Mechanism Of Action Of Sec1p-like Proteins In Membrane Trafficking.
Funder
National Health and Medical Research Council
Funding Amount
$440,250.00
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
Structural Characterisation Of SNARE Protein Complexes Involved In Insulin-regulated Glucose Transport
Funder
National Health and Medical Research Council
Funding Amount
$320,803.00
Summary
Insulin-regulated glucose transportation is defective in type 2 diabetes, a disease that is a major health problem worldwide and in some cases can lead to death. The aim of this work is to investigate the molecular structure and function of proteins critical to the transportation and delivery of glucose to muscle and fat cells, which will lead to the validation of new therapeutic targets and the development of new treatments for diabetes.
Structural Studies On SNARE Proteins Involved In Insulin Action
Funder
National Health and Medical Research Council
Funding Amount
$308,263.00
Summary
Diabetes mellitus, a disease characterised by high blood glucose levels, is caused by a relative or absolute deficiency in the activity of insulin. The blood-glucose lowering action of insulin is a result of its ability to stimulate glucose uptake by fat and muscle cells. A major goal of Professor James' laboratory is to identify molecules that are involved in this insulin-regulated uptake of glucose. Professor James has identified and characterised the glucose transporter, GLUT4, a protein that ....Diabetes mellitus, a disease characterised by high blood glucose levels, is caused by a relative or absolute deficiency in the activity of insulin. The blood-glucose lowering action of insulin is a result of its ability to stimulate glucose uptake by fat and muscle cells. A major goal of Professor James' laboratory is to identify molecules that are involved in this insulin-regulated uptake of glucose. Professor James has identified and characterised the glucose transporter, GLUT4, a protein that is normally stored inside muscle and fat cells. In response to insulin stimulation, GLUT4 moves to the cell surface where it functions to transport glucose into the cell. Over the past 5 years Professor James laboratory has, in conjunction with other groups, discovered several key proteins that are involved in the insulin-regulated movement of GLUT4 within the cell. We plan to exploit the therapeutic potential of this biological system by obtaining high resolution three dimensional structures of these key proteins. The resulting structural information will allow us to develop compounds that modify the function of these key proteins. Such compounds could prove useful as novel therapeutic agents in the treatment of diabetes. The purpose of this proposal is to begin to implement this goal. By combining the knowledge and reagents coming out of the work on insulin-regulated glucose transport in Professor James' laboratory with the molecular and structural biology expertise in Dr Martin's, Dr Halliday's and Prof Craik's laboratories we are in a unique position to achieve this highly significant goal.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
Analysis Of The Role Of Vesicle Docking/Fusion Proteins In Trafficking Of The Glut4 Glucose Transporter In Adipocytes
Funder
National Health and Medical Research Council
Funding Amount
$212,036.00
Summary
The objective of these studies is to understand the molecular mechanisms that are involved in the control of blood glucose levels by the hormone insulin. Elevated blood glucose levels following a meal stimulate the pancreas to release insulin into the circulation. Insulin acts to reduce blood sugar levels by stimulating the uptake of glucose into fat and muscle and suppressing glucose production by the liver. Defects in insulin action in these tissues are the primary cause of Type II diabetes. T ....The objective of these studies is to understand the molecular mechanisms that are involved in the control of blood glucose levels by the hormone insulin. Elevated blood glucose levels following a meal stimulate the pancreas to release insulin into the circulation. Insulin acts to reduce blood sugar levels by stimulating the uptake of glucose into fat and muscle and suppressing glucose production by the liver. Defects in insulin action in these tissues are the primary cause of Type II diabetes. The debilitating effects of Type II diabetes, the dramatic increase its incidence, and the expense of treating the symptoms of diabetic complications have lead to the realization that the disease represents a major health problem requiring substantial research and development efforts. The project will focus on insulin regulation of glucose uptake in fat cells. Insulin promotes glucose uptake into fat by activating an intracellular signaling pathway that triggers the translocation of a unique glucose transporter protein (Glut4) from storage sites inside the cell to the cell surface. Glut4 translocation is mediated by small membrane vesicles that function to sequester the glucose transporter inside cells in the absence of insulin, and to shuttle Glut4 to the cell surface in response to the hormone. Despite the central importance of this event to the maintenance of normal blood glucose levels, it is poorly understood. The studies will be directed towards investigating the cellular machinery involved in the latter stages of insulin-stimulated glucose uptake- the vesicle-mediated delivery of Glut4 to the cell surface. The objective of these studies is to better understand the molecular basis for Glut4 translocation, and regulation by the insulin signaling cascade. Accomplishment of this goal may suggest potential drug intervention strategies aimed at enhancing insulin-stimulated Glut4 translocation and promoting improved control of blood glucose levels in Type II diabetes.Read moreRead less
Insulin resistance (the inability of ordinarily insulin-sensitive tissues such as muscle and adipose tissue to respond to insulin) contributes to a number of diseases including diabetes and obesity. A key metabolic step in these tissues is the uptake of glucose from the blood stream. This step is accelerated by insulin thus allowing efficient clearance of glucose from the bloodstream after a meal. Our laboratory has played a major role in showing that insulin regulates glucose uptake into muscle ....Insulin resistance (the inability of ordinarily insulin-sensitive tissues such as muscle and adipose tissue to respond to insulin) contributes to a number of diseases including diabetes and obesity. A key metabolic step in these tissues is the uptake of glucose from the blood stream. This step is accelerated by insulin thus allowing efficient clearance of glucose from the bloodstream after a meal. Our laboratory has played a major role in showing that insulin regulates glucose uptake into muscle and adipose tissue by stimulating the movement of a glucose transport protein from inside the cell to the cell surface (see http:--www.imb.uq.edu.au-groups-james-glut4 for an animated description of this process). The purpose of this proposal is to dissect the molecular mechanisms by which this glucose transporter can be held inside the cell in the absence of insulin and then allowed to be released from this site moving to the surface in the presence of insulin. Our studies over the past 5 years have brought us much closer to understanding this process in detail. The identification of the molecules responsible for this regulatory step will not only aid our understanding of this process but it will also provide a valuable target for development of therapeutic agents that can be used to combat insulin resistance.Read moreRead less