Global mechanisms for control of the trypanosome proteome: Defining the composition, origins and roles of cullin E3 ligases.

Parasitic protozoa are major agents of disease, and afflict a major proportion of the global population. In evolutionary terms parasites are highly removed from their hosts, and this is frequently reflected in the presence of unique or unusual mechanisms that underpin their biology. This aspect presents multiple opportunities for the understanding of pathogenesis, the possibility of identifying therapeutic targets as well as offering a fascinating evolutionary perspective on many cellular processes.
African trypanosomes are totally reliant on antigenic variation for their longterm survival. The process is so successful that trypanosomes can infect a mammalian host for many months and in some cases years/decades. Additional immune evasion mechanisms, which defend the parasite against components of both the innate and acquired arms of the host immune response, require surface or endosome-located proteins. We have recently made three significant advances in understanding the cell biology that underpins these immune evasion processes;

1. Characterisation of the cell surface proteome, which revealed both remarkable diversity from higher eukaryotes and even from other trypanosomatids such as American trypanosomes,

2. Demonstration of partitioning of surface components into at least four distinct micro-domains - surface, flagellar pocket, flagellum and endosome, with some proteins exhibiting a combination between all four, and

3. Identifying components of the ubiquitylation machinery that control the expression levels of surface proteins.

These new insights open a route to understanding how the unique pathogenic surface of African trypanosomes is regulated at the molecular level, together with dissecting how such regulation contributes towards basic biology and pathogenesis. Coupled with this is the means to exploit proteomics technology and insights developed recently to understand in a more global manner regulation of the trypanosome cellular proteome.

We propose to interrogate in detail how a group of trypanosome ubiquitin ligases, called cullins, function in this context. Ubiquitin is a major mediator of protein turnover pathways, with the cullin group especially important due to roles in surface receptor turnover and modulation of cell cycle progression. Given the amenability of the trypanosome for genetic manipulation, the comparatively small size of the proteome and the emphasis on post-transcriptional and translation-linked mechanisms to control protein expression levels, we propose that the trypanosome provides a very attractive approach to understand these ubiquitylating complexes, their functional stratification and specificity, in addition to how they contribute towards trypanosome biology and infectivity.

Trypanosomatids evade the host immune response by multiple mechanisms, but for all species the parasite surface plays roles that are central to virulence. In Leishmania and the American trypanosome, surface-based mechanisms are focused on invasion and survival within host cells. By contrast, for African trypanosomes, where the parasite resides in the host bloodstream, the major mechanism is antigenic variation, but mounting evidence suggests that non-variant epitopes can be exposed to the immune system at the parasite surface. Some years ago we and others demonstrated that surface antibody is endocytosed and rapidly degraded, suggesting this as an additional component to antigenic variation in immune evasion. Furthermore, we also found that the major invariant surface glycoproteins (ISGs) were degraded by a ubiquitin-dependent reaction. However, we were unable to identify the proteins responsible for ubiquitylation of ISGs and hence could not advance our characterisation of the system. Significantly, ISGs are clearly immunogenic and are recognised by both human and bovine antibodies in serum from infected individuals.

Recent work implicated cullin-class ubiquitin ligases as at least some of these ISG-interacting factors. Evidence comes from two major findings. Firstly, Usp7 and Vdu1, two deubiquitylating enzymes of ISGs, and which interact in mammalian cells with cullin ligases, modulate ISG expression levels. Secondly, knockdown of Skp1, a cullin adaptor recognising substrate proteins, is also involved in control of ISG expression levels. We propose to characterise cullin ligases in trypanosomes, to identify those that are responsible for surface protein turnover and to assess their overall roles in regulating protein turnover in trypanosomes. Given that cullin ligases are currently being exposed as possible drug targets, these studies will also contribute to assessing if cullins can be therapeutically exploited in trypanosomes.

As a basic biology application, clearly most of the impact here is to individuals in directly related fields, and impact to the more general public is somewhat diluted and rather non-specific by nature. However, clearly the advancement of understanding of immune evasion and potential host interactions via control of the surface proteome and developmental changes is of significant interest to a broad audience.
The RA will certainly benefit hugely. The application is highly cross discipline in nature, and will expose the RA to a great range of methods and collaborative opportunities. Specifically, analysis using imaging, knockdown, proteomics, SILAC and biochemical approaches will provide opportunities to learn specific and advanced analytical methodology. Imaging will facilitate exposure to these methods. As the project will touch on many biological aspects, from singling to evolution and on to immune evasion mechanisms this will also provide great networking potential. The work will be presented at several international meetings, encompassing parasitology, cell singling and ubiquitylation again providing opportunity for networking, advancement and considerable broadening of expertise.
Most directly, research communities working with trypanosomes and their close relatives will see a major empowerment, and which includes the direct experimental community, parasitologists/trypanosomatid cell biologists working at the fundamental biology level, plus those working on direct impact of these organisms on health and agriculture. These individuals and their research goals will be aided in formulating improved hypotheses specifically targeting essential systems for study or therapeutic potential.
Next are researchers with interests in wider aspects of biology. Specifically here we consider the general protist and evolutionary biology communities. The analysis of divergent mechanisms for control of the proteome in trypanosomes is clearly of general impact, and the detailed mechanistic and novel methodological aspects of the work we seek to perform will broaden this further. As the UK has a particularly large parasitology and trypanosome research community this is therefore of specific benefit to the domestic scientific community.
Due to the broad geographical range of trypanosomes, beneficiaries will also include those with interests in ecology, environmental monitoring and human impact plus potentially climate change and agricultural management.
Further, individuals seeking to develop therapeutics will benefit from this work in the medium term - it is hoped that we will continue to refine our understanding of how the trypanosome E3 culling operate and to fully assess opportunities for the design, and possible execution of small molecule screens arising from the interactions that will be identified.
Finally, in public understanding and education: Trypanosomes and tropical diseases are organisms most school children and the general public encounter at some level, either more formally or in the media. Advances in understanding the biology of these organisms may increase the appeal and enthusiasm for basic and applied bioscience to the next generation and general public.