%0 Journal Article %J Mol. Cell %D 2023 %T A central role for regulated protein stability in the control of TFE3 and MITF by nutrients %A C. Nardone %A B. A. Palanski %A D. C. Scott %A R. T. Timms %A K. W. Barber %A X. Gu %A A. Mao %A Y. Leng %A E. V. Watson %A B. A. Schulman %A Cole, P. A. %A S. J. Elledge %B Mol. Cell %V 83 %P 57-73 %G eng %U https://www.sciencedirect.com/science/article/pii/S1097276522011704?via%3Dihub %0 Journal Article %J J. Am. Chem. Soc. %D 2023 %T Histone H2B Deacylation Selectivity: Exploring Chromatin’s Dark Matter with an Engineered Sortase %A Wang, Z. A. %A S. D. Whedon %A Wu, M. %A E.A. Brown %A A. Anmangandla %A L. Regan %A K. Lee %A J. Du %A J.Y. Hong %A Fairall, L. %A T. Kay %A H. Lin %A Zhao, Y. %A J.W.R. Schwabe %A Cole, P. A. %B J. Am. Chem. Soc. %V 144 %P 3360-3364 %G eng %U https://pubs.acs.org/doi/full/10.1021/jacs.1c13555 %0 Journal Article %J J. Am. Chem. Soc. %D 2023 %T Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination %A Iwase, R. %A Dempsey, D. R. %A S. D. Whedon %A H. Jiang %A B. A. Palanski %A Deng, B. %A Cole, P. A. %B J. Am. Chem. Soc. %V 145 %P 6039–6044 %G eng %U https://pubs.acs.org/doi/abs/10.1021/jacs.2c13871 %0 Journal Article %J Curr Opin Chem Biol %D 2022 %T Distinct biochemical properties of the class I histone deacetylase complexes %A K. Lee %A S. D. Whedon %A Wang, Z. A. %A Cole, P. A. %B Curr Opin Chem Biol %V 70 %P 102179 %G eng %0 Journal Article %J J. Biol. Chem. %D 2022 %T Enzymatic Analysis of WWP2 E3 Ubiquitin Ligase Using Protein Microarrays Identifies Autophagy-Related Substrates %A H. Jiang %A C. Chiang %A Chen, Z. %A S. Nathan %A G. D’Agostino %A J. Paulo %A Song, G. %A H. Zhu %A S.B. Gabelli %A Cole, P. A. %B J. Biol. Chem. %V 298 %P 101854 %G eng %0 Journal Article %J eLife %D 2022 %T PH domain-mediated autoinhibition and oncogenic activation of Akt %A Bae, H. %A Viennet, T. %A E. Park %A Chu, N. %A Salguero, A. %A Eck, M. J. %A Arthanari, H. %A Cole, P. A. %B eLife %V 11 %P e80148 %G eng %0 Journal Article %J Mol. Cancer Res. %D 2022 %T Epigenetic Silencing of BMP6 by Sin3A-HDAC1/2 Repressor Complex Drives Melanoma Metastasis via FAM83G/PAWS1 %A Min, D. %A Byun, J. %A Lee, E.H. %A Khan, A.A. %A C. Liu %A Loudig, O. %A W. Hu %A Zhao, Y. %A Herlyn, M. %A Tycko, B. %A Cole, P. A. %A Ryu, B. %B Mol. Cancer Res. %V 20 %P 217-230 %G eng %0 Journal Article %J J. Am. Chem. Soc. %D 2022 %T Histone H2B Deacylation Selectivity: Exploring Chromatin’s Dark Matter with an Engineered Sortase %A Wang, Z. A. %A S. D. Whedon %A Wu, M. %A S. Weng %A E.A. Brown %A A. Anmangandla %A L. Regan %A K. Lee %A J. Du %A J.Y. Hong %A Fairall, L. %A T. Kay %A H. Lin %A Zhao, Y. %A J.W.R. Schwabe %A Cole, P. A. %B J. Am. Chem. Soc. %V 144 %P 3360-3364 %G eng %0 Journal Article %J Curr. Opin. Chem. Biol. %D 2022 %T KATs off: Biomedical insights from lysine acetyltransferase inhibitors %A S. Whedon %A Cole, P. A. %B Curr. Opin. Chem. Biol. %V 72 %P 102255 %G eng %U https://www.sciencedirect.com/science/article/pii/S1367593122001405?via%3Dihub %0 Journal Article %J ACS Chem. Biol. %D 2022 %T Multifaceted Regulation of Akt by Diverse C-terminal Post-translational Modifications %A Salguero, Antonieta %A Chen, Maggie %A Balana, Aaron %A Chu, Nam %A Jiang, Hanjie %A Bae, Hwan %A Palanski, Brad %A Wright, Katharine %A Nathan, Sara %A Zhu, Heng %A Gabelli, Sandra %A Pratt, Matthew %A Cole, Philip A %B ACS Chem. Biol. %V 17 %P 68-76 %G eng %0 Journal Article %J Mol Cancer Res %D 2021 %T Chemical Screen Identifies Diverse and Novel Histone Deacetylase Inhibitors as Repressors of NUT Function: Implications for NUT Carcinoma Pathogenesis and Treatment %A Shiota, Hitoshi %A Alekseyenko, Artyom A %A Wang, Zhipeng A %A Filic, Ivona %A Knox, Tatiana M %A Luong, Nhi M %A Huang, Yeying %A Scott, David A %A Jones, Kristen L %A Gokhale, Prafulla C %A Lemieux, Madeleine E %A Cole, Philip A %A Kuroda, Mitzi I %A French, Christopher A %X NUT carcinoma (NC), characterized most commonly by the BRD4-NUTM1 fusion, is a rare, aggressive variant of squamous carcinoma with no effective treatment. BRD4-NUT drives growth and maintains the poorly differentiated state of NC by activating pro-growth genes such as MYC, through the formation of massive, hyperacetylated, superenhancer-like domains termed megadomains. BRD4-NUT-mediated hyperacetylation of chromatin is facilitated by the chromatin-targeting tandem bromodomains of BRD4, combined with NUT, which recruits the histone acetyltransferase, p300. Here, we developed a high-throughput small-molecule screen to identify inhibitors of transcriptional activation by NUT. In this dCAS9-based GFP-reporter assay, the strongest hits were diverse histone deacetylase (HDAC) inhibitors. Two structurally unrelated HDAC inhibitors, panobinostat and the novel compound, IRBM6, both repressed growth and induced differentiation of NC cells in proportion to their inhibition of NUT transcriptional activity. These two compounds repressed transcription of megadomain-associated oncogenic genes, such as MYC and SOX2, while upregulating pro-differentiation, non-megadomain-associated genes, including JUN, FOS, and key cell-cycle regulators, such as CDKN1A. The transcriptional changes correlate with depletion of BRD4-NUT from megadomains, and redistribution of the p300/CBP-associated chromatin acetylation mark, H3K27ac, away from megadomains toward regular enhancer regions previously populated by H3K27ac. In NC xenograft models, we demonstrated that suppression of tumor growth by panobinostat was comparable with that of bromodomain inhibition, and when combined they improved both survival and growth suppression. IMPLICATIONS: The findings provide mechanistic and preclinical rationale for the use of HDAC inhibitors, alone or combined with other agents, in the treatment of NUT carcinoma. %B Mol Cancer Res %V 19 %P 1818-1830 %8 2021 Nov %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/34285087?dopt=Abstract %R 10.1158/1541-7786.MCR-21-0259 %0 Journal Article %J Bioorg Med Chem Lett %D 2021 %T Discovery of spirohydantoins as selective, orally bioavailable inhibitors of p300/CBP histone acetyltransferases %A Ji, Zhiqin %A Clark, Richard F %A Bhat, Vikram %A Matthew Hansen, T %A Lasko, Loren M %A Bromberg, Kenneth D %A Manaves, Vlasios %A Algire, Mikkel %A Martin, Ruth %A Qiu, Wei %A Torrent, Maricel %A Jakob, Clarissa G %A Liu, Hong %A Cole, Philip A %A Marmorstein, Ronen %A Kesicki, Edward A %A Lai, Albert %A Michaelides, Michael R %K Administration, Oral %K Biological Availability %K CREB-Binding Protein %K Dose-Response Relationship, Drug %K Drug Discovery %K E1A-Associated p300 Protein %K Enzyme Inhibitors %K Humans %K Hydantoins %K Molecular Structure %K Spiro Compounds %K Structure-Activity Relationship %X p300 and CREB-binding protein (CBP) are essential for a multitude of cellular processes. Dysregulation of p300/CBP histone acetyltransferase activity is linked to a broad spectrum of human diseases including cancers. A novel drug-like spirohydantoin (21) has been discovered as a selective orally bioavailable inhibitor of p300/CBP histone acetyltransferase. Lead compound 21 is more potent than the first-in-class lead A-485 in both enzymatic and cellular assays and lacks the off-target inhibition of dopamine and serotonin transporters, that was observed with A-485. %B Bioorg Med Chem Lett %V 39 %P 127854 %8 2021 05 01 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/33631370?dopt=Abstract %R 10.1016/j.bmcl.2021.127854 %0 Journal Article %J Sci Rep %D 2021 %T HDAC2 targeting stabilizes the CoREST complex in renal tubular cells and protects against renal ischemia/reperfusion injury %A Aufhauser, David D %A Hernandez, Paul %A Concors, Seth J %A O'Brien, Ciaran %A Wang, Zhonglin %A Murken, Douglas R %A Samanta, Arabinda %A Beier, Ulf H %A Krumeich, Lauren %A Bhatti, Tricia R %A Wang, Yanfeng %A Ge, Guanghui %A Wang, Liqing %A Cheraghlou, Shayan %A Wagner, Florence F %A Holson, Edward B %A Kalin, Jay H %A Cole, Philip A %A Hancock, Wayne W %A Levine, Matthew H %K Animals %K Co-Repressor Proteins %K Endothelins %K Enzyme Inhibitors %K Female %K Gene Deletion %K Histone Deacetylase 1 %K Histone Deacetylase 2 %K Isoenzymes %K Kidney Tubules, Proximal %K Male %K Mice %K Mice, Knockout %K Reperfusion Injury %X Histone/protein deacetylases (HDAC) 1 and 2 are typically viewed as structurally and functionally similar enzymes present within various co-regulatory complexes. We tested differential effects of these isoforms in renal ischemia reperfusion injury (IRI) using inducible knockout mice and found no significant change in ischemic tolerance with HDAC1 deletion, but mitigation of ischemic injury with HDAC2 deletion. Restriction of HDAC2 deletion to the kidney via transplantation or PAX8-controlled proximal renal tubule-specific Cre resulted in renal IRI protection. Pharmacologic inhibition of HDAC2 increased histone acetylation in the kidney but did not extend renal protection. Protein analysis demonstrated increased HDAC1-associated CoREST protein in HDAC2-/- versus WT cells, suggesting that in the absence of HDAC2, increased CoREST complex occupancy of HDAC1 can stabilize this complex. In vivo administration of a CoREST inhibitor exacerbated renal injury in WT mice and eliminated the benefit of HDAC2 deletion. Gene expression analysis of endothelin showed decreased endothelin levels in HDAC2 deletion. These data demonstrate that contrasting effects of HDAC1 and 2 on CoREST complex stability within renal tubules can affect outcomes of renal IRI and implicate endothelin as a potential downstream mediator. %B Sci Rep %V 11 %P 9018 %8 2021 04 27 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/33907245?dopt=Abstract %R 10.1038/s41598-021-88242-3 %0 Journal Article %J Sci Adv %D 2021 %T The regulatory enzymes and protein substrates for the lysine β-hydroxybutyrylation pathway %A Huang, He %A Zhang, Di %A Weng, Yejing %A Delaney, Kyle %A Tang, Zhanyun %A Yan, Cong %A Qi, Shankang %A Peng, Chao %A Cole, Philip A %A Roeder, Robert G %A Zhao, Yingming %X Metabolism-mediated epigenetic changes represent an adapted mechanism for cellular signaling, in which lysine acetylation and methylation have been the historical focus of interest. We recently discovered a β-hydroxybutyrate-mediated epigenetic pathway that couples metabolism to gene expression. However, its regulatory enzymes and substrate proteins remain unknown, hindering its functional study. Here, we report that the acyltransferase p300 can catalyze the enzymatic addition of β-hydroxybutyrate to lysine (Kbhb), while histone deacetylase 1 (HDAC1) and HDAC2 enzymatically remove Kbhb. We demonstrate that p300-dependent histone Kbhb can directly mediate in vitro transcription. Moreover, a comprehensive analysis of Kbhb substrates in mammalian cells has identified 3248 Kbhb sites on 1397 substrate proteins. The dependence of histone Kbhb on p300 argues that enzyme-catalyzed acylation is the major mechanism for nuclear Kbhb. Our study thus reveals key regulatory elements for the Kbhb pathway, laying a foundation for studying its roles in diverse cellular processes. %B Sci Adv %V 7 %8 2021 Feb %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/33627428?dopt=Abstract %R 10.1126/sciadv.abe2771 %0 Journal Article %J Nat Struct Mol Biol %D 2021 %T The structural basis of PTEN regulation by multi-site phosphorylation %A Dempsey, Daniel R %A Viennet, Thibault %A Iwase, Reina %A Park, Eun Young %A Henriquez, Stephanie %A Chen, Zan %A Jeliazkov, Jeliazko R %A Palanski, Brad A %A Phan, Kim L %A Coote, Paul %A Gray, Jeffrey J %A Eck, Michael J %A Gabelli, Sandra B %A Arthanari, Haribabu %A Cole, Philip A %K Animals %K Ciona intestinalis %K Crystallography, X-Ray %K Fluorescence Polarization %K Humans %K Magnetic Resonance Spectroscopy %K Molecular Docking Simulation %K Phosphorylation %K PTEN Phosphohydrolase %X Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN's catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN's cellular functions. %B Nat Struct Mol Biol %V 28 %P 858-868 %8 2021 10 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/34625746?dopt=Abstract %R 10.1038/s41594-021-00668-5 %0 Journal Article %J Biochemistry %D 2021 %T Ubiquitin Ligase Activities of WWP1 Germline Variants K740N and N745S %A Jiang, Hanjie %A Dempsey, Daniel R %A Cole, Philip A %X WWP1 is an E3 ubiquitin ligase that has been reported to target the tumor suppressor lipid phosphatase PTEN. K740N and N745S are recently identified germline variants of WWP1 that have been linked to PTEN-associated cancers [Lee, Y. R., et al. (2020) ]. These WWP1 variants have been suggested to release WWP1 from its native autoinhibited state, thereby promoting enhanced PTEN ubiquitination as a mechanism for driving cancer. Using purified proteins and enzymatic assays, we investigate the possibility that K740N and N745S WWP1 possess enhanced ubiquitin ligase activity and demonstrate that these variants are similar to the wild type (WT) in both autoubiquitination and PTEN ubiquitination. Furthermore, K740N and N745S WWP1 show dependencies similar to those of WT in terms of allosteric activation by an engineered ubiquitin variant, upstream E2 concentration, and substrate ubiquitin concentration. Transfected WWP1 WT and mutants demonstrate comparable effects on cellular PTEN levels. These findings challenge the idea that K740N and N745S WWP1 variants promote cancer by enhanced PTEN ubiquitination. %B Biochemistry %8 2021 Jan 20 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/33470109?dopt=Abstract %R 10.1021/acs.biochem.0c00869 %0 Journal Article %J Curr Protoc Mol Bio %D 2021 %T N-Terminal Protein Labeling with N-Hydroxysuccinimide Esters and Microscale Thermophoresis Measurements of Protein-Protein Interactions Using Labeled Protein %A Jiang, Hanjie %A Cole, Philip A %X Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein-protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy-to-apply protocol for high-stoichiometric, site-specific labeling of a protein at its N-terminus with N-hydroxysuccinimide (NHS) esters as a means to measure protein-protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent-labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2-E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Labeling a protein of interest at its N-terminus with NHS esters through stepwise reaction Alternate Protocol: Labeling a protein of interest at its N-terminus with NHS esters through a one-pot reaction Basic Protocol 2: Purifying the N-terminal fluorescent-labeled protein and determining its concentration and labeling efficiency Basic Protocol 3: Using MST to determine the binding affinity of an N-terminal fluorescent-labeled protein to a binding partner. Basic Protocol 4: NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay. %B Curr Protoc Mol Bio %P e14 %8 2021 Jan %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/33484499?dopt=Abstract %R 10.1002/cpz1.14 %0 Journal Article %J JCI Insights %D 2021 %T P300 Suppresses the Transition of Myelodysplastic Syndrome to Acute Myeloid Leukemia %A Na, M. %A Martin, G.M. %A Karl, D.L. %A Sun, J. %A Torres-Martin, M. %A Itonaga, H. %A Martinez, C. %A Chen, S. %A Xu, Y. %A Duffort, S. %A Hamard, P.-J. %A Chen, C. %A Cimmino, L. %A Yang, F.-C. %A Zucconi, B. %A Cole, P. A. %A M. Xu %A Figueroa, M.E. %A Nimer, S.D. %B JCI Insights %V 6 %P e138478 %G eng %0 Journal Article %J Angewandte Chemie %D 2021 %T Site-specific 5-Formyl Cytosine Mediated DNA-Histone Crosslinks: Synthesis and Polymerase Bypass by Human DNA Polymerase η %A Pujari, S.S %A Wu, M. %A Thomforde, J. %A Wang, Z. A. %A Chao, C. %A Olson, N. %A Erber, L. %A Pomerantz, W. %A Cole, P. A. %A Tretyakova, N. %B Angewandte Chemie %V 60 %P 26489-26494 %G eng %0 Journal Article %J Cell Chem Biol %D 2020 %T The Chemical Biology of Reversible Lysine Post-translational Modifications %A Wang, Z. A. %A Cole, P. A. %X Lysine (Lys) residues in proteins undergo a wide range of reversible post-translational modifications (PTMs), which can regulate enzyme activities, chromatin structure, protein-protein interactions, protein stability, and cellular localization. Here we discuss the "writers," "erasers," and "readers" of some of the common protein Lys PTMs and summarize examples of their major biological impacts. We also review chemical biology approaches, from small-molecule probes to protein chemistry technologies, that have helped to delineate Lys PTM functions and show promise for a diverse set of biomedical applications. %B Cell Chem Biol %8 Jul %G eng %0 Journal Article %J Mol. Cancer Ther. %D 2020 %T Combined Targeting of the BRD4-NUT-p300 Axis in NUT Midline Carcinoma by Dual Selective Bromodomain Inhibitor, NEO2734 %A Morrison-Smith, C. D. %A Knox, T. M. %A Filic, I. %A Soroko, K. M. %A Eschle, B. K. %A Wilkens, M. K. %A Gokhale, P. C. %A Giles, F. %A Griffin, A. %A Brown, B. %A Shapiro, G. I. %A Zucconi, B. E. %A Cole, P. A. %A Lemieux, M. E. %A French, C. A. %X NUT midline carcinoma (NMC) is a rare, aggressive subtype of squamous carcinoma that is driven by the BRD4-NUT fusion oncoprotein. BRD4, a BET protein, binds to chromatin through its two bromodomains, and NUT recruits the p300 histone acetyltransferse (HAT) to activate transcription of oncogenic target genes. BET-selective bromodomain inhibitors have demonstrated on-target activity in patients with NMC, but with limited efficacy. P300, like BRD4, contains a bromodomain. We show that combining selective p300/CBP and BET bromodomain inhibitors, GNE-781 and OTX015, respectively, induces cooperative depletion of MYC and synergistic inhibition of NMC growth. Treatment of NMC cells with the novel dual p300/CBP and BET bromodomain-selective inhibitor, NEO2734, potently inhibits growth and induces differentiation of NMC cells in vitro; findings that correspond with potentiated transcriptional effects from combined BET and p300 bromodomain inhibition. In three disseminated NMC xenograft models, NEO2734 provided greater growth inhibition, with tumor regression and significant survival benefit seen in two of three models, compared with a lead clinical BET inhibitor or "standard" chemotherapy. Our findings provide a strong rationale for clinical study of NEO2734 in patients with NMC. Moreover, the synergistic inhibition of NMC growth by CBP/p300 and BET bromodomain inhibition lays the groundwork for greater mechanistic understanding of the interplay between p300 and BRD4-NUT that drives this cancer. %B Mol. Cancer Ther. %V 19 %P 1406–1414 %8 Jul %G eng %N 7 %0 Journal Article %J Elife %D 2020 %T Diverse nucleosome Site-Selectivity among histone deacetylase complexes %A Wang, Z. A. %A Millard, C. J. %A Lin, C. L. %A Gurnett, J. E. %A Wu, M. %A K. Lee %A Fairall, L. %A Schwabe, J. W. %A Cole, P. A. %X Histone acetylation regulates chromatin structure and gene expression and is removed by histone deacetylases (HDACs). HDACs are commonly found in various protein complexes to confer distinct cellular functions, but how the multi-subunit complexes influence deacetylase activities and site-selectivities in chromatin is poorly understood. Previously we reported the results of studies on the HDAC1 containing CoREST complex and acetylated nucleosome substrates which revealed a notable preference for deacetylation of histone H3 acetyl-Lys9 vs. acetyl-Lys14 (Wu et al, 2018). Here we analyze the enzymatic properties of five class I HDAC complexes: CoREST, NuRD, Sin3B, MiDAC and SMRT with site-specific acetylated nucleosome substrates. Our results demonstrate that these HDAC complexes show a wide variety of deacetylase rates in a site-selective manner. A Gly13 in the histone H3 tail is responsible for a sharp reduction in deacetylase activity of the CoREST complex for H3K14ac. These studies provide a framework for connecting enzymatic and biological functions of specific HDAC complexes. %B Elife %V 9 %8 Jun %G eng %0 Journal Article %J J. Clin. Invest. %D 2020 %T Essential Role of the CoREST Complex in Foxp3+ T-Regulatory Cell Functions %A Xiong, Y., Wang, L., Han, R., Belfer, U., Kalin, J., Cole, P. A., Hancock, W. %B J. Clin. Invest. %V 130 %P 1830-1842 %G eng %0 Journal Article %J Cell Reports %D 2020 %T Functional and Structural Coupling Between LSD1 and HDAC1 in the CoREST Corepressor Complex %A Song, Y., Dagil, L., Fairall, L., Robertson, N., Wu, M., Ragan, T. J., Savva, G. C., Morone, N., Kunze, M. B. A., Jamieson, A. G %B Cell Reports %V 30 %P 2699-2711 %G eng %0 Journal Article %J Mol. Cancer Res. %D 2020 %T Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer %A Miller, S. A. %A Policastro, R. A. %A Savant, S. S. %A Sriramkumar, S. %A Ding, N. %A Lu, X. %A Mohammad, H. P. %A Cao, S. %A Kalin, J. H. %A Cole, P. A. %A Zentner, G. E. %A O'Hagan, H. M. %X Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine-specific demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer and enhances cell migration. In this study, we determine that LSD1 expression is significantly elevated in patients with colorectal cancer with mutation of the catalytic subunit of PI3K, PIK3CA, compared with patients with colorectal cancer with WT PIK3CA. LSD1 enhances activation of the AKT kinase in colorectal cancer cells through a noncatalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. In addition, growth of PIK3CA-mutant colorectal cancer cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall, we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA-mutant cells. IMPLICATIONS: Our data support the hypothesis that inhibitors targeting the CoREST complex may be clinically effective in patients with colorectal cancer harboring PIK3CA mutations. %B Mol. Cancer Res. %V 18 %P 264–277 %8 02 %G eng %N 2 %0 Journal Article %J Methods Mol. Biol. %D 2020 %T Methods and Applications of Expressed Protein Ligation %A Wang, Z. A. %A Cole, P. A. %X Expressed protein ligation is a method of protein semisynthesis and typically involves the reaction of recombinant protein C-terminal thioesters with N-cysteine containing synthetic peptides in a chemoselective ligation. The recombinant protein C-terminal thioesters are produced by exploiting the action of nature's inteins which are protein modules that catalyze protein splicing. This chapter discusses the basic principles of expressed protein ligation and recent advances and applications in this protein semisynthesis field. Comparative strengths and weaknesses of the method and future challenges are highlighted. %B Methods Mol. Biol. %V 2133 %P 1–13 %G eng %0 Journal Article %J National Academy of Sciences %D 2020 %T Paul Talalay 1923-2019 A Biographical Memoir %A Cole, P. A. %A Shapiro, T. %B National Academy of Sciences %G eng %U http://www.nasonline.org/publications/biographical-memoirs/memoir-pdfs/talalay-paul.pdf %0 Journal Article %J Nat. Cell Biol. %D 2020 %T The protein kinase Akt acts as a coat adaptor in endocytic recycling %A Hsu, J. W. %A Bai, M. %A Li, K. %A Yang, J. S. %A Chu, N. %A Cole, P. A. %A Eck, M. J. %A Li, J. %A Hsu, V. W. %X Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins1-3. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling4-6. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event. %B Nat. Cell Biol. %V 22 %P 927–933 %8 Aug %G eng %N 8 %0 Journal Article %J Meth. Enzymol. %D 2020 %T Selective protein N-terminal labeling with N-hydroxysuccinimide esters %A H. Jiang %A D'Agostino, G. D. %A Cole, P. A. %A Dempsey, D. R. %X In order to gain detailed insight into the biochemical behavior of proteins, researchers have developed chemical tools to incorporate new functionality into proteins beyond the canonical 20 amino acids. Important considerations regarding effective chemical modification of proteins include chemoselectivity, near stoichiometric labeling, and reaction conditions that maintain protein stability. Taking these factors into account, we discuss an N-terminal labeling strategy that employs a simple two-step "one-pot" method using N-hydroxysuccinimide (NHS) esters. The first step converts a R-NHS ester into a more chemoselective R-thioester. The second step reacts the in situ generated R-thioester with a protein that harbors an N-terminal cysteine to generate a new amide bond. This labeling reaction is selective for the N-terminus with high stoichiometry. Herein, we provide a detailed description of this method and further highlight its utility with a large protein (>100kDa) and labeling with a commonly used cyanine dye. %B Meth. Enzymol. %V 639 %P 333–353 %G eng %0 Journal Article %J Elife %D 2020 %T The Structural Determinants of PH Domain-Mediated Regulation of Akt Revealed by Segmental Labeling %A Chu, N. %A Viennet, T. %A Bae, H. %A Salguero, A. %A Boeszoermenyi, A. %A Arthanari, H. %A Cole, P. A. %X Akt is a critical protein kinase that governs cancer cell growth and metabolism. Akt appears to be autoinhibited by an intramolecular interaction between its N-terminal pleckstrin homology (PH) domain and kinase domain, which is relieved by C-tail phosphorylation, but the precise molecular mechanisms remain elusive. Here we use a combination of protein semisynthesis, NMR, and enzymological analysis to characterize structural features of the PH domain in its autoinhibited and activated states. We find that Akt autoinhibition depends on the length/flexibility of the PH-kinase linker. We identify a role for a dynamic short segment in the PH domain that appears to regulate autoinhibition and PDK1-catalyzed phosphorylation of Thr308 in the activation loop. We determine that Akt allosteric inhibitor MK2206 drives distinct PH domain structural changes compared to baseline autoinhibited Akt. These results highlight how the conformational plasticity of Akt governs the delicate control of its catalytic properties. %B Elife %V 9 %8 Aug %G eng %0 Journal Article %J Curr. Opinion in Struct. Biol. %D 2020 %T Editorial Overview: Biological catalysis at the cross-roads of signaling and metabolism %A Cole, P. A., Mattevi, A. %B Curr. Opinion in Struct. Biol. %V 59 %P iii-iv %G eng %0 Journal Article %J Cancer Cell %D 2019 %T Re-programing Chromatin with a Bifunctional LSD1/HDAC Inhibitor Induces Therapeutic Differentiation in DIP %A Jamie N Anastas %A Zee, Barry M %A Kalin, Jay H %A Mirhee Kim %A Robyn Guo %A Sanda Alexandrescu %A Mario Andres Blanco %A Stefanie Giera %A Gillespie, Shawn M %A Das, Jayanta %A Wu, Muzhou %A Sarah Nocco %A Bonal, Dennis M %A Nguyen, Quang-De %A Mario L Suva %A Bernstein, Bradley E %A Rhoda Alani %A Golub, Todd R %A Cole, Philip A %A Filbin, Mariella G %A Shi, Yang %X H3K27M mutations resulting in epigenetic dysfunction are frequently observed in diffuse intrinsic pontine glioma (DIPGs), an incurable pediatric cancer. We conduct a CRISPR screen revealing that knockout of KDM1A encoding lysine-specific demethylase 1 (LSD1) sensitizes DIPG cells to histone deacetylase (HDAC) inhibitors. Consistently, Corin, a bifunctional inhibitor of HDACs and LSD1, potently inhibits DIPG growth in vitro and in xenografts. Mechanistically, Corin increases H3K27me3 levels suppressed by H3K27M histones, and simultaneously increases HDAC-targeted H3K27ac and LSD1-targeted H3K4me1 at differentiation-associated genes. Corin treatment induces cell death, cell-cycle arrest, and a cellular differentiation phenotype and drives transcriptional changes correlating with increased survival time in DIPG patients. These data suggest a strategy for treating DIPG by simultaneously inhibiting LSD1 and HDACs. %B Cancer Cell %G eng %U https://www.sciencedirect.com/science/article/abs/pii/S1535610819304258?via%3Dihub %0 Journal Article %J J Biol Chem %D 2019 %T Comparative analysis of the catalytic regulation of NEDD4-1 and WWP2 ubiquitin ligases. %A Jiang, Hanjie %A Stefani N. Thomas %A Chen, Zan %A Claire Y. Chiang %A Philip A. Cole %X NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities are important for controlling cellular protein homeostasis and their dysregulation may lead to cancer and other diseases. Previous work has implicated non-catalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecular interactions. Here, we explored the molecular mechanisms of these NEDD4-1 and WWP2 regulatory regions and their interplay with allosteric binding proteins such as Nedd4 family interacting protein (NDFIP1), engineered ubiquitin variants, and linker phosphomimics. We found that in addition to influencing catalytic activities, the WW domain linker regions in NEDD4-1 and WWP2 can impact product distribution, including the degree of polyubiquitination and Lys-48 versus Lys-63 linkages. We show that allosteric activation by NDFIP1 or engineered ubiquitin variants is largely mediated by relief of WW domain linker autoinhibition. WWP2-mediated ubiquitination of WW domain-binding protein 2 (WBP2), phosphatase and tensin homolog (PTEN), and p62 proteins by WWP2 suggests that substrate ubiquitination can also be influenced by WW linker autoinhibition, although to differing extents. Overall, our results provide a deeper understanding of the intricate and multifaceted set of regulatory mechanisms in the control of NEDD4-1 related ubiquitin ligases. %B J Biol Chem %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/?term=31578285 %0 Journal Article %J Curr Opin Struct Biol %D 2019 %T AKTivation mechanisms %A Cole, Philip A %A Chu, Nam %A Salguero, Antonieta L %A Bae, Hwan %X Akt1-3 (Akt) are a subset of the AGC protein Ser/Thr kinase family and play important roles in cell growth, metabolic regulation, cancer, and other diseases. We describe some of the roles of Akt in cell signaling and the biochemical and structural mechanisms of the regulation of Akt catalysis by the phospholipid PIP3 and by phosphorylation. Recent findings highlight a diverse set of strategies to control Akt catalytic activity to ensure its normal biological functions. %B Curr Opin Struct Biol %V 59 %P 47-53 %8 2019 Mar 19 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/30901610?dopt=Abstract %R 10.1016/j.sbi.2019.02.004 %0 Journal Article %J Chembiochem %D 2019 %T Analysis of Site-specific Phosphorylation of PTEN using Enzyme-Catalyzed Expressed Protein Ligation %A Henager, Samuel %A Henriquez, Stephanie %A Dempsey, Daniel %A Cole, Philip A %X The activity and localization of PTEN, a tumor suppressor lipid phosphatase that converts the phospholipid PIP3 to PIP2, is governed in part by phosphorylation on a cluster of four Ser and Thr residues near the C-terminus. Prior enzymatic characterization of the four mono-phosphorylated (1p) PTENs using classical expressed protein ligation (EPL) was complicated by the inclusion of a non-native Cys at the ligation junction (aa379), which may alter the properties of the semisynthetic protein. Here we apply subtiligase-mediated EPL to create wt 1p-PTENs. These PTENs are more autoinhibited than previously appreciated, consistent with Tyr379's role in driving autoinhibition. Alkaline phosphatase sensitivity analysis revealed that these autoinhibited 1p conformations are kinetically labile. In contrast to the Cys mutant 1p-PTENs, which are poorly recognized by an anti-phospho-PTEN Ab, three of the four wt 1p-PTENs are recognized by a commonly used anti-phospho-PTEN Ab. %B Chembiochem %8 2019 Jun 17 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31206229?dopt=Abstract %R 10.1002/cbic.201900316 %0 Journal Article %J Biochemistry %D 2019 %T Combination Targeting of the Bromodomain and Acetyltransferase Active Site of p300/CBP %A Zucconi, Beth E %A Makofske, Jessica L %A Meyers, David J %A Hwang, Yousang %A Wu, Mingxuan %A Kuroda, Mitzi I %A Cole, Philip A %X p300 and CBP are highly related histone acetyltransferase (HAT) enzymes that regulate gene expression, and their dysregulation has been linked to cancer and other diseases. p300/CBP is composed of a number of domains including a HAT domain, which is inhibited by the small molecule A-485, and an acetyl-lysine binding bromodomain, which was recently found to be selectively antagonized by the small molecule I-CBP112. Here we show that the combination of I-CBP112 and A-485 can synergize to inhibit prostate cancer cell proliferation. We find that the combination confers a dramatic reduction in p300 chromatin occupancy compared to the individual effects of blocking either domain alone. Accompanying this loss of p300 on chromatin, combination treatment leads to the reduction of specific mRNAs including androgen-dependent and pro-oncogenic prostate genes such as KLK3 (PSA) and c-Myc. Consistent with p300 directly affecting gene expression, mRNAs that are significantly reduced by combination treatment also exhibit a strong reduction in p300 chromatin occupancy at their gene promoters. The relatively few mRNAs that are up-regulated upon combination treatment show no correlation with p300 occupancy. These studies provide support for the pharmacologic advantage of concurrent targeting of two domains within one key epigenetic modification enzyme. %B Biochemistry %V 58 %P 2133-2143 %8 2019 Apr 23 %G eng %N 16 %1 http://www.ncbi.nlm.nih.gov/pubmed/30924641?dopt=Abstract %R 10.1021/acs.biochem.9b00160 %0 Journal Article %J J Biol Chem %D 2019 %T Comparative analysis of the catalytic regulation of NEDD4-1 and WWP2 ubiquitin ligases %A Jiang, Hanjie %A Thomas, Stefani N %A Chen, Zan %A Chiang, Claire Y %A Cole, Philip A %X NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities are important for controlling cellular protein homeostasis and their dysregulation may lead to cancer and other diseases. Previous work has implicated non-catalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecular interactions. Here, we explored the molecular mechanisms of these NEDD4-1 and WWP2 regulatory regions and their interplay with allosteric binding proteins such as Nedd4 family interacting protein (NDFIP1), engineered ubiquitin variants, and linker phosphomimics. We found that in addition to influencing catalytic activities, the WW domain linker regions in NEDD4-1 and WWP2 can impact product distribution, including the degree of polyubiquitination and Lys-48 versus Lys-63 linkages. We show that allosteric activation by NDFIP1 or engineered ubiquitin variants is largely mediated by relief of WW domain linker autoinhibition. WWP2-mediated ubiquitination of WW domain-binding protein 2 (WBP2), phosphatase and tensin homolog (PTEN), and p62 proteins by WWP2 suggests that substrate ubiquitination can also be influenced by WW linker autoinhibition, although to differing extents. Overall, our results provide a deeper understanding of the intricate and multifaceted set of regulatory mechanisms in the control of NEDD4-1 related ubiquitin ligases. %B J Biol Chem %8 2019 Oct 02 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31578285?dopt=Abstract %R 10.1074/jbc.RA119.009211 %0 Journal Article %J Cancers (Basel) %D 2019 %T Complementary Roles of GCN5 and PCAF in Foxp3+ T-Regulatory Cells %A Liu, Yujie %A Bao, Chunrong %A Wang, Liqing %A Han, Rongxiang %A Beier, Ulf H %A Akimova, Tatiana %A Cole, Philip A %A Dent, Sharon Y R %A Hancock, Wayne W %X Functions of the GCN5-related N-acetyltransferase (GNAT) family of histone/protein acetyltransferases (HATs) in Foxp3+ T-regulatory (Treg) cells are unexplored, despite the general importance of these enzymes in cell biology. We now show that two prototypical GNAT family members, GCN5 (general control nonrepressed-protein 5, lysine acetyltransferase (KAT)2a) and p300/CBP-associated factor (p300/CBP-associated factor (PCAF), Kat2b) contribute to Treg functions through partially distinct and partially overlapping mechanisms. Deletion of Gcn5 or PCAF did not affect Treg development or suppressive function in vitro, but did affect inducible Treg (iTreg) development, and in vivo, abrogated Treg-dependent allograft survival. Contrasting effects were seen upon targeting of each HAT in all T cells; mice lacking GCN5 showed prolonged allograft survival, suggesting this HAT might be a target for epigenetic therapy in allograft recipients, whereas transplants in mice lacking PCAF underwent acute allograft rejection. PCAF deletion also enhanced anti-tumor immunity in immunocompetent mice. Dual deletion of GCN5 and PCAF led to decreased Treg stability and numbers in peripheral lymphoid tissues, and mice succumbed to severe autoimmunity by 3-4 weeks of life. These data indicate that HATs of the GNAT family have contributions to Treg function that cannot be replaced by the functions of previously characterized Treg HATs (CBP, p300, and Tip60), and may be useful targets in immuno-oncology. %B Cancers (Basel) %V 11 %8 2019 Apr 18 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/31003455?dopt=Abstract %R 10.3390/cancers11040554 %0 Journal Article %J Mol Cell Biol %D 2019 %T CREB Promotes Beta Cell Gene Expression by Targeting Its Coactivators to Tissue-Specific Enhancers %A Van de Velde, Sam %A Wiater, Ezra %A Tran, Melissa %A Hwang, Yousang %A Cole, Philip A %A Montminy, Marc %X CREB mediates effects of cyclic AMP on cellular gene expression. Ubiquitous CREB target genes are induced following recruitment of CREB and its coactivators to promoter proximal binding sites. We found that CREB stimulates the expression of pancreatic beta cell-specific genes by targeting CBP/p300 to promoter-distal enhancer regions. Subsequent increases in histone acetylation facilitate recruitment of the coactivators CRTC2 and BRD4, leading to release of RNA polymerase II over the target gene body. Indeed, CREB-induced hyperacetylation of chromatin over superenhancers promoted beta cell-restricted gene expression, which is sensitive to inhibitors of CBP/p300 and BRD4 activity. Neurod1 appears critical in establishing nucleosome-free regions for recruitment of CREB to beta cell-specific enhancers. Deletion of a CREB-Neurod1-bound enhancer within the superenhancer disrupted the expression of both genes and decreased beta cell function. Our results demonstrate how cross talk between signal-dependent and lineage-determining factors promotes the expression of cell-type-specific gene programs in response to extracellular cues. %B Mol Cell Biol %V 39 %8 2019 Sep 01 %G eng %N 17 %1 http://www.ncbi.nlm.nih.gov/pubmed/31182641?dopt=Abstract %R 10.1128/MCB.00200-19 %0 Journal Article %J Curr. Opin. Struct. Biol. %D 2019 %T Editorial overview: Biological catalysis at the cross-roads of signaling and metabolism %A Cole, P. A. %A Mattevi, A. %B Curr. Opin. Struct. Biol. %V 59 %P iii-v %8 12 %G eng %0 Journal Article %J Molecules %D 2019 %T Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II %A Miller, Michelle S %A Maheshwari, Sweta %A Shi, Wuxian %A Gao, Yuan %A Chu, Nam %A Soares, Alexei S %A Cole, Philip A %A Amzel, L Mario %A Fuchs, Martin R %A Jakoncic, Jean %A Gabelli, Sandra B %K Crystallography, X-Ray %K Models, Molecular %K Phosphatidylinositol 3-Kinases %K Protein Conformation %K Proteins %K Pyrophosphatases %X Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II-AMX and FMX-deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins-Akt1, PI3Kα, and CDP-Chase-to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or "raster-collect", a more automated approach for a larger number of crystals (using CDP-Chase as an example). %B Molecules %V 24 %8 2019 Jan 30 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/30704096?dopt=Abstract %R 10.3390/molecules24030496 %0 Journal Article %J PLoS One %D 2019 %T Investigation into the use of histone deacetylase inhibitor MS-275 as a topical agent for the prevention and treatment of cutaneous squamous cell carcinoma in an SKH-1 hairless mouse model %A Kalin, Jay H %A Eroglu, Abdulkerim %A Liu, Hua %A Holtzclaw, W David %A Leigh, Irene %A Proby, Charlotte M %A Fahey, Jed W %A Cole, Philip A %A Dinkova-Kostova, Albena T %X Cutaneous squamous cell carcinomas are a common form of highly mutated keratinocyte skin cancers that are of particular concern in immunocompromised patients. Here we report on the efficacy of topically applied MS-275, a clinically used histone deacetylase inhibitor, for the treatment and management of this disease. At 2 mg/kg, MS-275 significantly decreased tumor burden in an SKH-1 hairless mouse model of UVB radiation-induced skin carcinogenesis. MS-275 was cell permeable as a topical formulation and induced histone acetylation changes in mouse tumor tissue. MS-275 was also effective at inhibiting the proliferation of patient derived cutaneous squamous cell carcinoma lines and was particularly potent toward cells isolated from a regional metastasis on an immunocompromised individual. Our findings support the use of alternative routes of administration for histone deacetylase inhibitors in the treatment of high-risk squamous cell carcinoma which may ultimately lead to more precise delivery and reduced systemic toxicity. %B PLoS One %V 14 %P e0213095 %8 2019 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/30865688?dopt=Abstract %R 10.1371/journal.pone.0213095 %0 Journal Article %J Cancer Res %D 2019 %T MITF Expression Predicts Therapeutic Vulnerability to p300 Inhibition in Human Melanoma %A Kim, Edward %A Zucconi, Beth E %A Wu, Muzhou %A Nocco, Sarah E %A Meyers, David J %A McGee, Jean S %A Venkatesh, Samantha %A Cohen, Daniel L %A Gonzalez, Estela C %A Ryu, Byungwoo %A Cole, Philip A %A Alani, Rhoda M %X Histone modifications, largely regulated by histone acetyltransferases (HAT) and histone deacetylases, have been recognized as major regulatory mechanisms governing human diseases, including cancer. Despite significant effort and recent advances, the mechanism by which the HAT and transcriptional coactivator p300 mediates tumorigenesis remains unclear. Here, we use a genetic and chemical approach to identify the microphthalmia-associated transcription factor (MITF) as a critical downstream target of p300 driving human melanoma growth. Direct transcriptional control of MITF by p300-dependent histone acetylation within proximal gene regulatory regions was coupled to cellular proliferation, suggesting a significant growth regulatory axis. Further analysis revealed forkhead box M1 (FOXM1) as a key effector of the p300-MITF axis driving cell growth that is selectively activated in human melanomas. Targeted chemical inhibition of p300 acetyltransferase activity using a potent and selective catalytic p300/CBP inhibitor demonstrated significant growth inhibitory effects in melanoma cells expressing high levels of MITF. Collectively, these data confirm the critical role of the p300-MITF-FOXM1 axis in melanoma and support p300 as a promising novel epigenetic therapeutic target in human melanoma. SIGNIFICANCE: These results show that MITF is a major downstream target of p300 in human melanoma whose expression is predictive of melanoma response to small-molecule inhibition of p300 HAT activity. %B Cancer Res %V 79 %P 2649-2661 %8 2019 May 15 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/30910803?dopt=Abstract %R 10.1158/0008-5472.CAN-18-2331 %0 Journal Article %J Biochemistry %D 2018 %T Analysis of Cellular Tyrosine Phosphorylation via Chemical Rescue of Conditionally Active Abl Kinase %A Wang, Zhihong %A Kim, Min-Sik %A Martinez-Ferrando, Isabel %A Koleske, Anthony J %A Pandey, Akhilesh %A Cole, Philip A %X Identifying direct substrates targeted by protein kinases is important in understanding cellular physiology and intracellular signal transduction. Mass spectrometry-based quantitative proteomics provides a powerful tool for comprehensively characterizing the downstream substrates of protein kinases. This approach is efficiently applied to receptor kinases that can be precisely, directly, and rapidly activated by some agent, such as a growth factor. However, nonreceptor tyrosine kinase Abl lacks the experimental advantage of extracellular growth factors as immediate and direct stimuli. To circumvent this limitation, we combine a chemical rescue approach with quantitative phosphoproteomics to identify targets of Abl and their phosphorylation sites with enhanced temporal resolution. Both known and novel putative substrates are identified, presenting opportunities for studying unanticipated functions of Abl under physiological and pathological conditions. %B Biochemistry %8 2018 Feb 06 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29341593?dopt=Abstract %R 10.1021/acs.biochem.7b01158 %0 Journal Article %J Nature %D 2018 %T Author Correction: Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours %A Lasko, Loren M %A Jakob, Clarissa G %A Edalji, Rohinton P %A Qiu, Wei %A Montgomery, Debra %A Digiammarino, Enrico L %A Hansen, T Matt %A Risi, Roberto M %A Frey, Robin %A Manaves, Vlasios %A Shaw, Bailin %A Algire, Mikkel %A Hessler, Paul %A Lam, Lloyd T %A Uziel, Tamar %A Faivre, Emily %A Ferguson, Debra %A Buchanan, Fritz G %A Martin, Ruth L %A Torrent, Maricel %A Chiang, Gary G %A Karukurichi, Kannan %A Langston, J William %A Weinert, Brian T %A Choudhary, Chunaram %A de Vries, Peter %A Kluge, Arthur F %A Patane, Michael A %A Van Drie, John H %A Wang, Ce %A McElligott, David %A Kesicki, Edward A %A Marmorstein, Ronen %A Sun, Chaohong %A Cole, Philip A %A Rosenberg, Saul H %A Michaelides, Michael R %A Lai, Albert %A Bromberg, Kenneth D %X In the originally published version of this Letter, the authors Arthur F. Kluge, Michael A. Patane and Ce Wang were inadvertently omitted from the author list. Their affiliations are: I-to-D, Inc., PO Box 6177, Lincoln, Massachusetts 01773, USA (A.F.K.); Mitobridge, Inc. 1030 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (M.A.P.); and China Novartis Institutes for BioMedical Research, No. 4218 Jinke Road, Zhangjiang Hi-Tech Park, Pudong District, Shanghai 201203, China (C.W.). These authors contributed to the interpretation of results and design of compounds. In addition, author 'Edward A. Kesicki' was misspelled as 'Ed Kesicki'. These errors have been corrected online. %B Nature %V 558 %P E1 %8 2018 Jun %G eng %N 7710 %1 http://www.ncbi.nlm.nih.gov/pubmed/29769713?dopt=Abstract %R 10.1038/s41586-018-0111-5 %0 Journal Article %J ACS Med Chem Lett %D 2018 %T Discovery of Spiro Oxazolidinediones as Selective, Orally Bioavailable Inhibitors of p300/CBP Histone Acetyltransferases %A Michaelides, Michael R %A Kluge, Arthur %A Patane, Michael %A Van Drie, John H %A Wang, Ce %A Hansen, T Matthew %A Risi, Roberto M %A Mantei, Robert %A Hertel, Carmen %A Karukurichi, Kannan %A Nesterov, Alexandre %A McElligott, David %A de Vries, Peter %A Langston, J William %A Cole, Philip A %A Marmorstein, Ronen %A Liu, Hong %A Lasko, Loren %A Bromberg, Kenneth D %A Lai, Albert %A Kesicki, Edward A %X p300 and its paralog CBP can acetylate histones and other proteins and have been implicated in a number of diseases characterized by aberrant gene activation, such as cancer. A novel, highly selective, orally bioavailable histone acetyltransferase (HAT) domain inhibitor has been identified through virtual ligand screening and subsequent optimization of a unique hydantoin screening hit. Conformational restraint in the form of a spirocyclization followed by substitution with a urea led to a significant improvement in potency. Replacement of the hydantoin moiety with an oxazolidinedione followed by fluoro substitution led to A-485, which exhibits potent cell activity, low clearance, and high oral bioavailability. %B ACS Med Chem Lett %V 9 %P 28-33 %8 2018 Jan 11 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/29348807?dopt=Abstract %R 10.1021/acsmedchemlett.7b00395 %0 Journal Article %J J Am Chem Soc %D 2018 %T Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action %A Bhat, Shridhar %A Hwang, Yousang %A Gibson, Matthew D %A Morgan, Michael T %A Taverna, Sean D %A Zhao, Yingming %A Wolberger, Cynthia %A Poirier, Michael G %A Cole, Philip A %X A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins. We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys that could be recognized selectively by relevant acyl-Lys modification antibodies. The acyl-Lys modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a function of modification type and site. We also installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study the SAGA deubiquitinating module's molecular recognition. These results suggest that acyl-Lys mimics offer a relatively simple and promising strategy to study the role of acyl-Lys modifications in the function, structure, and regulation of proteins and protein complexes. %B J Am Chem Soc %V 140 %P 9478-9485 %8 2018 Aug 01 %G eng %N 30 %1 http://www.ncbi.nlm.nih.gov/pubmed/29991262?dopt=Abstract %R 10.1021/jacs.8b03572 %0 Journal Article %J Elife %D 2018 %T Lysine-14 acetylation of histone H3 in chromatin confers resistance to the deacetylase and demethylase activities of an epigenetic silencing complex %A Wu, Mingxuan %A Hayward, Dawn %A Kalin, Jay H %A Song, Yun %A Schwabe, John W R %A Cole, Philip A %X The core CoREST complex (LHC) contains histone deacetylase HDAC1 and histone demethylase LSD1 held together by the scaffold protein CoREST. Here, we analyze the purified LHC with modified peptide and reconstituted semisynthetic mononucleosome substrates. LHC demethylase activity toward methyl-Lys4 in histone H3 is strongly inhibited by H3 Lys14 acetylation, and this appears to be an intrinsic property of the LSD1 subunit. Moreover, the deacetylase selectivity of LHC unexpectedly shows a marked preference for H3 acetyl-Lys9 versus acetyl-Lys14 in nucleosome substrates but this selectivity is lost with isolated acetyl-Lys H3 protein. This diminished activity of LHC to Lys-14 deacetylation in nucleosomes is not merely due to steric accessibility based on the pattern of sensitivity of the LHC enzymatic complex to hydroxamic acid-mediated inhibition. Overall, these studies have revealed how a single Lys modification can confer a composite of resistance in chromatin to a key epigenetic enzyme complex involved in gene silencing. %B Elife %V 7 %8 2018 Jun 05 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29869982?dopt=Abstract %R 10.7554/eLife.37231 %0 Journal Article %J Nucleic Acids Res %D 2018 %T N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA-dsDNA junctions %A Weiser, Brian P %A Rodriguez, Gaddiel %A Cole, Philip A %A Stivers, James T %X The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias for uracil located <10 bp from the junction, but only when the overhang had a 5' end. Biased targeting required the NTD and was not observed with the hUNG2 catalytic domain alone. Consistent with this requirement, the isolated NTD was found to bind weakly to ssDNA. These findings indicate that the NTD of hUNG2 targets the enzyme to ssDNA-dsDNA junctions using RPA-dependent and RPA-independent mechanisms. This structure-based specificity may promote efficient removal of uracils that arise from dUTP incorporation during DNA replication, or additionally, uracils that arise from DNA cytidine deamination at transcriptional R-loops during immunoglobulin class-switch recombination. %B Nucleic Acids Res %V 46 %P 7169-7178 %8 2018 Aug 21 %G eng %N 14 %1 http://www.ncbi.nlm.nih.gov/pubmed/29917162?dopt=Abstract %R 10.1093/nar/gky525 %0 Journal Article %J Methods Enzymol %D 2018 %T Protein Chemical Approaches to Understanding PTEN Lipid Phosphatase Regulation %A Dempsey, Daniel R %A Cole, Philip A %X Since the discovery of C-tail phosphorylation of PTEN almost 20 years ago, much progress has been made in understanding its regulatory influences on the cellular function of PTEN. Phosphorylation of Ser380, Thr382, Thr383, and Ser385 drives a PTEN conformational change from an open to closed state where catalytic function is impaired, plasma membrane binding is reduced, and cellular stability is enhanced. Despite these advances, a detailed structural and mechanistic model of how these phosphorylations impact PTEN function is lacking. We discuss here several recent approaches to analyzing PTEN phosphorylation and highlight several insights that have come from this work. We also discuss remaining challenges for the PTEN regulation field and potential directions for future research. %B Methods Enzymol %V 607 %P 405-422 %8 2018 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/30149868?dopt=Abstract %R 10.1016/bs.mie.2018.05.007 %0 Journal Article %J J Am Chem Soc %D 2018 %T Site-Specific Protein Labeling with N-Hydroxysuccinimide-Esters and the Analysis of Ubiquitin Ligase Mechanisms %A Dempsey, Daniel R %A Jiang, Hanjie %A Kalin, Jay H %A Chen, Zan %A Cole, Philip A %X N-Hydroxysuccinimide (NHS)-esters are widely used to label proteins nonselectively on free amino groups. Such broad labeling can be disadvantageous because it can interfere with protein structure or function and because stoichiometry is poorly controlled. Here we describe a simple method to transform NHS-esters into site-specific protein labeling on N-terminal Cys residues. MESNA addition converts NHS-esters to chemoselective thioesters for N-Cys modification. This labeling strategy was applied to clarify mechanistic features of the ubiquitin E3 ligase WWP2 including its interaction with one of its substrates, the tumor suppressor PTEN, as well as its autoubiquitination molecularity. We propose that this convenient protein labeling strategy will allow for an expanded application of NHS-esters in biochemical investigation. %B J Am Chem Soc %V 140 %P 9374-9378 %8 2018 Aug 01 %G eng %N 30 %1 http://www.ncbi.nlm.nih.gov/pubmed/30016585?dopt=Abstract %R 10.1021/jacs.8b05098 %0 Journal Article %J Nat Commun %D 2018 %T Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors %A Kalin, Jay H %A Wu, Muzhou %A Gomez, Andrea V %A Song, Yun %A Das, Jayanta %A Hayward, Dawn %A Adejola, Nkosi %A Wu, Mingxuan %A Panova, Izabela %A Chung, Hye Jin %A Kim, Edward %A Roberts, Holly J %A Roberts, Justin M %A Prusevich, Polina %A Jeliazkov, Jeliazko R %A Roy Burman, Shourya S %A Fairall, Louise %A Milano, Charles %A Eroglu, Abdulkerim %A Proby, Charlotte M %A Dinkova-Kostova, Albena T %A Hancock, Wayne W %A Gray, Jeffrey J %A Bradner, James E %A Valente, Sergio %A Mai, Antonello %A Anders, Nicole M %A Rudek, Michelle A %A Hu, Yong %A Ryu, Byungwoo %A Schwabe, John W R %A Mattevi, Andrea %A Alani, Rhoda M %A Cole, Philip A %X Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin's favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities. %B Nat Commun %V 9 %P 53 %8 2018 Jan 04 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/29302039?dopt=Abstract %R 10.1038/s41467-017-02242-4 %0 Journal Article %J Cell %D 2018 %T Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome %A Weinert, Brian T %A Narita, Takeo %A Satpathy, Shankha %A Srinivasan, Balaji %A Hansen, Bogi K %A Schölz, Christian %A Hamilton, William B %A Zucconi, Beth E %A Wang, Wesley W %A Liu, Wenshe R %A Brickman, Joshua M %A Kesicki, Edward A %A Lai, Albert %A Bromberg, Kenneth D %A Cole, Philip A %A Choudhary, Chunaram %X The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here, we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites and a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (<30 min) acetylation turnover, revealing a dynamic balance between acetylation and deacetylation. Quantification of acetylation, mRNA, and protein abundance after CBP/p300 inhibition reveals a kinetically competent network of gene expression that strictly depends on CBP/p300-catalyzed rapid acetylation. Collectively, our in-depth acetylome analyses reveal systems attributes of CBP/p300 targets, and the resource dataset provides a framework for investigating CBP/p300 functions and for understanding the impact of small-molecule inhibitors targeting its catalytic and bromodomain activities. %B Cell %V 174 %P 231-244.e12 %8 2018 Jun 28 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/29804834?dopt=Abstract %R 10.1016/j.cell.2018.04.033 %0 Journal Article %J Cell %D 2018 %T Akt Kinase Activation Mechanisms Revealed Using Protein Semisynthesis %A Chu, Nam %A Salguero, Antonieta L %A Liu, Albert Z %A Chen, Zan %A Dempsey, Daniel R %A Ficarro, Scott B %A Alexander, William M %A Marto, Jarrod A %A Li, Yana %A Amzel, L Mario %A Gabelli, Sandra B %A Cole, Philip A %X Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms. %B Cell %V 174 %P 897-907.e14 %8 2018 Aug 09 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/30078705?dopt=Abstract %R 10.1016/j.cell.2018.07.003 %0 Journal Article %J Curr Opin Chem Biol %D 2017 %T Allosteric regulation of epigenetic modifying enzymes %A Zucconi, Beth E %A Cole, Philip A %K Allosteric Regulation %K Amino Acid Sequence %K Animals %K Biocatalysis %K Enzyme Inhibitors %K Enzymes %K Epigenesis, Genetic %K Humans %K Protein Domains %X Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. %B Curr Opin Chem Biol %V 39 %P 109-115 %8 2017 Aug %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28689145?dopt=Abstract %R 10.1016/j.cbpa.2017.05.015 %0 Journal Article %J Mol Cell %D 2017 %T CBP Regulates Recruitment and Release of Promoter-Proximal RNA Polymerase II %A Boija, Ann %A Mahat, Dig Bijay %A Zare, Aman %A Holmqvist, Per-Henrik %A Philip, Philge %A Meyers, David J %A Cole, Philip A %A Lis, John T %A Stenberg, Per %A Mannervik, Mattias %K Acetylation %K Animals %K Cell Line %K Chromatin %K Chromatin Assembly and Disassembly %K DNA-Binding Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Gene Expression Regulation, Developmental %K Histones %K Nucleosomes %K p300-CBP Transcription Factors %K Promoter Regions, Genetic %K Protein Binding %K RNA Polymerase II %K Transcription Factor TFIIB %K Transcription Factors %K Transcription, Genetic %X Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is unclear. Here, we identify a novel activity of the histone acetyltransferase p300/CREB-binding protein (CBP) in regulating promoter-proximal paused Pol II. We find that Drosophila CBP inhibition results in "dribbling" of Pol II from the pause site to positions further downstream but impedes transcription through the +1 nucleosome genome-wide. Promoters strongly occupied by CBP and GAGA factor have high levels of paused Pol II, a unique chromatin signature, and are highly expressed regardless of cell type. Interestingly, CBP activity is rate limiting for Pol II recruitment to these highly paused promoters through an interaction with TFIIB but for transit into elongation by histone acetylation at other genes. Thus, CBP directly stimulates both Pol II recruitment and the ability to traverse the first nucleosome, thereby promoting transcription of most genes. %B Mol Cell %V 68 %P 491-503.e5 %8 2017 Nov 02 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/29056321?dopt=Abstract %R 10.1016/j.molcel.2017.09.031 %0 Journal Article %J Nature %D 2017 %T Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours %A Lasko, Loren M %A Jakob, Clarissa G %A Edalji, Rohinton P %A Qiu, Wei %A Montgomery, Debra %A Digiammarino, Enrico L %A Hansen, T Matt %A Risi, Roberto M %A Frey, Robin %A Manaves, Vlasios %A Shaw, Bailin %A Algire, Mikkel %A Hessler, Paul %A Lam, Lloyd T %A Uziel, Tamar %A Faivre, Emily %A Ferguson, Debra %A Buchanan, Fritz G %A Martin, Ruth L %A Torrent, Maricel %A Chiang, Gary G %A Karukurichi, Kannan %A Langston, J William %A Weinert, Brian T %A Choudhary, Chunaram %A de Vries, Peter %A Van Drie, John H %A McElligott, David %A Kesicki, Ed %A Marmorstein, Ronen %A Sun, Chaohong %A Cole, Philip A %A Rosenberg, Saul H %A Michaelides, Michael R %A Lai, Albert %A Bromberg, Kenneth D %X The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases. %B Nature %V 550 %P 128-132 %8 2017 10 05 %G eng %N 7674 %1 http://www.ncbi.nlm.nih.gov/pubmed/28953875?dopt=Abstract %R 10.1038/nature24028 %0 Journal Article %J ACS Chem Biol %D 2017 %T Disordered N-Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation %A Rodriguez, Gaddiel %A Esadze, Alexandre %A Weiser, Brian P %A Schonhoft, Joseph D %A Cole, Philip A %A Stivers, James T %K Binding Sites %K Biological Transport %K DNA %K DNA Glycosylases %K Humans %K Nuclear Localization Signals %K Protein Domains %X Nuclear human uracil-DNA glycosylase (hUNG2) initiates base excision repair (BER) of genomic uracils generated through misincorporation of dUMP or through deamination of cytosines. Like many human DNA glycosylases, hUNG2 contains an unstructured N-terminal domain that encodes a nuclear localization signal, protein binding motifs, and sites for post-translational modifications. Although the N-terminal domain has minimal effects on DNA binding and uracil excision kinetics, we report that this domain enhances the ability of hUNG2 to translocate on DNA chains as compared to the catalytic domain alone. The enhancement is most pronounced when physiological ion concentrations and macromolecular crowding agents are used. These data suggest that crowded conditions in the human cell nucleus promote the interaction of the N-terminus with duplex DNA during translocation. The increased contact time with the DNA chain likely contributes to the ability of hUNG2 to locate densely spaced uracils that arise during somatic hypermutation and during fluoropyrimidine chemotherapy. %B ACS Chem Biol %V 12 %P 2260-2263 %8 2017 09 15 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/28787572?dopt=Abstract %R 10.1021/acschembio.7b00521 %0 Journal Article %J Nat Commun %D 2017 %T Endotoxemia-mediated activation of acetyltransferase P300 impairs insulin signaling in obesity %A Cao, Jia %A Peng, Jinghua %A An, Hongying %A He, Qiyi %A Boronina, Tatiana %A Guo, Shaodong %A White, Morris F %A Cole, Philip A %A He, Ling %K Animals %K Cell Line, Tumor %K E1A-Associated p300 Protein %K Endoplasmic Reticulum Stress %K Endotoxemia %K Gene Expression Profiling %K Immunoblotting %K Insulin %K Insulin Resistance %K Lipopolysaccharides %K Liver %K Male %K Membrane Proteins %K Mice, Inbred C57BL %K Mice, Obese %K Obesity %K Protein-Serine-Threonine Kinases %K Receptor, Insulin %K Signal Transduction %K X-Box Binding Protein 1 %X Diabetes and obesity are characterized by insulin resistance and chronic low-grade inflammation. An elevated plasma concentration of lipopolysaccharide (LPS) caused by increased intestinal permeability during diet-induced obesity promotes insulin resistance in mice. Here, we show that LPS induces endoplasmic reticulum (ER) stress and protein levels of P300, an acetyltransferase involved in glucose production. In high-fat diet fed and genetically obese ob/ob mice, P300 translocates from the nucleus into the cytoplasm of hepatocytes. We also demonstrate that LPS activates the transcription factor XBP1 via the ER stress sensor IRE1, resulting in the induction of P300 which, in turn, acetylates IRS1/2, inhibits its association with the insulin receptor, and disrupts insulin signaling. Pharmacological inhibition of P300 acetyltransferase activity by a specific inhibitor improves insulin sensitivity and decreases hyperglycemia in obese mice. We suggest that P300 acetyltransferase activity may be a promising therapeutic target for the treatment of obese patients.Elevated plasma LPS levels have been associated with insulin resistance. Here Cao et al. show that LPS induces ER stress and P300 activity via the XBP1/IRE1 pathway. P300 acetylates IRS1/2 and inhibits its binding with the insulin receptor. The consequent impairment of insulin signaling can be rescued by pharmacological inhibition of P300. %B Nat Commun %V 8 %P 131 %8 2017 07 25 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/28743992?dopt=Abstract %R 10.1038/s41467-017-00163-w %0 Journal Article %J Nat Methods %D 2017 %T Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution %A Mo, Gary C H %A Ross, Brian %A Hertel, Fabian %A Manna, Premashis %A Yang, Xinxing %A Greenwald, Eric %A Booth, Chris %A Plummer, Ashlee M %A Tenner, Brian %A Chen, Zan %A Wang, Yuxiao %A Kennedy, Eileen J %A Cole, Philip A %A Fleming, Karen G %A Palmer, Amy %A Jimenez, Ralph %A Xiao, Jie %A Dedecker, Peter %A Jin Zhang %K Biosensing Techniques %K Cell Membrane %K Cyclic AMP-Dependent Protein Kinases %K Escherichia coli %K Fluorescence Resonance Energy Transfer %K Fluorescent Dyes %K Green Fluorescent Proteins %K HeLa Cells %K Humans %K Microscopy %K Molecular Imaging %K Mutagenesis, Site-Directed %K Protein Interaction Mapping %K Stochastic Processes %X Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with cellular compartmentalization. Here, we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to threefold better than the diffraction limit. These 'FLINC' biosensors use binding-induced changes in protein fluorescence dynamics to translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A protein kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membranes of living cells and to uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture whose structural and functional characteristics can be revealed by these new biosensors. %B Nat Methods %V 14 %P 427-434 %8 2017 Apr %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/28288122?dopt=Abstract %R 10.1038/nmeth.4221 %0 Journal Article %J Biophys J %D 2017 %T Investigation of N-Terminal Phospho-Regulation of Uracil DNA Glycosylase Using Protein Semisynthesis %A Weiser, Brian P %A Stivers, James T %A Cole, Philip A %K Catalysis %K DNA Glycosylases %K Escherichia coli %K Humans %K Mutation %K Phosphorylation %K Proliferating Cell Nuclear Antigen %K Protein Binding %K Protein Domains %K Protein Stability %K Replication Protein A %K Spectrometry, Mass, Electrospray Ionization %X Uracil DNA Glycosylase (UNG2) is the primary enzyme in humans that prevents the stable incorporation of deoxyuridine monophosphate into DNA in the form of U/A basepairs. During S-phase, UNG2 remains associated with the replication fork through its interactions with two proteins, Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RPA), which are critical for DNA replication and repair. In this work, we used protein semisynthesis and fluorescence anisotropy assays to explore the interactions of UNG2 with PCNA and RPA and to determine the effects of two UNG2 phosphorylation sites (Thr6 and Tyr8) located within its PCNA-interacting motif (PIP-box). In binding assays, we found that phosphorylation of Thr6 or Tyr8 on UNG2 can impede PCNA binding without affecting UNG2 catalytic activity or its RPA interaction. Our data also suggests that unmodified UNG2, PCNA, and RPA can form a ternary protein complex. We propose that the UNG2 N-terminus may serve as a flexible scaffold to tether PCNA and RPA at the replication fork, and that post-translational modifications on the UNG2 N-terminus disrupt formation of the PCNA-UNG2-RPA protein complex. %B Biophys J %V 113 %P 393-401 %8 2017 Jul 25 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/28746850?dopt=Abstract %R 10.1016/j.bpj.2017.06.016 %0 Journal Article %J Nucleic Acids Res %D 2017 %T Measurement of nanoscale DNA translocation by uracil DNA glycosylase in human cells %A Esadze, Alexandre %A Rodriguez, Gaddiel %A Weiser, Brian P %A Cole, Philip A %A Stivers, James T %K Cell Line %K DNA %K DNA Glycosylases %K Humans %K Uracil %X DNA 'sliding' by human repair enzymes is considered to be important for DNA damage detection. Here, we transfected uracil-containing DNA duplexes into human cells and measured the probability that nuclear human uracil DNA glycosylase (hUNG2) excised two uracil lesions spaced 10-80 bp apart in a single encounter without escaping the micro-volume containing the target sites. The two-site transfer probabilities were 100% and 54% at a 10 and 40 bp spacing, but dropped to only 10% at 80 bp. Enzyme trapping experiments suggested that site transfers over 40 bp followed a DNA 'hopping' pathway in human cells, indicating that authentic sliding does not occur even over this short distance. The transfer probabilities were much greater than observed in aqueous buffers, but similar to in vitro measurements in the presence of polymer crowding agents. The findings reveal a new role for the crowded nuclear environment in facilitating DNA damage detection. %B Nucleic Acids Res %V 45 %P 12413-12424 %8 2017 Dec 01 %G eng %N 21 %1 http://www.ncbi.nlm.nih.gov/pubmed/29036472?dopt=Abstract %R 10.1093/nar/gkx848 %0 Journal Article %J Mol Cell %D 2017 %T A Tunable Brake for HECT Ubiquitin Ligases %A Chen, Zan %A Jiang, Hanjie %A Xu, Wei %A Li, Xiaoguang %A Dempsey, Daniel R %A Zhang, Xiangbin %A Devreotes, Peter %A Wolberger, Cynthia %A Amzel, L Mario %A Gabelli, Sandra B %A Cole, Philip A %K Allosteric Regulation %K Endosomal Sorting Complexes Required for Transport %K Enzyme Activation %K Enzyme Stability %K HeLa Cells %K Humans %K Models, Molecular %K Mutation %K Nedd4 Ubiquitin Protein Ligases %K Phosphorylation %K Protein Domains %K Protein Processing, Post-Translational %K Proteolysis %K Repressor Proteins %K Structure-Activity Relationship %K Transfection %K Ubiquitin-Protein Ligases %X The HECT E3 ligases ubiquitinate numerous transcription factors and signaling molecules, and their activity must be tightly controlled to prevent cancer, immune disorders, and other diseases. In this study, we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities. Biochemical, structural, and cellular analyses have revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site. Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications, but complete removal of the brake can induce hyperactive autoubiquitination and E3 self destruction. These results clarify the mechanisms of several HECT protein cancer associated mutations and provide a new framework for understanding how HECT ubiquitin ligases must be finely tuned to ensure normal cellular behavior. %B Mol Cell %V 66 %P 345-357.e6 %8 2017 May 04 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/28475870?dopt=Abstract %R 10.1016/j.molcel.2017.03.020 %0 Journal Article %J Chem Rev %D 2016 %T Correction to Protein Lysine Acetylation by p300/CBP %A Dancy, Beverley M %A Cole, Philip A %B Chem Rev %V 116 %P 8314 %8 2016 07 27 %G eng %N 14 %1 http://www.ncbi.nlm.nih.gov/pubmed/27304234?dopt=Abstract %R 10.1021/acs.chemrev.6b00351 %0 Journal Article %J Biochemistry %D 2016 %T Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases %A Chen, Zan %A Thomas, Stefani N %A Bolduc, David M %A Jiang, Xuejun %A Zhang, Xiangbin %A Wolberger, Cynthia %A Cole, Philip A %K Chromatography, Liquid %K Endosomal Sorting Complexes Required for Transport %K Humans %K Immunoprecipitation %K Nedd4 Ubiquitin Protein Ligases %K Phosphorylation %K Protein Processing, Post-Translational %K PTEN Phosphohydrolase %K Tandem Mass Spectrometry %K Ubiquitin %K Ubiquitin-Protein Ligases %K Ubiquitination %K X-Linked Inhibitor of Apoptosis Protein %X PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growth. PTEN is subject to regulation by a variety of post-translational modifications, including phosphorylation on a C-terminal cluster of four Ser/Thr residues (380, 382, 383, and 385) and ubiquitylation by various E3 ligases, including NEDD4-1 and WWP2. It has previously been shown that C-terminal phosphorylation of PTEN can increase its cellular half-life. Using in vitro ubiquitin transfer assays, we show that WWP2 is more active than NEDD4-1 in ubiquitylating unphosphorylated PTEN. The mapping of ubiquitylation sites in PTEN by mass spectrometry showed that both NEDD4-1 and WWP2 can target a broad range of Lys residues in PTEN, although NEDD4-1 versus WWP2 showed a stronger preference for ubiquitylating PTEN's C2 domain. Whereas tetraphosphorylation of PTEN did not significantly affect its ubiquitylation by NEDD4-1, it inhibited PTEN ubiquitylation by WWP2. Single-turnover and pull-down experiments suggested that tetraphosphorylation of PTEN appears to weaken its interaction with WWP2. These studies reveal how the PTEN E3 ligases WWP2 and NEDD4-1 exhibit distinctive properties in Lys selectivity and sensitivity to PTEN phosphorylation. Our findings also provide a molecular mechanism for the connection between PTEN Ser/Thr phosphorylation and PTEN's cellular stability. %B Biochemistry %V 55 %P 3658-66 %8 2016 07 05 %G eng %N 26 %1 http://www.ncbi.nlm.nih.gov/pubmed/27295432?dopt=Abstract %R 10.1021/acs.biochem.6b00448 %0 Journal Article %J Nat Methods %D 2016 %T Enzyme-catalyzed expressed protein ligation %A Henager, Samuel H %A Chu, Nam %A Chen, Zan %A Bolduc, David %A Dempsey, Daniel R %A Hwang, Yousang %A Wells, James %A Cole, Philip A %K Animals %K Bacillus subtilis %K Blotting, Western %K Catalytic Domain %K Cells, Cultured %K Cysteine %K Escherichia coli %K Fibroblasts %K Mice %K Mutagenesis, Site-Directed %K Peptide Fragments %K Peptide Synthases %K Phosphorylation %K Protein Processing, Post-Translational %K PTEN Phosphohydrolase %K Recombinant Proteins %K Subtilisins %X Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-terminal cysteine (N-Cys)-containing peptides, but the requirement of a Cys residue at the ligation junction can limit the utility of this method. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN. %B Nat Methods %V 13 %P 925-927 %8 2016 Nov %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/27669326?dopt=Abstract %R 10.1038/nmeth.4004 %0 Journal Article %J Epigenetics Chromatin %D 2016 %T Erratum to: CBP binding outside of promoters and enhancers in Drosophila melanogaster %A Philip, Philge %A Boija, Ann %A Vaid, Roshan %A Churcher, Allison M %A Meyers, David J %A Cole, Philip A %A Mannervik, Mattias %A Stenberg, Per %X [This corrects the article DOI: 10.1186/s13072-015-0042-4.]. %B Epigenetics Chromatin %V 9 %P 38 %8 2016 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/27625704?dopt=Abstract %R 10.1186/s13072-016-0088-y %0 Journal Article %J Chembiochem %D 2016 %T An Fc-Small Molecule Conjugate for Targeted Inhibition of the Adenosine 2A Receptor %A Hsiao, Po-Yuan %A Kalin, Jay H %A Sun, Im-Hong %A Amin, Mohammed N %A Lo, Ying-Chun %A Chiang, Meng-Jung %A Giddens, John %A Sysa-Shah, Polina %A Gabrielson, Kathleen %A Wang, Lai-Xi %A Powell, Jonathan D %A Cole, Philip A %K Adenosine A2 Receptor Antagonists %K Animals %K Female %K Humans %K Immunoglobulin Fab Fragments %K Male %K Mice %K Mice, Inbred C57BL %K Mice, Knockout %K Models, Molecular %K Molecular Structure %K Receptor, Adenosine A2A %K Respiratory Tract Infections %K Triazines %K Triazoles %K Vaccinia virus %X The adenosine A2A receptor (A2A R) is expressed in immune cells, as well as brain and heart tissue, and has been intensively studied as a therapeutic target for multiple disease indications. Inhibitors of the A2A R have the potential for stimulating immune response, which could be valuable for cancer immune surveillance and mounting a response against pathogens. One well-established potent and selective small molecule A2A R antagonist, ZM-241385 (ZM), has a short pharmacokinetic half-life and the potential for systemic toxicity due to A2A R effects in the brain and the heart. In this study, we designed an analogue of ZM and tethered it to the Fc domain of the immunoglobulin IgG3 by using expressed protein ligation. The resulting protein-small molecule conjugate, Fc-ZM, retained high affinity for two Fc receptors: FcγRI and the neonatal Fc receptor, FcRn. In addition, Fc-ZM was a potent A2A R antagonist, as measured by a cell-based cAMP assay. Cell-based assays also revealed that Fc-ZM could stimulate interferon γ production in splenocytes in a fashion that was dependent on the presence of A2A R. We found that Fc-ZM, compared with the small molecule ZM, was a superior A2A R antagonist in mice, consistent with the possibility that Fc attachment can improve pharmacokinetic and/or pharmacodynamic properties of the small molecule. %B Chembiochem %V 17 %P 1951-1960 %8 2016 10 17 %G eng %N 20 %1 http://www.ncbi.nlm.nih.gov/pubmed/27432157?dopt=Abstract %R 10.1002/cbic.201600337 %0 Journal Article %J Leuk Res %D 2016 %T Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin %A Robert, Carine %A Nagaria, Pratik K %A Pawar, Nisha %A Adewuyi, Adeoluwa %A Gojo, Ivana %A Meyers, David J %A Cole, Philip A %A Rassool, Feyruz V %K Acetylation %K Benzamides %K Chromatin %K DNA Breaks, Double-Stranded %K DNA End-Joining Repair %K Histone Deacetylase Inhibitors %K Humans %K Ku Autoantigen %K Leukemia, Myeloid, Acute %K Poly (ADP-Ribose) Polymerase-1 %K Pyridines %K Tumor Cells, Cultured %X Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP "trapping". Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP "trapping", which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells. %B Leuk Res %V 45 %P 14-23 %8 2016 Jun %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/27064363?dopt=Abstract %R 10.1016/j.leukres.2016.03.007 %0 Journal Article %J Biochemistry %D 2016 %T Interaction with the DNA Repair Protein Thymine DNA Glycosylase Regulates Histone Acetylation by p300 %A Henry, Ryan A %A Mancuso, Pietro %A Kuo, Yin-Ming %A Tricarico, Rossella %A Tini, Marc %A Cole, Philip A %A Bellacosa, Alfonso %A Andrews, Andrew J %K Acetylation %K Animals %K Cell Line %K Cells, Cultured %K DNA Repair %K E1A-Associated p300 Protein %K Histones %K Mice %K Mice, Knockout %K Thymine DNA Glycosylase %X How protein-protein interactions regulate and alter histone modifications is a major unanswered question in epigenetics. The histone acetyltransferase p300 binds thymine DNA glycosylase (TDG); utilizing mass spectrometry to measure site-specific changes in histone acetylation, we found that the absence of TDG in mouse embryonic fibroblasts leads to a reduction in the rate of histone acetylation. We demonstrate that TDG interacts with the CH3 domain of p300 to allosterically promote p300 activity to specific lysines on histone H3 (K18 and K23). However, when TDG concentrations approach those of histones, TDG acts as a competitive inhibitor of p300 histone acetylation. These results suggest a mechanism for how histone acetylation is fine-tuned via interaction with other proteins, while also highlighting a connection between regulators of two important biological processes: histone acetylation and DNA repair/demethylation. %B Biochemistry %V 55 %P 6766-6775 %8 2016 Dec 13 %G eng %N 49 %1 http://www.ncbi.nlm.nih.gov/pubmed/27951654?dopt=Abstract %R 10.1021/acs.biochem.6b00841 %0 Journal Article %J Biochemistry %D 2016 %T Modulation of p300/CBP Acetylation of Nucleosomes by Bromodomain Ligand I-CBP112 %A Zucconi, Beth E %A Luef, Birgit %A Xu, Wei %A Henry, Ryan A %A Nodelman, Ilana M %A Bowman, Gregory D %A Andrews, Andrew J %A Cole, Philip A %K Acetylation %K Bromine Compounds %K Cell Proliferation %K Crystallography, X-Ray %K E1A-Associated p300 Protein %K Histones %K Humans %K Leukemia %K Male %K Models, Molecular %K Mutagenesis, Site-Directed %K Nucleosomes %K p300-CBP Transcription Factors %K Prostatic Neoplasms %K Protein Binding %K Protein Conformation %K Tumor Cells, Cultured %X The histone acetyltransferase (HAT) enzymes p300 and CBP are closely related paralogs that serve as transcriptional coactivators and have been found to be dysregulated in cancer and other diseases. p300/CBP is a multidomain protein and possesses a highly conserved bromodomain that has been shown to bind acetylated Lys residues in both proteins and various small molecules, including I-CBP112 and CBP30. Here we show that the ligand I-CBP112 can stimulate nucleosome acetylation up to 3-fold while CBP30 does not. Activation of p300/CBP by I-CBP112 is not observed with the isolated histone H3 substrate but requires a nucleosome substrate. I-CBP112 does not impact nucleosome acetylation by the isolated p300 HAT domain, and the effects of I-CBP112 on p300/CBP can be neutralized by CBP30, suggesting that I-CBP112 likely allosterically activates p300/CBP through bromodomain interactions. Using mass spectrometry and Western blots, we have found that I-CBP112 particularly stimulates acetylation of Lys18 of histone H3 (H3K18) in nucleosomes, an established in vivo site of p300/CBP. In addition, we show that I-CBP112 enhances H3K18 acetylation in acute leukemia and prostate cancer cells in a concentration range commensurate with its antiproliferative effects. Our findings extend the known pharmacology of bromodomain ligands in the regulation of p300/CBP and suggest a novel approach to modulating histone acetylation in cancer. %B Biochemistry %V 55 %P 3727-34 %8 2016 07 12 %G eng %N 27 %1 http://www.ncbi.nlm.nih.gov/pubmed/27332697?dopt=Abstract %R 10.1021/acs.biochem.6b00480 %0 Journal Article %J J Biol Chem %D 2016 %T Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation %A Chen, Zan %A Dempsey, Daniel R %A Thomas, Stefani N %A Hayward, Dawn %A Bolduc, David M %A Cole, Philip A %K Amino Acid Sequence %K Humans %K Phosphorylation %K PTEN Phosphohydrolase %X PTEN is a tumor suppressor that functions to negatively regulate the PI3K/AKT pathway as the lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate. Phosphorylation of a cluster of Ser/Thr residues (amino acids 380-385) on the C-terminal tail serves to alter the conformational state of PTEN from an open active state to a closed inhibited state, resulting in a reduction of plasma membrane localization and inhibition of enzyme activity. The relative contribution of each phosphorylation site to PTEN autoinhibition and the structural basis for the conformational closure is still unclear. To further the structural understanding of PTEN regulation by C-terminal tail phosphorylation, we used protein semisynthesis to insert stoichiometric and site-specific phospho-Ser/Thr(s) in the C-terminal tail of PTEN. Additionally, we employed photo-cross-linking to map the intramolecular PTEN interactions of the phospho-tail. Systematic evaluation of the PTEN C-tail phospho-cluster showed autoinhibition, and conformational closure was influenced by the aggregate effect of multiple phospho-sites rather than dominated by a single phosphorylation site. Moreover, photo-cross-linking suggested a direct interaction between the PTEN C-tail and a segment in the N-terminal region of the catalytic domain. Mutagenesis experiments provided additional insights into how the PTEN phospho-tail interacts with both the C2 and catalytic domains. %B J Biol Chem %V 291 %P 14160-9 %8 2016 Jul 01 %G eng %N 27 %1 http://www.ncbi.nlm.nih.gov/pubmed/27226612?dopt=Abstract %R 10.1074/jbc.M116.728980 %0 Journal Article %J Epigenetics Chromatin %D 2015 %T CBP binding outside of promoters and enhancers in Drosophila melanogaster %A Philip, Philge %A Boija, Ann %A Vaid, Roshan %A Churcher, Allison M %A Meyers, David J %A Cole, Philip A %A Mannervik, Mattias %A Stenberg, Per %X BACKGROUND: CREB-binding protein (CBP, also known as nejire) is a transcriptional co-activator that is conserved in metazoans. CBP plays an important role in embryonic development and cell differentiation and mutations in CBP can lead to various diseases in humans. In addition, CBP and the related p300 protein have successfully been used to predict enhancers in both humans and flies when they occur with monomethylation of histone H3 on lysine 4 (H3K4me1). RESULTS: Here, we compare CBP chromatin immunoprecipitation sequencing data from Drosophila S2 cells with modENCODE data and show that CBP is bound at genomic sites with a wide range of functions. As expected, we find that CBP is bound at active promoters and enhancers. In addition, we find that the strongest CBP sites in the genome are found at Polycomb response elements embedded in histone H3 lysine 27 trimethylated (H3K27me3) chromatin, where they correlate with binding of the Pho repressive complex. Interestingly, we find that CBP also binds to most insulators in the genome. At a subset of these, CBP may regulate insulating activity, measured as the ability to prevent repressive H3K27 methylation from spreading into adjacent chromatin. CONCLUSIONS: We conclude that CBP could be involved in a much wider range of functions than has previously been appreciated, including Polycomb repression and insulator activity. In addition, we discuss the possibility that a common role for CBP at all functional elements may be to regulate interactions between distant chromosomal regions and speculate that CBP is controlling higher order chromatin organization. %B Epigenetics Chromatin %V 8 %P 48 %8 2015 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26604986?dopt=Abstract %R 10.1186/s13072-015-0042-4 %0 Journal Article %J Bioorg Chem %D 2015 %T Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs %A Taylor, Martin S %A Dempsey, Daniel R %A Hwang, Yousang %A Chen, Zan %A Chu, Nam %A Boeke, Jef D %A Cole, Philip A %K Acyltransferases %K Amino Acid Sequence %K Animals %K Baculoviridae %K Biotin %K Cell Line %K Enzyme Assays %K Genetic Vectors %K Ghrelin %K Hydrogen-Ion Concentration %K Mice %K Models, Molecular %X Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates the metabolism-regulating peptide hormone ghrelin at Ser3 and may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Protein octanoylation is unique to ghrelin in humans, and little is known about the mechanism of GOAT or of related protein-O-acyltransferases HHAT or PORC. In this study, we explored an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features. Measurement of Km for 10-mer, 27-mer, and synthetic Tat-peptide-containing ghrelin substrates provided evidence for a role of charge interactions in substrate binding. Ghrelin substrates with amino-alanine in place of Ser3 demonstrated that GOAT can catalyze the formation of an octanoyl-amide bond at a similar rate compared with the natural reaction. A pH-rate comparison of these substrates revealed minimal differences in acyltransferase activity across pH 6.0-9.0, providing evidence that these reactions may be relatively insensitive to the basicity of the substrate nucleophile. The conserved His338 residue was required both for Ser3 and amino-Ala3 ghrelin substrates, suggesting that His338 may have a key catalytic role beyond that of a general base. %B Bioorg Chem %V 62 %P 64-73 %8 2015 Oct %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26246082?dopt=Abstract %R 10.1016/j.bioorg.2015.07.003 %0 Journal Article %J Chem Rev %D 2015 %T Protein lysine acetylation by p300/CBP %A Dancy, Beverley M %A Cole, Philip A %K Acetylation %K Animals %K Humans %K Lysine %K Models, Molecular %K p300-CBP Transcription Factors %B Chem Rev %V 115 %P 2419-52 %8 2015 Mar 25 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/25594381?dopt=Abstract %R 10.1021/cr500452k %0 Journal Article %J Elife %D 2015 %T Switching immune signals on and off %A Chu, Nam %A Cole, Philip A %K Animals %K Phytic Acid %K Protein-Tyrosine Kinases %B Elife %V 4 %8 2015 Apr 02 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/25838130?dopt=Abstract %R 10.7554/eLife.07204 %0 Journal Article %J Curr Opin Chem Biol %D 2015 %T Synthetic approaches to protein phosphorylation %A Chen, Zan %A Cole, Philip A %K Animals %K Humans %K Mutagenesis, Site-Directed %K Phosphoproteins %K Phosphorylation %K Protein Transport %K Recombinant Proteins %X Reversible protein phosphorylation is critically important in biology and medicine. Hundreds of thousands of sites of protein phosphorylation have been discovered but our understanding of the functions of the vast majority of these post-translational modifications is lacking. This review describes several chemical and biochemical methods that are under development and in current use to install phospho-amino acids and their mimics site-specifically into proteins. The relative merits of total chemical synthesis, semisynthesis, and nonsense suppression strategies for studying protein phosphorylation are discussed in terms of technical simplicity, scope, and versatility. %B Curr Opin Chem Biol %V 28 %P 115-22 %8 2015 Oct %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26196731?dopt=Abstract %R 10.1016/j.cbpa.2015.07.001 %0 Journal Article %J Elife %D 2015 %T YcgC represents a new protein deacetylase family in prokaryotes %A Tu, Shun %A Guo, Shu-Juan %A Chen, Chien-Sheng %A Liu, Cheng-Xi %A Jiang, He-Wei %A Ge, Feng %A Deng, Jiao-Yu %A Zhou, Yi-Ming %A Czajkowsky, Daniel M %A Li, Yang %A Qi, Bang-Ruo %A Ahn, Young-Hoon %A Cole, Philip A %A Zhu, Heng %A Tao, Sheng-Ce %K Amidohydrolases %K Escherichia coli %K Escherichia coli Proteins %K Lysine %K Protein Processing, Post-Translational %K Substrate Specificity %K Transcription Factors %X Reversible lysine acetylation is one of the most important protein posttranslational modifications that plays essential roles in both prokaryotes and eukaryotes. However, only a few lysine deacetylases (KDACs) have been identified in prokaryotes, perhaps in part due to their limited sequence homology. Herein, we developed a 'clip-chip' strategy to enable unbiased, activity-based discovery of novel KDACs in the Escherichia coli proteome. In-depth biochemical characterization confirmed that YcgC is a serine hydrolase involving Ser200 as the catalytic nucleophile for lysine deacetylation and does not use NAD(+) or Zn(2+) like other established KDACs. Further, in vivo characterization demonstrated that YcgC regulates transcription by catalyzing deacetylation of Lys52 and Lys62 of a transcriptional repressor RutR. Importantly, YcgC targets a distinct set of substrates from the only known E. coli KDAC CobB. Analysis of YcgC's bacterial homologs confirmed that they also exhibit KDAC activity. YcgC thus represents a novel family of prokaryotic KDACs. %B Elife %V 4 %8 2015 Dec 30 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26716769?dopt=Abstract %R 10.7554/eLife.05322 %0 Journal Article %J Methods Enzymol %D 2014 %T Catalytic mechanisms and regulation of protein kinases %A Wang, Zhihong %A Cole, Philip A %K Adenosine Triphosphate %K Animals %K Biocatalysis %K Humans %K Models, Molecular %K Mutation %K Phosphorylation %K Protein Conformation %K Protein Kinase Inhibitors %K Protein Kinases %K Protein Processing, Post-Translational %K Substrate Specificity %X Protein kinases transfer a phosphoryl group from ATP onto target proteins and play a critical role in signal transduction and other cellular processes. Here, we review the kinase kinetic and chemical mechanisms and their application in understanding kinase structure and function. Aberrant kinase activity has been implicated in many human diseases, in particular cancer. We highlight applications of technologies and concepts derived from kinase mechanistic studies that have helped illuminate how kinases are regulated and contribute to pathophysiology. %B Methods Enzymol %V 548 %P 1-21 %8 2014 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/25399640?dopt=Abstract %R 10.1016/B978-0-12-397918-6.00001-X %0 Journal Article %J Anal Chem %D 2014 %T Dissecting the binding mode of low affinity phage display peptide ligands to protein targets by hydrogen/deuterium exchange coupled to mass spectrometry %A Leurs, Ulrike %A Lohse, Brian %A Ming, Shonoi %A Cole, Philip A %A Clausen, Rasmus P %A Kristensen, Jesper L %A Rand, Kasper D %K Binding Sites %K Deuterium Exchange Measurement %K Histone Demethylases %K Humans %K Jumonji Domain-Containing Histone Demethylases %K Ligands %K Mass Spectrometry %K Models, Molecular %K Molecular Structure %K Peptides %X Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize interactions of low affinity peptides with their cognate protein targets. The HDX-MS workflow was optimized to accurately detect low-affinity peptide-protein interactions by use of ion mobility, electron transfer dissociation, nonbinding control peptides, and statistical analysis of replicate data. We show that HDX-MS can identify regions in the two epigenetic regulator proteins KDM4C and KDM1A that are perturbed through weak interactions with PD-identified peptides. Two peptides cause reduced HDX on opposite sides of the active site of KDM4C, indicating distinct binding modes. In contrast, the perturbation site of another PD-selected peptide inhibiting the function of KDM1A maps to a GST-tag. Our results demonstrate that HDX-MS can validate and map weak peptide-protein interactions and pave the way for understanding and optimizing the binding of peptide scaffolds identified through PD and similar ligand discovery approaches. %B Anal Chem %V 86 %P 11734-41 %8 2014 Dec 02 %G eng %N 23 %1 http://www.ncbi.nlm.nih.gov/pubmed/25325890?dopt=Abstract %R 10.1021/ac503137u %0 Journal Article %J J Am Chem Soc %D 2014 %T An Fc domain protein-small molecule conjugate as an enhanced immunomodulator %A Chiang, Meng-Jung %A Holbert, Marc A %A Kalin, Jay H %A Ahn, Young-Hoon %A Giddens, John %A Amin, Mohammed N %A Taylor, Martin S %A Collins, Samuel L %A Chan-Li, Yee %A Waickman, Adam %A Hsiao, Po-Yuan %A Bolduc, David %A Leahy, Daniel J %A Horton, Maureen R %A Wang, Lai-Xi %A Powell, Jonathan D %A Cole, Philip A %K Adenosine %K Animals %K CD4-Positive T-Lymphocytes %K Immunoconjugates %K Immunoglobulin Fc Fragments %K Immunologic Factors %K Mice %K Models, Molecular %K Phenethylamines %K Protein Conformation %X Proteins as well as small molecules have demonstrated success as therapeutic agents, but their pharmacologic properties sometimes fall short against particular drug targets. Although the adenosine 2a receptor (A(2A)R) has been identified as a promising target for immunotherapy, small molecule A(2A)R agonists have suffered from short pharmacokinetic half-lives and the potential for toxicity by modulating nonimmune pathways. To overcome these limitations, we have tethered the A(2A)R agonist CGS-21680 to the immunoglobulin Fc domain using expressed protein ligation with Sf9 cell secreted protein. The protein small molecule conjugate Fc-CGS retained potent Fc receptor and A(2A)R interactions and showed superior properties as a therapeutic for the treatment of a mouse model of autoimmune pneumonitis. This approach may provide a general strategy for optimizing small molecule therapeutics. %B J Am Chem Soc %V 136 %P 3370-3 %8 2014 Mar 05 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/24533830?dopt=Abstract %R 10.1021/ja5006674 %0 Journal Article %J Elife %D 2014 %T How IGF-1 activates its receptor %A Kavran, Jennifer M %A McCabe, Jacqueline M %A Byrne, Patrick O %A Connacher, Mary Katherine %A Wang, Zhihong %A Ramek, Alexander %A Sarabipour, Sarvenaz %A Shan, Yibing %A Shaw, David E %A Hristova, Kalina %A Cole, Philip A %A Leahy, Daniel J %K Amino Acid Sequence %K Animals %K Conserved Sequence %K HEK293 Cells %K Humans %K Insulin-Like Growth Factor I %K Ligands %K Mice %K Models, Molecular %K Molecular Sequence Data %K Mutation %K Phosphorylation %K Protein Binding %K Protein Multimerization %K Protein Structure, Tertiary %K Receptor, IGF Type 1 %K Receptor, Insulin %X The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation. %B Elife %V 3 %8 2014 Sep 25 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/25255214?dopt=Abstract %R 10.7554/eLife.03772 %0 Journal Article %J J Biol Chem %D 2014 %T Regulation of S-adenosylhomocysteine hydrolase by lysine acetylation %A Wang, Yun %A Kavran, Jennifer M %A Chen, Zan %A Karukurichi, Kannan R %A Leahy, Daniel J %A Cole, Philip A %K Acetylation %K Adenosylhomocysteinase %K Amino Acid Sequence %K Catalysis %K Crystallography, X-Ray %K Humans %K Hydrogen Bonding %K Lysine %K Methylation %K Models, Molecular %K Molecular Sequence Data %K Mutagenesis, Site-Directed %K NAD %K Plasmids %K Protein Binding %K Protein Processing, Post-Translational %K Protein Structure, Tertiary %K Recombinant Proteins %K Sequence Homology, Amino Acid %K Structure-Activity Relationship %X S-Adenosylhomocysteine hydrolase (SAHH) is an NAD(+)-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys(401) and Lys(408) but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys(401) and Lys(408) and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD(+) binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns. %B J Biol Chem %V 289 %P 31361-72 %8 2014 Nov 07 %G eng %N 45 %1 http://www.ncbi.nlm.nih.gov/pubmed/25248746?dopt=Abstract %R 10.1074/jbc.M114.597153 %0 Journal Article %J ACS Chem Biol %D 2014 %T A selective phenelzine analogue inhibitor of histone demethylase LSD1 %A Prusevich, Polina %A Kalin, Jay H %A Ming, Shonoi A %A Basso, Manuela %A Givens, Jeffrey %A Li, Xin %A Hu, Jianfei %A Taylor, Martin S %A Cieniewicz, Anne M %A Hsiao, Po-Yuan %A Huang, Rong %A Roberson, Heather %A Adejola, Nkosi %A Avery, Lindsay B %A Casero, Robert A %A Taverna, Sean D %A Qian, Jiang %A Tackett, Alan J %A Ratan, Rajiv R %A McDonald, Oliver G %A Feinberg, Andrew P %A Cole, Philip A %K Animals %K Blotting, Western %K Cell Survival %K Cells, Cultured %K DNA Methylation %K Embryo, Mammalian %K Enzyme Inhibitors %K Fetus %K Histone Demethylases %K Histones %K Humans %K Monoamine Oxidase %K Neurons %K Phenelzine %K Rats %K Rats, Sprague-Dawley %X Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor in vitro and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1-/- cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease. %B ACS Chem Biol %V 9 %P 1284-93 %8 2014 Jun 20 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/24707965?dopt=Abstract %R 10.1021/cb500018s %0 Journal Article %J Oncotarget %D 2014 %T Structural basis of nSH2 regulation and lipid binding in PI3Kα %A Miller, Michelle S %A Schmidt-Kittler, Oleg %A Bolduc, David M %A Brower, Evan T %A Chaves-Moreira, Daniele %A Allaire, Marc %A Kinzler, Kenneth W %A Jennings, Ian G %A Thompson, Philip E %A Cole, Philip A %A Amzel, L Mario %A Vogelstein, Bert %A Gabelli, Sandra B %K Amino Acid Sequence %K Animals %K Binding Sites %K Boron Compounds %K Models, Molecular %K Molecular Sequence Data %K Phosphatidylinositol 3-Kinases %K Protein Binding %K Protein Conformation %K Sf9 Cells %K Signal Transduction %K Spodoptera %K src Homology Domains %X We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP₂, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP₂ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant. %B Oncotarget %V 5 %P 5198-208 %8 2014 Jul 30 %G eng %N 14 %1 http://www.ncbi.nlm.nih.gov/pubmed/25105564?dopt=Abstract %R 10.18632/oncotarget.2263 %0 Journal Article %J Biochemistry %D 2014 %T Structure of the p300 histone acetyltransferase bound to acetyl-coenzyme A and its analogues %A Maksimoska, Jasna %A Segura-Peña, Dario %A Cole, Philip A %A Marmorstein, Ronen %K Acetyl Coenzyme A %K Catalytic Domain %K Coenzyme A %K Humans %K Models, Molecular %K p300-CBP Transcription Factors %K Protein Binding %K Protein Conformation %X The p300 and CBP transcriptional coactivator paralogs (p300/CBP) regulate a variety of different cellular pathways, in part, by acetylating histones and more than 70 non-histone protein substrates. Mutation, chromosomal translocation, or other aberrant activities of p300/CBP are linked to many different diseases, including cancer. Because of its pleiotropic biological roles and connection to disease, it is important to understand the mechanism of acetyl transfer by p300/CBP, in part so that inhibitors can be more rationally developed. Toward this goal, a structure of p300 bound to a Lys-CoA bisubstrate HAT inhibitor has been previously elucidated, and the enzyme's catalytic mechanism has been investigated. Nonetheless, many questions underlying p300/CBP structure and mechanism remain. Here, we report a structural characterization of different reaction states in the p300 activity cycle. We present the structures of p300 in complex with an acetyl-CoA substrate, a CoA product, and an acetonyl-CoA inhibitor. A comparison of these structures with the previously reported p300/Lys-CoA complex demonstrates that the conformation of the enzyme active site depends on the interaction of the enzyme with the cofactor, and is not apparently influenced by protein substrate lysine binding. The p300/CoA crystals also contain two poly(ethylene glycol) moieties bound proximal to the cofactor binding site, implicating the path of protein substrate association. The structure of the p300/acetonyl-CoA complex explains the inhibitory and tight binding properties of the acetonyl-CoA toward p300. Together, these studies provide new insights into the molecular basis of acetylation by p300 and have implications for the rational development of new small molecule p300 inhibitors. %B Biochemistry %V 53 %P 3415-22 %8 2014 Jun 03 %G eng %N 21 %1 http://www.ncbi.nlm.nih.gov/pubmed/24819397?dopt=Abstract %R 10.1021/bi500380f %0 Journal Article %J ACS Chem Biol %D 2014 %T Substrate- and cofactor-independent inhibition of histone demethylase KDM4C %A Leurs, Ulrike %A Lohse, Brian %A Rand, Kasper D %A Ming, Shonoi %A Riise, Erik S %A Cole, Philip A %A Kristensen, Jesper L %A Clausen, Rasmus P %K Amino Acid Sequence %K Catalytic Domain %K Cell Line %K Coenzymes %K Deuterium Exchange Measurement %K Enzyme Inhibitors %K High-Throughput Screening Assays %K Histone Demethylases %K Humans %K Inhibitory Concentration 50 %K Jumonji Domain-Containing Histone Demethylases %K Molecular Sequence Data %K Peptide Library %X Inhibition of histone demethylases has within recent years advanced into a new strategy for treating cancer and other diseases. Targeting specific histone demethylases can be challenging, as the active sites of KDM1A-B and KDM4A-D histone demethylases are highly conserved. Most inhibitors developed up-to-date target either the cofactor- or substrate-binding sites of these enzymes, resulting in a lack of selectivity and off-target effects. This study describes the discovery of the first peptide-based inhibitors of KDM4 histone demethylases that do not share the histone peptide sequence or inhibit through substrate competition. Through screening of DNA-encoded peptide libraries against KDM1 and -4 histone demethylases by phage display, two cyclic peptides targeting the histone demethylase KDM4C were identified and developed as inhibitors by amino acid replacement, truncation, and chemical modifications. Hydrogen/deuterium exchange mass spectrometry revealed that the peptide-based inhibitors target KDM4C through substrate-independent interactions located on the surface remote from the active site within less conserved regions of KDM4C. The sites discovered in this study provide a new approach of targeting KDM4C through substrate- and cofactor-independent interactions and may be further explored to develop potent selective inhibitors and biological probes for the KDM4 family. %B ACS Chem Biol %V 9 %P 2131-8 %8 2014 Sep 19 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/25014588?dopt=Abstract %R 10.1021/cb500374f %0 Journal Article %J Mol Cell Biol %D 2014 %T Two histone/protein acetyltransferases, CBP and p300, are indispensable for Foxp3+ T-regulatory cell development and function %A Liu, Yujie %A Wang, Liqing %A Han, Rongxiang %A Beier, Ulf H %A Akimova, Tatiana %A Bhatti, Tricia %A Xiao, Haiyan %A Cole, Philip A %A Brindle, Paul K %A Hancock, Wayne W %K Animals %K Cell Survival %K Colitis %K CREB-Binding Protein %K Cyclic AMP Response Element-Binding Protein %K E1A-Associated p300 Protein %K Epigenesis, Genetic %K Female %K Forkhead Transcription Factors %K GATA3 Transcription Factor %K Humans %K Male %K Mice %K Mice, Inbred C57BL %K Promoter Regions, Genetic %K Sequence Deletion %K T-Lymphocytes, Regulatory %X T-regulatory (Treg) cells are important to immune homeostasis, and Treg cell deficiency or dysfunction leads to autoimmune disease. A histone/protein acetyltransferase (HAT), p300, was recently found to be important for Treg function and stability, but further insights into the mechanisms by which p300 or other HATs affect Treg biology are needed. Here we show that CBP, a p300 paralog, is also important in controlling Treg function and stability. Thus, while mice with Treg-specific deletion of CBP or p300 developed minimal autoimmune disease, the combined deletion of CBP and p300 led to fatal autoimmunity by 3 to 4 weeks of age. The effects of CBP and p300 deletion on Treg development are dose dependent and involve multiple mechanisms. CBP and p300 cooperate with several key Treg transcription factors that act on the Foxp3 promoter to promote Foxp3 production. CBP and p300 also act on the Foxp3 conserved noncoding sequence 2 (CNS2) region to maintain Treg stability in inflammatory environments by regulating pCREB function and GATA3 expression, respectively. Lastly, CBP and p300 regulate the epigenetic status and function of Foxp3. Our findings provide insights into how HATs orchestrate multiple aspects of Treg development and function and identify overlapping but also discrete activities for p300 and CBP in control of Treg cells. %B Mol Cell Biol %V 34 %P 3993-4007 %8 2014 Nov %G eng %N 21 %1 http://www.ncbi.nlm.nih.gov/pubmed/25154413?dopt=Abstract %R 10.1128/MCB.00919-14 %0 Journal Article %J Anal Biochem %D 2014 %T Using S-adenosyl-L-homocysteine capture compounds to characterize S-adenosyl-L-methionine and S-adenosyl-L-homocysteine binding proteins %A Brown, Lindsey J %A Baranowski, Matthias %A Wang, Yun %A Schrey, Anna K %A Lenz, Thomas %A Taverna, Sean D %A Cole, Philip A %A Sefkow, Michael %K Catechol O-Methyltransferase %K DNA-Binding Proteins %K Fluorescence Polarization %K Histone-Lysine N-Methyltransferase %K Humans %K Hydrolases %K Nuclear Proteins %K S-Adenosylhomocysteine %K S-Adenosylmethionine %K Transcription Factors %X S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH-CC with biotin used in conjunction with streptavidin-horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the Kd values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established Kd values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs. %B Anal Biochem %V 467 %P 14-21 %8 2014 Dec 15 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/25172130?dopt=Abstract %R 10.1016/j.ab.2014.08.013