@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{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{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}

}