10779/crick.12651911.v1 Vesela Encheva Vesela Encheva Clémence Foltz Clémence Foltz Ambrosius P Snijders Ambrosius P Snijders Eva-Maria Frickel Eva-Maria Frickel Murine Gbp1 and Gbp2 are ubiquitinated independent of Toxoplasma gondii infection The Francis Crick Institute 2020 Guanylate binding proteins Toxoplasma gondii Ubiquitin Ubiquitinated substrates Animals Embryo, Mammalian Fibroblasts GTP-Binding Proteins Mice Mice, Inbred C57BL Toxoplasmosis Ubiquitination Frickel FC001076 PRT 1199 Other Medical and Health Sciences 0601 Biochemistry and Cell Biology Bioinformatics 2020-07-15 11:27:26 Journal contribution https://crick.figshare.com/articles/journal_contribution/Murine_Gbp1_and_Gbp2_are_ubiquitinated_independent_of_Toxoplasma_gondii_infection/12651911 OBJECTIVE: The intracellular parasite Toxoplasma gondii can invade any nucleated cell residing inside a parasitophorous vacuole (PV). Upon infection, the cytokine interferon gamma (IFNγ) is produced and elicits host defence mechanisms able to recognise the PV and destroy the parasite. Hereby, Guanylate binding proteins, ubiquitin and the E3 ubiquitin ligases Tripartite Motif Containing 21 (TRIM21) and TNF receptor associated factor 6 are targeted to the murine PV leading to its destruction. This study is the side product of research aiming to identify ubiquitinated substrates in a TRIM21-dependent fashion in murine cells infected with Toxoplasma. RESULTS: We infected IFNγ-stimulated murine embryonic fibroblasts (MEFs) from either C57BL/6×129 wild-type (WT) mice or C57BL/6 TRIM21-/- mice with Toxoplasma. Using mass spectrometry, we analysed proteins in both cell backgrounds presenting with the di-glycine remnant of ubiquitination. In addition, we compared peptide levels between WT and TRIM21-/- cells. In line with earlier reports, Gbp1 was expressed to higher levels in the C57BL/6×129 WT MEFs compared to the C57BL/6-only background TRIM21-/- MEFs. Protein expression differences in these different murine backgrounds thus precluded identification of TRIM21-dependent ubiquitinated substrates. Nevertheless, we identified and confirmed Gbp1 and Gbp2 as being ubiquitinated in a Toxoplasma-infection independent manner.