AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions
- Ehime University, Japan
- University of Shizuoka, Japan
- Tokushima University, Japan
Abstract
Proximity biotinylation based on Escherichia coli BirA enzymes like BioID (BirA*) and TurboID is a key technology for identifying proteins interacting with a target protein in a cell or organism. However, there have been some improvements in the enzymes for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins like AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-IκBα biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analysing protein–protein interactions.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 3, 4, and 6.
Article and author information
Author details
Funding
Japan Agency for Medical Research and Development (JP19am0101077)
- Tatsuya Sawasaki
Japan Society for the Promotion of Science (JP16H06579)
- Tatsuya Sawasaki
Japan Society for the Promotion of Science (JP16H04729)
- Tatsuya Sawasaki
Japan Society for the Promotion of Science (JP19H03218)
- Tatsuya Sawasaki
Japan Society for the Promotion of Science (18KK0229)
- Hidetaka Kosako
Japan Society for the Promotion of Science (19H04966)
- Hidetaka Kosako
Takeda Science Foundation
- Tatsuya Sawasaki
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Volker Dötsch, Goethe University, Germany
Version history
- Received: January 8, 2020
- Accepted: May 7, 2020
- Accepted Manuscript published: May 11, 2020 (version 1)
- Version of Record published: June 18, 2020 (version 2)
Copyright
© 2020, Kido et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 22,740
- views
-
- 3,152
- downloads
-
- 73
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions
eLife 9:e54983.
https://doi.org/10.7554/eLife.54983
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single-domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here, we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast et al., 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.
-
- Biochemistry and Chemical Biology
Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.
