Modulation of Cullin-RING E3 ubiquitin ligase-dependent ubiquitination by small molecule compounds.

Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a subcomplex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes the modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33, and suramin, which target the CRL core ligases. We show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4CRBN. However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of ß-catenin by CRL1ß-TrCP and Nedd8-CRL1ß-TrCP almost equally. Thus, neddylation of CRL1ß-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high-resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators.

Monoubiquitination empowers ubiquitin chain elongation.

Ubiquitination often generates lysine 48-linked polyubiquitin chains that signal proteolytic destruction of the protein target. A significant subset of ubiquitination proceeds by a priming/extending mechanism, in which a substrate is first monoubiquitinated with a priming E2-conjugating enzyme or a set of E3 ARIH/E2 enzymes specific for priming. This is then followed by ubiquitin (Ub) chain extension catalyzed by an E2 enzyme capable of elongation. This report provides further insights into the priming/extending mechanism. We employed reconstituted ubiquitination systems of substrates CK1α (casein kinase 1α) and ß-catenin by Cullin-RING E3 Ub ligases (CRLs) CRL4CRBN and CRL1ßTrCP, respectively, in the presence of priming E2 UbcH5c and elongating E2 Cdc34b (cell division cycle 34b). We have established a new "apyrase chase" strategy that uncouples priming from chain elongation, which allows accurate measurement of the decay rates of the ubiquitinated substrate with a defined chain length. Our work has revealed highly robust turnover of monoubiquitinated ß-catenin that empowers efficient polyubiquitination. The results of competition experiments suggest that the interactions between the ubiquitinated ß-catenin and CRL1ßTrCP are highly dynamic. Moreover, ubiquitination of the Ub-modified ß-catenin appeared more resistant to inhibition by competitors than the unmodified substrate, suggesting tighter binding with CRL1ßTrCP. These findings support a role for conjugated Ub in enhancing interactions with E3.

CRL4b Inhibition Ameliorates Experimental Autoimmune Encephalomyelitis Progression.

Multiple sclerosis, and its murine model experimental autoimmune encephalomyelitis (EAE), is a neurodegenerative autoimmune disease of the CNS characterized by T cell influx and demyelination. Similar to other autoimmune diseases, therapies can alleviate symptoms but often come with side effects, necessitating the exploration of new treatments. We recently demonstrated that the Cullin-RING E3 ubiquitin ligase 4b (CRL4b) aided in maintaining genome stability in proliferating T cells. In this study, we examined whether CRL4b was required for T cells to expand and drive EAE. Mice lacking Cul4b (Cullin 4b) in T cells had reduced EAE symptoms and decreased inflammation during the peak of the disease. Significantly fewer CD4+ and CD8+ T cells were found in the CNS, particularly among the CD4+ T cell population producing IL-17A, IFN-γ, GM-CSF, and TNF-α. Additionally, Cul4b-deficient CD4+ T cells cultured in vitro with their wild-type counterparts were less likely to expand and differentiate into IL-17A- or IFN-γ-producing effector cells. When wild-type CD4+ T cells were activated in vitro in the presence of the recently developed CRL4 inhibitor KH-4-43, they exhibited increased apoptosis and DNA damage. Treatment of mice with KH-4-43 following EAE induction resulted in stabilized clinical scores and significantly reduced numbers of T cells and innate immune cells in the CNS compared with control mice. Furthermore, KH-4-43 treatment resulted in elevated expression of p21 and cyclin E2 in T cells. These studies support that therapeutic inhibition of CRL4 and/or CRL4-related pathways could be used to treat autoimmune disease.

Targeting Cullin-RING E3 Ubiquitin Ligase 4 by Small Molecule Modulators.

Cullin-RING E3 ubiquitin ligase 4 (CRL4) plays an essential role in cell cycle progression. Recent efforts using high throughput screening and follow up hit-to-lead studies have led to identification of small molecules 33-11 and KH-4-43 that inhibit E3 CRL4's core ligase complex and exhibit anticancer potential. This review provides: 1) an updated perspective of E3 CRL4, including structural organization, major substrate targets and role in cancer; 2) a discussion of the challenges and strategies for finding the CRL inhibitor; and 3) a summary of the properties of the identified CRL4 inhibitors as well as a perspective on their potential utility to probe CRL4 biology and act as therapeutic agents.

A Novel Strategy to Track Lysine-48 Ubiquitination by Fluorescence Resonance Energy Transfer.

Posttranslational modification of protein by lysine-48 (K48) linked ubiquitin (Ub) chains is the major cellular mechanism for selective protein degradation that critically impacts biological processes such as cell cycle checkpoints. In this chapter, we describe an in vitro biochemical approach to detect a K48-linked di-Ub chain by fluorescence resonance energy transfer (FRET). To this end, we detail methods for the preparation of the relevant enzymes and substrates, as well as for the execution of the reaction with high efficiency. Tracking K48 polyubiquitination using this sensitive and highly reproducible format provides an opportunity for high-throughput screening that leads to identification of small molecule modulators capable of changing ubiquitination for improving human health.

Inhibitors of cullin-RING E3 ubiquitin ligase 4 with antitumor potential.

Cullin-RING (really intersting new gene) E3 ubiquitin ligases (CRLs) are the largest E3 family and direct numerous protein substrates for proteasomal degradation, thereby impacting a myriad of physiological and pathological processes including cancer. To date, there are no reported small-molecule inhibitors of the catalytic activity of CRLs. Here, we describe high-throughput screening and medicinal chemistry optimization efforts that led to the identification of two compounds, 33-11 and KH-4-43, which inhibit E3 CRL4 and exhibit antitumor potential. These compounds bind to CRL4's core catalytic complex, inhibit CRL4-mediated ubiquitination, and cause stabilization of CRL4's substrate CDT1 in cells. Treatment with 33-11 or KH-4-43 in a panel of 36 tumor cell lines revealed cytotoxicity. The antitumor activity was validated by the ability of the compounds to suppress the growth of human tumor xenografts in mice. Mechanistically, the compounds' cytotoxicity was linked to aberrant accumulation of CDT1 that is known to trigger apoptosis. Moreover, a subset of tumor cells was found to express cullin4 proteins at levels as much as 70-fold lower than those in other tumor lines. The low-cullin4-expressing tumor cells appeared to exhibit increased sensitivity to 33-11/KH-4-43, raising a provocative hypothesis for the role of low E3 abundance as a cancer vulnerability.

A new FRET-based platform to track substrate ubiquitination by fluorescence.

Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence-based techniques in ubiquitination studies remain underexplored at least partly because of challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, FRET diubiquitination, to track substrate ubiquitination by fluorescence. This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The diubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET diubiquitination relies on Ub-substrate fusion, which can be implemented using either one of the two validated strategies. Method 1 is the use of recombinant substrate-Ub fusion, applicable to all substrate peptides that can bind to E3. Method 2 is a chemoenzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based diubiquitination system provides a timely technology of potential to advance both basic research and translation sciences.

Methylation of EZH2 by PRMT1 regulates its stability and promotes breast cancer metastasis.

Enhancer of zeste homolog 2 (EZH2), a key histone methyltransferase and EMT inducer, is overexpressed in diverse carcinomas, including breast cancer. However, the molecular mechanisms of EZH2 dysregulation in cancers are still largely unknown. Here, we discover that EZH2 is asymmetrically dimethylated at R342 (meR342-EZH2) by PRMT1. meR342-EZH2 was found to inhibit the CDK1-mediated phosphorylation of EZH2 at T345 and T487, thereby attenuating EZH2 ubiquitylation mediated by the E3 ligase TRAF6. We also demonstrate that meR342-EZH2 resulted in a decrease in EZH2 target gene expression, but an increase in breast cancer cell EMT, invasion and metastasis. Moreover, we confirm the positive correlations among PRMT1, meR342-EZH2 and EZH2 expression in the breast cancer tissues. Finally, we report that high expression levels of meR342-EZH2 predict a poor clinical outcome in breast cancer patients. Our findings may provide a novel diagnostic target and promising therapeutic target for breast cancer metastasis.

Gossypol inhibits cullin neddylation by targeting SAG-CUL5 and RBX1-CUL1 complexes.

Cullin-RING E3 ligase (CRL) is the largest family of E3 ubiquitin ligase, responsible for ubiquitylation of ∼20% of cellular proteins. CRL plays an important role in many biological processes, particularly in cancers due to abnormal activation. CRL activation requires neddylation, an enzymatic cascade transferring small ubiquitin-like protein NEDD8 to a conserved lysine residue on cullin proteins. Recent studies have validated that neddylation is an attractive anticancer target. In this study, we report the establishment of an Alpha-Screen-based high throughput screen (HTS) assay for in vitro CUL5 neddylation, and screened a library of 17,000 compounds including FDA approved drugs, natural products and synthetic drug-like small-molecule compounds. Gossypol, a natural compound derived from cotton seed, was identified as an inhibitor of cullin neddylation. Biochemical studies showed that gossypol blocked neddylation of both CUL5 and CUL1 through direct binding to SAG-CUL5 or RBX1-CUL1 complex, and CUL5-H572 plays a key role for gossypol binding. On cellular level, gossypol inhibited cullin neddylation in a variety of cancer cell lines and selectively caused accumulation of NOXA and MCL1, the substrates of CUL5 and CUL1, respectively, in multiple cancer cell lines. Combination of gossypol with specific MCL1 inhibitor synergistically suppress growth of human cancer cells. Our study revealed a previously unknown anti-cancer mechanism of gossypol with potential to develop a new class of neddylation inhibitors.

Cullin-RING E3 Ubiquitin Ligase 7 in Growth Control and Cancer.

CRL7Fbxw8 is an E3 ubiquitin ligase complex, containing cullin7 (CUL7) as a scaffold, the F-box protein Fbxw8 as a substrate receptor, the Skp1 adaptor, and the ROC1/Rbx1 RING finger protein for working with E2 enzyme to facilitate ubiquitin transfer. This chapter provides an update on studies linking CRL7Fbxw8 to hereditary human growth retardation disease, as at least 64 cul7 germ line mutations were found in patients with autosomal recessive 3-M syndrome. CRL7Fbxw8 interacts with two additional 3-M associated proteins OBSL1 and CCDC8, leading to subcellular localization of the E3 complex to regions including plasma membrane, centrosome, and Golgi. At least ten mammalian cellular proteins were identified or implicated as CRL7Fbxw8 substrates. Discussion focuses on the possible impact of CRL7Fbxw8-mediated proteolytic or non-proteolytic pathways in growth control and cancer.

IKK-Mediated Regulation of the COP9 Signalosome via Phosphorylation of CSN5.

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex, which controls protein degradation through deneddylation and inactivation of cullin-RING ubiquitin E3 ligases (CRLs). Recently, the CSN complex has been linked to the NF-κB signaling pathway due to its association with the IKK complex. However, how the CSN complex is regulated in this signaling pathway remains unclear. Here, we have carried out biochemical experiments and confirmed the interaction between the CSN and IKK complexes. In addition, we have determined that overexpression of IKKα or IKKß leads to enhanced phosphorylation of CSN5, the catalytic subunit for CSN deneddylase activity. Mutational analyses have revealed that phosphorylation at serine 201 and threonine 205 of CSN5 impairs CSN-mediated deneddylation activity in vitro. Interestingly, TNF-α treatment not only enhances the interaction between CSN and IKK but also induces an IKK-dependent phosphorylation of CSN5 at serine 201, linking CSN to TNF-α signaling through IKK. Moreover, TNF-α treatment affects the CSN interaction network globally, especially the associations of CSN with the proteasome complex, eukaryotic translation initiation factor complex, and CRL components. Collectively, our results provide new insights into IKK-mediated regulation of CSN associated with the NF-κB signaling pathway.

SCFFBXO22 targets HDM2 for degradation and modulates breast cancer cell invasion and metastasis.

Human homolog of mouse double minute 2 (HDM2) is an oncogene frequently overexpressed in cancers with poor prognosis, but mechanisms of controlling its abundance remain elusive. In an unbiased biochemical search, we discovered Skp1-Cullin 1-FBXO22-ROC1 (SCFFBXO22) as the most dominating HDM2 E3 ubiquitin ligase from human proteome. The results of protein decay rate analysis, ubiquitination, siRNA-mediated silencing, and coimmunoprecipitation experiments support a hypothesis that FBXO22 targets cellular HDM2 for ubiquitin-dependent degradation. In human breast cancer cells, FBXO22 knockdown (KD) increased cell invasiveness, which was driven by elevated levels of HDM2. Moreover, mouse 4T1 breast tumor model studies revealed that FBXO22 KD led to a significant increase of breast tumor cell metastasis to the lung. Finally, low FBXO22 expression is correlated with worse survival and high HDM2 expression in human breast cancer. Altogether, these findings suggest that SCFFBXO22 targets HDM2 for degradation and possesses inhibitory effects against breast cancer tumor cell invasion and metastasis.

A potent small-molecule inhibitor of the DCN1-UBC12 interaction that selectively blocks cullin 3 neddylation.

The Cullin-RING E3 ubiquitin ligases (CRLs) regulate homeostasis of ~20% of cellular proteins and their activation require neddylation of their cullin subunit. Cullin neddylation is modulated by a scaffolding DCN protein through interactions with both the cullin protein and an E2 enzyme such as UBC12. Here we report the development of DI-591 as a high-affinity, cell-permeable small-molecule inhibitor of the DCN1-UBC12 interaction. DI-591 binds to purified recombinant human DCN1 and DCN2 proteins with K i values of 10-12 nM, and disrupts the DCN1-UBC12 interaction in cells. Treatment with DI-591 selectively converts cellular cullin 3 into an un-neddylated inactive form with no or minimum effect on other cullin members. Our data firmly establish a previously unrecognized specific role of the DCN1-UBC12 interaction for cellular neddylation of cullin 3. DI-591 is an excellent probe compound to investigate the role of the cullin 3 CRL ligase in biological processes and human diseases.

Extracellular LDLR repeats modulate Wnt signaling activity by promoting LRP6 receptor endocytosis mediated by the Itch E3 ubiquitin ligase.

The LOW-density lipoprotein related protein 6 (LRP6) receptor is an important effector of canonical Wnt signaling, a developmental pathway, whose dysregulation has been implicated in various diseases including cancer. The membrane proximal low-density lipoprotein (LDL) receptor repeats in LRP6 exhibit homology to ligand binding repeats in the LDL receptor (LDLR), but lack known function. We generated single amino acid substitutions of LRP6-LDLR repeat residues, which are highly conserved in the human LDLR and mutated in patients with Familial Hypercholesteremia (FH). These substitutions negatively impacted LRP6 internalization and activation of Wnt signaling. By mass spectrometry, we observed that the Itch E3 ubiquitin ligase associated with and ubiquitinated wild type LRP6 but not the LDLR repeat mutants. These findings establish the involvement of LRP6-LDLR repeats in the regulation of canonical Wnt signaling.

Neddylation E2 UBE2F Promotes the Survival of Lung Cancer Cells by Activating CRL5 to Degrade NOXA via the K11 Linkage.

Purpose: Recent studies have shown that the process of protein neddylation was abnormally activated in several human cancers. However, it is unknown whether and how UBE2F, a less characterized neddylation E2, regulates lung cancer cell survival, and whether and how NOXA, a proapoptotic protein, is ubiquitylated and degraded by which E3 and via which ubiquitin linkage.Experimental Design: Methods of immunohistochemistry and immunoblotting were utilized to examine UBE2F protein expression. The biological functions of UBE2F were evaluated by in vitro cell culture and in vivo xenograft models. The in vivo complex formation among UBE2F-SAG-CUL5-NOXA was measured by a pulldown assay. Polyubiquitylation of NOXA was evaluated by in vivo and in vitro ubiquitylation assays.Results: UBE2F is overexpressed in non-small cell lung cancer (NSCLC) and predicts poor patient survival. While UBE2F overexpression promotes lung cancer growth both in vitro and in vivo, UBE2F knockdown selectively inhibits tumor growth. By promoting CUL5 neddylation, UBE2F/SAG/CUL5 tri-complex activates CRL5 (Cullin-RING-ligase-5) to ubiquitylate NOXA via a novel K11, but not K48, linkage for targeted proteasomal degradation. CRL5 inactivation or forced expression of K11R ubiquitin mutant caused NOXA accumulation to induce apoptosis, which is rescued by NOXA knockdown. Notably, NOXA knockdown rescues the UBE2F silencing effect, indicating a causal role of NOXA in this process. In lung cancer tissues, high levels of UBE2F and CUL5 correlate with a low level of NOXA and poor patient survival.Conclusions: By ubiquitylating and degrading NOXA through activating CRL5, UBE2F selectively promotes lung cancer cell survival and could, therefore, serve as a novel cancer target. Clin Cancer Res; 23(4); 1104-16. ©2016 AACR.

Suramin inhibits cullin-RING E3 ubiquitin ligases.

Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3's cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1's conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2-E3 interface through small-molecule modulators.

PinX1 serves as a potential prognostic indicator for clear cell renal cell carcinoma and inhibits its invasion and metastasis by suppressing MMP-2 via NF-κB-dependent transcription.

PIN2/TRF1-interacting telomerase inhibitor 1 (PinX1) is a novel cloned gene which has been identified as a major haploinsufficient tumor suppressor essential for maintaining telomerase activity, the length of telomerase and chromosome stability. This study explored the clinical significance and biological function of PinX1 in human clear cell renal cell carcinoma (ccRCC). The clinical relevance of PinX1 in ccRCC was evaluated using tissue microarray and immunohistochemical staining in two independent human ccRCC cohorts. Our data demonstrated that PinX1 expression was dramatically decreased in ccRCC tissues compared with normal renal tissues and paired adjacent non-tumor tissues. Low PinX1 expression was significantly correlated with depth of invasion, lymph node metastasis and advanced TNM stage in patients, as well as with worse overall and disease-specific survival. Cox regression analysis revealed that PinX1 expression was an independent prognostic factor for ccRCC patients. Moreover, PinX1 inhibited the migration and invasion of ccRCC by suppressing MMP-2 expression and activity via NF-κB-dependent transcription in vitro. In vivo studies confirmed that PinX1 negatively regulated ccRCC metastasis and the expression of MMP-2 and NF-κB-p65. These findings indicate that PinX1 suppresses ccRCC metastasis and may serve as a ccRCC candidate clinical prognostic marker and a potential therapeutic target.

Decreased expression of CHIP leads to increased angiogenesis via VEGF-VEGFR2 pathway and poor prognosis in human renal cell carcinoma.

CHIP (c-terminal Hsp70-interacting protein) is an E3 ligase which may play different roles in different cancers. The elucidation of the VHL-HIF-1α (hypoxia inducible factor-1α)-VEGF (vascular endothelial growth factor) pathway has led to the development of targeted therapy in renal cell carcinoma (RCC). However, little is known about the role of CHIP and the relationship between CHIP and VEGF-VEGFR2 (VEGF receptor 2) pathway in RCC. In this study, we found that the expression of CHIP was downregulated and significantly correlated with pT status (P = 0.022) and TNM stage (P = 0.022) in 304 RCC and 35 normal renal tissues using tissue microarray. Moreover, low expression of CHIP is a strong and independent negative prognostic value for RCC. In vitro, CHIP negatively regulated RCC cell migration, invasion and angiogenesis. In addition, ELISA tests showed that restoration of CHIP inhibited, while knockdown promoted, the secreted level of VEGF. Furthermore, western blot indicated that the VEGFR2 protein level was reduced after CHIP overexpression. Our findings demonstrate for the first time that CHIP may be involved in RCC angiogenesis through regulating VEGF secretion and expression of VEGFR2. CHIP may serve as promising prognostic biomarker of angiogenesis and may constitute a potential therapeutic target in RCC.

PinX1 inhibits the invasion and metastasis of human breast cancer via suppressing NF-κB/MMP-9 signaling pathway.

BACKGROUND: PinX1 (PIN2/TRF1-interacting telomerase inhibitor 1) was suggested to be correlated with tumor progression. This study was designed to evaluate the role of PinX1 in human breast cancer. METHODS: To evaluate the function of PinX1 in breast cancer, we used a tissue microarray (TMA) of 405 human breast cancer patients and immunohistochemistry to analyze the correlation between PinX1 expression and clinicopathologic variables and patient survival. We also detected the abilities of cell migration and invasion in breast cancer by performing cell migration and invasion assay, gelatin zymography and western blot analysis. Lastly, we set up the nude mice model by Tail vein assay to exam the functional role of PinX1 in breast cancer metastasis. RESULTS: We found that low PinX1 expression was associated with lymph node metastasis (P = 0.002) and histology grade (P = 0.001) in patients, as well as with poorer overall and disease-specific survival (P = 0.010 and P = 0.003, respectively). Moreover, we identified that PinX1 inhibited the migration and invasion of breast cancer by suppressing MMP-9 expression and activity via NF-κB-dependent transcription in vitro. Finally, our mice model confirmed that PinX1 suppressed breast cancer metastasis in vivo. CONCLUSIONS: Our data revealed that low PinX1 expression was an independent negative prognostic factor for breast cancer patients. These findings suggested that PinX1 might be function as a tumor metastasis suppressor in the development and progression of breast cancer by regulating the NF-κB/MMP-9 signaling pathway, and might be a prognostic marker as well as a therapeutic target for breast cancer.

Generation of a proteolytic signal: E3/E2-mediated polyubiquitination of IκBα.

A key regulatory node in NF-κB signaling is the removal of the IκBα inhibitor, whose levels are tightly controlled by the ubiquitin-proteasome system. In response to signal activation and transmission, ubiquitin E1, E2, and E3 enzymes are employed to generate a lysine 48-linked ubiquitin chain that triggers degradation of IκBα by the proteasome. In this chapter we describe an in vitro biochemical approach to reconstitute the ubiquitination system. To do so, we detail methods for the preparation of the relevant enzymes and substrate, as well as for the execution of the reaction with high efficiency. This sensitive and highly reproducible readout can be applied to the study of proteins, small molecules, and other factors that modulate IκBα ubiquitination, thereby producing outcomes that impact NF-κB signaling to advance the course of improving human health.