2017
Weiser, Brian P; Stivers, James T; Cole, Philip A
Investigation of N-Terminal Phospho-Regulation of~Uracil DNA Glycosylase Using Protein Semisynthesis Journal Article
In: Biophys J, vol. 113, no. 2, pp. 393-401, 2017, ISSN: 1542-0086.
Abstract | Links | BibTeX | Tags: 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
@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}
}
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.
Mo, Gary C H; Ross, Brian; Hertel, Fabian; Manna, Premashis; Yang, Xinxing; Greenwald, Eric; Booth, Chris; Plummer, Ashlee M; Tenner, Brian; Chen, Zan; Wang, Yuxiao; Kennedy, Eileen J; Cole, Philip A; Fleming, Karen G; Palmer, Amy; Jimenez, Ralph; Xiao, Jie; Dedecker, Peter; Zhang, Jin
Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution Journal Article
In: Nat Methods, vol. 14, no. 4, pp. 427-434, 2017, ISSN: 1548-7105.
Abstract | Links | BibTeX | Tags: 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
@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}
}
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.
2016
Henager, Samuel H; Chu, Nam; Chen, Zan; Bolduc, David; Dempsey, Daniel R; Hwang, Yousang; Wells, James; Cole, Philip A
Enzyme-catalyzed expressed protein ligation Journal Article
In: Nat Methods, vol. 13, no. 11, pp. 925-927, 2016, ISSN: 1548-7105.
Abstract | Links | BibTeX | Tags: 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
@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}
}
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.
2015
Tu, Shun; Guo, Shu-Juan; Chen, Chien-Sheng; Liu, Cheng-Xi; Jiang, He-Wei; Ge, Feng; Deng, Jiao-Yu; Zhou, Yi-Ming; Czajkowsky, Daniel M; Li, Yang; Qi, Bang-Ruo; Ahn, Young-Hoon; Cole, Philip A; Zhu, Heng; Tao, Sheng-Ce
YcgC represents a new protein deacetylase family in prokaryotes Journal Article
In: Elife, vol. 4, 2015, ISSN: 2050-084X.
Abstract | Links | BibTeX | Tags: Amidohydrolases, Escherichia coli, Escherichia coli Proteins, Lysine, Post-Translational, Protein Processing, Substrate Specificity, Transcription Factors
@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}
}
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.