scholarly journals Genome-wide protein-DNA interaction site mapping using a double strand DNA-specific cytosine deaminase

2021 ◽  
Author(s):  
Larry A. Gallagher ◽  
Elena Velazquez ◽  
S. Brook Peterson ◽  
James C. Charity ◽  
FoSheng Hsu ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Cytosine Deaminase ◽  
Dna Interaction ◽  
Uracil Dna Glycosylase ◽  
Cellular Processes ◽  
Site Mapping ◽  
Genome Wide ◽  
Functional Consequences ◽  
Natural Inhibitor

DNA-protein interactions (DPIs) are central to such fundamental cellular processes as transcription and chromosome maintenance and organization. The spatiotemporal dynamics of these interactions dictate their functional consequences; therefore, there is great interest in facile methods for defining the sites of DPI within cells. Here, we present a general method for mapping DPI sites in vivo using the double stranded DNA-specific cytosine deaminase toxin DddA. Our approach, which we term DddA-sequencing (3D-seq), entails generating a translational fusion of DddA to a DNA binding protein of interest, inactivating uracil DNA glycosylase, modulating DddA activity via its natural inhibitor protein, and DNA sequencing for genome-wide DPI detection. We successfully applied this method to three Pseudomonas aeruginosa transcription factors that represent divergent protein families and bind variable numbers of chromosomal locations. 3D-seq offers several advantages over existing technologies including ease of implementation and the possibility to measure DPIs at single-cell resolution.

scholarly journals Assay Systems for Profiling Deubiquitinating Activity

2020 ◽  
Vol 21 (16) ◽  
pp. 5638
Author(s):  
Jinhong Cho ◽  
Jinyoung Park ◽  
Eunice EunKyeong Kim ◽  
Eun Joo Song
Keyword(s):  
Protein Degradation ◽  
Protein Interactions ◽  
Live Cells ◽  
Deubiquitinating Enzyme ◽  
Protein Protein Interactions ◽  
Deubiquitinating Enzymes ◽  
Cell Lysates ◽  
Cellular Processes

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

scholarly journals Diverse control of metabolism and other cellular processes in Streptomyces coelicolor by the PhoP transcription factor: genome-wide identification of in vivo targets

2012 ◽  
Vol 40 (19) ◽  
pp. 9543-9556 ◽  
Author(s):  
Nicholas E. E. Allenby ◽  
Emma Laing ◽  
Giselda Bucca ◽  
Andrzej M. Kierzek ◽  
Colin P. Smith
Keyword(s):  
Transcription Factor ◽  
Streptomyces Coelicolor ◽  
Cellular Processes ◽  
Genome Wide

scholarly journals Application of a bacterial two-hybrid system for the analysis of protein–protein interactions between FemABX family proteins

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2733-2738 ◽  
Author(s):  
Susanne Rohrer ◽  
Brigitte Berger-Bächi
Keyword(s):  
Hybrid System ◽  
Protein Interactions ◽  
Gel Filtration ◽  
Protein Protein Interactions ◽  
Peptidoglycan Biosynthesis ◽  
Cellular Processes ◽  
Gram Positive Bacteria ◽  
Pulldown Assay ◽  
Two Hybrid

Protein–protein interactions play an important role in all cellular processes. The development of two-hybrid systems in yeast and bacteria allows for in vivo assessment of such interactions. Using a recently developed bacterial two-hybrid system, the interactions of the Staphylococcus aureus proteins FemA, FemB and FmhB, members of the FemABX protein family, which is involved in peptidoglycan biosynthesis and β-lactam resistance of numerous Gram-positive bacteria, were analysed. While FmhB is involved in the addition of glycine 1 of the pentaglycine interpeptide of S. aureus peptidoglycan, FemA and FemB are specific for glycines 2/3 and 4/5, respectively. FemA–FemA, FemA–FemB and FemB–FemB interactions were found, while FmhB exists solely as a monomer. Interactions detected by the bacterial two-hybrid system were confirmed using the glutathione S-transferase-pulldown assay and gel filtration.

scholarly journals Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrea Mair ◽  
Shou-Ling Xu ◽  
Tess C Branon ◽  
Alice Y Ting ◽  
Dominique C Bergmann
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Cell Types ◽  
Specific Protein ◽  
Cell Type ◽  
Subcellular Compartment ◽  
Cellular Processes ◽  
Highly Active

Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and Nicotiana benthamiana and versatile vectors enable customization by plant researchers.

scholarly journals Pseudouridines on Trypanosoma brucei spliceosomal small nuclear RNAs and their implication for RNA and protein interactions

2019 ◽  
Vol 47 (14) ◽  
pp. 7633-7647 ◽  
Author(s):  
K Shanmugha Rajan ◽  
Tirza Doniger ◽  
Smadar Cohen-Chalamish ◽  
Dana Chen ◽  
Oz Semo ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Interactions ◽  
Elevated Temperatures ◽  
Mammalian Host ◽  
U2 Snrna ◽  
Genome Wide ◽  
A Genome ◽  
Small Nuclear Rnas ◽  
Different Temperatures

Abstract The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an insect and a mammalian host. Here, we investigated the presence of pseudouridines (Ψs) on the spliceosomal small nuclear RNAs (snRNAs), which may enable growth at the very different temperatures characterizing the two hosts. To this end, we performed the first high-throughput mapping of spliceosomal snRNA Ψs by small RNA Ψ-seq. The analysis revealed 42 Ψs on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding Ψ on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA Ψs are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth at elevated temperatures. We further demonstrate that Ψ strengthens U4/U6 RNA–RNA and U2B"/U2A’ proteins-U2 snRNA interaction at elevated temperatures. The existence of single hairpin RNAs that modify both the spliceosome and ribosome RNAs is unique for these parasites, and may be related to their ability to cycle between their two hosts that differ in temperature.

scholarly journals Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions

2010 ◽  
Vol 189 (4) ◽  
pp. 739-754 ◽  
Author(s):  
Nina C. Hubner ◽  
Alexander W. Bird ◽  
Jürgen Cox ◽  
Bianca Splettstoesser ◽  
Peter Bandilla ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Small Scale ◽  
Mitotic Spindles ◽  
Cellular Processes ◽  
Green Fluorescent ◽  
Highly Sensitive Detection ◽  
Interaction Proteomics

Protein interactions are involved in all cellular processes. Their efficient and reliable characterization is therefore essential for understanding biological mechanisms. In this study, we show that combining bacterial artificial chromosome (BAC) TransgeneOmics with quantitative interaction proteomics, which we call quantitative BAC–green fluorescent protein interactomics (QUBIC), allows specific and highly sensitive detection of interactions using rapid, generic, and quantitative procedures with minimal material. We applied this approach to identify known and novel components of well-studied complexes such as the anaphase-promoting complex. Furthermore, we demonstrate second generation interaction proteomics by incorporating directed mutational transgene modification and drug perturbation into QUBIC. These methods identified domain/isoform-specific interactors of pericentrin- and phosphorylation-specific interactors of TACC3, which are necessary for its recruitment to mitotic spindles. The scalability, simplicity, cost effectiveness, and sensitivity of this method provide a basis for its general use in small-scale experiments and in mapping the human protein interactome.

scholarly journals Theory of site-specific DNA-protein interactions in the presence of nucleosome roadblocks

2017 ◽  
Author(s):  
R. Murugan
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
Specific Binding ◽  
First Passage Time ◽  
Free Energy Barrier ◽  
Site Specific ◽  
Specific Binding Sites ◽  
Genome Wide ◽  
Diffusion Dynamics

AbstractWe show that nucleosomes can efficiently control the relative search times spent by transcription factors (TFs) on one- (1D) and three-dimensional (3D) diffusion routes towards locating their cognate sites on DNA. Our theoretical results suggest that the roadblock effects of nucleosomes are dependent on the relative position on DNA with respect to TFs and their cognate sites. Especially, nucleosomes exert maximum amount of hindrance to the 1D diffusion dynamics of TFs when they are positioned in between TFs and their cognate sites. The effective 1D diffusion coefficient (χTF) associated with the dynamics of TFs in the presence of nucleosome decreases with the free energy barrier (µ) associated the sliding dynamics of nucleosomes as . Subsequently the mean first passage time (ηL) that is required by TFs to scan L number of binding sites on DNA via 1D diffusion increases with μ as . When TFs move close to nucleosomes then they exhibit a typical sub-diffusive dynamics. Nucleosomes can enhance the search dynamics of TFs when TFs present in between nucleosomes and transcription factor binding sites (TFBS). The level of enhancement effects of nucleosomes seems to be much lesser than the level of retardation effects when nucleosomes present in between TFs and their cognate sites. These results suggest that nucleosome depleted regions around the cognate sites of TFs is mandatory for an efficient site-specific interactions of TFs with DNA. Remarkably the genome wide positioning pattern of TFs shows maximum at their specific binding sites and the positioning pattern of nucleosome shows minimum at the specific binding sites of TFs under in vivo conditions. This seems to be a consequence of increasing level of breathing dynamics of nucleosome cores and decreasing levels of fluctuations in the DNA binding domains of TFs as they move across TFBS. Since the extent of breathing dynamics of nucleosomes and fluctuations in the DBDs of TFs are directly linked with their respective 1D diffusion coefficients, the dynamics of TFs becomes slow as they approach their cognate sites so that TFs form tight site-specific complex. Whereas the dynamics of nucleosomes becomes rapid so that they pass through the cognate sites of TFs. Several in vivo datasets on genome wide positioning pattern of nucleosomes as well as TFs seem to agree well with our arguments. We further show that the condensed conformational state of DNA can significantly decrease the retarding effects of nucleosome roadblocks. The retarding effects of nucleosomes on the 1D diffusion dynamics of TFs can be nullified when the degree of condensation of the genomic DNA is such that it can permit a jump size associated with the dynamics of TFs beyond k > 150 bps.

scholarly journals AGC kinase members phosphorylate ubiquitin

2020 ◽  
Author(s):  
Carl-Christian Kolbe ◽  
Eicke Latz
Keyword(s):  
Protein Interactions ◽  
Posttranslational Modification ◽  
Kinase Domain ◽  
Agc Kinases ◽  
Peptide Specificity ◽  
Cellular Processes ◽  
Ubiquitinated Proteins ◽  
Protein Ubiquitination ◽  
Genome Wide

AbstractThe posttranslational modification of proteins with ubiquitin controls most cellular processes, such as protein degradation or transport, cell signaling, or transcription1–5. Ubiquitin can be phosphorylated at multiple sites, which likely further modulates the function of protein ubiquitination6,7. However, except for PINK18–10, the kinases involved in ubiquitin phosphorylation remain unknown, which hampers our understanding of phospho-ubiquitin signaling. In this study, we performed genome-wide in vitro kinase screenings and discovered that AGC kinases phosphorylate ubiquitin. Ubiquitin phosphorylation by members of the PKA, PKC, PKG and RSK families as well as by less well-characterized kinases, such as SGK2, was not solely dependent on peptide specificity but required additional kinase recruitment to ubiquitin. The stabilization of the kinase interaction with ubiquitin resulted in phosphorylation of suboptimal kinase motifs on ubiquitin, suggesting that ubiquitin phosphorylation is dictated primarily through the recruitment of kinases to the ubiquitinated proteins. Hence, we identify AGC kinase members as enzymes that can phosphorylate ubiquitin in a mechanism regulated by protein interactions outside of the catalytic kinase domain and are only applicable to specific subsets of ubiquitinated proteins.

scholarly journals Proximity labeling of protein complexes and cell type-specific organellar proteomes in Arabidopsis enabled by TurboID

2019 ◽  
Author(s):  
Andrea Mair ◽  
Shou-Ling Xu ◽  
Tess C. Branon ◽  
Alice Y. Ting ◽  
Dominique C. Bergmann
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Cell Types ◽  
Specific Protein ◽  
Cell Type ◽  
Subcellular Compartment ◽  
Cellular Processes ◽  
Highly Active

AbstractDefining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurboID), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurboID in Arabidopsis and N. benthamiana and versatile vectors enable customization by plant researchers.

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