Covalent Modification of the NF-κB Essential Modulator (NEMO) by a Chemical Compound Can Regulate Its Ubiquitin Binding Properties in Vitro

Abstract

Background: NEMO is a known ubiquitin-binding protein that functions as a key regulator of NF-B activation. Results: WA is able to covalently modify NEMO to induce a gain-of-function binding to long Lys-48-linked polyubiquitin chains. Conclusion: WA can change the NEMO specificity for polyubiquitin chain interaction. Significance: This study reveals a novel drug class to target the UBD:ubiquitin interaction.

Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

“…Plasmids were transformed into the BL21 Rosetta 2 E. coli strain and induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside, followed by lysis and purification by glutathione-agarose beads (G4510, Pierce) and elution by 50 mM reduced glutathione at pH 8.0. Removal of GST from GST-H1-P1 to generate H1-P1c was done by incubation with GST-fused PreScission protease (GE Healthcare) as described previously (59).…”

Potential Anticancer Properties and Mechanisms of Action of Withanolides

“…WFA also preferentially promotes NEMO to bind to long Lys48-linked polyubiquitin chains but not to tetra-ubiquitin species. These data indicate that WFA may alter the ubiquitin interaction properties of NEMO, an ubiquitin-binding protein [88], and its reorganization [89]. It will be of interest to examine the effect of WFA on other ubiquitin and ubiquitin-binding proteins.…”

Identification of protein adduction using mass spectrometry: Protein adducts as biomarkers and predictors of toxicity mechanisms

“…Human serum albumin is the major target of amoxicillin, and haptenation at Lys‐190 was identified in ex vivo samples from healthy subjects dosed with amoxicillin, which is helpful in delineating the mechanisms and developing new diagnostic tools for the allergic effects. Withaferin, a steroidal lactone, was demonstrated to covalently modify NF‐κB Essential Modulator (NEMO), a key polyubiquitin chain‐binding protein in canonical NF‐κB signaling . The modification occurred at Cys‐397 in the C‐terminal zinc finger (ZF) domain of NEMO.…”

“…For rats orally dosed with 0.5 mg MMS/kg (n = 5) for 4 days, the mean concentration of alkylated hemoglobin was 10.02 pmol/mg. [104] Protein adducts with other xenobiotics Important findings have been achieved with MS towards the identification/characterization of protein adducts by other xenobiotics, such as amoxicillin, [107] withaferin, [108] diclofenac, [109] fipexide, [105] mycophenolic acid [112] and monocrotaline pyrrole. [106] Such findings give deeper insights Figure 5.…”

“…Withaferin, a steroidal lactone, was demonstrated to covalently modify NF-κB Essential Modulator (NEMO), a key polyubiquitin chain-binding protein in canonical NF-κB signaling. [109] The modification occurred at Cys-397 in the C-terminal zinc finger (ZF) domain of NEMO. This modification led to an alteration in NEMO's interaction with polyubiquitin chains both in vitro and in vivo, directing the binding to long Lys-48-linked polyubiquitin chains but not tetra-ubiquitin species, thereby shedding light on the design of novel drugs to modulate ubiquitin-based cellular processes.…”