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A screening system for identifying interacting proteins using biomolecular fluorescence complementation and transposon gene trap.

ABSTRACT: We have established a new screening system for identifying interacting proteins by combining biomolecular fluorescence complementation (BiFC) and a transposon gene trap system. This system requires creation of a bait strain that stably expresses a fusion product of part of the fluorescent monomeric Kusabira-Green (mKG) protein to a protein of interest. A PiggyBac transposon vector is then introduced into this strain, and a sequence encoding the remainder of mKG is inserted into the genome and fused randomly with endogenous genes. The binding partner can be identified by isolating cells that fluoresce when BiFC occurs. Using this system, we screened for interactors of p65 (also known as RELA), an NF-κB subunit, and isolated a number of mKG-positive clones. 5'- or 3'-RACE to produce cDNAs encoding mKG-fragment fusion genes and subsequent reconstitution assay identified PKM, HSP90AB1, ANXA2, HSPA8, and CACYBP as p65 interactors. All of these, with the exception of CACYBP, are known regulators of NF-κB. Immunoprecipitation assay confirmed endogenously expressed CACYBP and p65 formed a complex. A reporter assay revealed that CACYBP enhanced 3κB reporter activation under TNFα stimulation. This screening system therefore represents a valuable method for identifying interacting factors that have not been identified by other methods.

SUBMITTER: Miyakura H

PROVIDER: S-EPMC8121353 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundTheileria annulata can infect monocytes/macrophages and B lymphocytes and causes severe lymphoproliferative disease in ruminants. Meanwhile, infection by T. annulata leads to the permanent proliferation of cell population through regulating signaling pathways of host cells. Cysteine proteinases (CPs) are one kind of protein hydrolase and usually play critical roles in parasite virulence, host invasion, nutrition and host immune response. However, the biological function of T. annulata CP (TaCP) is still unclear. In this study, a yeast-two-hybrid assay was performed to screen host proteins interacting with TaCP, to provide information to help our understanding of the molecular mechanisms between T. annulata and host cells.MethodsThe cDNA from purified bovine B cells was inserted into pGADT7-SfiI vector (pGADT7-SfiI-BcDNA, Prey plasmid) for constructing the yeast two-hybrid cDNA library. TaCP was cloned into the pGBKT7 vector (pGBKT7-TaCP) and was considered as bait plasmid after evaluating the expression, auto-activation and toxicity tests in the yeast strain Y2HGold. The yeast two-hybrid screening was carried out via co-transforming bait and prey plasmids into yeast strain Y2HGold. Sequences of positive preys were analyzed using BLAST, Gene Ontology, UniProt and STRING.ResultsTwo host proteins, CRBN (Bos taurus cereblon transcript variant X2) and Ppp4C (Bos indicus protein phosphatase 4 catalytic subunit) were identified to interact with TaCP. The results of functional analysis showed that the two proteins were involved in many cellular processes, such as ubiquitylation regulation, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs.ConclusionsThis study is the first to screen the host proteins of bovine B cells interacting with TaCP, and 2 proteins, CRBN and Ppp4C, were identified using yeast two-hybrid technique. The results of functional analysis suggest that the two proteins are involved in many cellular processes, such as ubiquitylation regulating, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs. The interaction with CRBN and Ppp4C indicate that TaCP possibly is involved in regulating signaling pathways and cell proliferation, which is helpful for understanding the interaction between T. annulata and host cells.

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A screening system for identifying interacting proteins using biomolecular fluorescence complementation and transposon gene trap.

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