A Functional Interaction Between Domains Of The Flavivirus NS5 Protein Presents A New Target For Antiviral Therapy
Funder
National Health and Medical Research Council
Funding Amount
$502,891.00
Summary
Mosquito-transmitted flaviviruses such as dengue, yellow fever, Japanese encephalitis and West Nile infect hundreds of millions of people and cause debilitating and fatal diseases. Developing anti-viral treatments against these diseases is a high priority. Our strategy is to develop small molecules that can bind to specific sites on viral proteins and prevent the virus from replicating and causing disease.
Engineered Histones As DNA Carriers With Application In Therapeutic Gene Delivery
Funder
National Health and Medical Research Council
Funding Amount
$417,750.00
Summary
We intend to apply our knowledge of protein transport to the nucleus to enhance the delivery of DNA to target cells. This relates to the use of gene therapy to treat genetic defects such as inborn errors of metabolism, where a disease-causing lack-of-function mutation can be overcome by engineering cells within the organism which express, in the necessary quantities and in response to the appropriate regulatory signals, the particular component which is lacking. A limiting factor in gene therapy ....We intend to apply our knowledge of protein transport to the nucleus to enhance the delivery of DNA to target cells. This relates to the use of gene therapy to treat genetic defects such as inborn errors of metabolism, where a disease-causing lack-of-function mutation can be overcome by engineering cells within the organism which express, in the necessary quantities and in response to the appropriate regulatory signals, the particular component which is lacking. A limiting factor in gene therapy approaches is the low efficiency of nuclear uptake of introduced DNA, where it has been estimated that < 1% of the DNA taken up is actually expressed. Our proposal seeks to develop approaches to enhance non-viral-mediated gene delivery, in particular by optimising this critical, limiting step of the delivery of exogenous DNA to the nucleus. We intend to apply knowledge from studies of nuclear targeting and chromatin assembly to improve gene transfer technologies. We will build on our work showing that specific signals for nuclear import - nuclear targeting signals (NTSs) - can be used to enhance nuclear gene delivery and expression. Since DNA in the normal cellular context is in the form of chromatin - a specific complex with proteins such as histones - we intend to use reconstituted chromatin as the transfecting DNA, whereby histones engineered to include NTSs and other modular sequence elements will be used. Chromatin should not only enable NTSs and other sequence modules to be linked to the DNA but also protect against nuclease-mediated degradation prior to nuclear entry, condense the DNA to enable more efficient cellular-nuclear entry, and ensure expression of the transfected reporter gene by presenting it to the cell in a physiological context. Our approaches should contribute to bringing gene therapy closer to reality in the clinic.Read moreRead less
Characterisation Of Putative Targets Of The Ubiquitin-protein Ligase, Nedd4
Funder
National Health and Medical Research Council
Funding Amount
$258,055.00
Summary
Cellular proteins are synthesised and degraded depending on the metabolic state of the cell. The normal turnover of a number of cellular proteins is mediated by a complex pathway involving a highly conserved polypeptide called ubiquitin. Ubiquitin-dependent proteolysis of a number of proteins is essential for the maintenance of the health of a cell. Many cell cycle proteins, membrane channels, receptors and products of some oncogenes are known targets of the ubiquitin-dependent turnover. Clearly ....Cellular proteins are synthesised and degraded depending on the metabolic state of the cell. The normal turnover of a number of cellular proteins is mediated by a complex pathway involving a highly conserved polypeptide called ubiquitin. Ubiquitin-dependent proteolysis of a number of proteins is essential for the maintenance of the health of a cell. Many cell cycle proteins, membrane channels, receptors and products of some oncogenes are known targets of the ubiquitin-dependent turnover. Clearly, a defect in this tightly regulated mechanism for the downregulation of proteins can result in a pathological condition and therefore it is important to understand how this pathway is regulated at molecular level. In the multistep ubiquitin pathway, some component enzymes called E3 are required for specifying the targets to be degraded. We discovered one such enzyme Nedd4. One of the proteins regulated by Nedd4 is epithelial sodium channel (ENaC). Loss of Nedd4-mediated regulation of ENaC results in Liddle's Syndrome, a genetic form of hypertension. Since Nedd4 is expressed in many tissues and during development, we predicted that Nedd4 may regulate other important proteins in addition to ENaC. We have recently identified several proteins which interact with Nedd4. Some of these proteins are likely to play important roles in cellular regulation and during development. The proposed project is designed to characterise these proteins. We believe that by studying these proteins we will learn a great deal about the cellular regulatory pathways. In summary, Nedd4 is an important protein involved in cellular regulation and has a proven role in human disease. A study of characterising targets of Nedd4 will be vital in understanding the molecular basis of cell regulation and its implication in disease.Read moreRead less
Heterogeneous Nuclear Ribonucleoprotein A2-dependent MRNA Trafficking In The Cytoplasm Of Cells.
Funder
National Health and Medical Research Council
Funding Amount
$346,650.00
Summary
Control of the use of DNA, gene expression, is vital to all living organisms, especially in development and disease. The information in the genes of DNA is transferred to an intermediate molecule, mRNA, in a process called transcription. The genetic information in the mRNA is subsequently used to make the protein encoded by the original gene. The switching on and off of DNA appears to be most frequently controlled at the transcription step but recently it has become apparent that there are many ....Control of the use of DNA, gene expression, is vital to all living organisms, especially in development and disease. The information in the genes of DNA is transferred to an intermediate molecule, mRNA, in a process called transcription. The genetic information in the mRNA is subsequently used to make the protein encoded by the original gene. The switching on and off of DNA appears to be most frequently controlled at the transcription step but recently it has become apparent that there are many post-transcriptional events that govern how efficiently the genetic information is ultimately converted to protein molecules. The RNA molecules may be confined to a small region of the cell, resulting in the localization of the protein produced from it; the RNA may be rapidly degraded or stabilized; and the efficiency of production of the protein from its RNA precursor may be modulated by other molecules. It had previously been shown that the mRNA encoding a protein that is an essential structural component of central nervous system myelin is selectively transported to the regions in the cell where the myelin is made. We have identified the molecule that recognises this RNA as a protein called hnRNP A2 and shown how it selects the RNA molecules that have to be transported from the myriad of RNA moleclues in the cell at any given time. hnRNP A2 was previously thought to be confined to the nuclei of cells, but we have shown that it is also present outside the nucleus and is involved in RNA transport in a variety of cell types. hnRNP A2 appears to be directly involved in rheumatoid arthritis, lung cancer and other cancers. It has been proposed as a diagnostic test for cancer as elevated intracellular levels and circulating antibodies against this protein appear before the cancerous cells are visible under the microscope. The major aim of the proposed project is to explore the molecular mechanism by which hnRNP A2 transports RNA molecules in cells.Read moreRead less
Role Of The Microtubule Network In Nuclear Transport: Potential Use In Gene Delivery
Funder
National Health and Medical Research Council
Funding Amount
$491,767.00
Summary
Transport of key proteins that regulate growth and other processes, into and out of the nucleus, the site of gene transcription, is central to the function of eukaryotic cells. Knowledge of the process is largely based on studies using semi-intact cell systems, however, meaning that the role of cytoskeletal elements in nuclear transport has been largely overlooked. Intriguingly, in vivo studies, including our own relating to signalling molecules important in the regulation of cancer, clearly imp ....Transport of key proteins that regulate growth and other processes, into and out of the nucleus, the site of gene transcription, is central to the function of eukaryotic cells. Knowledge of the process is largely based on studies using semi-intact cell systems, however, meaning that the role of cytoskeletal elements in nuclear transport has been largely overlooked. Intriguingly, in vivo studies, including our own relating to signalling molecules important in the regulation of cancer, clearly implicate the cellular microtubule (MT) network as playing an integral role in nuclear import. We propose to carry out a detailed examination of the mechanistic basis of the dependence of nuclear import on the MT network of 3 molecules regulating cancer. We will compare the properties of these molecules to those of other nuclear localizing molecules, as well as examine the ability to mediate nuclear import of similar MT-associating sequences from viral and other proteins. The results will establish for the first time, the generality of the dependence of nuclear protein import on cytoskeletal elements. Since the movement of large DNA molecules by simple diffusion is a limiting factor in non-viral gene delivery approaches, the possibility of applying this knowledge to facilitate the transport of DNA encoding therapeutic gene products to the nucleus of target cells, will also be assessed for the first time. We will use the modular sequences that confer interaction with the MT network to assist gene transfer by including them in modular constructs we have designed with this in mind. We will thus be able to test directly for the first time whether MT-interaction can enhance the delivery of DNA to the nucleus and reporter gene expression, with obvious application in gene therapy approaches.Read moreRead less
The Role Of Molecular Chaperones And Proteases In Mitochondrial Function
Funder
National Health and Medical Research Council
Funding Amount
$432,750.00
Summary
Mitochondria are essential organelles providing the cell with essential molecules and being the source of oxidative energy in the cell. They are at the centre of many clinical conditions, ranging from genetic to common neurological diseases and other conditions related to ageing. We have been defining the way in which mammalian cells respond to the accumulation of unfolded proteins within the mitochondrial compartment and have found this produces what we have called the Mitochondrial Stress Resp ....Mitochondria are essential organelles providing the cell with essential molecules and being the source of oxidative energy in the cell. They are at the centre of many clinical conditions, ranging from genetic to common neurological diseases and other conditions related to ageing. We have been defining the way in which mammalian cells respond to the accumulation of unfolded proteins within the mitochondrial compartment and have found this produces what we have called the Mitochondrial Stress Response, a process that results in the selective upregulation of a suite of genes encoding mitochondrial stress proteins. This application deals with the question of the consequences to the cell of the creation of proteolytic environment. We have found that the two major proteases of the mitochondrion are upregulated and that this results in a marked increase in the rate of degradation of mitochondrial proteins. We aim to determine the specific roles of individual proteases in this process and the consequences of this proteolysis on the efflux of peptides from the mitochondria. This question has important medical implications, as one of the consequences of defects in mitochondrial function is the loss of cells from the affected tissue. We will also address the question of how mitochondrial biogenesis is regulated. We have recently found that the cytosolic molecular chaperone Hsp90 is required for protein import into mitochondria in mammalian cells. Since Hsp90 has hitherto been shown to be a key regulatory component in the steroid hormone and tyrosine kinase signalling pathways, this finding raises the possibility that protein import and thereby mitochondrial biogenesis may be regulated via the involvement of Hsp90.Read moreRead less
The Tumour Cell-specific Nuclear Targeting Properties Of Chicken Anaemia Virus VP-3: Potential For Anti-tumour Therapy
Funder
National Health and Medical Research Council
Funding Amount
$465,210.00
Summary
Current trends indicate that cancer will cause 40% of all deaths in Australia by 2012, meaning that new anti-cancer strategies are urgently required. Our proposal intends to examine the subcellular targeting abilities of the unique tumour-cell specific agent apoptin (VP3 - viral protein 3), a small protein encoded by the genome of the chicken anaemia virus. Using various strategies, we have identified part of the apoptin molecule that confers efficient localisation in the nucleus of tumour cells ....Current trends indicate that cancer will cause 40% of all deaths in Australia by 2012, meaning that new anti-cancer strategies are urgently required. Our proposal intends to examine the subcellular targeting abilities of the unique tumour-cell specific agent apoptin (VP3 - viral protein 3), a small protein encoded by the genome of the chicken anaemia virus. Using various strategies, we have identified part of the apoptin molecule that confers efficient localisation in the nucleus of tumour cells, but not non-tumour cells. Our experimental program intends to define this tumour cell-specific targeting signal in detail, and determine the molecular basis of the differential subcellular localisation of apoptin in tumour compared to normal cells. This should contribute fundamental new information regarding the differences between cancer and normal cells. Additionally, we intend to optimise the targeting signal and perform initial experiments to test its efficacy in targeting anti-tumour drugs to the nucleus of tumour cells. Our long-term aim is to use the apoptin tumour cell-specific nuclear targeting signal as part of modular constructs to combat cancer efficiently, and above all, with minimal damage to normal cells and tissues.Read moreRead less