Anti-Ubiquitin+1 monoclonal antibody, clone 41C4

We report a general method for amino acid-type specific O-17-labeling of recombinant proteins in Escherichia coli. In particular, we have prepared several [1-C-13,O-17]-labeled yeast ubiquitin (Ub) samples including Ub-[1-C-13,O-17]Gly, Ub-[1-C-13,O-17]Tyr, and Ub-[1-C-13,O-17]Phe using the auxotrophic E. coli strain DL39 GlyA lambda DE3 (aspC(-) tyrB(-) ilvE(-) glyA(-) lambda DE3). We have also produced Ub-[eta-O-17]Tyr, in which the phenolic group of Tyr59 is O-17-labeled. We show for the first time that O-17 NMR signals from protein terminal residues and side chains can be readily detected in aqueous solution. We also reported solid-state O-17 NMR spectra for Ub-[1-C-13,O-17]Tyr and Ub-[1-C-13,O-17]Phe obtained at an ultrahigh magnetic field, 35.2 T (1.5 GHz for H-1). This work represents a significant advance in the field of O-17 NMR studies of proteins.

The transcription factor SOX9 is frequently amplified in diverse advanced-stage human tumors. Its stability has been shown to be tightly controlled by ubiquitination-dependent proteasome degradation. However, the exact underlying molecular mechanisms remain unclear. This work reports that SOX9 protein abundance is regulated by the Cullin 3-based ubiquitin ligase KEAP1 via proteasome-mediated degradation. Loss-of-function mutations in KEAP1 compromise polyubiquitination-mediated SOX9 degradation, leading to increased protein levels, which facilitate tumorigenesis. Moreover, the loss of critical ubiquitination residues in SOX9, by either a SOX9 (Delta K2) truncation or K249R mutation, leads to elevated protein stability. Furthermore, it is shown that the KEAP1/SOX9 interaction is modulated by CKI gamma-mediated phosphorylation. Importantly, it is demonstrated that DNA damage drugs, topoisomerase inhibitors, can trigger CKI activation to restore the KEAP1/SOX9 interaction and its consequent degradation. Collectively, herein the findings uncover a novel molecular mechanism through which SOX9 protein stability is negatively regulated by KEAP1 to control tumorigenesis. Thus, these results suggest that mitigating SOX9 resistance to KEAP1-mediated degradation can represent a novel therapeutic strategy for cancers with KEAP1 mutations.