The Role Of Cholesterol In Patched/hedgehog Signalling During Mammalian Development.
Funder
National Health and Medical Research Council
Funding Amount
$198,660.00
Summary
Fluctuations in levels of cholesterol during development of the mammalian embryo have been shown to have catastrophic affects leading to gross deformities particularly in terms of brain and facial development. The requirement of the developing embryo for cholesterol has been linked to the patched-hedgehog signalling pathway which we have previously shown to be central to mammalian development as well as tumour formation. In particular, the patched protein which is responsible for regulating sign ....Fluctuations in levels of cholesterol during development of the mammalian embryo have been shown to have catastrophic affects leading to gross deformities particularly in terms of brain and facial development. The requirement of the developing embryo for cholesterol has been linked to the patched-hedgehog signalling pathway which we have previously shown to be central to mammalian development as well as tumour formation. In particular, the patched protein which is responsible for regulating signalling through this complex cascade of protein interactions has a domain similar to that which in other proteins has been shown to detect and respond to intracellular levels of cholesterol. The patched protein binds to hedgehog at the surface of the cell and mediates the transduction of the the hedgehog signal into the cell. By analogy to the role of sterol sensing domains in other proteins, we hypothesise that this domain in patched detects fluctuations in intracellular cholesterol levels which in turn alter trafficking of patched to the cell surface where it can participate in the hedgehog receptor complex. This hypothesis is supported by our preliminary data which suggests that patched is normally localised both at the cell surface and intracellularly. We are proposing a series of experiments to test our hypothesis, most of which deal with determing the localisation of patched in a cell culure system exposed to agents aimed at varying the intracellular levels of cholesterol. Subcellular localisation of patched will be analysed by immunofluorescence, electron microscopy and immunoblotting analysis. We will also test the ability of patched to aggregate at the cell surface with other molecules important in receiving and sending the hedgehog signal. The experiments in this proposal are likely to give the first clues as to the function of the sterol sensing domain in patched and its role in mediating the vital link between cholesterol and embryonic development.Read moreRead less
Use Of Novel Transfection Protocols To Study Protein Trafficking In Malaria-infected Erythrocytes
Funder
National Health and Medical Research Council
Funding Amount
$211,527.00
Summary
Malaria kills between 1 and 3 million children each year. In addition, the disease debilitates the adult population in malaria-endemic areas, thereby contributing to the cycle of poverty in many third world countries. As resistance to existing antimalarial drugs increases, there is an urgent need to understand the workings of the parasite at a molecular level to enable the development of alternative antimalarial strategies. During part of its life cycle, the malaria parasite infects the erythroc ....Malaria kills between 1 and 3 million children each year. In addition, the disease debilitates the adult population in malaria-endemic areas, thereby contributing to the cycle of poverty in many third world countries. As resistance to existing antimalarial drugs increases, there is an urgent need to understand the workings of the parasite at a molecular level to enable the development of alternative antimalarial strategies. During part of its life cycle, the malaria parasite infects the erythrocytes of its human host. The parasite transports proteins to the erythrocyte membrane so as to modify the properties of its adopted cellular residence. The parasite proteins that are deposited at or in the erythrocyte membrane increase the leakiness and the stickiness of the parasitised erythrocytes. This allows more efficient uptake of nutrients and allows the parasitised erythrocytes to adhere to blood vessel walls, thereby avoiding passage through the spleen. Adherence of parasitised erythrocytes to capillaries in the brain is thought to lead to the development of the complication known as cerebral malaria. This complication is responsible for most of the deaths due to malaria. In order to traffic the adherence proteins to the erythrocyte surface, the parasite establishes a novel transport pathway for moving proteins across the erythrocyte cytoplasm. As the uninfected erythrocyte has no means, nor requirement, for moving proteins, this novel transport mechanism may represent a target for drugs that kill the malaria parasite without being toxic to humans. The pathways for the movement of proteins around the infected erythrocyte are largely unknown. We propose to use techniques to introduce foreign genes into malaria-infected erythrocytes to unravel the details of the molecular machinery and the ticketing system that the parasite uses to traffic proteins to their correct destinations in its adopted home.Read moreRead less
Protein Trafficking In Malaria Parasite-infected Erythrocytes
Funder
National Health and Medical Research Council
Funding Amount
$417,750.00
Summary
Malaria kills between 1 and 3 million children each year. In addition, the disease debilitates the adult population in malaria-endemic areas, thereby contributing to the cycle of poverty in many third world countries. As resistance to existing antimalarial drugs increases, there is an urgent need to understand the workings of the parasite at a molecular level to enable the development of alternative antimalarial strategies. During part of its life cycle, the malaria parasite infects the erythroc ....Malaria kills between 1 and 3 million children each year. In addition, the disease debilitates the adult population in malaria-endemic areas, thereby contributing to the cycle of poverty in many third world countries. As resistance to existing antimalarial drugs increases, there is an urgent need to understand the workings of the parasite at a molecular level to enable the development of alternative antimalarial strategies. During part of its life cycle, the malaria parasite infects the erythrocytes of its human host. The parasite transports proteins to the erythrocyte membrane so as to modify the properties of its adopted cellular residence. The parasite proteins that are deposited at or in the erythrocyte membrane increase the leakiness and the stickiness of the parasitised erythrocytes. This allows more efficient uptake of nutrients and allows the parasitised erythrocytes to adhere to blood vessel walls, thereby avoiding passage through the spleen. Adherence of parasitised erythrocytes to capillaries in the brain is thought to lead to the development of the complication known as cerebral malaria. This complication is responsible for most of the deaths due to malaria. In order to traffic the adherence proteins to the erythrocyte surface, the parasite establishes novel transport pathways for moving proteins across the erythrocyte cytoplasm. As the uninfected erythrocyte has no means, nor requirement, for moving proteins, this novel transport mechanism may represent a target for drugs that kill the malaria parasite without being toxic to humans. The pathways for the movement of proteins around the infected erythrocyte are largely unknown. We propose to use cell biology techniques and techniques to introduce foreign genes into malaria-infected erythrocytes to unravel the details of the molecular machinery and the ticketing system that the parasite uses to traffic proteins to their correct destinations in its adopted home.Read moreRead less
Regulation Of Nuclear Import Of Viral Oncoproteins And Transcription Factors By Protein-protein Interactions
Funder
National Health and Medical Research Council
Funding Amount
$650,383.00
Summary
The present application examines the controls that exerted over proteins that localize in the nucleus of eukaryotic cells. This relates relates integrally to cellular processes such as growth, development and oncogenesis. This research area is not represented elsewhere in Australia, and the particular experimental strategies to approach the problem, revolving around the use of special quantitative microscopic techniques are novel internationally. One part of the application seeks to examine tran ....The present application examines the controls that exerted over proteins that localize in the nucleus of eukaryotic cells. This relates relates integrally to cellular processes such as growth, development and oncogenesis. This research area is not represented elsewhere in Australia, and the particular experimental strategies to approach the problem, revolving around the use of special quantitative microscopic techniques are novel internationally. One part of the application seeks to examine transport within the cell of complexes of interacting proteins, rather than single proteins, under as close as possible to physiologically relevant conditions. This will be truly unique, and of great importance to our comprehension of eukaryotic cell function. This application examines particular types of negative control over protein nuclear localization. Since many proteins show such regulation, and in particular important proteins controlling cell growth and division, the results are fundamentally important to our understanding of how cells function in general. Further, this understanding may be applied in disease situations, such as viral-mediated oncogenesis. In the work we propose to do, viral proteins with functions relating to cancer will be examined in detail, as well as a cellular protein which is recognised by them - the tumor suppressor Rb. We intend to examine several viral oncoproteins which target Rb; one is a protein (E7) from the Human Papilloma Virus which has been frequently associated with cervical carcinomas and other cancers. Accordingly, the results may have direct application to viral-induced cancer, and our work may lead to understanding of the regulation of protein transport to the nucleus. This may thus afford a new approach at the pharmacological level to combat transformation.Read moreRead less