Identification of novel interacting proteins for proprotein convertase furin in T cells

Background and aims: Proprotein convertase furin cleaves inactive proproteins into their biologically active forms and regulates the bioavailability of many cellular proteins. Recently, furin was connected to immunology as one of the key regulators of peripheral immunotolerance in T cells. This far the mechanisms beyond the action of furin are, however, undiscovered and the aim of this project is to find novel furin interaction candidates from T cells in order to clarify the role of furin in the regulation of cell mediated immunity. Methods: Strep-tagged forms of wild type and inactive furin were cloned to pcDNA3.1 plasmids and transfected to Jurkat T cells in order to generate stable cell lines. Furin and its interaction partners were purified with Strep-tag based affinity chromatography. Collected fractions were compared in silver stained gels and differential bands were extracted and analyzed with mass spectrometry. Potential interaction candidates were discussed further based on literature. Results: Stable furin expressing Jurkat T cell lines were successfully generated and furin and interacting proteins were effectively enriched from cell lysates with Strep-tag purification. Mass spectrometry based protein identification gave a list of possible interaction partners, from which two candidates, Ras protein family members RAC1/2 and RAP1, were the most potential mediators of furin action in T cell function. Conclusions: Potential furin interacting candidate proteins RAC1/2 and RAP1 could serve as regulators of T cell signaling and trafficking. However, additional replicates or other interaction authentication methods and functional assays must be performed prior final conclusions. In the future, same Strep-tag mediated purification of the furin interactors will be utilized, but the protein identifications will be done with quantitative mass spectrometry in order to obtain more reliable and comprehensive results.