RNA-DNA interactome of a prokaryotic cell uncovered by proximity ligation
Ontology highlight
ABSTRACT:
ORGANISM(S): Escherichia coli Bacillus subtilis Thermofilum adornatum
PROVIDER: GSE209901 | GEO | 2023/04/07
REPOSITORIES: GEO
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Project description:RNA-DNA interactome of a prokaryotic cell uncovered by proximity ligation
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Project description:Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein Tissue inhibitors of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.
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Project description:Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase?A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site-specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity-based sortase-mediated ligation (PBSL), which improves the ligation efficiency to over 95?% by linking the target protein to SrtA using the SpyTag-SpyCatcher peptide-protein pair. By expressing the target protein with SpyTag C-terminal to the SrtA recognition motif, it can be covalently captured by an immobilized SpyCatcher-SrtA fusion protein during purification. Following the ligation reaction, SpyTag is cleaved off, rendering PBSL traceless, and only the labeled protein is released, simplifying target protein purification and labeling to a single step.
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2015-06-27 | E-GEOD-70342 | biostudies-arrayexpress
Project description:Chaperones have essential role in assist nascent peptides folding, prevent proteins aggregation and maintain cellular protein homeostasis. Considering spatial and temporal features of chaperones regulating in vivo, changes in single or combined chaperone-depleted E.coli strain is needed to be put into understand at transcriptional level. Here, we utilized expression microarrays to investigate global transcriptional response upon deletion of single or multiple chaperones in E. coli for understanding the transcriptional network affected by chaperones. To identify prokaryotic expression profiles in deletion of chaperones, several E.coli mutants were constructed in the following: Z116 (△tig 37℃), Z125(△dnaK37℃), Z625(△tig△dnaK 37℃ or 30℃), Z629(△tig△dnaK 30℃), NM (C-domain of tig was deleted 37℃), MC (N-domain of tig was deleted 37℃), NC (M-domain of tig was deleted 37℃). Two types of cDNA mixtures containing Cy3-labeled (or Cy5-labeled) control DNA (from BW25113) and Cy5-labeled (or Cy3-labeled) DNA targets (from mutant strain) were hybridized with E.coli microarrays in a dye-swap strategy. E.coli gene expression data of chaperone DnaK deletant compared control WT (BW25113). Please note that other mutant microarray data will be added in the future.
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