Recycling Endosomes Governing Cell Polarity And Cytokine Secretion.
Funder
National Health and Medical Research Council
Funding Amount
$958,412.00
Summary
Cytokines are chemical messengers released by cells to mount inflammatory responses to fight infections. The timing and direction of cytokine release must be tightly regulated. We investigate the cellular compartments and molecules that control cytokine secretion using sophisticated live cell imaging. Uncontrolled cytokine release is the main cause of ongoing inflammation in arthritis and inflammatory bowel disease and our studies aim to identify cellular targets for new drug development.
Cytokine Secretion: A Model For Protein Trafficking.
Funder
National Health and Medical Research Council
Funding Amount
$204,111.00
Summary
TNF-a is an inflammatory cytokine with important roles in host defense, tumour regulation and energy homeostatis, however the oversecretion of TNF-a is also a major cause of septic shock, rheumatoid arthritis, Chron?s disease and the cachexia of cancer. TNF-a synthesis and its release from the surface of cells are relatively well understood. However little is known about its trafficking through the secretory pathway of cells. Understanding this process has the potential to provide new ways of co ....TNF-a is an inflammatory cytokine with important roles in host defense, tumour regulation and energy homeostatis, however the oversecretion of TNF-a is also a major cause of septic shock, rheumatoid arthritis, Chron?s disease and the cachexia of cancer. TNF-a synthesis and its release from the surface of cells are relatively well understood. However little is known about its trafficking through the secretory pathway of cells. Understanding this process has the potential to provide new ways of controlling the secretion of TNF-a. In previous work we have characterized transport vesicles and cytoskeletal proteins involved in secretory pathways of epithelial cells. We now propose to focus on the characterization of transport vesicles, and the roles of actin and myosins involved in TNF-a secretion in macrophages. These studies will rely on introducing new technology to this line of research. Fluorescent tagged constructs of TNF-a will be expressed and viewed in living cells to analyse the secretory pathway and measure the transport of TNF-a from its site of accumulation in the Golgi complex to the cell surface. This work aims to identify membrane-bound vesicles and vesicle-associated proteins that target TNF-a for secretion. We will begin to investigate the role of actin and myosins, using drugs and microinjected peptides to block their function. Overall these studies will provide important cell biological information about protein trafficking in cells. Cytokine secretion is important in immunity and cancer, information important to both fields will be gained from these studies.Read moreRead less
Physiological Significance Of Cellular Translocation Of The Intestine-specific Homeodomain Protein Cdx2
Funder
National Health and Medical Research Council
Funding Amount
$196,527.00
Summary
Ulcerative colitis and Crohn's disease are debilitating inflammatory diseases of the bowel. Conservative estimates (Australian Crohn's and Colitis Association) suggest that at least 23,000 Australians are affected (>1 in 1000). Ten years after onset, there is an estimated risk of 0.5-1.0% per year of pancolitis patients developing full-blown bowel cancer. Current therapies for colon cancer are not very effective and the median survival for patients with metastatic disease is poor at 7-12 mont ....Ulcerative colitis and Crohn's disease are debilitating inflammatory diseases of the bowel. Conservative estimates (Australian Crohn's and Colitis Association) suggest that at least 23,000 Australians are affected (>1 in 1000). Ten years after onset, there is an estimated risk of 0.5-1.0% per year of pancolitis patients developing full-blown bowel cancer. Current therapies for colon cancer are not very effective and the median survival for patients with metastatic disease is poor at 7-12 months. It is therefore important to increase our understanding of the biology underlying these inflammatory conditions so that more effective treatments may be developed and fewer patients proceed to the cancerous stage. We have recently demonstrated a novel interaction between two proteins that may be relevant to intestinal inflammation. Surprisingly, the two proteins would not normally be expected to coincide with each other because of their different localisations within cells and tissues. The first protein, Cdx2, is only synthesised by intestinal lining cells and normally resides in the nucleus where it activates genes that play a role in the highly specialised absorptive functions of the intestine. The other protein, acrogranin-granulin, is more widely distributed in the body and is generally transported out of cells shortly after it has been made. It has been shown to interact with receptors on epithelial cells and blood cells and promotes their growth. In this proposal we will be investigating whether the complex formed between Cdx2 and granulin is important for normal physiology. Moreover since elevated levels of granulin are associated with inflammation, we aim to determine whether the Cdx2-granulin complex is formed during the active phase of ulcerative colitis and Crohn's disease. Specifically, we will test the hypothesis that the Cdx2-granulin complex plays an important role in repairing the damage caused to the lining of the intestine during inflammation.Read moreRead less
Mechanism Of Action Of Sec1p-like Proteins In Membrane Trafficking
Funder
National Health and Medical Research Council
Funding Amount
$234,936.00
Summary
One of the most important evolutionary changes that has occurred is the development of intracellular compartments. All eukaryotic cells possess numerous membrane-encased structures which provide the basis for intracellular specialisation. For example, in order to degrade unwanted components cells have developed degradative enzymes. It is vital for the cell that these enzymes are sequestered away from other cellular components to avoid destruction of valuable molecules. In addition, the cell has ....One of the most important evolutionary changes that has occurred is the development of intracellular compartments. All eukaryotic cells possess numerous membrane-encased structures which provide the basis for intracellular specialisation. For example, in order to degrade unwanted components cells have developed degradative enzymes. It is vital for the cell that these enzymes are sequestered away from other cellular components to avoid destruction of valuable molecules. In addition, the cell has developed a complex assembly line of modifications that are added to proteins in a specific order as they travel to their final destination within the cell. This necessitates the accurate passage of molecules between compartments, a process known as vesicle transport. To orchestrate the complex network of vesicular transport steps between all of the various intracellular compartments it is necessary to employ complex machinery to guide and check that these steps occur with high fidelity. The goal of our research proposal is to define the function of one of the molecules involved in this control process, the so-called Sec1p proteins. The strength of our proposal lies in the diversity of our approach. We intend to explore the molecular advantages of a relatively simple eukaryotic organism, a yeast cell, and apply the findings obtained from this cell to a more complex but highly related vesicular transport process; that of the insulin-regulated movement of a glucose transporter in mammalian fat and muscle cells. While we intend to apply our findings to the treatment of patients with diabetes, it is our ultimate goal to be able to learn more about this fundamental cell biological process so that we can apply our knowledge to understanding many different disease states.Read moreRead less
LPS-regulated SNAREs And Control Of Cytokine Secretion In Macrophages.
Funder
National Health and Medical Research Council
Funding Amount
$470,750.00
Summary
TNF(tumour necrosis factor alpha) is a potent proinflammatory cytokine secreted by immune activated macrophages. TNF has essential roles in host defense, tumour killing and energy metabolism. Excessive secretion of TNF in acute and chronic inflammatory conditions, such as septic shock, Crohn s disease, rheumatoid arthritis and in cancer has many severe, even fatal, consequences. Improved anti-TNF therapeutics are needed for clinical management in all of these conditions. Our studies are focused ....TNF(tumour necrosis factor alpha) is a potent proinflammatory cytokine secreted by immune activated macrophages. TNF has essential roles in host defense, tumour killing and energy metabolism. Excessive secretion of TNF in acute and chronic inflammatory conditions, such as septic shock, Crohn s disease, rheumatoid arthritis and in cancer has many severe, even fatal, consequences. Improved anti-TNF therapeutics are needed for clinical management in all of these conditions. Our studies are focused on investigating how macrophages synthesize and secrete TNF, with the ultimate goal of characterizing the molecules and vesicles in the TNF secretory pathway. Our recent findings show the expression of SNARE proteins, part of the vesicle docking and fusion machinery, is regulated in concert with cytokine secretion and other trafficking changes in activated macrophages. We identified the proteins Syntaxin4, Munc-18c and SNAP-23 as the specific t-SNARE complex that regulates TNF delivery to the cell surface. In the proposed studies we will investigate how SNAREs are regulated during macrophage activation by studying their gene expression and protein modifications. We have developed a single-cell assay to measure TNF trafficking in macrophages; this allows the identification of molecules with roles in TNF secretion and it will be used in a series of experiments to identify the specific v-SNARE proteins that partner the t-SNARE for TNF delivery. Finally we will use live cell imaging to investigate how and where TNF is delivered to the macrophage cell surface and membrane fractionation to examine a role for membrane microdomains in organizing SNARE-mediated TNF secretion. Manipulation of SNAREs, using data generated by these studies, holds potential for the development of new anti-TNF therapies.Read moreRead less