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One of the themes of the Division is Protein Structure and Function. The Membrane Biochemistry Group is a major participant in the School’s Membrane Biology Program. This year Professors Gage and Dulhunty, who are also participants in the program, transferred to the Division from the Division of Neuroscience. The Membrane Biology Program studies the function of membrane ion channels involved in energy transduction, nerve conduction and in viruses. The latter work is creating new opportunities for controlling the replication of viruses such as HIV. The Division also has a strong group working on structural predictions, The Computational Molecular Biology and Drug Design Group.
The Medical Molecular Biology Group has continued its collaborative study on the structure of the interleukin-5 receptor with the X-ray Crystallography Group of the Research School of Chemistry as part of the Centre for Molecular Structure and Function. Interleukin-5 is a new drug target for asthma. This work also interfaces with the research of the Leukocyte Signalling and Regulation Laboratory and the Gene Targeting Laboratory which collaborate in using transgenic animals to study allergic lung disease in mice.
The Divisional theme on Gene Regulation and Cell Signalling has been strengthened by the participation of Dr Frances Shannon working on cytokine gene transcription. This adds to the existing strengths in the Division on nuclear translocation and chromatin function creating a strong focus on gene transcription.
The Division has continued to participate in projects of the Centre for Molecular Structure and Function such as the Functional Genomics and Molecules to Memory initiatives with the Research School of Biological Sciences. The University’s Biomolecular Resource Facility, which provides a wide range of services to molecular biologists, is also housed in the Division. It has continued to develop in 1998 and has expanded the services available.
Expertise in gene-targeting and transgenic animal research is provided by the Gene Targeting Facility and is also present in the Autoimmunity/Gene Manipulation Laboratory. Such approaches are necessary in generating the much-needed link between molecular studies and integrative whole animal research.
Professor Ian Young, Head of Division
The overall aim of this project is to determine the precise molecular steps involved in the transcriptional activation of a eukaryotic gene within a native chromatin environment. Elucidation of these mechanisms, at the molecular level, is crucial to our understanding of gene expression and regulation during cellular differentiation and to understand disease states such as cancer where normal gene expression patterns become altered. In addition, understanding how chromatin contributes to the regulation of gene expression is essential for understanding the pathogenesis of retrovirus, such as HIV-1, infection. HIV-1 must integrate a DNA copy of their genome into chromosomal DNA of newly infected cells. Moreover, independent of the site of integration, the virus assembles into a highly sterospecific chromatin structure indicating that this assembly process is important for its life cycle. Using HIV-1 as a model system, the specific aims of this work are to understand (1) the mechanism by which DNA is assembled into chromatin, (2) how chromatin architecture contributes to the regulation of transcription and (3) how chromatin is remodelled to allow transcription factor access. This will involve looking at nucleosome displacement, positioning, exclusion and modification.
Background:
A multicellular organism is made up of many different cell types; each cell type performing a unique function. The information that determines the function or behavior of a cell is stored in its genes (DNA). An important unanswered question in biology is how can different cells develop, carrying out different functions, when every cell within an organism contains the same genes?
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Genes are copied into RNA in a process called transcription and then, this RNA is translated into protein (it is the type of protein made that determines the function of a cell). The transcription of gene is a complex process requiring many 'DNA binding' proteins. These proteins are called transcription factors. The binding of transcription factors to one gene but not to another is one mechanism by which a subset of genes can be expressed enabling a cell type to carry out its specific function.
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Research:
In order to understand how the chromatin structure of a gene is altered to allow transcription, it is first necessary to understand how DNA is assembled into chromatin. Fractionation of the oocyte extract, and the eventual purification of all chromatin assembly components, will reveal the mechanism by which chromatin is assembled. Current fractionation work has shown that the assembly of DNA into chromatin is a sequential, time dependent process occurring in at least four steps. Two steps are required for the formation of the nucleosome, and a further two steps are needed to space these nucleosomes in order to generate a regular array.
These experiments have led to the identification of a novel nucleosome spacing factor that requires ATP to function. This protein has recently been purified and is a new type of chromosomal protein. A similar functional protein has been purified from mammalian cells. Mechanistic studies have shown that this mammalian protein spaces nucleosomes by interacting with both the nucleosome and the linker DNA. Other work has characterised the protein domains required for this interaction. Most interestingly, the binding of this nucleosome spacing protein facilitates the binding of transcription factors to DNA by modifying the structure of chromatin. Based on this and other fractionation work, it appears that during the chromatin assembly process, a time window exists which enables transcription factors to bind DNA before the formation of mature inactive chromatin. In addition, during this time window, the mammalian spacing protein can bind DNA to modify the structure of chromatin which further facilitates the transcriptional activation process. There are also other mechanisms involved in chromatin disruption. For example, transcription factors themselves may be able to modify or alter chromatin to allow transcription. We have also shown that a transcription factor known as AP-1 can completely disrupt a nucleosome to allow the subsequent binding of other transcription factors. Most interestingly, this disruption process is reversible since removal of AP-1 allows the reformation of the nucleosome. The ability of histones to rapidly refold into a nucleosome upon removal of AP-1 from the nucleoprotein complex provides an attractive mechanism by which an inducible promoter can be rapidly inactivated upon cessation of the activation signal. Importantly, more recent findings have demonstrated that this nucleosome disruption is dependent on a histone modification (acetylation). This suggests that the nucleosome itself may be an impotant target for signal pathways.
This work is particularly relevant to HIV-1 transcription. The replication rate of integrated HIV-1 is mainly controlled at the level transcription. The long terminal repeats, located at both ends of the integrated virus, contains all of the transcription binding factor sites needed for transcription initiation. Notable, the promoter region contains three binding sites for the transcription factor AP-1. Most interestingly, during the activation of the HIV-1 promoter in vivo, a nucleosome that covers these three AP-1 binding sites is specifically disrupted. Therefore, our finding that AP-1 can directly disrupt the structure of nucleosome in vitro may provide a molecular explanation for this in vivo observation. This hypothesis is currently being tested.
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One of the main points of control for the production of these protein lies at the level of gene transcription, ie the point at which a messenger RNA is made from the gene. Thus, the genes encoding the cytokines, chemokines and cell surface molecules of the immune system represent one of the best examples of highly regulated inducible gene transcription. Understanding the molecular nature of this highly regulated and transient gene transcription is of fundamental biological importance as well as having great significance in understanding the molecular steps involved in an immune response.
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The major research interest of the laboratory is to understand the molecular mechanisms involved in the transcription of cytokine genes in T cells, a major cell type of the immune system. T cells recognize foreign antigens when they are presented to them by so called antigen presenting cells (APC). The T cells not only recognize the antigen with specific cell surface receptors but also need to receive other signals from the APCs in order to produce sufficient cytokines for a productive response. In the absenteeism of these signals, which involve cell:cell interaction between molecules known as B7 (on the APCs) and CD28 (on the T cells), the T cells become unresponsive (anergic) or undergo programmed cell death (apoptosis). Our aim is to understand the signals sent to the cell by the CD28:B7 interactions that generate the "correct level" of cytokine gene transcription.
Signals that originate at the cell surface are transmitted to the cell nucleus by a cascade of events that results in the activation of proteins known as transcription factors that in turn form active transcription complexes on the promoters or enhancers of the appropriate genes and lead to gene transcription. We have previously identified the essential regions of two cytokine genes (interleuki-2 (IL-2) and GM-CSF) that respond to the CD28 signal and we have also shown that the response is controlled by the interplay of several transcription factors and DNA architectural proteins. We are now using this information to approach two important questions. Firstly, we need to understand not only how the transcription factors bind to DNA but more importantly how they form an active transcriptional complex in the presence of chromatin and other architectural features of the gene. The DNA is generally encased in protein complexes known as nucleosomes (made up of histone proteins) that package the DNA into a structure known as chromatin. We are developing methodologies to assemble nucleosomes on the IL-2 or GM-CSF genes in vitro (with Dr David Tremethick) and then ask what are the requirements for the formation of an active transcription complex on these genes using in vitro transcription systems. The second question is whether the control mechanims that we have identified in vitro are also functional in vivo. To answer this question we have developed transgenic mouse models of GM-CSF gene transcription and hope to determine the requirements for CD28 activation of GM-CSF in vivo. We also hope to use these transgenic mouse systems together with mice where one or more transcription factors have been deleted to ask what factors are essential to respond to CD28.
Viruses which infect the immune system, eg Human Immunodeficiency Virus (HIV) and Human T Leukemia Virus (HTLV) rely on interaction of their own regulatory proteins with the cellular transcriptional machinery to perturb cellular gene transcription and maximize their own survival. HIV infection is well known to lead to severe immune dysregulation and immunopathogenesis. One of the mechanisms by which HIV infection can perturb the immune system is by destroying the tight control exerted over cytokine gene transcription. The virus produces a protein known as Tat that is required for viral transcription but also interacts with the cellular transcription machinery and can lead to either increases or decreases in cellular gene expression. While there is a good understanding of how HIV Tat affects virus gene transcription, there is little information on the mechanism by which it alters cytokine gene transcription. It has been suggested that its affect is mediated through the CD28 responsive regions of IL-2 and GM-CSF We are, therefore, attempting to investigate how Tat may interact with the IL-2 and GM-CSF transcriptional machinery. We have shown that Tat physically and functionally interacts with transcriptional coactivator known as PC4 to lead to increased transcription from both the HIV and GM-CSF promoters. We are now attempting to integrate this information into a model of Tat function of the cytokine genes.
HIV infects macrophages as well as T cells and the infected macrophages are thought to be an important reservoir of the virus during the latent stages of infection. The interaction of HIV-infected macrophages with uninfected T cells can lead to T cell death (apoptosis) and this is thought to be an important component of the massive destruction of the immune system seen at the latter stages of HIV infection. One of the possible mechanisms by which HIV-infected macrophages destroy T cells is related to the lack of correct cell:cell interaction such as that mediated by DC28:B7 interactions.HIV infected macrophages have been shown to be deficient in B7 expression. It has also been shown that activating the T cells simultaneously through DC28 leads to a block in the apoptotic response. This may relate to cytokine production or to the production of other anti-apoptotic molecules in the cell. We are currently establishing systems to analyze the CD28 signals that are involved.
In summary, the majority of our efforts are directed at understanding the signals within the T cell that are required for the orchestration of a controlled immune response and how these mechanisms are perturbed by HIV infection. It is also possible that this understanding will lead to the identification of new pathways or molecules that may represent possible drug targets to alter immune responses.
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We have been trying to get a more complete understanding of the role of IL-5 in vivo through studies with a mouse engineered by gene targeting so that it is unable to produce IL-5. This work involves a continuing collaboration with K. Matthaei (Gene Targeting Laboratory, JCSMR ). The results of experiments with P. Foster and A. Ramsay (JCSMR) have previously shown a central role for IL-5 in lung damage and airways hyperresponsiveness in a mouse asthma model. More recently, different strains of inbred mice have been prepared carrying the IL-5 deficiency as well as an eotaxin /IL-5 double knockout mouse.
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Evidence for a role of IL-5 and eosinophils in host defence against parasites has been obtained in collaboration with C. Behm, K. Ovington and E. Milbourne (The Faculties, ANU) and S. Collins (Mc Master University, Ontario) and a role for IL-5 in mucosal immunity has been shown with B. Bao and A. Husband (University of Sydney).
Studies have continued on the mechanisms regulating IL-5 expression in T lymphocytes. This expression is both tissue-specific and inducible and is very relevant to the eosinophil-mediated tissue damage which occurs in asthma and allergy. Structural studies using X-ray crystallography on the IL-5 beta receptor in collaboration with P. Carr and D. Ollis (Research School of Chemistry) have now reached an exciting stage and we hope to gain new insights into the mechanism of receptor activation using this approach in the near future. The role of nuclear localisation in IL-5 signalling is being examined in collaboration with D. Jans.
Two other projects are under way in the area of functional genomics. With H. Campbell (RSBS) the function of two interesting genes concerned with development and behaviour in Drosophila are being investigated in mammals via gene targeting in mice. We are also seeking to identify genes involved in the formation of long-term memory using chickens as a model system. This project is interfaced with a parallel project on memory formation in honey bees involving R. Maleska and M. Srinivasan (RSBS).
The worldwide incidence, morbidity, and mortality of allergic asthma are increasing at a dramatic rate. In the USA alone 15 million people are thought to suffer from asthma and this disorder is now the most common cause of childhood absence from school. Deaths from asthma have now reached over 180,000 worldwide annually.
A predominant feature of the asthmatic lung is a persistent inflammation of the airway wall (infiltration of the airways with various white blood cells). Currently, it is thought that inflammatory cells induce asthma by releasing substances that damage the lining of the airways and induce constriction (narrowing of the airways). The inflammatory response in the asthmatic lung is a very complex mixture of cells and molecules and it is not clear which factors play the major role in inducing disease. Clinical investigations suggest that the inappropriate inflammatory response in the lung is driven by a lymphocyte known as a TH-2 cell. This cell is thought to direct the inflammatory response by releasing factors called cytokines that recruit and activate other white blood cells to the airways. In particular, the recruitment of the eosinophil to the lung is thought to play a major role in the development and initiation of asthma by releasing toxic mediators onto the lining of the airways which results in tissue damage and constriction of the airway.
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Research in our laboratory focuses on two major areas 1) identifying the key cells and molecules which induce disease and 2) developing strategies that will direct the immune response away from the harmful TH2 inflammatory response to that which is protective or non-responsive. The long-term goal of this research is to identify novel therapeutic approaches for the treatment of asthma.
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The second focus of our work is the employment of specific molecules that have the potential to act as vaccines to deviate the immune system away from the harmful TH-2 response to allergens. These strategies employ specific structures of allergens in association with molecules that promote non-responsiveness of the immune system.
This work is conducted in collaboration with research groups at the JCSMR, the ANU, nationally and internationally.
The second focus of our research is on malignant hyperpyrexia (MH). MH occurs in individuals with a specific inherited disorder of muscle, and presents clinically as a syndrome of life-threatening complications during general anaesthesia. The MH myopathy may also present clinically as heat stroke, death after taking neuroleptic drugs for psychiatric disorders (the neuroleptic malignant syndrome) and the sudden infant death syndrome (SIDS). The association between MH and SIDS occurs through overheating, which is an important clinical feature of MH and is also an important predisposing factor for SIDS While the exact aetiological mechanism which predisposes to the occurrence of MH is unknown, it is now widely accepted that an MH episode results from an elevated level of calcium ions in the myoplasm, which leads to accelerated muscle metabolism, hyperpyrexia and the clinical manifestation of the syndrome. In collaboration (Drs Taske and Cavanaugh, The Canberra Hospital and Drs Denborough and Milburn, JCSMR) the genes which predispose to MH are being indentified and characterized, in Australian families. The aim of this research is to define the molecular basis of MH and develop a simple non-invasive genetic test to screen infants at birth for susceptibility to MH and SIDS, and individuals before general anaesthesia. This should eliminate deaths from MH, and reduce sudden infant deaths from this cause.
We continue to act as a national reference centre for the diagnosis of MH susceptibility.
| Eukaryotic cells possess a nucleus in which the genetic information, the DNA, is stored, separated from the rest of the cell. Protein synthesis occurs in the cytoplasm so that proteins which are required in the nucleus need to be specifically transported from the cytoplasm into the nucleus. Our work is focussed on the mechanisms regulating protein transport to the nucleus, which relates to many important cellular processes such as differentiation, transformation, signal transduction and the regulation of transcription and cell metabolism. |
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We are using the techniques of microinjection and quantitative fluorescence microscopy to examine nuclear protein import in living cells, and thereby identify the mechanisms of regulation of this important process. We have also developed reconstituted in vitro systems to analyse nuclear protein transport at the single cell level. Our work has shown that whilst nuclear localisation is dependent on targeting sequences called nuclear localisation signals (NLSs), phosphorylation (the covalent attachment of phosphate groups to proteins) at sites near the NLSs can act as additional signals determining the rate and maximal level of nuclear transport of a particular protein. Hormonal signals can thus modulate gene expression through phosphorylation at such sites, thereby controlling the nuclear entry of particular TFs. We are currently attempting to identify and isolate the cellular proteins interacting with phosphorylation-regulated NLSs. We have also used our quantitative approaches to characterise several novel signal-mediated nuclear inport pathways. These include DNA-binding proteins, proteins of RNA viruses such as HIV-1, and granzymes, the serine proteases involved in eliciting apoptosis of virus infected cells.
Understanding of the mechanisms regulating nuclear protein import may ultimately enable their application in targeting molecules of interest to the nucleus in a research or clinical setting. In the latter case, efficient and tightly regulated nuclear uptake of DNA will be very useful in gene therapy applications (eg., the introduction of normal gene copies into appropriate cells harbouring a genetically conferred error of metabolism), or alternatively, toxins can be efficiently targeted to the nucleus of tumour cells in cancer therapy applications. We are currently developing a strategy of modular conjugate molecules containing modified NLSs which has thus far proved successful in the case of both of these applications.
| A major aim of modern biology is to understand how normal gene activities give rise to the structure and behavior of complex organisms. In particular it is important to study the function of genes and their derangement's involved in human diseases. In most cases however, it is impossible to achieve these studies directly in the human. It is easier therefore, to carry out such studies in a more manipulable system such as the mouse. |
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Gene targeting involves firstly the use of recombinant DNA technology to modify a cloned gene (usually to stop the function of the gene). At the same time a cultured cell line of embryonic stem (ES) cells is generated by culturing cells from an early mouse embryo (a blastocyst). The ES cells are to tipotent and can be used to regenerate live normal animals (i.e. it is possible to select a single ES cell and produce a whole mouse from that cell, see below). Whilst in tissue culture the modified gene is introduced into the ES cells and the normal gene is replaced by the mutated (functionally inactive) gene. The modified ES cells are then micro-injected into another mouse embryo and the ES cells become integrated. These blastocysts are re-implanted in to pseudo-pregnant mice and give rise to live chimæric offspring which consist of the modified injected cells as well as the normal cells. Since the injected cells can also contribute to the testis of these mice, the breeding of a chimæra with a normal mouse gives rise to an animal carrying half of the genes of the modified stem cell including the mutated gene. Interbreeding of the heterozygous (F1) siblings finally yields transgenic animals homozygous for the desired mutation (usually a deletion or a "gene knockout" mouse). In this way co-isogenic animals can be generated, i.e. animals which are identical to the original mouse strain except that the function of a single gene has been deleted thereby allowing the study of the loss of this gene in vivo.
Gene targeting therefore allows for the first time in a mammal the ability to study the function of a cloned gene in the context of the whole organism by creating mutants defective in that gene. This is particularly important since, with gene targeting, mouse models can be created for studying human genetic diseases and also provides a powerful approach to the development of somatic gene therapy.
Our laboratory has generated a number of different "knockout" mouse mutants using C57BL/6 or BALB/c cells which are at different stages of investigation. These include mouse models of asthma, nerve re-generation, xenograft rejection, parasite-hostrelationships, drug de-toxification and cancer.
Through the potential of the crelox system to target genes in vivo it is possible to delete class I expression specifically from the beta cells while leaving class I expression in other tissues unchanged. This involves making NOD ES cells for targeting the b-2M gene in such a way that it carries recombination sequences (lox sites) flanking the gene. NOD mice resulting from ES cells targeted in this way will be crossed to transgenic NOD mice which express the cre enzyme in the beta-cells of the pancreas. Expression of the cre enzyme in the beta cells of NOD mice which carry lox sites flanking ß-2M will result in the tissue specific recombination and deletion of beta-2M These mice will lack class I expression in the b cells, but express class I normally elsewhere. It should then be possible to separate the roles of class I expression on beta cells vs on antigen presenting cells and to gain an insight into the class I-restricted CD8 mediated autoimmune destruction of the islets in IDDM
The in vivo gene targeting system will not only be used for the study of the role of class I in IDDM, but also the role of beta cell antigens. Clearly this technology has many applications for the study of other organ specific diseases as well as in non-disease states for the understanding of the normal functioning of genes. This extends beyond the tissue specific control of gene expression to the temporal control with the use of inducible promotors.
A molecular biology and microinjection laboratory has been established. Constructs for the generation of transgenic mice and for the transfection of embryonic stem (ES) cells have been completed. 18 transgenic founder lines have been produced. Intercrossing of these lines indicates that cre is able to mediate recombination at lox sites in vivo.
The Membrane Biochemistry Group has joined the Membrane Physiology and Biophysics Group and the Muscle Research Group (both from Division of Neuroscience) to form the 'Membrane Biology' program. We are using a combination of advanced biophysical, biochemical and molecular biological techniques to probe the molecular mechanisms of two such membrane systems:
1) Viroporins - small virus encoded membrane proteins that form ion channels - such as the M2 and NB proteins of influenza virus, and the VPU and VPR proteins of HIV-1.
The viral proteins M2, NB, VPR, VPU have all been expressed as fusion proteins in bacteria. The purified proteins have been incorporated into planar lipid bilayers and their ion channel properties analysed and probed with site-directed mutations. These four proteins have also been expressed as unfused proteins in Escherichia coli and their effects on membrane-associated functions correlated with the ion-channel properties in the bilayer system. One of the aims of our research is to find inhibitors of these viral ion channels that may allow alternative anti-viral strategies to be developed. In that vein, we have identified a chemical (ANU 9) that inhibits the Vpu ion channel activity and have shown that ANU 9 also inhibits replication of HIV-1 in cultured human blood monocytes and macrophages.
We are also investigating viroporin-like proteins from various alphaviruses and flaviviruses to see whether they too form ion channels that have important roles for the replication of these viruses. Members of these virus groups are responsible for a wide range of diseases in humans (eg Dengue Fever and Murray Valley Encephalitis) that are currently very difficult to treat. It is our hope that drugs targeting the ion channels in these viruses will prove useful for combating the associated diseases.
2) The human GABAA receptor.
Gamma-amino-butyric acid (GABAA) is the major neuroinhibitory transmitter in the brain and is the target for many drugs. We are using an insect cell culture system and a genetically engineered virus to express the human GABAA receptor in a functional form on the cell surface. Substitution of amino acid residues in the hydrophobic domains of the subunits has had significant effects on ion conduction and on the process of desensitization. The effects of drugs have also been modified by these mutations.
| The surface membrane is an extremely important region of a cell, being responsible for a broad spectrum of functions such as transmission of electrical signals in nervous systems, initiation of immune responses and cell division. These phenomena depend on the presence of specialised proteins called ion channels that are embedded in the surface or internal membranes of a cell and allow movements of ions across lipid bilayer membranes. Ion channels are fundamental to cell function. They are present in every cell and are responsible for transfer of signals from the outside environment into the cell interior. Ion channels are the "portholes" of the cell. |
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The research of the Group is dedicated to understanding the cellular mechanisms that underlie changes in cytoplasmic calcium concentration in general, and more specifically those mechanisms which trigger contraction following an electrical signal on the surface membrane of skeletal and cardiac muscle fibres. Several different approaches are used to tackle this problem. These include:
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biochemical isolation and modification of ion channel proteins, molecular biology of ion channel proteins and proteins that regulate the calcium release channels
NMR studies of protein structure and immunoelectron microscopic studies of the distribution of proteins in membrane systems
We are examining the regulation calcium ion flow through the ryanodine receptor by studying the currents through single channels incorporated into artificial lipid bilayers. Our specific interests are the modulation of channel activity by calcium and magnesium ions, following sulfhydryl reduction and oxidation (by oxidants such as NO), by FK-506 binding proteins (FKBPs), by co-proteins like triadin and by protein-protein interactions with the skeletal muscle L-type calcium channel (an essential step in excitation-contraction coupling), which is also known as a dihydropyridine receptor (DHPR). We have so far identified basic mechanisms in (a) calcium magnesium regulation sites, (b) redox state and (c) FKBP in controlling the "gating" of the ion channel. Our studies have shown for the first time that small peptides, corresponding to a sequence in the DHPR, both activate and inhibit single ryanodine receptor channels, and that the activation is modified by FKBP12. These studies are continuing. Future studies will investigate the sequences in the ryanodine receptor and co-proteins, and the structural constraints, that allow regulatory interactions to proceed. Our studies include ryanodine receptors from skeletal and cardiac muscle. These are the predominant isoforms of the ryanodine receptor in striated muscle and in brain. We are also examining the effects of the ryanodine receptor mutation in malignant hyperthermia on single channel activity. The distribution of the ryanodine receptor protein in the sarcoplasmic reticulum of skeletal and cardiac muscle fibres is being examined using immunoelectron microscopic techniques. We have shown that there are an unexpectedly large number of ryanodine receptors in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres. These studies are continuing in both skeletal and cardiac muscle and we are investigating the important functional implications of extrajunctional ryanodine receptors in calcium regulation.
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However, it is important to remember that a full understanding of biological processes requires understanding at a number of levels - whole organism, cellular, sub-cellular, molecular and genetic - and how these processes and functions are integrated. The unifying theme of the Group's research is the study of the relationship between protein structure and function at the molecular level. |
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While the Group specialises in the use of computer-based methods, the emphasis is on using computer-based methods as a complement to experiment. Thus, the computer-based methods are being used to investigate aspects not directly amenable to experimental study or more convenient or efficient to study by computer-based methods, and to provide predictions testable by experiment. Complementary experimental work is being undertaken either within the Group or by collaboration. An increasing advantage of computer-based methods is in utilising the wealth of information now available in databases of protein structure and sequence, and for quickly and conveniently testing out ideas, especially for drug design. The Group's research is focussed in three areas: enzymic reaction mechanisms, protein-ligand recognition and drug design, and mechanisms of action of modular proteins, in particular extracellular receptors. An indication of the scope and goals of current projects is given below.
At the most detailed level, we are investigating how the construction of the active site of enzymes, the place where molecules such as substrates bind, facilitates the particular reaction for which the enzyme has evolved. While the roles of speeding up reactions are well known, the other essential role of enzymes in preventing unwanted reactions has received little attention. Enzymes have a key role in reaction selectivity from a possibly very large number of possible reactions of the biochemical "soup". Other unresolved issues which are particularly suitable for computer-based study, as they are largely "invisible" by experiment, are the roles of enzyme-bound water molecules and protons. Three enzymes for which high resolution x-ray structure and other sequence/function data are available are under study. Rubisco (D-ribulose 1,5-bisphosphate carboxylase-oxygenase), the enzyme catalysing the fixation of CO2 in photosynthesis is being studied in a collaboration with Professor John Andrews in the Research School of Biological Sciences. The enzyme lactate dehydrogenase (LDH) is involved in energy-producing cycles in the cell. Dihydrofolate reductase (DHFR) is a key enzyme in DNA biosynthesis and production of tetrahydrofolate cofactors. As the work requires very large amounts of computing time, we are collaborating with the ANU Supercomputer Facility and Fujitsu Japan to develop computer programs which can fully utilise the power of vector and parallel supercomputers.
Our program to design and develop new inhibitors of the enzyme dihydrofolate reductase incorporates themes from both the enzyme mechanism and recognition areas. Inhibitors are small molecules which bind to the enzyme active site and stop it from working by preventing its normal substrates from binding. We have successfully designed and tested new classes of compounds which bind tightly to the enzyme and are cytotoxic to cancer or malarial cells. We are now designing further compounds with improved pharmacological and binding properties, and predicting how strongly and in which orientations they bind to the enzyme. However, it is now known that protein structure "relaxes" to optimise the binding of different small molecules. A major challenge in our work is to simulate these changes by computer. This allows us to predict reliably the relative binding ability for series of compounds. In turn, this information is used to suggest the best compounds to synthesise, and to understand their biological effectiveness.
The area of protein structure prediction is one which is expanding very rapidly with the explosion of the amount of information in databases. In the emerging area of bioinformatics, computer-based methods may be used for "mining" protein structure and sequence databases to extract new insights into structure/function relationships and protein evolution. Although it is not possible to predict a priori structure from sequence, these methods attempt to use existing knowledge to do this. For sequences of unknown proteins, such as those emerging from genome projects, it may even be possible to predict their functions if a structure can be deduced. Although the resulting 3-D structural models are currently of variable reliability, they are proving to be very useful for interpreting existing experimental data and for designing further experiments.
Our particular goals in this area are to develop novel approaches combining computer-based and experimental methods to predict and model the structure of modular proteins or large protein complexes, for which high-resolution structural data from x-ray crystallography or nmr is difficult to obtain. In particular we are interested in extracellular (EC) receptors, which are bound to the cell membrane by an embedded anchor, or are attached via a linker which traverses the membrane to an intracellular component. Although such EC proteins constitute ~60% of cellular proteins, relatively few have been characterised by high-resolution structural methods, and how they work mechanistically and organisationally is largely unknown. In a "divide and conquer" strategy, computer-based methods (especially protein-fold prediction, homology modelling, protein-domain boundary prediction and sequence analysis) are coupled with medium-resolution (cryo electron microscopy) and low-resolution (CD, FTIR, ultracentrifuge, fluorescence spectroscopy) structural methods to build a complete model, which may be complemented with high-resolution structural data for separate domains or subunits if available.
We have three current projects in this area. We are developing models for the structure and interactions of a number of sequence regions of prion protein (PrP), the unusual protein implicated in such brain diseases as Creutzfeldt-Jacob and "mad-cow" diseases. Surprisingly the normal function of this protein is still uncertain, although a bewildering number of different possibilities have now been suggested. The models developed provide coherent explanations for a large range of data and offer many predictions, and possible hints for therapies. In collaboration with Professor Ian Young we are attempting to develop structure/function models for the cytokine interleukin-5 (IL-5) and its receptor. In an exploratory study using electron microscopy we have visualized a form of the interleukin-5 receptor and are attempting to define the relative orientations of its component modules with a view to developing a 3-D model using x-ray and other structural data for "homologous" receptors. Homologous receptors are those deduced from comparisons of sequences to belong to the same family as the IL-5 receptor and, hence, likely to have a similar 3-D structure. For the transmitter-gated ion channels, such as glycine and GABA (gamma-amino butyric acid) receptors, an important class of receptors which bind neurotransmitters, we have predicted a 3-D model for the EC region which is now being tested by Dr Peter Schofield from the Garvan Institute.
In general, research in the protein structure prediction and modelling area is very challenging with many of the models produced being quite speculative. However, application of bioinformatic approaches to complex biomolecular problems, especially those defying conventional structural analysis, offers immense potential for discovering unexpected homologies with known proteins (some of which will ultimately turn out to be correct!) and prompting new types of experiments or new theories of the evolution of protein structure and function. Recent advances in computing power and computational chemistry methodology have allowed us recently to perform quantum chemical calculations on whole enzymes (dihydrofolate reductase), i.e. molecules of several thousand atoms rather than the mere hundred or so possible previously. Extension of these methods to the multidomain protein receptor problems we have been working on so far only with bioinformatic, modelling and dynamical simulation methods will open up completely new lines of investigation into mechanisms of receptor action.
One of the primary roles of the biological NMR laboratory is to foster and develop collaborative ties with members of the JCSMR community. The laboratory is well equipped having access to two high-field NMR spectrometers (Varian Inova 500 & 600) and a network of Silicon Graphics workstations. A number of projects are in progress involving drug binding, mechanistic studies of enzyme systems and the structural study of membrane related proteins. Since many of the projects are collaborative efforts within and outside the School, an integrated approach involving other techniques complementary to NMR such as molecular biology, kinetics and molecular modelling are employed.
One enzyme under investigation is dihydrofolate reductase which is an important target for anticancer, antibacterial and antimalarial drugs. Its role in the cell is to catalyse the conversion of dihydrofolate to tetrahydrofolate using NADPH as the coenzyme. Although this enzyme has been the focus of numerous kinetic and structural studies, the manner in which it functions remains unknown. Our goal is to combine protein engineering and NMR to examine the drug binding and mechanistic properties of this enzyme and ultimately be in a position to design novel therapeutic agents.
Structural information of membrane-associated proteins is often difficult to obtain, however, NMR spectroscopy can be a versatile tool in exploring such systems. As part of the "Membrane Biology" program we have undertaken NMR studies on three very different and challenging membrane related proteins.
One of these involves the dihydropyridine receptor (DHPR) which is important for muscle excitation-contraction. In skeletal muscle it is known that DHPR interacts with the ryanodine receptor (RyR) triggering release of Ca2+ from the sarcoplasmic reticulum. A cytoplasmic loop region on DHPR has been identified as being essential for RyR activation and excitation-contraction coupling. We are using high resolution NMR spectroscopy to probe the structure of several loop fragments of DHPR ranging from the entire loop of 12 kD to small fragments of approximately 20 amino acids. Results indicate that specific peptides with well defined structures are capable of interacting with RyR The region of RyR which interacts with DHPR has been recently identified raising the prospect of examining protein-protein type interactions between these two membrane systems using NMR methods.
The viral proteins Vpu and Vpr of the human immunodeficiency virus have been reported to possess ion channel activity. Although not essential for replication, it is thought that these proteins do play some role in replication and/or pathogenesis in vivo. Peptide fragments of these viral proteins are capable of maintaining their ion-pore capabilities and are being structurally examined in model membrane systems by NMR
| Sedimentation analysis was the first and is still the best method for defining the size of proteins in the conditions where they are actually active , that is aqueous solutions and at the physiologically appropriate concentrations, which in the case of hormones and enzymes may be very low, or in the case of oxygen binding proteins, very high. |
 Click to enlarge photo |
In the protein interactions laboratory we have a state of the art instrument for studying proteins in this way, the Beckman Optima-XLA analytical ultracentrifuge. The XLA with its superb optics and electronics can collect and process data from three cells run simultaneously at speeds up to 60000rpm. Centrifugal forces generated at these speeds are up to 250000g's and force molecules to sediment at rates dependent on their sizes and shapes in velocity experiments, upon their sizes only in equilibrium experiments. Measurements of sedimentation rates can give gross information about shapes of macromolecules while measurements of concentration distributions at sedimentation equilibrium give absolute molecular weights under defined solution conditions independent of molecular shape. Only 100 microlitres of solution containing 100 micrograms of material are needed and the technique is non-destructive.
Proteins studied during this year were subunits of the IL 5 receptor and ATP synthase and an Anti-freeze Protein
The present finding helped explain a previous observation of a large peak seen in reverse-phase chromatographic separations of sow whey and coincides with the current attempts by K Nicholas and S Mailer at the Victorian Institute of Animal Science, to isolate factors influencing lactation in the pig. It has now been shown by internal sequencing, after pre-treatment with formic acid, that the peak is PRP If the HPLC separation is performed with a shallow gradient it is possible to isolate several components of PRP with varying degrees of glycosylation. In addition, during the study of lactation factors, some forms of PRP have been isolated which have a sequencable N-terminus and in these repeating sequences characteristic of PRP's, are most evident.
A Perkin Elmer (Applied Biosystems) Procise Carboxyl-terminal Protein Sequencer has been purchased with part of the funds from the ARC under its Research Infrastructure (Equipment and Facilities) Program (RIEFP) to establish an ultra-high sensitivity, high resolution protein analytical resource as a collaborative initiative between the Biomolecular Resource Facility at the ANU, the Biomedical Mass Spectrometry Unit at the University of New South Wales and the Sydney University and Prince Alfred Hospital Macromolecular Analysis Centre at the University of Sydney. C-terminal sequencing has proved particularly useful in completing the amino acid sequence of some spider toxins isolated by GF King's group at the University of Sydney. Professor King writes "The venoms of numerous aquatic and terrestrial animals have been shown to contain toxins that specifically interact with and alter the conductance properties of various ion channels. In addition to being useful pharmacological tools for exploring the functional diversity of ion channels, these toxins can also provide leads for the design of novel pharmaceuticals and insecticides. It is in this context that we have begun to examine toxins derived from Australian funnel web spiders, a group of venomous arachnids confined to the south eastern coast of Australia." C-terminal sequencing is also of value in fully characterising recombinant over-expressed proteins, especially in systems where gylcosylation can occur. Interpretation of mass data is greatly facilitated by knowing precisely both the N- and C-terminal sequences.
School Technical Manager:
R Ayling
Divisional Administrator:
JM Lee
Assistant Administrator:
S Lavender
Technical Officer:
R Taylor
Laboratory Stores Attendant:
J Burum
Autoimmunity/Genetic Manipulation Laboratory
Leader:
R Slattery, PhD
Laboratory Technician:
S Palmer, BSc (Hons)
Laboratory Assistant:
Jessica Stapley, BSc (Hons)
Biological NMR Laboratory
Research Fellow and Leader
MG Casarotto, Bsc (Hons) (Melb), PhD (Melb)
Visiting Fellows:
WLF Armarego, PhD, DSc (Lond), FRSC, FRACI
JF Morrison, BSc (Syd), MSc (Qld), Dphil (Oxford), DSc, (Protein Biochemistry)
Chromatin and Transcriptional Regulation Laboratory
Fellow and Leader:
D Tremethick, BSc (Hons) (Syd), PhD (Macq)
Technical Officers:
L Hyman BSc (UC)
M Clarkson BSc (Hons) (Uni. Adelaide)
Computational Molecular Biology & Drug Design Group
Senior Fellow and Leader:
JE Gready, BSc (Hons) PhD (USyd) FRACI
Research Fellow:
PL Cummins, BSc (Hons) PhD (USyd)
Postdoctoral Fellows:
SP Greatbanks, BSc (UMIST) PhD (Manc) [funded by ANUSF/Fujitsu]
PJ Harvey, BAppSc (QUT) BSc (Hons) PhD (Griff) [funded by Canberra Hospital Private Practice Fund]
R Hornig, BSc Dipl (Basel) PhD (ETH Zur) (from September)
WA King, BSc PhD (UNSW) [Interschool collaborative PDF with RSBS]
TA Morton, BSc (UQld) PhD (Georgia) [Collaborative PDF with Medical Molecular Biology] (until July)
SJ Ohms, MBChB Dipl (Obstet) BEng MEng PhD (Auck) [funded by Comm. Dept. of Health]
RK Schmidt, BS (Nebraska) PhD (Cornell) (ARC Postdoctoral Fellow)
J Zuegg, Dipl-Ing Dr (Graz)
Visiting Fellows:
M Ngu-Schwemlein, BSc (La Trobe) PhD (ANU)
APL Rendell, BSc (Durham) PhD (USyd)
Research Officers:
DL Diedrich, BS (Mich State) PhD (Penn State)
J Fan, BSc MSc (Fudan) PhD (Auck) (from March)
Research Assistant:
C McKinlay, Chem Cert. (Woll) BSc (Woll)
Cytokine Gene Transcription Laboratory
Senior Fellow and Leader:
MF Shannon, BSc (Hons), PhD
Postdoctoral Fellows:
A Holloway, BSc (Hons), PhD
R Mital, BSc, MSc, PhD, (from November)
Technical Officer:
D Woltring, Ass Dip Biology (CIT), Ass Dip Pathology (CIT) (from June)
Leukocyte Signalling and Regulation Laboratory
Fellow and Leader:
Dr P Foster, BSc(Hons) (WA), PhD (ANU)
Postdoctoral Fellows:
Dr S Hogan, BSc(Hons), PhD (ANU) (until April)
Dr D Webb, PhD (UC) (from December)
Visiting Fellows:
Dr M Denborough, MD, ChB (Capetown), MD (Melb), DPhil (Oxon), DSc (Melb), FRCP
Dr N Taske, BSc (Hons) (Qld), PhD (ANU)
Dr Ming Yang, West China Uni. of Medical Science, (Bachelor of Medicine)
EPI Section Henan Provincial Instit. of Hygiene and Anti Epidemic, PhD
Laboratory Technician:
A Koskinen, Assoc. Dip. Med. Sci.
Gene Targeting Laboratory
Fellow and Leader:
KI Matthaei, BSc (Hons) (UNSW), PhD (ANU)
Laboratory Technicians:
VW Damcevski, Ass Dip App Science (CIT) (from April).
HI Taylor
MJ Newhouse BSc, Grad Dip (ANU)
S Young (part time)
Medical Molecular Biology
Professor and Leader
IG Young, MSc (Melb), PhD (ANU)
Postdoctoral Fellow:
VL Ross, BA (LittB), PhD
Research Assistant:
S Ford, BA, MSc (Qld)
Senior Technical Officer:
D Mann BSc (Hons)
Technical Officers:
A Church (Lord) BSc (Hons)
S Gustin, Ass Dip Biology (CIT), Ass Dip Pathology (CIT), BSc (Hons
D Woltring, Ass Dip Biology (CIT), Ass Dip Pathology (CIT) (until May)
Membrane Physiology and Biophysics
Professor and Group Leader
P W Gage, MB ChB (NZ), PhD (ANU)
DSc (NSW), FAA
Research Fellow
B Birnir, BS (Wash), PhD (UCLA)
Postdoctoral Fellow
A K M Hamarström, BSc (Hons),
PhD (Monash) (funded by NHF)
Research Assistant
J Curmi, (BOptom)(UNSW)
Laboratory Technician A Everitt BSc (ANU)
Administrative Assistant
L Hardy (part time)
Membrane Biochemistry Group
Group Leader (Acting) & Research Fellow:
G Ewart, BSc (Hons), PhD (ANU)
Professor:
GB Cox, BSc, PhD (Melb), FAA
Laboratory Technicians:
T Sutherland, BApp. Sc. (Charles Sturt Uni) until October 1998
B Matheson (from January)
Visiting Fellows:
FWE Gibson, BSc, DSc (Melb), MA, DPhil (Oxon), FAA, FRS
B Cromer, BSc (Hons) ANU
L Tierney PhD (ANU)
Muscle Research Group
Professor and Group Leader
A F Dulhunty, BSc (Syd), PhD, DSc (NSW)
Postdoctoral Fellow
J Hart, BSc (Hons),
PhD (Monash)
Visiting Fellows
E Gallant, PhD (USA)
DLaver, BSc (Hons), PhD (UNSW), (ARC)
K Eager BSc (NZ), PhD (ANU) (from June)
Senior Technical Officer
S Pace, BSc (UTS)
Technical Officer
S M Curtis, BSc, PLTC (half time)
Laboratory Technician
J Stivala
Administrative Assistant
L Hardy (part time)
Nuclear Signalling Laboratory
Senior Fellow and Leader:
DA Jans, BSc (Hons) (Melb), PhD (ANU)
Technical Officer:
P Jans, Technical Diploma (Lausanne) (part-time)
Laboratory Technician:
C Barton,
Visiting Fellow:
R Lixin, MSc (China), BM (MD, Wuhu Anhui), MSc (Taiyuan, Shanxi)
Protein Interaction Laboratory
Fellow and Head:
PD Jeffrey, BSc (Hons), PhD (Adel)
Protein Chemistry Laboratory
Fellow and Head:
DC Shaw, BSc (WA), PhD (Cantab)
Technical Officer:
H Gajardo, BRTC
Molecules to Memory Project
(jointly with Prof S Redman)
Postdoctoral Fellow:
F Freeman, BSc(Hons) (Nottingham), PhD (Open)
Technical Officer:
U Wiedemann, Technical College Certificate (Germany)
Joint appointment with The Faculties
Fellow:
BH van Leeuwen, BSc, PhD (Monash)