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https://www.readbyqxmd.com/read/29735996/c-berst-defining-subnuclear-proteomic-landscapes-at-genomic-elements-with-dcas9-apex2
#1
Xin D Gao, Li-Chun Tu, Aamir Mir, Tomás Rodriguez, Yuehe Ding, John Leszyk, Job Dekker, Scott A Shaffer, Lihua Julie Zhu, Scot A Wolfe, Erik J Sontheimer
Mapping proteomic composition at distinct genomic loci in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. C-BERST enables the high-throughput identification of proteins associated with specific sequences, thereby facilitating annotation of these factors and their roles.
May 7, 2018: Nature Methods
https://www.readbyqxmd.com/read/29712893/linear-mitochondrial-dna-is-rapidly-degraded-by-components-of-the-replication-machinery
#2
Viktoriya Peeva, Daniel Blei, Genevieve Trombly, Sarah Corsi, Maciej J Szukszto, Pedro Rebelo-Guiomar, Payam A Gammage, Alexei P Kudin, Christian Becker, Janine Altmüller, Michal Minczuk, Gábor Zsurka, Wolfram S Kunz
Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown. Here, we show that, in cellular models of restriction endonuclease-induced mtDNA double-strand breaks, linear mtDNA is eliminated within hours by exonucleolytic activities. Inactivation of the mitochondrial 5'-3'exonuclease MGME1, elimination of the 3'-5'exonuclease activity of the mitochondrial DNA polymerase POLG by introducing the p...
April 30, 2018: Nature Communications
https://www.readbyqxmd.com/read/29679029/author-correction-optimizing-the-fragment-complementation-of-apex2-for-detection-of-specific-protein-protein-interactions-in-live-cells
#3
Miaomiao Xue, Junjie Hou, Linlin Wang, Dongwan Cheng, Jingze Lu, Li Zheng, Tao Xu
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
April 20, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29414245/bifunctional-fusion-proteins-containing-the-sequence-of-the-bradykinin-homologue-maximakinin-activities-at-the-rat-bradykinin-b-2-receptor
#4
Xavier Charest-Morin, Robert Lodge, François Marceau
To support bradykinin (BK) B2 receptor (B2 R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B2 Rs (immunoblots, epifluorescence microscopy)...
February 7, 2018: Canadian Journal of Physiology and Pharmacology
https://www.readbyqxmd.com/read/29275994/a-proximity-labeling-strategy-provides-insights-into-the-composition-and-dynamics-of-lipid-droplet-proteomes
#5
Kirill Bersuker, Clark W H Peterson, Milton To, Steffen J Sahl, Victoria Savikhin, Elizabeth A Grossman, Daniel K Nomura, James A Olzmann
Lipid droplet (LD) functions are regulated by a complement of integral and peripheral proteins that associate with the bounding LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to two different cell types identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins...
January 8, 2018: Developmental Cell
https://www.readbyqxmd.com/read/29238746/determining-the-target-protein-localization-in-3d-using-the-combination-of-fib-sem-and-apex2
#6
Yang Shi, Li Wang, Jianguo Zhang, Yujia Zhai, Fei Sun
Determining the cellular localization of proteins of interest at nanometer resolution is necessary for elucidating their functions. Besides super-resolution fluorescence microscopy, conventional electron microscopy (EM) combined with immunolabeling or clonable EM tags provides a unique approach to correlate protein localization information and cellular ultrastructural information. However, there are still rare cases of such correlation in three-dimensional (3D) spaces. Here, we developed an approach by combining the focus ion beam scanning electron microscopy (FIB-SEM) and a promising clonable EM tag APEX2 (an enhanced ascorbate peroxidase 2) to determine the target protein localization within 3D cellular ultrastructural context...
2017: Biophysics Reports
https://www.readbyqxmd.com/read/29213341/ciapex2-and-cip0-candidates-of-ap-endonucleases-in-ciona-intestinalis-have-3-5-exonuclease-activity-and-contribute-to-protection-against-oxidative-stress
#7
Masafumi Funakoshi, Daisuke Nambara, Yuichiro Hayashi, Qiu-Mei Zhang-Akiyama
Apurinic/apyrimidinic (AP) sites are one of the most frequent DNA lesions. AP sites inhibit transcription and DNA replication, and induce cell death. AP endonucleases are key enzymes in AP site repair. Several types of AP endonucleases have been reported, such as AP endonuclease 2 (APEX2) and ribosomal protein P0 (P0). However, it is not known how the functions and roles differ among AP endonucleases. To clarify the difference of roles among AP endonucleases, we conducted biochemical analysis focused on APEX2 and P0 homologues in Ciona intestinalis ...
2017: Genes and Environment: the Official Journal of the Japanese Environmental Mutagen Society
https://www.readbyqxmd.com/read/29026164/production-and-evaluation-of-parathyroid-hormone-receptor-1-ligands-with-intrinsic-or-assembled-peroxidase-domains
#8
Xavier Charest-Morin, Patrice E Poubelle, François Marceau
Parathyroid hormone (PTH) can be C-terminally extended without significant affinity loss for the PTH1 receptor (PTHR1 ). We developed fusion protein ligands with enzymatic activity to probe PTHR1 s at the cell surface. Two fusion proteins were generated by linking PTH to the N-terminus of either horseradish peroxidase (PTH-HRP) or the genetically modified soybean peroxidase APEX2 (PTH-APEX2). Alternatively, myc-tagged PTH (PTH-myc) was combined with antibodies, some of which HRP-conjugated, in the extracellular fluid...
October 12, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28956770/genome-wide-mutagenesis-of-dengue-virus-reveals-plasticity-of-the-ns1-protein-and-enables-generation-of-infectious-tagged-reporter-viruses
#9
Nicholas S Eyre, Stephen M Johnson, Auda A Eltahla, Maria Aloi, Amanda L Aloia, Christopher A McDevitt, Rowena A Bull, Michael R Beard
Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture...
December 1, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28955036/optimizing-the-fragment-complementation-of-apex2-for-detection-of-specific-protein-protein-interactions-in-live-cells
#10
Miaomiao Xue, Junjie Hou, Linlin Wang, Dongwan Cheng, Jingze Lu, Li Zheng, Tao Xu
Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. Recently, an ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. In this study, we tested whether APEX2 could be employed for PFC...
September 27, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28881650/apex2-enhanced-electron-microscopy-distinguishes-sigma-1-receptor-localization-in-the-nucleoplasmic-reticulum
#11
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
https://www.readbyqxmd.com/read/28808085/sub-mitochondrial-localization-of-the-genetic-tagged-mitochondrial-intermembrane-space-bridging-components-mic19-mic60-and-sam50
#12
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. By using the novel visualizable genetic tags miniSOG and APEX2 in cultured mouse cardiac and human astrocyte cell lines and performing electron tomography, we have mapped at nanoscale resolution three key MIB components, Mic19, Mic60 and Sam50 (also known as CHCHD3, IMMT and SAMM50, respectively), in the environment of structural landmarks such as cristae and crista junctions (CJs)...
October 1, 2017: Journal of Cell Science
https://www.readbyqxmd.com/read/28796234/electron-microscopy-using-the-genetically-encoded-apex2-tag-in-cultured-mammalian-cells
#13
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
https://www.readbyqxmd.com/read/28720733/dengue-virus-hijacks-a-noncanonical-oxidoreductase-function-of-a-cellular-oligosaccharyltransferase-complex
#14
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
https://www.readbyqxmd.com/read/28572528/apex2-enhanced-electron-microscopy-distinguishes-sigma-1-receptor-localization-in-the-nucleoplasmic-reticulum
#15
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
https://www.readbyqxmd.com/read/28528629/correlative-light-and-electron-microscopic-detection-of-gfp-labeled-proteins-using-modular-apex
#16
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
https://www.readbyqxmd.com/read/28261569/development-of-a-proximity-labeling-system-to-map-the-chlamydia-trachomatis-inclusion-membrane
#17
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
https://www.readbyqxmd.com/read/27484787/enzyme-mediated-polymerization-inside-engineered-protein-cages
#18
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
https://www.readbyqxmd.com/read/27274088/proximity-dependent-biotin-labelling-in-yeast-using-the-engineered-ascorbate-peroxidase-apex2
#19
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
https://www.readbyqxmd.com/read/27216209/rac-tagging-recombineering-and-cas9-assisted-targeting-for-protein-tagging-and-conditional-analyses
#20
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...
May 24, 2016: Scientific Reports
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