Cell-selective labelling of proteomes in Drosophila melanogaster

Erdmann, Ines; Marter, Kathrin; Kobler, Oliver; Niehues, Sven; Abele, Julia; Müller, Anke; Bussmann, Julia; Storkebaum, Erik; Ziv, Tamar; Thomas, Ulrike; Dieterich, Daniela C.

doi:10.1038/ncomms8521

Cited by 95 publications

(123 citation statements)

References 58 publications

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“…Indeed, prior studies have shown that DNA polymerases cannot undergo the necessary conformational changes needed for catalysis when dG•dT is in a wobble conformation 7 and all available structures of catalytically active polymerases with bound mismatches within the active site feature WC-like dG•dT or dA•dC geometries 6,7 . Similarly, WC-like rG•rU mismatches have been shown to form in the first and second codon positions of catalytically active ribosomes 9 , in which wobbles are typically rejected 5 , potentially helping to explain translational error hotspots 41 .…”

Section: Tautomerization/ionization During Misincorporationmentioning

confidence: 99%

“…There is also evidence that both tautomeric 6–13 (Fig. 1b) and anionic 7–9,14,15 (Fig. 1c) WC-like mismatches can evade such fidelity checkpoints and give rise to replication 6,7 and translation errors 16 .…”

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confidence: 96%

“…1). Protons are generally invisible to X-ray crystallography and cryo-EM 12 , and consequently it has not been possible to unambiguously resolve the identity of WC-like mismatches captured within active sites of polymerases 6,7,15,23 and the ribosome decoding site 9,24 . Moreover, WC-like mismatches are predicted to exist in rapid tautomeric (G enol •T/U⇌G•T enol /U enol ) 25,26 equilibria (Fig.…”

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confidence: 99%

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Dynamic basis for dG•dT misincorporation via tautomerization and ionization

Kimsey

Szymanski

Zahurancik

et al. 2018

Nature

134

183

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Tautomeric and anionic Watson-Crick-like mismatches play important roles in replication and translation errors through mechanisms that are not fully understood. Using NMR relaxation dispersion, we resolved a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson-Crick mismatches consisting of two rapidly exchanging tautomeric species (Genol•T/U⇌G•Tenol/Uenol; population <0.4%) and one anionic species (G•T−/U−; population ≈0.001% at neutral pH). Inserting the sequence-dependent tautomerization/ionization step into a minimal kinetic mechanism for correct incorporation during replication following initial nucleotide binding leads to accurate predictions of dG•dT misincorporation probability across different polymerases, pH conditions, and for a chemically modified nucleotide, and provides mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization/ionization accounts for ≈10−2−10−3–fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol with contributions from anionic dG•dT− dominating at pH ≥8.4 or for some mutagenic nucleotides.

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Section: Tautomerization/ionization During Misincorporationmentioning

confidence: 99%

mentioning

confidence: 96%

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confidence: 99%

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Dynamic basis for dG•dT misincorporation via tautomerization and ionization

Kimsey

Szymanski

Zahurancik

et al. 2018

Nature

134

183

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“…Fig. 2c) as recently shown by us for different cell types including glial and neuronal cells in Drosophila melanogaster [7]. This technique relies on a mutated methionine tRNA synthetase (MetRSLtoG), which incorporates, instead of methionine, the noncanonical amino acid azidonorleucine (ANL) into nascent proteins.…”

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confidence: 85%

Dissecting the regional diversity of glial cells by applying -omic technologies

Dieterich

Rossner²

2015

E-Neuroforum

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With an estimated number of approximately 10,000 different proteins in each single mammalian cell [16], in-depth identification of a cell's entire proteome Dissecting the regional diversity of glial cells by applying -omic technologies Abstract Neuronal as well as glial cells contribute to higher order brain functions. Many observations show that neurons and glial cells are not only physically highly intermingled but are physiologically tightly connected and mutually depend at various levels on each other. Moreover, macroglia classes like astrocytes, NG2 cells and oligodendrocytes are not at all homogenous cell populations but do possess a markedly heterogeneity in various aspects similar to neurons. The diversity of differences in morphology, functionality and, cellular activity has been acknowledged recently and will be integrated into a concept of brain function that pictures a neural rather than a puristical neuronal world. With the recent progress in "omic" technologies, an unbiased and exploratory approach toward an enhanced understanding of glial heterogeneity has become possible. Here, we provide an overview on current technical transcriptomic and proteomic approaches used to dissect glial heterogeneity of the brain. is still a major challenge for modern proteomic technologies. In addition, the total number of different/unique proteins per synapse is estimated to be at approximately 2000-2500 [17], which are often under control of different activity-dependent turnover rates. While today's state-of-theart MS instruments routinely sequence single purified proteins with subfemtomolar sensitivity, the effective identification of low-abundance proteins is orders of magnitude lower in complex mixtures due to limited dynamic range and sequencing speed and due to the common, strong bias toward acquiring MS/MS data on higher abundance molecules. Hence, to tackle activity-dependent proteome alterations entire, functionally heterogeneous brain regions with different subtypes of neurons and macroglial cells are usually being sampled at the loss of cell-type specificity and spatial resolution. The characterization of a proteome is an even more difficult challenge if temporal and spatial aspects of a proteome or a subpopulation of the proteome have to be taken into consideration. Thus, the separation and enrichment of the subproteome in question is key for its successful characterization. To overcome the above mentioned limitations, several cellular and biochemical (subcellular) enrichment strategies combined with proteomics have been developed to increase sensitivity and selectivity for the analysis of neuronal and glial proteomes, and to finally dissolve cellular heterogeneity of neural cells in the brain (. Fig. 2a-b). KeywordsConcerning cellular selectivity, intracellular proteomes and secretomes from cultured primary astrocytes and astroglial cell lines have been characterized in detail by several labs as proteomic approaches as indicated above require a much larger quantity than transcriptomic approaches. Analys...

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“…Abb. 2c ), wie aktuell von uns für verschiedene Zelltypen, einschließ-lich Glia-und Nervenzellen, in Drosophila melanogaster gezeigt wurde [ 7 ]. Diese Technik basiert auf einer mutierten Methionin -tRNA -Synthetase (MetRSLtoG), welche anstelle von Methionin die nichtkanonische Aminosäure Azidonorleucine (ANL) in neusynthetisierte Proteine einbaut.…”

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Entschlüsselung glialer Funktionen mittels OMICs-Technologien

Dieterich

Rossner²

2015

E-Neuroforum

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ZusammenfassungSowohl neuronale als auch gliale Zellen tragen zu höheren Hirnfunktionen bei. Viele Beobachtungen zeigen, dass Neurone und Gliazellen nicht nur physische Berührungspunkte besitzen, sondern auch physiologisch eng verbunden sind und auf vielen Ebenen wechselseitig voneinander abhängen. Zudem stellen Macroglia wie Astrozyten, NG2 - Zellen und Oligodendrocyten keine homogenen Zellpopulationen dar, sondern besitzen, ahnlich wie Neurone, merkliche Unterschiede in verschiedenen Aspekten. Diese Vielzahl der Unterschiede in Morphologie, Funktionalität und zellularer Aktivität wurden jungst anerkannt und in ein generelles Konzept der Gehirnfunktion integriert, welches eine neurale, statt einer ausschließlich neuronalen Welt zeichnet. Mit den neusten Fortschritten der ‚OMICs‘ Technologie wird ein unvoreingenommener und explorativer Ansatz möglich, über welchen ein detailliertes Verständnis des glialen Heterogenitätsprofils in Reichweite gelangt. In diesem Artikel geben wir einen Überblick über aktuelle Transkriptomics-und Proteomicstechniken, die benutzt werden, um die gliale Heterogenität des Gehirns zu analysieren.

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Cell-selective labelling of proteomes in Drosophila melanogaster

Cited by 95 publications

References 58 publications

Dynamic basis for dG•dT misincorporation via tautomerization and ionization

Dynamic basis for dG•dT misincorporation via tautomerization and ionization

Dissecting the regional diversity of glial cells by applying -omic technologies

Entschlüsselung glialer Funktionen mittels OMICs-Technologien

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