Read by QxMD icon Read


Timur A Mavlyutov, Huan Yang, Miles L Epstein, Arnold E Ruoho, Jay Yang, Lian-Wang Guo
The sigma-1 receptor (Sig1R) is an endoplasmic reticulum chaperonin that is attracting tremendous interest as a potential anti-neurodegenerative target. While this membrane protein is known to reside in the inner nuclear envelope (NE) and influences transcription, apparent Sig1R presence in the nucleoplasm is often observed, seemingly contradicting its NE localization. We addressed this confounding issue by applying an antibody-free approach of electron microscopy (EM) to define Sig1R nuclear localization. We expressed APEX2 peroxidase fused to Sig1R-GFP in a Sig1R-null NSC34 neuronal cell line generated with CRISPR-Cas9...
August 1, 2017: Oncotarget
Mira Sastri, Manjula Darshi, Mason Mackey, Ranjan Ramachandra, Saeyeon Ju, Sebastien Phan, Stephen Adams, Kathryn Stein, Christopher R Douglas, Jiwan John Kim, Mark H Ellisman, Susan S Taylor, Guy A Perkins
Each mitochondrial compartment contains varying protein compositions that underlie a diversity of localized functions. Insights into the localization of mitochondrial intermembrane space bridging (MIB) components will have an impact on our understanding of mitochondrial architecture, dynamics and function. Using the novel visualizable genetic tags miniSOG and APEX2 in cultured mouse cardiac and human astrocyte cell lines and electron tomography, we have mapped at nanoscale resolution three key MIB components: Mic19, Mic60 and Sam50, in the environment of structural landmarks such as cristae and crista junctions (CJs)...
August 14, 2017: Journal of Cell Science
Jeffrey D Martell, Thomas J Deerinck, Stephanie S Lam, Mark H Ellisman, Alice Y Ting
Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest...
September 2017: Nature Protocols
David L Lin, Natalia A Cherepanova, Leonia Bozzacco, Margaret R MacDonald, Reid Gilmore, Andrew W Tai
Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation...
July 18, 2017: MBio
Timur A Mavlyutov, Huan Yang, Miles L Epstein, Arnold E Ruoho, Jay Yang, Lian-Wang Guo
The sigma-1 receptor (Sig1R) is an endoplasmic reticulum chaperonin that is attracting tremendous interest as a potential anti-neurodegenerative target. While this membrane protein is known to reside in the inner nuclear envelope (NE) and influences transcription, apparent Sig1R presence in the nucleoplasm is often observed, seemingly contradicting its NE localization. We addressed this confounding issue by applying an antibody-free approach of electron microscopy (EM) to define Sig1R nuclear localization. We expressed APEX2 peroxidase fused to Sig1R-GFP in a Sig1R-null NSC34 neuronal cell line generated with CRISPR-Cas9...
May 16, 2017: Oncotarget
Nicholas Ariotti, Thomas E Hall, Robert G Parton
The use of green fluorescent protein (GFP) and related proteins has revolutionized light microscopy. Here we describe a rapid and simple method to localize GFP-tagged proteins in cells and in tissues by electron microscopy (EM) using a modular approach involving a small GFP-binding peptide (GBP) fused to the ascorbate peroxidase-derived APEX2 tag. We provide a method for visualizing GFP-tagged proteins by light and EM in cultured cells and in the zebrafish using modular APEX-GBP. Furthermore, we describe in detail the benefits of this technique over many of the currently available correlative light and electron microscopy approaches and demonstrate APEX-GBP is readily applicable to modern three-dimensional techniques...
2017: Methods in Cell Biology
Elizabeth A Rucks, Macy G Olson, Lisa M Jorgenson, Rekha R Srinivasan, Scot P Ouellette
Chlamydia grows within a membrane-bound vacuole termed an inclusion. The cellular processes that support the biogenesis and integrity of this pathogen-specified parasitic organelle are not understood. Chlamydia secretes integral membrane proteins called Incs that insert into the chlamydial inclusion membrane (IM). Incs contain at least two hydrophobic transmembrane domains flanked by termini, which vary in size and are exposed to the host cytosol. In addition, Incs are temporally expressed during the chlamydial developmental cycle...
2017: Frontiers in Cellular and Infection Microbiology
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
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"