Structure-function relationships of kinesin molecular motors in Plasmodium parasites
Lead Research Organisation:
Birkbeck, University of London
Department Name: Biological Sciences
Abstract
Strategic Research Priority: World Class Bioscience
Abstract
Kinesins are ATP-dependent microtubule-based motors with multiple essential roles in eukaryotes, including parasitic protozoa. The intracellular Plasmodium parasite infects >200 million people annually, killing nearly 1 million. The complex parasite life cycle presents numerous challenges to text-book concepts of cell biology. The few drugs available and emerging drug resistance also mean that novel drug targets are urgently needed. Mitotic kinesins are targets for cancer therapeutics and could form attractive targets for anti-malarials. Little is known about kinesins in Plasmodium, so the aim of this project is to combine structural, biochemical and parasitology approaches to mechanistically and functionally characterize these motors.
Project
This project will elucidate the structure-function relationship of Plasmodium kinesins, with a focus on kinesin-13 and kinesin-8 motors. Their molecular mechanisms, biological roles, and sensitivity to inhibition, primarily in P. falciparum, will be investigated.
Microtubules in all eukaryotes, including Plasmodium, undergo dramatic cell cycle-dependent reorganisation, particularly during cell division. A subset of kinesins - principally kinesin-13s and kinesin-8s - are implicated in microtubule remodeling and reflect an important, but poorly understood, divergence of the typical kinesin stepping mechanism. Kinesin-13s are known to be microtubule depolymerisers, using ATP to shorten microtubules; we have shown that recombinant subdomains of kinesin-13 motors from both P. falciparum and humans depolymerise microtubules. However, the specifics of their activities suggest there are mechanistic differences between motors from each organism. The kinesin-8 mechanism is more controversial.
It is essential to discover how sequence homology/divergence between Plasmodium kinesins and those from higher organisms is reflected mechanistically. Differences potentially render parasite enzymes susceptible to specific inhibition by anti-malaria treatments. We will use cryo-electron microscopy to structurally characterize motor mechanisms. Another central question is what role individual kinesins play in the parasite. Novel technologies for manipulating the otherwise genetically challenging P. falciparum - including Zinc Finger Nuclease Technology - will enable efficient assessment of the role of kinesins in Plasmodium.
The objectives of the project are: 1) Purify recombinant P. falciparum kinesin-13 and kinesin-8 motor domains; 2) Biochemically/structurally characterize them and their microtubule interaction using cryo-electron microscopy; 3) Screen for inhibitors using small molecule libraries; 4) Determine expression profiles of kinesin-13/8 to understand their biological roles; 5) Compare sequences of kinesin-13/8 across Plasmodium species; 6) Generate transgenic parasite lines of modified kinesin-13/8 in P. falciparum to specify their functions.
Thus, this project will involve the study of both the molecular mechanisms and functional contribution of kinesins in intracellular parasites.
Abstract
Kinesins are ATP-dependent microtubule-based motors with multiple essential roles in eukaryotes, including parasitic protozoa. The intracellular Plasmodium parasite infects >200 million people annually, killing nearly 1 million. The complex parasite life cycle presents numerous challenges to text-book concepts of cell biology. The few drugs available and emerging drug resistance also mean that novel drug targets are urgently needed. Mitotic kinesins are targets for cancer therapeutics and could form attractive targets for anti-malarials. Little is known about kinesins in Plasmodium, so the aim of this project is to combine structural, biochemical and parasitology approaches to mechanistically and functionally characterize these motors.
Project
This project will elucidate the structure-function relationship of Plasmodium kinesins, with a focus on kinesin-13 and kinesin-8 motors. Their molecular mechanisms, biological roles, and sensitivity to inhibition, primarily in P. falciparum, will be investigated.
Microtubules in all eukaryotes, including Plasmodium, undergo dramatic cell cycle-dependent reorganisation, particularly during cell division. A subset of kinesins - principally kinesin-13s and kinesin-8s - are implicated in microtubule remodeling and reflect an important, but poorly understood, divergence of the typical kinesin stepping mechanism. Kinesin-13s are known to be microtubule depolymerisers, using ATP to shorten microtubules; we have shown that recombinant subdomains of kinesin-13 motors from both P. falciparum and humans depolymerise microtubules. However, the specifics of their activities suggest there are mechanistic differences between motors from each organism. The kinesin-8 mechanism is more controversial.
It is essential to discover how sequence homology/divergence between Plasmodium kinesins and those from higher organisms is reflected mechanistically. Differences potentially render parasite enzymes susceptible to specific inhibition by anti-malaria treatments. We will use cryo-electron microscopy to structurally characterize motor mechanisms. Another central question is what role individual kinesins play in the parasite. Novel technologies for manipulating the otherwise genetically challenging P. falciparum - including Zinc Finger Nuclease Technology - will enable efficient assessment of the role of kinesins in Plasmodium.
The objectives of the project are: 1) Purify recombinant P. falciparum kinesin-13 and kinesin-8 motor domains; 2) Biochemically/structurally characterize them and their microtubule interaction using cryo-electron microscopy; 3) Screen for inhibitors using small molecule libraries; 4) Determine expression profiles of kinesin-13/8 to understand their biological roles; 5) Compare sequences of kinesin-13/8 across Plasmodium species; 6) Generate transgenic parasite lines of modified kinesin-13/8 in P. falciparum to specify their functions.
Thus, this project will involve the study of both the molecular mechanisms and functional contribution of kinesins in intracellular parasites.
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M009513/1 | 01/10/2015 | 31/03/2024 | |||
| 1618885 | Studentship | BB/M009513/1 | 01/10/2015 | 30/09/2019 | Fiona Shilliday |
| Description | Two publications have resulted from this award with two more in the writing / preparation phase. The student (myself Fiona Shilliday) successfully submitted and defended my PhD thesis because of this award and has moved to a new role as a Scientist at AstraZeneca in Electron Microscopy, due to experience gained through this funding. |
| Exploitation Route | The publications and thesis will hopefully aid future researchers under the supervision of Carolyn Moores and inform other researchers in the microtubule / cryo-EM field. |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| Description | Instruct ERIC - Nanobody Discovery |
| Amount | £0 (GBP) |
| Funding ID | PID: 6545 |
| Organisation | European Commission |
| Sector | Public |
| Country | European Union (EU) |
| Start | 01/2019 |
| End | 09/2019 |
| Description | Collaboration with Steinmetz laboratory from PSI Switzerland |
| Organisation | Paul Scherrer Institute |
| Country | Switzerland |
| Sector | Academic/University |
| PI Contribution | - Cryo-EM expertise to aid PSI collaborators with solving the structure of their tubulin from a diverse species. - TIRF microscopy expertise to develop an assay to solve their biological questions about their sample. |
| Collaborator Contribution | The collaborators at PSI supply all the protein material and the expertise to obtain a pure sample for further processing. |
| Impact | - Sub-nanometre resolution structure of this novel tubulin in its polymerised form as a microtubule, this will soon be added into a manuscript. - Optimisation of conditions for TIRF microscopy assay using currently available resources to aid further development when additional resources become available. |
| Start Year | 2018 |
| Description | Collaboration with the Tewari laboratory |
| Organisation | University of Nottingham |
| Department | University of Nottingham Museum |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Sharing of research to compare similar parasite species, therefore providing robustness to results. Contribution of research to an upcoming publication. Expertise on protein biochemistry and specific knowledge about the type of proteins and their characteristics. |
| Collaborator Contribution | Expertise on parasite cell biology, culturing parasite samples. |
| Impact | This collaboration is multi-disciplinary bringing together the Moores laboratory at Birkbeck with expertise on protein biochemistry, kinesin biology and electron microscopy and the Tewari laboratory at University of Nottingham with expertise in parasite cell biology. |
| Start Year | 2018 |
| Description | Using cryo-EM to study the structure of a kinesin-5 chimeric protein bound to microtubules |
| Organisation | Louisiana State University Health Sciences Center New Orleans |
| Country | United States |
| Sector | Hospitals |
| PI Contribution | Structural studies by cryo-EM of a chimeric kinesin protein bound to microtubules. |
| Collaborator Contribution | - Protein cloning, expression and purification of the chimeric kinesin protein, |
| Impact | - Sub-nanometre cryo-EM reconstruction of this chimeric kinesin protein bound to microtubules in one nucleotide state. |
| Start Year | 2015 |
| Description | Recruitment video for Birkbeck Masters course |
| Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Undergraduate students |
| Results and Impact | Aiding a filming expert at Birkbeck College to film the laboratory facilities and demonstrate a few basic techniques. Film material was used to create a short recruitment video for one of the new Birkbeck Masters Courses. |
| Year(s) Of Engagement Activity | 2018 |
| Description | School visit to see Birkbeck College Electron Microscopy Research facilities |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | Met several times with a small team of researchers from Birkbeck College Electron Microscopy facility and developed a half-day activity session. This was designed for around 30 Year 10/11 GCSE students to visit Birkbeck University and find out more about research at university in general but also focus on the use of electron microscopy. The topics discussed were linked to the National curriculum as much as possible, looking at the structure of cells, having an idea of scale and particular proteins involved in important cellular processes and diseases which they may focus on. |
| Year(s) Of Engagement Activity | 2019 |