Protein-protein interactions (PPIs) play a crucial role in most cellular and biological processes, such as DNA replication, signal transduction, and immune defense. Various in vivo and in vitro methods have been developed to identify PPIs, including commonly used techniques such as yeast two-hybrid screening, co-immunoprecipitation, pull-down assays, and protein microarrays.
Traditional affinity purification-mass spectrometry (AP-MS) techniques are limited in effectively capturing transient or weak protein interactions. They are also unsuitable for low-abundance proteins or hydrophobic membrane proteins and cannot resolve the cellular compartmentalization information of protein interactions, presenting several constraints.
The TurboID proximity labeling technology overcomes many of these limitations. It offers significant advantages over traditional AP-MS in studying the interactomes of low-abundance proteins and rare, transient cell types, as well as subcellular proteomes. TurboID proximity labeling technology is a high-throughput protein interaction detection method that has been widely used in academic research and biotechnology in recent years.
Here are some case studies.
Case 1 : TurboID Screening Identifies Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis[1]
Glaesserella parasuis is a common pathogen in the porcine upper respiratory tract and may induce intracellular killing through phagocytosis by alveolar macrophages. However, highly virulent strains of Glaesserella parasuis can resist phagocytosis by alveolar macrophages. The outer membrane protein P2 (OmpP2) of G. parasuis, belonging to the porin family, is a prominent protein on the outer membrane, characterized by eight loops that are exposed on the surface.
To reveal the host proteins interacting with OmpP2 through TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage (iPAM) cells, His-OmpP2 and TurboID recombinant proteins were expressed and purified at concentrations of 0.1 mg/mL and 2.58 mg/mL, respectively. Mass spectrometry analysis identified 948 and 758 iPAM cell proteins that interact with His-TurboID-OmpP2 and His-TurboID, respectively. After removing background proteins by comparison with the TurboID-treated group, 240 unique interacting proteins were identified in the TurboID-OmpP2-treated group. Membrane proteins are typically involved in the recognition and phagocytosis of pathogens by iPAM cells, thus the authors focused particularly on membrane proteins. Among the 240 proteins, only CAV1, ARF6, PPP2R1A, and AP2M1 were membrane proteins, and co-IP results indicated that AP2M1 does not interact with OmpP2. Therefore, the authors further investigated the roles of CAV1, ARF6, and PPP2R1A in iPAM cell adhesion and phagocytosis of Glaesserella parasuis.
Bioinformatics analysis of host cell proteins putatively identified as interacting with TurboID-OmpP2 proteins.(Jiang,Microbiology spectrum,2022)
This study identified 240 unique interacting proteins in the His-TurboID-OmpP2 group. Among them, only four membrane proteins—CAV1, ARF6, PPP2R1A, and AP2M1—were identified. Co-IP assays further confirmed that CAV1, ARF6, and PPP2R1A directly interact with Glaesserella parasuis.
Case 2 : A novel mechanism of plant virus replication was revealed by proximity labeling based on TurboID[2]
Beet black scorch virus (BBSV) is a positive-sense RNA virus belonging to the Betanecrovirus genus of the Tombusviridae family. It causes beet black scorch disease in the field. The infection of positive-sense RNA viruses in host cells can be divided into several stages, including replication, virus particle assembly, intracellular and intercellular movement, with replication being one of the key steps in establishing infection. This process is primarily facilitated by virus-induced remodeling of intracellular membranes, which forms viral replication complexes (VRCs).
The replication-associated protein p23 of BBSV plays a crucial role in the formation of BBSV VRCs by inducing the remodeling of the endoplasmic reticulum (ER) membrane. To identify functional connections between proteins in proximity to p23, a TurboID-based proximity labeling technique was employed, revealing several potential interacting proteins and constructing their interaction network. These proteins are involved in the endoplasmic reticulum system, transport systems (including the endosomal sorting complexes required for transport, such as ESCRT), and protein folding systems. This suggests that BBSV replication involves multiple intracellular metabolic pathways and biological processes, which are interconnected to form a complex regulatory network.
A proximity-based interaction network for some of the proteins enriched in p23-Citrine-TurboID purification.(Zhang,The Plant cell ,2023)
This study systematically analyzed the composition of BBSV VRCs using TurboID-based proximity labeling technology, identifying a new component of the BBSV replication complex—reticulon-like protein B2 (RTNLB2)—and revealing its role in the formation of the viral replication factories.
References
- Jiang, Changsheng et al. "TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells." Microbiology spectrum vol. 10,5 (2022): e0230722.
- Zhang, Qianshen et al. "RETICULON-LIKE PROTEIN B2 is a proviral factor co-opted for the biogenesis of viral replication organelles in plants." The Plant cell vol. 35,8 (2023): 3127-3151.
