BioID technology revolves around the utilization of a biotin ligase enzyme, BirA, derived from Escherichia coli. The core principle involves biotinylating nearby proteins within live cells, thereby facilitating the identification of interacting protein networks. The introduction of mutated forms of BirA, such as BirA*, has enhanced the efficiency of biotinylation, leading to improved catalytic activity and specificity.
Creative Proteomics has been at the forefront of utilizing BioID technology in diverse protein interaction studies. From identifying enzyme interaction proteins to elucidating membrane protein networks, BioID has proven instrumental. Notably, the technology has enabled the discovery of novel protein interactions, shedding light on cellular processes ranging from metabolism to signaling.
A Schematic diagram of BioID labelling strategy. B HeLa cells, either wild type or stably expressing mBidWT–BirA*, mBidS66A–BirA*, mBidG94E–BirA* or venus BirA*, were grown with 50 µM biotin for 16 h in the presence (+) or absence (−) of nocodazole. C Single-cell-fate profiles of HeLa cells in the presence or absence of nocodazole, imaged over 48 h. D Volcano plot of mean- fold change of biotinylated protein abundance for mBidWT–BirA* vs. venus-BirA* control for unsynchronised and nocodazole-treated samples (Pedley et al., 2020).
Identification of Membrane Protein Interaction Proteins
Membrane proteins are integral to a plethora of cellular processes, including substance transport, energy conversion, and signal transduction. However, their hydrophobic nature poses significant challenges for traditional biochemical methods aimed at studying protein interactions. Conventional approaches often struggle to maintain the integrity and solubility of membrane proteins outside their native lipid environment, hindering the identification of their interaction partners.
In this context, BioID technology emerges as a groundbreaking solution, revolutionizing the study of membrane protein interactions. By leveraging the proximity-dependent biotinylation mechanism within live cells, BioID enables the specific labeling of proteins in close proximity to a target membrane protein of interest. This proximity labeling occurs through the fusion of the target membrane protein with a promiscuous biotin ligase, such as BirA*.
The fusion protein, comprising the target membrane protein and the biotin ligase, is expressed in the cellular context, allowing for the selective biotinylation of proteins in the immediate vicinity of the membrane protein. Notably, this labeling occurs in situ, preserving the native membrane environment and ensuring the detection of physiologically relevant interactions.
Following biotinylation, the labeled proteins can be isolated using streptavidin affinity purification methods. Subsequent mass spectrometry analysis of the isolated protein complex unveils the identity of the interaction partners associated with the target membrane protein. This approach circumvents the challenges posed by the hydrophobicity of membrane proteins, as the interactions are captured within the cellular membrane milieu.
Moreover, BioID technology offers unparalleled sensitivity, enabling the detection of weak or transient interactions that may evade traditional biochemical techniques. By providing a comprehensive snapshot of the membrane protein interactome, BioID facilitates the elucidation of complex signaling cascades and regulatory networks governing membrane protein function.
For instance, Bareja et al. used BioID to explore the interaction network of the insulin-like growth factor 1 receptor (IGF1R), revealing novel interaction proteins involved in protein transport. They focused on the biological effects of the interaction between sorting nexin 6 and IGF1R, shedding light on sorting nexin 6's role in the IGF1R signaling pathway. Similarly, Haugsten et al. engineered a FGFR4-BirA* fusion protein and applied BioID to systematically study the dynamic protein interactome of FGFR4. This effort identified 291 interacting proteins, including transiently interacting proteins and insoluble membrane proteins, providing insights into the intracellular trafficking and signaling processes of FGFR4.
Moreover, BioID technology has successfully identified interaction protein groups of various other membrane proteins such as RAB GTPases (RAB10 and RAB13), E-cadherin, tight junction protein ZO-1, dopamine and glutamate transporters, and potassium channel proteins. These studies revealed novel interaction partners associated with their respective functions, uncovering new functionalities of these membrane proteins.
Identifying Enzyme Interaction Proteins
Enzymes, as essential catalysts in biological processes, play a crucial role in virtually all life functions. They function by interacting with other proteins, most notably substrates, in order to facilitate various biological reactions. However, these interactions can often be weak or fleeting, which makes it a challenging task to precisely identify the potential substrates that an enzyme might interact with using conventional techniques such as immunoprecipitation-mass spectrometry.
This is where BioID technology has been proven to be incredibly beneficial. In contrast to other methodologies, BioID has the capability to overcome this limitation as it works by biotinylating target proteins within living cells. This facilitates the identification of weak and transient interaction proteins. In this realm, it acts as a significant asset for researchers working on identifying enzyme substrates.
For example, Coyaud and colleagues used BioID to discover substrates of two E3 ubiquitin ligases, SCFβ-TrCP1 and SCFβ-TrCP2, demonstrating its superiority over conventional methods. They identified 77 substrates with BioID compared to only 41 using traditional techniques. Similarly, Jahan et al. applied BioID to uncover interaction partners of the ubiquitin-specific peptidase Usp12, revealing various nearby and transient interactions. Furthermore, BioID aided in the identification of Caspase-1 substrates, revealing its interaction with p62 and its role in inflammation regulation.
In cancer research, identifying functionally relevant interacting proteins of enzymes like carbonic anhydrase IX (CA IX) in cancer cells is challenging. Swayampakula et al. used BioID to explore the interaction network of CA IX in breast cancer cells, identifying several interacting proteins involved in tumor invasion and migration, shedding light on CA IX's role in these processes.
Unveiling Transcription Factor Interaction Proteins
Transcription factors are essential proteins that regulate gene expression by binding to specific DNA sequences, either individually or in complexes with other proteins. Traditional methods of identifying these complexes face challenges due to insolubility issues. However, BioID technology has emerged as a solution, successfully used to study various protein interaction networks associated with transcription factors.
For example, researchers engineered a fusion protein system called MYC-BirA* in HEK293 cells, allowing them to detect over 100 high-confidence interaction proteins of MYC. This approach led to the discovery of previously unknown interactions, such as MYC-CHD8. In another study, BioID revealed a novel interaction between MYC and protein phosphatase 1 (PP1) in HeLa cells, shedding light on MYC's regulatory mechanisms.
Similarly, BioID technology was employed to compare the interaction protein groups of different variants of MYC in HEK293 cells, leading to the identification of potential binding partners for specific regions of MYC. Despite variations in cell systems, BioID consistently revealed overlapping proteins, indicating its reliability in identifying transcription factor interaction protein networks.
In lung cancer research, BioID uncovered interaction partners of the transcription factor SOX2, including histone acetyltransferase EP300. This interaction, undetectable by traditional methods, suggested new therapeutic strategies targeting SOX2.
Furthermore, BioID studies of ZEB1, a transcription factor implicated in cancer progression, identified co-regulatory molecules that contribute to its function. By employing BioID with fusion proteins containing BirA* at different locations in ZEB1, researchers revealed interaction proteins that traditional methods could not detect, providing insights into ZEB1's role in cancer.
Overall, BioID technology offers a valuable tool for studying protein interaction networks associated with transcription factors, providing insights into gene regulation and potential therapeutic targets in various diseases.
Discovering Structural Protein Interaction Proteins
Within cells, a variety of structural proteins play crucial roles in maintaining cell morphology and processes like cell movement. These proteins, often insoluble, pose challenges for traditional methods in detecting their interaction networks. BioID technology initially applied to identify the interaction network of nuclear lamina protein Lamin A, has since become increasingly prevalent.
For instance, Chojnowski et al. revealed insights into the pathogenesis of premature aging syndromes and the regulatory functions of nuclear lamina in proliferation and chromatin remodeling by studying the interaction network of premature aging proteins.
Similarly, studies in multiple myeloma cells using BioID uncovered the interaction network of tropomyosin-4 (Tm4), shedding light on how actin and intermediate filament-binding proteins collectively regulate processes like cell migration and morphogenesis.
Furthermore, Firat-Karalar and Arslanhan systematically elucidated the interaction protein network of 58 centrosome-associated proteins using BioID. Among over 7,000 high-confidence protein networks generated, more than 1,700 were specific proteins, including 213 already reported interaction proteins. Through immunoprecipitation validation, they confirmed interactions among 30 previously unreported proteins, revealing novel structural and functional aspects of the centrosome.
These studies demonstrate that BioID serves as an effective method for identifying interaction networks of structural proteins like those associated with the centrosome.
Studying Other Protein Interaction Proteins
Within cells, numerous critical signaling molecules play essential roles in regulating various activities such as proliferation, differentiation, aging, and apoptosis. These molecules are of utmost importance in the formation and development of tumors. Investigating the interaction networks of signaling molecules can further elucidate the mechanisms underlying tumor pathogenesis and provide potential therapeutic targets. However, these proteins are often structurally complex, undergo post-translational modifications, and their mechanisms of action remain unclear. BioID technology serves as an effective method for identifying dynamic or transient interaction protein networks. Currently, researchers have successfully utilized BioID technology to identify protein interaction networks such as those related to the Hippo signaling pathway, the factor inhibiting hypoxia-inducible factor 1 (FIH-1), K-Ras, and p38α. This has led to the discovery of new interacting proteins and the revelation of novel regulatory mechanisms.
Additionally, BioID technology has been employed to identify the molecular microenvironment of coronavirus replication/transcription complexes, HIV viral protein interaction networks, host-bacteria recognition, and interactions of proteins related to mitosis. BioID technology has also been applied in plant systems, with successful identification of protein interaction groups in rice cells and Arabidopsis leaf tissues by modifying BirA*. In recent years, the application scope of BioID technology has expanded continuously, spanning from mammalian cells to plant cells, from eukaryotes to prokaryotes, and even viruses. This expansion is of significant importance for studying various life processes and regulatory mechanisms.
Reference
- Pedley, Robert, et al. "BioID-based proteomic analysis of the Bid interactome identifies novel proteins involved in cell-cycle-dependent apoptotic priming." Cell death & disease 11.10 (2020): 872.
