title: Breaking Ub with Leishmania mexicana: a ubiquitin activating enzyme as a novel therapeutic target for leishmaniasis
Creators: name: Harris, Daniel James
Publisher: name: University of Glasgow
Year: 2022

description: Leishmaniasis is a neglected tropical disease, which inflicts a variety of gruesome pathologies on humans. The number of individuals afflicted with leishmaniasis is thought to vary between 0.7 and 1.2 million annually, of whom it is estimated that 20 to 40 thousand die. This problem is exemplary of inequality in healthcare – current leishmaniasis treatments are inadequate due to toxicity, cost, and ineffectiveness, so there is an urgent need for improved chemotherapies. Ubiquitination is a biochemical pathway that has received attention in cancer research. It is the process of adding the ubiquitin protein as a post-translational modification to substrate proteins, using an enzymatic cascade comprised of enzymes termed E1s, E2s, and E3s. Ubiquitination can lead to degradation of substrate proteins, or otherwise modulate their function. As the name suggests, this modification can be found across eukaryotic cell biology. As such, interfering with ubiquitination may interfere with essential biological processes, which means ubiquitination may present a new therapeutic target for leishmaniasis. Before ubiquitination inhibitors can be designed, components of the ubiquitination system must be identified. To this end, a bioinformatic screening campaign employed BLASTs and hidden Markov models, using characterised orthologs from model organisms as bait, to screen publicly-available Leishmania mexicana genome sequence databases, searching for genes encoding putative E1s, E2s, and E3s. To confirm some of these identifications on a protein level, activity-based probes, protein pulldowns, and mass spectrometry were used. Using an activity-based probe that emulates the structure of adenylated ubiquitin, E1s were identified, and their relative abundance quantified. A chemical crosslinker extended the reach of this probe, allowing the identification of an E2 (LmxM.33.0900). It is noted that L. mexicana has two E1s – unusual for a single celled organism. Of these E1s, LmxM.34.3060 was considerably more abundant than LmxM.23.0550 in both major life cycle stages of the in vitro Leishmania cultures. It is important to describe the wider context of these enzymes – what is their interactome, what are their substrates? To study this, CRISPR was used to fuse a proximity-based labelling system, BioID, on genes of interest – LmxM.34.3060 and LmxM.33.0900. The E2 (LmxM.33.0900) was shown to interact with the E1 (LmxM.34.3060), validating the results from the activity-based probe and crosslinker experiments. Due to sequence homology with characterised orthologs, the E2 was hypothesised to function in the endoplasmic reticulum degradation pathway. Immunoprecipitations of a ubiquitin motif, diglycine, were conducted with a view to gathering information on the substrates of ubiquitin. Anti-diglycine peptides included some of those identified by BioID. Experiments examining ubiquitin’s role in the DNA damage response were also initiated, as were improvements to the proximity-based labelling system, however these were not followed to completion due to a lack of time and resources. To examine the possibility of finding novel drug targets in the ubiquitination cascade, recombinant proteins were expressed. LmxM.34.3060 was expressed in a functional form, while a putative SUMO E2 (LmxM.02.0390) was functional after refolding. Expressed LmxM.33.0900 was not functional and could not be refolded into a functional form. Drug assays were conducted on LmxM.34.3060, which found an inhibitor of the human ortholog, TAK-243, to be 20-fold less effective against the Leishmania enzyme. Additional assays found an inhibitor that was 50-fold more effective at inhibiting the Leishmania enzyme as opposed to its human equivalent - 5'O-sulfamoyl adenosine. Furthermore, a new mechanism of action, inhibiting the E1, for was identified for drugs previously characterised to inhibit protein synthesis. LmxM.34.3060 underwent biophysical characterisation, with structural information obtained using SAXS and protein crystallography. A crystal structure was solved to 3.1 Å, with the in-solution SAXS structure complementary to this. TAK-243 was modelled into the LmxM.34.3060 structure and clashes were predicted, concurring with TAK-243’s reduced efficacy against the Leishmania enzyme in the drug assays. This project aimed to characterise the potential of an understudied biochemical system to provide novel therapeutic targets for a neglected tropical pathogen. To achieve this aim it presents the identifications of two E1s, an interactome, a structure, and a potent, selective inhibitor of a Leishmania ubiquitin activating enzyme.