Anti-UBR2 (aa 45-338) polyclonal antibody

C/EBP beta is a key mediator of cancer-induced skeletal muscle wasting. However, the signaling mechanisms that activate C/EBP beta in the cancer milieu are poorly defined. Here, we report cancer-induced muscle wasting requires the transcriptional cofactor p300, which is critical for the activation of C/EBP beta. Conditioned media from diverse types of tumor cells as well as recombinant HSP70 and HSP90 provoked rapid acetylation of C/EBP beta in myotubes, particularly at its Lys39 residue. Overexpression of C/EBP beta with mutated Lys39 impaired Lewis lung carcinoma (LLC)-induced activation of the C/EBP beta-dependent catabolic response, which included upregulation of E3 ligases UBR2 and atrogin1/MAFbx, increased LC3-II, and loss of muscle proteins both in myotubes and mouse muscle. Silencing p300 in myotubes or overexpressing a dominant negative p300 mutant lacking acetyltransferase activity in mouse muscle attenuated LLC tumor-induced muscle catabolism. Administration of pharmacologic p300 inhibitor C646, but not PCAF/GCN5 inhibitor CPTH6, spared LLC tumor-bearing mice from muscle wasting. Furthermore, mice with muscle-specific p300 knockout were resistant to LLC tumor-induced muscle wasting. These data suggest that p300 is a key mediator of LLC tumor-induced muscle wasting whose acetyltransferase activity may be targeted for therapeutic benefit in this disease. Significance: These findings demonstrate that tumor-induced muscle wasting in mice is abrogated by knockout, mutation of Lys39 or Asp1399, and pharmacologic inhibition of p300. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1331/F1.large.jpg.

The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the Rpn4 transcription factor upregulates the proteasome genes and is rapidly degraded by the proteasome. Previous work has identified Ubr2 and Rad6 as the cognate E3 and E2 enzymes for Rpn4 ubiquitylation. However, our recent attempts to ubiquitylate Rpn4 using purified Ubr2 and Rad6 proteins in a reconstitution system have been unsuccessful, suggesting that an additional factor is required for Rpn4 ubiquitylation. Here, We screened the entire collection of the single-gene-deletion yeast mutants generated by the Saccharomyces Genome Deletion Project and identified the mubl Delta mutant defective in ubiquitin-dependent degradation of Rpn4. An in vitro reconstitution ubiquitylation assay confirms that Mub1 is the missing factor for Rpn4 ubiquitylation. We further show that Mub1 directly interacts with Ubr2 and Rpn4. The MYND domain of Mub1 may play an important role in Rpn4 ubiquitylation. Interestingly, Mub1 itself is a short-lived protein and its degradation is dependent on the Ubr2/Rad6 ubiquitin ligase. Together, these data suggest that Mub1 and Ubr2 cooperate to transfer ubiquitin to Rpn4 from Rad6 and that Mub1 may switch from a partner to a substrate of the Ubr2/Rad6 ubiquitin ligase.