Overview of TurboID Technology
TurboID technology represents a significant leap forward in proximity labeling (PL) methodologies, offering unparalleled speed, sensitivity, and spatial resolution in identifying protein-protein interactions (PPIs). Unlike conventional PL techniques such as BioID and APEX, TurboID harnesses the catalytic power of a mutated biotin ligase derived from BirA, termed "TurboID," to rapidly biotinylate proximal proteins within living cells. This expedited labeling process, facilitated by the enhanced kinetics of TurboID, significantly reduces the labeling time from hours to mere minutes, enabling real-time interrogation of dynamic protein interaction networks.
The key innovation driving TurboID technology lies in its engineered biotin ligase variant, which boasts an elevated catalytic efficiency and substrate turnover rate compared to its predecessors. By fusing TurboID to target proteins of interest, researchers can efficiently biotinylate neighboring proteins within cellular microenvironments, capturing transient or weak interactions that may evade detection using traditional PL methods. Furthermore, TurboID's compact size and minimal steric hindrance enhance its versatility, allowing for labeling of insoluble proteins, membrane-associated complexes, and rare or short-lived protein interactions with unprecedented precision.
TurboID technology operates on the principle of proximity-dependent biotinylation, wherein the biotin ligase enzyme conjugates biotin molecules to lysine residues of nearby proteins within a defined radius. Upon biotinylation, these labeled proteins can be selectively enriched and identified using affinity purification coupled with mass spectrometry (AP-MS), facilitating comprehensive characterization of protein interaction landscapes in diverse cellular contexts. Moreover, TurboID's rapid labeling kinetics and improved signal-to-noise ratio minimize background noise and false positives, enhancing the reliability and specificity of PPI detection.
In recent years, TurboID technology has undergone continuous refinement and optimization, giving rise to novel variants such as miniTurboID and split-TurboID. These innovative derivatives offer tailored solutions to specific experimental requirements, further expanding the utility of TurboID across a broad spectrum of biological studies. As TurboID continues to evolve, fueled by ongoing advancements in protein engineering and chemical biology, its impact on disease proteomics research is poised to grow exponentially, unlocking new avenues for therapeutic discovery and precision medicine.
Development of TurboID Technology
The development of TurboID technology marks a significant milestone in the evolution of proximity labeling methodologies, driven by the need for enhanced speed, sensitivity, and spatial resolution in characterizing protein-protein interactions (PPIs). Building upon the foundation laid by earlier PL techniques such as BioID and APEX, TurboID represents a paradigm shift in the field, offering unprecedented efficiency and versatility in labeling proximal proteins within living cells.
The conceptualization of TurboID emerged from a synthesis of insights gleaned from previous PL methodologies and advancements in protein engineering. Researchers sought to address the limitations inherent in conventional PL techniques, including prolonged labeling times, low signal-to-noise ratios, and restricted applicability to specific cellular compartments or protein complexes. Leveraging the catalytic power of biotin ligases, particularly BirA, as the enzymatic catalyst for proximity labeling, TurboID was designed to overcome these challenges by enhancing catalytic efficiency and substrate turnover rates.
The engineering of TurboID involved the rational design and optimization of the biotin ligase enzyme to augment its labeling kinetics while maintaining specificity and compatibility with cellular environments. Through directed evolution and structure-guided mutagenesis, researchers identified key residues and structural motifs within BirA responsible for its catalytic activity and substrate recognition. By introducing targeted mutations and modifications, such as amino acid substitutions and domain truncations, they were able to enhance the enzymatic performance of BirA, culminating in the creation of TurboID.
The iterative refinement of TurboID technology involved iterative cycles of design, experimentation, and validation, aimed at fine-tuning its performance characteristics and expanding its utility across diverse biological applications. Initial proof-of-concept studies demonstrated the feasibility of TurboID for rapid and selective biotinylation of proximal proteins in various cellular contexts, paving the way for its widespread adoption by the scientific community.
Subsequent iterations of TurboID, including miniTurboID and split-TurboID, further diversified the toolkit available to researchers, offering tailored solutions to specific experimental needs. MiniTurboID, for instance, capitalized on the modular nature of TurboID to create a compact fusion enzyme with enhanced spatial resolution and reduced steric hindrance, enabling labeling of smaller protein complexes and subcellular structures. Split-TurboID, on the other hand, introduced a modular split enzyme system that could be reconstituted in situ to enable compartmentalized labeling of distinct cellular compartments or organelles.
The evolution of TurboID technology continues unabated, fueled by ongoing advancements in protein engineering, chemical biology, and bioinformatics. As researchers continue to refine and optimize TurboID and its derivatives, the scope of its applications in disease proteomics, drug discovery, and systems biology is expected to expand exponentially, heralding a new era of precision medicine and therapeutic innovation.
The reconstituted activity of Split-TurboID in neurons and astrocytes in vitro (Takano et al., 2020)
Applications of TurboID Technology in Disease Research
TurboID technology has emerged as a powerful tool for unraveling the complex molecular mechanisms underlying various diseases, offering unprecedented insights into disease pathogenesis, biomarker discovery, and therapeutic intervention strategies. Leveraging its ability to systematically interrogate protein-protein interactions (PPIs) within cellular environments, TurboID has been instrumental in elucidating the dysregulated signaling networks and aberrant protein complexes associated with a wide spectrum of diseases, ranging from cancer to infectious disorders.
Cancer Research
In cancer research, TurboID has revolutionized our understanding of oncogenic signaling pathways, tumor suppressor mechanisms, and therapeutic resistance mechanisms. By mapping the interactome of key cancer-related proteins, such as mutant p53 variants and dysregulated signaling kinases, TurboID has provided critical insights into the molecular basis of tumorigenesis, metastasis, and drug resistance. For example, studies have utilized TurboID to delineate the interactome of mutant p53(R175H) and wild-type p53, shedding light on the distinct protein networks and cellular processes modulated by these variants. The identification of novel protein interactors and signaling nodes associated with mutant p53 has opened new avenues for targeted cancer therapy and biomarker development.
Moreover, TurboID technology has been instrumental in elucidating the mechanisms of action of anticancer drugs and targeted therapeutics. By profiling the interactome of drug targets or resistance factors, researchers can uncover novel biomarkers of drug response and resistance, as well as identify potential combination therapies to overcome treatment resistance. For instance, studies have utilized TurboID to elucidate the molecular mechanisms underlying the antitumor effects of small molecule inhibitors, such as dasatinib and sorafenib, by mapping their interactomes and downstream signaling cascades. These findings not only enhance our understanding of drug action but also inform the development of personalized treatment strategies for cancer patients.
Infectious Disease Research
In infectious disease research, TurboID has emerged as a valuable tool for studying host-pathogen interactions and viral pathogenesis. By profiling the interactome of viral proteins within host cells, TurboID enables the systematic identification of host factors involved in viral replication, immune evasion, and pathogenicity. For example, studies have utilized TurboID to elucidate the interactome of viral proteins from diverse pathogens, including influenza virus, dengue virus, and SARS-CoV-2, providing insights into the molecular mechanisms of viral infection and host response.
Furthermore, TurboID technology has facilitated the discovery of novel antiviral targets and therapeutic strategies by identifying host factors essential for viral replication or pathogenesis. By screening the interactome of viral proteins against drug libraries or clinically approved compounds, researchers can identify potential antiviral agents and repurposable drugs for the treatment of viral infections. For instance, studies have utilized TurboID to screen for host factors that interact with viral proteins from SARS-CoV-2, identifying candidate drugs with potential efficacy against COVID-19. These findings not only accelerate the development of antiviral therapies but also provide insights into the host factors and pathways targeted by viral pathogens.
Neurological Disorders
In neurological disorders, TurboID has emerged as a powerful tool for dissecting the molecular mechanisms underlying neurodegenerative diseases, neuropsychiatric disorders, and developmental abnormalities. By profiling the interactome of disease-associated proteins within neuronal cells or brain tissues, TurboID enables the systematic identification of protein complexes, signaling pathways, and regulatory networks implicated in disease pathogenesis. For example, studies have utilized TurboID to map the interactome of proteins involved in Alzheimer's disease, Parkinson's disease, and autism spectrum disorders, revealing novel insights into disease etiology and progression.
Moreover, TurboID technology has facilitated the discovery of potential therapeutic targets and biomarkers for neurological disorders by identifying disease-specific protein interactors and signaling pathways. By screening the interactome of disease-associated proteins against drug libraries or gene expression databases, researchers can identify candidate drugs or molecular targets for therapeutic intervention. For instance, studies have utilized TurboID to identify novel interactors of disease-associated proteins, such as tau and α-synuclein, providing valuable insights into disease mechanisms and potential avenues for drug discovery. These findings not only advance our understanding of neurological disorders but also hold promise for the development of novel treatments and diagnostic markers.
Reference
- Takano, Tetsuya, et al. "Chemico-genetic discovery of astrocytic control of inhibition in vivo." Nature 588.7837 (2020): 296-302.
