@article{1332026,

title = {Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action},

author = {Shridhar Bhat and Yousang Hwang and Matthew D Gibson and Michael T Morgan and Sean D Taverna and Yingming Zhao and Cynthia Wolberger and Michael G Poirier and Philip A Cole},

doi = {10.1021/jacs.8b03572},

issn = {1520-5126},

year  = {2018},

date = {2018-08-01},

journal = {J Am Chem Soc},

volume = {140},

number = {30},

pages = {9478-9485},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{1332028,

title = {Author Correction: Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours},

author = {Loren M Lasko and Clarissa G Jakob and Rohinton P Edalji and Wei Qiu and Debra Montgomery and Enrico L Digiammarino and T Matt Hansen and Roberto M Risi and Robin Frey and Vlasios Manaves and Bailin Shaw and Mikkel Algire and Paul Hessler and Lloyd T Lam and Tamar Uziel and Emily Faivre and Debra Ferguson and Fritz G Buchanan and Ruth L Martin and Maricel Torrent and Gary G Chiang and Kannan Karukurichi and J William Langston and Brian T Weinert and Chunaram Choudhary and Peter Vries and Arthur F Kluge and Michael A Patane and John H Van Drie and Ce Wang and David McElligott and Edward A Kesicki and Ronen Marmorstein and Chaohong Sun and Philip A Cole and Saul H Rosenberg and Michael R Michaelides and Albert Lai and Kenneth D Bromberg},

doi = {10.1038/s41586-018-0111-5},

issn = {1476-4687},

year  = {2018},

date = {2018-06-01},

journal = {Nature},

volume = {558},

number = {7710},

pages = {E1},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{1332029,

title = {Lysine-14 acetylation of histone H3 in chromatin confers resistance to the deacetylase and demethylase activities of an epigenetic silencing complex},

author = {Mingxuan Wu and Dawn Hayward and Jay H Kalin and Yun Song and John W R Schwabe and Philip A Cole},

doi = {10.7554/eLife.37231},

issn = {2050-084X},

year  = {2018},

date = {2018-06-01},

journal = {Elife},

volume = {7},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{1299782,

title = {Analysis of Cellular Tyrosine Phosphorylation via Chemical Rescue of Conditionally Active Abl Kinase},

author = {Zhihong Wang and Min-Sik Kim and Isabel Martinez-Ferrando and Anthony J Koleske and Akhilesh Pandey and Philip A Cole},

doi = {10.1021/acs.biochem.7b01158},

issn = {1520-4995},

year  = {2018},

date = {2018-02-01},

journal = {Biochemistry},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{1299932,

title = {Discovery of Spiro Oxazolidinediones as Selective, Orally Bioavailable Inhibitors of p300/CBP Histone Acetyltransferases},

author = {Michael R Michaelides and Arthur Kluge and Michael Patane and John H Van Drie and Ce Wang and T Matthew Hansen and Roberto M Risi and Robert Mantei and Carmen Hertel and Kannan Karukurichi and Alexandre Nesterov and David McElligott and Peter Vries and J William Langston and Philip A Cole and Ronen Marmorstein and Hong Liu and Loren Lasko and Kenneth D Bromberg and Albert Lai and Edward A Kesicki},

doi = {10.1021/acsmedchemlett.7b00395},

issn = {1948-5875},

year  = {2018},

date = {2018-01-01},

journal = {ACS Med Chem Lett},

volume = {9},

number = {1},

pages = {28-33},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{1299936,

title = {Measurement of nanoscale DNA translocation by uracil DNA glycosylase in human cells},

author = {Alexandre Esadze and Gaddiel Rodriguez and Brian P Weiser and Philip A Cole and James T Stivers},

doi = {10.1093/nar/gkx848},

issn = {1362-4962},

year  = {2017},

date = {2017-12-01},

journal = {Nucleic Acids Res},

volume = {45},

number = {21},

pages = {12413-12424},

abstract = {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.},

keywords = {Cell Line, DNA, DNA Glycosylases, Humans, Uracil},

pubstate = {published},

tppubtype = {article}

}

@article{1299940,

title = {Investigation of N-Terminal Phospho-Regulation of~Uracil DNA Glycosylase Using Protein Semisynthesis},

author = {Brian P Weiser and James T Stivers and Philip A Cole},

doi = {10.1016/j.bpj.2017.06.016},

issn = {1542-0086},

year  = {2017},

date = {2017-07-01},

journal = {Biophys J},

volume = {113},

number = {2},

pages = {393-401},

abstract = {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.},

keywords = {Catalysis, DNA Glycosylases, Electrospray Ionization, Escherichia coli, Humans, Mass, Mutation, Phosphorylation, Proliferating Cell Nuclear Antigen, Protein Binding, Protein Domains, Protein Stability, Replication Protein A, Spectrometry},

pubstate = {published},

tppubtype = {article}

}

@article{1299944,

title = {A Tunable Brake for HECT Ubiquitin Ligases},

author = {Zan Chen and Hanjie Jiang and Wei Xu and Xiaoguang Li and Daniel R Dempsey and Xiangbin Zhang and Peter Devreotes and Cynthia Wolberger and L Mario Amzel and Sandra B Gabelli and Philip A Cole},

doi = {10.1016/j.molcel.2017.03.020},

issn = {1097-4164},

year  = {2017},

date = {2017-05-01},

journal = {Mol Cell},

volume = {66},

number = {3},

pages = {345-357.e6},

abstract = {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.},

keywords = {Allosteric Regulation, Endosomal Sorting Complexes Required for Transport, Enzyme Activation, Enzyme Stability, HeLa Cells, Humans, Models, Molecular, Mutation, Nedd4 Ubiquitin Protein Ligases, Phosphorylation, Post-Translational, Protein Domains, Protein Processing, Proteolysis, Repressor Proteins, Structure-Activity Relationship, Transfection, Ubiquitin-Protein Ligases},

pubstate = {published},

tppubtype = {article}

}

@article{1299945,

title = {Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution},

author = {Gary C H Mo and Brian Ross and Fabian Hertel and Premashis Manna and Xinxing Yang and Eric Greenwald and Chris Booth and Ashlee M Plummer and Brian Tenner and Zan Chen and Yuxiao Wang and Eileen J Kennedy and Philip A Cole and Karen G Fleming and Amy Palmer and Ralph Jimenez and Jie Xiao and Peter Dedecker and Jin Zhang},

doi = {10.1038/nmeth.4221},

issn = {1548-7105},

year  = {2017},

date = {2017-04-01},

journal = {Nat Methods},

volume = {14},

number = {4},

pages = {427-434},

abstract = {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.},

keywords = {Biosensing Techniques, Cell Membrane, Cyclic AMP-Dependent Protein Kinases, Escherichia coli, Fluorescence Resonance Energy Transfer, Fluorescent Dyes, Green Fluorescent Proteins, HeLa Cells, Humans, Microscopy, Molecular Imaging, Mutagenesis, Protein Interaction Mapping, Site-Directed, Stochastic Processes},

pubstate = {published},

tppubtype = {article}

}

@article{1299948,

title = {Interaction with the DNA Repair Protein Thymine DNA Glycosylase Regulates Histone Acetylation by p300},

author = {Ryan A Henry and Pietro Mancuso and Yin-Ming Kuo and Rossella Tricarico and Marc Tini and Philip A Cole and Alfonso Bellacosa and Andrew J Andrews},

doi = {10.1021/acs.biochem.6b00841},

issn = {1520-4995},

year  = {2016},

date = {2016-12-01},

journal = {Biochemistry},

volume = {55},

number = {49},

pages = {6766-6775},

abstract = {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.},

keywords = {Acetylation, Animals, Cell Line, Cells, Cultured, DNA Repair, E1A-Associated p300 Protein, Histones, Knockout, Mice, Thymine DNA Glycosylase},

pubstate = {published},

tppubtype = {article}

}

@article{1299949,

title = {Enzyme-catalyzed expressed protein ligation},

author = {Samuel H Henager and Nam Chu and Zan Chen and David Bolduc and Daniel R Dempsey and Yousang Hwang and James Wells and Philip A Cole},

doi = {10.1038/nmeth.4004},

issn = {1548-7105},

year  = {2016},

date = {2016-11-01},

journal = {Nat Methods},

volume = {13},

number = {11},

pages = {925-927},

abstract = {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.},

keywords = {Animals, Bacillus subtilis, Blotting, Catalytic Domain, Cells, Cultured, Cysteine, Escherichia coli, Fibroblasts, Mice, Mutagenesis, Peptide Fragments, Peptide Synthases, Phosphorylation, Post-Translational, Protein Processing, PTEN Phosphohydrolase, Recombinant Proteins, Site-Directed, Subtilisins, Western},

pubstate = {published},

tppubtype = {article}

}

@article{1299966,

title = {Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation},

author = {Zan Chen and Daniel R Dempsey and Stefani N Thomas and Dawn Hayward and David M Bolduc and Philip A Cole},

doi = {10.1074/jbc.M116.728980},

issn = {1083-351X},

year  = {2016},

date = {2016-07-01},

journal = {J Biol Chem},

volume = {291},

number = {27},

pages = {14160-9},

abstract = {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.},

keywords = {Amino Acid Sequence, Humans, Phosphorylation, PTEN Phosphohydrolase},

pubstate = {published},

tppubtype = {article}

}

@article{1299967,

title = {Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin},

author = {Carine Robert and Pratik K Nagaria and Nisha Pawar and Adeoluwa Adewuyi and Ivana Gojo and David J Meyers and Philip A Cole and Feyruz V Rassool},

doi = {10.1016/j.leukres.2016.03.007},

issn = {1873-5835},

year  = {2016},

date = {2016-06-01},

journal = {Leuk Res},

volume = {45},

pages = {14-23},

abstract = {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.},

keywords = {Acetylation, Acute, Benzamides, Chromatin, Cultured, DNA Breaks, DNA End-Joining Repair, Double-Stranded, Histone Deacetylase Inhibitors, Humans, Ku Autoantigen, Leukemia, Myeloid, Poly (ADP-Ribose) Polymerase-1, Pyridines, Tumor Cells},

pubstate = {published},

tppubtype = {article}

}

@article{1299963,

title = {Modulation of p300/CBP Acetylation of Nucleosomes by Bromodomain Ligand I-CBP112},

author = {B. E. Zucconi and B. Luef and W. Xu and R. A. Henry and I. M. Nodelman and G. D. Bowman and A. J Andrews and P. A. Cole},

doi = {10.1021/acs.biochem.6b00480},

issn = {1520-4995},

year  = {2016},

date = {2016-00-00},

journal = {Biochemistry},

volume = {55},

number = {27},

pages = {3727-34},

abstract = {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.},

keywords = {Acetylation, Bromine Compounds, Cell Proliferation, Crystallography, Cultured, E1A-Associated p300 Protein, Histones, Humans, Leukemia, Male, Models, Molecular, Mutagenesis, Nucleosomes, p300-CBP Transcription Factors, Prostatic Neoplasms, Protein Binding, Protein Conformation, Site-Directed, Tumor Cells, X-Ray},

pubstate = {published},

tppubtype = {article}

}

@article{1299968,

title = {YcgC represents a new protein deacetylase family in prokaryotes},

author = {Shun Tu and Shu-Juan Guo and Chien-Sheng Chen and Cheng-Xi Liu and He-Wei Jiang and Feng Ge and Jiao-Yu Deng and Yi-Ming Zhou and Daniel M Czajkowsky and Yang Li and Bang-Ruo Qi and Young-Hoon Ahn and Philip A Cole and Heng Zhu and Sheng-Ce Tao},

doi = {10.7554/eLife.05322},

issn = {2050-084X},

year  = {2015},

date = {2015-12-01},

journal = {Elife},

volume = {4},

abstract = {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.},

keywords = {Amidohydrolases, Escherichia coli, Escherichia coli Proteins, Lysine, Post-Translational, Protein Processing, Substrate Specificity, Transcription Factors},

pubstate = {published},

tppubtype = {article}

}

@article{1299970,

title = {Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs},

author = {Martin S Taylor and Daniel R Dempsey and Yousang Hwang and Zan Chen and Nam Chu and Jef D Boeke and Philip A Cole},

doi = {10.1016/j.bioorg.2015.07.003},

issn = {1090-2120},

year  = {2015},

date = {2015-10-01},

journal = {Bioorg Chem},

volume = {62},

pages = {64-73},

abstract = {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.},

keywords = {Acyltransferases, Amino Acid Sequence, Animals, Baculoviridae, Biotin, Cell Line, Enzyme Assays, Genetic Vectors, Ghrelin, Hydrogen-Ion Concentration, Mice, Models, Molecular},

pubstate = {published},

tppubtype = {article}

}

@article{1299971,

title = {Synthetic approaches to protein phosphorylation},

author = {Zan Chen and Philip A Cole},

doi = {10.1016/j.cbpa.2015.07.001},

issn = {1879-0402},

year  = {2015},

date = {2015-10-01},

journal = {Curr Opin Chem Biol},

volume = {28},

pages = {115-22},

abstract = {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.},

keywords = {Animals, Humans, Mutagenesis, Phosphoproteins, Phosphorylation, Protein Transport, Recombinant Proteins, Site-Directed},

pubstate = {published},

tppubtype = {article}

}

@article{1299972,

title = {Switching immune signals on and off},

author = {Nam Chu and Philip A Cole},

doi = {10.7554/eLife.07204},

issn = {2050-084X},

year  = {2015},

date = {2015-04-01},

journal = {Elife},

volume = {4},

keywords = {Animals, Phytic Acid, Protein-Tyrosine Kinases},

pubstate = {published},

tppubtype = {article}

}

@article{1299973,

title = {Protein lysine acetylation by p300/CBP},

author = {Beverley M Dancy and Philip A Cole},

doi = {10.1021/cr500452k},

issn = {1520-6890},

year  = {2015},

date = {2015-03-01},

journal = {Chem Rev},

volume = {115},

number = {6},

pages = {2419-52},

keywords = {Acetylation, Animals, Humans, Lysine, Models, Molecular, p300-CBP Transcription Factors},

pubstate = {published},

tppubtype = {article}

}

@article{1299969,

title = {CBP Binding Outside of Promoters and Enhancers in Drosophila Melanogaster},

author = {P. Philip and A. Boija and R. Vaid and A. M. Churcher and D. J. Meyers and P. A. Cole and M. Mannervik and P. Stenberg},

doi = {10.1186/s13072-015-0042-4},

issn = {1756-8935},

year  = {2015},

date = {2015-00-00},

urldate = {2015-00-00},

journal = {Epigenetics Chromatin},

volume = {8},

pages = {48},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{1299978,

title = {Using S-adenosyl-L-homocysteine capture compounds to characterize S-adenosyl-L-methionine and S-adenosyl-L-homocysteine binding proteins},

author = {Lindsey J Brown and Matthias Baranowski and Yun Wang and Anna K Schrey and Thomas Lenz and Sean D Taverna and Philip A Cole and Michael Sefkow},

doi = {10.1016/j.ab.2014.08.013},

issn = {1096-0309},

year  = {2014},

date = {2014-12-01},

journal = {Anal Biochem},

volume = {467},

pages = {14-21},

abstract = {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.},

keywords = {Catechol O-Methyltransferase, DNA-Binding Proteins, Fluorescence Polarization, Histone-Lysine N-Methyltransferase, Humans, Hydrolases, Nuclear Proteins, S-Adenosylhomocysteine, S-Adenosylmethionine, Transcription Factors},

pubstate = {published},

tppubtype = {article}

}

@article{1299975,

title = {Dissecting the binding mode of low affinity phage display peptide ligands to protein targets by hydrogen/deuterium exchange coupled to mass spectrometry},

author = {Ulrike Leurs and Brian Lohse and Shonoi Ming and Philip A Cole and Rasmus P Clausen and Jesper L Kristensen and Kasper D Rand},

doi = {10.1021/ac503137u},

issn = {1520-6882},

year  = {2014},

date = {2014-12-01},

journal = {Anal Chem},

volume = {86},

number = {23},

pages = {11734-41},

abstract = {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.},

keywords = {Binding Sites, Deuterium Exchange Measurement, Histone Demethylases, Humans, Jumonji Domain-Containing Histone Demethylases, Ligands, Mass Spectrometry, Models, Molecular, Molecular Structure, Peptides},

pubstate = {published},

tppubtype = {article}

}

@article{1299979,

title = {Two histone/protein acetyltransferases, CBP and p300, are indispensable for Foxp3+ T-regulatory cell development and function},

author = {Yujie Liu and Liqing Wang and Rongxiang Han and Ulf H Beier and Tatiana Akimova and Tricia Bhatti and Haiyan Xiao and Philip A Cole and Paul K Brindle and Wayne W Hancock},

doi = {10.1128/MCB.00919-14},

issn = {1098-5549},

year  = {2014},

date = {2014-11-01},

journal = {Mol Cell Biol},

volume = {34},

number = {21},

pages = {3993-4007},

abstract = {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.},

keywords = {Animals, Cell Survival, Colitis, CREB-Binding Protein, Cyclic AMP Response Element-Binding Protein, E1A-Associated p300 Protein, Epigenesis, Female, Forkhead Transcription Factors, GATA3 Transcription Factor, Genetic, Humans, Inbred C57BL, Male, Mice, Promoter Regions, Regulatory, Sequence Deletion, T-Lymphocytes},

pubstate = {published},

tppubtype = {article}

}

@article{1299977,

title = {Regulation of S-adenosylhomocysteine hydrolase by lysine acetylation},

author = {Yun Wang and Jennifer M Kavran and Zan Chen and Kannan R Karukurichi and Daniel J Leahy and Philip A Cole},

doi = {10.1074/jbc.M114.597153},

issn = {1083-351X},

year  = {2014},

date = {2014-11-01},

journal = {J Biol Chem},

volume = {289},

number = {45},

pages = {31361-72},

abstract = {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.},

keywords = {Acetylation, Adenosylhomocysteinase, Amino Acid, Amino Acid Sequence, Catalysis, Crystallography, Humans, Hydrogen Bonding, Lysine, Methylation, Models, Molecular, Molecular Sequence Data, Mutagenesis, NAD, Plasmids, Post-Translational, Protein Binding, Protein Processing, Protein Structure, Recombinant Proteins, Sequence Homology, Site-Directed, Structure-Activity Relationship, Tertiary, X-Ray},

pubstate = {published},

tppubtype = {article}

}

@article{1299976,

title = {How IGF-1 activates its receptor},

author = {Jennifer M Kavran and Jacqueline M McCabe and Patrick O Byrne and Mary Katherine Connacher and Zhihong Wang and Alexander Ramek and Sarvenaz Sarabipour and Yibing Shan and David E Shaw and Kalina Hristova and Philip A Cole and Daniel J Leahy},

doi = {10.7554/eLife.03772},

issn = {2050-084X},

year  = {2014},

date = {2014-09-01},

journal = {Elife},

volume = {3},

abstract = {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.},

keywords = {Amino Acid Sequence, Animals, Conserved Sequence, HEK293 Cells, Humans, IGF Type 1, Insulin, Insulin-Like Growth Factor I, Ligands, Mice, Models, Molecular, Molecular Sequence Data, Mutation, Phosphorylation, Protein Binding, Protein Multimerization, Protein Structure, Receptor, Tertiary},

pubstate = {published},

tppubtype = {article}

}

@article{1299981,

title = {Substrate- and cofactor-independent inhibition of histone demethylase KDM4C},

author = {Ulrike Leurs and Brian Lohse and Kasper D Rand and Shonoi Ming and Erik S Riise and Philip A Cole and Jesper L Kristensen and Rasmus P Clausen},

doi = {10.1021/cb500374f},

issn = {1554-8937},

year  = {2014},

date = {2014-09-01},

journal = {ACS Chem Biol},

volume = {9},

number = {9},

pages = {2131-8},

abstract = {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.},

keywords = {Amino Acid Sequence, Catalytic Domain, Cell Line, Coenzymes, Deuterium Exchange Measurement, Enzyme Inhibitors, High-Throughput Screening Assays, Histone Demethylases, Humans, Inhibitory Concentration 50, Jumonji Domain-Containing Histone Demethylases, Molecular Sequence Data, Peptide Library},

pubstate = {published},

tppubtype = {article}

}

@article{1299980,

title = {Structural basis of nSH2 regulation and lipid binding in PI3Kα},

author = {Michelle S Miller and Oleg Schmidt-Kittler and David M Bolduc and Evan T Brower and Daniele Chaves-Moreira and Marc Allaire and Kenneth W Kinzler and Ian G Jennings and Philip E Thompson and Philip A Cole and L Mario Amzel and Bert Vogelstein and Sandra B Gabelli},

doi = {10.18632/oncotarget.2263},

issn = {1949-2553},

year  = {2014},

date = {2014-07-01},

journal = {Oncotarget},

volume = {5},

number = {14},

pages = {5198-208},

abstract = {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-PIP$_2$, 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 PIP$_2$ 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.},

keywords = {Amino Acid Sequence, Animals, Binding Sites, Boron Compounds, Models, Molecular, Molecular Sequence Data, Phosphatidylinositol 3-Kinases, Protein Binding, Protein Conformation, Sf9 Cells, Signal Transduction, Spodoptera, src Homology Domains},

pubstate = {published},

tppubtype = {article}

}

@article{1299983,

title = {A selective phenelzine analogue inhibitor of histone demethylase LSD1},

author = {Polina Prusevich and Jay H Kalin and Shonoi A Ming and Manuela Basso and Jeffrey Givens and Xin Li and Jianfei Hu and Martin S Taylor and Anne M Cieniewicz and Po-Yuan Hsiao and Rong Huang and Heather Roberson and Nkosi Adejola and Lindsay B Avery and Robert A Casero and Sean D Taverna and Jiang Qian and Alan J Tackett and Rajiv R Ratan and Oliver G McDonald and Andrew P Feinberg and Philip A Cole},

doi = {10.1021/cb500018s},

issn = {1554-8937},

year  = {2014},

date = {2014-06-01},

journal = {ACS Chem Biol},

volume = {9},

number = {6},

pages = {1284-93},

abstract = {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.},

keywords = {Animals, Blotting, Cell Survival, Cells, Cultured, DNA Methylation, Embryo, Enzyme Inhibitors, Fetus, Histone Demethylases, Histones, Humans, Mammalian, Monoamine Oxidase, Neurons, Phenelzine, Rats, Sprague-Dawley, Western},

pubstate = {published},

tppubtype = {article}

}

@article{1299982,

title = {Structure of the p300 histone acetyltransferase bound to acetyl-coenzyme A and its analogues},

author = {Jasna Maksimoska and Dario Segura-Peña and Philip A Cole and Ronen Marmorstein},

doi = {10.1021/bi500380f},

issn = {1520-4995},

year  = {2014},

date = {2014-06-01},

journal = {Biochemistry},

volume = {53},

number = {21},

pages = {3415-22},

abstract = {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.},

keywords = {Acetyl Coenzyme A, Catalytic Domain, Coenzyme A, Humans, Models, Molecular, p300-CBP Transcription Factors, Protein Binding, Protein Conformation},

pubstate = {published},

tppubtype = {article}

}

@article{1299984,

title = {An Fc domain protein-small molecule conjugate as an enhanced immunomodulator},

author = {Meng-Jung Chiang and Marc A Holbert and Jay H Kalin and Young-Hoon Ahn and John Giddens and Mohammed N Amin and Martin S Taylor and Samuel L Collins and Yee Chan-Li and Adam Waickman and Po-Yuan Hsiao and David Bolduc and Daniel J Leahy and Maureen R Horton and Lai-Xi Wang and Jonathan D Powell and Philip A Cole},

doi = {10.1021/ja5006674},

issn = {1520-5126},

year  = {2014},

date = {2014-03-01},

journal = {J Am Chem Soc},

volume = {136},

number = {9},

pages = {3370-3},

abstract = {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.},

keywords = {Adenosine, Animals, CD4-Positive T-Lymphocytes, Immunoconjugates, Immunoglobulin Fc Fragments, Immunologic Factors, Mice, Models, Molecular, Phenethylamines, Protein Conformation},

pubstate = {published},

tppubtype = {article}

}

@article{1299974,

title = {Catalytic mechanisms and regulation of protein kinases},

author = {Z. Wang and P. A. Cole},

doi = {10.1016/B978-0-12-397918-6.00001-X},

issn = {1557-7988},

year  = {2014},

date = {2014-00-00},

journal = {Methods Enzymol.},

volume = {548},

pages = {1-21},

abstract = {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.},

keywords = {Adenosine Triphosphate, Animals, Biocatalysis, Humans, Models, Molecular, Mutation, Phosphorylation, Post-Translational, Protein Conformation, Protein Kinase Inhibitors, Protein Kinases, Protein Processing, Substrate Specificity},

pubstate = {published},

tppubtype = {article}

}

@article{1299939,

title = {Disordered N-Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation},

author = {Gaddiel Rodriguez and Alexandre Esadze and Brian P Weiser and Joseph D Schonhoft and Philip A Cole and James T Stivers},

doi = {10.1021/acschembio.7b00521},

issn = {1554-8937},

journal = {ACS Chem Biol},

volume = {12},

number = {9},

pages = {2260-2263},

abstract = {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.},

keywords = {Binding Sites, Biological Transport, DNA, DNA Glycosylases, Humans, Nuclear Localization Signals, Protein Domains},

pubstate = {published},

tppubtype = {article}

}

@article{1299941,

title = {Endotoxemia-mediated activation of acetyltransferase P300 impairs insulin signaling in obesity},

author = {Jia Cao and Jinghua Peng and Hongying An and Qiyi He and Tatiana Boronina and Shaodong Guo and Morris F White and Philip A Cole and Ling He},

doi = {10.1038/s41467-017-00163-w},

issn = {2041-1723},

journal = {Nat Commun},

volume = {8},

number = {1},

pages = {131},

abstract = {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.},

keywords = {Animals, Cell Line, E1A-Associated p300 Protein, Endoplasmic Reticulum Stress, Endotoxemia, Gene Expression Profiling, Immunoblotting, Inbred C57BL, Insulin, Insulin Resistance, Lipopolysaccharides, Liver, Male, Membrane Proteins, Mice, Obese, Obesity, Protein-Serine-Threonine Kinases, Receptor, Signal Transduction, Tumor, X-Box Binding Protein 1},

pubstate = {published},

tppubtype = {article}

}

@article{1299964,

title = {Correction to Protein Lysine Acetylation by p300/CBP},

author = {Beverley M Dancy and Philip A Cole},

doi = {10.1021/acs.chemrev.6b00351},

issn = {1520-6890},

journal = {Chem Rev},

volume = {116},

number = {14},

pages = {8314},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{1299965,

title = {Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases},

author = {Zan Chen and Stefani N Thomas and David M Bolduc and Xuejun Jiang and Xiangbin Zhang and Cynthia Wolberger and Philip A Cole},

doi = {10.1021/acs.biochem.6b00448},

issn = {1520-4995},

journal = {Biochemistry},

volume = {55},

number = {26},

pages = {3658-66},

abstract = {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.},

keywords = {Chromatography, Endosomal Sorting Complexes Required for Transport, Humans, Immunoprecipitation, Liquid, Nedd4 Ubiquitin Protein Ligases, Phosphorylation, Post-Translational, Protein Processing, PTEN Phosphohydrolase, Tandem Mass Spectrometry, Ubiquitin, Ubiquitin-Protein Ligases, Ubiquitination, X-Linked Inhibitor of Apoptosis Protein},

pubstate = {published},

tppubtype = {article}

}

@article{1299962,

title = {Erratum to: CBP binding outside of promoters and enhancers in Drosophila melanogaster},

author = {Philge Philip and Ann Boija and Roshan Vaid and Allison M Churcher and David J Meyers and Philip A Cole and Mattias Mannervik and Per Stenberg},

doi = {10.1186/s13072-016-0088-y},

issn = {1756-8935},

journal = {Epigenetics Chromatin},

volume = {9},

number = {1},

pages = {38},

abstract = {[This corrects the article DOI: 10.1186/s13072-015-0042-4.].},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{1299961,

title = {An Fc-Small Molecule Conjugate for Targeted Inhibition of the Adenosine 2A Receptor},

author = {Po-Yuan Hsiao and Jay H Kalin and Im-Hong Sun and Mohammed N Amin and Ying-Chun Lo and Meng-Jung Chiang and John Giddens and Polina Sysa-Shah and Kathleen Gabrielson and Lai-Xi Wang and Jonathan D Powell and Philip A Cole},

doi = {10.1002/cbic.201600337},

issn = {1439-7633},

journal = {Chembiochem},

volume = {17},

number = {20},

pages = {1951-1960},

abstract = {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.},

keywords = {Adenosine A2 Receptor Antagonists, Adenosine A2A, Animals, Female, Humans, Immunoglobulin Fab Fragments, Inbred C57BL, Knockout, Male, Mice, Models, Molecular, Molecular Structure, Receptor, Respiratory Tract Infections, Triazines, Triazoles, Vaccinia virus},

pubstate = {published},

tppubtype = {article}

}

@article{1624368,

title = {Discovery of spirohydantoins as selective, orally bioavailable inhibitors of p300/CBP histone acetyltransferases},

author = {Zhiqin Ji and Richard F Clark and Vikram Bhat and T Matthew Hansen and Loren M Lasko and Kenneth D Bromberg and Vlasios Manaves and Mikkel Algire and Ruth Martin and Wei Qiu and Maricel Torrent and Clarissa G Jakob and Hong Liu and Philip A Cole and Ronen Marmorstein and Edward A Kesicki and Albert Lai and Michael R Michaelides},

doi = {10.1016/j.bmcl.2021.127854},

issn = {1464-3405},

journal = {Bioorg Med Chem Lett},

volume = {39},

pages = {127854},

abstract = {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.},

keywords = {Administration, Biological Availability, CREB-Binding Protein, Dose-Response Relationship, Drug, Drug Discovery, E1A-Associated p300 Protein, Enzyme Inhibitors, Humans, Hydantoins, Molecular Structure, Oral, Spiro Compounds, Structure-Activity Relationship},

pubstate = {published},

tppubtype = {article}

}

@article{1624367,

title = {HDAC2 targeting stabilizes the CoREST complex in renal tubular cells and protects against renal ischemia/reperfusion injury},

author = {David D Aufhauser and Paul Hernandez and Seth J Concors and Ciaran O’Brien and Zhonglin Wang and Douglas R Murken and Arabinda Samanta and Ulf H Beier and Lauren Krumeich and Tricia R Bhatti and Yanfeng Wang and Guanghui Ge and Liqing Wang and Shayan Cheraghlou and Florence F Wagner and Edward B Holson and Jay H Kalin and Philip A Cole and Wayne W Hancock and Matthew H Levine},

doi = {10.1038/s41598-021-88242-3},

issn = {2045-2322},

journal = {Sci Rep},

volume = {11},

number = {1},

pages = {9018},

abstract = {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.},

keywords = {Animals, Co-Repressor Proteins, Endothelins, Enzyme Inhibitors, Female, Gene Deletion, Histone Deacetylase 1, Histone Deacetylase 2, Isoenzymes, Kidney Tubules, Knockout, Male, Mice, Proximal, Reperfusion Injury},

pubstate = {published},

tppubtype = {article}

}