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VSG Exclusion Protein 2 (VEX2) affinity purification combined with LC-MS/MS analysis in Trypanosoma brucei

ABSTRACT: UPF1-like helicases play roles in telomeric heterochromatin formation and X-chromosome inactivation and we focus here on variant surface glycoprotein (VSG) exclusion-factor-2 (VEX2), a UPF1-related protein in the African trypanosome. This bloodstream parasite evades host immunity by employing monogenic and switchable VSG expression, a form of antigenic variation which involves transcription of one telomeric VSG gene at a time. A trans-splicing compartment and the single VSG transcription factory are juxtaposed and enriched for VEX1 and VEX2, respectively, but our understanding of the VSG exclusion mechanism remains otherwise incomplete. Here, we sought VEX2-interacting partners, using cryomilling and affinity purification followed my LC-MS/MS analysis. We confirmed previous observations that VEX2 interacts with VEX1 and CAF-1, but also found evidence for weaker interactions and/or spatial proximity to telomere binding proteins.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Michele Tinti

LAB HEAD: Joana R. Correia Faria

PROVIDER: PXD040686 | Pride | 2023-11-20

REPOSITORIES: Pride

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Project description:Variant Surface Glycoproteins (VSG) coat parasitic African trypanosomes and underpin antigenic variation and immune evasion. This VSG is a super-abundant virulence factor that is subject to post-transcriptional gene expression controls mediated via the VSG 3’-untranslated region (3’-UTR). To identify positive VSG regulators in bloodstream form cells, we used prior genome-scale screening data to prioritise mRNA binding protein (mRBPs) knockdowns that phenocopy VSG mRNA knockdown, displaying both loss-of-fitness and pre-cytokinesis accumulation. The top three candidate VSG regulators were CFB2 (cyclin F-box protein 2, Tb927.1.4650), MKT1 (Tb927.6.4770) and PBP1 (polyadenylate binding-protein binding-protein, Tb927.8.4540). Notably, CFB2 was recently found to regulate VSG transcript stability, and all three proteins were found to associate with each other. We used data-independent acquisition for accurate label-free quantification and deep proteome coverage to quantify expression profiles following depletion of each mRBP. Only CFB2 knockdown significantly reduced VSG expression and the expression of a reporter under the control of a VSG 3’-untranslated region (3’-UTR). CFB2 knockdown also triggered depletion of cytoplasmic ribosomal proteins, consistent with translation arrest observed when VSG synthesis is blocked. In contrast, PBP1 knockdown triggered depletion of CFB2, MKT1, and other components of the PBP1-complex. Finally, all three knockdowns triggered depletion of cytokinesis initiation factors, consistent with the cytokinesis defect that was confirmed here for all three knockdowns. Thus, genome-scale knockdown datasets facilitate the triage and prioritisation of candidate regulators. Quantitative proteomic analysis confirms 3’-UTR dependent positive control of VSG expression by CFB2 and interactions with additional mRBPs. Our results also reveal connections between VSG expression control by CFB2, ribosomal protein expression, and cytokinesis.

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VSG Exclusion Protein 2 (VEX2) affinity purification combined with LC-MS/MS analysis in Trypanosoma brucei

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