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Raphael Frey, Takahiro Hayashi, Donald Hilvert
Engineered variants of the capsid-forming enzyme lumazine synthase, AaLS, were used as nanoreactors for an enzyme-mediated polymerization. Oxidation of 3,3-diaminobenzidine (DAB) by the engineered ascorbate peroxidase APEX2 encapsulated in AaLS capsids resulted in templated formation of polyDAB-capsid nanoparticles of homogeneous size and shape.
August 16, 2016: Chemical Communications: Chem Comm
Jiwon Hwang, Peter J Espenshade
The engineered ascorbate peroxidase (APEX2) has been effectively employed in mammalian cells to identify protein-protein interactions. APEX2 fused to a protein of interest covalently tags nearby proteins with biotin-phenol (BP) when H2O2 is added to the cell culture medium. Subsequent affinity purification of biotinylated proteins allows for identification by MS. BP labelling occurs in 1 min, providing temporal control of labelling. The APEX2 tool enables proteomic mapping of subcellular compartments as well as identification of dynamic protein complexes, and has emerged as a new methodology for proteomic analysis...
August 15, 2016: Biochemical Journal
Oliver Baker, Ashish Gupta, Mandy Obst, Youming Zhang, Konstantinos Anastassiadis, Jun Fu, A Francis Stewart
A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags...
2016: Scientific Reports
Nicholas S Eyre, Rachel J Hampton-Smith, Amanda L Aloia, James S Eddes, Kaylene J Simpson, Peter Hoffmann, Michael R Beard
Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components...
April 2016: Virology
Victoria Hung, Namrata D Udeshi, Stephanie S Lam, Ken H Loh, Kurt J Cox, Kayvon Pedram, Steven A Carr, Alice Y Ting
This protocol describes a method to obtain spatially resolved proteomic maps of specific compartments within living mammalian cells. An engineered peroxidase, APEX2, is genetically targeted to a cellular region of interest. Upon the addition of hydrogen peroxide for 1 min to cells preloaded with a biotin-phenol substrate, APEX2 generates biotin-phenoxyl radicals that covalently tag proximal endogenous proteins. Cells are then lysed, and biotinylated proteins are enriched with streptavidin beads and identified by mass spectrometry...
March 2016: Nature Protocols
Aline Camporez Crispim, Matthew John Kelly, Simone Eliza Facioni Guimarães, Fabyano Fonseca e Silva, Marina Rufino Salinas Fortes, Raphael Rocha Wenceslau, Stephen Moore
Understanding the genetic architecture of beef cattle growth cannot be limited simply to the genome-wide association study (GWAS) for body weight at any specific ages, but should be extended to a more general purpose by considering the whole growth trajectory over time using a growth curve approach. For such an approach, the parameters that are used to describe growth curves were treated as phenotypes under a GWAS model. Data from 1,255 Brahman cattle that were weighed at birth, 6, 12, 15, 18, and 24 months of age were analyzed...
2015: PloS One
Jisu Lee, Eun Kyung Song, Yoonji Bae, Junseon Min, Hyun-Woo Rhee, Tae Joo Park, Moonil Kim, Sebyung Kang
A recombinant target-specific signal amplifier was constructed by genetically fusing the enhanced ascorbate peroxidase 2 (APEX2) and an antibody-binding domain (ABD). The fusion protein APEX2-ABD possessed the peroxidase activity and the antibody-binding capability simultaneously and replaced the conventional HRP-conjugated secondary antibodies in a TSA assay for amplifying fluorescence signals.
July 11, 2015: Chemical Communications: Chem Comm
Stephanie S Lam, Jeffrey D Martell, Kimberli J Kamer, Thomas J Deerinck, Mark H Ellisman, Vamsi K Mootha, Alice Y Ting
APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins...
January 2015: Nature Methods
Jeremy Willis, Yogin Patel, Barry L Lentz, Shan Yan
The base excision repair pathway is largely responsible for the repair of oxidative stress-induced DNA damage. However, it remains unclear how the DNA damage checkpoint is activated by oxidative stress at the molecular level. Here, we provide evidence showing that hydrogen peroxide (H2O2) triggers checkpoint kinase 1 (Chk1) phosphorylation in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3-related]-dependent but ATM-independent manner in Xenopus egg extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) endonuclease 2 (APE2, APN2, or APEX2), is required for the generation of replication protein A (RPA)-bound single-stranded DNA, the recruitment of a checkpoint protein complex [ATR, ATR-interacting protein (ATRIP), and Rad9] to damage sites, and H2O2-induced Chk1 phosphorylation...
June 25, 2013: Proceedings of the National Academy of Sciences of the United States of America
Vrajesh Karkhanis, Li Wang, Sookil Tae, Yu-Jie Hu, Anthony N Imbalzano, Saïd Sif
Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2...
August 24, 2012: Journal of Biological Chemistry
Wenyi Wang, Peidong Shen, Sreedevi Thiyagarajan, Shengrong Lin, Curtis Palm, Rita Horvath, Thomas Klopstock, David Cutler, Lynn Pique, Iris Schrijver, Ronald W Davis, Michael Mindrinos, Terence P Speed, Curt Scharfe
A common goal in the discovery of rare functional DNA variants via medical resequencing is to incur a relatively lower proportion of false positive base-calls. We developed a novel statistical method for resequencing arrays (SRMA, sequence robust multi-array analysis) to increase the accuracy of detecting rare variants and reduce the costs in subsequent sequence verifications required in medical applications. SRMA includes single and multi-array analysis and accounts for technical variables as well as the possibility of both low- and high-frequency genomic variation...
January 2011: Nucleic Acids Research
Jessica M Grunda, John Fiveash, Cheryl A Palmer, Alan Cantor, Hassan M Fathallah-Shaykh, L Burt Nabors, Martin R Johnson
PURPOSE: Previous preclinical studies suggested that concurrent capecitabine and radiation could be an effective new treatment modality for glioblastoma (GBM). In the current study, we investigate toxicity and response to this regimen and explore associations between gene expression and patient outcome. EXPERIMENTAL DESIGN: Eighteen newly diagnosed GBM patients received concurrent capecitabine at 625 mg/m2 BID (25% escalation) and irradiation (60 Gy total) for 6 weeks followed by 4 weeks of capecitabine only...
May 15, 2010: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Zahra Sabouri, Il-Mi Okazaki, Reiko Shinkura, Nasim Begum, Hitoshi Nagaoka, Daisuke Tsuchimoto, Yusaku Nakabeppu, Tasuku Honjo
The DNA cleavage step in both the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID). However, the detailed mechanisms of the DNA strand cleavage in SHM and CSR are still largely unknown. Recently, the apurinic/apyrimidinic endonucleases, Apex1 and Apex2, were reported to be involved in the DNA cleavage step of CSR. Here, we examined the role of Apex2 in SHM using Apex2-deficient mice and found that the Apex2 deficiency caused a drastic reduction in the frequency of SHM and the number of mutations per mutated clone without affecting the pattern of base substitution...
August 2009: International Immunology
Sean Fortier, Xiaojie Yang, Yi Wang, Richard A O Bennett, Phyllis R Strauss
The base excision repair (BER) pathway recognizes and repairs most nonbulky lesions, uracil and abasic (AP) sites in DNA. Several participants are embryonic lethals in knockout mice. Since the pathway has never been investigated during embryogenesis, we characterized the first three steps of BER in zebrafish extracts from unfertilized eggs, embryos at different developmental stages, and adults. Using a 45-mer double-stranded substrate with a U/G mispair at position 21, we showed that extracts from all stages are capable of performing BER...
June 16, 2009: Biochemistry
Yukihiko Dan, Yutaka Ohta, Daisuke Tsuchimoto, Mizuki Ohno, Yasuhito Ide, Manabu Sami, Tomomasa Kanda, Kunihiko Sakumi, Yusaku Nakabeppu
A second class II AP endonuclease, APEX2, possesses strong 3'-5' exonuclease and 3'-phosphodiesterase activities but only very weak AP-endonuclease activity. APEX2 associates with proliferating cell nuclear antigen (PCNA), and the progression of S phase of the cell cycle is accompanied by its expression. APEX2-null mice exhibit severe dyslymphopoiesis in thymus as well as moderate dyshematopoiesis and growth retardation. Comparative gene expression profiling of wild-type and APEX2-null mice using an oligonucleotide microarray revealed that APEX2-null thymus has significantly altered gene expression profiles, reflecting its altered populations of thymocytes...
September 1, 2008: DNA Repair
Ezra C Wood, Scott C Herndon, Michael T Timko, Paul E Yelvington, Richard C Miake-Lye
Measurements of nitrogen oxides from a variety of commercial aircraft engines as part of the JETS-APEX2 and APEX3 campaigns show that NOx (NOx [triple bond] NO + NO2) is emitted primarily in the form of NO2 at idle thrust and NO at high thrust. A chemical kinetics combustion model reproduces the observed NO2 and NOx trends with engine power and sheds light on the relevant chemical mechanisms. Experimental evidence is presented of rapid conversion of NO to NO2 in the exhaust plume from engines at low thrust...
March 15, 2008: Environmental Science & Technology
Yasuhito Ide, Daisuke Tsuchimoto, Yohei Tominaga, Manabu Nakashima, Takeshi Watanabe, Kunihiko Sakumi, Mizuki Ohno, Yusaku Nakabeppu
APEX2/APE2 is a secondary mammalian apurinic/apyrimidinic endonuclease that associates with proliferating cell nuclear antigen (PCNA), and the progression of S phase of the cell cycle is accompanied by its expression. To determine the biologic significance of APEX2, we established APEX2-null mice. These mice were about 80% the size of their wild-type littermates and exhibited a moderate dyshematopoiesis and a relatively severe defect in lymphopoiesis. A significant accumulation of both thymocytes and mitogen-stimulated splenocytes in G(2)/M phase was seen in APEX2-null mice compared with the wild type, indicating that APEX2 is required for proper cell cycle progression of proliferating lymphocytes...
December 15, 2004: Blood
Yusaku Nakabeppu, Daisuke Tsuchimoto, Akimasa Ichinoe, Mizuki Ohno, Yasuhito Ide, Seiki Hirano, Daisuke Yoshimura, Yohei Tominaga, Masato Furuichi, Kunihiko Sakumi
In mammalian cells, more than one genome in a single cell has to be maintained throughout the entire life of the cell, namely, one in the nucleus and the other in the mitochondria. The genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are inevitably generated as a result of the respiratory function in mitochondria. To counteract such oxidative damage in nucleic acids, cells are equipped with several defense mechanisms. Modified nucleotides in the nucleotide pools are hydrolyzed, thus avoiding their incorporation into DNA or RNA...
April 2004: Annals of the New York Academy of Sciences
Yasuhito Ide, Daisuke Tsuchimoto, Yohei Tominaga, Yukihide Iwamoto, Yusaku Nakabeppu
We isolated a mouse cDNA encoding APEX2 protein and demonstrated that APEX2 binds to PCNA. The level of Apex2 mRNA was high in the thymus, bone marrow, spleen, and kidney in adult mice. Apex2 consists of six exons and is flanked on the 3' end by Alas2 on X chromosome 63.0. Furthermore, Apex2 is flanked on the 5' end by a novel gene with a 106-bp intergenic sequence. We disrupted Apex2 in embryonic stem cells derived from a male mouse, and a 55-kDa APEX2 protein was detected in the nuclei of Apex2(+) but not Apex2-disrupted cells...
January 2003: Genomics
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