The cytosolic carboxypeptidase 6 (CCP6) catalyzes the deglutamylation of polyglutamate side chains, a post-translational modification that affects proteins such as tubulins or nucleosome assembly proteins. CCP6 is involved in several cell processes, such as spermatogenesis, antiviral activity, embryonic development, and pathologies like renal adenocarcinoma. In the present work, the cellular role of CCP6 has been assessed by BioID, a proximity labeling approach for mapping physiologically relevant protein-protein interactions (PPIs) and bait proximal proteins by mass spectrometry. We used HEK 293 cells stably expressing CCP6-BirA* to identify 37 putative interactors of this enzyme. This list of CCP6 proximal proteins displayed enrichment of proteins associated with the centrosome and centriolar satellites, indicating that CCP6 could be present in the pericentriolar material. In addition, we identified cilium assembly-related proteins as putative interactors of CCP6. In addition, the CCP6 proximal partner list included five proteins associated with the Joubert syndrome, a ciliopathy linked to defects in polyglutamylation. Using the proximity ligation assay (PLA), we show that PCM1, PIBF1, and NudC are true CCP6 physical interactors. Therefore, the BioID methodology confirms the location and possible functional role of CCP6 in centrosomes and centrioles, as well as in the formation and maintenance of primary cilia.

Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase belonging to the insulin receptor superfamily. In addition to its well-studied role in cancer, numerous studies have revealed that Alk signaling is associated with a variety of complex traits such as: regulation of growth and metabolism, hibernation, regulation of neurotransmitters, synaptic coupling, axon targeting, decision making, memory formation and learning, alcohol use disorder, as well as steroid hormone metabolism. In this study, we used BioID-based in vivo proximity labeling to identify molecules that interact with Alk in the Drosophila central nervous system (CNS). To do this, we used CRISPR/Cas9 induced homology-directed repair (HDR) to modify the endogenous Alk locus to produce first and next generation Alk::BioID chimeras. This approach allowed identification of Alk proximitomes under physiological conditions and without overexpression. Our results show that the next generation of BioID proteins (TurboID and miniTurbo) outperform the first generation BirA* fusion in terms of labeling speed and efficiency. LC-MS3-based BioID screening of AlkTurboID and AlkminiTurbo larval brains revealed an extensive neuronal Alk proximitome identifying numerous potential components of Alk signaling complexes. Validation of Alk proximitome candidates further revealed co-expression of Stardust (Sdt), Discs large 1 (Dlg1), Syntaxin (Syx) and Rugose (Rg) with Alk in the CNS and identified the protein-tyrosine-phosphatase Corkscrew (Csw) as a modulator of Alk signaling.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is mutated in approximately 10% of pediatric neuroblastoma (NB). To shed light on ALK-driven signaling processes, we employed BioID-based in vivo proximity labeling to identify molecules that interact intracellularly with ALK. NB-derived SK-N-AS and SK-N-BE(2) cells expressing inducible ALK-BirA* fusion proteins were generated and stimulated with ALKAL ligands in the presence and absence of the ALK tyrosine kinase inhibitor (TKI) lorlatinib. LC/MS-MS analysis identified multiple proteins, including PEAK1 and SHP2, which were validated as ALK interactors in NB cells. Further analysis of the ALK-SHP2 interaction confirmed that the ALK-SHP2 interaction as well as SHP2-Y542 phosphorylation was dependent on ALK activation. Use of the SHP2 inhibitors, SHP099 and RMC-4550, resulted in inhibition of cell growth in ALK-driven NB cells. In addition, we noted a strong synergistic effect of combined ALK and SHP2 inhibition that was specific to ALK-driven NB cells, suggesting a potential therapeutic option for ALK-driven NB.

Cellular compartmentalization of proteins and protein complex formation allow cells to tightly control biological processes. Therefore, understanding the subcellular localization and interactions of a specific protein is crucial to uncover its biological function. The advent of proximity labeling (PL) has reshaped cellular proteomics in infection biology. PL utilizes a genetically modified enzyme that generates a \"labeling cloud\" by covalently labeling proteins in close proximity to the enzyme. Fusion of a PL enzyme to a specific antibody or a \"bait\" protein of interest in combination with affinity enrichment mass spectrometry (AE-MS) enables the isolation and identification of the cellular proximity proteome, or proxisome. This powerful methodology has been paramount for the mapping of membrane or membraneless organelles as well as for the understanding of hard-to-purify protein complexes, such as those of transmembrane proteins. Unsurprisingly, more and more infection biology research groups have recognized the potential of PL for the identification of host-pathogen interactions. In this chapter, we introduce the enzymes commonly used for PL labeling as well as recent promising advancements and summarize the major achievements in organelle mapping and nucleic acid PL. Moreover, we comprehensively describe the research on host-pathogen interactions using PL, giving special attention to studies in the field of virology.

Western blot (protein immunoblot) is a widely used analytical technique in molecular biology. Utilizing the specific recognizing primary antibody, proteins immobilized on various matrix are investigated by subsequent visualization steps, for example, by the horse radish peroxidase conjugated secondary antibody incubation. Methods to improve the sensitivity in protein identification or quantification are appreciated by biochemists. Herein, we report a new strategy to amplify Western blot signals by constructing a probe with proximal labeling and IgG targeting abilities. The R118G mutation attenuated the biotin-AMP binding affinity of the bacterial biotin ligase BirA*, offering a proximity-dependent labeling ability, which could be used as a signal amplifier. We built a BirA*-protein A fusion protein (BioEnhancer) that specifically binds to IgG and adds biotin tags to its proximal amine groups, enhancing the immunosignal of target proteins. In our experiments, the BioEnhancer system amplified the immunosignal by tenfold compared to the standard western blot. Additionally, our strategy could couple with other signal enhancement methods to further increase the western blot sensitivity.

RNA biology is orchestrated by the dynamic interactions of RNAs and RNA-binding proteins (RBPs). In the present study, we describe a new method of proximity-dependent protein labeling to detect RNA-protein interactions [RNA-bound protein proximity labeling (RBPL)]. We selected the well-studied RNA-binding protein PUF to examine the current proximity labeling enzymes birA* and APEX2. A new version of birA*, BASU, was used to validate that the PUF protein binds its RNA motif. We further optimized the RBPL labeling system using an inducible expression system. The RBPL (λN-BASU) labeling experiments exhibited high signal-to-noise ratios. We subsequently determined that RBPL (λN-BASU) is more suitable than RBPL (λN-APEX2) for the detection of RNA-protein interactions in live cells. Interestingly, our results also reveal that proximity labeling is probably capable of biotinylating proximate nascent peptide.

Toxoplasma gondii is an opportunistic pathogen infecting humans and a variety of vertebrate animals. Secretory dense-granule proteins (GRAs) play diverse roles in the mediation of host-parasite interactions and facilitate parasitism, but many of them still remain to be identified. Here, we used two proximity-based protein labeling techniques to identify novel GRA proteins. Taking GRA1 as bait, transgenic strains expressing GRA1-BirA* or GRA1-APEX were constructed to biotinylate GRAs. Using these methods, a total of 46 proteins were identified, 20 of which were known GRA proteins. Among these 46, 17 were identified by both strategies, and 14 out of the 17 were known GRAs. The other three were all confirmed to localize to dense granules. Nonetheless a significant portion of the proteins were only identified by either APEX or BirA*, indicating that there are differences between these methods. Of the 26 novel GRAs, 5 were validated as bona fide GRAs by localization studies. The majority of these novel GRAs are only present in coccidian parasites and are likely dispensable for parasite growth in vitro; they may play roles during animal infections. The identification of novel GRAs laid the foundation for further studies investigating the mechanisms underlying parasite-host interactions.