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Poster
115 |
Structural and functional dissection of VSG-Exclusion Protein 2 in African trypanosomes |
African Trypanosomes are extracellular parasites that evade the host immune system by periodic switching their homogenous coat made of a variant-surface-glycoprotein (VSG). Trypanosomes have a vast repertoire of > 2600 VSG genes and pseudogenes, but VSG genes can only be expressed from a limited subset of transcription-units referred to as ‘expression-sites’, however, only one is active at a time.
VEX2, a large (224 kDa) member of the Superfamily 1 (SF1) helicases has been shown to be critical to sustain singular VSG expression. Indeed, the loss of VEX2 results in multiple VSG expression at the single cell level. VEX2 colocalizes with the ESB and interacts with VEX1, which is associated with the Spliced-Leader-(SL)-array thereby forming an inter-chromosomal bridge between those nuclear compartments. So far there is little molecular understanding of those interactions and how they are controlling monoallelic expression.
Firstly, we aim to understand VEX2 substrate specificity and the potential role of the N-terminal domain in the regulation of the helicase activity. To that end, we are currently recombinantly expressing VEX2 full-length and AlphaFold guided helicase-domain constructs in different expression systems including Leishmania tarentolae (LEXSY), to pursue a biochemical and structural characterization, the latter using single-particle cryoEM. So far, a recombinant helicase core has been produced in baculovirus-infected insect cells showing that VEX2 is an ATP-dependent RNA:DNA helicase. Notably, precision editing of key residues using CRISPR/Cas9 indicated that the helicase activity is required for parasite survival and VSG expression control.
Secondly, VEX2 is an orthologue of human Senataxin, which is an RNA:DNA helicase involved in R-Loop metabolism. Whether VEX2 shows similar activity in context of R-Loop resolution at the active-VSG expression-site remains elusive, therefore, we performed DRIP-Seq before and after VEX2 depletion and found interesting and unexpected patterns.
Thirdly, we are currently investigating key post-translational modifications with a focus on SUMOylation and phosphorylation via LC-MS/MS analysis in both bloodstream and procyclic cells to understand their impact on the regulation of VEX2 function and localisation during different cell cycle stages.
