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Biomolecular analysis of the Leishmania mRNA translation machinery to identify potential targets for drug therapy

Principal Supervisor: Professor John McCarthy, School of Life Sciences

Co-supervisor: Professor Alex Cameron

PhD project title: Biomolecular analysis of the LeishmaniamRNA translation machinery to identify potential targets for drug therapy

University of Registration: University of Warwick

Project outline:

This biomedical project will examine the molecular structure and properties of the cellular machinery responsible for protein synthesis in a globally significant pathogenic protozoan with a view to establishing a new strategy for developing drugs that could help tens of millions of people worldwide.

The eukaryotic translation machinery comprises ribosomes and tRNAs plus a host of translation factors that promote mRNA recruitment and AUG (start codon) recognition, as well as polypeptide elongation and termination1. While it has been evident for some time that there are variations in terms of the structural and functional properties of eukaryotic translation factors across the animals, plants and fungi2, recent work has highlighted particularly distinctive features of the trypanosomatid translation machinery. These differences are of special interest, not only in the context of our fundamental understanding of biology, but also because trypanosomatids are a worldwide threat to human health and analysis of distinctive molecular features may help identify potential drug targets. Approximately 37 million people are thought to be infected collectively with Trypanosoma brucei (African sleeping sickness), Trypanosoma cruzi (Chagas disease) and Leishmania species (responsible for multiple forms of leishmaniasis), and many more people are at risk of infection3.

One remarkable feature of trypanosomatid translation machineries is the involvement of an exceptionally large number of isomers of both of the translation factors eIF4E and eIF4G. The eIF4E isomers bind to the highly modified cap4 structure (m7Gpppm36,6,2’Apm2’Apm2’Cpm23,2’U) that is added to the 5’end of the monocistronic mRNAs during processing, and are thought to fulfill differentiated functions over the Leishmania life cycle. Another distinctive feature is that two of the eIF4E isomers (3 and 4) are cytoplasmic proteins with long N-terminal extensions that are not evident in the shared architecture of the eIF4E counterparts that have been characterised in animals, plants and fungi.
Interactions between proteins that associate with the 5’ end and the 3’ end of mRNA have been identified in animals, plants and fungi. Up until recently, research in this area has focused on the role of eIF4G as a bridge between the cap-binding protein eIF4E and the poly(A) binding protein PABP. Overall, these results convey a picture of mutual enhancement of many of the interactions in the molecular chain mRNA5’-m7G-eIF4E-eIF4G-PABP-poly(A)-3’mRNA.
However, trypanosomatids are different. Using techniques of NMR, x-ray crystallography, fluorescence anisotropy, microscale thermophoresis and surface plasmon resonance we have recently been able to develop a quantitative understanding of the structures and interactions underpinning the trypanosomal version of this molecular chain4. We have elucidated a uniquely pivotal structural role for a novel trypanosomal eIF4E4-PABP1 interaction. This unique interaction, as opposed to the eIF4G-PABP interaction found in other organisms, takes over the role of mediating ‘circularisation’ of mRNAs by linking the mRNA 5’ and 3’ ends.

Based on this platform of recently established knowledge, the student working on this project will be able to harness a wide range of biochemical, genetic and biophysical technologies to drive a further step-change in our understanding of trypanosomal translation initiation. The overall aim will be to identify further interactions of the core cap-binding complex involving, for example, eIF4A and mRNA. An exciting new direction will be to utilise cryo-EM to elucidate the structure of these larger complexes. In addition, biomolecular interaction and inhibition assays will be developed as a basis for identifying potent inhibitory molecules that can block trypanosomal translation.

References:

  1. Jackson et al (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nature Rev Mol Cell Biol, 11, 113-127.
  2. Gallie (2014) The role of the poly(A) binding protein in the assembly of the cap-binding complex during translation initiation in plants. Translation 2, e959378.
  3. Lopes et al (2010) Trypanosomatids: Odd Organisms, Devastating Diseases. The Open Parasitology J 4, 30-59.
  4. Rodrigues et al (2018) The Leishmania PABP1-eIF4E4 interface: a novel 5’-3’ interaction architecture for trans-spliced mRNAs. Nucleic Acids Research.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

The student will be able to receive training in, and make use of, an exceptionally wide range of powerful techniques, including x-ray crystallography, NMR, cryo-EM, biophysical interaction measurement techniques (microscale thermophoresis, isothermal calorimetry, surface plasmon resonance, fluorescence anisotropy), and molecular modeling, as well as CRISPR, synthetic DNA synthesis and genomic integration.

Contact: Professor John McCarthy, University of Warwick