Characterizing Protein Dynamics of Protein-Ligand Interactions by Hydrogen-Deuterium Exchange Mass Spectrometry

The study of protein-ligand and protein-protein interactions is of paramount importance to the understanding of their biological function. Whereas this area of research has been largely dominated by conventional structural biology techniques, such as NMR and X-ray crystallography, an emerging methodology that relies on the implementation of hydrogen deuterium exchange (HDX) powered by MS-based analysis holds the potential to greatly expand on our ability to probe the protein dynamics of fundamental biological processes. In this work, the entire HDX workflow for site-specific analysis of protein dynamics was integrated onto a concerted microfluidic device and applied to the interrogation of the dynamic changes that accompany protein-ligand interactions. This application is described for two model systems: the binding of glutathione (GSH) by Glutathione-S-Transferase (GST), and the binding of three novel salicylic acid-based inhibitors of Signal Transducer and Activator of Transcription 3 (STAT3) to its SH2 domain. This work extends the application of time-resolved electrospray ionization mass spectrometry (TRESI-MS) HDX to the study of protein ligand interaction dynamics and ligand-binding site mapping

MYC Deregulation in Primary Human Cancers

MYC regulates a complex biological program by transcriptionally activating and repressing its numerous target genes. As such, MYC is a master regulator of many processes, including cell cycle entry, ribosome biogenesis, and metabolism. In cancer, the activity of the MYC transcriptional network is frequently deregulated, contributing to the initiation and maintenance of disease. Deregulation often leads to constitutive overexpression of MYC, which can be achieved through gross genetic abnormalities, including copy number alterations, chromosomal translocations, increased enhancer activity, or through aberrant signal transduction leading to increased MYC transcription or increased MYC mRNA and protein stability. Herein, we summarize the frequency and modes of MYC deregulation and describe both well-established and more recent findings in a variety of cancer types. Notably, these studies have highlighted that with an increased appreciation for the basic mechanisms deregulating MYC in cancer, new therapeutic vulnerabilities can be discovered and potentially exploited for the inhibition of this potent oncogene in cancer

MYC dephosphorylation by the PP1/PNUTS phosphatase complex regulates chromatin binding and protein stability.

The c-MYC (MYC) oncoprotein is deregulated in over 50% of cancers, yet regulatory mechanisms controlling MYC remain unclear. To this end, we interrogated the MYC interactome using BioID mass spectrometry (MS) and identified PP1 (protein phosphatase 1) and its regulatory subunit PNUTS (protein phosphatase-1 nuclear-targeting subunit) as MYC interactors. We demonstrate that endogenous MYC and PNUTS interact across multiple cell types and that they co-occupy MYC target gene promoters. Inhibiting PP1 by RNAi or pharmacological inhibition results in MYC hyperphosphorylation at multiple serine and threonine residues, leading to a decrease in MYC protein levels due to proteasomal degradation through the canonical SCFFBXW7 pathway. MYC hyperphosphorylation can be rescued specifically with exogenous PP1, but not other phosphatases. Hyperphosphorylated MYC retained interaction with its transcriptional partner MAX, but binding to chromatin is significantly compromised. Our work demonstrates that PP1/PNUTS stabilizes chromatin-bound MYC in proliferating cells

MYC dephosphorylation by the PP1/PNUTS phosphatase complex regulates chromatin binding and protein stability.

The c-MYC (MYC) oncoprotein is deregulated in over 50% of cancers, yet regulatory mechanisms controlling MYC remain unclear. To this end, we interrogated the MYC interactome using BioID mass spectrometry (MS) and identified PP1 (protein phosphatase 1) and its regulatory subunit PNUTS (protein phosphatase-1 nuclear-targeting subunit) as MYC interactors. We demonstrate that endogenous MYC and PNUTS interact across multiple cell types and that they co-occupy MYC target gene promoters. Inhibiting PP1 by RNAi or pharmacological inhibition results in MYC hyperphosphorylation at multiple serine and threonine residues, leading to a decrease in MYC protein levels due to proteasomal degradation through the canonical SCFFBXW7 pathway. MYC hyperphosphorylation can be rescued specifically with exogenous PP1, but not other phosphatases. Hyperphosphorylated MYC retained interaction with its transcriptional partner MAX, but binding to chromatin is significantly compromised. Our work demonstrates that PP1/PNUTS stabilizes chromatin-bound MYC in proliferating cells