluster, which is responsible for synthesis of the compound, suggest that early steps of the biosynthetic pathway, up to production of the intermediate 2,3-dihydroxypropylphosphonic acid (DHPPA) are identical to those of the unrelated phosphonate natural product valinophos. This conclusion was strongly supported by the observation of biosynthetic intermediates from the shared pathway in spent media from two phosphonothrixin producing strains. Biochemical characterization of ftx-encoded proteins confirmed these early steps, as well as subsequent steps involving the oxidation of DHPPA to 3-hydroxy-2-oxopropylphosphonate and its conversion to phosphonothrixin by the combined action of an unusual heterodimeric, thiamine-pyrophosphate (TPP)-dependent ketotransferase and a TPP-dependent acetolactate synthase. The frequent observation of ftx-like gene clusters within actinobacteria suggests that production of compounds related to phosphonothrixin is common within these bacteria. IMPORTANCE Phosphonic acid natural products, such as phosphonothrixin, have great potential for biomedical and agricultural applications; however, discovery and development of these compounds requires detailed knowledge of the metabolism involved in their biosynthesis. The studies reported here reveal the biochemical pathway phosphonothrixin production, which enhances our ability to design strains that overproduce this potentially useful herbicide. This knowledge also improves our ability to predict the products of related biosynthetic gene clusters and the functions of homologous enzymes.

Differentiating radiation necrosis from tumor recurrence: a systematic review and diagnostic meta-analysis comparing imaging modalities.

PURPSOSE: Cerebral radiation necrosis (RN) is often a delayed phenomenon occurring several months to years after the completion of radiation treatment. Differentiating RN from tumor recurrence presents a diagnostic challenge on standard MRI. To date, no evidence-based guidelines exist regarding imaging modalities best suited for this purpose. We aim to review the current literature and perform a diagnostic meta-analysis comparing various imaging modalities that have been studied to differentiate tumor recurrence and RN. METHODS: A systematic search adherent to PRISMA guidelines was performed using Scopus, PubMed/MEDLINE, and Embase. Pooled sensitivities and specificities were determined using a random-effects or fixed-effects proportional meta-analysis based on heterogeneity. Using diagnostic odds ratios, a diagnostic frequentist random-effects network meta-analysis was performed, and studies were ranked using P-score hierarchical ranking. RESULTS: The analysis included 127 studies with a total of 220 imaging datasets, including the following imaging modalities: MRI (n = 10), MR Spectroscopy (MRS) (n = 28), dynamic contrast-enhanced MRI (n = 7), dynamic susceptibility contrast MRI (n = 36), MR arterial spin labeling (n = 5), diffusion-weighted imaging (n = 13), diffusion tensor imaging (DTI) (n = 2), PET (n = 89), and single photon emission computed tomography (SPECT) (n = 30). MRS had the highest pooled sensitivity (90.7%). DTI had the highest pooled specificity (90.5%). Our hierarchical ranking ranked SPECT and MRS as most preferable, and MRI was ranked as least preferable. CONCLUSION: These findings suggest SPECT and MRS carry greater utility than standard MRI in distinguishing RN from tumor recurrence.

Trends in Technology for Pedicle Screw Placement: A Temporal Meta-Analysis.

STUDY DESIGN: Systematic Review. OBJECTIVE: We sought to determine which method of the pedicle screw (PS) placement is most accurate and understand how the development of subsequent generations of robotic systems has changed placement accuracy over time. SUMMARY OF BACKGROUND DATA: Previous studies have demonstrated the success of robotic PS placement, but how this accuracy compares to other methods is unclear. METHODS: A systematic review following PRISMA Guidelines was performed on articles published between January 2000 and August 2021, comparing PS insertion methods with at least 10 screws per study arm. Single and multiple-arm trials were included. Data were extracted for patient outcomes, including optimal PS placement, misplacement, and accuracy. The logit-event rate of misplacement was calculated for each study. P values were adjusted for multiple comparisons using the Tukey post hoc correction. RESULTS: Our search revealed 127 studies, and 156 comparative arms, with 77,360 pedicle screws placed using five different modalities. Meta-regression of pooled accuracy revealed no significant changes in PS accuracy over time for freehand, 2D fluoroscopic navigation, 3D fluoroscopic navigation, and computed tomography navigation. Robotic navigation had a significant increase in accuracy over time ( P =0.036). Pooled misplacement rates were also compared across all modalities. Robotics was found to have the lowest rates of misplacement for PS compared to freehand ( P =0.0015) and 2D fluoroscopic navigation ( P =0.026). CONCLUSION: Our analysis is the largest study to date on pedicle screw placement. Pedicle screw placement through robotics was found to be superior due to its low misplacement rates compared with other modalities. Intraoperative 3D fluoroscopic navigation was found to have comparable misplacement rates. In addition, pedicle screw placement accuracy with robotics has continued to improve over time. This speaks to both the stability of the technology and the potential for continued improvement with new and more accurate robotic systems.

Anterior versus Posterior Ventricular Catheter Placement in Pediatric Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: Ventriculoperitoneal shunt placement is the mainstay of treatment for hydrocephalus, but there are relatively high rates of malfunction. Shunt catheter entry can be performed anteriorly or posteriorly, with the body of evidence from randomized controlled trials and retrospective studies suggesting conflicting findings. METHODS: A systematic review of PubMed, Medline, Scopus, and Web of Science was performed adherent to PRISMA guidelines, searching for clinical studies examining outcomes for anterior or frontal and posterior or occipital ventriculoperitoneal shunt placement. A random-effects model meta-analysis was performed on R. RESULTS: Six studies (2 randomized controlled trials and 4 retrospective cohort studies) comprising 1808 patients were identified. There were no statistically significant differences between anterior and posterior ventriculoperitoneal shunt placement for the outcomes of poor catheter placement (odds ratio [OR], 0.74; P = 0.6) and shunt infections (OR, 1.01; P = 0.9). Posterior shunts trended toward greater number of shunt revisions (OR, 0.72; P = 0.06). Six and 12 months shunt survival was comparable between anterior and posterior approaches (P > 0.05). There were significant differences between long-term shunt survival (2 and 5 years shunt survival), favoring anterior shunt placement with greater odds of survival (OR, 1.91 and OR, 1.62, respectively; P < 0.05). CONCLUSIONS: We show that although anteriorly and posteriorly placed shunts have mostly comparable outcomes, shunt survival at 2-year and 5-year intervals favors anteriorly placed shunts. Additional well-designed clinical trials are needed to validate the findings of greater late shunt failure in posteriorly placed shunts, with more time-dependent statistical measures.

Management of brain metastasis. Surgical resection versus stereotactic radiotherapy: a meta-analysis.

Background: Treatment of metastatic brain tumors often involves radiotherapy with or without surgical resection as the first step. However, the indications for when to use surgery are not clearly defined for certain tumor sizes and multiplicity. This study seeks to determine whether resection of brain metastases versus exclusive radiotherapy provided improved survival and local control in cases where metastases are limited in number and diameter. Methods: According to PRISMA guidelines, this meta-analysis compares outcomes from treatment of a median number of brain metastases ≤ 4 with a median diameter ≤ 4 cm with exclusive radiotherapy versus surgery followed by radiotherapy. Four randomized control trials and 11 observational studies (1693 patients) met inclusion criteria. For analysis, studies were grouped based on whether radiation involved stereotactic radiosurgery (SRS) or whole-brain radiotherapy (WBRT). Results: In both analyses, there was no difference in survival between surgery ± SRS versus SRS alone two years after treatment (OR 1.89 (95% CI: 0.47-7.55, P = .23) or surgery + WBRT versus radiotherapy alone (either WBRT and/or SRS) (OR 1.18 (95% CI: 0.76-1.84, P = .46). However, surgical patients demonstrated greater risk for local tumor recurrence compared to SRS alone (OR 2.20 (95% CI: 1.49-3.25, P < .0001)) and compared to WBRT/SRS (OR 2.93; 95% CI: 1.68-5.13, P = .0002). Conclusion: The higher incidence of local tumor recurrence for surgical patients suggests that more prospective studies are needed to clarify outcomes for treatment of 1-4 metastasis less than 4 cm diameter.

Comparison of fluorescein sodium, 5-ALA, and intraoperative MRI for resection of high-grade gliomas: A systematic review and network meta-analysis.

High grade gliomas (HGGs) are aggressive brain tumors associated with poor prognosis despite advances in surgical treatment and therapy. Navigated tumor resection has yielded improved outcomes for patients. We compare 5-ALA, fluorescein sodium (FS), and intraoperative MRI (IMRI) with no image guidance to determine the best intraoperative navigation method to maximize rates of gross total resection (GTR) and outcomes. A frequentist network meta-analysis was performed following standard PRISMA guidelines (PROSPERO registration CRD42021268659). Surface-under-the-cumulative ranking (SUCRA) analysis was executed to hierarchically rank modalities by the outcomes of interest. Heterogeneity was measured by the I2 statistic. Publication bias was assessed by funnel plots and the use of Egger's test. Statistical significance was determined by p < 0.05. Twenty-three studies were included for analysis with a total of 2,643 patients. Network meta-analysis comparing 5-ALA, IMRI, and FS was performed. The primary outcome assessed was the rate of GTR. Analysis revealed the superiority of all intraoperative navigation to control (no navigation). SUCRA analysis revealed the superiority of IMRI + 5-ALA, IMRI alone, followed by FS, and 5-ALA. Overall survival (OS) and progression free survival (PFS) were also examined. FS (vs. control) was associated with improved OS, while IMRI was associated with improved PFS (vs. control, FS, and 5-ALA). Intraoperative navigation using IMRI, FS, and 5-ALA lead to greater rates of GTR in HGGs. FS and 5-ALA also yielded improvement in OS and PFS. Further studies are needed to evaluate differences in survival benefit, operative duration, and cost.

Robotic external ventricular drain placement for acute neurosurgical care in low-resource settings: feasibility considerations and a prototype design.

OBJECTIVE: Emergency neurosurgical care in lower-middle-income countries faces pronounced shortages in neurosurgical personnel and infrastructure. In instances of traumatic brain injury (TBI), hydrocephalus, and subarachnoid hemorrhage, the timely placement of external ventricular drains (EVDs) strongly dictates prognosis and can provide necessary stabilization before transfer to a higher-level center of care that has access to neurosurgery. Accordingly, the authors have developed an inexpensive and portable robotic navigation tool to allow surgeons who do not have explicit neurosurgical training to place EVDs. In this article, the authors aimed to highlight income disparities in neurosurgical care, evaluate access to CT imaging around the world, and introduce a novel, inexpensive robotic navigation tool for EVD placement. METHODS: By combining the worldwide distribution of neurosurgeons, CT scanners, and gross domestic product with the incidence of TBI, meningitis, and hydrocephalus, the authors identified regions and countries where development of an inexpensive, passive robotic navigation system would be most beneficial and feasible. A prototype of the robotic navigation system was constructed using encoders, 3D-printed components, machined parts, and a printed circuit board. RESULTS: Global analysis showed Montenegro, Antigua and Barbuda, and Seychelles to be primary candidates for implementation and feasibility testing of the novel robotic navigation system. To validate the feasibility of the system for further development, its performance was analyzed through an accuracy study resulting in accuracy and repeatability within 1.53 ± 2.50 mm (mean ± 2 × SD, 95% CI). CONCLUSIONS: By considering regions of the world that have a shortage of neurosurgeons and a high incidence of EVD placement, the authors were able to provide an analysis of where to prioritize the development of a robotic navigation system. Subsequently, a proof-of-principle prototype has been provided, with sufficient accuracy to target the ventricles for EVD placement.

Evaluating robotic pedicle screw placement against conventional modalities: a systematic review and network meta-analysis.

OBJECTIVE: Several approaches have been studied for internal fixation of the spine using pedicle screws (PSs), including CT navigation, 2D and 3D fluoroscopy, freehand, and robotic assistance. Robot-assisted PS placement has been controversial because training requirements, cost, and previously unclear benefits. This meta-analysis compares screw placement accuracy, operative time, intraoperative blood loss, and overall complications of PS insertion using traditional freehand, navigated, and robot-assisted methods. METHODS: A systematic review was performed of peer-reviewed articles indexed in several databases between January 2000 and August 2021 comparing ≥ 2 PS insertion methods with ≥ 10 screws per treatment arm. Data were extracted for patient outcomes, including PS placement, misplacement, and accuracy; operative time, overall complications, intraoperative blood loss, postoperative hospital length of stay, postoperative Oswestry Disability Index (ODI) score, and postoperative visual analog scale (VAS) score for back pain. Risk of bias was assessed using the Newcastle-Ottawa score and Cochrane tool. A network meta-analysis (NMA) was performed to estimate PS placement accuracy as the primary outcome. RESULTS: Overall, 78 studies consisting of 6262 patients and > 31,909 PSs were included. NMA results showed that robot-assisted and 3D-fluoroscopy PS insertion had the greatest accuracy compared with freehand (p < 0.01 and p < 0.001, respectively), CT navigation (p = 0.02 and p = 0.04, respectively), and 2D fluoroscopy (p < 0.01 and p < 0.01, respectively). The surface under the cumulative ranking (SUCRA) curve method further demonstrated that robot-assisted PS insertion accuracy was superior (S = 0.937). Optimal screw placement was greatest in robot-assisted (S = 0.995) placement, and misplacement was greatest with freehand (S = 0.069) approaches. Robot-assisted placement was favorable for minimizing complications (S = 0.876), while freehand placement had greater odds of complication than robot-assisted (OR 2.49, p < 0.01) and CT-navigation (OR 2.15, p = 0.03) placement. CONCLUSIONS: The results of this NMA suggest that robot-assisted PS insertion has advantages, including improved accuracy, optimal placement, and minimized surgical complications, compared with other PS insertion methods. Limitations included overgeneralization of categories and time-dependent effects.

Ubiquitin ligation to F-box protein targets by SCF-RBR E3-E3 super-assembly.

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates1,2. However, rather than functioning individually, many neddylated cullin-RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family-which together account for nearly half of all ubiquitin ligases in humans-form E3-E3 super-assemblies3-7. Here, by studying CRLs in the SKP1-CUL1-F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3-E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3-E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3-E3 super-assembly may therefore underlie widespread ubiquitylation.

Linkage-specific ubiquitin chain formation depends on a lysine hydrocarbon ruler.

Virtually all aspects of cell biology are regulated by a ubiquitin code where distinct ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C terminus of their transiently linked donor ubiquitin and a specific nucleophilic amino acid on the acceptor ubiquitin, yet it is unknown whether the fundamental feature of most acceptors-the lysine side chain-affects catalysis. Here, use of synthetic ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side chain specifying reactive amine geometry is a determinant of the ubiquitin code, through unanticipated and complex reliance of many distinct ubiquitin-carrying enzymes on a canonical acceptor lysine.

NEDD8 nucleates a multivalent cullin-RING-UBE2D ubiquitin ligation assembly.

Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation1, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD82-6. However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1ß-TRCP promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.

Mapping low-affinity/high-specificity peptide-protein interactions using ligand-footprinting mass spectrometry.

Short linear peptide motifs that are intracellular ligands of folded proteins are a modular, incompletely understood molecular interaction language in signaling systems. Such motifs, which frequently occur in intrinsically disordered protein regions, often bind partner proteins with modest affinity and are difficult to study with conventional structural biology methods. We developed LiF-MS (ligand-footprinting mass spectrometry), a method to map peptide binding sites on folded protein domains that allows consideration of their dynamic disorder, and used it to analyze a set of D-motif peptide-mitogen-activated protein kinase (MAPK) associations to validate the approach and define unknown binding structures. LiF-MS peptide ligands carry a short-lived, indiscriminately reactive cleavable crosslinker that marks contacts close to ligand binding sites with high specificity. Each marked amino acid provides an independent constraint for a set of directed peptide-protein docking simulations, which are analyzed by agglomerative hierarchical clustering. We found that LiF-MS provides accurate ab initio identification of ligand binding surfaces and a view of potential binding ensembles of a set of D-motif peptide-MAPK associations. Our analysis provides an MKK4-JNK1 structural model, which has thus far been crystallographically unattainable, a potential alternate binding mode for part of the NFAT4-JNK interaction, and evidence of bidirectional association of MKK4 peptide with ERK2. Overall, we find that LiF-MS is an effective noncrystallographic way to understand how short linear motifs associate with specific sites on folded protein domains at the level of individual amino acids.

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.

UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN.

Ring-between-ring (RBR) E3 ligases have been implicated in autoimmune disorders and neurodegenerative diseases. The functions of many RBR E3s are poorly defined, and their regulation is complex, involving post-translational modifications and allosteric regulation with other protein partners. The functional complexity of RBRs, coupled with the complexity of the native ubiquitination reaction that requires ATP and E1 and E2 enzymes, makes it difficult to study these ligases for basic research and therapeutic purposes. To address this challenge, we developed novel chemical probes, ubiquitin C-terminal fluorescein thioesters UbMES and UbFluor, to qualitatively and quantitatively assess the activity of the RBR E3 ligase PARKIN in a simple experimental setup and in real time using fluorescence polarization. First, we confirmed that PARKIN does not require an E2 enzyme for substrate ubiquitination, lysine selection, and polyubiquitin chain formation. Second, we confirmed that UbFluor quantitatively detects naturally occurring activation states of PARKIN caused by Ser65 phosphorylation (pPARKIN) and phosphorylated ubiquitin (pUb). Third, we showed that both pUb and the ubiquitin-accepting substrate contribute to maximal pPARKIN ubiquitin conjugation turnover. pUb enhances the transthiolation step, whereas the substrate clears the pPARKIN∼Ub thioester intermediate. Finally, we established that UbFluor can quantify activation or inhibition of PARKIN by structural mutations. These results demonstrate the feasibility of using UbFluor for quantitative studies of the biochemistry of RBR E3s and for high-throughput screening of small-molecule activators or inhibitors of PARKIN and other RBR E3 ligases.

UbFluor: A Fluorescent Thioester to Monitor HECT E3 Ligase Catalysis.

HECT E3 ubiquitin ligases (∼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1∼Ub, E2∼Ub, and HECT E3∼Ub (where ∼ indicates a thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes, UbFluor, that act as mechanistically relevant pseudosubstrates of HECT E3s. These probes undergo a direct transthiolation reaction with the catalytic cysteine of HECT E3s, producing the catalytically active HECT E3∼Ub thioester accompanied by fluorophore release. Thus, a fluorescence polarization assay can continuously monitor UbFluor consumption by HECT E3s, and changes in UbFluor consumption rendered by biochemical point mutations or small molecule modulation of HECT E3 activity. © 2017 by John Wiley & Sons, Inc.

UbFluor: A Mechanism-Based Probe for HECT E3 Ligases.

Homologous to E6AP Carboxyl Terminus E3 ubiquitin ligases (HECT, ~28 known) are genetically implicated in cancer, neurological, hypertensive, and autoimmune disorders, and are potential drug targets to treat these diseases. The major bottleneck in the field of HECT E3s is a lack of simple assays to quantify the enzymatic activity of these enzymes in the presence of small molecules. Typical assays require E1, E2, HECT E3, ubiquitin (Ub), ATP and additional reagents to detect the resulting free poly-ubiquitin chains. To address this need, we developed UbFluor, a fluorescent thioester conjugate between the C-terminus of Ub and fluorescein-thiol (Fluor-SH). UbFluor is a mechanism-based probe that undergoes a direct transthiolation reaction with the catalytic cysteine of the model HECT E3 ligase Rsp5, producing the catalytically active Rsp5~Ub (~ indicates thioester) accompanied by release of Fluor-SH. The kinetics of this two-component reaction can be easily monitored with real-time fluorescence polarization (FP) assays. Importantly, UbFluor eliminates the need to use SDS-PAGE, ATP, E1, E2 enzymes, and extra poly-ubiquitin chain detection reagents. Although the developed system lacks ATP, E1 and E2 enzymes, we show that UbFluor can recapitulate the native ubiquitination reaction by detecting and quantifying defects in transthiolation and isopeptide ligation of Rsp5 HECT E3 alanine mutants. Based on our findings, we show that UbFluor can be utilized to conduct high-throughput screens (HTS) of small molecules against HECT ligases.

Catalytically Important Residues of E6AP Ubiquitin Ligase Identified Using Acid-Cleavable Photo-Cross-Linkers.

Inactivation of the E6AP E3 ubiquitin ligase (UBE3A gene) causes Angelman syndrome, while aberrant degradation of p53 by E6AP is implicated in cervical cancers. Herein, we describe the development of photo-cross-linkers to discover catalytic residues of E6AP. Using these cross-linkers, we identified covalent modifications of the E6AP catalytic cysteine and two lysines: Lys(847) and Lys(799). Lys(847) is required for the formation of Lys(48)-linked polyubiquitin chains, while the K799A E6AP mutant was more active at producing Lys(48)-linked polyubiquitin chains. Thus, opposing roles of Lys(799) and Lys(847) pave the path forward to pharmacological inhibitors or activators of E6AP for therapeutic purposes.

Protein ubiquitination and formation of polyubiquitin chains without ATP, E1 and E2 enzymes.

Studying protein ubiquitination is difficult due to the complexity of the E1-E2-E3 ubiquitination cascade. Here we report the discovery that C-terminal ubiquitin thioesters can undergo direct transthiolation with the catalytic cysteine of the model HECT E3 ubiquitin ligase Rsp5 to form a catalytically active Rsp5∼ubiquitin thioester (Rsp5∼Ub). The resulting Rsp5∼Ub undergoes efficient autoubiquitination, ubiquitinates protein substrates, and synthesizes polyubiquitin chains with native Ub isopeptide linkage specificity. Since the developed chemical system bypasses the need for ATP, E1 and E2 enzymes while maintaining the native HECT E3 mechanism, we named it "Bypassing System" (ByS). Importantly, ByS provides direct evidence that E2 enzymes are dispensable for K63 specific isopeptide bond formation between ubiquitin molecules by Rsp5 in vitro. Additionally, six other E3 enzymes including Nedd4-1, Nedd4-2, Itch, and Wwp1 HECT ligases, along with Parkin and HHARI RBR ligases processed Ub thioesters under ByS reaction conditions. These findings provide general mechanistic insights on protein ubiquitination, and offer new strategies for assay development to discover pharmacological modulators of E3 enzymes.