Dynamic phosphorylation in the post-transcriptional regulation of the Trypanosoma brucei cell cycle

How do cells make sure that they duplicate themselves correctly? When cells grow and divide, they must coordinate the duplication and division of their content between the two daughter cells in an ordered process known as the cell cycle. Understanding the many processes that regulate the cell cycle is important as many diseases such as cancer and diabetes involve a breakdown in this regulation. This project will study an important part of the cell cycle control mechanism using an organism called Trypanosoma brucei as a simplified system.

Proteins are produced by transcribing DNA into messenger RNA, and then translating the messenger RNA into proteins. Regulation can occur at many different points during this process to control when and how much protein is produced within the cell cycle. Modification of proteins by adding or removing a phosphate group, a process called dynamic phosphorylation, is an important contol mechanism in cell cycle regulation. We are studying Trypanosoma brucei as unusually it does not regulate the transcription of DNA. Instead, all the regulation occurs after transcription, allowing us to focus on these events. The way that T. brucei uses dynamic phosphorylation to regulate the cell cycle is not understood, but is likely to be novel.

To understand how Trypanosoma brucei is able to regulate its cell cycle, we will first measure how much of each protein is produced at different time points in the cell cycle, and whether they are phosphorylated. By breaking proteins down into smaller chains called peptides, we can measure many thousands of peptides and phosphorylation sites using sophisticated machine called mass spectrometers. These measurements can be used to determine how much of each protein and phosphorylation site is present at each time point. This will produce a description of the cell cycle that can be interpreted to make a model explaining how it is regulated. We will then conduct experiments to see if what the model predicts is correct. If our experiments show the model is not right, we can use this new information to improve the model until the predictions agree with our experiments.

As Trypanosoma brucei is a parasite of cattle and a pathogen of humans, what we learn in this study will contribute to the development of new drugs against the parasite. Better understanding of the fundamental processes regulating the cell cycle will also have an impact on other important diseases such as cancer and diabetes.

In the divergent eukaryote Trypanosoma brucei, gene expression occurs in polycistronic units and is regulated post-transcriptionally by the many RNA binding proteins (RBPs). The cell cycle is highly organised, coordinating nuclear and kinetoplast DNA replication and the segregation of single copy organelles. The genome contains many identifiable cell cycle regulators but lacks some cell cycle check points, and the signalling mechanisms coordinating the cell cycle are largely undefined. Two RBPs have been identified that that coordinate the cell cycle enrichment of sets of transcripts. My recent work has identified phosphorylation sites on these RBPs which may control their RNA binding, as has been observed in the related kinetoplastid Crithidia fasiculata. My hypothesis is that dynamic phosphorylation of RBPs contributes to the post-transcriptional regulation of the trypanosome cell cycle.

To test this hypothesis, I propose a data-driven biology approach to examine cell cycle regulation in two lifecycle stages of T. brucei using quantitative proteomic and phosphoproteomic analysis of synchronised cell cultures. The experimental approach will combine the SILAC isotopic labelling approach that I have pioneered in T. brucei with double-cut centrifugal elutriation or hydroxyurea treatment to obtain synchronised cell populations. Selected observations will be experimentally validated by in situ tagging, western blotting, immunofluorescence microscopy, and targeted proteomics. Bioinformatic analysis of the temporal profiles will be combined with morphological observations and data from model organisms to produce a descriptive model of the role of dynamic phosphorylation in post-transcriptional regulation and cell cycle control. The model will be experimentally tested by perturbing components of the system and observing the effect. The data produced will improve the understanding of the trypanosome cell cycle, even if my hypothesis does not prove correct.

The parasite Trypanosoma brucei is transmitted by the bite of the tsetse fly, and is able to form a chronic infection in domestic cattle known as Nagana. This not only causes animal suffering, but also severely reduces cattle productivity in endemic areas thereby causing economic hardship. My proposed research will identify novel drug targets that may be exploitable therapeutically by subsequent researchers to develop drug therapies to treat the parasitic infections in cattle, improving animal welfare and productivity. Such an impact would directly contribute toward wealth creation and economic prosperity in endemic regions of sub-Saharan Africa through regeneration and economic development. Additionally, two other species of T. brucei are human pathogens and such targets and may also provide improved therapies for human infections, benefitting health and wellbeing.

T. brucei is the best characterised of the trypanosomatids which include the clinically relevant T. cruzi and Leishmania species. Mechanisms uncovered in the current work are likely to be conserved in these closely related species, and may also be therapeutically exploitable. Whilst T. cruzi is traditionally confined to South America, and Leishmania is mainly found in South American and Asia, migration and climate change are increasing their distribution and transmission range, making these truly global diseases.

Post-transcriptional regulation of gene expression is involved in genetic disorders, metabolic diseases and cancer. The research will lead to an improved understanding of the underlying molecular mechanism of post-transcriptional regulation, and exploitation of this basic scientific knowledge in academia and the pharmaceutical industry may impact on efforts to tackle these human diseases.