Ub-ProT reveals global length and composition of protein ubiquitylation in cells.
Hikaru TsuchiyaDaocharad BuranaFumiaki OhtakeNaoko AraiAi KaihoMasayuki KomadaKeiji TanakaYasushi SaekiPublished in: Nature communications (2018)
Protein ubiquitylation regulates diverse cellular processes via distinct ubiquitin chains that differ by linkage type and length. However, a comprehensive method for measuring these properties has not been developed. Here we describe a method for assessing the length of substrate-attached polyubiquitin chains, "ubiquitin chain protection from trypsinization (Ub-ProT)." Using Ub-ProT, we found that most ubiquitylated substrates in yeast-soluble lysate are attached to chains of up to seven ubiquitin molecules. Inactivation of the ubiquitin-selective chaperone Cdc48 caused a dramatic increase in chain lengths on substrate proteins, suggesting that Cdc48 complex terminates chain elongation by substrate extraction. In mammalian cells, we found that ligand-activated epidermal growth factor receptor (EGFR) is rapidly modified with K63-linked tetra- to hexa-ubiquitin chains following EGF treatment in human cells. Thus, the Ub-ProT method can contribute to our understanding of mechanisms regulating physiological ubiquitin chain lengths and composition.
Keyphrases
- epidermal growth factor receptor
- small molecule
- tyrosine kinase
- small cell lung cancer
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- advanced non small cell lung cancer
- induced apoptosis
- cell cycle
- gene expression
- combination therapy
- dna methylation
- cell cycle arrest
- cell death
- binding protein
- cell proliferation
- heat stress
- hiv testing
- heat shock
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