The MYC oncoprotein directly interacts with its chromatin cofactor PNUTS to recruit PP1 phosphatase.

Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remain an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation, chromatin occupancy, and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. By NMR we identified a distinct peptide motif within MB0 that interacts with PNUTS residues 1-148, a functional unit, here termed PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its MYC-binding patch. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo, leading to elevated MYC phosphorylation. These data demonstrate that the MB0 region of MYC directly interacts with the PAD of PNUTS, which provides new insight into the control mechanisms of MYC as a regulator of gene transcription and a pervasive cancer driver.

Multiple direct interactions of TBP with the MYC oncoprotein.

Transcription factor c-MYC is a potent oncoprotein; however, the mechanism of transcriptional regulation via MYC-protein interactions remains poorly understood. The TATA-binding protein (TBP) is an essential component of the transcription initiation complex TFIID and is required for gene expression. We identify two discrete regions mediating MYC-TBP interactions using structural, biochemical and cellular approaches. A 2.4 -Å resolution crystal structure reveals that human MYC amino acids 98-111 interact with TBP in the presence of the amino-terminal domain 1 of TBP-associated factor 1 (TAF1TAND1). Using biochemical approaches, we have shown that MYC amino acids 115-124 also interact with TBP independently of TAF1TAND1. Modeling reveals that this region of MYC resembles a TBP anchor motif found in factors that regulate TBP promoter loading. Site-specific MYC mutants that abrogate MYC-TBP interaction compromise MYC activity. We propose that MYC-TBP interactions propagate transcription by modulating the energetic landscape of transcription initiation complex assembly.

Cytotoxic unsaturated electrophilic compounds commonly target the ubiquitin proteasome system.

A large number of natural products have been advocated as anticancer agents. Many of these compounds contain functional groups characterized by chemical reactivity. It is not clear whether distinct mechanisms of action can be attributed to such compounds. We used a chemical library screening approach to demonstrate that a substantial fraction (~20%) of cytotoxic synthetic compounds containing Michael acceptor groups inhibit proteasome substrate processing and induce a cellular response characteristic of proteasome inhibition. Biochemical and structural analyses showed binding to and inhibition of proteasome-associated cysteine deubiquitinases, in particular ubiquitin specific peptidase 14 (USP14). The results suggested that compounds bind to a crevice close to the USP14 active site with modest affinity, followed by covalent binding. A subset of compounds was identified where cell death induction was closely associated with proteasome inhibition and that showed significant antineoplastic activity in a zebrafish embryo model. These findings suggest that proteasome inhibition is a relatively common mode of action by cytotoxic compounds containing Michael acceptor groups and help to explain previous reports on the antineoplastic effects of natural products containing such functional groups.