protein fragment complementation
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2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Orhi Barroso-Gomila ◽  
Fredrik Trulsson ◽  
Veronica Muratore ◽  
Iñigo Canosa ◽  
Laura Merino-Cacho ◽  
...  

AbstractThe fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We develop an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allow us to identify interactors of SUMOylated SALL1, a less characterized SUMO substrate. Furthermore, using TP53 as a substrate, we identify SUMO1, SUMO2 and Ubiquitin preferential interactors. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.


2021 ◽  
Author(s):  
Daniel Evans-Yamamoto ◽  
François D Rouleau ◽  
Piyush Nanda ◽  
Koji Makanae ◽  
Yin Liu ◽  
...  

Barcode fusion genetics (BFG) utilizes deep sequencing to improve the throughput of protein-protein interaction (PPI) screening in pools. BFG has been implemented in Yeast two-hybrid (Y2H) screens (BFG-Y2H). While Y2H requires test protein pairs to localize in the nucleus for reporter reconstruction, Dihydrofolate Reductase Protein-Fragment Complementation Assay (DHFR-PCA) allows proteins to localize in broader subcellular contexts and proves to be largely orthogonal to Y2H. Here, we implemented BFG to DHFR-PCA (BFG-PCA). This plasmid-based system can leverage ORF collections across model organisms to perform comparative analysis, unlike the original DHFR-PCA that requires yeast genomic integration. The scalability and quality of BFG-PCA were demonstrated by screening human and yeast interactions of >11,000 protein pairs. BFG-PCA showed high-sensitivity and high-specificity for capturing known interactions for both species. BFG-Y2H and BFG-PCA capture distinct sets of PPIs, which can partially be explained based on the domain orientation of the reporter tags. BFG-PCA is a high-throughput protein interaction technology to interrogate binary PPIs that exploits clone collections from any species of interest, expanding the scope of PPI assays.


Author(s):  
Ewa Blaszczak ◽  
Natalia Lazarewicz ◽  
Aswani Sudevan ◽  
Robert Wysocki ◽  
Gwenaël Rabut

Protein–protein interactions (PPIs) orchestrate nearly all biological processes. They are also considered attractive drug targets for treating many human diseases, including cancers and neurodegenerative disorders. Protein-fragment complementation assays (PCAs) provide a direct and straightforward way to study PPIs in living cells or multicellular organisms. Importantly, PCAs can be used to detect the interaction of proteins expressed at endogenous levels in their native cellular environment. In this review, we present the principle of PCAs and discuss some of their advantages and limitations. We describe their application in large-scale experiments to investigate PPI networks and to screen or profile PPI targeting compounds.


2021 ◽  
Author(s):  
Luca Piccinini ◽  
Stefano Cazzaniga ◽  
Sergio Iacopino ◽  
Matteo Ballottari ◽  
Beatrice Giuntoli ◽  
...  

ABSTRACTSynthetic biology approaches to engineer light‐responsive system are widely used, but their applications in plants are still limited, due to the interference with endogenous photoreceptors. Cyanobacteria, such as Synechocystis spp., possess a soluble carotenoid associated protein named Orange Carotenoid binding Protein (OCP) that, when activated by blue‐green light, undergoes reversible conformational changes that enable photoprotection of the phycobilisomes. Exploiting this system, we developed a new chloroplast‐localized synthetic photoswitch based on a photoreceptor‐associated protein‐fragment complementation assay (PCA). Since Arabidopsis thaliana does not possess the prosthetic group needed for the assembly of the OCP2 protein, we implemented the carotenoid biosynthetic pathway with a bacterial β‐carotene ketolase enzyme (crtW), to generate keto‐carotenoids producing plants. The novel photoswitch was tested and characterized in Arabidopsis protoplasts with experiments aimed to uncover its regulation by light intensity, wavelength, and its conversion dynamics. We believe that this pioneer study establishes the basis for future implementation of plastid optogenetics to regulate organelle responses, such as gene transcription or enzymatic activity, upon exposure to specific light spectra.One-sentence summaryInspired by the light-driven conformational transitions of orange carotenoid proteins of cyanobacteria, we generated a molecular device able to switch its dimeric state in response to blue light.


2020 ◽  
Author(s):  
Orhi Barroso-Gomila ◽  
Fredrik Trulsson ◽  
Veronica Muratore ◽  
Iñigo Canosa ◽  
Ana Rosa Cortazar ◽  
...  

ABSTRACTThe fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We developed an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allowed us to identify novel interactors of SUMOylated SALL1, a less characterized SUMO substrate. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.


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