leupeptins and pevonedistat

The F-box protein is the substrate recognition subunit of SCF (SKP1/CUL1/F-box) E3 ubiquitin ligase complex, a multicomponent RING-type E3 ligase involved in the regulation of numerous cellular processes by targeting critical regulatory proteins for ubiquitination. However, whether and how F-box proteins are regulated is largely unknown. Here we report that FBXO28, a poorly characterized F-box protein, is a novel substrate of SCF E3 ligase. Pharmaceutical or genetic inhibition of neddylation pathway that is required for the activation of SCF stabilizes FBXO28 and prolongs its half-life. Meanwhile, FBXO28 is subjected to ubiquitination and cullin1-based SCF complex promotes FBXO28 degradation. Moreover, deletion of F-box domain stabilizes FBXO28 and knockdown of endogenous FBXO28 strongly upregulates exogenous FBXO28 expression. Taken together, these data reveal that SCF

Topics: Antineoplastic Agents; Cell Line; Chromatography, Liquid; Cullin Proteins; Cyclopentanes; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; F-Box Proteins; Humans; Leupeptins; Proteasome Endopeptidase Complex; Proteome; Pyrimidines; Signal Transduction; SKP Cullin F-Box Protein Ligases; Tandem Mass Spectrometry; Transcriptional Activation; Ubiquitination

Modification of proteins with ubiquitin and ubiquitin-like molecules is involved in the regulation of almost every biological process. Historically, each conjugation pathway has its unique set of E1, E2 and E3 enzymes that lead to activation and conjugation of their cognate molecules. Here, we present the unexpected finding that under stress conditions, the ubiquitin E1 enzyme Ube1 mediates conjugation of the ubiquitin-like molecule NEDD8. Inhibition of the 26S proteasome, heat shock and oxidative stress cause a global increase in NEDDylation. Surprisingly, this does not depend on the NEDD8 E1-activating enzyme, but rather on Ube1. A common event in the tested stress conditions is the depletion of "free" ubiquitin. A decrease in "free" ubiquitin levels in the absence of additional stress is sufficient to stimulate NEDDylation through Ube1. Further analysis on the NEDD8 proteome shows that the modified NEDDylated proteins are simultaneously ubiquitinated. Mass spectrometry on the complex proteome under stress reveals the existence of mixed chains between NEDD8 and ubiquitin. We further show that NEDDylation of the p53 tumor suppressor upon stress is mediated mainly through Ube1. Our studies reveal an unprecedented interplay between NEDD8 and ubiquitin pathways operating in diverse cellular stress conditions.

Topics: Amino Acid Sequence; Blotting, Western; Cell Line, Tumor; Cyclopentanes; Enzyme Activation; Gene Knockdown Techniques; Humans; Leupeptins; Mass Spectrometry; Molecular Sequence Data; NEDD8 Protein; Oxidative Stress; Proteasome Endopeptidase Complex; Pyrimidines; Time Factors; Transfection; Tumor Suppressor Protein p53; Ubiquitin-Activating Enzymes; Ubiquitins