@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{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{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{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{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}

}