Functional Characterisation Of N4WBP5 And N4WBP5A, Novel Nedd4-interacting Proteins
Funder
National Health and Medical Research Council
Funding Amount
$480,750.00
Summary
The proteins that make up a cell must be correctly localised in order to perform their normal function. Specialised cellular activities are carried out in distinct compartments within a cell and proteins must correctly localise in them and traffic between them. Intracellular protein trafficking is a highly regulated process involving many components. Recent findings have shown that intracellular trafficking is regulated in many cases by distinct protein modifications. One such modification is ta ....The proteins that make up a cell must be correctly localised in order to perform their normal function. Specialised cellular activities are carried out in distinct compartments within a cell and proteins must correctly localise in them and traffic between them. Intracellular protein trafficking is a highly regulated process involving many components. Recent findings have shown that intracellular trafficking is regulated in many cases by distinct protein modifications. One such modification is tagging of a small protein called ubiquitin to proteins that are being trafficked. A focus of research in our laboratory is the study of a protein, called Nedd4, which directly tags proteins with ubiquitin. We have recently identified two novel proteins that interact with Nedd4 and localise to distinct subcellular compartments that are sites for the correct sorting and delivery of proteins trafficking within the cell. The main aim of our proposal is to characterise how these proteins function. We propose that these proteins are involved in intracellular trafficking and that they may function by targeting Nedd4 to the cellular trafficking machinery. This may be required for Nedd4 to tag molecules with ubiquitin that are involved in intracellular trafficking. Our experiments will test the functional relationship between Nedd4 and the novel proteins and determine the particular trafficking pathways in which these proteins are involved. Defects in cellular processes regulated by Nedd4 and other similar proteins cause a number of human diseases including an inherited form of hypertension and a specific group of cancers. In addition, a large number of human diseases result directly from defects which disrupt intracellular trafficking pathways. The results of this study will provide further insight into this essential cellular process and may ultimately contribute to the development of therapies for diseases resulting from defects in intracellular trafficking.Read moreRead less
Protein / Protein Interactions Important For AMP-activated Protein Kinase Regulation
Funder
National Health and Medical Research Council
Funding Amount
$242,545.00
Summary
The AMP-activated protein kinase (AMPK) is an enzyme that monitors the energy levels of the body. When oxygen and nutrient levels decrease, the energy levels of a cell also decrease leading to activation of the AMPK. This results in activation of energy-producing pathways and inhibition of energy-consuming pathways, allowing cells to match supply with demand to ensure their survival. The AMPK comprises of three proteins that together form a functional enzyme. In this application I aim to obtain ....The AMP-activated protein kinase (AMPK) is an enzyme that monitors the energy levels of the body. When oxygen and nutrient levels decrease, the energy levels of a cell also decrease leading to activation of the AMPK. This results in activation of energy-producing pathways and inhibition of energy-consuming pathways, allowing cells to match supply with demand to ensure their survival. The AMPK comprises of three proteins that together form a functional enzyme. In this application I aim to obtain a thorough understanding of the molecular basis of how the AMPK functions. I will determine how and where the three proteins interact with each other and determine where in a cell at any given time the AMPK can be found. This is an important question to answer because many proteins are inactive within the cytoplasm but when they are bound to the plasma membrane they are active. I have previously found the AMPK to be localized to the cytoplasm, membrane and nuclear compartments of the cell, but little is known about the AMPK s function in these different locations. Activation of the AMPK is known to depend on another protein that is also activated when cellular energy levels decrease. This protein has remained elusive to many researchers over the past few years. I plan to identify this protein using new bioinformatics together with the vast amount of information provided by the sequencing of the human genome. Exercise and reduced caloric intake activate the AMPK, these are associated with health benefits and reduce the risk of cardiovascular and neurodegenerative diseases, diabetes and obesity. For these reasons information on the role of the AMPK may improve our understanding of the reasons these diseases develop.Read moreRead less
Inside our cells is a complex traffic system. The vehicles are vesicles that come in different shapes and sizes and travel to specific destinations in the cell to deliver cargo such as: surface growth factor receptors that are to have their signalling terminated, proteins and lipids destined for the cell wall for growth or development (like neurite outgrowth) and proteins and hormones destined for secretion (like neurotransmitter release). More than 100 human genetic disorders map to defects in ....Inside our cells is a complex traffic system. The vehicles are vesicles that come in different shapes and sizes and travel to specific destinations in the cell to deliver cargo such as: surface growth factor receptors that are to have their signalling terminated, proteins and lipids destined for the cell wall for growth or development (like neurite outgrowth) and proteins and hormones destined for secretion (like neurotransmitter release). More than 100 human genetic disorders map to defects in one of the components of this system. Proteins called small GTPases provide order for this traffic and allow specific cargo to reach specific destinations. They regulate cell functions by acting as switches, turning biochemical processes on and off inside the cell. Ral is a small GTPase enzyme found in brain and broadly distributed in other cells. We have discovered that Ral is part of a large signalling complex. When activated Ral stimulates effectors, either the exocyst or RalBP1. In turn, mild oxidative stress controls a Ral inhibitor protein called ERp57. The research proposed aims to establish the functional role for the Ral signalling complex in cells. We will determine with which vesicle trafficking events Ral is associated, which effector it utilises in that pathway, and how that effector directs the traffic. We will also map the steps that may lead to inactivation of Ral via ERp57 in cells, and propose that this is mediated by mild oxidative stress. Techniques of molecular biology, biochemistry, molecular biology, proteomics and microscopy will be used to establish these functions. The research will lead to increased knowledge of the significance of this protein to cellular and particularly neuronal cell function. This forms the basis for understanding normal cell function and for identification of further factors causing diseases of vesicle transport. In time, such research aids in the development of specific therapies for sufferers of such diseases.Read moreRead less
The regulation of signalling molecules in Saccharomyces Cerevisiae by inositol polyphosphate 5-phosphatases. Phosphoinositide signalling molecules regulate the actin cytoskeleton, secretion, vesicular trafficking and cell growth and death. We have identified, cloned and characterised a family of signal terminating enzymes called inositol polyphosphate 5-phosphatases (5-phosphatases) that regulate phosphoinositide signalling molecules. We have cloned and characterised four distinct 5-phosphatases ....The regulation of signalling molecules in Saccharomyces Cerevisiae by inositol polyphosphate 5-phosphatases. Phosphoinositide signalling molecules regulate the actin cytoskeleton, secretion, vesicular trafficking and cell growth and death. We have identified, cloned and characterised a family of signal terminating enzymes called inositol polyphosphate 5-phosphatases (5-phosphatases) that regulate phosphoinositide signalling molecules. We have cloned and characterised four distinct 5-phosphatases in the yeast Saccharomyces Cerevisiae and demonstrated by both deletion and overexpression studies that these enzymes regulate the actin cytoskeleton, endocytosis and secretion. This research proposal aims to investigate the signalling complexes the 5-phosphatases form with specific actin binding and or regulatory proteins, investigate the complex interactions of phosphoinositide lipid phosphatases and the roles they play in regulating secretion from the endoplasmic reticulum and finally characterize a novel 5-phosphatase that we have recently identified. Collectively the outcome of these studies will provide novel information about the functionallly significant signalling pathways regulated by this important enzyme family.Read moreRead less
The role of PtdIns(4,5)P2 in cellular responses in Saccharomyces cerevisiae. This grant application falls under the criteria of frontier technologies in genomics/phenomics and complex systems. We are characterizing a highly conserved network of signaling molecules regulated by complex large families of enzymes that regulate the bending of membranes, and cellular events including cell division in plants, yeast and mammalian cells. We have developed cutting edge novel technologies to localize sign ....The role of PtdIns(4,5)P2 in cellular responses in Saccharomyces cerevisiae. This grant application falls under the criteria of frontier technologies in genomics/phenomics and complex systems. We are characterizing a highly conserved network of signaling molecules regulated by complex large families of enzymes that regulate the bending of membranes, and cellular events including cell division in plants, yeast and mammalian cells. We have developed cutting edge novel technologies to localize signaling on specific intracellular membranes and visualise the role cellular lipids play in forming tubules in cells. This project will result in the presentation of Australian research at international forums and support the training of PhD students.Read moreRead less
The Interactions Of Dengue Virus RNA Dependent RNA Polymerase (NS5) With Other Viral And Host Factors.
Funder
National Health and Medical Research Council
Funding Amount
$170,165.00
Summary
Dengue fever is a mosquito-borne disease that is prevalent in tropical countries. It is estimated that 40% of the global population is at risk of dengue infection. Classical dengue fever is not life threatening. However, the more serious disease, dengue haemorrhagic fever-shock syndrome requires intensive medical attention to prevent fatality. A significant number of deaths are recorded each year especially in the underdeveloped countries. Dengue is periodically also a problem in northern Austra ....Dengue fever is a mosquito-borne disease that is prevalent in tropical countries. It is estimated that 40% of the global population is at risk of dengue infection. Classical dengue fever is not life threatening. However, the more serious disease, dengue haemorrhagic fever-shock syndrome requires intensive medical attention to prevent fatality. A significant number of deaths are recorded each year especially in the underdeveloped countries. Dengue is periodically also a problem in northern Australia. There is no cure for dengue fever. The present research aims to use a knowledge-based approach to develop novel antiviral strategies based on preventing the critical protein interactions required for the normal virus life cycle. Two of the most important proteins involved in dengue virus replication are called the NS3 and NS5 proteins. The protein-protein interaction (contact) that occurs between NS5 and NS3 is crucial for the replication of the virus. Little is known about this interaction at present, and the studies we propose will directly address this issue. We have previously shown that a 37 amino acid in the middle of NS5 contains a nuclear localisation signal that can target the normally cytoplasmic protein to the nucleus of the infected cell. What the function of this protein is in the nucleus is not known. We will use a technique called the yeast two-hybrid test to address the question of dengue virus protein interactions in the common bakers yeast. This method is very sensitive and powerful and will provide important insights that will contribute to the development of a rapid high-throughput test to screen the extensive extract collection from Australia's marine biodiversity, held by the Australian Institute of Marine Sciences, to discover suitable inhibitors of NS3-NS5 interaction.Read moreRead less
FHA Domain-dependent Functions Of Cell Cycle Checkpoint Kinases
Funder
National Health and Medical Research Council
Funding Amount
$235,500.00
Summary
Human chromosomes as carriers of the genetic information are constantly subjected to DNA damage. This usually occurs spontaneously, simply as a result of oxidation of DNA residues as a byproduct of cellular energy consumption or as a result of errors during chromosome duplication in growing cells, and is compounded by chemical or physical agents, for example carcinogens, UV rays or X-rays. DNA damage can have severe consequences if not properly repaired, leading to genomic instability with loss ....Human chromosomes as carriers of the genetic information are constantly subjected to DNA damage. This usually occurs spontaneously, simply as a result of oxidation of DNA residues as a byproduct of cellular energy consumption or as a result of errors during chromosome duplication in growing cells, and is compounded by chemical or physical agents, for example carcinogens, UV rays or X-rays. DNA damage can have severe consequences if not properly repaired, leading to genomic instability with loss of vast tracts of DNA or inappropriate genome rearrangements, that may ultimately give rise to cancer. To prevent such dire consequences, all organisms from yeast to man contain molecular checkpoints that sense the presence of DNA damage and then activate a cellular response program that includes damage repair and prevention of cell division while damage persists. These molecular checkpoints are highly conserved throughout evolution which allows us to analyse the details involved in simple organisms such as yeast, to draw general conclusions on their function in more complex human cells. Along these lines, we are studying the function of two yeast proteins that are similar to the human Chk2 protein, a tumour suppressor that is mutated in a subset of families suffering from the Li-Fraumeni multi-cancer syndrome. We have identified new pathways by which these proteins contribute to the survival of cells after treatment with DNA damaging agents and will further charaterise these in the present proposal.Read moreRead less
Inhibition Of Nef-activated Src-family Kinases By CHK
Funder
National Health and Medical Research Council
Funding Amount
$514,307.00
Summary
HIV hijacks infected blood cells to produce its own proteins. Nef is one of these proteins and Nef alone is sufficient to cause an AIDS-like disease. Recently, we discovered that a protein called CHK can inhibit Nef. Our research will determine how CHK inhibits Nef and test the feasibility of drugs based on CHK. Such drugs would slow AIDS progression, assisting conventional therapies and patients' immune systems to combat the infection, leading to longer, healthier, more productive lives.