Biochemical Analysis Of Akt 3-specific Signal Transduction
Funder
National Health and Medical Research Council
Funding Amount
$349,375.00
Summary
The Akt family of enzymes consists of 3 protein kinases (Akt 1,2 and 3) and has been shown to regulate many normal cellular processes such as cell proliferation, growth, survival and motility, as well as the growth of new blood vessels. All these processes are critical for cancers to grow. However, few studies have distinguished the roles of the individual family members. Our preliminary data revealed Akt3 is far more active than the other two forms. Furthermore, using our unique Akt3 specific a ....The Akt family of enzymes consists of 3 protein kinases (Akt 1,2 and 3) and has been shown to regulate many normal cellular processes such as cell proliferation, growth, survival and motility, as well as the growth of new blood vessels. All these processes are critical for cancers to grow. However, few studies have distinguished the roles of the individual family members. Our preliminary data revealed Akt3 is far more active than the other two forms. Furthermore, using our unique Akt3 specific antibody, we find Akt 3 protein and activity levels are high in rapidly proliferating ovarian cancer cell lines and in primary ovarian tumours. The aim of this proposal is to characterise the mode and role of signalling via Akt3, including the identification of targeted substrates and signaling pathways and the outcomes of Akt3 driven signaling on cellular properties. These studies will provide important clues to understanding how this family member functions in both health and disease. Elucidation of the basis of Akt3 dependent signalling will open the possibility for the development of drugs that interfere with Akt3 function (for example in high Akt 3 expressing tumours like those of the ovary). In the long term, extension of our profiling studies to other tumour types will give a novel insight into the extent of Akt3 de-regulation as a key mediator of cancer formation.Read moreRead less
Regulation Of SRC-Family And Focal Adhesion Kinase Function
Funder
National Health and Medical Research Council
Funding Amount
$381,338.00
Summary
Cells in our bodies stick to one another and to the cementing material called extracellular matrix surrounding them. An ezyme called focal adhesion kinase (FAK) is a major regulator of cell stickiness. It can catalyze the covalent attachment of a chemical group called phosphate to specific cellular protein. This proposal aims at studying how FAK is regulated by insulin stimulation and how FAK is regulated by a tumour suppressor called PTEN. Results of the study will shed light on how abberration ....Cells in our bodies stick to one another and to the cementing material called extracellular matrix surrounding them. An ezyme called focal adhesion kinase (FAK) is a major regulator of cell stickiness. It can catalyze the covalent attachment of a chemical group called phosphate to specific cellular protein. This proposal aims at studying how FAK is regulated by insulin stimulation and how FAK is regulated by a tumour suppressor called PTEN. Results of the study will shed light on how abberrations in the regulation and PTEN contribute to the development of development defects, heart attack, and the spreading of cancer cells.Read moreRead less
Multi-domain Regulation Of DNA Damage Response Kinases
Funder
National Health and Medical Research Council
Funding Amount
$313,427.00
Summary
DNA damage plays a key role in the onset of cancer and the response to cancer therapies. Mutations in the Chk2 DNA damage response kinase are associated with increased cancer risk. We will study detailed mechanisms how phosphorylation of Chk2-like kinases contributes to normal copying of our DNA every time a cell divides, and how it regulates how Chk2 is activated. The studies will improve our understanding how cancer may originate and how cancer cells respond to chemo- or radiation therapy.
Regulation Of The Tumour Suppressor PTEN By Phosphorylation And Oligomerization
Funder
National Health and Medical Research Council
Funding Amount
$241,650.00
Summary
The tumour suppressor PTEN is an enzyme involved in controlling cell growth, cell death, and cell migration. PTEN was identified as a tumour suppressor because many tumour cells were found to carry mutations in the PTEN gene that cause the loss of PTEN protein or the loss of PTEN enzyme activity. Hereditary mutations of the PTEN gene are the causes of a rare genetic disease called Cowden's disease. Cowden's disease patients are predisposed to developing skin, thyroid, and breast cancers. In labo ....The tumour suppressor PTEN is an enzyme involved in controlling cell growth, cell death, and cell migration. PTEN was identified as a tumour suppressor because many tumour cells were found to carry mutations in the PTEN gene that cause the loss of PTEN protein or the loss of PTEN enzyme activity. Hereditary mutations of the PTEN gene are the causes of a rare genetic disease called Cowden's disease. Cowden's disease patients are predisposed to developing skin, thyroid, and breast cancers. In laboratory conditions, increasing the abundance of PTEN in tumour cells such as brain and prostate tumour cells can suppress their growth, hence its role as a tumour suppressor. In addition to its role as a tumour suppressor, PTEN controls cancer cell spreading. Although much is known about the involvement of PTEN in cancer formation and the spreading of cancer cells, how PTEN suppresses tumour cell growth and spreading is not fully understood. The enzyme activity of PTEN enhances the removal of a chemical group called phosphate group from proteins and the fat-soluble compounds called phospholipids in the cell membrane. The ability of PTEN to suppress cell growth and spreading is due to its enzyme activity. However, exactly how the enzyme activity of PTEN is regulated is not well understood. In order for PTEN to efficiently enhance the removal of phosphate group from specific cellular proteins and phospholipids, PTEN needs to be located in close vicinity to these proteins and phospholipids. However, exactly how PTEN moves to the locations where these proteins and phospholipids are present remains elusive. This proposal aims at studying the regulation of PTEN enzyme activity and movement inside the cells. Results of the proposed studies will shed new light on how PTEN gene mutations contribute to cancer formation and the spreading of cancer cells and may facilitate the search for the cure of cancers.Read moreRead less
Enhancing The Cardioprotective Effect Of Diadenosine Tetraphosphate: Designing Inhibitors Against Ap4A Hydrolase
Funder
National Health and Medical Research Council
Funding Amount
$442,500.00
Summary
Ischemia describes the condition where blood flow in the blood vessels of the heart is decreased or blocked, preventing delivery of oxygen and nutrients to the heart. Ischemic preconditioning is a phenomenon where short bursts of ischemia, followed by reperfusion, actually protect the heart from a subsequent longer period of ischemia. The biochemical signalling events involved in preconditioning are complex and incompletely defined, but most likely involve multiple pathways, although the mitocho ....Ischemia describes the condition where blood flow in the blood vessels of the heart is decreased or blocked, preventing delivery of oxygen and nutrients to the heart. Ischemic preconditioning is a phenomenon where short bursts of ischemia, followed by reperfusion, actually protect the heart from a subsequent longer period of ischemia. The biochemical signalling events involved in preconditioning are complex and incompletely defined, but most likely involve multiple pathways, although the mitochondrial ATP-dependent potassium channel may be in common with most pathways. Pretreatment with the compound diadenosine tetraphosphate (Ap4A) mimics ischemic preconditioning with noticeable reductions in tissue necrosis (cell death). This treatment has been shown in experimental work to protect the heart during periods of stress such as in heart surgery or recovery from an ischemic event. The biological site of action by Ap4A may be the mitochondria ATP-dependent potassium channel or an associated protein. Ap4A can be degraded by enzymes located inside and on the outside of heart cells, notably by two forms of Ap4A hydrolase. We will use antibody assays to understand the specific localization and amount of Ap4A hydrolase before and after ischemia and after ischemic preconditioning in human heart muscle and blood vessels. We propose to determine the structure of the enzyme and use novel computer methods to screen databases for potential inhibitors. These inhibitors of Ap4A hydrolase activity could aid the design of a potent inhibitor that would prevent Ap4A hydrolase from degrading Ap4A and therefore enhance the cardioprotective properties of Ap4A as well as minimizing side effects from the break down of Ap4A. We will also use these inhibitors and other known non-degradable Ap4A analogues in bioassays to test the relative significance of Ap4A hydrolase present in different cellular locations.Read moreRead less
Characterization Of The 72 KDa Inositol Polyphosphate 5-phosphatase
Funder
National Health and Medical Research Council
Funding Amount
$454,050.00
Summary
Cells respond to external signals and the enviroment to undergo cell growth, secretion and or other specialized functions including control of cell death and or cell size. We have identified a new enzyme (72 kDa 5-phosphatase) which resides inside the cell, which we have evidence plays a role in regulating both the movement of intracellular vesicles and also lipid signals stimulated by insulin. We have characterised the phospholipids that the enzyme cleaves and demonstrated the generation of new ....Cells respond to external signals and the enviroment to undergo cell growth, secretion and or other specialized functions including control of cell death and or cell size. We have identified a new enzyme (72 kDa 5-phosphatase) which resides inside the cell, which we have evidence plays a role in regulating both the movement of intracellular vesicles and also lipid signals stimulated by insulin. We have characterised the phospholipids that the enzyme cleaves and demonstrated the generation of new cell signals at specific subcellular localizations on intracellular membranes. We predict the generation of these specific lipid signals may play a significant role in controlling the transport of intracellular cargo to specific sites in the cell. In this grant proposal we aim to examine the regulation of specialised cargo called the glucose transporter, which is found in fat and muscle cells, and also the mannose 6-phosphate receptor, which regulates the trafficking of specific enzymes which mediate digestion of proteins. These studies include the clarification of which phospholipid signals the enzyme terminates and where in the cell this occurs. Secondly, we will examine the movement of the glucose transporter GLUT-4 in unstimulated cells and in response to insulin and furthermore how expression of the novel enzyme regulates its movement. We will also examine the movement of the mannose 6-phosphate receptor and the specific phospholipid signals which control the route the receptor traffics, using inhibitors of lipid signals and expression of lipid phosphatases and kinases. We will also examine how our novel enzyme forms complexes with other molecules in the cell and characterise these novel molecules using basic biochemical assessment of enzyme activity and function. Finally we will examine the regulation of intracellular messages by our novel enzyme following insulin stimulation, which facilitates glucose uptake into the cell.Read moreRead less
Development Of Purine Nucleoside Phosphonates As Anti-malarial Drugs Targeting Nuceloside Synthesis In Plasmodium
Funder
National Health and Medical Research Council
Funding Amount
$428,917.00
Summary
Malaria is one of the most serious infectious diseases today. Because of its location in a malaria endemic region, the tropical regions (above 19 S in latitude) of Australia face an emerging threat. The causative agent of the disease is the parasite, Plasmodium. Because of increasing resistance to existing medicines, new drugs are now needed. The drugs we will develop target the parasites replication cycle and are related in structure to those in use to treat viral infections including AIDS.
Regulation Of The Cardiovascular Disease-Associated Protease Inhibitor Cystatin C For Therapeutic Application
Funder
National Health and Medical Research Council
Funding Amount
$498,505.00
Summary
Proteases can contribute to atherosclerosis, so they are normally controlled by the endogenous inhibitor, Cystatin C (Cst C). Some conditions cause reduction in Cst C levels and hence disease. On the other hand, excess Cst C can form toxic aggregates. In this project, we will identify mechanisms controlling Cst C expression and aggregation to find therapeutic strategies to treat cardiovascular diseases associated with Cst C.
Regulation Of Synthesis, Dimerisation And Secretion Of The Amyloidogenic Protease Inhibitor Cystatin C
Funder
National Health and Medical Research Council
Funding Amount
$423,565.00
Summary
The cells that compose our tissues are embedded in a complex mesh of extracellular proteins (for example collagen) that provide support, strenght and elasticity to the tissues. This extracellular matrix is not static; it is constantly remodelled when, for example, the cells of the immune system move through interstitial spaces to monitor the healthiness of the tissues. When infections or injuries occur, the inflammatory reactions that develop, and the processes involved in tissue repair, also in ....The cells that compose our tissues are embedded in a complex mesh of extracellular proteins (for example collagen) that provide support, strenght and elasticity to the tissues. This extracellular matrix is not static; it is constantly remodelled when, for example, the cells of the immune system move through interstitial spaces to monitor the healthiness of the tissues. When infections or injuries occur, the inflammatory reactions that develop, and the processes involved in tissue repair, also involve profound changes in the composition of the extracellular matrix. Such processes are also important for tumour growth; the cancer cells need to clear their way through interstitial space to escape to circulation and metastasize. During all these processes, the cells release to the extracellular space proteases that degrade collagen and the other components of the extracellular matrix. Obviously, these proteases must be tightly regulated to prevent them running out of control, so the cells also produce inhibitors of the proteases. The amount of proteases and inhibitors contained in the extracellular space must be maintained properly. If this equilibrium is disrupted, this can lead to pathology For instance, atherosclerosis is caused in part by excessive proteolysis of the blood vessel wall. In this project we want to study the mechanisms of one of the most abundant and important inhibitors of extracellular proteolysis: Cystatin C. We have discovered that certain cells of the immune system called dendritic cells posses interesting mechanisms to regulate how much Cystatin C they secrete. Furthermore, one of this mechanisms, which consists of pairing the protein to produce inactive dimers, may be the cause of some diseases characterised by accumulation of Cystatin C in the extracellular space. Our study may allow us to design therapies for the treatment of pathologies associated with defective or excessive production of Cystatin C.Read moreRead less
The Biosynthesis Of Mycobactin T, A Virulence Factor From Mycobacterium Tuberculosis.
Funder
National Health and Medical Research Council
Funding Amount
$211,527.00
Summary
Mycobacterium tuberculosis is the causative agent of tuberculosis. The drug isoniazid led to a dramatic and sustained decline in mortality due to tuberculosis. This led to it being described in medical literature in 1988 as a disappearing disease which was now fairly easy to treat. However, the advent of HIV and the rapid rise of multidrug resistant M. tuberculosis led to dramatic changes. The risk that an HIV infected individual will develop active tuberculosis is 7% per year, compared to a lif ....Mycobacterium tuberculosis is the causative agent of tuberculosis. The drug isoniazid led to a dramatic and sustained decline in mortality due to tuberculosis. This led to it being described in medical literature in 1988 as a disappearing disease which was now fairly easy to treat. However, the advent of HIV and the rapid rise of multidrug resistant M. tuberculosis led to dramatic changes. The risk that an HIV infected individual will develop active tuberculosis is 7% per year, compared to a lifetime risk of 10% for an immunocompetent person. Similarly, the prevalence of drug resistant strains of M. tuberculosis is over 5% in many regions, including SE asia. Mycobacterial infections are regarded as the leading cause of morbidity and mortality world wide and WHO estimates that 30 million deaths will occur in the next decade due to these infections. Clearly, new drugs are required to combat the rising menace of this organism. The aim of this project is to detail the unique metabolic pathways in M. tuberculosis that produce Mycobactin T, essential to the virulence of this organism. Mycobactin T helps the bacteria obtain iron, an essential nutrient. These factors make the mycobaction pathway an ideal drug target and an understanding of its biochemistry is essential to its eventual exploitation for intervention in M. tuberculosis infections. We hypothesise that it may already provide the unknown site of action of a clinically employed, antituberculosis drug para-aminosalicylate (PAS). This project will i) fully define the structure of mycobactin T; ii) clone and overexpress key genes which catalyse the first three steps of mycobactin formation; iii) purify and characterise the overexpressed proteins with respect to their biochemical function; iv) examine the interaction of PAS with the proteins likely to be targeted by this antimycobacterial agent. The results of this work will provide the basis for the development of future anti-tuberculosis drugs.Read moreRead less