Division of Molecular Bioscience
Membrane Proteins in Health and Disease
Membrane Physiology and Biophysics | Muscle
Research | Biomolecular Structure
Computational Proteomics and Therapy Design
Computational Proteomics and Therapy Design
Cytokines and Inflammation
Allergy and Inflammation Research | Cytokine
Molecular Biology and Signalling | Gene Targeting
Gene Regulation in Development and Immunity
Cytokine Gene Expression | Chromatin
and Transcriptional Regulation |
Autoimmunity and Genetics
Autoimmunity and Genetics
Genetics
Molecular Genetics | Ubiquitin
Laboratory
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The research projects of the Division of Molecular Bioscience have the aim of gaining an understanding at the molecular level of the life processes occurring in living cells and of the derangements in these processes which result in disease. Using cutting-edge technologies, we are seeking knowledge of the structures of life and the chemistry of health. Professor Ian Young |
The projects relate to clinical medicine in areas such as the control of viral diseases, in understanding asthma and allergy, in anaesthetic complications and in diabetes.
The Membrane Proteins in Health and Disease Program studies the function of membrane ion channels involved in nerve conduction, the control of muscle contraction and in the replication of viruses. This work includes the examination of how the bioactivity of these channels is governed by molecular shape and recognition. The program is also investigating new approaches for controlling HIV. The Division also has a strong group working on the prediction of protein structure, the Computational Proteomics and Therapy Design Group.
In the Cytokines and Inflammation Program, the Cytokine Molecular Biology and Signalling Group has continued its collaborative studies on the structure of the cytokine receptor system involved in IL-3, IL-5 and GM-CSF signalling with the X-ray Crystallography Group of the Research School of Chemistry. The work of this group also interfaces with that of the Allergy and Inflammation Research Group and the Gene Targeting Laboratory which use transgenic animal models to study the key cytokines and cells involved in asthma and allergy.
The Division also has a strong focus on how genes are regulated, with high quality research on nuclear translocation, chromatin function and cytokine gene transcription. This work, which is carried out by the Gene Regulation in Development and Immunity Program, includes studies on the signals which regulate gene expression during the early development of an organism and during immune responses to foreign antigens.
A new Genetics program has been added to the Division this year, which brings additional high quality expertise in molecular genetics. One of the major thrusts of this program is the study of enzymes (GST's) which detoxify environmentally derived toxins and therapeutic drugs. Deficiencies of such enzymes can be a risk factor in lung, stomach and skin cancers whereas high levels can be associated with resistance to cancer chemotherapy.
A novel protein regulating the cytoskeleton of cells, which is essential for early embryonic development, is being studied in a collaborative project between members of the Division and the Research School of Biological Sciences. This project in functional genomics exploits the high conservation of the fundamental life processes between simpler organisms like the fruit fly and more complex mammalian species to give a better understanding of early mammalian development.
Expertise in gene-targeting and transgenic animal research is provided by the Gene Targeting Laboratory and the Autoimmunity and Genetics Laboratory. Such approaches are necessary in generating the much-needed link between molecular studies and integrative whole animal research. The Gene Targeting Laboratory is involved in collaborations with other scientists in the School and elsewhere involving research into asthma, nerve growth, hypertension and embryonic development. The Autoimmunity and Genetics Laboratory has pioneered the use of a new genetic engineering tool called cre-lox recombination and is using it to investigate the causation of diabetes.
Professor Ian Young, Head of Division
Membrane Proteins in Health and
Disease
Membrane Physiology and Biophysics
Professor Peter W Gage
Our research is focused on the structure and function of ion channels that allow ions to pass between the outside and inside of cells. They are like windows and doors in a house. Ion channels are vital for the proper functioning of all cells and some of the most prescribed drugs in medicine act on ion channels. For example, drugs such as calcium channel blockers and anaesthetics interact specifically with ion channels but we do not yet know how they change channel function. Many major pharmaceutical companies are showing increasing interest in ion channels as a new drug target. Research in our group is aimed at understanding several very different kinds of ion channels.
Ion channels in the brain
The ubiquitous inhibitory neurotransmitter, gamma-aminobutyric acid
(GABA), binds to GABA receptors present on most nerve cells and opens
channels that are targets for clinically important drugs such as barbiturates,
benzodiazepines and some general anaesthetics. We are studying the
properties of individual "native" channels in nerve cells,
with particular interest in how drugs such as general anaesthetics,
tranquillisers and anti-epileptics affect their behaviour. We are
also synthesizing these channels by introducing the cDNA into foreign
cells that then make the GABA receptor protein and fuctioning channels.
In this way we can introduce changes in single amino acids and study
their effect. We are finding that small changes can have major effects
on channel function and on the response of the receptors to drugs.
Our results are helping us to understand how the normal GABA receptor
functions and to find new and better drugs to produce anaesthesia
or relief from anxiety.
Ion channels formed by virus proteins
It has been known for about 10 years that a small protein from Influenza
A virus forms ion channels essential for viral reproduction and that
a clinically useful anti-influenza drug stops the virus by blocking
these ion channels. We have been exploring the idea that small proteins
from other viruses may form ion channels that play an important role
in the virus life cycle. The proteins are expressed in bacteria, purified
and inserted into artificial membranes. We have also developed other
ways of testing for ion channel function. We have found that proteins
from several viruses (influenza and HIV-1) form channels that allow
movement of sodium, potassium and other cations through them. Our
aim is to determine how these "simole" proteins form ion
channels. It may then be possible to find drugs that interact with
these sites and stop virus replication. It is also hoped that the
simple nature of these proteins will give us more information about
how ion channels work in general.
Ion channels that open during oxygen-deprivation
Following a heart attack (coronary occlusion), and in some other abnormal
conditions, the electrical currents that produce an orderly sequence
of contractions in the heart can become erratic but we don't really
know why. The heart beats irregularly and patients can die because
the heart no longer pumps sufficient blood to vital organs. This is
a major cause of death following a heart attack. Some antiarrhythmic
drugs can help subdue the arrhythmias but none yet available is considered
ideal: they have unwanted side effects. We have demonstrated the presence
of a new kind of sodium channel in heart muscle that we believe has
an important role in initiating electrical impulses. This channel
is blocked by many currently used antiarrhythmic drugs and becomes
more active during hypoxia. This may be the cause of abnormal electrical
impulses and arrhythmias during hypoxia or ischaemia e.g. following
coronary artery occlusion. We are now looking for the link between
hypoxia and hyper-activity of this sodium channel. If we are successful,
our work will provide a new kind of target for new, more selective,
antiarrhythmic drugs.
Muscle Research Group
Professor Angela Dulhunty
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: electrophysiological studies of currents through single ion channel proteins and of contraction in isolated bundles of intact muscle fibres and in skinned segments of single fibres; 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.
The Ryanodine Receptor Calcium Release Channel
The ubiquitous ryanodine receptor calcium release channel is found in the membranes of intracellular calcium stores and is the major calcium release pathway from these stores in many cell types. Although regulation of cytoplasmic calcium concentrations is basic to the function of all cells, the mechanisms controlling ryanodine receptor activity are not well understood.
We are examining the regulation of 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 calsequestrin 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 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. 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.
Counterion Channels in the Sarcoplasmic Reticulum Membrane
The ability of internal calcium stores to sequester and release calcium depends on: 1, a calcium uptake mechanism; 2, a calcium release mechanism; 3, channels to allow a counter current to flow with calcium, to prevent large changes in the potential difference across the membrane, which would impede the calcium fluxes; 4, phosphate concentrations in the store lumen. All four components are essential for the proper regulation of cytoplasmic calcium concentrations. The counterion channels, although essential, have been studied far less than the calcium uptake and release pathways. We have recently discovered that there are two types of anion channels in sarcoplasmic reticulum. One of these channels is highly regulated by a number of substances which are present in vivo, including cytoplasmic calcium concentration, inositol polyphosphates, ATP and pH. This highly regulated channel also conducts sulphate and phosphate ions. This is the first description of a divalent anion channel in sarcoplasmic reticulum and an ion channel which provides a pathway for phosphate movement in and out of the calcium store.
Excitation Contraction Coupling
The Muscle Research Group was largely responsible for much of the basic work on voltage-dependence of excitation-contraction coupling in mammalian skeletal muscle. However, the basic mechanism of excitation-contraction coupling in skeletal muscle is still not properly understood. We know that depolarization of the surface membrane activates a voltage sensor which is a part of the dihydropyridine receptor in the transverse tubule membrane. The loop between the second and third transmembrane segment of the dihydropyridine receptor is thought to be involved in transmitting the depolarisation-evoked signal to the ryanodine receptor and one serine residue in the 2/3 loop appears to be essential for activation of ryanodine receptors. We are currently looking at the ability of peptides containing different sequences to specifically activate skeletal and cardiac ryanodine receptor ion channels. Future experiments will examine the interactions between activating peptides and other co-proteins, especially the FKBPs and triadin, so that a model can be developed of the in vivo activation of the ryanodine receptor by the dihydropyridine receptor during excitation-contraction coupling.
Biomolecular Structure Laboratory
Dr Marco G. Casarotto
One of the fascinating properties of biological molecules is their remarkable ability to trigger a range of biological responses by adopting distinct three-dimensional structures. The range of structural diversity forms the basis of a wide range of scientific disciplines ranging from molecular recognition and drug design through to protein folding and design. The Biomolecular Structure Laboratory at the JCSMR seeks to carry out research that explores how the structural properties of biological molecules can impact on biological process involved in health and disease. Through our close ties with biomedical researchers and clinicians at the JCSMR our laboratory is perfectly placed to examine how bioactivity is governed by molecular shape and recognition.
Although the main focus of our research is from a structural perspective,
an integrated approach involving complementary techniques such as
molecular biology, kinetics and molecular modeling are routinely employed
in the laboratory. Research projects currently under investigation
relate to a wide range of diseases and applications including cancer,
malaria, heart disease, muscular dystrophy and virus related illnesses
such as AIDS and Ross River fever.
A number of projects are currently the focus of our research efforts;
these include (1) the structure, specificity and mechanism of enzyme
systems including dihydrofolate reductase, glutathione-S-transferases
and chitinases (2) structural and functional studies of muscle related
proteins (3) the role of ion channels in virus associated proteins.
(1) The mechanism by which enzyme systems function is central to
the development of effective therapies associated with these systems.
The enzyme dihydrofolate reductase is the target for a extensive ranges
of diseases such as cancer, malaria, and bacterial infections and
we have used NMR structural data, molecular biology and enzymology
to determine how this enzyme functions. Enzymes, such as glutathione
S-transferase, are involved in the metabolism of chemical toxins and
mutagens as well as of therapeutic agents. A detailed understanding
of their specificity and mechanism is crucial if one is to be able
to predict the metabolism of foreign compounds.
Chitinases are sugar degrading enzymes that specifically target chitin.
Both chitin and chitinase are widespread in nature, occurring in a
range of organisms and are consequently, of major biotechnological
interest. We are actively involved in the structural study of a chitin
binding domain and chitinase with the view of investigating its binding
and inhibitory properties.
(2) For skeletal and heart muscle to function properly careful regulation
of calcium levels must occur. In skeletal muscle two proteins, the
dihydropyridine and ryanodine receptors interact, triggering the release
of calcium. We are using high resolution NMR spectroscopy to determine
how these proteins function by firstly determining the structure of
various regions of these proteins and then using this structural information
to determine how they interact. As a result of this work we have designed
a series of peptides and peptido-mimetic analogues which have the
ability to regulate calcium levels in both skeletal and cardiac muscle.
Such peptide therapies may hold the key to designing new drugs which
may be beneficial in the treatment of a range of muscle -related diseases
such as heart failure, malignant hypothermia and muscular dystrophy.
(3) Many membrane proteins are essential components for the survival
of viruses and we are targeting several proteins which form ion channels.
The aim is to design "blockers" of the ion channels based
on a structural knowledge of these ion channels. In one case, we have
identified a drug which slows the replication of the AIDS virus. Work
is currently underway to chemically and structurally optimise the
effectiveness of this drug. This approach will give rise to a new
generation of drugs to treat diseases such as HIV AIDS, hepatitis
C and Ross River Fever.
Facilities
The laboratory is well equipped with access to state-of-the-art facilities
including two high field Nuclear Magnetic Resonance spectrometers
(Varian, Inova 500 & 600) and a network of Silicon Graphics workstations
running the latest structure related software. The ANU is also due
to take delivery of an 800 MHz NMR spectrometer (2004). Other facilities
include stopped-flow instrumentation capable of performing CD, fluorescence
and UV kinetic analysis which is housed in a well appointed molecular
biology laboratory.
Computational Proteomics and
Therapy Design
Dr Jill Gready
The Group's research aims to understand how the structure and function of proteins have evolved together, and how the molecular details of their structure, interactions, energetics and dynamics enable proteins to perform their functions. Such understanding can be developed only by undertaking detailed studies on particular protein problems, and then developing more general conclusions through comparative studies. While such studies have always used information from the literature, the scale and richness of data coming from the discovery-driven "Xomics" revolution - genomics, structural genomics, functional genomics and proteomics - in biological science is transforming the way in which protein structure and function problems are framed and undertaken. A particular goal of our research is to couple computer-based methods and experiment in study of particular topics within our two main themes, enzyme mechanisms and extracellular receptors. The Group also undertakes development of theory, methods and programs in the area of computational simulations, including in collaboration with Dr Andrey Bliznyuk and Dr Ivan Rostov in the ANU Supercomputer Facility and Fujitsu Japan, as part of our participation in a Research and Development contract with ANU.
Theme 1: Mechanisms of enzyme reactions:
In this area, our aim is to predict the energetics and mechanisms
of enzymic reactions. Of particular interest is how the enzyme environment
binds substrates, products and transition states, and facilitates
the reaction. This requires definition of factors such as the roles
of protein sidechains in the active site, electrostatics of the whole
enzyme, and enzyme-bound solvent and protons. Also of interest, is
how this environment is modified and conserved by protein evolution.
Much of this information, particularly energetic contributions, cannot
be obtained easily or unambiguously from experiment. We are using
a suite of computational modeling methods, including high-level and
more approximate quantum mechanical (QM) methods, linear-scaling QM,
molecular mechanics (MM) and dynamics (MD), and hybrid QM/MM potentials
within MD simulations. We are currently applying these to the study
of two enzymes.
Dihydrofolate reductase (DHFR) has become a paradigm test case for understanding both its catalytic mechanism, for which critical aspects have long remained elusive, and ligand binding, which is of significant interest as DHFR is a key enzyme in DNA biosynthesis and a major target for the antifolate class of cytotoxic drugs, which includes anti-cancer and antimicrobial agents. Our mechanistic work addresses fundamental questions in the catalysis and forms the basis of a more sophisticated approach to design of enzyme inhibitors, some of which we have already successfully tested experimentally.
Rubisco. The photosynthetic enzyme, D-ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) is responsible for virtually all carbon fixation (from carbon dioxide) in the biosphere. However, it also has the dubious honour of being the most abundant protein on earth, a consequence of its woeful catalytic efficiency, being both very slow and poorly selective for its substrate CO2, as well as binding oxygen (O2) and catalysing a competing wasteful oxygenation reaction. Understanding the reasons offers huge potential as a basis for reengineering Rubisco; even modest improvements in efficiency have major implications for improving light, water and nutrient utilization by plants, and, hence, applications for better agricultural crops, greening deserts and degraded land, and for soaking up green-house gases. Our work again exploits computational methods for obtaining information not available from experiment for the complex multi-step reaction, in particular the states and roles of the "invisibles" - protons and water molecules - which appear to hold the key to the issues of efficiency and selectivity. The work is being undertaken in collaboration with Professor John Andrews in the Research School of Biological Sciences.
Theme 2: Mechanisms of action of modular extracellular (EC) proteins:
In this second area, our aim is to understand how evolutionary factors
have produced the spectrum of EC proteins, in particular membrane-bound
receptors and proteins of the extracellular matrix, and how their
structures and ligand-binding properties are related to their specialized
functions in multicellular organisms. These proteins characteristically
have several discrete domains, often with independent ligand- or protein-binding
capacities, and are the result of recent rapid evolution involving
extensive duplication of genes and recombination of the domains as
modules. This has produced large families and superfamilies of proteins
divergent in both structure and function. EC receptor functions are
required for intercellular communication, and defence and repair systems
in multicellular organisms.
Understanding the molecular basis of dysfunction in these systems of protein families is important as they underlie many of the human diseases which are most difficult to treat. This is because the complex interactions among these systems, especially overlapping specificities for ligands producing redundancy in their actions, complicates the design of selective therapeutic agents. By contrast for enzyme inhibitors, the principle of one molecule (i.e. drug) to target one site or mode of action (i.e. the enzyme) usually works well. Our work addresses these issues using both a comparative genomics approach, which seeks to understand how the families and superfamilies observed in higher vertebrate genomes (currently human and mouse) have arisen from lower vertebrates (currently Fugu and other fish genomes) and invertebrates, and by detailed studies on particular mammalian proteins. For this work we are using a strongly coupled combination of experimental and computer-based methods, focusing on two main problems.
Prion protein (PrP) is the unusual protein for which an aberrant conformational form (PrPSc) is associated with diseases such as mad-cow disease (BSE) and Creuzfeldt-Jakob disease (CJD) in humans. We have been studying the conformational and ligand-binding properties of the N-terminal repeat region of PrP using biophysical and computational methods, and the effect of glycosylation of the C-terminal folded region on conformational stability, unfolding pathways, and disease-associated mutants using MD simulations. Such information is critical to understanding the potential of PrP to interact with other proteins and ligands, which underlies its normal, but as-yet-still-unknown, function, and its capacity to change into other conformational states with apparently neurotoxic or, unprecedented, infectious properties. Our second approach is to attempt to track the evolutionary origin of the PrP gene, and the development of its function in higher vertebrates, by identification and analysis of homologues in fish. This has been facilitated by the availability of several fish genome databases (Fugu, zebrafish, tetraodon) in 2002. We are studying the relationships of mammalian and fish PrP-like genes and proteins by bioinformatic and molecular biology methods, in collaboration with Professor Jenny Graves in the Research School of Biological Sciences, ANU and Professor Tatjana Simonic at the University of Milan.
C-type lectin-like domains (CTLDs). Our second area of research
is the structural and functional evolution of proteins containing
CTLDs. This is a large diverse superfamily of proteins with functions
in intercellular signalling, matrix interactions, and both the innate
and adaptive immune systems. This diversity appears to have been enabled
by properties of the CTLD fold which is unusually "plastic"
structurally, allowing rapid evolutionary adaptation to bind many
different ligand types (sugars, proteins, lipids, ice, CaCO3)
with several surfaces of the CTLD having been recruited for binding,
sometimes simultaneously in the same CTLD. We are studying this diversity
at whole-genome level (human, mouse, C. elegans, Drosophila, Fugu)
by sequence and phylogenetic analysis, and by comparative structural
analysis of solved structures. Combining sequence and structure, we
have predicted and ranked the major CTLD groups in the C. elegans
genome and a representative structure of each is being solved in a
structural genomics collaboration with RIKEN Genomic Sciences Center,
Yokohama. Some individual CTLD-containing proteins are also being
investigated by a combination of experimental, bioinformatic and modeling
methods, in particular some novel sugar-binding members expressed
on dendritic cells, in collaboration with Dr Mark Hulett in JCSMR
and Dr Nick Dixon in the Research School of Chemistry, ANU.
Cytokines and Inflammation
Allergy and Inflammation Research Group
Dr Paul Foster
The aim of this research is to understand the cellular and molecular networks that underlie the development of disease processes associated with asthma and allergic disorders. We also focus on understanding how respiratory viral and bacterial infections regulate the immune system and their role in protecting or predisposing to allergic disorders and chronic respiratory infections.
The worldwide incidence, morbidity, and mortality of allergic asthma and allergies are increasing at a dramatic rate. Deaths from asthma have now reached over 180,000 worldwide annually. 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. Allergic disease of the gastrointestinal tract in response to food allergens is also of growing concern in developed countries. One of the major precipitating factors of asthma, particularly in children, is viral infection of the respiratory tract.
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A predominant clinical feature of allergic disease is a persistent inflammation at the site of disease. In asthma, inflammation is localized to the airway wall. Currently, it is thought that inflammatory cells (white blood cells) induce asthma by releasing substances that damage the lining of the airways and induce constriction (narrowing of the airways), mucus production and remodeling of tissue. Dr Dianne Webb |
The inflammatory response in the asthmatic lung and in other allergic diseases is a very complex mixture of cells and molecules and it is not clear which factors play the major role in inducing disease.
Allergic asthma is recognized as a chronic inflammatory disease of the airways that is characterized by reversible airways obstruction in association with aberrant CD4+T helper 2 (Th2 cell) lymphocyte responses to common environmental stimuli. Indeed, the hallmark features of allergic asthma, elevated serum immunoglobulin E (IgE), mucus hypersecretion, eosinophilia and enhanced bronchial reactivity (airways hyperreactivity [AHR]) to non-specific spasmogenic stimuli have all been linked to the effector functions of Th2 cytokines (e.g. interleukin- (IL)-4, 5, 9, 10 and 13). Collectively, it is these pathogenic processes that are thought to promote airways obstruction in asthma, which predisposes to wheezing, shortness of breath and life-threatening limitations in airflow. Th2 mediated immune processes have also linked to the pathogenesis of allergic diseases of the gastrointestinal tract. Viral infections of the lung play a key role in exacerbating asthma and eosinophils and T -lymphocytes are also thought to underpin aspects of viral induced asthmatic episodes.
Research in our Group focuses on two major areas:
1. Identifying the key cells and molecules, which underpin the pathogenesis of allergic disease of the lung, skin and gastrointestinal tracts and those that underpin the pathogenesis of respiratory viral infections.
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 and allergy and viral-induced disease of the lung.
Our experimental approach is integrative, employing state-of-the-art molecular genetic techniques in association with model systems to identify the role of inflammatory cells and molecules in the events that underpin disease. Projects within the group focus on the role of individual Th2 cytokines in disease pathogenesis and characterization of the downstream signaling pathways employed by theses molecules to induce disease.
Cytokine
Molecular Biology and Signalling Group
Professor Ian Young
Interleukin-5 (IL-5) is a hormone-like protein which plays a key role in the regulation of immune responses to helminth parasites and to allergens. This cytokine regulates inducible white blood cell formation in response to infection by helminth parasites or to allergen exposure and is principally responsible for controlling the eosinophilic inflammation characteristic of asthma and allergy. This inflammatory response is primarily orchestrated by the inducible production of IL-5 by T lymphocytes which results in enhanced production of eosinophils in the bone marrow. IL-5 then cooperates with other cytokines like eotaxin which promote the migration of the eosinophils to the affected site. Our group has had a long term interest in defining the biological role, regulation and signalling mechanism for IL-5. It is expected that these fundamental studies will provide opportunities for the development of novel therapeutic approaches for the treatment of allergic disease and also information relevant to understanding some leukemias.
Collaborative studies with the Allergy and Inflammation Research Group and the Gene Targeting Laboratory on the role of IL-5 in allergic lung disease have continued. The present emphasis is to define the roles played by IL-5 and eotaxin in regulating eosinophilic inflammation and airways hyperresponsiveness using mice deficient in one or both cytokines. The results indicate that eosinophils play a key role in the orchestration of allergic inflammation and airways dysfunction. These and other studies carried out by the Cytokines and Inflammation Group, have formed the basis of our NH&MRC Program Grant on the Molecular Mechanisms in the Regulation of Allergy and Inflammation which starts in 2003.
Studies have also continued on the mechanisms regulating IL-5 gene 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. Jun Wang established transient expression systems in both mouse and human T cells and carried out comprehensive transactivation studies to identify the transcription factors and the binding sites involved in the regulation of the mouse and human IL-5 genes. The results suggest the formation of an enhanceosome type complex in the proximal promoter region involving the transcription factors GATA-3, AP-1 and Ets and possibly also HMGI(Y) which regulates inducible and tissue-specific gene expression. The involvement of MAP kinase pathways in IL-5 induction has also been studied.
The receptor system for IL-5 is shared with two related cytokines, IL-3 and GM-CSF, which are also involved in the regulation of blood cell formation and inflammation. One of the major problems in cell signalling is to understand how these cytokines regulate blood cell growth and function by binding and activating their cell-surface receptors. In a collaborative project with Paul Carr and David Ollis (Research School of Chemistry, ANU) using X-ray crystallography we have determined the structure of the complete extracellular domain of the beta common receptor which is the major signalling entity of the IL-5 receptor and is central to the signalling of all three cytokines. The receptor was expressed in insect cells and its crystallization and derivatization involved extensive use of site-directed mutagenesis to improve crystal quality and to solve the phase problem. The novel dimer configuration of the receptor gives new insights into receptor activation. James Murphy is using site-directed mutagenesis to define the residues of the beta common receptor which are involved in forming the activated receptor complex. Peter Fineran, Alice Church, Sally Ford, Janine Inggs and Jane Olsen have prepared the activated complex of the closely related beta-IL-3 receptor. Further structural studies should give a better understanding of the process of receptor activation and provide opportunities to develop drugs capable of controlling this important receptor system. Such drugs could be useful in treating asthma, allergy or cancer.
The group has also participated in another project in the area of
functional genomics with Hugh Campbell (Research School of Biological
Sciences, ANU) and the Gene Targeting Laboratory, JCSMR. In this work,
the functions of the mammalian homologues of two interesting genes,
flightless and small optic lobes, with functions in
development and behaviour in the fruit fly Drosophila are being
investigated by gene targeting in mice. Investigations to date have
shown a function for mammalian flightless in early embryonic
development, remarkably analogous to its role in Drosophila.
This cross-organism approach uses information gained from studies
with the simpler fruit fly to better understand development and brain
function in mammals.
Gene Targeting
Dr Klaus Matthaei
Creating mice with a pre-determined genetic makeup
A major aim of modern biology is to understand how normal gene activities
give rise to the structure and behaviour of complex organisms. In
particular it is important to study the function of genes and their
derangements 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. However, natural mutations occur in a serendipitous
manner, ie by chance. To find a mutation that mimics a particular
human disease is therefore difficult. However, given a knowledge of
the nucleotide sequence of a gene, it is now possible to make changes
to the corresponding endogenous gene of an embryonal stem cell and
to produce a mouse that is homozygous for the desired mutation. This
procedure is called gene targeting.
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 totipotent and can be used to regenerate live normal
animals (ie 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 into
pseudo-pregnant mice and give rise to live chimæric offspring
that 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 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, ie. 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 the ability to study the function
of a cloned gene in the context of the whole mammal by creating mutant
mice defective in specific genes. This is particularly important since,
with gene targeting, mouse models can be created for studying human
genetic diseases which provides a powerful approach to the development
of somatic gene therapy. Moreover, it is possible to also add genes
using similar processes resulting in "transgenic" mice.
Our laboratory has generated a number of different "knockout"
and "transgenic" mouse mutants using C57BL/6 or BALB/c ES
cells which are at different stages of investigation. These include
mouse models of asthma, nerve re-generation, xenograft rejection,
parasite-host relationships, hypertension, drug de-toxification and
cancer.
Gene Regulation in Development
and Immunity
Cytokine
Gene Expression Laboratory
Dr Frances Shannon
The immune system fights infection by activating a variety of cell types, which interact in a complex way to control and eliminate the infection. One of the cell types involved is the T lymphocyte, which when activated, produces a variety of secreted proteins known as cytokines. Cytokines play an important role in cell:cell communication in an immune response.
Cytokines are made in T cells in response to activation of the T cell receptor by MHC:antigen complexes on antigen presenting cells and through other cell:cell interactions known as costimulatory signals. Inside the cell, these signals are fed, through a series of intermediate signalling molecules, into molecular switches that control the level of expression from a specific set of genes. These molecular switches are composed of large protein complexes that assemble on specific DNA sequences upstream of genes (promoter or enhancer regions) and control the activity of the RNA polymerase at that gene. This in turn controls the amount of RNA and protein made from the gene.
The promoter and enhancer regions of many of the cytokine genes are very well defined but there are still a number of important questions that need to be answered if we are to fully understand the activation of genes in an immune response or eventually control the function of these molecular switches. Firstly, we need to know the entire protein composition of these molecular switches. We are applying a functional proteomics approach by purifying the complexes and then identifying the components using mass spectrometry (see Project 1). We will then map the binding sites of these proteins on cytokine genes in vivo using chromatin immunoprecipitation. Secondly, we need to know how endogenous gene expression takes place in the context of packaged DNA in the nucleus. We have developed assays that allow us to measure changes in this packaging (chromatin remodelling) on cytokine genes in primary T cells. We are using these assays to determine the role of specific transcription factors in chromatin remodelling (see Project 2). We are also carrying out structure function analysis of one of the key molecules (c-Rel) involved in cytokine gene transcription in T cells in order to elucidate its mode of action (see Project 2). Thirdly, we are investigating how the signals impinging on the T cell are relayed through intermediate signalling molecules to the molecular switches on the DNA (see Project 3). Finally, we are using a functional genomics approach to determine the overall patterns of gene expression in T cells following activation. This involves expression profiling using Affymetrix microarrays followed by computational analysis of gene control regions to identify common patterns of activity for the molecular switches (see Project 4).
Project 1. A functional proteomics analysis of transcription complexes
on the GM-CSF gene promoter (Dr Torsten Juelich, Ms Xinxin Chen, Mr
Ian Parish in collaboration with Dr Adele Holloway)
According to our current model of transcriptional regulation, so-called
multi-subunit co-regulator complexes have evolved in the cell to act
as an interface between gene specific factors and the general transcriptional
apparatus in order to direct transcriptional initiation by the RNA
polymerase II machinery. These complexes can either interact directly
with the transcriptional machinery (eg mediators), or be directed
towards chromatin in the vicinity of responsive genes to either modify
histones (eg histone acetyltransferase complexes) or to remodel
the structure of nucleosomes (eg ATP-dependent remodelling
complexes).
Whilst an ever-growing number of co-regulators is being identified,
little is known about the nature of such complexes and their role
for correct temporal and spatial activation of the cytokine genes
such as the human GM-CSF gene.
The aim of this project is to understand firstly, what co-activator
complexes interact with transcription factors known to be important
for GM-CSF gene activation, and secondly, in what order those complexes
are being recruited to the gene in the nucleus.
To analyse coactivator complexes involved in this process, a functional
proteomics approach is being utilized. Different regions of the human
GM-CSF promoter are produced by PCR using biotinylated 5' primers
and immobilized to streptavidin-conjugated sepharose beads. Bead/oligo
conjugates are then incubated with nuclear extracts of either resting
or activated Jurkat T cells and samples subjected to 1D or 2D gel
electrophoresis, followed by identification of regulator proteins
by mass spectrometry. The in vivo binding of proteins identified
in these in vitro approaches will be verified using chromatin
immunoprecipitation in T cells. Subsequently, this method will be
employed for nucleosome-assembled DNA templates of the GM-CSF promoter
to be able to closely mimic physiological conditions.
In order to generate nucleosome-assembled templates that mimic the
physiological structure, we are also mapping chromatin structure across
the GM-CSF gene in T cells. Using a number of nuclease accessibility
approaches, we have positioned a nucleosome at approx -175 to +24bp
across the transcription start point, and found that the accessibility
of this region increased following T cell activation, which indicates
some structural change in this region of the chromatin. The region
spans the transcription start site as well as many of the functionally
significant transcription factor binding sites and may play an important
role in controlling GM-CSF transcription.
Project 2. c-Rel is essential for regulation of interleukin-2 gene
transcription in T cells. (Dr Sudha Rao, Ms Donna Woltring, Ms Karen
Bunting, Mr Ziyad Jhumka in collaboration with Dr Steve Gerondakis
and Dr Tom Parks)
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The structure of chromatin and its remodeling following activation
are important aspects of the control of inducible gene transcription.
The interleukin-2 (IL-2) gene is an important cytokine that
drives the proliferation of T-cells, B-cells and natural killer
cells. The expression of this cytokine is induced in a cell
specific manner in T-cells following an antigenic stimulus.
We have previously shown that one of the critical events leading
to increased IL-2 transcription is an alteration in chromatin
structure across the 300bp region of the IL-2 gene. Recently,
we have shown that IL-2 transcription in CD4+ primary
T cells is dependent on c-Rel but not RelA. |
We found that c-Rel is essential for global changes in chromatin
structure across the 300bp IL-2 promoter in response to CD3/CD28 in
primary CD4+ T cells but not in response to the pharmacological
signals, paralleling the requirement for c-Rel in IL-2 mRNA accumulation.
Interestingly, measurement of activation-induced localized changes
in chromatin accessibility revealed that accessibility close to the
c-Rel binding site is specifically dependent on c-Rel. These results
suggest a non-redundant role for c-Rel in generating a correctly remodeled
chromatin state across the IL-2 promoter and imply that the strength
of the signal determines the requirement for c-Rel.
Precisely how c-Rel carries out these important functions during T
cell activation is not well understood. To further dissect the role
of c-Rel in cytokine gene transcription and chromatin remodelling
we are undertaking a structure/function analysis of the c-Rel protein.
So far, we have identified a number of mutations in the human c-Rel
protein which confer dominant negative activity in a cell line reporter
assay. We plan, firstly, to use these mutants to examine what effects
they have on IL-2 and GM-CSF gene transcription and chromatin remodelling
in activated T cells. Secondly, we will use a biochemical approach
to determine how these single residue substitutions have altered the
capacity of these mutant proteins to fold correctly, to form dimers
with wild-type c-Rel and other members of the NF-kB family, to bind
DNA and/or to interact with transcription coactivator proteins. Using
these molecular approaches, we hope to elucidate the biochemical properties
of c-Rel and its role in IL-2 and GM-CSF gene transcription.
Project 3. Coordinated kinase signalling pathways involved in interleukin-2
gene regulation. (Ms Anna Moore, Dr Sudha Rao, Ms Donna Woltring)
Signals derived from the T cell receptor and costimulatory receptors
are mediated by two major kinase signalling pathways; the PI3-Kinase/Akt
pathway and the Protein Kinase C (PKC) pathway predominantly through
the PKC theta (PKC
)
isoform. Activation of these signalling pathways has been linked to
the downstream activation of the NFkappaB/Rel family of transcription
factors. Recent evidence pinpoints the NF-kappaB family member, c-Rel,
as critical for IL-2 gene transcription following T cell activation.
We hypothesise a direct signalling link from PKC
to c-Rel to control cytokine gene transcription.
Using a specific PKC
inhibitor, Rottlerin, we have shown that both c-Rel mRNA synthesis
as well as c-Rel translocation to the nucleus require PKC
in primary CD4+ T cells. In contrast, c-Rel mRNA synthesis
is not dependent on PKC
in a mouse T cell line, EL-4, implying a signalling defect in these
cells. Inhibition of PKC
also leads to reduced chromatin remodelling of the IL-2 proximal promoter
after stimulation in both the EL-4 T cell line and primary CD4+
T cells implying that chromatin remodelling is at least partially
dependent on PKC
signalling. Interestingly, in CD4+ T cells the PKC
message is down-regulated following stimulation and this effect is
enhanced by Rottlerin treatment. These data support the essential
role of PKC
and c-Rel in IL-2 gene activation and suggests PKC
signals upstream of c-Rel leading to IL-2 gene activation. On the
other hand, the PI3-kinase/Akt pathway does not appear to play a role
in chromatin remodelling but is critical for the overall rate of transcription.
Mice where the PKC
gene has been deleted will now be used to further dissect the link
between PKC
and c-Rel.
Project 4. Deciphering the role of c-Rel in T cells by a genome-wide
analysis approach. (Dr Sudha Rao, Ms Donna Woltring in collaboration
with Dr Steve Gerondakis and Dr Gareth Denyer)
We are currently attempting to understand the global roles of c-Rel in T-cells by using Affymetrix murine U74A arrays to measure gene expression of CD4+ T-cells from WT and rel-/- mice treated with CD3/CD28 or pharmacological agents. Using k-means clustering analysis, we have identified a cohort of genes whose expression pattern is similar to IL-2. Interestingly, we have found that c-Rel appears to function as both an activator and repressor of transcription in T-cells. In the near future, our goal is to understand c-Rel regulatory networks in T-cells by performing comparative promoter analysis on the groups of co-regulated genes we have identified from our array analysis. This approach would allow us to determine the direct targets of c-Rel in T-cells.
Chromatin
and Transcriptional Regulation Laboratory
Dr David Tremethick
Chromatin and transcriptional regulation during development
The fate of the eukaryotic cell at all stages of its life cycle is
dependent upon the accurate readout of genes encoded by DNA. For example,
the development of a single cell into multicellular organism requires
precise temporal and spatial regulation of gene transcription. Consequently,
certain diseases and developmental disorders are often associated
with, and likely to be caused by, aberrant gene expression. It has
become increasingly clear over the last decade that eukaryotic gene
regulation at the level of transcription is strictly connected to
the structural organisation of the genome. The basis for this structural
organisation is the nucleosome. Our overall aim is to understand how
chromatin structure contributes to the regulation of transcription
during development.
Remarkably, a typical eukaryotic cell contains approximately two meters of DNA, which can be squeezed into a nucleus of about 20 µm in diameter. This packaging of DNA is achieved by a hierarchical scheme of folding and compaction into a protein-DNA ensemble called chromatin. At the first level of organisation, approximately two superhelical turns of DNA are wrapped around a protein complex consisting of eight histone molecules. This complete unit, the nucleosome, forms the basic building block of chromatin and is further reorganised into a regular array to form a chromatin fibre. How this fibre subsequently folds into higher order structures is not yet understood. This protein-induced folding of DNA into a complex three-dimensional structure has profound implications with respect to understanding how gene expression is regulated.
As a result of this compaction of the eukaryotic genome, the conformation and accessibility of DNA is dramatically altered. The compaction of a gene into chromatin clearly impedes the transcription process. The cell has therefore devised mechanisms, which reversibly de-compact or remodel chromatin to allow transcription factor binding by altering the stability of protein-DNA interactions in underlying nucleosomes. The multi-subunit structure of the nucleosome (consisting of four histone-dimer subunits) is ideally suited for performing these opposing roles; nucleosomes are both stable enough to compact DNA while at the same time labile enough to allow access of DNA to transcription factors. This lability can be enhanced by targeted modification of the histone proteins or by changing the biochemical composition of the nucleosome. The hypothesis that we are examining is that the stability of protein-DNA interactions in underlying nucleosomes can be altered by the replacement of major histone types with specific histone variants.
There are two important stages with regard to the accurate transcriptional regulation of a gene. (1) The establishment of gene activity, which for most genes occurs during early mammalian development, and (2) the subsequent maintenance of this gene activity throughout many rounds of cell division during the life of the organism. Major global transitions in chromosome and chromatin structure occur early in development when cell lineage and tissue-specific transcriptional patterns of gene expression are established. Very little is known about these structural changes and the mechanisms by which these changes differentially regulate gene transcription. However, it is clear that chromatin plays a fundamental causal role in determining patterns of gene activity.
An important way to control chromatin function is to alter the biochemical
make-up of the nucleosome by replacing an individual histone with
a histone variant. Interestingly, most histone variants belong to
the histone H2A family implying that H2A plays a unique role in the
nucleosome. Modulating nucleosomal and higher-order chromatin structure
through variation in H2A will have an impact on all nuclear functions.
Our work focuses on a variant of histone H2A referred to as H2A.Z.
The presence and high level of conservation from yeast to man (across
species, the amino acid sequence of H2A.Z is more conserved than the
amino acid sequence of major H2A) shows that H2A.Z plays an important
and specific role in chromosome function. This function is essential
since in Drosophila and Tetrahymena null mutants die.
We also found that the H2A.Z gene is essential for mouse survival
with the defect occurring early in development around the time of
implantation. However, despite being essential, nothing is known about
the specific functional and structural consequences of having H2A.Z
incorporated into chromatin.
To begin to understand why H2A.Z is essential for survival, we adopted an in vivo approach. To search for the unique feature(s) of H2A.Z required for its function, we performed amino acid swap experiments in which residues unique to Drosophila H2A.Z were replaced with equivalently positioned histone H2A residues. Mutated H2A.Z genes encoding modified versions of this histone were transformed into Drosophila and tested for their ability to rescue null mutant lethality. Most interestingly, we discovered that the unique and essential feature of H2A.Z lies outside the histone fold in the carboxy-terminal domain. This C-terminal region maps to a short alpha-helix in H2A that is buried deep inside the nucleosome. A region immediately adjacent to this short alpha-helix, located at the surface of the nucleosome, was also found to be important for adult Drosophila survival. Together, this region forms part of a docking domain, a domain involved in stabilising the interaction between the H2A/H2B dimer with the H3/H4 tetramer and in contributing to surface features of the nucleosome. Based on these results, our prediction was that H2A.Z would alter the stability of the nucleosome potentially weakening the interaction between the H2A.Z/H2B dimer with the H3/H4 tetramer and/or modifying the surface of the nucleosome.
Recently, we tested this prediction, in part, by solving the crystal structure of a nucleosome containing H2A.Z (in collaboration with Karolin Luger from Colorado State University). The overall structure is similar to the previously reported structure containing major H2A. In part, consistent with our prediction, distinct localised changes in the docking domain result in a subtle destabilisation between the dimer and the tetramer. However, there is also a stablisation between the two H2A.Z molecules at the back of the nucleosome. Interestingly, salt dissociation experiments indicate that H2A.Z actually increases the overall stability of the nucleosome. Further experiments are in progress to determine whether this is indeed the case. Potentially even more significant, the amino changes in the docking domain of H2A.Z results in an altered nucleosomal surface that includes a metal ion, a more extensive acidic patch, and a larger hole in the centre of the nucleosome. Our current favoured hypothesis is that these surface changes may create a highly specific interaction interface for other nuclear proteins like chromatin remodelling factors and/or modulate nucleosome-nucleosome interactions.
Most recently we tested the prediction that incorporation of H2A.Z into chromatin alters intra- and/or inter- nucleosomal interactions. This was determined by examining whether this histone variant alters the folding pathway from an extended nucleosomal array to highly compacted heterochromatin. In summary, we carried out the first biochemical analysis of a homogenous preparation of H2A.Z-containing nucleosomal arrays. H2A.Z uniquely affects chromatin condensation; intra-nucleosomal interactions are accentuated while fibre-fibre interactions and the subsequent formation of condensed heterochromatin is inhibited. These data suggest that a major function of H2A.Z is to generate a conformational intermediate in the chromatin-folding pathway poised to be assembled into a specialised functional chromosomal domain.
The prediction, from these structural studies, that H2A.Z functions
to establish a specialised higher-order chromatin domain was recently
tested. Using early mouse embryos at a time when H2AZ null mice die,
confocal immunofluorescence experiments demonstrated that H2A.Z was
located at constitutive heterochromatin. This is the first description
of a function of H2A.Z. Current work is directed to determine, at
the molecular level, the role of H2AZ at this specialised domain.
Autoimmunity
and Genetics Laboratory
Dr Robyn Slattery
Type I diabetes affects 0.3% of people in Australia. The strongest inherited trait associated with this disease is the expression of certain markers on cells in the body known as HLA (or MHC in mice). These markers usually act as traffic lights to the immune system telling it when to stop and when to go. Normally these markers signal a red-light to the immune system so that it does not attack our own cells, and only when there is an infection do these markers send a green signal to the immune system that the infected cells should be destroyed. For reasons which are not well understood, the immune system in diabetes patients perceives these markers on the insulin-producing cells as sending a green signal when there is no apparent infection. This results in the immune system attacking and destroying the body's own insulin-producing cells. When this happens the body is unable to absorb sugar and so a patient becomes diabetic.
The research carried out in the AGM laboratory aims to determine where the faulty signal is initiated i.e. is the 'faulty' signal sent by the beta-cells of the pancreas to a normal immune system, or alternatively, are normal beta-cells being attacked by a 'faulty' immune system? The answers to these questions are of fundamental importance to understanding whether disease may recur in patients transplanted with healthy islets, and therefore whether transplantation needs to be combined with modification of the immune system by vaccination to prevent disease recurrence.
By using a very new genetic engineering tool called the cre-lox recombination system, it is now possible to generate mice which have lost the traffic signal molecule (HLA) from pancreatic beta-cells. Previous genetic engineering methods have concentrated on deleting HLA genes from all cells of the animal. The unique approach of the cre-lox recombination system is that it allows us to distinguish between the role of the HLA molecules on the beta-cells versus on the cells of the immune system.
Through the generation of such mice it has been possible to determine that the disease is initiated, and progresses through all but the final stages of beta cell destruction in the absence of any 'faulty traffic signal' on the target cell. It appears that disease is not initiated directly by the beta cells. Further studies are now focused on understanding which compartment of the immune systyem might be responsible for disease initiation.
During the course of these studies it was discovered that beta 2
microglobulin (ß2M), which forms the light chain which pairs
with the HLA molecule, is a type 1 susceptibility gene in mice. This
identification is the first unequivocal identification of a single
diabetes susceptibility locus.
Molecular Genetics Group
Professor Philip Board
Because human beings are constantly exposed to a variety of environmentally derived chemicals that effect the normal function of our cells, tissues and organs, we have evolved a complex group of enzymes that detoxify these compounds and provide an important layer of protection against their deleterious effects. It is now very clear that an individual's genetically determined complement of detoxication enzymes has a significant influence on their response to a variety of therapeutic drugs and environmentally derived toxins. The work of the Molecular Genetics Group is aimed at gaining a fundamental understanding of the molecular and biochemical mechanisms that underlie individual responses to such compounds. One of the major research interests of the Molecular Genetics Group is the role played by the glutathione-linked enzymes such as the glutathione transferases (GSTs) in the metabolism and detoxification of therapeutic drugs and environmentally derived carcinogens and toxins.
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The GSTs are a large family of enzymes and previous studies have shown that they can be subdivided into a number of different classes that have characteristic structural variations, substrate preferences and sites of expression.
Dr Anna Robinson |
The GSTs function by conjugating glutathione to the target chemical thereby making it more water soluble and making it recognisable by an export pump that expels glutathione conjugates from cells.
Genetically determined deficiency in the expression of some GSTs can be a risk factor for lung, stomach and skin cancer. In contrast, over expression of GSTs has been associated with resistance to cancer chemotherapy. Genetic variations that cause subtle changes in GST function can be clinically important. For example, we found that a variant form of glutathione transferase GSTP1 that works with different substrates was associated with the occurrence of Parkinson's disease in patients who had been exposed to pesticides.
To gain a comprehensive understanding of the genetic diversity in response to environmental toxins and therapeutic drugs it will be necessary to identify all the enzymes involved in detoxification processes and to identify the common genetic variants of these enzymes that contribute to functional differences. The recent expansion of the Expressed Sequence Tag database (EST) to include more than a million DNA sequences encoding copies of most active human genes has provided a remarkable resource for the identification of new genes and polymorphisms. We have developed novel screening strategies that have successfully identified several new glutathione transferase gene families and a number of novel polymorphisms.
The new enzymes discovered by this data mining approach have been shown to catalyse unique detoxification reactions and to participate in metabolic pathways not previously attributed to the action of glutathione transferases. For example we recently discovered that a GST we have termed Omega can inhibit ryanodine receptor calcium release channels in the heart. This enzyme also plays a role in the metabolism of arsenic. Another GST we have discovered and called Zeta is involved in the metabolism of compounds such as dichloracetic acid (DCA) which is known to cause cancer in mice. Significantly, DCA is a contaminant of chlorinated drinking water
The strategies we have developed for screening the EST database can
be readily applied to other genes and gene families and will be of
great value in the identification of new genes and polymorphic variants
of pharmacogenetic interest.
Ubiquitin Laboratory
Dr Rohan Baker
The Ubiquitin Laboratory investigates the small protein ubiquitin; its role in the destruction of other proteins (proteolysis) in the cell; and in the consequences of abnormal proteolysis due to defects in the ubiquitin system.
Complex processes such as cell growth, development, and gene expression, are controlled by proteins that are only required to function for a short time, and must be rapidly destroyed to control their activity. This is accomplished by attaching multiple ubiquitin proteins to the target, which "marks" it for destruction by a large proteolytic complex called the proteasome. Most of our research focuses on a family of enzymes called ubiquitin-specific proteases (USPs), that have the ability to cleave ubiquitin from such marked proteins, and thus slow or prevent their destruction. In this context, some USPs can be considered "proof-reading" components of the system, safeguarding a target from inappropriate ubiquitination (and thus destruction), and imposing a further level of regulation on the pathway. Other USPs function to recycle ubiquitin after protein degradation at the proteasome.
We use mice as a model organism to study human disease. We have previously identified a USP that can cause cancer when overproduced, while others have linked the human version of this USP with certain types of lung cancer. We and our collaborators have shown that this USP interacts with the Retinoblastoma tumour-suppressor protein and other related tumour suppressors, which function to prevent unregulated cell growth by keeping transcription factors inactive. We have found that this USP shuttles in and out of the nucleus in normal cells, but in cancer cells, it becomes trapped in the nucleus. Interfering with tumour suppressor protein levels can also change its location in the cell. Our current efforts are aimed at determining how the nuclear localisation of this USP is linked to cancer, and how it traffics in and out of the nucleus. We are also studying other novel USPs identified by the genome project. One of these seems to have a role in muscle differentiation, and may regulate signalling pathways in this process.
We also study USP enzymes using yeast as a model system, both because it is very amenable to genetic and biochemical studies, and also because the ubiquitin system is so highly conserved between yeast and humans, that insights we gain in yeast can be applied to our studies on mouse and human enzymes. We have found that Ubp6, one of a family of 16 USPs in yeast, is a previously unknown integral part of the proteasome, and its major job appears to be in removing ubiquitin chains from proteins as they are destroyed, thus allowing ubiquitin recycling. Interestingly, recent work from another lab has shown that defects in the mouse version of this enzyme result in neurodegenerative disease, suggesting that a lack of free ubiquitin is at fault.
Our results show that USPs can play important roles in regulating
the ubiquitin pathway, and through this, processes such as cell growth
and cell development. Aberrations in the function of these USPs may
lead to cancer and disease.