Development Of BRET Detection Systems: Tools For Functional Proteomics And Drug Discovery
Funder
National Health and Medical Research Council
Funding Amount
$376,320.00
Summary
The internal structure of articular cartilage is critical to its biomechanical function. Cartilage is one of the most intricate and difficult tissues to examine in-vivo. Maintenance of its functional characteristics depends heavily of the internal microstructure of the tissue, while conventional arthroscopy can only give a view of the surface and provides no information on the internal structure. Biopsy examination can also destroy the integrity of the tissue, making it impossible to concurrentl ....The internal structure of articular cartilage is critical to its biomechanical function. Cartilage is one of the most intricate and difficult tissues to examine in-vivo. Maintenance of its functional characteristics depends heavily of the internal microstructure of the tissue, while conventional arthroscopy can only give a view of the surface and provides no information on the internal structure. Biopsy examination can also destroy the integrity of the tissue, making it impossible to concurrently examine the structure and function of the tissue. The structure-function relationship is thus critical to the study and the advancement of clinical treatment techniques for cartilage disorders. Osteoarthritis is characterized by severe disruption to the cartilage matrix. The emergence of autologous chondrocyte implant (ACI) therapy as a method for repairing cartilage defects has further increased interest in clinical techniques for the examination of cartilage structure and function. The development of confocal microscopy facilitates internal examination of loaded tissue for the first time, enabling direct examination of the association between structure and function of the tissue. A prototype confocal arthroscope has been developed to facilitate clinical examination of cartilage structure. This, in turn, allows the functional characteristics of the tissue to be deduced. Cartilage exhibits little intrinsic repair making biopsies undesirable. Thus, with respect to cartilage in particular, the developed technologies promise to enable examination to a level of detail which was previously impossible. The current prototype arthroscope has demonstrated the feasibility of a genuine clinical instrument. This grant application seeks funds to conduct initial clinical trials in order to gain sufficient practical feedback to enable design and construction of a clinically ready system.Read moreRead less
Following a meal glucose circulates in the blood and is taken up into cells via movement of an intracellular glucose transporter from the inside of the cell to fuse with the cell membrane and subsequent transfer of the glucose into the cell. This process is triggered by insulin. One of the commonest diseases resulting from a failure of this cellular process is diabetes. A common form of diabetes which occurs in many adults in Australia results from insulin resistance, whereby the effects of insu ....Following a meal glucose circulates in the blood and is taken up into cells via movement of an intracellular glucose transporter from the inside of the cell to fuse with the cell membrane and subsequent transfer of the glucose into the cell. This process is triggered by insulin. One of the commonest diseases resulting from a failure of this cellular process is diabetes. A common form of diabetes which occurs in many adults in Australia results from insulin resistance, whereby the effects of insulin are diminished and cells become increasingly unable to uptake glucose. Recent studies have demonstrated that a novel enzyme known as SHIP-2 may play a role in regulating insulin action in cells. Deletion of SHIP-2 in mice results in these animals have increased sensitivity to insulin, low blood glucose levels, and a greatly enhanced ability to take up glucose in cells in response to low dose insulin. Our laboratory has been working on the cellular mechanisms regulating SHIP-2 function. We have recently revealed the intracellular location of SHIP-2 and also demonstrated how SHIP-2 is localized in the cell. These studies have shown that SHIP-2, via interactions with other proteins, regulates the actin cytoskeleton immediately beneath the cell membrane and this may be a mechanism for facilitating cellular glucose uptake. This research proposal aims to determine how SHIP-2 facilitates glucose uptake into cells. We will make cell lines and transgenic animals which express high levels of this enzyme and determine the functional consequences on insulin stimulated glucose uptake. Collectively these studies in the long term may facilitate better treatment strategies for diabetic patients.Read moreRead less
Analysis Of The C-terminal Hypervariable Region Of Ras Proteins
Funder
National Health and Medical Research Council
Funding Amount
$419,241.00
Summary
In human cancers one or more of the signaling pathways leading from growth factor receptors at the cell surface to the nucleus where cell division is initiated are subverted. For example, a protein called Ras, that regulates one major signaling pathway, is mutated in 90% of pancreatic cancers, 50% of colon cancers and 30% of acute leukaemias. This leaves Ras and the signaling pathway permanently switched on causing uncontrolled cell proliferation. The clinical impact of drugs that could neutrali ....In human cancers one or more of the signaling pathways leading from growth factor receptors at the cell surface to the nucleus where cell division is initiated are subverted. For example, a protein called Ras, that regulates one major signaling pathway, is mutated in 90% of pancreatic cancers, 50% of colon cancers and 30% of acute leukaemias. This leaves Ras and the signaling pathway permanently switched on causing uncontrolled cell proliferation. The clinical impact of drugs that could neutralise Ras function in these tumours is potentially enormous. Our previous work demonstrated that Ras must be attached to the inner surface of the cell membrane in order to function properly. This project now seeks to understand exactly how Ras gets to and attaches to the cell membrane. Once we understand this mechanism drugs can be designed to block Ras getting to the membrane. Such drugs should neutralize the effect of Ras in tumours and control cell proliferation. In fact, our previous study has already led to the identification of the first generation of anti-Ras drugs that work on this principle.Read moreRead less
Identification And Characterization Of Novel PI3-kinase Signal Transducing Elements In Platelets
Funder
National Health and Medical Research Council
Funding Amount
$457,500.00
Summary
Platelets play an important role in blood clotting and blood vessel repair. Upon vessel injury, platelets rapidly adhere to the site of damage where they undergo dramatic shape change to spread over the site of injury. Activation and regulation of these processes relies on a complex network of signal transduction processes, involving the integration of multiple receptors and pathways. One pathway demonstrated to play a role in regulating platelet responses is the enzyme phosphatidylinositol 3-ki ....Platelets play an important role in blood clotting and blood vessel repair. Upon vessel injury, platelets rapidly adhere to the site of damage where they undergo dramatic shape change to spread over the site of injury. Activation and regulation of these processes relies on a complex network of signal transduction processes, involving the integration of multiple receptors and pathways. One pathway demonstrated to play a role in regulating platelet responses is the enzyme phosphatidylinositol 3-kinase (PI3-kinase) and its lipid products PtdIns(3,4,5)P3 and PtdIns(3,4)P2. However, very little is known about exactly how PI3-kinase and its products regulate the platelet responses. Our research studies aim to gain a deeper understanding into the molecular mechanisms of PI3-kinase signal transduction in platelets, through the identification and characterization of novel platelet proteins that bind to PI3-kinase lipid products, and to define what role these proteins play in platelet PI3-kinase dependent responses.Read moreRead less
The Role Of Plasma Membrane Microdomains In Regulating Ras-dependent Raf Activation
Funder
National Health and Medical Research Council
Funding Amount
$216,100.00
Summary
In human cancers one or more of the signaling pathways leading from growth factor receptors at the cell surface to the nucleus where cell division is initiated are subverted. For example, a protein called Ras, that regulates a series of major signaling pathways, is mutated in 25% of all human tumours. This leaves Ras and the signaling pathways permanently switched on causing uncontrolled cell proliferation. Our previous work has demonstrated that Ras must be attached to the inner surface of the ....In human cancers one or more of the signaling pathways leading from growth factor receptors at the cell surface to the nucleus where cell division is initiated are subverted. For example, a protein called Ras, that regulates a series of major signaling pathways, is mutated in 25% of all human tumours. This leaves Ras and the signaling pathways permanently switched on causing uncontrolled cell proliferation. Our previous work has demonstrated that Ras must be attached to the inner surface of the cell membrane in order to function properly. This project now seeks to understand exactly how Ras attaches to and interacts with specific sites in the plasma membrane. Its is becoming clear that different isoforms of Ras, called H-, N- and K-ras have different functions in the cell which may in turn result from their different sites of attachment to the cell membrane. This is important because by understanding the precise micro-environment in which the different Ras proteins operate and how they activate subsequent proteins in their signaling networks we will be in a good position to design drugs that selectively compromise the function of each specific Ras isoform. A highly relevant example is provided by K-ras which is mutated in 90% of all pancreatic cancers and 50% of all colon cancers. Clearly the clinical impact of a drug that could selectively neutralise K-Ras function in these tumours is potentially enormous.Read moreRead less
Ras Signalling And Cholesterol Efflux From Late Endosomes
Funder
National Health and Medical Research Council
Funding Amount
$276,598.00
Summary
Accumulation of cholesterol is a hallmark of early atherosclerotic lesions, known as foam cell formation. Hence the stimulation of cholesterol removal (efflux) from macrophages has great therapeutic potential. High Density Lipoproteins (HDL) and apolipoprotein A-I (apoA-I) stimulate efflux via activation of HDL-apoA-I receptors and poorly understood signalling pathways. This application is investigating the role of the Ras-MAPK signalling pathway in promoting efflux from late endosomes.
Functional Characterization Of Caveolae And Caveolins
Funder
National Health and Medical Research Council
Funding Amount
$140,660.00
Summary
This project aims to study the cellular machinery that allows a cell to respond to its external environment. Specifically, this project focusses on the function of a family of membrane proteins, called caveolins, which are the major protein components of caveolae small pits which cover the surface of many mammalian cells. Caveolins are believed to regulate signalling from the external environment to the cell interior and loss of this regulation leads to uncontrolled growth leading to cancer. Sig ....This project aims to study the cellular machinery that allows a cell to respond to its external environment. Specifically, this project focusses on the function of a family of membrane proteins, called caveolins, which are the major protein components of caveolae small pits which cover the surface of many mammalian cells. Caveolins are believed to regulate signalling from the external environment to the cell interior and loss of this regulation leads to uncontrolled growth leading to cancer. Signalling from the cell surface relies on organisation of signalling components into modules. Our studies suggest that these modules are dependent on specific lipid molecules which form discrete patches, called lipid rafts, on the cell surface. We have hypothesised that caveolins control the lipid molecules associated with lipid rafts and so, indirectly, control signalling pathways. In particular, we have shown that caveolin is important in the regulation of cellular cholesterol, a vital molecule involved in maintaining the function of lipid raft domains. As numerous human diseases are associated with cholesterol imbalance, studies of caveolins can give fundamental new insights into this process, and the previously unidentified links between the cellular lipid balance and signal transduction. This project aims to use mutant caveolin molecules to disrupt caveolin function and so determine the role of caveolin in lipid regulation and in signal transduction. We will then use a lower vertebrate model system, which is amenable to experimental manipulation, to determine the role of caveolins and rafts in the development of the whole embryo.Read moreRead less
Development Of Resonance Energy Transfer Technologies To Detect GPCR Heterodimer Complexes In Living Cells
Funder
National Health and Medical Research Council
Funding Amount
$205,555.00
Summary
G-protein coupled receptors are proteins at the surface of most cells in the body. They bind to drugs, transmitting signals into cells that change what cells are doing. Recent research indicates that different types of these proteins can interact with each other and when one of these protein combinations binds a drug, it acts differently to when the proteins act separately. The aim of our project is to find out which protein combinations exist and to find drugs that bind to them specifically.
Identification And Characterization Of Substrates Of Tyrosine Kinases Involved In Hematopoiesis And Leukemia
Funder
National Health and Medical Research Council
Funding Amount
$241,527.00
Summary
The development and maintenance of tissues in mammals are tightly controlled and complex processes involving the growth, maturation and survival of vast numbers of cells of various types. In cancer, the cell's capacity to faithfully regulate these processes is diminished or lost. Many of the proteins that are essential for growth control are produced by an important class of genes called proto-oncogenes; literally, the prototypes of cancer-causing genes. Naturally occurring mutations in these ge ....The development and maintenance of tissues in mammals are tightly controlled and complex processes involving the growth, maturation and survival of vast numbers of cells of various types. In cancer, the cell's capacity to faithfully regulate these processes is diminished or lost. Many of the proteins that are essential for growth control are produced by an important class of genes called proto-oncogenes; literally, the prototypes of cancer-causing genes. Naturally occurring mutations in these genes have been identified in man and are likely to play a major role in the initiation and progression of distinct human malignancies. A significant number of proto-oncogenes are enzymes called protein tyrosine kinases (PTKs). Research has shown that the function of PTKs is to relay growth signals or other regulatory signals from the outer surface of the cell to specific target proteins inside the cell. These target proteins are needed to relay the signal to other target molecules and so on. This highly ordered process, involving a specific sequence of proteins, ensures that cells respond appropriately to a given signal. Our research focuses on identifying and studying the immediate targets of PTKs with the broad aim of understanding how PTKs control growth in normal and cancerous cells. We have recently developed a method that has enabled us to identify a new protein that may regulate the growth of blood cells. The research proposed here aims to extend our preliminary observations showing that the growth of specific types of blood cells is inhibited by this protein. We also plan to search for new targets of a PTK that is involved in leukemia. The findings of this research will provide important insight into how blood cells are regulated in health and disease.Read moreRead less
Molecular Mechanisms Underlying G Protein Coupled Receptor Signaling
Funder
National Health and Medical Research Council
Funding Amount
$596,956.00
Summary
The maintenance of optimum health and function of living cells, and consequently that of the whole organism, depends on how cells respond to a multitude of physical and chemical stimuli that continually bombard them. The majority of the chemical stimuli such as hormones and neurotransmitters impart their actions not by directly entering the cell, but instead, by binding to a specific receiver protein at the cell surface called a receptor. In one class of such receptors called G protein-coupled r ....The maintenance of optimum health and function of living cells, and consequently that of the whole organism, depends on how cells respond to a multitude of physical and chemical stimuli that continually bombard them. The majority of the chemical stimuli such as hormones and neurotransmitters impart their actions not by directly entering the cell, but instead, by binding to a specific receiver protein at the cell surface called a receptor. In one class of such receptors called G protein-coupled receptors, the transmission of the message to the interior of the cell involves yet another protein called G protein. These receptors are the most abundant type of cell surface receptors and form the targets for nearly 50% of currently used therapeutic drugs. It is, therefore, extremely important to unravel how each of these components works, and in particular to know how they work in living cells. This project utilizes state-of-the-art methodologies to examine interactions between receptors and their cognate G proteins, in living cells and in real-time. The work will answer fundamental questions about the nature of G protein-coupled receptor signaling and will aid in the future development of more effective therapeutic agents.Read moreRead less