Origins and composition of the cullin-RING E3 ligases in trypanosomes

Abstract

Ubiquitylation is a post-translation modification essential for protein homeostasis that controls the fate of the targeted proteins by inducing their degradation, controlling enzymatic activity or changing cellular localisation following the covalent attachment of ubiquitin to a lysine residue of the substrate. The modification is a three-step process that requires activation of ubiquitin by E1 enzymes, its conjugation to the E2s and eventually their recruitment to an E3 ligase that will transfer it to the substrate. Cullin-RING is a prominent family of E3 ligases responsible for ubiquitylation of up to 20% of the human proteome. In contrast with the rest of the class, cullin-RINGs perform their function as multi-subunit complexes that comprise substrate adaptors, RINGs for the transference of ubiquitin and cullins that serve as scaffold for the complex.

Some cullin-RING E3 ligases and other elements of the ubiquitylation network have been described as resistance-associated genes in Trypanosoma brucei to compounds used to treat the disease it causes, African trypanosomiasis. Moreover, some components have also been described as essential for the pathogenesis of T. brucei and its cell cycle. Beyond this, not much is known about the system in the Kinetoplastida supergroup that T. brucei and other important pathogens like T. cruzi or Leishmania sp. belong to.

Here, I present the systemic analysis and characterisation of the cullin-RING family of E3 ligases in Trypanosoma brucei. Through comparative genomics we were able to identify seven cullins, five SKP1-like protein adaptors and three RBX proteins in Kinetoplastida with many of them revealed to be exclusive to the group via phylogenetic reconstruction. The composition of each of the complexes they form was studied by immunoprecipitating endogenously tagged cullins and using mass spectrometry to identify their partners. This led to the discovery of two potential new classes of substrate adaptors and the description of cullin-RING complexes unique to Kinetoplastida termed ‘private’ and other shared among eukaryotes or ‘public’.

An initial assessment of the role of two cullins, one public and one private, was performed using RNA interference. This revealed the role of Kinetoplastida-specific cullins in the cell cycle of the parasite as well as unveiling novel functions acquired by conserved cullin-RING complexes, including the degradation of ornithine decarboxylase which is a target of an antitrypanosomal drug.

Date of Award	2020
Original language	English
Supervisor	Mark Field (Supervisor)