We use the techniques of structural biology to research in several different areas. Some examples are:
- Mutations in a number of proteins are implicated in the neurodegenerative disease Familial Amyotrophic Lateral Sclerosis, aka Motor Neuron Disease. We study their structural features in order to understand how these lead to the disease.
- One third of the global population is now estimated to be infected with Mycobacterium tuberculosis. As a part of an international consortium, we study tuberculosis genes as potential drug targets.
- Malaria is a widespread disease, especially in Africa, Asia and South America. Anti-malarial drug resistance is a growing problem and our research is directed towards finding mechanisms that may allow the development of new drugs to combat the disease.
- In collaboration with the MRC Prion Unit, we study prion proteins in order to understand the basis of prion pathologies such as in BSE and CJD.
Environmental – the Nitrogen Cycle
All living organisms require a source of nitrogen to survive but most cannot use atmospheric N2. The biological process that restores N2 to an accessible form is nitrogen fixation, where microorganisms reduce N2 to NH4-, and subsequently to NO3-. Fixing atmospheric nitrogen into ammonia and nitrates is thus essential for plant growth. Our research programme is centred on several proteins associated with denitrification - the process of re-cycling the fixed nitrogen to the atmosphere.
Throughout the 20th century the increasing use of nitrogen fertilizer to boost food production has resulted in an inbalance of the Nitrogen Cycle resulting in the accumulation of N-oxide levels in soil and surface waters. Denitrifiying microbes utilize these N-oxides as electron acceptors in the anaerobic oxidation of organic matter. This respiratory pathway has N2 as the final product resulting in losses of terrestrial fixed nitrogen to the atmosphere. The greenhouse and ozone-depleting gas N2O is a significant by-product in this process, so denitrification impacts on both agronomy and the environment.
In collaboration with computational and theoretical science groups at Daresbury Laboratory, we use molecular dynamics and quantum chemistry calculations to understand and predict the electronic structures and molecular behavior of proteins involved in neurodegenerative disease, the nitrogen cycle and electron-transfer processes. Results from our experiments provide the background for these theoretical studies.
Current Group Projects
Amyotrophic Lateral Sclerosis (Motor Neuron Disease)
One of our themes is the study of the structure of mutated proteins that have been identified with the development of the inherited forms of the neurological disease Amyotrophic Lateral Sclerosis (ALS, better known in the UK as Motor Neuron Disease, MND), where they are responsible for 5%-10% of cases. In ALS, motor neurons progressively die, eventually leading to the death of the sufferer. As yet, there is no cure for the disease and only one drug, riluzole, that has been shown to delay the progression of the disease by a few months. Our objective is to understand how mutations in certain proteins interfere with their correct operation and thus how they lead to the disease.
Fernanda Sala, Mike Capper, Gareth Wright and Svetlana Antonyuk are studying the interaction of the chaperone protein hCCS with a variety of mutated forms of copper-zinc superoxide dismutase (SOD1). Mutated SOD1 is one of the causes of MND/ALS (see above). SOD1 is responsible for breaking down the free radical superoxide (an oxygen molecule carrying an additional electron) which is produced in muscle-controlling neurons. Superoxide is a normal product of the operation of the neuron but, in common with other free radicals, it is potentially disruptive to the chemistry of a cell and its quantity must be carefully controlled by SOD1. The formation of SOD1 requires the assistance of the hCCS (the human form of the copper chaperone for superoxide dismutase, CCS) to bind the copper ion it needs to be able function. Our work suggests that mutations in SOD1 can prevent hCCS doing its job properly and we aim to understand why this is the case.
Extracellular Superoxide Dismutase
Varunya Chantadul is studying a different form of superoxide dismutase, known as SOD3. Being distinct from other SODs, SOD3 or EcSOD (extracellular superoxide dismutase) is the only anti-oxidant enzyme which is normally localized on the cell surface. To protect cells from surrounding free radicals, SOD3 anchors to heparan sulphate, type I collagen and fibulin-5 through its unique extracellular matrix (ECM) binding domain. SOD3 is usually found in blood vessel walls, thereby protecting the vessels from being damaged. This anti-oxidant enzyme also maintains the level of nitric oxide which is a potent vasodilator. As a result, it plays critical roles in blood pressure regulation and cardiovascular diseases such as atherosclerosis and hypertension.
R213G SOD3 mutation has been shown to be associated with cardiovascular diseases as well as oxidative damage in several organs. While the other common mutated SOD3, A40T, is considered as a genetic marker for diabetes, its clinical and structural significances remain elusive. The crystal structure of SOD3 has already been determined except for the C-terminus where the ECM binding domain is located and N-terminus which is important for its tetrameric structure. Therefore, our research focuses on the study of the missing parts of SOD3 and its mutated forms in relation to human diseases.
Malaria and Toxoplasmosis
Kangsa Amporndanai and Nopphon Petchyam are working to develop new therapies against Apicomplexan parasites: Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis). Malaria is a lethal mosquito-borne disease in tropical areas, especially Africa and Asia. It has developed significant drug resistance and is threatening global health systems – new drugs are needed. Toxoplasmosis is a parasitic infection and there are currently no treatments that can eradicate latent parasites in patients. Cytochrome bc1 is an essential component that enables biochemical generation of ATP within mitochondria and pyrimidine synthesis in Apicomplexan parasites. Many drugs have been developed to prevent cytochrome bc1 function and kill the parasite. However, some drugs failed in safety tests due to their unexpected binding to human, as well as parasite, cytochrome bc1. Kangsa is attempting to visualise the interactions between cytochrome bc1 and drug molecules by x-ray crystallography. This information could be beneficial for the design of new drugs for greater efficiency and enhanced selectivity.
In addition, Kangsa is interested in the possibility of malaria vaccine development based on the merozoite surface protein 1 (MSP1). MSP1 is an important vaccine target in the entire life-cycle of the blood-stage parasite. Rusticyanin is an extremely acidic-stable copper protein from Thiobacillus ferroxidans. It is known to have antimalarial activity through interaction with MSP1, but the mechanism is still unclear. Kangsa is also applying x-ray crystallography to reveal how rusticyanin inhibits MSP1. This discovery may provide an alternative route for the development of novel vaccines for the prevention of malaria.
Thanalai Poonsiri is studying the structure of a protein called Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) as a potential target for drugs to combat the disease. JEV is a member of the Flaviviridae family and Flavivirus genus. It is closely related to West Nile virus and other important viruses in the same genus, for example, Dengue virus, yellow fever virus, tick-borne encephalitis virus, and Zika virus. JEV is mediated by mosquitoes and is a major cause of viral encephalitis in Asia. The disease incidence was estimated in 2011 to be up to 67,900 cases per year and 20-30% fatality with no specific treatment. NS1 is a protein encoded by the virus, but is not a part of the virus particle itself. It co-localizes with the replication complex and is involved in viral replication. The protein is also secreted out of the infected cells and interacts with our immune system. NS1 is believed to be the key antigen that makes us sick. NS1 protein is highly conserved (i.e. varies little) between members of the Flavivirus genus and several studies (including our study) show a similarity in its structure. However, more and more evidence indicates that NS1 of different members of Flavivirus function differently. We are seeking a better understanding of this protein in the hope that it will be useful for diagnostic and therapeutic development in the future.
Crossing the Blood-Brain Barrier
George Chiduza is studying transport across the blood-brain barrier using a structural biology approach. The brain is a remarkably intricate organ, requiring a finely controlled internal environment in order to perform its functions. Evolution's means of ensuring the controlled exchange is the highly selectively permeable blood-brain barrier (BBB). Cells that compose the BBB express proteins that prevent movement of substances around them by forming a physical barrier and across them by actively pumping them out. There are also transporter proteins present in the BBB that enable the absorption of essential nutrients and metabolites into the brain. Despite the benefits of the BBB's effectiveness as a barrier, it presents a significant challenge for the development of therapeutic molecules that require access to the brain to have their effect. Drugs such levodopa, used in the treatment of Parkinson's disease, and gabapentin, useful for the treatment of neurological pain, cross the BBB via the transport protein LAT1.
However, the fact that these drugs can cross the BBB is serendipitous rather than by design. Structural biology techniques, such as macromolecular crystallography and cryo-electron microscopy, are essential tools for increasing the understanding of LAT1 which will aid in the design of potential therapeutics that can penetrate into the brain. This will be important for treatment of neurodegenerative diseases, which are becoming more common in the advanced economies of the world as a result of increasingly ageing populations. In addition, the insights gained into the LAT1 function will contribute to our knowledge of the uptake of essential amino acids and metabolites into the brain, in which the transporter is involved, and into tumours, a number of which have been demonstrated to have LAT1 in higher than normal amounts.
More projects to follow....