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Identification of TbUnc119-interacting proteins by proximity-based biotinylation

ABSTRACT: Both intraflagellar transport (IFT) and lipidated intraflagellar transport (LIFT) pathways are essential for cilia/flagella biogenesis, motility and sensory functions. In the LIFT pathway, lipidated cargoes are transported into the cilia through the coordinated actions of cargo carrier proteins such as Unc119 or PDE6δ, as well as small GTPases Arl13b and Arl3 in the cilium. Our previous studies revealed a single Arl13b ortholog in the evolutionarily divergent Trypanosoma brucei. TbArl13 catalyses two TbArl3 homologs, TbArl3A and TbArl3C, suggesting the presence of a conserved LIFT pathway in these protozoan parasites. Only a single homolog to the cargo carrier protein Unc119 was identified in T. brucei genome, but its function in lipidated protein transport has not been characterized. In this study, we exploited the proximity-based biotinylation approach to identify binding partners of TbUnc119 using an improved version of biotin ligase BioID2. The BioID2 tag was fused to either the N-terminus (3HA-BioID2-TbUnc119) or the C-terminus (TbUnc119-BioID2-HA) of TbUnc119 and expressed using a cumate-inducible expression system in the procyclic cells of T. brucei. We performed LC-MS/MS on the affinity-purified biotinylated samples followed by evaluation of emPAI score.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Trypanosoma Brucei

TISSUE(S): Cell Suspension Culture

SUBMITTER: Maneesha Pandey

LAB HEAD: Cynthia Y. He

PROVIDER: PXD019488 | Pride | 2020-06-25

REPOSITORIES: Pride

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Source:

			Action	DRS
	3HA-BioID2-TbUnc119.dat	Other
	3HA-BioID2-TbUnc119.group	Other
	3HA-BioID2-TbUnc119.mgf	Mgf
	3HA-BioID2-TbUnc119.wiff	Wiff
	3HA-BioID2-TbUnc119.wiff.scan	Wiff

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Project description:Study Smoking and COPD are associated with decreased mucociliary clearance and healthy smokers have shorter cilia in the large airway than nonsmokers. Intraflagellar transport (IFT) is the process by which cilia are produced and maintained. We assessed expression of IFT-related genes in smokers and nonsmokers and evaluated cilia length in the large and small airway of nonsmokers, healthy smokers, and smokers with COPD. Methods Airway epithelium was obtained via bronchoscopic brushing. Affymetrix microarrays were used to evaluate IFT gene expression in 2 independent data sets from large and small airway. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject. Results All 40 IFT genes were expressed in the human large and small airway epithelium. In the large airway, 10 IFT genes were down-regulated and 1 up-regulated in smokers. In the small airway, 11 genes were down-regulated and 3 up-regulated in smokers. A set of 8 IFT genes was down-regulated in both data sets. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. Answer to the Question These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies. Gene expression was assessed for 40 intraflagellar transport related genes in the LAE of nonsmokers (n=21) and healthy smokers (n=31) and the SAE of an independent group of nonsmokers (n=28) and healthy smokers (n=69). Cilia length was assessed in a total of 228 airway epithelium samples, including 120 LAE samples and 108 SAE samples; a subset of the 228 samples is represented among the 149 samples in this Series.

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Identification of TbUnc119-interacting proteins by proximity-based biotinylation

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