@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{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{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{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{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}

}