21 results on '"Peng, G."'
Search Results
2. In Vitro Activity of Neisseria meningitidis PglL O-Oligosaccharyltransferase with Diverse Synthetic Lipid Donors and a UDP-activated Sugar
- Author
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Musumeci, Matias A., Hug, Isabelle, Scott, Nichollas E., Ielmini, M.Veronica, Foster, Leonard J., Wang, Peng G., and Feldman, Mario F.
- Published
- 2013
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Catalog
3. Defining Function of Lipopolysaccharide O-antigen Ligase WaaL Using Chemoenzymatically Synthesized Substrates
- Author
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Han, Weiqing, Wu, Baolin, Li, Lei, Zhao, Guohui, Woodward, Robert, Pettit, Nicholas, Cai, Li, Thon, Vireak, and Wang, Peng G.
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- 2012
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4. Specificity and Mechanism of Metal Ion Activation in UDP-galactose:β-Galactoside-α-1,3-galactosyltransferase
- Author
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Zhang, Yingnan, Wang, Peng G., and Brew, Keith
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- 2001
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5. Production of homogeneous glycoprotein with multisite modifications by an engineered N-glycosyltransferase mutant.
- Author
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Qitao Song, Zhigang Wu, Yueyuan Fan, Woran Song, Peiru Zhang, Li Wang, Faxing Wang, Yangyang Xu, Wang, Peng G., and Jiansong Cheng
- Subjects
- *
GLYCOSYLTRANSFERASES , *GLYCOPROTEINS , *PROTEIN engineering , *GLYCANS , *PROTEIN structure - Abstract
Naturally occurring N-glycoproteins exhibit glycoform heterogeneity with respect to N-glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). However, access to well-defined glycoproteins is always important for both basic research and therapeutic purposes. As a result, there has been a substantial effort to identify and understand the catalytic properties of N-glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study we found that ApNGT, a newly discovered cytoplasmic N-glycosyltransferase from Actinobacillus pleuropneumoniae, has strict selectivitytowardthe residuesaroundtheAsnofN-glycosylation sequon by screening a small library of synthetic peptides. The inherent stringency was subsequently demonstrated to be closely associated with a critical residue (Gln-469) of ApNGT which we propose hinders the access of bulky residues surrounding the occupied Asn into the active site. Site-saturated mutagenesis revealed that the introduction of small hydrophobic residues at the site cannot only weaken the stringency of ApNGT but can also contribute to enormous improvement of glycosylation efficiency against both short peptides and proteins. We then employed the most efficient mutant (Q469A) other than the wild-type ApNGT to produce a homogeneous glycoprotein carrying multiple (up to 10) N-glycans, demonstrating that this construct is a promising biocatalyst for potentially addressing the issue of macroheterogeneity in glycoprotein preparation. [ABSTRACT FROM AUTHOR] more...
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- 2017
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6. Defining Function of Lipopolysaccharide O-antigen Ligase WaaL Using Chemoenzymatically Synthesized Substrates.
- Author
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Weiqing Han, Baolin Wu, Lei Li, Guohui Zhao, Woodward, Robert, Pettit, Nicholas, Li Cai, Thon, Vireak, and Wang, Peng G.
- Subjects
- *
LIPOPOLYSACCHARIDES , *CHROMOGENIC compounds , *O antigens , *ESCHERICHIA coli , *ORGANIC acids , *ADENOSINE triphosphate - Abstract
The WaaL-mediated ligation of O-antigen onto the core region of the lipid A-core block is an important step in the lipopolysaccharide (LPS) biosynthetic pathway. Although the LPS biosynthesis has been largely characterized, only a limited amount of in vitro biochemical evidence has been established for the ligation reaction. Such limitations have primarily resulted from the barriers in purifying WaaL homologues and obtaining chemically defined substrates. Accordingly, we describe herein a chemical biology approach that enabled the reconstitution of this ligation reaction. The O-antigen repeating unit (O-unit) of Escherichia coli O86 was first enzymatically assembled via sequential enzymatic glycosylation of a chemically synthesized GalNAc-pyrophosphate-undecaprenyl precursor. Subsequent expression of WaaL through use of a chaperone co-expression system then enabled the demonstration of the in vitro ligation between the synthesized donor (O-unit-pyrophosphate-undecaprenyl) and the isolated lipid A-core acceptor. The previously reported ATP and divalent metal cation dependence were not observed using this system. Further analyses of other donor substrates revealed that WaaL possesses a highly relaxed specificity toward both the lipid moiety and the glycan moiety of the donor. Lastly, three conserved amino acid residues identified by sequence alignment were found essential for the WaaL activity. Taken together, the present work represents an in vitro systematic investigation of the WaaL function using a chemical biology approach, providing a system that could facilitate the elucidation of the mechanism of WaaL-catalyzed ligation reaction. [ABSTRACT FROM AUTHOR] more...
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- 2012
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7. Structural comparisons of host and African swine fever virus dUTPases reveal new clues for inhibitor development.
- Author
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Liang R, Wang G, Zhang D, Ye G, Li M, Shi Y, Shi J, Chen H, and Peng G
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- African Swine Fever Virus drug effects, African Swine Fever Virus physiology, Amino Acid Sequence, Animals, Antiviral Agents chemistry, Catalytic Domain, Crystallography, X-Ray, Drug Development, Enzyme Inhibitors chemistry, Host-Pathogen Interactions, Macrophages virology, Plasmodium falciparum enzymology, Protein Conformation, Swine, Virus Replication drug effects, African Swine Fever Virus enzymology, Antiviral Agents pharmacology, Enzyme Inhibitors pharmacology, Pyrophosphatases antagonists & inhibitors, Pyrophosphatases chemistry
- Abstract
African swine fever, caused by the African swine fever virus (ASFV), is among the most significant swine diseases. There are currently no effective treatments against ASFV. ASFV contains a gene encoding a dUTPase (E165R), which is required for viral replication in swine macrophages, making it an attractive target for inhibitor development. However, the full structural details of the ASFV dUTPase and those of the comparable swine enzyme are not available, limiting further insights. Herein, we determine the crystal structures of ASFV dUTPase and swine dUTPase in both their ligand-free and ligand-bound forms. We observe that the swine enzyme employs a classical dUTPase architecture made up of three-subunit active sites, whereas the ASFV enzyme employs a novel two-subunit active site. We then performed a comparative analysis of all dUTPase structures uploaded in the Protein Data Bank (PDB), which showed classical and non-classical types were mainly determined by the C-terminal β-strand orientation, and the difference was mainly related to the four amino acids behind motif IV. Thus, our study not only explains the reason for the structural diversity of dUTPase but also reveals how to predict dUTPase type, which may have implications for the dUTPase family. Finally, we tested two dUTPase inhibitors developed for the Plasmodium falciparum dUTPase against the swine and ASFV enzymes. One of these compounds inhibited the ASFV dUTPase at low micromolar concentrations (K
d = 15.6 μM) and with some selectivity (∼2x) over swine dUTPase. In conclusion, our study expands our understanding of the dUTPase family and may aid in the development of specific ASFV inhibitors., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.) more...- Published
- 2021
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8. A conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence.
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Shen Z, Wang G, Yang Y, Shi J, Fang L, Li F, Xiao S, Fu ZF, and Peng G
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- Alphacoronavirus pathogenicity, Animals, Betacoronavirus, Host Microbial Interactions genetics, Host Microbial Interactions physiology, Humans, Middle East Respiratory Syndrome Coronavirus genetics, Porcine epidemic diarrhea virus genetics, Protein Biosynthesis, RNA-Dependent RNA Polymerase genetics, RNA-Dependent RNA Polymerase metabolism, RNA-Dependent RNA Polymerase ultrastructure, Severe acute respiratory syndrome-related coronavirus genetics, Swine, Synapsins metabolism, Transmissible gastroenteritis virus genetics, Viral Nonstructural Proteins genetics, Viral Nonstructural Proteins ultrastructure, Virulence, Virus Replication physiology, Alphacoronavirus genetics, Viral Nonstructural Proteins metabolism, Virus Replication genetics
- Abstract
Coronaviruses are enveloped, single-stranded RNA viruses that are distributed worldwide. They include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and the human coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), many of which seriously endanger human health and well-being. Only alphacoronaviruses and betacoronaviruses harbor nonstructural protein 1 (nsp1), which performs multiple functions in inhibiting antiviral host responses. The role of the C terminus of betacoronavirus nsp1 in virulence has been characterized, but the location of the alphacoronavirus nsp1 region that is important for virulence remains unclear. Here, using TGEV nsp1 as a model to explore the function of this protein in alphacoronaviruses, we demonstrate that alphacoronavirus nsp1 inhibits host gene expression. Solving the crystal structure of full-length TGEV at 1.85-Å resolution and conducting several biochemical analyses, we observed that a specific motif (amino acids 91-95) of alphacoronavirus nsp1 is a conserved region that inhibits host protein synthesis. Using a reverse-genetics system based on CRISPR/Cas9 technology to construct a recombinant TGEV in which this specific nsp1 motif was altered, we found that this mutation does not affect virus replication in cell culture but significantly reduces TGEV pathogenicity in pigs. Taken together, our findings suggest that alphacoronavirus nsp1 is an essential virulence determinant, providing a potential paradigm for the development of a new attenuated vaccine based on modified nsp1., (© 2019 Shen et al.) more...
- Published
- 2019
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9. Insight into the evolution of nidovirus endoribonuclease based on the finding that nsp15 from porcine Deltacoronavirus functions as a dimer.
- Author
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Zheng A, Shi Y, Shen Z, Wang G, Shi J, Xiong Q, Fang L, Xiao S, Fu ZF, and Peng G
- Subjects
- Amino Acid Sequence, Arterivirus chemistry, Arterivirus classification, Arterivirus genetics, Arterivirus metabolism, Binding Sites, Cloning, Molecular, Coronavirus classification, Coronavirus genetics, Coronavirus metabolism, Crystallography, X-Ray, Endoribonucleases genetics, Endoribonucleases metabolism, Escherichia coli genetics, Escherichia coli metabolism, Evolution, Molecular, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Models, Molecular, Nidovirales classification, Nidovirales genetics, Nidovirales metabolism, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Multimerization, Protein Subunits genetics, Protein Subunits metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Severe acute respiratory syndrome-related coronavirus chemistry, Severe acute respiratory syndrome-related coronavirus classification, Severe acute respiratory syndrome-related coronavirus genetics, Severe acute respiratory syndrome-related coronavirus metabolism, Sequence Alignment, Sequence Homology, Amino Acid, Viral Nonstructural Proteins genetics, Viral Nonstructural Proteins metabolism, Virus Replication genetics, Coronavirus chemistry, Endoribonucleases chemistry, Nidovirales chemistry, Protein Subunits chemistry, Viral Nonstructural Proteins chemistry
- Abstract
Nidovirus endoribonucleases (NendoUs) include nonstructural protein 15 (nsp15) from coronaviruses and nsp11 from arteriviruses, both of which have been reported to participate in the viral replication process and in the evasion of the host immune system. Results from a previous study of coronaviruses SARS-CoV, HCoV-229E, and MHV nsp15 indicate that it mainly forms a functional hexamer, whereas nsp11 from the arterivirus PRRSV is a dimer. Here, we found that porcine Deltacoronavirus (PDCoV) nsp15 primarily exists as dimers and monomers in vitro Biological experiments reveal that a PDCoV nsp15 mutant lacking the first 27 amino acids of the N-terminal domain (Asn-1-Asn-27) forms more monomers and displays decreased enzymatic activity, indicating that this region is important for its dimerization. Moreover, multiple sequence alignments and three-dimensional structural analysis indicated that the C-terminal region (His-251-Val-261) of PDCoV nsp15 is 10 amino acids shorter and forms a shorter loop than that formed by the equivalent sequence (Gln-259-Phe-279) of SARS-CoV nsp15. This result may explain why PDCoV nsp15 failed to form hexamers. We speculate that NendoUs may have originated from XendoU endoribonucleases (XendoUs) forming monomers in eukaryotic cells, that NendoU from arterivirus gained the ability to form dimers, and that the coronavirus variants then evolved the capacity to assemble into hexamers. We further propose that PDCoV nsp15 may be an intermediate in this evolutionary process. Our findings provide a theoretical basis for improving our understanding of NendoU evolution and offer useful clues for designing drugs and vaccines against nidoviruses., (© 2018 Zheng et al.) more...
- Published
- 2018
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10. Transcriptome analysis reveals determinant stages controlling human embryonic stem cell commitment to neuronal cells.
- Author
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Li Y, Wang R, Qiao N, Peng G, Zhang K, Tang K, Han JJ, and Jing N
- Subjects
- Embryonic Stem Cells chemistry, Eye Proteins physiology, Homeodomain Proteins physiology, Humans, Nerve Tissue Proteins physiology, Transcription Factors genetics, Homeobox Protein SIX3, Cell Differentiation genetics, Embryonic Stem Cells metabolism, Neurons cytology, Transcriptome
- Abstract
Proper neural commitment is essential for ensuring the appropriate development of the human brain and for preventing neurodevelopmental diseases such as autism spectrum disorders, schizophrenia, and intellectual disorders. However, the molecular mechanisms underlying the neural commitment in humans remain elusive. Here, we report the establishment of a neural differentiation system based on human embryonic stem cells (hESCs) and on comprehensive RNA sequencing analysis of transcriptome dynamics during early hESC differentiation. Using weighted gene co-expression network analysis, we reveal that the hESC neurodevelopmental trajectory has five stages: pluripotency (day 0); differentiation initiation (days 2, 4, and 6); neural commitment (days 8-10); neural progenitor cell proliferation (days 12, 14, and 16); and neuronal differentiation (days 18, 20, and 22). These stages were characterized by unique module genes, which may recapitulate the early human cortical development. Moreover, a comparison of our RNA-sequencing data with several other transcriptome profiling datasets from mice and humans indicated that Module 3 associated with the day 8-10 stage is a critical window of fate switch from the pluripotency to the neural lineage. Interestingly, at this stage, no key extrinsic signals were activated. In contrast, using CRISPR/Cas9-mediated gene knockouts, we also found that intrinsic hub transcription factors, including the schizophrenia-associated SIX3 gene and septo-optic dysplasia-related HESX1 gene, are required to program hESC neural determination. Our results improve the understanding of the mechanism of neural commitment in the human brain and may help elucidate the etiology of human mental disorders and advance therapies for managing these conditions., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.) more...
- Published
- 2017
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11. Structural and Molecular Evidence Suggesting Coronavirus-driven Evolution of Mouse Receptor.
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Peng G, Yang Y, Pasquarella JR, Xu L, Qian Z, Holmes KV, and Li F
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- Amino Acid Sequence, Animals, Binding Sites, Carcinoembryonic Antigen chemistry, Crystallography, X-Ray, Membrane Fusion, Mice, Murine hepatitis virus physiology, Mutation, Protein Conformation, Receptors, Virus chemistry, Sequence Homology, Amino Acid, Carcinoembryonic Antigen metabolism, Murine hepatitis virus metabolism, Receptors, Virus metabolism
- Abstract
Hosts and pathogens are locked in an evolutionary arms race. To infect mice, mouse hepatitis coronavirus (MHV) has evolved to recognize mouse CEACAM1a (mCEACAM1a) as its receptor. To elude MHV infections, mice may have evolved a variant allele from the Ceacam1a gene, called Ceacam1b , producing mCEACAM1b, which is a much poorer MHV receptor than mCEACAM1a. Previous studies showed that sequence differences between mCEACAM1a and mCEACAM1b in a critical MHV-binding CC' loop partially account for the low receptor activity of mCEACAM1b, but detailed structural and molecular mechanisms for the differential MHV receptor activities of mCEACAM1a and mCEACAM1b remained elusive. Here we have determined the crystal structure of mCEACAM1b and identified the structural differences and additional residue differences between mCEACAM1a and mCEACAM1b that affect MHV binding and entry. These differences include conformational alterations of the CC' loop as well as residue variations in other MHV-binding regions, including β-strands C' and C'' and loop C'C''. Using pseudovirus entry and protein-protein binding assays, we show that substituting the structural and residue features from mCEACAM1b into mCEACAM1a reduced the viral receptor activity of mCEACAM1a, whereas substituting the reverse changes from mCEACAM1a into mCEACAM1b increased the viral receptor activity of mCEACAM1b. These results elucidate the detailed molecular mechanism for how mice may have kept pace in the evolutionary arms race with MHV by undergoing structural and residue changes in the MHV receptor, providing insight into this possible example of pathogen-driven evolution of a host receptor protein., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.) more...
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- 2017
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12. Dynamic Heterogeneity of Brachyury in Mouse Epiblast Stem Cells Mediates Distinct Response to Extrinsic Bone Morphogenetic Protein (BMP) Signaling.
- Author
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Song L, Chen J, Peng G, Tang K, and Jing N
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- Animals, Cell Differentiation drug effects, Endoderm cytology, Epidermis drug effects, Epidermis metabolism, Epithelial-Mesenchymal Transition drug effects, Gene Expression Regulation, Developmental drug effects, Green Fluorescent Proteins metabolism, Mesoderm cytology, Mice, Models, Biological, Mouse Embryonic Stem Cells drug effects, Pluripotent Stem Cells cytology, Pluripotent Stem Cells drug effects, Pluripotent Stem Cells metabolism, Signal Transduction drug effects, Bone Morphogenetic Protein 4 pharmacology, Fetal Proteins metabolism, Germ Layers cytology, Germ Layers metabolism, Mouse Embryonic Stem Cells cytology, T-Box Domain Proteins metabolism
- Abstract
Mouse pluripotent cells, such as embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), provide excellent in vitro systems to study imperative pre- and postimplantation events of in vivo mammalian development. It is known that mouse ESCs are dynamic heterogeneous populations. However, it remains largely unclear whether and how EpiSCs possess heterogeneity and plasticity similar to that of ESCs. Here, we show that EpiSCs are discriminated by the expression of a specific marker T (Brachyury) into two populations. The T-positive (T(+)) and the T-negative (T(-)) populations can be interconverted within the same culture condition. In addition, the two populations display distinct responses to bone morphogenetic protein (BMP) signaling and different developmental potentials. The T(-) EpiSCs are preferentially differentiated into ectoderm lineages, whereas T(+) EpiSCs have a biased potential for mesendoderm fates. Mechanistic studies reveal that T(+) EpiSCs have an earlier and faster response to BMP4 stimulation than T(-) EpiSCs. Id1 mediates the commitment of T(-) EpiSCs to epidermal lineage during BMP4 treatment. On the other hand, Snail modulates the conversion of T(+) EpiSCs to mesendoderm fates with the presence of BMP4. Furthermore, T expression is essential for epithelial-mesenchymal transition during EpiSCs differentiation. Our findings suggest that the dynamic heterogeneity of the T(+)/T(-) subpopulation primes EpiSCs toward particular cell lineages, providing important insights into the dynamic development of the early mouse embryo., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.) more...
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- 2016
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13. The Steroidogenic Enzyme AKR1C3 Regulates Stability of the Ubiquitin Ligase Siah2 in Prostate Cancer Cells.
- Author
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Fan L, Peng G, Hussain A, Fazli L, Guns E, Gleave M, and Qi J
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- 3-Hydroxysteroid Dehydrogenases antagonists & inhibitors, 3-Hydroxysteroid Dehydrogenases metabolism, Aldo-Keto Reductase Family 1 Member C3, Androgens metabolism, Animals, Cell Line, Tumor, Enzyme Stability, Humans, Hydroxyprostaglandin Dehydrogenases antagonists & inhibitors, Hydroxyprostaglandin Dehydrogenases metabolism, Male, Mice, Mice, Nude, Neoplasm Transplantation, Nuclear Proteins antagonists & inhibitors, Nuclear Proteins metabolism, Prostate pathology, Prostate-Specific Antigen genetics, Prostate-Specific Antigen metabolism, Prostatic Neoplasms genetics, Prostatic Neoplasms pathology, Proteasome Endopeptidase Complex metabolism, Protein Binding, Proteolysis, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Receptors, Androgen metabolism, Signal Transduction, Ubiquitin-Protein Ligases antagonists & inhibitors, Ubiquitin-Protein Ligases metabolism, Ubiquitination, 3-Hydroxysteroid Dehydrogenases genetics, Gene Expression Regulation, Neoplastic, Hydroxyprostaglandin Dehydrogenases genetics, Nuclear Proteins genetics, Prostate enzymology, Prostatic Neoplasms enzymology, Receptors, Androgen genetics, Ubiquitin-Protein Ligases genetics
- Abstract
Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.) more...
- Published
- 2015
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14. Phosphorylation of the BRCA1 C terminus (BRCT) repeat inhibitor of hTERT (BRIT1) protein coordinates TopBP1 protein recruitment and amplifies ataxia telangiectasia-mutated and Rad3-related (ATR) Signaling.
- Author
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Zhang B, Wang E, Dai H, Shen J, Hsieh HJ, Lu X, and Peng G
- Subjects
- Ataxia Telangiectasia Mutated Proteins genetics, Ataxia Telangiectasia Mutated Proteins metabolism, Carrier Proteins antagonists & inhibitors, Carrier Proteins genetics, Cell Cycle Proteins, Cell Line, Cell Line, Tumor, Cytoskeletal Proteins, DNA Damage, DNA-Binding Proteins antagonists & inhibitors, DNA-Binding Proteins genetics, Humans, Mammary Glands, Human cytology, Mammary Glands, Human metabolism, Nerve Tissue Proteins antagonists & inhibitors, Nerve Tissue Proteins genetics, Nuclear Proteins antagonists & inhibitors, Nuclear Proteins genetics, Osteoblasts cytology, Osteoblasts metabolism, Phosphorylation, Protein Structure, Tertiary, Protein Transport, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Ubiquitin-Protein Ligases chemistry, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Carrier Proteins metabolism, DNA-Binding Proteins metabolism, Nerve Tissue Proteins metabolism, Nuclear Proteins metabolism, Protein Processing, Post-Translational, Signal Transduction genetics
- Abstract
The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase functions as a central node in the DNA damage response signaling network. The mechanisms by which ATR activity is amplified and/or maintained are not understood. Here we demonstrate that BRIT1/microcephalin (MCPH1), a human disease-related protein, is dispensable for the initiation but essential for the amplification of ATR signaling. BRIT1 interacts with and recruits topoisomerase-binding protein 1 (TopBP1), a key activator of ATR signaling, to the sites of DNA damage. Notably, replication stress-induced ataxia telangiectasia-mutated or ATR-dependent BRIT1 phosphorylation at Ser-322 facilitates efficient TopBP1 recruitment. These results reveal a mechanism that ensures the continuation of ATR-initiated DNA damage signaling. Our study uncovers a previously unknown regulatory axis of ATR signaling in maintaining genomic integrity, which may provide mechanistic insights into the perturbation of ATR signaling in human diseases such as neurodevelopmental defects and cancer., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.) more...
- Published
- 2014
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15. Crystal structure of bovine coronavirus spike protein lectin domain.
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Peng G, Xu L, Lin YL, Chen L, Pasquarella JR, Holmes KV, and Li F
- Subjects
- Animals, Binding Sites, Cattle, Coronavirus, Bovine genetics, Coronavirus, Bovine metabolism, Crystallography, X-Ray, Humans, Lectins chemistry, Lectins genetics, Lectins metabolism, Membrane Glycoproteins genetics, Membrane Glycoproteins metabolism, Mice, Neuraminic Acids, Protein Structure, Secondary, Protein Structure, Tertiary, Spike Glycoprotein, Coronavirus, Structure-Activity Relationship, Viral Envelope Proteins genetics, Viral Envelope Proteins metabolism, Coronavirus, Bovine chemistry, Evolution, Molecular, Membrane Glycoproteins chemistry, Viral Envelope Proteins chemistry
- Abstract
The spike protein N-terminal domains (NTDs) of bovine coronavirus (BCoV) and mouse hepatitis coronavirus (MHV) recognize sugar and protein receptors, respectively, despite their significant sequence homology. We recently determined the crystal structure of MHV NTD complexed with its protein receptor murine carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which surprisingly revealed a human galectin (galactose-binding lectin) fold in MHV NTD. Here, we have determined at 1.55 Å resolution the crystal structure of BCoV NTD, which also has the human galectin fold. Using mutagenesis, we have located the sugar-binding site in BCoV NTD, which overlaps with the galactose-binding site in human galectins. Using a glycan array screen, we have identified 5-N-acetyl-9-O-acetylneuraminic acid as the preferred sugar substrate for BCoV NTD. Subtle structural differences between BCoV and MHV NTDs, primarily involving different conformations of receptor-binding loops, explain why BCoV NTD does not bind CEACAM1 and why MHV NTD does not bind sugar. These results suggest a successful viral evolution strategy in which coronaviruses stole a galectin from hosts, incorporated it into their spike protein, and evolved it into viral receptor-binding domains with altered sugar specificity in contemporary BCoV or novel protein specificity in contemporary MHV. more...
- Published
- 2012
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16. Mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus.
- Author
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Wu K, Peng G, Wilken M, Geraghty RJ, and Li F
- Subjects
- Angiotensin-Converting Enzyme 2, Animals, Cell Line, Disease Reservoirs virology, Disease Vectors, Evolution, Molecular, Humans, Models, Molecular, Mutation, Peptidyl-Dipeptidase A chemistry, Peptidyl-Dipeptidase A genetics, Protein Binding, Protein Structure, Tertiary, Receptors, Virus chemistry, Receptors, Virus genetics, Severe acute respiratory syndrome-related coronavirus chemistry, Severe acute respiratory syndrome-related coronavirus genetics, Severe acute respiratory syndrome-related coronavirus isolation & purification, Severe Acute Respiratory Syndrome genetics, Severe Acute Respiratory Syndrome virology, Viral Proteins chemistry, Viral Proteins genetics, Viral Proteins metabolism, Viverridae genetics, Viverridae metabolism, Peptidyl-Dipeptidase A metabolism, Receptors, Virus metabolism, Severe acute respiratory syndrome-related coronavirus metabolism, Severe Acute Respiratory Syndrome metabolism, Viverridae virology
- Abstract
The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals. more...
- Published
- 2012
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17. Chromodomain helicase DNA-binding protein 4 (CHD4) regulates homologous recombination DNA repair, and its deficiency sensitizes cells to poly(ADP-ribose) polymerase (PARP) inhibitor treatment.
- Author
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Pan MR, Hsieh HJ, Dai H, Hung WC, Li K, Peng G, and Lin SY
- Subjects
- Autoantigens genetics, BRCA1 Protein metabolism, Breast cytology, Breast Neoplasms, Cell Cycle Proteins, Cell Line, Chromatin physiology, Cytoskeletal Proteins, DNA Damage physiology, Female, Homologous Recombination drug effects, Humans, Mi-2 Nucleosome Remodeling and Deacetylase Complex deficiency, Mi-2 Nucleosome Remodeling and Deacetylase Complex genetics, Nerve Tissue Proteins metabolism, Poly (ADP-Ribose) Polymerase-1, Poly(ADP-ribose) Polymerases metabolism, RNA, Small Interfering pharmacology, Replication Protein A metabolism, Autoantigens metabolism, DNA Repair physiology, Homologous Recombination physiology, Mi-2 Nucleosome Remodeling and Deacetylase Complex metabolism, Phthalazines pharmacology, Piperazines pharmacology, Poly Adenosine Diphosphate Ribose metabolism, Poly(ADP-ribose) Polymerase Inhibitors
- Abstract
To ensure genome stability, cells have evolved a robust defense mechanism to detect, signal, and repair damaged DNA that is generated by exogenous stressors such as ionizing radiation, endogenous stressors such as free radicals, or normal physiological processes such as DNA replication. Homologous recombination (HR) repair is a critical pathway of repairing DNA double strand breaks, and it plays an essential role in maintaining genomic integrity. Previous studies have shown that BRIT1, also known as MCPH1, is a key regulator of HR repair. Here, we report that chromodomain helicase DNA-binding protein 4 (CHD4) is a novel BRIT1 binding partner that regulates the HR repair process. The BRCA1 C-terminal domains of BRIT1 are required for its interaction with CHD4. Depletion of CHD4 and overexpression of the ATPase-dead form of CHD4 impairs the recruitment of BRIT1 to the DNA damage lesions. As a functional consequence, CHD4 deficiency sensitizes cells to double strand break-inducing agents, reduces the recruitment of HR repair factor BRCA1, and impairs HR repair efficiency. We further demonstrate that CHD4-depleted cells are more sensitive to poly(ADP-ribose) polymerase inhibitor treatment. In response to DNA damage induced by poly(ADP-ribose) polymerase inhibitors, CHD4 deficiency impairs the recruitment of DNA repair proteins BRIT1, BRCA1, and replication protein A at early steps of HR repair. Taken together, our findings identify an important role of CHD4 in controlling HR repair to maintain genome stability and establish the potential therapeutic implications of targeting CHD4 deficiency in tumors. more...
- Published
- 2012
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18. DNA damage response is suppressed by the high cyclin-dependent kinase 1 activity in mitotic mammalian cells.
- Author
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Zhang W, Peng G, Lin SY, and Zhang P
- Subjects
- Ataxia Telangiectasia Mutated Proteins, BRCA1 Protein genetics, BRCA1 Protein metabolism, CDC2 Protein Kinase genetics, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Cyclin-Dependent Kinase 5 genetics, Cyclin-Dependent Kinase 5 metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, HeLa Cells, Histones genetics, Histones metabolism, Humans, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Phosphorylation physiology, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Tumor Suppressor Proteins genetics, Tumor Suppressor Proteins metabolism, Tumor Suppressor p53-Binding Protein 1, CDC2 Protein Kinase metabolism, DNA Damage physiology, Mitosis physiology
- Abstract
DNA damage response (DDR) is vital for genomic stability, and its deficiency is linked to tumorigenesis. Extensive studies in interphase (G(1)-S-G(2)) mammalian cells have revealed the mechanisms of DDR in great detail; however, how mitotic cells respond to DNA damage remains less defined. We report here that a full DDR is suppressed in mitotic mammalian cells until telophase/cytokinesis. Although early DDR markers such as the phosphorylations of ataxia telangiectasia mutated (ATM) and histone H2A.x (H2AX) can be readily detected, the ionizing radiation-induced foci (IRIF) formation of late DDR markers such as breast cancer type 1 susceptibility protein (BRCA1) and p53-binding protein 1 (53BP1) are absent until the telophase/cytokinesis stage. We further showed that the IR-induced ubiquitination cascade around DNA damage sites did not occur in mitotic cells, which explains, at least in part, why BRCA1 and 53BP1 cannot be recruited to the damaged sites. These observations indicate that DDR is suppressed in mitotic cells after the step of γH2AX formation. Not surprisingly, we found that the absence of a full DDR in mitotic cells was associated with the high cyclin-dependent kinase 1 (CDK1) activities. More 53BP1 IRIF could be detected when the irradiated mitotic cells were treated with a CDK1 inhibitor. Further, the activation of CDK5 in interphase cells impedes the formation of 53BP1 IRIF. Together, these results suggest that the DDR is suppressed by the high CDK1 activity in mitotic mammalian cells. more...
- Published
- 2011
- Full Text
- View/download PDF
19. Monoubiquitination of H2AX protein regulates DNA damage response signaling.
- Author
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Pan MR, Peng G, Hung WC, and Lin SY
- Subjects
- Adaptor Proteins, Signal Transducing, Amino Acid Substitution, Animals, Cell Cycle Proteins, Checkpoint Kinase 2, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Histones genetics, Humans, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Mice, Mice, Knockout, Mutation, Missense, Nuclear Proteins genetics, Nuclear Proteins metabolism, Phosphorylation genetics, Polycomb Repressive Complex 1, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins metabolism, Repressor Proteins genetics, Repressor Proteins metabolism, Trans-Activators genetics, Trans-Activators metabolism, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, DNA Breaks, Double-Stranded, Histones metabolism, Signal Transduction, Ubiquitination
- Abstract
Double strand breaks (DSBs) are the most deleterious of the DNA lesions that initiate genomic instability and promote tumorigenesis. Cells have evolved a complex protein network to detect, signal, and repair DSBs. In mammalian cells, a key component in this network is H2AX, which becomes rapidly phosphorylated at Ser(139) (γ-H2AX) at DSBs. Here we show that monoubiquitination of H2AX mediated by the RNF2-BMI1 complex is critical for the efficient formation of γ-H2AX and functions as a proximal regulator in DDR (DNA damage response). RNF2-BMI1 interacts with H2AX in a DNA damage-dependent manner and is required for monoubiquitination of H2AX at Lys(119)/Lys(120). As a functional consequence, we show that the H2AX K120R mutant abolishes H2AX monoubiquitination, impairs the recruitment of p-ATM (Ser(1981)) to DSBs, and thereby reduces the formation of γ-H2AX and the recruitment of MDC1 to DNA damage sites. These data suggest that monoubiquitination of H2AX plays a critical role in initiating DNA damage signaling. Consistent with these observations, impairment of RNF2-BMI1 function by siRNA knockdown or overexpression of the ligase-dead RNF2 mutant all leads to significant defects both in accumulation of γ-H2AX, p-ATM, and MDC1 at DSBs and in activation of NBS1 and CHK2. Additionally, the regulatory effect of RNF2-BMI1 on γ-H2AX formation is dependent on ATM. Lacking their ability to properly activate the DNA damage signaling pathway, RNF2-BMI1 complex-depleted cells exhibit impaired DNA repair and increased sensitivity to ionizing radiation. Together, our findings demonstrate a distinct monoubiquitination-dependent mechanism that is required for H2AX phosphorylation and the initiation of DDR. more...
- Published
- 2011
- Full Text
- View/download PDF
20. SIP30 is regulated by ERK in peripheral nerve injury-induced neuropathic pain.
- Author
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Peng G, Han M, Du Y, Lin A, Yu L, Zhang Y, and Jing N
- Subjects
- Animals, Cyclic AMP genetics, Extracellular Signal-Regulated MAP Kinases physiology, Neuralgia etiology, PC12 Cells, Rats, Response Elements, Spinal Cord, Transcription Factors, Up-Regulation, Chromosomal Proteins, Non-Histone genetics, Extracellular Signal-Regulated MAP Kinases metabolism, Nerve Tissue Proteins genetics, Neuralgia metabolism, Peripheral Nerve Injuries
- Abstract
ERK plays an important role in chronic neuropathic pain. However, the underlying mechanism is largely unknown. Here we show that in chronic constriction injury-treated rat spinal cords, up-regulation of SIP30 (SNAP25-interacting protein 30), which is involved in the development and maintenance of chronic constriction injury-induced neuropathic pain, correlates with ERK activation and that the up-regulation of SIP30 is suppressed by intrathecal delivery of the MEK inhibitor U0126. In PC12 cells, up-regulation of SIP30 by nerve growth factor is also dependent on ERK activation. We found that there is an ERK-responsive region in the rat sip30 promoter. Activation of ERK promotes the recruitment of the transcription factor cyclic AMP-response element-binding protein to the sip30 gene promoter. Taken together, our results provide a potential downstream target of ERK activation-mediated neuropathic pain. more...
- Published
- 2009
- Full Text
- View/download PDF
21. Mechanistic coupling of bacteriophage T4 DNA packaging to components of the replication-dependent late transcription machinery.
- Author
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Black LW and Peng G
- Subjects
- Bacteriophage T4 chemistry, DNA Replication, DNA, Superhelical genetics, DNA, Viral genetics, Gene Expression Regulation, Viral, Models, Biological, Models, Genetic, Plasmids metabolism, Bacteriophage T4 genetics, DNA Packaging, Transcription, Genetic
- Abstract
Regulation of the terminal stage of viral DNA development, DNA packaging, is poorly understood. A new phage T4 in vitro DNA packaging assay employed purified proheads, terminase (gp17 + gp16), and ATP to encapsidate DNA resistant to nuclease. Mature phage T4 DNA and linearized plasmid DNAs containing or lacking a cloned T4 gene were packaged with high (approximately 10%) efficiency. Supercoiled, relaxed covalently closed, and nicked circular plasmid DNAs were packaged inefficiently, if at all, by these components. However, efficient packaging is achieved for nicked circular plasmid DNA, but not covalently closed plasmid DNA, upon addition to packaging mixtures of the purified T4 late transcription-replication machinery proteins: gp45 (sliding clamp), gp44/gp62 (clamp loader complex), gp55 (late sigma-factor), and gp33 (transcriptional co-activator). The small terminase subunit (gp16) is inhibitory for packaging linear DNAs, but enhances the transcription-replication protein packaging of nicked plasmid DNA. Taken together with genetic and biochemical evidence of a requirement for gp55 for concatemer packaging to assemble active wild-type phage particles (1), the plasmid packaging results show that initiation of phage T4 packaging on "endless" concatemeric DNA in vivo by terminase depends upon interaction with the DNA loaded gp45 coupled late transcription-replication machinery. The results suggest a close mechanistic connection in vivo between DNA packaging and developmentally concurrent replication-dependent late transcription. more...
- Published
- 2006
- Full Text
- View/download PDF
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