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Free keywords:
Amino Acid Sequence
Blotting, Western
Cell Line, Tumor
Cyclopentanes/pharmacology
Enzyme Activation
Gene Knockdown Techniques
Humans
Leupeptins/pharmacology
Mass Spectrometry
Molecular Sequence Data
*Oxidative Stress
Proteasome Endopeptidase Complex/drug effects/metabolism
Pyrimidines/pharmacology
Time Factors
Transfection
Tumor Suppressor Protein p53/metabolism
Ubiquitin-Activating Enzymes/genetics/*metabolism
Ubiquitins/genetics/*metabolism
Abstract:
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.
