@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{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{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{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{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{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}

}