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
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 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
The Interaction Between CD46 And PSD-95/Dlg-4: Roles In Cell Polarisation And CD46 Signalling.
Funder
National Health and Medical Research Council
Funding Amount
$70,000.00
Summary
Immune defence against pathogens is primarily achieved by the activities of a range of blood cells, including T cells. T cells have specialised functions involving direct killing of the pathogen, and recruitment and activation of other immune cells. Many of these functions require the lymphocyte to become polarised, or asymmetric, in order to concentrate the appropriate cellular machinery towards the site of activity. Examples of polarisation in lymphocytes includes (i) the formation of a single ....Immune defence against pathogens is primarily achieved by the activities of a range of blood cells, including T cells. T cells have specialised functions involving direct killing of the pathogen, and recruitment and activation of other immune cells. Many of these functions require the lymphocyte to become polarised, or asymmetric, in order to concentrate the appropriate cellular machinery towards the site of activity. Examples of polarisation in lymphocytes includes (i) the formation of a single protrusion, or uropod, that forms the basis for cell-cell interactions, (ii) the formation of an immune synapse which allows a T cell to recognise a pathogen, and (iii) the direction of the cellular killing machinery towards the target. The process of cell polarisation is best characterised in neurons and epithelial cells, both of which are asymmetric. In each cell type, a major mechanism of regulating polarisation is the expression and targeting of a family of proteins containing regions called PDZ domains. PDZ domains mediate protein-protein interactions and so allow the assembly of large molecular scaffolds which hold proteins in specific cell sites. The loss of cell polarity in some cells is thought to cause uncontrolled proliferation and tumour progression, and some of the PDZ-containing proteins are tumour suppressors. We have identified a PDZ-containing protein that is polarised in T cells, and have evidence that this protein interacts with and controls the polarisation of a cell surface receptor whose functions include the regulation of T cell function and proliferation. The aim of this proposal is to determine the mechanisms and functional consequences of polarisation of these two proteins in T cells, and to determine whether their interaction or polarisation is important for T cell proliferation.Read moreRead less
Immune defence against pathogens is primarily achieved by the activities of a range of blood cells, including T cells. T cells have specialised functions involving direct killing of the pathogen, and recruitment and activation of other immune cells. Many of these functions require the lymphocyte to become polarised, or asymmetric, in order to concentrate the appropriate cellular machinery towards the site of activity. Examples of polarisation in lymphocytes includes (i) the formation of a single ....Immune defence against pathogens is primarily achieved by the activities of a range of blood cells, including T cells. T cells have specialised functions involving direct killing of the pathogen, and recruitment and activation of other immune cells. Many of these functions require the lymphocyte to become polarised, or asymmetric, in order to concentrate the appropriate cellular machinery towards the site of activity. Examples of polarisation in lymphocytes includes (i) the formation of a single protrusion, or uropod, that forms the basis for cell-cell interactions, (ii) the formation of an immune synapse which allows a T cell to recognise a pathogen, and (iii) the direction of the cellular killing machinery towards the target. The process of cell polarisation is best characterised in neurons and epithelial cells, both of which are asymmetric. In each cell type, a major mechanism of regulating polarisation is the expression and targeting of a family of proteins containing regions called PDZ domains. PDZ domains mediate protein-protein interactions and so allow the assembly of large molecular scaffolds which hold proteins in specific cell sites. The loss of cell polarity in some cells is thought to cause uncontrolled proliferation and tumour progression, and some of the PDZ-containing proteins are tumour suppressors. We have identified a PDZ-containing protein that is polarised in T cells, and have evidence that this protein interacts with and controls the polarisation of a cell surface receptor whose functions include the regulation of T cell function and proliferation. The aim of this proposal is to determine the mechanisms and functional consequences of polarisation of these two proteins in T cells, and to determine whether their interaction or polarisation is important for T cell proliferation.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