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

Lead Research Organisation: Lancaster University
Department Name: Division of Biomedical and Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description We have developed a new method of producing cell cycle synchronized populations of both bloodstream and procyclic (insect) form Trypanosoma brucei cells, allowing us to study process at different points in the cell division cycle, and have used this technique to improve the model of the relative timing and duration of the T. brucei cell cycle.
We have combined the synchronization method with advanced mass spectrometry to identify protein and phosphorylation sites that alter in abundance over the T. brucei cell cycle, allowing us to identify proteins involved in the regulation of the cell cycle that act in novel ways. Such novel regulators may be suitable drug targets for development of therapeutic agents against this human and animal parasite.
Exploitation Route We have identified proteins involved in regulating the T. brucei cell cycle that act in novel ways, mainly through post-transcriptional regulation. Such post-transcriptional regulators may be suitable drug targets for development of therapeutic agents against this human and animal parasite. In addition, the improved understanding of novel mechanisms of post-transcriptional regulation may be relevant to other diseases that feature aberrant post-transcriptional regulation such as cancers and metabolic diseases.
Sectors Agriculture, Food and Drink,Healthcare

 
Description Our detailed study of cell cycle regulation in Trypanosoma brucei has provided the basic biomedical knowledge which is essential to underpin successful efforts to develop effective therapies against trypanosomiasis and related diseases. The identification of cell cycle regulated phosphorylation sites on key cell cycle regulators has contributed to the understanding of drug mode of action and assisted pre-clinical development of drug candidates by academic and pharmaceutical partnerships in the UK. The topic of research has formed the basis for public outreach and school engagement activities, and the results of the research have been disseminated to the general public via conventional and social media routes, helping to create an informed public and fostering an interest in STEM subjects amongst young people.
First Year Of Impact 2019
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Title Cell cycle synchronization by Centrifugal counter flow elutriation of Trypanosoma brucei 
Description We have produced an optimized centrifugal counter-flow elutriation protocol for the rapid and direct isolation of G1 cell cycle synchronized populations of both the procyclic and bloodstream stages of Trypanosoma brucei that yields viable and proliferative cells 
Type Of Material Biological samples 
Year Produced 2017 
Provided To Others? Yes  
Impact We have used temporal observations of the synchronized cell populations to construct a model of the relative timing and duration of the nuclear and kinetoplast cell cycle that differs from the current model 
 
Title Proteomic and phosphoproteomic analysis of cell cycle regulation in Trypanosoma brucei 
Description Proteomic and phosphoproteomic analysis of cell cycle regulation in Trypanosoma brucei; Raw and processed mass spectrometry data published in DOI:10.1371/journal.ppat.1008129 and publicly available here for re-analysis and or meta analysis. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact This data underpins publication and future work. 
URL https://www.ebi.ac.uk/pride/archive/projects/PXD013488
 
Description Typanosoma cruzi cell cycle analysis 
Organisation University of the Republic
Country Uruguay 
Sector Academic/University 
PI Contribution Hosting a visiting scientist Santigo Chavez from the group of Dr Maria Ana Duhagon (University of the Republic, Uruguay) in Lancaster University and providing training in proteomic sample preparation, acquisition and analysis. We could lever our expertise in cell cycle analysis in Trypanosoma brucei to provide training to a local scientist working in an country where the related parasite Trypanosoma cruzi is an endemic disease.
Collaborator Contribution The partners provided expertise in cell cycle synchronization of Trypanosoma cruzi cells, material for analysis and ribosome footprinting data. The partner institution provided funding to cover the travel, subsistence and data acquiaition.
Impact The results of the collaboration will shortly be submitted for publication and further funding will be sort.
Start Year 2018
 
Description Parasites in a Box school outreach project 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I have received School - University Partnership Initiative (SUPI) funding through the Research in Box initiative to design and create a 'Parasites in a Box' resource that can be loaned to UK schools, which highlights my research of trypanosome parasites. Duplicate boxes have also been sent to collaborators in Ghana and Brazil, where the parasites are endemic, to be used in local schools for education and to foster international links. The box is regularly used by local schools and additionally as a demonstration for University open days and applicant visit days, and we have hosted a teacher CPD training day on 26th February 2018. Feedback from participants (pupils and teachers) has reported increased interest in sciences and change in opinion towards negelected tropical diseases.
Year(s) Of Engagement Activity 2016,2017,2018,2019