Human epidemiological and human descriptive studies of tuberculosis and HIV infection
An important aspect of our work is discovery of novel therapeutic interventions that promote immune mediated control of chronic overwhelming infections. The ability to test these experimental therapies in pre-clinical models is a prerequisite to clinical translation.
The transition from basic science discoveries to clinical trials underpins our work but it is equally important to examine human epidemiological data and human correlates of disease that provide profound insights into pathogenesis and drive further basic science research.
To this end we are establishing collaborative field studies in Papua New Guinea and Uganda that will allow us to define population and host variables that impact on infectious disease outcomes in Mycobacterium tuberculosis and HIV infection. This will allow us to generate new hypotheses that can be tested in our basic science and pre-clinical models.
Testing therapeutic interventions in pre-clinical hepatitis B virus, HIV and tuberculosis infection
We have developed several unique modifications to generate human immune system (HIS)-reconstituted mice that allow an unprecedented ability to modulate genetic pathways during the course of HIV infection.
HIS mice are animals that have been transplanted with human haemopoietic stem cells capable of recapitulating the human haemopoietic system, particularly the human lymphoid compartment. Additionally we have engineered the animals to express the human IL-7 cytokine at levels that maintain physiological proportions of T cells, B cells and myeloid cells. Prior to transplantation the human haemopoietic stem cells can be transduced with genetic vectors that permit reversible silencing of genes in vivo.
Using this system we can analyse the human immune response to HIV and the virological consequences that follow the transient silencing of human immune cell signalling pathways. Similar preclinical in vivo infection models allow us to examine the role of genes that regulate immunity to HBV and MTb infection.
Identifying host genes that abrogate immunity
Using an extensive array of gene-targeted mice and innovatively engineered haemopoietic chimeric animals, in which genes can be reversibly silenced mimicking a therapeutic drug, we are able to interrogate which signalling pathways promote or obstruct immune mediated clearance of chronic overwhelming infections.
Signalling pathways that are being examined include cytokine networks, NFκB, JNK, pattern recognition receptor signalling, apoptosis and necroptosis pathways. Utilising virological, molecular and immunological techniques we are able to determine which genetic pathways are most amenable to therapeutic targeting and offer the greatest translatable potential to promote immune mediated control of human chronic overwhelming infections.
Protein translocation to the mitochondria
About 30% of all cellular proteins are found in membranes, transported there as precursors by vesicular transport or tightly regulated translocase systems. In eukaryotic cells, mitochondrial membrane proteins undergo a sequence of chaperoned transfers across a dual membrane system prior to final assembly, utilising dedicated mitochondrial translocases. Transport of membrane-targeted precursors presents specific issues, most notably non-specific aggregation of transmembrane segments. At the same time, internal targeting signals must remain accessible to engage translocase receptors during serial changeovers.
The targeting and transfer of membrane-spanning transporters through the periplasm-like environment of the intermembrane space requires specialised translocase components. Our structures of Tim9-Tim10 revealed a modular propeller-like fold, suggesting a previously unanticipated mode of chaperone activity and serving as a model for deriving function. Achieving our objective of experimentally defining the interactions of mitochondrial carrier family precursors with translocase components would represent a significant step toward elucidating the pathway, and provides a direct means of establishing novel chaperone function.
Mechanisms controlling current in ion channels
Potassium currents are an essential part of electrical signalling in all cells, responsible for a functioning central nervous system as well as the activity of cardiac, renal and other organs.
Potassium conduction across cell membranes occurs through the highly selective pore of K+ channels, and is regulated by cellular and electrical signals. The flow of ions is controlled by means of molecular gates in the permeation pathway that alter the energetics of permeation.
Our main objective is to determine the nature of these gate(s) and pin down the mechanisms that cause the channel to switch between conducting and non-conducting states. While the ‘gate’ has historically been ascribed to a narrow cytoplasmic aperture at the junction of four transmembrane helices (one from each subunit), we have provided novel evidence attesting to greater complexity within the system, showing that conduction status is correlated to subtle conformational changes in a conserved intracellular assembly, irrespective of aperture width.
Targeting inflammation during islet transplantation
For some individuals with type 1 diabetes, being glucose unaware is a persistent life threatening condition. One definitive treatment is islet transplantation, which is effectively a cure for type 1 diabetes. Currently islet transplantation is limited by poor engraftment of islets from multiple donors, and the rejection of these islets after a number of years. Both of these processes are linked to the innate immune system and inflammation.
One substance that could be contributing to the failure of islet transplantation is islet amyloid polypeptide (IAPP), which is produced by beta cells when they make insulin and is found near transplanted islets [3]. We have discovered that IAPP oligomers activate the innate immune receptor Nlrp3, making the pro-inflammatory cytokine IL-1b [2]. It has also been recently shown that blocking IL-1b facilitates islet transplantation in mice [1].
In this project we propose to dissect the molecular basis for this and, in collaboration with industry, test neutralising antibodies targeting these inflammatory pathways. We will use mice with genetic deletions of innate immune receptors in models of islet transplant, and perform pre-clinical trials using new biologic reagents in humanised mouse models of disease.
The primary outcome of this project will be to improve islet transplantation as a potential cure for type 1 diabetes. Because this disease afflicts almost half a million children between the ages of 0-14, the potential to provide these individuals with decades of improved quality of life is particularly appealing.
Relevant references
1. Westwell-Roper, C, Dai, DL, Soukhatcheva, G, Potter, KJ, van Rooijen, N, Ehses, JA, and Verchere, CB. IL-1 blockade attenuates islet amyloid polypeptide-induced proinflammatory cytokine release and pancreatic islet graft dysfunction. J Immunol, 2011. PMID: 21813778
2. Masters, SL, Dunne, A, Subramanian, SL, Hull, RL, Tannahill, GM, Sharp, FA, Becker, C, Franchi, L, Yoshihara, E, Chen, Z, Mullooly, N, Mielke, LA, Harris, J, Coll, RC, Mills, KH, Mok, KH, Newsholme, P, Nunez, G, Yodoi, J, Kahn, SE, Lavelle, EC, and O'Neill, LA. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1beta in type 2 diabetes. Nat Immunol, 2010. 11: 897-904. PMID: 20835230
3. Westermark, GT, Westermark, P, Berne, C, and Korsgren, O. Widespread amyloid deposition in transplanted human pancreatic islets. N Engl J Med, 2008. 359: 977-9. PMID: 18753660
Screening for virus miRNA that target the host innate immune response and inflammation
The body’s first line of defense towards pathogens and tissue damage is the inflammatory response. One important class of regulators that limit inflammation are micro-RNAs (miRNAs), small nucleic acid-based molecules encoded in the human genome. It has also been discovered that some viruses produce miRNA [4], and this project will be the first to screen for how these might limit host inflammation to give the virus an advantage.
To do this we will take the following approaches:
1. Debilitating viruses with many miRNA, such as Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein Barr Virus (EBV), are difficult to work with and do not infect mice. Therefore we will transfer the KSHV/EBV miRNAs to the closely related Murine gammaherpesvirus 68 (MHV-68) [1] and infect cell lines and live mice to look for effects on the innate immune response and inflammation.
2. Our second screening approach involves herpes simplex virus (HSV-1). We can deplete the miRNA which are expressed by HSV-1 using 'sponge' technology [3]. Therefore we can create a cell population in which the HSV-1 miRNA are non-functional, and we can compare the inflammatory response to control cells.
3. Finally we will take individual miRNA from KSHV, EBV or HSV-1 and determine the exact inflammatory pathway they control, and the specific target that they regulate.
We expect the outcome of this research to be a definitive catalogue of those miRNA in EBV, KSHV and HSV that regulate innate immune pathways and inflammation. Herpes viruses are responsible for extensive death and disease worldwide [2]. Some of these viruses are also implicated in the pathogenesis of lymphomas, such as Kaposi’s Sarcoma (KS). Learning how viruses use miRNAs to prevent inflammation will help develop new strategies to treat, vaccinate and prevent the spread of these diseases.
Relevant references
1. Mount, AM, Masson, F, Kupresanin, F, Smith, CM, May, JS, van Rooijen, N, Stevenson, PG, and Belz, GT, Interference with dendritic cell populations limits early antigen presentation in chronic gamma-herpesvirus-68 infection. J Immunol, 2010. 185: 3669-76. PMID: 20720208
2. Koelle, DM and Corey, L. Herpes simplex: insights on pathogenesis and possible vaccines. Annu Rev Med, 2008. 59: 381-95. PMID: 18186706
3. Ebert, MS, Neilson, JR, and Sharp, PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods, 2007. 4: 721-6. PMID: 17694064
4. Stern-Ginossar, N, Elefant, N, Zimmermann, A, Wolf, DG, Saleh, N, Biton, M, Horwitz, E, Prokocimer, Z, Prichard, M, Hahn, G, Goldman-Wohl, D, Greenfield, C, Yagel, S, Hengel, H, Altuvia, Y, Margalit, H, and Mandelboim, O. Host immune system gene targeting by a viral miRNA. Science, 2007. 317: 376-81. PMID: 17641203
Role of master regulators in the mammary gland
Recent studies have revealed that mammary stem cells are highly responsive to steroid hormones despite lacking the oestrogen and progesterone receptors. These findings provide a cellular mechanism for the established link between sustained exposure to ovarian hormones and breast cancer risk.
Expression profiling analyses of different epithelial populations has led to the identification of potential effectors of hormone action such as the RANK signalling pathway. Current work is addressing whether blockade of this pathway can prevent breast tumorigenesis or inhibit tumor growth.
The identification of “cells of origin” and cancer stem cells
We are delineating nuclear regulators of mammary gland development in order to understand how their deregulation contributes to carcinogenesis. The analysis of different mouse models of mammary tumorigenesis and human breast tissue has revealed potential ‘cells of origin’ of cancer and cancer stem cells in certain tumours.
Together with the establishment of a large cohort of human breast cancer xenografts in mice, these studies have the potential of identifying molecules that could serve as novel prognostic markers or therapeutic targets in breast cancer.
The identification and characterisation of normal stem and progenitor cells in breast tissue
Key areas of research in the breast cancer laboratory include the identification of normal breast cell types, of molecular regulators that control cell proliferation and maturation, and cell types that are predisposed to carcinogenesis.
One of our working hypotheses is that the different sub-types of breast cancer arise in different types of breast epithelial cells. Recently, we identified stem, progenitor and mature cells in human breast tissue and showed that they share remarkable functional properties with those in mouse mammary gland.
In addition, molecular profiling of the different subsets of cells showed that a large number of genes are conserved across species, highlighting the utility of mouse models to understand human breast cancer. Using the cellular hierarchy as a framework, we have discovered that ductal progenitors are the likely culprits for breast cancers that develop in women carrying a BRCA1 mutation. The cells were found to be expanded and exhibited aberrant growth properties.
In collaboration with Professor Gordon Smyth’s group in the Bioinformatics division, we further showed that the gene profiles of basal breast cancers (an aggressive subtype) were most similar to that of ductal progenitor cells, suggesting that these cells go awry in individuals that develop basal breast cancer. This study also revealed a number of markers overexpressed by the perturbed progenitor cells, potentially providing novel therapeutic targets to be tested in the preclinical models of breast cancer that we have developed in our laboratory.
Inhibitors of apoptosis proteins (IAPs) antagonists
The effectors of the cell death program are enzymes called caspases, that degrade essential proteins within the cell and effectively digest the cell from the inside. Capsases can be regulated directly or indirectly by the so called inhibitor of apoptosis proteins (IAPs). It has been shown that some tumour types, such as hepatocarcinoma, are frequently associated with amplification of cIAP1 and that the tumour cells require cIAP1 to progress to full blown tumours (Zender, L., et al. Cell (2006)).
The discovery of natural mammalian IAP antagonists (by Verhagen et al (2000); Du et al (2000)) and their mode of action led directly to the development of IAP-targeting drugs that mimicked the action of the natural IAP antagonists. Our lab has been fortunate to collaborate with one of the companies involved in the development of such drugs; Tetralogic Pharmaceuticals. Together with Tetralogic Pharmaceuticals, our lab has described how these novel compounds are able to kill cancer cells and this exciting work was published in the prestigious Journal Cell (Silke, J., et al Cell (2007)).
One particularly exciting aspect of these drugs is that they specifically target cancer cells but leave normal cells relatively unaffected and we are focused on trying to discover why the drugs are so selective with the aim of further improving the utility of these new drugs.
Inducible oncogenes
We are investigating the function of characteristic, or pathognomic cancer-related fusion proteins. We want to know how necessary and sufficient these translocations are for tumour development and progression.
We use both in vitro models and are developing in vivo models to study some of the rarer translocations in paediatric solid tumours. The current project focuses on the EWS-WT1 fusion which arises in Desmoplastic Small Round Cell Sarcoma.
Analysis of Hox gene function in myeloid progenitor cells
We generate IL-3- or GM-CSF-dependent cell lines by co-transfecting haematopoietic stem cells with Hox genes (HoxB8 or HoxA9). We use inducible expression systems which allow us to turn expression of the Hox genes on and off.
Hox gene expression blocks progenitor cell differentiation, but also regulates cell survival. How they do this, and how Hox gene expression impacts on the proteins that regulate cell survival, is another major project within the lab.
We are trying to identify genes specifically regulated by cancer-associated Hox genes and to validate a therapeutic approach which targets Hox proteins or Hox protein targets.
How does AKT signaling regulate cell survival?
AKT is a serine/threonine kinase activated by IL-3 signalling and is thought to play a role in the direct regulation of apoptosis pathways. Constitutive activation of the AKT pathway is frequently observed in malignancy, even in the absence of activating mutations.
Using an inducible expression system, we are studying how different AKT isoforms regulate the response to cytokine deprivation. We have also generated cytokine dependent cells from AKT-deficient mice to study the requirement for AKT in IL-3 receptor signaling.
Understanding the molecular signal cascade which is activated by IL-3
We have generated a comprehensive panel of IL-3-dependent myeloid progenitor cell lines from mice lacking Bcl-2 family member genes (which regulate the intrinsic cell death pathway) or genes involved in IL-3 signalling. We use this model to determine which molecules are required for apoptosis when IL-3 is removed and which are required for normal survival signalling when IL-3 is restored.
We identified a key role for the Bcl-2 family member, Puma/Bbc3 in the apoptotic response to cytokine deprivation and showed transcriptional upregulation of Puma in response to IL-3 withdrawal requires p53. We are now working to understand the molecular links between loss of IL-3 signalling and p53 activation. More recently, we are focusing on a novel mechanisms of Puma regulation involving post-translational modification.
Control of haematopoietic stem cell self-renewal
During the majority of foetal life, haematopoietic progenitor/stem cell (HSCs) are restricted to the liver. In the liver, HSCs are thought to be able to rapidly expand in number whilst driving the production of committed haematopoietic lineages, a process known as definitive haematopoiesis.
For this to happen the HSC population must be able to undergo both expanding and maintaining self-renewals (shown in (A) in the figure below). How these decisions are controlled during foetal development is not clearly understood.
We have been able to gain a mechanistic insight into this process using a mouse line harbouring a loss-of-function mutation in the gene Erg; in the absence of functional ERG foetal HSCs fail to efficiently self-renewal.
We have identified two in vivo targets of ERG, Gata2 and Runx1, both of which are dramatically downregulated in Erg-mutant foetal livers, resulting in haematopoietic exhaustion (shown in (B) in the figure below). We are currently investigating how both GATA2 and RUNX1 cooperate in vivo to direct fetal HSC self-renewal.
Haematopoietic stem cell formation
At the core of the question of how haematopoietic progenitor/stem cell (HSCs) are formed is understanding what molecular pathways distinguish HSCs from non-stem cell haematopoietic cells.
In the aorta-gonad-mesonephros (AGM) region of the E11.5 mouse embryo, a population of pre-HSCs exists that co-express endothelial and haematopoietic markers and are spatially localised to intra-aortic clusters within the dorsal aorta (shown in (A) in the figure below.
These cells are committed to haematopoietic fate but do not posses the ability of HSCs to reconstitute the haematopoietic system upon transplantation. However, these cells are poised to mature into fully functional HSCs.
We are currently exploring strategies that can be used to define the factors required and sufficient for the transition of a pre-HSC to a HSC state (shown in (B) in the figure below).
Directing fate through lineage interactions
From embryonic day (E) 7.0 haematopoietic cells appear; this occurs first in the yolk sac. At E10.5, although mature haematopoietic cells and haematopoietic progenitors are present in both the yolk sac and within multiple tissues of the embryo proper, the yolk sac remains a focus of haematopoiesis. We are interested in understanding why this is.
Within the yolk sac haematopoietic cells are in close contact with multiple cell types, particularly the endothelium (shown in (A) in the figure below).
We are currently investigating if/how these multi-lineage associations control the decision of haematopoietic progenitors to either expand their numbers or undergo haematopoietic differentiation (shown in (B) in the figure below).
Haematopoietic development in the embryo
The embryo provides an invaluable resource for tackling the problems of understanding the processes of HSC formation and self-renewal. Embryonic development is the only period in life when the processes of HSC formation and physiological expansion of HSCs occurs. If we can tease apart how the embryo instructs cells to become a HSC and then control when these stem cells self-renew or differentiate to produce functional mature blood cells we should be able to recapitulate these processes in the laboratory, and eventually apply these cells to the clinic.
Haematopoietic development in the embryo occurs in a sequential process (shown below), during which primitive erythropoiesis and progenitor formation occurs in the yolk sac. HSCs are then formed (a process that likely involves the AGM region, yolk sac and placenta) and expand in the placenta and liver. During foetal life the majority of HSCs reside within the liver, where HSCs continue to expand and initiate definitive haematopoisis (a sustained haematopoietic system driven by a self-renewing HSC).
Our laboratory is interested in understanding three events that take place in the embryo:
- how haematopoietic progenitor activity is regulated in the embryo yolk sac;
- the molecular regulation of de novo HSC formation;
- the molecular pathways that underpin self-renewal of foetal HSCs.
Red blood cell development
The demand for banked blood continues to outpace the supply. Due to this lack of donor blood and the risk of transferring infectious blood to patients, a longstanding dream in the field has been to produce fully functional red blood cells (RBCs) in vitro in a large scale.
Human embryonic stem (hES) cells represent an ideal source to overcome this limitation; hES cells can be cultivated in vitro indefinitely providing a potentially inexhaustible and donorless source. In addition, hES cells can be generated pathogenic free without the use of animal serum or animal feeder cells9, 10 and can additionally be screened for any pathogens before differentiation thus avoiding the risk of transferring diseases.
Demonstrating the feasibility of this approach, we have established an efficient yet easy strategy to generate mass cultures of pure, immature erythroid progenitors from mouse ES cells (ES-EP) using serum-free medium plus recombinant cytokines and hormones specific for the outgrowth and proliferation of adult erythroid progenitors.
Importantly, ES-EP exhibit a much longer life span (>90 days) than primary erythroid cells derived from fetal liver or bone marrow without losing cytokine dependency or ability to differentiate into mature RBCs.
Currently we are studying the mechanisms that allow mouse ES cell-derived EP cells to self-renewal long-term and establishing similar erythroid cultures from human ES cells. We hope that these studies allow us to improve our understanding of red blood cell homeostasis and ultimately provide a platform to generate universal human red blood cells for blood transfusion.
Relevant references
- Carotta S*, Pilat S*, , Schiedlmeier B, et al. HOXB4 enforces equivalent fates of ES-cell-derived and adult hematopoietic cells. Proc Natl Acad Sci U S A. 2005;102:12101-12106. (* equal contribution). PMID: 16093308
- Carotta S, Pilat S, Mairhofer A, et al. Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells. Blood. 2004;104:1873-1880. PMID: 15166028
- Kolbus A, Blazquez-Domingo M, Carotta S, et al. Cooperative signaling between cytokine receptors and the glucocorticoid receptor in the expansion of erythroid progenitors: molecular analysis by expression profiling. Blood. 2003;102:3136-3146. PMID: 12869505
Natural killer cell development
Natural killer (NK) cells are innate immune effector cells that are important in immunosurveillance to eliminate cancerous cells or virally infected cells.
The main focus of our group is to understand how NK cell development is regulated on the transcriptional level.
We are employing several mouse models and novel in vitro systems to analyse the role of key transcription factors at the different developmental stages of NK cell maturation.
Relevant references
- Kallies A, Carotta S, Huntington ND, et al. A role for Blimp-1 in the transcriptional network controlling natural killer cell maturation. Blood. 2010. Dec 3. [Epub ahead of print] PMID: 21131593
- Brady J, Carotta S, Thong RP, et al. The interactions of multiple cytokines control NK cell maturation. J Immunol. 2010;185:6679-6688. PMID: 20974986
- Carotta S, Brady J, Wu L, Nutt SL. Transient Notch signaling induces NK cell potential in Pax5-deficient pro-B cells. Eur J Immunol. 2006;36:3294-3304. PMID: 17111353
Book chapter
Nutt SL, Carotta S, Kallies A; Cytotoxic lymphocyte function and natural killer cells, Clinical Immunology, 3rd edition 2008.
Transcriptional regulation of haematopoietic stem cell development
All mature haematopoietic cells develop from a small pool of hematopoietic stem cells that reside in the bone marrow. Blood homeostasis is a tightly regulated process as deregulation of this process can lead to either life threatening anemia or the formation of cancer.
Although several transcription factors have been identified to play important roles in hematopoietic stem cell self-renewal and/or differentiation, it is largely unknown how they do so.
Our group aims to identify the role of the key transcription factors PU.1 during hematopoietic stem cell self-renewal and lineage commitment. We have discovered that PU.1 is a key regulator of early hematopoiesis and dendritic cell development by directly regulating the cytokine receptor Flt3.
We are currently studying the role of PU.1 during hematopoietic stem cell self-renewal using conditional mouse models and ChIP-seq and RNA-seq approaches.
Relevant references
- Carotta S, Dakic A, D'Amico A, et al. The transcription factor PU.1 controls dendritic cell development and Flt3 cytokine receptor expression in a dose-dependent manner. Immunity. 2010;32:628-641. PMID: 20510871
- Naik SH, Sathe P, Park HY, et al. Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. Nat Immunol. 2007;8:1217-1226. PMID: 17922015
- Holmes ML, Carotta S, Corcoran LM, Nutt SL. Repression of Flt3 by Pax5 is crucial for B-cell lineage commitment. Genes Dev. 2006;20:933-938. PMID: 16618805
- Pilat S, Carotta S, Schiedlmeier B, et al. HOXB4 enforces equivalent fates of ES-cell-derived and adult hematopoietic cells. Proc Natl Acad Sci U S A. 2005;102:12101-12106. PMID: 16093308
- Carotta S, Pilat S, Mairhofer A, et al. Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells. Blood. 2004;104:1873-1880. PMID: 15166028
Understanding how Bak and Bax damage mitochondria to cause cell death
Although structures of the inactive forms of Bak and Bax have been solved, little is known regarding the structures of the activated, oligomerised forms. As a consequence, how Bak and Bax puncture the mitochondrial outer membrane during apoptosis remains enigmatic.
We have identified important aspects of the activated conformer of Bak and Bax and provided the first insight into how these proteins oligomerise during cell death (Dewson et al, Mol Cell 2008; Dewson et al, Mol Cell 2009). We aim to build on these novel findings to fully understand how Bak and Bax coalesce to form the deadly “apoptotic pore”.
Using innovative confocal and electron microscopy approaches we plan to visualise this process as it occurs in cells as they die by apoptosis, and thereby gain important understanding of how Bak and Bax kill cells.
Elucidating how Bak and Bax are activated in response to apoptotic stimuli
The effectors of apoptosis, Bak and Bax, remain dormant until a cell receives an apoptotic stimulus. This sets in motion a complex interplay between the members of the Bcl-2 family of proteins that culminates in triggering the activation of Bak and Bax.
Bak and Bax activation involves stepwise changes in conformation that facilitates their self-association into large complexes that are thought responsible for damaging mitochondria and killing the cell.
Interactions with Bcl-2 proteins (and non-Bcl-2 proteins) appear key in governing whether the deadly function of Bak and Bax is unleashed thereby deciding the fate of the cell. However, which are the important interactions and how they occur is unclear.
Using a combination of cell biology and biochemical techniques, we aim to elucidate these interactions and delineate the consequent activation steps in Bak and Bax. Understanding the complexities of Bak and Bax regulation is critical if the apoptotic pathway is to be targeted effectively for therapy.
SOCS proteins and inflammation
Small, secreted proteins called cytokines mediate regulation of the hematopoietic system and the immune response.
Cytokine signalling is initiated through interaction with specific trans-membrane receptor subunits. The subsequent receptor oligomerisation results in activation of either an intrinsic kinase domain or receptor associated JAK kinases, and the following cascade of intracellular phosphorylation and signal transduction culminates in an appropriate cellular response. This cascade requires exquisite cellular control and loss of regulation can promote tumorigenesis and chronic inflammation.
The expression of SOCS proteins can be induced by cytokine stimulation, and they serve to interfere with signalling not only from the inducing cytokine in a classic “negative-feedback” loop, but also to regulate signalling downstream of other cytokines, a process known as “cross-talk”.
Ubiquitination involves the transfer of a ubiquitin molecule via an E1-E2-E3 enzyme cascade, where the E3 ligase functions as both a substrate recognition molecule and a catalyst for the transfer of ubiquitin to a lysine in the substrate protein
We are interested in SOCS proteins and their role in inflammation.
Autoinflammatory disease in SHP1 mutant mice: roles for interleukin-1 and neutrophils
Mammals have evolved an array of mechanisms to sense viral, bacterial, fungal and parasitic infection, serving to initiate an immune response. These immune sentinels must distinguish self from non-self, and at times they must respond to danger signals released from damaged, infected host cells to activate a circumscribed immune response.
This research examines molecules that are critical in prevention of systemic inflammatory responses that damage host tissue and promote chronic inflammation. Building a more comprehensive picture of the machinery required to extinguish an immune response will be important in the prevention and treatment of acute and chronic inflammatory diseases.
The IL-1β-dependent inflammatory disease developing in SHP1Y208N/Y208N mutant mice is independent of caspase 1. The literature suggested that proteinase 3 and neutrophil elastase, proteases that are abundantly expressed in neutrophils, could process pro-IL-1β in the absence of caspase 1. However, we generated SHP1 mutant mice deficient in proteinase 3 and neutrophil elastase and found that inflammatory disease was unaffected.
Furthermore, we generated triple mutant neutrophils deficient in caspase 1, proteinase 3 and neutrophil elastase and found that processing of pro-IL-1β was normal. This indicated that functional redundancy between caspase 1, proteinase 3 and neutrophil elastase could not explain the processing of pro-IL-1β in neutrophils deficient in only caspase 1 or proteinase 3 and neutrophil elastase. This indicates that an unknown mechanism exists to process pro-IL-1β to the bioactive IL-1β in neutrophils.
This research investigates the role of neutrophils in the development of a caspase 1-independent inflammatory disease in SHP1Y208N/Y208N mutant mice.
References
- BA Croker, RS Lewis, JJ Babon, JD Mintern, DE Jenne, D Metcalf, JG Zhang, LH Cengia, JA O’Donnell, AW Roberts. Neutrophils require SHP1 to regulate IL-1β production and prevent inflammatory skin disease. J Immunology 2011, 186:1131-9. PMID: 21160041
- BA Croker, BR Lawson, M Berger, C Eidenschenk, AL Blasius, EY Moresco, S Sovath, L Cengia, LD Shultz, AN Theofilopoulos, S Pettersson, BA Beutler. Inflammation and autoimmunity caused by a SHP1 mutation depend on IL-1, Myd88, and a microbial trigger. PNAS 2008, 105:15028. PMID: 18806225
Mechanisms of sepsis in Kir6.1-deficient mice
Sepsis is a systemic immune response associated with infection. It is the leading cause of death in critically ill patients. In the United States, 900,000 cases are reported annually and over 200,000 people die from sepsis.
Microbial infection is thought to trigger the disease but infection is not always detectable in sepsis patients. In addition, comparable levels of bacteria or virus may be fatal to one patient but well tolerated by another and it is thought that genetic changes in the human population account for this disparity in patient outcome.
Patients with sepsis can show a change in body temperature, an increased heart rate, a high breathing rate, an increase in white blood cells, a decrease in blood pH and oxygen levels, increased death of immune cells, decreased platelet numbers, organ failure and an irreversible drop in blood pressure. Autopsy studies have not revealed why patients die from sepsis however cardiovascular collapse is commonly associated with septic shock.
The release of cytokines from immune cells may trigger sepsis but the overall levels of cytokines are not a reliable indicator of survival in sepsis patients. Therapies aimed at blocking cytokines in the hospital have not been successful in treating sepsis.
There are significant differences between human sepsis and currently available animal models of sepsis. Our research identified a novel mouse model of sepsis that is highly susceptible to bacterial and viral infection (Croker et al., Nature Genetics 2007, 39:1453). These mice are deficient in the Kir6.1 potassium channel and are up to 20,000-fold more susceptible to cardiovascular collapse precipitated by infection.
We aim to delineate the physiological mechanisms that lead to inflammation-induced cardiovascular collapse in Kir6.1-deficient mice using genetic and pharmacological approaches.
Reference
1. BA Croker, K Crozat, M Berger, Y Xia, S Sovath, I Eleftherianos, JL Imler, B Beutler. ATP-sensitive potassium channels mediate survival during infection in mammals and insects. Nature Genetics 2007, Dec 39: 1453-60. PMID: 18026101
Modulating the inflammasome
To counter viral, bacterial, fungal and parasitic infections, mammals have evolved an array of cytoplasmic, cell surface and extracellular factors that detect the presence of foreign pathogens. They include:
- the Toll-like receptors, cell surface and endosomal proteins that are critical for the recognition of microbial ligands;
- the cytoplasmic nucleotide-binding domain leucine-rich repeat containing (NLR) family of receptors, which include the inflammasome, a multi-protein complex activated by ATP, RNA, DNA, flagellin and uric acid crystals; and
- cytoplasmic RNA helicases, responsible for sensing of foreign nucleic acids.
These host factors must discriminate self from non-self if the host is to avoid not only autoinflammation (inflammation in the absence of microbes, or sterile inflammation), but also autoimmunity and tumourigenesis.
Whilst these host pathogen receptors and their specific microbial ligands are now relatively well defined, the proteins that restrict unwanted initiation of innate immune responses by modulating these pathogen sensors remain largely unexplored.
This project utilises both forward and reverse genetic approaches to investigate novel regulators of the inflammasome.
References
- SL Masters, LA Mielke, AL Cornish, CE Sutton, J O’Donnell, LH Cengia, AW Roberts, IP Wicks, KHG Mills, BA Croker. Regulation of IL-1β by IFNγ is species-specific, limited by SOCS1 and influences IL-17 production. EMBO reports 2010, 11:640-6. PMID: 20596075
- BA Croker, RS Lewis, JJ Babon, JD Mintern, DE Jenne, D Metcalf, JG Zhang, LH Cengia, JA O’Donnell, AW Roberts. Neutrophils require SHP1 to regulate IL-1β production and prevent inflammatory skin disease. J Immunology 2011, 186:1131-9. PMID: 21160041
- BA Croker, BR Lawson, M Berger, C Eidenschenk, AL Blasius, EY Moresco, S Sovath, L Cengia, LD Shultz, AN Theofilopoulos, S Pettersson, BA Beutler. Inflammation and autoimmunity caused by a SHP1 mutation depend on IL-1, Myd88, and a microbial trigger. PNAS 2008, 105:15028. PMID: 18806225
- Masters, SL, Latz, E, and O'Neill, LA. The inflammasome in atherosclerosis and type 2 diabetes. Sci Transl Med, 2011. 3: 81ps17. PMID: 21543720
- Masters, SL and O'Neill, LA. Disease-associated amyloid and misfolded protein aggregates activate the inflammasome. Trends Mol Med, 2011. 17: 276-82. PMID: 21376667
- Masters, SL, Dunne, A, Subramanian, SL, Hull, RL, Tannahill, GM, Sharp, FA, Becker, C, Franchi, L, Yoshihara, E, Chen, Z, Mullooly, N, Mielke, LA, Harris, J, Coll, RC, Mills, KH, Mok, KH, Newsholme, P, Nunez, G, Yodoi, J, Kahn, SE, Lavelle, EC, and O'Neill, LA. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1beta in type 2 diabetes. Nat Immunol, 2010. 11: 897-904. PMID: 20835230
Neutrophil apoptosis
A circumscribed neutrophilic inflammatory response requires regulation of neutrophil lifespan. The variation of neutrophil lifespan is likely to reflect a balancing act by the host, allowing neutrophils time to eliminate pathogens before an apoptotic pathway is triggered.
A deviation in the programmed neutrophil lifespan during infection is likely to be detrimental, affecting the efficiency of pathogen clearance, the number of apoptotic neutrophils in an inflamed tissue, the ability of phagocytic cells to clear apoptotic bodies, and the potential for a chronic inflammatory response to be initiated.
Altered lifespan of neutrophils has been associated with many diseases including acute lung injury, systemic inflammatory response syndrome (SIRS), bronchiolitis obliterans organizing pneumonia (BOOP), acute respiratory distress syndrome (ARDS) and pathogenic infections, including influenza, Streptococcus pneumoniae, RSV, Herpes Simplex Virus and human cytomegalovirus.
During homeostatic conditions, approximately 100 billion neutrophils are produced daily in humans, and they display a remarkably constant, and short, lifespan following exit from the bone marrow into the peripheral circulation. Without activation, neutrophils exit the circulation and are removed by reticuloendothelial cells in the spleen, liver, lung and bone marrow.
Following infection or tissue damage, the lifespan of a neutrophil will change as it enters damaged or inflamed tissues, and is exposed to inflammatory mediators. Our research seeks to identify the key regulators of neutrophil lifespan during inflammatory responses.
References
- BA Croker, D Metcalf, L Robb, W Wei, S Mifsud, L DiRago, LA Cluse, KD Sutherland, L Hartley, E Williams, JG Zhang, DJ Hilton, NA Nicola, WS Alexander, AW Roberts. SOCS3 is a critical physiological negative regulator of G-CSF signaling and emergency granulopoiesis. Immunity 2004, 20, 153-165. PMID: 14975238
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Neutrophils undergoing apoptosis. (Green: Annexin V, which stains apoptotic cells. Red: Propidium Iodide, which stains dead cells)Identification of antigens and host responses involved in immunity to human malaria
Antibodies against blood forms of the Plasmodium parasite such as merozoites are thought to play an important role in mediating protective immunity. However, the specific antigens recognised by immune individuals are unknown. Moreover, the protective effector mechanisms by which naturally acquired antibodies facilitate control of parasite burden are poorly understood.
To grow and divide merozoites invade red blood cells via a complex process involving different molecules. These proteins are located on the apical tip of the parasite, appear to be secreted to the surface just before the invasion event and constitute important candidate targets of protective immunity.
Using mutant parasites deficient in critical molecules required for parasite invasion into the red blood cell, we are investigating if the acquisition of antibody responses able to inhibit parasite growth is associated with reduced symptomatic malaria and protective immunity in individuals residing in malaria-endemic areas of Papua New Guinea.
Understanding these processes is vital to formulate novel candidates for anti-malaria vaccine development.
Understanding the interplay between JAK2 and SOCS3 in myeloproliferative disease
Three closely related myeloproliferative disorders, Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are characterised by over-proliferation of erythrocytes, thrombocytes and granulocytes which leads to thrombosis, increased risk of haemorrhage, stroke and heart attack, as well as a tendency to progress to leukaemia.
In 2005 these three disorders were shown to be associated with a single point mutation in JAK2, (V617F). This mutation results in constitutively active JAK2 and cytokine independent activation of downstream signalling pathways, resulting in over-proliferation of myeloid cells. Interestingly, JAK2V617F is susceptible to inhibition by SOCS3 and therefore SOCS3 potentially regulates both the onset and the severity of the disease.
Despite being intensively studied for more than a decade, the molecular details of JAK signalling, and its suppression by SOCS3, remains incomplete. We have the molecular tools required to dissect the SOCS/JAK interaction and aim to provide a full molecular description of the regulation of JAK signalling by SOCS. This includes dissecting the contributions of ubiquitination, proteolysis, and kinase inhibition to SOCS-mediated JAK suppression.
In addition, we aim to determine the structural details of the SOCS-JAK and SOCS-JAK-Receptor interactions in order to provide a scaffold for the future development of a new class of selective JAK kinase inhibitors. Finally, we aim to perform a complete biochemical and biophysical analysis of JAK itself, the interaction between its various domains, its substrate specificity and enzymatic efficiency.
All this information is crucial for understanding the progression and mitigation, both intrinsic and therapeutic, of myeloproliferative disorders. Given that aberrant JAK signalling is seen in a wide variety of human cancers it also highlights that an in depth understanding of the mechanism of signal transduction through the JAK/STAT pathway, will benefit knowledge of a wide range of human pathologies.
Figure legend: The two key suppressors of cytokine signalling (SOCS1 and SOCS3) associate with JAK whilst it is attached to a cytokine receptor and then inhibit its catalytic activity.
Gates open on understanding potassium channel controls
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Biomolecular solutions for hearing loss
The problems: Age-related hearing loss (presbycusis) is a significant public health issue and is predicted to become increasingly so given Australia's ageing population. Noise- and drug-induced hearing loss are also common. The molecular mechanisms resulting in these conditions are poorly understood. However, it is clear that cell death (apoptosis) within the cochlear is often involved. The structure of the cochlear and the auditory system as a whole is exquisitely complex. There are many points at which failure on a molecular level within this organ during development or ageing can lead to hearing loss.
The project: This project consists of three streams of work investigating the genetic and biological basis of hearing loss, and the possibility of using chemical inhibition to modulate the molecular pathways leading to deafness:
Stream 1 – Genetics: ENU mutagenesis screens to identify genes involved in hearing loss and prevention of hearing loss.
Stream 2 – Biology: Investigation of the intrinsic pathway of apoptosis in development and maintenance of hearing, utilising the extensive catalogue of engineered strains of mice at the institute.
Stream 3- Chemistry: Development and preclinical testing of small molecule inhibitors of apoptosis to prevent age-related and environmentally-induced hearing loss.
The outcomes: It is anticipated that molecular manipulation of apoptotic pathways within the cochlea will protect against hearing loss. This is a radical and exciting prospect given Australia's ageing population.
This work is funded by the Hearing Co-operative Research Centre.
Investigation of the genetics of platelet disorders
The problems: Research carried out in mice has implicated several genes in the regulation of platelet numbers. It is clear that in mice, mutations in some genes can lead to either a marked deficiency of platelets (thrombocytopenia) or a gross excess of platelets (thrombocytosis). Platelet disorders are commonly diagnosed in humans. However, the underlying reasons for the conditions are often unknown. Patients with thrombocytopenia are at increased risk of prolonged bleeding because of a decreased ability to form blood clots. Patients with thrombocytosis may have an increased tendency for their blood to clot, and so can be at an increased risk of stroke, heart attack or blood vessel blockage. It is possible that many thrombocytopenic and thrombocytotic patients have mutations in the genes involved in regulation of platelet numbers.
The project: Dr Benjamin Kile and I have established the International Platelet Genetics Consortium. This network of clinical collaborators is actively recruiting individuals and families with platelet disorders that fall into several different categories. We have recruited approximately 300 individuals with Idiopathic Thrombocytopenia Purpura (ITP) for mutation detection in BCL2L1 and related apoptotic regulators. We have also initiated recruitment of extended family pedigrees with syndromic and/or persistent thrombocytopenia, for non-hypothesis-driven genetic linkage analysis.
The outcomes: The current treatments for thrombocytopenia include platelet transfusion, and therapies directed at combating autoimmunity. These treatments are invasive, expensive and have significant side-effects. There is a pressing need for the development of better diagnostic tools to distinguish different categories of platelet disorder. We have already contributed to this by discovering a novel MYH9 mutation that is now being screened for in patients. A longer-term goal is to identify therapeutic targets and drive shifts in clinical management and treatment of patients with these disorders.
Epithelial ovarian cancer program
Ovarian cancer is difficult to diagnose early, usually presenting at late clinical stage and is often resistant to current therapies. The most lethal type is epithelial ovarian cancer, accounting for 90 per cent of ovarian tumours. It is not clear whether this type of ovarian cancer arises from the surface lining of the ovary or from cells outside the ovary, which have the ability to mimic the ovarian surface cells, such as cells from the fallopian tube.
Despite efforts to develop screening tools, 80 per cent of potentially lethal ovarian cancers are diagnosed after they have spread beyond the ovary and 70 per cent are generally incurable. New forms of therapy targeted to underlying abnormalities within the ovarian cancer cells are urgently needed. The development of improved mouse models of potentially lethal ovarian cancer is essential.
Exploring the origin (cell type in which the cancer starts) of ovarian cancers in mice will allow different approaches to be undertaken to complement those that are possible with human ovarian cancer samples and cell lines. In addition, mouse models of ovarian cancer can be used to test new approaches to therapy for ovarian cancer. The development and utility of new mouse models of epithelial ovarian cancer will be the focus of this research program. (Hartley, Wakefield and Scott)
DNA damage-induced oocyte apoptosis & fertility loss requires p63-mediated induction of Puma & Noxa
The pro-apoptotic effectors of the p53 family member, p63, have not previously been defined in a genetically controlled system. As TAp63a is the predominant p53 family member expressed in primordial follicle oocytes and is essential for their death following genotoxic stress, we are exploring the role of p63-mediated induction of Puma and Noxa following g-irradiation. We utilised gene-targeted mice to show that p63-mediated apoptosis of oocytes and infertility post-DNA damage are prevented by loss of Puma and that Noxa can contribute. We aim to understand the complexities of these BH3-only protein responses and their impact on oocyte quality (Kerr, Hutt, Michalak, Cook, Findlay, Strasser and Scott).
Role of NK cells in malarial pathogenesis
The Natural Killer Complex (NKC) is a conserved genetic region, which encodes several receptors involved in the inhibition or activation of NK and NKT cell function. Previous work from our group has established that the differential expression of NKC alleles in mice modulates the induction of several malarial syndromes including cerebral pathology, pulmonary oedema and severe anaemia.
Thus, our research established that the NKC is a genetic determinant of malaria pathogenesis. Because NKC receptors are mainly expressed on NK cells, in more recent work we investigated the contribution of this cell population to the development of cerebral malaria. We found that NK cells stimulate recruitment of inflammatory CXCR3+ T cells to the brain of cerebral malaria-affected mice in an IFN-γ-dependent manner. The precise mechanism by which NK cells stimulate T cell function during malaria is not understood.
It is accepted that conventional dendritic cells (DC) are the main antigen-presenting cell population required for the induction of T cell responses involved in experimental cerebral pathogenesis malaria. Therefore, we are currently investigating whether NK cells are required for the DC-mediated priming of T cells involved in malarial pathogenesis.
Splenic architecture and induction of protective immunity to malaria
Interferon-γ-inducible protein 10 (IP-10) has been identified as a biomarker strongly associated with severe malaria and mortality in human populations.
Studies from our group revealed that neutralization as well as genetic deletion of IP-10 reduces brain intravascular inflammation and protects Plasmodium berghei-ANKA-infected mice from lethal cerebral malaria. Moreover, lack of IP-10 during experimental infection reduces overall parasite burden, suggesting that this factor may have a detrimental effect in the induction of immune responses required to control parasitemia.
Both human and rodent acute malaria infections are accompanied by dramatic changes in splenic architecture including, dissolution of the marginal zone, loss of T cells from the white pulp and abnormal germinal centre structure. Emerging experimental evidence indicates that some inflammatory factors may alter the structure of secondary lymphoid organs, compromising the induction of protective immunity.
Using imaging and flow cytometry approaches, we are investigating whether IP-10 and other inflammatory molecules produced in response to infection are responsible for the loss splenic architecture characteristic of fatal malaria. The consequences of these processes for the induction of T cell and B cell responses involved in anti-malaria protective immunity are being investigated.
Screening for novel epigenetic modifiers
One of our key interests is understanding how the different components of the epigenetic machinery interact to bring about transcriptional silencing or activation. A major stumbling block is that we do not know the identity of all of the factors involved in epigenetic control, and therefore our understanding of the molecular mechanisms of epigenetic control will necessarily be incomplete. We are performing an in vitro RNAi screen for novel epigenetic modifiers in an attempt to identify more epigenetic modifiers in the genome. We have put together a list of known or potential epigenetic modifiers and we are now building a targeted retroviral shRNA library against these potential epigenetic modifiers. Our preliminary screen using this library will be in differentiating female ES cells where we will use X inactivation as a model epigenetic system to screen for novel epigenetic modifiers. Positive hits will then be further characterised for their role in X inactivation, and more broadly studied in development and disease.
The role of Smchd1 in cancer
Our previous work identified Smchd1 as a novel gene critically involved in X inactivation: the silencing of one of the two X chromosomes in female mammals to achieve dosage compensation between females and males. Smchd1 is a very large chromosomal protein and functions as a transcriptional repressor. Smchd1 has some homology to the SMC proteins that are involved in chromosome cohesion and condensation during cell division and DNA repair. Given that many epigenetic modifiers are aberrantly expressed in cancer, we are testing the role of Smchd1 in several models of cancer, using both our 2 null alleles of Smchd1 and retroviral knockdown approaches.
The role of polycomb group proteins in haematopoietic stem cells
Polycomb group (PcG) proteins are epigenetic repressors that are frequently overexpressed or mutated in cancer. PcG proteins function as three distinct complexes: Polycomb Repressive Complex 1 (PRC1), PRC2 and PhoRC. These complexes have roles in proliferation, apoptosis, skeletal development and stem cell biology. The predominant model for PRC action suggests that the PRCs act sequentially to elicit gene silencing; PhoRC directs PRC2 via its sequence specificity, PRC1 is then recruited and mediates transcriptional repression. This hierarchical recruitment model for PRC action is based on evidence from Drosophila, and mammalian ES and fibroblast cell lines. However, it is becoming clear that this model does not always hold true. In haematopoietic stem cells, a clinically relevant cell type, we know that PRC1 acts to enhance HSC activity, while PRC2 acts to restrict activity. These apparently opposing roles suggest that the complexes do not work in unison in these primary stem cells, and there is now evidence to suggest that the relationship is also unclear in leukaemia and breast cancer. We are studing the interaction between the three complexes using key knockouts or knockdowns of each complex, their molecular roles in terms of chromatin structure and gene expression and their functional consequences on HSC activity.
In collaboration with Dr Ian Majewski, Netherlands Cancer Institute; Professor Warren Alexander, Cancer and Haematology division; Associate Professor Gordon Smyth, Dr Matthew Ritchie and Dr Alicia Oshlack, Bioinformatics division.
How naturally-occurring mutations within the thrombopoietin receptor, c Mpl, cause CAMT
The molecular basis of how naturally-occurring mutations within the thrombopoietin receptor, c Mpl, cause chronic amegakaryocytic thrombocytopenia (CAMT)
CAMT is a very rare congenital disease, which leads to a chronic deficit in circulating platelet numbers in affected patients. Many causative mutations have been identified in patients and our studies are directed towards understanding the molecular basis for their effects using cell culture and mouse models.
These studies are being performed in collaboration with Professor Warren Alexander (Cancer and Haematology division) and Dr Ben Kile (Molecular Medicine division).
The mechanism of activation of the receptors for IL-3, IL-5 and GM-CSF
Using X-ray crystallography, NMR spectroscopy and cell-based studies, we have sought to understand how the related cytokines, IL-3, IL-5 and GM-CSF, can bind a common β-receptor yet elicit distinct cellular responses.
Our studies have focused on a related β-receptor (denoted βIL-3) present in mice, but not humans, which is specific for IL-3.
βIL 3 offers a unique opportunity for insight into the molecular basis of βIL-3’s specificity for IL 3 over IL-5 and GM-CSF, and how the receptor is able to bind IL 3 directly whilst the related βc cannot.
These studies are being performed in collaboration with Professor Ian Young (John Curtin School of Medical Research, Australian National University, Canberra) and Dr Shenggen Yao (Structural Biology Division).
Characterisation of a novel haematopoietic regulator Mlkl
Understanding the molecular mechanisms by which the pseudokinase domain-containing proteins, the Janus kinases and Mlkl, regulate blood cell production
Pseudokinase domains resemble protein kinase domains, but are predicted to lack phosphotransfer activity due to the absence of key catalytic motifs. Recent studies have illustrated that pseudokinases are not merely inert scaffolds, but instead are critical signal transducers whose mutation underlies many human pathologies.
Our studies are directed towards understanding the activities of the Janus kinases (JAK1, JAK2, JAK3 and TYK2) and a novel pseudokinase, Mixed lineage kinase domain-like (Mlkl), as downstream effectors of cytokine signalling using a spectrum of structural biology, biochemical, biological, proteomic and biophysical studies.
These studies are being performed in collaboration with Dr Jeff Babon, Professor Peter Colman, Dr Peter Czabotar (Structural Biology Division), Professors Nick Nicola, Warren Alexander (Cancer and Haematology Division) and Doug Hilton (Molecular Medicine Division), and Dr Ren Dobson (University of Melbourne).
Spatiotemporal dynamics of host cell invasion
We are probing how signal transduction events occur in a spatiotemporal manner using high-resolution imaging. By accessing the Walter and Eliza Hall Institute's high-end imaging workstations, in combination with the latest calcium and phosphoinositide biosensors, we will be able to reconstruct the steps that lead to invasion and how this occurs in space and time.
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Movie A
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Movie B
Conditional Toxoplasma invasion and egress mutant. A. A vacuole full of Toxoplasma parasites egressing (bursting out) from a host cell under permissive conditions. B. Under non-permissive conditions Toxoplasma parasites cannot exit host cells due to the key gene being turned off.
Phosphorylation networks mediating apicomplexan host cell invasion
Toxoplasma proteins are phosphorylated upon calcium signaling. 2DE gel of 32P radiolabelled Toxoplasma proteins. Red arrows point to proteins that are phosphorylated upon calcium pathway stimulation.
We are using the latest proteomics techniques to reveal how protein phosphorylation mediates parasite invasion. We have developed quantitative measures of phosphorylation which we are applying to the whole proteome to measure changes upon Toxoplasma and Plasmodium host cell invasion. The combination of these procedures with mutants that we create in Characterisation of molecules controlling the initiation of host cell invasion will enable us to pinpoint the exact role of particular molecules in phosphorylation networks that modulate host cell invasion.
Characterisation of molecules controlling the initiation of host cell invasion
A model for activation of invasion in Toxoplasma. A. Host cell recognition first occurs by a unknown parasite receptor. Upon binding, proteins analogous to heterotrimeric G proteins are activated and released, which then bind and sequester phospholipase C (PLC) to the membrane. B. PLC then hydrolyses the plasma membrane phosphoinositide PIP2 to DAG and soluble IP3. C. The phosphoinositide IP3 then can activate the IP3 Receptor (IP3R) to release calcium from the endoplasmic reticulum. D. Cytoplasmic calcium then activates calcium and calmodulin-dependent protein kinases. E. Phosphorylation by these kinases activates substrates to ultimately release micronemes (which contain high affinity host cell receptors) and activate the actin-myosin-based invasion motor.
Intracellular calcium signal transduction events modulate several steps leading to host cell invasion. We are looking at several molecules that we believe are involved in this process in Toxoplasma. These include a group of protein kinases that are novel to apicomplexa and that represent excellent drug targets. Further, we have evidence that suggests that phosphoinositide signaling events modulate invasion and are one of the first signals that initiate host cell invasion.
Cytoskeletal dynamics across the malaria parasite lifecycle
Cell movement occurs throughout the malaria parasite lifecycle. However, each stage has unique properties that make them of interest to study separate questions relating to the process of general motility or cell invasion. Merozoites, blood stage parasites, really only invade with little evidence that they can glide across substrates. Ookinetes - the stage in the mosquito midgut - glide beautifully but do not invade the midgut wall, instead traversing it on their way to forming an extra-epithelial cyst. Sporozoites, the stage responsible for re-infection of the human host, are jacks-of-all-trades, being able to glide and invade cells. Using the mouse malaria Plasmodium berghei as a model for human malaria, we are undertaking fluorescence imaging of the core actin regulators and exploring their localisation and function throughout the lifecycle. This work is being undertaken in collaboration with the laboratory of Professor Geoff McFadden at the University of Melbourne School of Botany and Professor Robert Sinden at Imperial College London, UK.
The Developmental Genetics Laboratory
In the last few years the discovery of histone acetyltransferases has revolutionised research into the regulation of gene expression and how the genome is organised. Current models propose that the transcription machinery used for protein coding genes requires co-activator complexes to link DNA binding transcription factors to the basal transcriptional apparatus. Co-activator complexes are potent regulators of current and future patterns of gene expression and therefore directly control proliferation and lineage determination. Not surprisingly de-regulation of these molecules lead to diseases such as leukaemia.
Co-activator complexes typically contain proteins that regulate the organization of chromatin by covalent modification of histones. One of the most important types of modification is the addition of acetyl groups to lysine residues in the N-terminal tail of histones. This is catalysed by histone acetyltransferases and co-activator complexes typically contain one or more of these enzymes. This shows that modification of chromatin structure is an essential aspect of the regulation of gene expression.
A simplified model of how histone acetylation mediated by Myst family transferases may act to regulate gene expression (A). Acetylation can occur on the lysine residues within the N-terminal "tail" of histones, as shown for histone 3 and histone 4 (B). The structural relationship between different MYST family members (C).
Since the ability to dramatically change gene expression patterns upon receiving external signals is the key property of stem cells, it follows that in order to understand the biology of stem cells it is necessary to identify the important co-activators in these cells and define their function.
Cells of the early embryo lacking the MYST histone acetyltransferase Mof show reduced levels of acetylation on histone 4 lysine 16, which results in abnormally condensed chromatin (compare D and E with A and B). Note that in the mutant cells there are reduced levels of histone 3 lysine 14 acetylation, a marker of transcriptionally active chromatin (F vs. C). Figure from Thomas et al. MCB 2008.
Our aim is to define the role of co-activators of transcription in stem and progenitors cells during embryonic development and in adults. We are particularly interested in the function of the MYST family of histone acetyltransferases in stem cell populations. We have shown that Moz is essential for the development of haematopoietic stem cells whereas Qkf has an essential role in adult neural stem cells. We are currently investigating the function of the MYST family, particularly Moz and Qkf, during embryonic development and in adult stem cell populations.
Cyton Calculator
The Cyton Calculator is free software that reads CFSE data from a file and calculates cyton model parameters.
For information on the cyton model see the following publication:
- Hawkins ED, Turner ML, Dowling MR, van Gend C, Hodgkin PD.
A model of immune regulation as a consequence of randomized lymphocyte division and death times.
Proc Natl Acad Sci U S A. 2007 Mar 20;104(12):5032-7. Epub 2007 Mar 14. PMID: 17360353 [PubMed - indexed for MEDLINE]
Cyton Calculator comes in two verions. One for Mac OS X (v10.3.9 or later) and one for Windows and other operating systems. Regardless of the type of computer you will need to have a copy of Java installed. This can be freely downloaded from the Sun web site.
To obtain a copy of the software, please complete the online form and licence agreement here. Once you have received your copy follow the instructions below.
Mac OS X (v10.3.9 or later)
Open disc image and copy the CytonCalculator application to its final resting place and then double click to launch.
Windows and other
Use Winzip or similar to decompress it. Copy the CytonCalculator application to its final resting place and then double click to launch.
Documentation and Sample Data
Here are some instructions on how to use the CytonCalculator
Here is a speadsheet containing sample data that CytonCalculator can read
Here is a sample file in CytonCalculator format
Measuring lymphocyte proliferation
Measuring lymphocyte proliferation, survival and differentiation using CFSE time series data
If you label a cell with CFSE then you can tell how many times it has divided since it was labelled. This quantity is called its division. For example cells which have not divided are said to be in division 0, those which have divided once, division 1 and so on. If you CFSE label a population of cells then you can measure how they are distributed between different divisions. If you repeat this experiment at different times then you can measure the distribution as a function of both division and time. From this data it is possible to work backwards and estimate the underlying statistical properties of the cells. This page explains two methods for doing so.
The Precursor Cohort Method
The precursor cohort method is a graphical method requiring no special software that can be used to determine the parameters of a simplified model of cell proliferation.
Download the Precursor cohort method and accompanying material here.
Cyton Calculator
The cyton model gives better agreement with CFSE data than the cohort method above. However, it is a more complicated model and so it is not possible to determine its parameters using graphical techniques.
Download the "Cyton Calculator" tool and accompanying material here.
Precursor Cohort Method
The precursor cohort method is a graphical method that can be used to determine the parameters of a simplified model of cell proliferation.
The details of the precursor cohort method are presented in a paper: Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. (2007) Hawkins et al. Proc. Natl. Acad. Sci. 2:2057.
A simple Step-by-Step worksheet for Excel
We have created an Excel spreadsheet to implement the precursor cohort method. Due to differences between Microsoft Office on Mac and PC it comes in two versions.
Here is a link to the Mac version
Here is a link to the PC version
Here is a link to a guide to using the worksheet (PDF)
Supporting Material
Victorian Breast Cancer Research Consortium (VBCRC) laboratory
Translational Research and Collaborations
Our laboratory forms the WEHI node of the Victorian Breast Cancer Research Consortium, which is an "Institute-without-walls", comprising a number of laboratories throughout Melbourne focusing on breast cancer research. Our groups meet on a regular basis.
We have strong links to basic, clinical and translational research efforts, which include:
- The VBCRC-supported Royal Melbourne Hospital Tissue Bank
- The Royal Melbourne Hospital Department of Medical Oncology and Familial Cancer Centre
- The Royal Womens Hospital and Royal Melbourne Hospital combined Breast Service
- The Australian New Zealand Breast Cancer Trials Group
- kConFab, a national consortium providing a resource for the study of multiple-case breast cancer families
- Cancer Trials Australia
- Victorian Cancer Agency
Research Support
We gratefully acknowledge current and previous research support from:
- The Victorian Breast Cancer Research Consortium
- The National Health and Medical Research Council (Australia)
- The National Breast Cancer Foundation
- The Cancer Council Victoria
- The Australian Stem Cell Centre
- US Department of Defense
- Susan Komen Foundation (USA)
Breast Cancer Facts
Breast cancer is the most common malignancy to affect women, and is the most common cause of cancer related death amongst Australian women. Approximately 1 in 11 women will develop breast cancer before age 75, and approximately 20% will die from the disease. Since the early 1990's mortality figures have begun to fall (by approximately 2% per year), largely due to the introduction of improved medical treatment (including adjuvant chemotherapy and tamoxifen) and better early detection. Approximately 5% of breast cancer is attributable to high penetrance hereditary predisposition genes (such as BRCA1 and BRCA2).
For information
The Medicinal Chemistry Group
The Walter and Eliza Hall Institute Medicinal Chemistry Group, led by Associate Professor Jonathan Baell, Dr Guillaume Lessene and Dr Keith Watson, conducts research aimed at developing new therapeutically useful molecules. Combining synthetic organic chemistry with a knowledge of structural biology and principles of drug design, we focus on small molecules and their biological applications.
Projects in the laboratory currently focus on cancer and parasitic diseases such as malaria, human African trypanosomiasis, leishmaniasis and Chagas’ disease. Within some of these areas - such as cancer - there are several projects, which have grown from different origins, both in terms of the source of the initial hit molecule and the target with which it is interacting.
Approaches to finding hit molecules
Within the lab, our target molecules are derived from one of three approaches: (i) structure-based design; (ii) natural products with known activity; and (iii) high throughput chemical screening. Once a hit is identified and validated, we adopt standard medicinal chemistry techniques such as structure-activity relationships in the quest to enhance the potency and improve the properties of the chosen molecules with the ultimate aim of producing a drug that can enter clinical trials.
(i) Structure-based drug design
Advances in structural biology have given chemists the power to rationally design drug-like molecules that, theoretically, will bind to a specific receptor or protein of interest. In the area of peptidomimetics, molecular modeling enables the selection of a backbone scaffold, which will project the residue mimics in the necessary direction in space. Beyond converting these calculations into the chemical reality of a biologically active hit, we aim to optimise the potency and selectivity of our molecules through structure-activity relationships. In some instances, thorough conformational studies are used to determine whether the molecules in fact adopt the designed shape.
(ii) Natural products and their derivatives
Molecules found in nature have evolved over billions of years to meet the needs of the plant or organism that produces it. Typically, this involves interaction with proteins or enzymes within a biological system. It follows that these molecules, while adapted to use in its original environment, might find similar interactions inside the human body. We are interested in natural products that have been reported to have promising biological activity, such that we can then work towards enhancing this activity in the search for therapeutic utility.
(iii) High-throughput chemical screening (HTCS)
With a library of over 100,000 commercially available synthetic compounds - and soon to be expanded towards 200,000 compounds, our colleagues in the HTCS group are able to rapidly test ‘lead-like’ molecules against many biological targets. Selected hits from a variety of assays are then used to initiate synthetic chemistry programs.
The Lab
Major equipment
NMR - Bruker Avance II, 300 MHz
HPLC - Waters Alliance HT 2795
LCMS - Finnigan LCQ Advantage MAX
Prep-LCMS - Waters
Infrared - Bruker Tensor27 FT-IR
Automated Flash Chromatography System - Biotage FlashMaster II
Microwave - CEM Discover Lab Mate
MBRAUN Solvent Purification System
Collaborations
Our chemists work closely with world-leading biologists and biochemists, both internal and external, creating a dynamic environment of drug discovery.
Internal divisions
Other Institutions
The University of Melbourne
La Trobe University
The Ludwig Institute for Cancer Research
The Howard Florey Institute
The CRC for Cancer Therapeutics
Private collaborations
Genentech Inc
Abbott Laboratories
Bionomics Ltd
Murigen
DNDi
The Lab
Down the lab's main corridor - plenty of natural light...
Major equipment:
NMR - Bruker Avance II, 300 MHz
HPLC - Waters Alliance HT 2795
LCMS - Finnigan LCQ Advantage MAX
Prep-LCMS - Waters
Infrared - Bruker Tensor27 FT-IR
Automated Flash Chromatography System - Biotage FlashMaster II
Microwave - CEM Discover Lab Mate
MBRAUN Solvent Purification System
Collaborations
WEHI Divisions:
Molecular Genetic of Cancer
Molecular Medicine
Infection and Immunity
Other Institutions:
The University of Melbourne
La Trobe University
The Ludwig Institute for Cancer Research
The Howard Florey Institute
The CRC for Cancer Therapeutics
Private collaborations:
Genentech, Inc.
Abbot Pharmaceuticals
Bionomics, Ltd.
Murigen
DNDi
The Group
Nick, Guillaume, Wilco, Nurul, Keith, Georgina, Angela, Andrew, Julia, Carl, Sanji, Dana, Brad, JB
Approaches to Finding Hit Molecules
A typical screening campaign
Within the lab, our target molecules are derived from one of three approaches: (i) structure-based design; (ii) natural products with known activity; and (iii) high throughput chemical screening. Once a hit is identified and validated, we adopt standard medicinal chemistry techniques such as structure-activity relationships in the quest to enhance the potency and improve the properties of the chosen molecules with the ultimate aim of producing a drug that can enter clinical trials.
(i) Structure Based Drug Design
Advances in structural biology have given chemists the power to rationally design drug-like molecules that, theoretically, will bind to a specific receptor or protein of interest. In the area of peptidomimetics, molecular modeling enables the selection of a backbone scaffold, which will project the residue mimics in the necessary direction in space. Beyond converting these calculations into the chemical reality of a biologically active hit, we aim to optimise the potency and selectivity of our molecules through structure-activity relationships. In some instances, thorough conformational studies are used to determine whether the molecules in fact adopt the designed shape.
(ii) Natural Products and their derivatives
Molecules found in nature have evolved over billions of years to meet the needs of the plant or organism that produces it. Typically, this involves interaction with proteins or enzymes within a biological system. It follows that these molecules, while adapted to use in its original environment, might find similar interactions inside the human body. We are interested in natural products that have been reported to have promising biological activity, such that we can then work towards enhancing this activity in the search for therapeutic utility.
(iii) High Throughput Chemical Screening
With a library of over 100,000 commercially available synthetic compounds - and soon to be expanded towards 200,000 compounds, our colleagues in the HTCS group are able to rapidly test ‘lead-like’ molecules against many biological targets. Selected hits from a variety of assays are then used to initiate synthetic chemistry programs.
Type 1 Insulin-like Growth Factor Receptor
The IGF signalling system can be briefly summarised as follows. Signalling by IGF I is mediated by IGF-1R, which binds IGF I with high affinity. Signalling by IGF II is also mediated by IGF-1R (which binds IGF II with ~20% affinity for IGF I) and by the exon 11 minus isoform (IR A) of IR. An expanding repertoire of intra cellular proteins act as substrates/effectors of ligand activated IGF-1R signalling. The bioavailability of IGF I and IGF II is regulated by six soluble IGF binding proteins IGFBP 1, 2, 3, 4, 5 and 6 whilst the bioavailability of IGF II is further regulated by the Type 2 IGF receptor (IGF 2R).
IGF-1R is involved in normal growth and development. However, there is a compelling body of evidence for the involvement of the IGF signalling system in cancer progression. The critical role of IGF-1R in tumorigenesis is highlighted by the fact that fibroblasts from IGF-1R null mice are resistant to oncogenic transformation. Over expression of IGF-1R in mice has been shown to accelerate the invasion and growth of tumors and mice expressing constitutively active IGF-1R have been shown to develop invasive adenocarcinomas. It has been shown that humans with levels of circulating IGF I at the high end of the population distribution have an increased risk of cancer. Over-expression of IGF I, IGF II and/or IGF-1R has been detected in a number of different tumor types. Taken together, these results strongly suggest that antagonists of IGF-1R and the hybrid receptors may be anti cancer therapeutics.
We now are seeking to use structural biology techniques to determine the three dimensional structure of the IGF-1R ectodomain both in its native and ligand bound conformations.
Insulin Receptor
The X-ray structure of the insulin receptor ectodomain complexed with four antibody fragments
The insulin receptor (IR) is a member of the tyrosine kinase receptor superfamily. Its major roles in higher organisms are the regulation of lipid, protein and carbohydrate metabolism including the maintenance of glucose homeostasis whereas the closely related insulin-like growth factor receptor (IGF-1R) is involved in normal growth and development. Of the two insulin receptor isoforms IR-A and IR-B, the former acts as a high-affinity receptor for insulin-like growth factor-II (IGF-II) and has been implicated, along with IGF-1R, in malignant transformation. There is a third member of the the IR family, the orphan insulin receptor related receptor (IRR). IR family members which are all homodimers, can also function as hybrid heterodimers.
Despite the fact that the atomic structure of insulin has been known for forty years and the primary sequence of its receptor for twenty-two years, detail at the molecular level of the interaction between insulin and its receptor is remarkably limited, as is detail about the way in which ligand binding effects signal transduction. A breakthrough occurred during 2006 with the determination by Ward, Lawrence and co-workers of the three-dimensional structure of the entire IR ectodomain in its apo state at a resolution of 3.8 Å. This structure provided the first insight into the likely structure of the intact insulin binding site, suggesting it to be formed by a juxtaposition of the first leucine-rich repeat domain of one monomer with the first fibronectin domain of the alternate monomer. The crystallographic electron density maps also indicated the possible location the C-terminus of the receptor ï¡-chain, a segment (CT) which is known to be essential for ligand binding. The methodological key to the structure of the receptor was to crystallise it in combination with four Fabs molecules (Fig. 1a), which significantly decreased the need for crystal contacts to involve the receptor's N-linked glycans.
We are now seeking to determine the structure of the ligand-bound insulin receptor in both high- and low-affinity states and to determine the nature of the structural transitions that occur upon ligand binding.
Caspase Recruitment Domains
Caspase recruitment domains, or CARDs, are small (~90 residue) protein-protein interaction motifs that mediate many signalling pathways through CARD-CARD interactions. Structurally, CARD domains consist of a helical bundle of six short helices, known as the death fold and this fold is shared with the death domain, death effector and pyrin domains. CARDs and structurally related domains are not only important in mediating the apoptotic response but also in regulating inflammation and innate immunity. Mutations in proteins containing CARD or related domains are important in diseases such as Crohn’s disease, Muckle-Wells syndrome and Familial Mediterranean Fever. Understanding the structures and interactions of CARD domains may allow us to design therapeutics in diseases where CARDs mediate signalling pathways.
Two views of the CARD domain from Apaf1.
The Inhibitor of Apoptosis family
The ‘inhibitor of apoptosis’ (IAP) proteins inhibits apoptosis through blocking the action of caspases, a family of cysteine aspartyl proteases that dismantle the cell, and act downstream of the Bcl-2 family. All IAPs contain at least one BIR (baculoviral IAP repeat) domain, a ~70 residue zinc-binding protein-protein interaction domain which we were the first to describe at atomic resolution. IAPs contain multiple BIRs together with a RING (really interesting new gene 1) domain, and some contain caspase recruitment domains. It has been shown recently that these other domains play important roles in regulating the IAPs and we are interested in determining the structures of protein-protein complexes formed by these domains to elucidate their interactions.
Structure of BIR3 domain.
The Bcl-2 family
Several pathways leading to cell death converge on a family of pro-survival molecules known as the Bcl-2 family. These molecules play an important role in determining the fate of a cell and the interactions between them determines whether a cell lives or dies. The mechanisms by which Bcl-2 proteins regulate cell death are still being clarified, but a key interaction is the inhibition of the pro-survival activity of Bcl-2 family members by interaction with the death inducing BH3-only proteins. The over-expression of pro-survival proteins such as Bcl-2 (B cell lymphoma-2) and Mcl-1 (Myeloid cell leukaemia-1) in cancer has made these proteins the target of structure directed drug design to discover antagonists to their survival activity. To this end we have focused on structural characterization of these proteins and their complexes and have solved structures of Bcl-w and Mcl-1.
Solution structures of Mcl-1 and Bcl-w.
Defining Pathways
Through our genetic screens we have identified suites of mutations that have similar effects - for example, Plt3, Plt4, Plt6, Plt8, Plt10, Plt11 and Plt15 all elevate the platelet count of Mpl-/- mice. Plt3 and Plt4 mice harbour independent mutations in the gene encoding the transcription factor c-Myb, while Plt6 mice contain a mutation in the gene for the histone acetyl transferase and c-Myb binding partner, p300. Consistent with the function of Myb and p300 in the same complex, Plt3, Plt4 and Plt6 mice show stem cell defects involving an increased commitment to the megakaryocyte lineage at the expense of red cell and B cell production. Plt8 and Plt10 in contrast contain a mutation in a gene that is a component of the polycomb repressive complex 2 (PRC2) which acts by methylating lysine 27 of Histone 3. Many interesting questions arise when analysing these mutants;
- Do each of the mutations exert their effect using the same biological mechanism - i.e. does mutation of Suz(12) or the mutations in the Plt11 and Plt15 mice, also bias differentiation down the megakaryocyte lineage?
- Do c-Myb, p300 and Suz(12) act in the same molecular pathway or separate pathways?
- As has occurred successfully in lower organisms, can we use epistasis analyses in which the phenotype of double mutants are compared to single mutants to infer pathway relationships.
- ENU-induced mutants provide finger-holds in biologically important pathways; can we combine genetics, genomics, proteomics and bioinformatic to flesh out other components of these pathways?
The haematopoietic hierarchy.
Stem cells shown at the left of the diagram are the ultimate source of all blood cells. The key property of stem cells, which distinguishes them from progenitors, is their capacity for self-renewal and their ability to produce all lineages of blood. Some more primitive progenitor cells retain the ability to form several lineages of cells. These are known as multipotent progenitors (CMP=common myeloid progenitor, MEP=megakaryocyte-erythroid progenitor, GMP=granulocyte-macrophage progenitor, CLP=common lymphoid progenitor) and they give rise to progenitors with restricted potential known as colony-forming cells or CFCs.
Mouse Genetics
Genetic Dissection Of Blood Cell Production
Genetics has been successfully employed in lower organisms including yeast, nematodes, insects and fish to unravel the molecular regulation of complex processes such as development, cell division and cell death. Two factors have combined to make large-scale mouse genetics feasible and accessible to even small laboratories; (1) the demonstration in the early 1980s that ENU is a powerful germ cell mutagen in the mouse and (2) the completion early this century of the mouse genome project.
Mouse mutation identification
Unlike many large national mouse mutagenesis centres, which have broad programs examining many different phenotypes, our approach has been to focus almost exclusively on the haematopoietic system. We have also borrowed a number of tricks, (e.g. modifier and sensitised screens) developed by geneticists using lower organisms such as yeast. Rather than beginning with wild type mice and identifying mutants with an aberrant phenotype, modifier screens commence with a mouse with a pre-existing phenotype and aim to identify mutations that make this phenotype more severe (enhancers) or less severe (suppressors). We are one of the first laboratories to employ this strategy in vertebrates and are particularly excited about this approach because, not only may it shed light on the in vivo regulation of complex biological such haematopoiesis, but also because of its potential to pin-point new targets for the treatment of human disease. Just as most ENU-induced mutations cause loss of function, most small molecule therapies also reduce the function of proteins to which they bind. Accordingly, screens for genes that lead to amelioration of disease when mutated should provide genome-wide access to novel in vivo validated targets for pharmaceutical discovery.
In collaboration with the laboratories of Professor Warren Alexander in the Division of Cancer and Haematology and Dr Benjamin Kile in this Division, our focus has been on dissecting platelet formation; a process that includes elements common to other blood cell lineages, including self-renewal of haematopoietic stem cells and lineage commitment, as well as unique events like polyploidization via endoreduplication, pro-platelet formation and platelet shedding. Many of our studies have commenced with a mouse model of human inherited thrombocytopenia, in which the gene encoding the thrombopoietin receptor, Mpl, has been mutated by homologous recombination in ES cells. Mpl-/- mice have approximately 10% of the normal number of platelets and a corresponding decrease in megakaryocytes, megacaryocyte progenitors and stem cells. Despite their stem cell deficit, Mpl-/- mice have normal numbers of other peripheral blood cells, implying the presence of a compensation mechanism within the haematopoietic system. We have used Mpl-/- in an ENU mutagensis screen to identify suppressors of thrombocytopenia and to find enhancers of the stem cell phenotype that manifest as a multi-lineage defect in the peripheral blood. In addition, we have carried out a conventional ENU mutagenesis screen using Mpl+/+ wild type mice and identified pedigrees with thrombocytopenia and thrombocytosis. To date we have identified more than 30 mutant pedigrees with defects in stem cells and/or the platelet lineage. The causative mutation in 12 of these has been identified and in an additional 10 cases genes in the candidate interval are currently being sequenced. These pedigrees are being intensively studied within our laboratory at both the biological and molecular level.
The SOCS Box; A link between generic and substrate-specific components of E3 ubiquitin ligases
The eight SOCS family members
In addition to the canonical SOCS proteins that contain a central SH2 domain, we found several large and poorly characterized protein families that contain a C-terminal SOCS box and a variety of other protein-protein interaction domains including SPRY domains, WD40 repeats and ankyrin repeats. We hypothesize that each of these SOCS box containing proteins targets a specific protein for polyubiquitylation and degradation and are taking a variety of proteomic and genetic approaches to identify these substrates.
Interactions of LIF
Limiting cytokine action: Suppressors Of Cytokine Signalling (SOCS) Proteins
Many cytokines are a double-edged sword - having beneficial effects in the body’s fight against infection, yet playing a role in the onset and development of inflammatory disease. This implied to us that the body must have mechanisms by which it keeps the effects of cytokines under strict control. Using an expression cloning approach we discovered a family of SH2 domain containing proteins, which we named the Suppressors Of Cytokine Signalling or SOCS proteins, that have proven to be the major negative regulators of cytokine action. Studies in collaboration with the Division of Cancer and Haematology and many other laboratories have established that SOCS proteins are expressed rapidly following stimulation of the JAK/STAT pathway by cytokines. Once produced, SOCS proteins terminate JAK/STAT signalling by binding to signalling complexes. Inhibition of signal transduction occurs both through interaction of the SH2 domain of SOCS proteins with key phosphotyrosine residues in activated signalling components and the recruitment of ubiquitin ligase machinery via a conserved 40 amino acid motif that we named the SOCS Box. This leads to polyubiquitylation of signalling proteins and their degradation by the proteasome.
The JAK/STAT pathway
In order to understand the physiological role of the SOCS proteins we have generated or obtained null alleles of all eight family members. Six of these have been created in collaboration with Professor Warren Alexander in the Division of Cancer and Haematology, one was obtained from Prof. James Ihle and one was obtained using a TILLING approach. Analysis of these mice has yielded important and surprising insights into the specificity of SOCS proteins. SOCS1 for example plays a crucial role in limiting the production and action of IFNγ, SOCS2 controls body size through regulation of growth hormone action and SOCS3 acts as a feedback inhibitor of the LIF/IL6 group of cytokines and of G-CSF. In contrast, the roles of SOCS4, SOCS5, SOCS6 and SOCS7 have been more difficult to define. Our current studies are focussed on understanding the redundancy in this system, by generating and analysing mice that lack two or more functional SOCS genes.
Pharmacologically Active Toxins: Chronic Pain and Multiple Sclerosis
K Khoo, BJ Smith, RS Norton (Structural Biology), in collaboration with G Bulaj, BM Olivera, D Yoshikami (University of Utah), MW Pennington (Bachem Biosciences) and KG Chandy (University of California, Irvine) [Khoo KK, Feng Z-F, Smith BJ, Zhang M-M, Yoshikami D, Olivera BM, Bulaj G & Norton RS (2009) Structure of the analgesic μ-conotoxin KIIIA and effects on structure and function of disulfide deletion. Biochemistry; 213. Beeton C, Smith BJ, Sabo JK, Crossley G, Nugent D, Khaytin I, Chi V, Chandy KG, Pennington MW & Norton RS (2008) The D-diastereomer of ShK toxin selectively blocks voltage-gated K+ channels and inhibits T lymphocyte proliferation. Journal of Biological Chemistry 283, 988-997]
Venomous creatures produce a wealth of interesting peptide and protein toxins as components of their venoms. Many of these toxins are potent and highly selective blockers or modulators of ion channel function, and as such are valuable pharmacological tools, as well as being potentially useful leads in the development of new human therapeutics. We focus on peptide and protein toxins that block or modulate the activity of sodium or potassium channels. The sodium channel blockers have potential applications as new analgesics against chronic (neuropathic) pain, while the potassium channel blockers are effective in ameliorating multiple sclerosis symptoms in animal models of this debilitating disease.
Zhihe, Ronelle, Shenggen, Jennifer, Ray, Alfreda, Jeff, Zhi-Ping, Andrew, Pengfei, Indu, Xuecheng, Daxiu, Xiaodong
Cytokines Receptors and Signal Transduction
Multi-functional cytokines and haemopoietin receptors.
Cytokines are secreted proteins that allow cells to communicate with one another. Cytokines act by binding to multi-subunit receptors expressed on the surface of responsive cells and activating the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) pathway. While some cytokines, such as G-CSF and TPO affect a single lineage of blood cells; others, like leukaemia inhibitory factor (LIF) have diverse effects. For example, while we purified LIF on the basis of its ability to induce the macrophage differentiation of blood cells, we also found that it could inhibit the differentiation of embryonic stem (ES) cells, leading to its sale as ESGRO and use in laboratories throughout the world. We have also identified a number of cytokine receptors including the interleukin-11 receptor and a shared component of the interleukin-4 and interleukin-13 receptors. In collaboration with Zenyth Therapeutics we produced a panel of antagonistic human antibodies to the human IL-13 receptor, which are currently being developed as a treatment for asthma in conjunction with Merck. Using a similar strategy we are also collaborating with Professor Ian Wicks to determine whether inhibitors of G-CSF action have a place in the treatment of inflammatory diseases such as arthritis. This project is being commercialised by Zenyth Therapeutics and Murigen Therapeutics.
Structures of malaria vaccine candidates
ZP Feng, DW Keizer, S Yao, JJ Babon, RA Stevenson, RS Norton, in collaboration with RF Anders, VJ Murphy, CG Adda (La Trobe University) [J Mol Biol. Jul 22;350(4):641-56 PMID:15964019 220. Zhang X, Perugini MA, Yao S, Adda CG, Murphy VJ, Low A, Anders RF & Norton RS (2008) Solution conformation, backbone dynamics and lipid interactions of the intrinsically unstructured malaria surface protein MSP2. Journal of Molecular Biology 379, 105–121.
Apical membrane antigen 1 (AMA1), a merozoite surface protein found in all species of Plasmodium, is a strong candidate for inclusion in a malarial vaccine. The 62-kDa ectodomain of AMA1 consists of three disulfide-stabilised domains, and the disulfide-bond stabilised conformation is essential for protection, as the antigen is not an effective vaccine after reduction and alkylation. Determining the structure of the ectodomain will provide a basis for understanding its interaction with inhibitory molecules and enhancing its properties as a vaccine.
We have determined the structures of domains II and III of AMA1. In domain II (Figure) two disulfide bonds link the N- and C-terminal regions of the molecule, which form a four-stranded beta-sheet linked to a short helix. A long loop linking the N- and C-terminal regions contains four other alpha-helices, the locations of which are not fixed relative to the beta-sheet core, even though they are well-defined locally. Importantly, this region of domain II contains the epitope recognised by the invasion-inhibitory antibody 4G2, even though it does not contain any of the polymorphisms that are regarded as having arisen in response to the pressure of immune recognition.
We are currently developing small molecule and peptide inhibitors of AMA1.
Merozoite surface protein 2, MSP2, is also a vaccine candidate. It is an intrinsically unstructured protein that forms amyloid-like fibrils upon storage. We have used NMR to examine peptides corresponding to the conserved N-terminal region of MSP2 for the presence of well-ordered local structure and the ability to form fibrils related to those formed by full-length MSP2. In the N-terminal region residues 8-15 formed a hairpin-like structure, with the remainder of this region being unstructured. An 8-residue peptide corresponding to residues 8-15 also formed this hairpin-like structure and produced amyloid fibrils similar to those formed by the N-terminal region of MSP2.
Structure and interactions of SSB-2
Z Kuang, S Yao, A Low, JJ Babon, TJP Garrett, RS Norton (Structural Biology), SL Masters, TA Willson, J-G Zhang, NA Nicola, SE Nicholson (Cancer and Haematology) [Nat Struct Mol Biol 2006 Jan;13(1):77-84 PMID:16369487 229. Kuang Z, Yao S, Xu Y, Lewis RS, Low A, Masters SL, Willson TA, Kolesnik TB, Nicholson SE, Garrett TJP & Norton RS (2009) SPRY domain-containing SOCS box protein 2: crystal structure and residues critical for protein binding. Journal of Molecular Biology 386, 662–674.
The SPRY domain-containing SOCS box protein 2 (SSB-2) is one of a sub-family of four proteins (SSB-1 to -4) within the SOCS protein family. Like other central domains in SOCS proteins, the SPRY domain is expected to mediate protein-protein interactions. It has since been shown that SSB proteins interact directly with hepatocyte growth factor (HGF) c-Met. Furthermore, interactions with Par-4 (prostate apoptosis response-4) through the SPRY domain of SSB proteins have also been observed. We have solved the structure of SSB2 in solution using NMR spectroscopy and subsequently by X-ray crystallography. The SSB-2 structure represents the first 3D structure of a SPRY domain. Residues responsible for Par-4 binding are located predominantly in two loop regions. Further studies are underway to map the interactive surfaces with other partners onto our 3D structure.
Recently we have identified another biologically relevant binding partner for the SSB proteins and are exploring the role of this interaction in controlling the body’s response to infectious diseases.
Two views of the SSB-2 structure
Structure and interactions of SOCS3
JJ Babon, S Yao, D Saunders, RS Norton (Structural Biology), NA Nicola (Cancer and Haematology) [Mol Cell.2006 Apr 21;22(2):205-16, PMID: 16630890 225. Babon JJ, Sabo JK, Soetopo A, Yao S, Bailey MF, Zhang J-G, Nicola NA & Norton RS (2008) The SOCS box domain of SOCS3: structure and interaction with the elonginBC-cullin5 ubiquitin ligase. Journal of Molecular Biology 381, 928-940
Cytokine signalling is essential for cells to communicate within the body. Cells must also regulate the duration of their responses to cytokines in order to maintain proper function. SOCS3 is a member of a family of proteins that suppress the signalling of a variety of cytokines, including growth hormone, LIF, G-CSF and members of the interleukin family. Abnormalities in SOCS3 expression are associated with a variety of haematopoietic and inflammatory diseases. We have determined the 3D structure of SOCS3 using NMR. SOCS3 contains a classic SH2 domain which in turn harbours a large, unstructured insertion. This insertion can be removed without affecting SOCS3 activity; it fits the description of a PEST motif, which is known to be important in regulating protein destruction by the proteasome. Adjacent to the SH2 domain is a short helix that forms part of the kinase inhibitory region, which is crucial for the activity of SOCS3. The proximity of the KIR and the pTyr binding site suggests a mechanism for cross-talk between these two important regions of the protein. We have also established that the SH2 domain binds to its intracellular partner, the gp130 receptor, through a series of hydrophobic interactions as well as an electrostatic interaction between the pTyr and a positively-charged patch on SOCS3. The SOCS box, a region responsible for directing the degradation of cytokine signaling intermediates, proved to be unstructured in the absence of any intracellular binding partners. Our structure is the first determined for a SOCS proteins, and it provides a basis for understanding how SOCS3 functions at the molecular level as well as for drug design programs. Future work is focused on the structure of the SOCS box in the presence of its intracellular partners and the roles of SOCS3 in inhibition of JAK kinase activity and receptor degradation.
Structure of SOCS3 showing the PEST motif important for protein turnover
gp130 receptor peptide binding to SOCS3
Statistical Genetics
The Statistical Genetics group focuses on the development of algorithms to perform statistical analysis to facilitate the mapping of human and murine (mouse) genes. We have collaborations with researchers and clinicians from around Australia, on mapping genes for both simple and complex human diseases such as deafness, muscular neuropathies, leukaemia and lymphomas, multiple sclerosis (MS), Leber's hereditary optical neuropathy, haemochromatosis and epilepsy. In particular we have a strong collaboration with the Menzies Research Institute, Hobart. We work together not only on mapping projects but also statistical and algorithmic development.
We also work closely with the Division of Molecular Medicine on the mapping and identification of ENU mutants and we have been able to help with the idenfication of several of the mutants. We also have a long association with the Australian Genome Research Facility (AGRF). This collaboration provides the important connection between the production of high quality genotyping data and its analysis, which is vital for the effective mapping of genes. Researchers also benefit substantially from this collaboration with our development of analysis methods and tools, especially for new genotyping technologies such as SNP chips.
We are always interested in new mapping projects and encourage clinician researchers to contact us to discuss potential projects. We have considerable experience in the use of both microsatellite and SNP chip mapping for both linkage and association studies.
Research projects are available for BSc and PhD students in Statistical Genetics. For further information on studies in Bioinformatics, visit the Bioinformatics web page. Projects involve analysis of DNA marker data from human pedigrees that display inherited disease patterns with mathematical and computational techniques to identify the responsible genetic loci. DNA marker data generated from crosses between mouse strains that show differences in genetically influenced traits are also analysed to identify genetic loci that contribute to these differences and also provide potential for Honours and PhD studies. Refer to the Prospective Students web page for further information on studying at the institute and contact Dr Melanie Bahlo (details at top of page) for further details on statistical genetics, in particular if you have specific questions or wish to discuss projects.
A family segregating deafness. The family’s DNA samples were used to identify a genomic region in the human genome that is likely to contain the faulty gene causing the deafness. Squares represent males, circles females. Shaded symbols indicate that the individual is deaf. Individuals in grey with a question mark may still become deaf with age.
We also work closely with the Division of Molecular Medicine on the mapping and identification of ENU mutants and we have been able to help with the idenfication of several of the mutants. We also have a long association with the Australian Genome Research Facility (AGRF). This collaboration provides the important connection between the production of high quality genotyping data and its analysis, which is vital for the effective mapping of genes. Researchers also benefit substantially from this collaboration with our development of analysis methods and tools, especially for new genotyping technologies such as SNP chips.
A plot of the genome wide scan results for the family shown in the pedigree above. A clear peak can be seen on chromosome 22 indicating that the faulty gene lies on this chromosome
We are always interested in new mapping projects and encourage clinician researchers to contact us to discuss potential projects. We have considerable experience in the use of both microsatellite and SNP chip mapping for both linkage and association studies.
Research projects are available for BSc and PhD students in Statistical Genetics. For further information on studies in Bioinformatics, visit the Bioinformatics web page. Projects involve analysis of DNA marker data from human pedigrees that display inherited disease patterns with mathematical and computational techniques to identify the responsible genetic loci. DNA marker data generated from crosses between mouse strains that show differences in genetically influenced traits are also analysed to identify genetic loci that contribute to these differences and also provide potential for Honours and PhD studies. Refer to the Prospective Students web page for further information on studying at the institute and contact Dr Melanie Bahlo (details at top of page) for further details on statistical genetics, in particular if you have specific questions or wish to discuss projects.
Approaches to Finding Hit Molecules
A typical screening campaign
Within the lab, our target molecules are derived from one of three approaches: (i) structure-based design; (ii) natural products with known activity; and (iii) high throughput chemical screening. Once a hit is identified and validated, we adopt standard medicinal chemistry techniques such as structure-activity relationships in the quest to enhance the potency and improve the properties of the chosen molecules with the ultimate aim of producing a drug that can enter clinical trials.
(i) Structure Based Drug Design
Advances in structural biology have given chemists the power to rationally design drug-like molecules that, theoretically, will bind to a specific receptor or protein of interest. In the area of peptidomimetics, molecular modeling enables the selection of a backbone scaffold, which will project the residue mimics in the necessary direction in space. Beyond converting these calculations into the chemical reality of a biologically active hit, we aim to optimise the potency and selectivity of our molecules through structure-activity relationships. In some instances, thorough conformational studies are used to determine whether the molecules in fact adopt the designed shape.
(ii) Natural Products and their derivatives
Molecules found in nature have evolved over billions of years to meet the needs of the plant or organism that produces it. Typically, this involves interaction with proteins or enzymes within a biological system. It follows that these molecules, while adapted to use in its original environment, might find similar interactions inside the human body. We are interested in natural products that have been reported to have promising biological activity, such that we can then work towards enhancing this activity in the search for therapeutic utility.
(iii) High Throughput Chemical Screening
With a library of over 100,000 commercially available synthetic compounds - and soon to be expanded towards 200,000 compounds, our colleagues in the HTCS group are able to rapidly test ‘lead-like’ molecules against many biological targets. Selected hits from a variety of assays are then used to initiate synthetic chemistry programs.
The Lab
Down the lab's main corridor - plenty of natural light...
Major equipment:
NMR - Bruker Avance II, 300 MHz
HPLC - Waters Alliance HT 2795
LCMS - Finnigan LCQ Advantage MAX
Prep-LCMS - Waters
Infrared - Bruker Tensor27 FT-IR
Automated Flash Chromatography System - Biotage FlashMaster II
Microwave - CEM Discover Lab Mate
MBRAUN Solvent Purification System
Collaborations
WEHI Divisions:
Molecular Genetic of Cancer
Molecular Medicine
Infection and Immunity
Other Institutions:
The University of Melbourne
La Trobe University
The Ludwig Institute for Cancer Research
The Howard Florey Institute
The CRC for Cancer Therapeutics
Private collaborations:
Genentech, Inc.
Abbot Pharmaceuticals
Bionomics, Ltd.
Murigen
DNDi
The Group
Nick, Guillaume, Wilco, Nurul, Keith, Georgina, Angela, Andrew, Julia, Carl, Sanji, Dana, Brad, JB
Insulin Receptor
Dr Mike Lawrence and Dr Colin Ward are interested in understanding the way in which insulin binds to its receptor on the surface of cells. Their work has recently led to the first structure of the intact extra-cellular part of the insulin receptor which revealed the likely way in which the insulin binding site is constructed. The challenge is now to find out exactly how insulin binds to this site and to understand what structural transitions occur within the receptor when insulin binds. The techniques employed in this work include both protein X-ray crystallography and small-angle X-ray scattering (SAXS). Their work includes extensive used of synchrotron radiation.
The insulin receptor (IR) is also part of the broader IGF signalling system which includes the insulin-like growth factor type 1 receptor (IGF-1R). IGF-1R and IR are closely related in structure. Aberrant signalling within this system has been shown to play a role in the development and progression of cancer. There is thus a need to understand the way in which insulin-like growth factors types 1 and 2 binds to IGF-1R and in which insulin-like growth factors type 2 binds to the IR. To this end the structure of the ligand-bound IGF-1R is also being pursued.
The X-ray structure of the insulin receptor ectodomain complexed with four antibody fragments
Statistical Genetics
The Statistical Genetics group focuses on the development of algorithms to perform statistical analysis to facilitate the mapping of human and murine (mouse) genes. We have collaborations with researchers and clinicians from around Australia, on mapping genes for both simple and complex human diseases such as deafness, muscular neuropathies, leukaemia and lymphomas, multiple sclerosis (MS), Leber's hereditary optical neuropathy, haemochromatosis and epilepsy. In particular we have a strong collaboration with the Menzies Research Institute, Hobart. We work together not only on mapping projects but also statistical and algorithmic development.
We also work closely with the Division of Molecular Medicine on the mapping and identification of ENU mutants and we have been able to help with the idenfication of several of the mutants. We also have a long association with the Australian Genome Research Facility (AGRF). This collaboration provides the important connection between the production of high quality genotyping data and its analysis, which is vital for the effective mapping of genes. Researchers also benefit substantially from this collaboration with our development of analysis methods and tools, especially for new genotyping technologies such as SNP chips.
We are always interested in new mapping projects and encourage clinician researchers to contact us to discuss potential projects. We have considerable experience in the use of both microsatellite and SNP chip mapping for both linkage and association studies.
Research projects are available for BSc and PhD students in Statistical Genetics. For further information on studies in Bioinformatics, visit the Bioinformatics web page. Projects involve analysis of DNA marker data from human pedigrees that display inherited disease patterns with mathematical and computational techniques to identify the responsible genetic loci. DNA marker data generated from crosses between mouse strains that show differences in genetically influenced traits are also analysed to identify genetic loci that contribute to these differences and also provide potential for Honours and PhD studies. Refer to the Prospective Students web page for further information on studying at the institute and contact Dr Melanie Bahlo (details at top of page) for further details on statistical genetics, in particular if you have specific questions or wish to discuss projects.
A family segregating deafness. The family’s DNA samples were used to identify a genomic region in the human genome that is likely to contain the faulty gene causing the deafness. Squares represent males, circles females. Shaded symbols indicate that the individual is deaf. Individuals in grey with a question mark may still become deaf with age.
We also work closely with the Division of Molecular Medicine on the mapping and identification of ENU mutants and we have been able to help with the idenfication of several of the mutants. We also have a long association with the Australian Genome Research Facility (AGRF). This collaboration provides the important connection between the production of high quality genotyping data and its analysis, which is vital for the effective mapping of genes. Researchers also benefit substantially from this collaboration with our development of analysis methods and tools, especially for new genotyping technologies such as SNP chips.
A plot of the genome wide scan results for the family shown in the pedigree above. A clear peak can be seen on chromosome 22 indicating that the faulty gene lies on this chromosome
We are always interested in new mapping projects and encourage clinician researchers to contact us to discuss potential projects. We have considerable experience in the use of both microsatellite and SNP chip mapping for both linkage and association studies.
Research projects are available for BSc and PhD students in Statistical Genetics. For further information on studies in Bioinformatics, visit the Bioinformatics web page. Projects involve analysis of DNA marker data from human pedigrees that display inherited disease patterns with mathematical and computational techniques to identify the responsible genetic loci. DNA marker data generated from crosses between mouse strains that show differences in genetically influenced traits are also analysed to identify genetic loci that contribute to these differences and also provide potential for Honours and PhD studies. Refer to the Prospective Students web page for further information on studying at the institute and contact Dr Melanie Bahlo (details at top of page) for further details on statistical genetics, in particular if you have specific questions or wish to discuss projects.
Negative regulation of cytokine signaling in inflammation
The regulation of haematopoiesis involves intricate communication between blood cells and their environment. Cytokines are proteins that act as messengers between cells, influence the proliferation, survival, maturation and functional activation of blood cells. These blood cell “hormones” are crucial in maintaining appropriate cell numbers and ensuring effective cell function. The positive influences of cytokines must be balanced by mechanisms to prevent excessive responses. Over the last decade, the Laboratory has used gene knockout technology to define the biological roles of the Suppressors of Cytokine Signalling (SOCS), a family of proteins that attenuate cellular cytokine responses to cytokines. In collaboration with Doug Hilton in the Division of Molecular Medicine and the other Laboratory Heads within the Cancer and Haematology Division, we have established that SOCS1 is an indispensable regulator of interferon responses, ensuring that the beneficial immune effects of this cytokine can be manifest without collateral damage to healthy tissues. Similarly, SOCS3 regulate responses to the clinically important cytokine G-CSF and, along with controlling interleukin-6 (IL-6), is essential to prevent spontaneous inflammatory disease.
Our current studies are focused on understanding the cellular basis for the essential role SOCS3 plays in preventing inflammatory disease in vivo. We have shown that SOCS3 is essential within the blood cells themselves to prevent excess production of specialized inflammatory cells called neutrophils and to ward of pathological inflammation, but expression in other cell types or tissues seems also to be important. Identifying these other cells and understanding how deficiency of SOCS3 perturbs the development of neutrophils is an important priority.
Within the SOCS protein family, 4 members: SOCS1, SOCS2, SOCS3 and CIS, have structural similarities and are implicated in regulating similar cytokines. This raises the possibility that SOCS proteins may act in concert to regulate cytokine responses. To determine whether there are shared or overlapping physiological roles of SOCS-1, SOCS2, SOCS-3 and CIS, we are examining cytokine responses and inflammation in the absence of functional genes for 2 or more of these proteins. As key regulators of inflammatory cytokines, defining the specific biological roles of the SOCS proteins will inform potential use of SOCS proteins or their inhibitors in inflammatory disease.
Refer to the Prospective Students web page for information about student projects available in the Alexander Laboratory.
Genetic dissection of blood cell production and function
Genetics tools such as specific gene knockouts or subtly modified alleles are used to probe physiological gene function and, in collaboration with colleagues Professor Doug Hilton and Benjamin Kile in the Division of Molecular Medicine, large scale ENU mutagenesis screens are in place for discovery of new blood cell controlling genes.
The process of blood cell formation, or haematopoiesis, is a dynamic system responsible for production of blood cells throughout life. Intricately controlled to allow precise numerical replacement of the billions of blood cells used every day of normal life, the haematopoietic system is also capable of rapid blood cell production in times of acute need, such as infection or severe bleeding. When these regulatory systems break down, health is compromised: the production of too few or ineffective blood cells can result in anaemia, susceptibility to infection or bleeding and conversely, production of too many or overactive cells can lead to leukaemia, inflammation or autoimmune disease. Understanding the molecules that regulate blood cell production and function has already led to major advances in treating and preventing disease and further advances will continue improve the outlook of patients with diseases of the blood. The Laboratory’s recent focus has been in two major areas of blood cell regulation: production of megakaryocytes and platelets, and control of haematopoietic stem cells.
The blood platelets are small cell fragments produced from specialized cells in the bone marrow called megakaryocytes. Platelets are necessary for blood clotting and situations in which platelet numbers are abnormally low (called thrombocytopenia), such as in some autoimmune diseases or in cancer patients receiving chemotherapy, there is a risk of serious haemorrhage. A major focus of the Laboratory is the use of genetics to define the molecular regulators of megakaryocyte and platelet production. Our knockout studies of the gene encoding c-Mpl, the receptor for thrombopoietin (TPO) contributed to defining the key role of TPO in maintaining normal circulating platelet numbers.
Over recent years we have used ENU mutagenesis in large-scale screens for regulators of platelet number and isolated over 30 mutations affecting this process. Wild-type screens have yielded new models of thrombocytopenia and mutagenesis on a genetically low platelet background has been used to search for mutations that cure thrombocytopenia, in an effort to streamline discovery of novel therapeutic targets. This suppressor screen has yielded multiple alleles that increase platelet numbers in thrombocytopenia including mutations in the genes encoding the components of the c-Myb/p300 transcriptional regulatory complex, and Suz12, an epigenetic regulator.
We have also undertaken sensitized genetic screens for regulators of stem cell function. Since compensatory mechanisms can overcome stem cell deficits allowing production of normal numbers of mature blood cells, mutations affecting stem cells may not always be evident in the blood. We reasoned that genetic screens on a stem cell-sensitized genetic background might overcome this compensation. Indeed, using this approach, we have isolated the first loss of function allele of the transcription factor Erg in a pedigree with multi-lineage haematopoietic deficiencies emanating from profound stem cell defects.
Our ongoing analyses of the mutant alleles and pedigrees emerging from these mutagenesis screens provide unique opportunities to make novel biological and molecular insights into the regulation of blood cell production and function in health and disease.
C3G: a signal transduction protein integrating extracellular signals to control neural progenitor pr
The Ras signalling pathway regulates a number of important cellular functions, such as cell proliferation, migration and differentiation in health and disease. The guanine exchange factor, C3G, is a regulator of Ras family proteins. C3G predominantly activates Rap1 and does so when signalling occurs through growth factor receptors and extracellular matrix receptors, particularly integrin β1 (Figure 4).
Figure 4: Schematic diagram of C3G function based on data published by our group and others (left) and the protein domain structure of C3G (right). C3G has a carboxy-terminal catalytic domain for GTP exchange on Ras family members and internal proline-rich domains, which convey binding to the focal adhesion molecule p130cas and to the adapter molecule Crk.
The C3G gene is expressed widely at low levels and at higher levels in the developing nervous system. Studying C3G deficient mice we have shown that, amongst a number of functions in different tissues, this protein has a major role in regulating the growth of the precursor cell population, which forms the cerebral cortex (Figure 5). We observed an overproliferation of the C3G deficient cerebrocortical neuroepithelium. Labelling of cells in the cell cycle and additional labelling of cells in DNA synthesis phase showed that C3G deficient cells are retained in the cell cycle instead of exiting the cell cycle normally.
Figure 5: C3G is required to restrict neural precursor proliferation. Expression of a lacZ reporter gene in the C3G locus shows C3G gene activity in the neural tube (blue) at embryonic day 9.5 (A). In the C3G deficient cerebrocortical neuroepithelium (Ctx in C) compared to wild type controls (B) neural precursor cells overproliferate, such that the neuroepithelium forms folds (arrow in C). C3G deficient neuroepithelium (VZ in E) fails to generate differentiating neurons (labelled with the DNA synthesis marker BrdU (green) 24 h earlier and marked with asterix in the control (D). Increase in phosphorylated Akt/PKB (pAkt) and Erk2 (pErk2) in C3G deficient neural precursor cells (MT) as compared to control cells (WT) indicate overactivation (F, H) and increase in phosphoryated Gsk3β (pGsk3β) indicates inhibition (F, G) in response to growth factor stimulation.
In C3G deficient cultured neural precursor cells we observed a significant overactivation of the Ras signalling pathway components Erk1/2 and Akt/PKB with a concomitant inhibition of Gsk3β, as shown by increased levels of phosphorylation on these proteins (Figure 5). Our data show that C3G promotes cell cycle exit and that signalling through FGF2 and the EGF-receptor, besides activating proliferation-promoting pathways, concomitantly activate a proliferation-restricting mechanism, which crucially requires C3G. Thus C3G is a critical regulator of the balance between neural precursor proliferation and differentiation.
Structure and function of the Bcl-2 protein family
Crystal structures of Mcl-1 bound to the BH3 domains of the BH3-only proteins Bim and Noxa have been determined, together with an NMR structure for the Noxa complex. The structure, and subsequent mutagenesis analysis, has shed light on the observation that Mcl-1 protein levels in the cell are stabilized through interaction with Bim but Mcl-1 is targeted for degradation when complexed with Noxa. A characteristic sequence in the C-terminal part of the Noxa BH3 domain has been implicated in triggering the degradation of Mcl-1. Through the use of a novel BH3 sequence derived from the BH3 domain of Bim, we have shown that cell death can be initiated even when Mcl-1 protein levels are high, provided that Mcl-1 is prevented from engaging the pro-apoptotic Bcl-2 family member Bak.
Our structure of the Bcl-2 antagonist ABT-737 bound to Bcl-xL has revealed some unexpected features of the complex, and suggested why the compound is unable to engage the Bcl-2 family member Mcl-1. We have recently derived a first ever image of a so-called ‘foldamer’, a peptide containing a mixture of alpha- and beta- amino acids, bound to its protein target, in this case Bcl-xL. The structure confirms the design features of the foldamer and supports the notion that such peptides could be tailored to bind a number of important drug targets.
Ongoing studies are directed at further understanding the interactions between pro-survival and pro-apoptotic members of the Bcl-2 family as a basis for discovering compounds that are better able to trigger the death of unwanted (e.g. tumor) cells.
Structures of (A) hMcl-1:hBim BH3 , (B) hMcl-1:mNoxaB BH3. The BH1, BH2 and BH3 regions of Mcl-1 are coloured blue, yellow and red respectively. The hBim BH3 is coloured green and the mNoxaB BH3 is coloured purple.
MYST family histone acetyltransferases in nervous system development and neural stem cells
Acetylation of lysine residues in histone amino-terminal tails is a hallmark of transcriptionally active gene loci in mammals. Histone acetylation is performed by histone acetyltransferases and thought to keep the chromatin in an open configuration (Figure 1A). The MYST family, characterised by its MYST histone acetyltransferase domain, has five members (Figure 1B). Our findings show that two of its members, Querkopf (Qkf, Morf, Myst4, Kat6b) and Monocytic leukaemia zinc finger protein (Moz, Myst3, Kat6a) are proteins that regulate stem cell dynamics at the transcriptional level.
Figure 1: Histone acetyltransferases acetylate lysine residues in the amino-terminal tails of histones, thereby rendering the chromatin accessible for transcriptional activation modelled in (A). The MYST histone acetyltransferase family consists of 5 members in mammals (B) characterised by their MYST domain.
The Qkf gene is initially expressed at low levels throughout the early embryo, but at E10.5 of development a strong domain of expression emerges in the dorsal telencephalon, the earliest cerebral cortex primordium. High levels of expression are maintained in this region throughout cortical development, both in the proliferating ventricular zone cells (the ‘stem cells’ of the cortex) and in the differentiating neuroblasts that migrate away from the ventricular zone to form the cortical plate (Figure 2).
The cortical plate is where further maturation of the newly born neurons takes place to ultimately form the adult six-layered cortex. Qkf has a dynamic pattern of expression and during the latter stages of development strong domains of expression also arise in the ventral telencephalon, which forms the caudate-putamen, and in the olfactory bulbs.
Figure 2: Expression of a lacZ reporter gene in the Qkf locus shows strong Qkf gene activity in the dorsal telencephalon at embryonic day 12.5 (dTel, blue, A) and RNA/RNA in situ hybridisation documents continuous strong expression of the endogenous Qkf gene in the ventricular germinal zone and the cerebral cortex primordium, the cortical plate at embryonic day 15.5 (Ctx, B). BrdU labelling of cells in DNA synthesis phase (brown, C) shows that Qkf deficient ventricular germinal zone cells (mt, VZ in C) proliferate less than controls (wt in C). Moreover, the Qkf deficient ventricular germinal zone gives rise to fewer differentiating neurons, which is manifest in the smaller cortical plate (CP in C).
We generated a Qkf deficient mouse strain. Mice deficient in the expression of Qkf have a reduction in the number of proliferating cells in the ventricular zone and reduced numbers of newly born neurons in the cortical plate at all stages of cortical neurogenesis (Figure 2). This suggests that Qkf is involved in the balance between proliferation and differentiation. As a result of these developmental defects the adult Qkf deficient mice have a reduction in the size of the cerebral cortex. In addition, Qkf deficient mice have reduced numbers of interneurons, cells that migrate into the cortex from the ventral telencephalon.
Adult mice lacking Qkf have a reduction in the number of neural stem cells in the forebrain, a stem cell population that continually gives rise to new neurons throughout life. Consequently, adult Qkf deficient mice fail to generate normal numbers of new neurons during adulthood (Figure 3).
Interestingly, while examining a mouse strain deficient in Moz, we found that Moz deficient mice lack long-term reconstituting haematopoietic stem cells. Therefore the closely related proteins Qkf and Moz perform essential corresponding roles in diverse stem cell populations.
Figure 3: Adult forebrain neural stem cells can be labelled by virtue of their ability to retain BrdU incorporated into the DNA in the long-term due to their long cell cycle time (A). Immunohistochemistry of BrdU (black) and the neural stem cell marker GFAP (brown) in a brain section (B). Enumeration of adult neural stem cells at 6 rostro-caudal brain levels (C) in Qkf deficient mice and wild type controls (D)
The structure and function of viral Bcl-2 homologues
Our description of the 3-d structures of myxoma virus M11L and vaccinia virus F1L revealed that both share structural homology with the mammalian Bcl-2 pro-survival proteins, although neither has any sequence similarity to their mammalian counterparts. We were also able to directly show for the first time that viral proteins of this class bind to BH3 only proteins. More recently we have extended this observation to the Epstein Barr Virus pro-survival protein BHRF1 and captured its structure in complex with the BH3 only protein Bim. Ongoing studies are directed at elucidating the critical protein-protein interactions in which these viral proteins engage in order to prolong the life of the infected cell.
Ribbon diagram of the M11L : Bak BH3 complex (blue and red) including the molecular surface (grey) on the left, and M11L on its own on the right (blue).
Why mice don’t have webbed feet
Removal of interdigital webbing during embryonic development is one of the best known examples of developmental apoptosis. How this process occurs however is still a mystery. We have undertaken a genetic analysis aimed at understanding the basis of this phenomenon.
Research interests
Dr Bouillet joined the Molecular Genetics of Cancer Division in 1997 and has used genetic approaches to study the physiological role of members of the Bcl-2 family in the regulation of apoptosis. Genetic ablation of the Bim gene in mice has shown the role of this protein in the maintenance of homeostasis in the hematopoietic system, in the elimination of autoreactive thymocytes and B cells. He later showed that loss of Bim could prevent all the degenerative diseases that develop in Bcl-2 knockout animals, a model that he used to show that transgenic RNA interference could be used in vivo to prevent the development of the polycystic kidney disease that develops in the absence of Bcl-2. The bim gene was also shown to have important role in the termination of immune responses.
Dr Bouillet is presently developing mouse models in which subtle mutations are introduced into genes of the Bcl-2 family to study certain aspects of their regulation and their functional interactions.
Polycomb genes
The Polycomb Group of epigenetic regulators silences genes involved in many cellular processes, including apoptosis, proliferation and senescence. Two of these proteins, Bmi1 and Cbx7, promote lymphomagenesis in mice, and elevated CBX7 expression in human follicular lymphoma correlates with both high Myc expression and advanced tumour grade (Scott et al PNAS 2007 PMID: 17374722). As p53 is important in promoting apoptosis in response to anti-cancer drugs, we are investigating the ability of Cbx7 and Bmi1 to cause drug resistance in Myc-driven lymphomas and are searching for downstream targets (Adisa, Happo, Sugiana and Scott, unpublished).
Bcl-2 family
It had been widely thought that all oncogenic changes enhance cell proliferation. This view was overturned in 1988 when we showed that bcl-2, the gene activated by the t14;18 translocation in follicular lymphoma, promotes cell survival rather than proliferation (Vaux, Cory and Adams Nature 1988). Further, we found that myc and bcl-2 were highly synergistic in driving leukemogenesis (Strasser et al Nature 1990), due to Bcl-2 countering the apoptosis driven by Myc when cytokines become limiting. Recently, we found that Bcl-2-promoted development of follicular lymphoma requires a proliferative stimulus provided by T-cell help (Egle et al Blood 2004). The concept that disturbances to programs controlling cell survival and proliferation are both essential steps in oncogenesis is now widely accepted.
In collaboration with other laboratories in the Molecular Genetics of Cancer Division, we are currently exploiting mouse genetics to explore how opposing factions of the Bcl-2 family of proteins control apoptosis, particularly during haemopoiesis; how mutations affecting this life/death switch can contribute to the development of malignancies; and how different family members influence the cytotoxic responses to chemotherapy and radiation. Key findings include the following:
- The Bcl-2 antagonist ABT-737 developed by Abbott Laboratories targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized (van Delft et al Cancer Cell 2006 PMID: 17097561)
- The BH3 mimetic ABT-737 can be highly efficacious in treating aggressive Myc-driven lymphomas, especially in combination therapy (Mason, Vandenberg et al PNAS 2008 PMID: 19004807)
- The BH3-only proteins Puma and, to a lesser extent, Noxa, are suppressors of Myc-induced lymphomagenesis (Michalak et al Cell Death Differentiation 2009 PMID: 19148184)
- Apoptosis of human tumour cell lines harbouring a mutant B-RAF that is induced with a MEK inhibitor requires the BH3-only protein Bim and is enhanced by the BH3-mimetic ABT-737 (Cragg et al J Clin Invest 2008 PMID: 18949058)
- Pan-haemopoietic expression of Mcl-1, a relative of Bcl-2, confers resistance to diverse cytotoxic signals and promotes lymphoma development (Campbell et al Blood 2010, Epub ahead of print)
Myc
In the 80s, we discovered that chromosome translocations in both human Burkitt lymphomas and mouse plasmacytomas link an immunoglobulin gene to the myc proto-oncogene (Adams et al PNAS 1983). Using mice bearing a myc transgene expressed under the control the Ig heavy chain gene enhancer Eï, we went on to directly demonstrate that constitutive myc expression during B lymphopoiesis is highly oncogenic (Adams et al Nature 1985). Exploration of the preneoplastic state revealed that myc had directly induced hyperproliferation and retarded B lymphoid differentiation, but full-fledged malignancy relied on somatic mutations in rare myc-driven cells (Langdon et al Cell 1986). The Eï-myc transgenic mice have proved to be an invaluable tool for cancer researchers around the world.
More recently, we have utilized a vector having regulatory sequences from the vav gene to explore the consequences of pan-haemopoietic expression of myc. The vavP vector directs transgene expression in every nucleated haemopoietic cell type we have examined, including both differentiated and progenitor cells. Intriguingly, we found that both the kinetics and nature of the malignancy were markedly influenced by the level of Myc expression. Aggressive T-cell lymphomas rapidly overwhelm mice expressing high levels of the VavP-myc gene, whereas late-onset monocytic tumors predominate in lines with lower expression (Smith et al Oncogene 2005, Blood 2006). In collaboration with Dr Peter Hurlin (Oregon Health & Science University), we are currently evaluating the tumorigenic impact of loss of the Myc antagonist Mnt (Campbell, Scott and Cory, unpublished).
Bak and Bax regulation by other Bcl-2 family proteins.
The killing function of Bak and Bax is blocked by Bcl-2 proteins such as Bcl-2, Bcl-xL and Mcl–1, although the molecular mechanisms involved remain the topic of significant debate. Based on our recent finding that Bak starts to become an activated killer protein by exposing its BH3 domain, we are testing whether this domain can be captured by the prosurvival proteins to prevent the cell from dying. This project also involves a range of molecular and cellular techniques, including cysteine cross-linking as described above.
Understanding how the pro-apoptotic Bak and Bax proteins interact with their prosurvival guardians is important for the current development of anti-cancer agents that target the Bcl-2 protein family e.g. BH3-mimetics.
BAK ACTIVATION DURING APOPTOSIS
What are the important conformation changes in Bak and Bax that allow pore formation in mitochondria
To define the major structural changes in Bak and Bax that occur during apoptotic pore formation, we are using a range of biochemical and structure-based approaches. We recently reported that a crucial initial step in Bak-mediated apoptosis involves exposure of the BH3 domain, which then binds to the hydrophobic groove of a second Bak molecule to form symmetric dimers. How dimers then interact to form the large complexes thought necessary to form a pore in the mitochondrial outer membrane is being investigated. Conformation changes in other regions of Bak and Bax are also being analysed by several techniques including exposure of antibody epitopes and cross-linking of cysteine residues placed at specific positions in the Bak protein. To complement our biochemical studies, we are collaborating with the Structural Biology Division to obtain structures of activated Bak and Bax.
These studies aim to identify each step in the formation of mitochondrial pores by Bak and Bax, the point of no return in apoptotic cell death. Each step is a potential target for therapeutic intervention in certain cancers and degenerative diseases.
Alternative splicing, phosphorylation and stability of Bim in vivo
Three major isoforms of Bim mRNA (EL, L and S) are detected in mouse and human cells. Whereas the corresponding proteins can be detected in human lymphocytes, BimS protein is often not detected in mouse cells. To establish the role of each isoform in vivo, we are generating mice that can produce only one Bim isoform.
Bim is also regulated by phosphorylation which promotes its ubiquitination and proteasomal degradation, but whether this is a general regulatory mechanism or holds only in certain cell types or with particular cell stimuli remains unclear. To explore the physiological role of this process of Bim regulation, we have created mice expressing a Bim mutant that cannot be ubiquitinated or cannot be phosphorylated by ERK1/2.
BH3 binding specificity ‘in vivo’
The affinities of BH3-only proteins for their pro-survival targets varies greatly: Whereas Bim and Puma bind all five pro-survival Bcl-2 family members with high (low nM) affinity, Bad binds only Bcl-2, Bcl-xL and Bcl-w, whereas Noxa binds only Mcl-1 and A1. Since the binding specificity of BH3-only proteins greatly affects their ability to kill cells, we are modifying the mouse Bim gene in situ by replacing its BH3 domain with that of Bad, Noxa or Puma, to determine how changing its binding specificity affects the biological activity of Bim in vivo
Targeting Pro-survival Bcl-2 Proteins
With other laboratories in the Division and Structural Biology colleagues, we are exploring how impaired apoptosis contributes to cancer development and impacts on therapy. In particular, to create better anti-cancer therapies, we are collaborating in attempts to develop small molecules that target pro-survival Bcl-2 proteins. Such ‘BH3 mimetics’ have shown considerable promise in pre-clinical studies (van Delft et al, 2006; Adams and Cory, 2007) and one developed by Abbott is in early phase clinical trials, including a trial in Melbourne on chronic lymphocytic leukaemia.
Molecular Control of Apoptosis
One key issue about the control of apoptosis is how the BH3-only proteins trigger the death programme. Studies together with David Huang have led us to propose that they act primarily by engaging pro-survival relatives (Chen et al, 2005; Willis et al, 2005; Willis et al, 2007; Uren et al, 2007), rather than by directly activating Bax and Bak as suggested by others. We think that apoptosis ensues if and only if the BH3-only proteins neutralize the pro-survival Bcl-2 relatives engaging Bak or Bax.
We are also exploring how the pro-survival proteins engage Bax and Bak and whether this occurs in healthy cells or only after apoptosis begins. We think the pro-survival proteins sequester a minor sub-population of Bax molecules with their BH3 domain exposed (Fletcher et al, 2008). We are also studying how Bax and Bak are activated: how their conformational changes are controlled, what drives Bax from the cytosol into the membranes, and how Bax and Bak form the oligomers (Dewson et al, 2008) that perforate the mitochondrial outer membrane.
Investigations into the mechanisms that control programmed cell death during development of the mous
Using our panel of gene-targeted mice, we are investigating the roles of pro- as well as anti-apoptotic Bcl-2 family members in mouse development. We are particularly interested in identifying the mechanisms that govern the deletion of potentially dangerous lymphocytes and myeloid cells during their development and during the shut-down of immune responses. Results from these studies are expected to provide valuable information for basic research and will assist translational and clinical research by identifying potential therapeutic targets for autoimmune or inflammatory disorders.
Which pro-apoptotic BH3-only Bcl-2 family members are critical for anti-cancer therapy induced tumou
Using our panel of gene-targeted mice and transgenic mouse models of tumour development or panels of human tumour-derived cell lines and RNAi interference, we are investigating which pro-apoptotic BH3-only Bcl-2 family members are critical for the tumour cell killing effects of which anti-cancer therapeutics. Results from these studies are expected to provide valuable information for basic research and will also assist translational and clinical research.
Which anti-apoptotic Bcl-2 protein family members are critical for the development and sustained gro
Description of research project goes here – Using our panel of gene-targeted mice and transgenic mouse models of tumour development, we are investigating which anti-apoptotic Bcl-2 protein family members (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1) are critical for the development and the sustained growth of tumours. Results from these studies are expected to provide valuable information for basic research and will assist translational and clinical research by pinpointing the proteins that need to be targeted for cancer therapy.
Design therapeutics to transiently manipulate host genes to facilitate viral clearance
Manipulating host genes to promote immune mediated viral clearance
Our approach is to manipulate host genes rather than viral genes to promote an immune response in our LCMV clone 13 infection model. We use several validated techniques to manipulate candidate host genes and promote immune clearance of virus in our mouse model of chronic infection. These techniques include linking siRNA to single chain antibodies to target host genes in specific cells in vivo. We also target genes that we have identified as being important in dendritic cells (DCs) and macrophages with customized siRNA–loaded nanoparticles that also capture and neutralize virus to promote antigen presentation. Our work focuses on how genetic manipulation of mouse and human DCs affects their functional capacity and their ability to survive and prime T cells. Similarly we investigate how genetic manipulation of human and mouse T cells affects their ability to produce cytokines, degranulate and kill target cells in the face of persistent infection.
Challenge the paradigm of T cell memory and dissect its failure in preventing
Factors impeding effective T cell memory responses
Despite tremendous efforts, vaccines targeting pathogens that require T cell immune responses for control of disease, such as HIV, hepatitis C and TB, are effective. All our efficacious vaccines rely solely on B cell memory and antibody mediated immunity. Notwithstanding this, T cell memory remains the central dogma behind the development of vaccines for these diseases. The majority of immune memory work has been performed in mouse models were it is extremely difficult to dissect the relevance of T versus B cell memory in repeated challenge with the identical pathogen in the same host. Consequently, what appear to be effective memory T cell responses in the mouse have not translated to effective responses in humans. We take the unconventional view that a substantial failing is not a vaccines ability to prime a response but rather the inherent ineffectiveness of memory T cells in preventing disease. Using a novel model that does not interfere with T and B cell collaboration, we are dissecting the effectiveness of memory T cells alone and examine which host genes need to be manipulated to make a T cell response effective in a therapeutic setting.
Which host genes abrogate immunity to chronic infections?
Some host genes regulating T cell mediated viral clearance
Current therapies have had a tremendous impact on chronic viral infections and diseases like HIV and hepatitis C but these treatments are rarely curative. A major obstacle is the high mutation rate of these organisms, which renders them refractory to treatments that directly target their viability. One of the greatest challenges is developing treatments that cure these diseases and that are impervious to resistance mechanisms used by the pathogens to escape eradication. We focus our attention on modulating immune responses in mouse models of chronic viral infection to promote viral clearance. Targeting host molecular pathways, which the pathogen is dependent on for persistence, rather than viral proteins, circumvents a major resistance mechanism. Using an array of conditionally gene targeted mice we can interrogate the relevance of many genes implicated in immune regulation and test their relevance in immunity to persistent pathogens. We aim to identify host pathways that upon manipulation will permit clearance of persistent pathogens. These studies are particularly relevant to HIV infections where the immune system fails to clear virus and eventually succumbs to uncontrolled viral turnover.
Trial of intermittent treatment of malaria in infants in Papua New Guinea
In Papua New Guinea, malaria is one of the leading causes of death and morbidity in young children. We are working with the PNG Institute of Medical Research to conduct a trial of intermittent malaria treatment that aims to reduce the burden of malaria and anemia in infants and young children at the time of greatest susceptibility. Infants are given curative doses of anti-malarial medications every 3 months, at the same time as they receive their childhood vaccinations, until 12 months of age. A key issue with the regular administration of antimalarials is the potential impairment of acquired immunity. Integrated into the trial is a comprehensive research program on immunity to malaria that will examine this issue in detail and more broadly aims to understand the acquisition of immunity to malaria from birth and identify key targets and mediators of protective immunity.
Mugil Health Centre and staff from the PNG Institute of Medical Research
Collaborating centres in malaria-endemic countries
KEMRI-Wellcome Trust Collaborative Research Program
Centre for Geographic Medicine Research, Coast
Kenya Medical Research Institute, Kilifi, Kenya
www.kemri-wellcome.org
Papua New Guinea Institute of Medical Research
www.pngimr.org.pg
Malawi-Liverpool-Wellcome Trust Clinical Research Program
College of Medicine, University of Malawi
Queen Elizabeth Central Hospital, Malawi
Antibodies that inhibit malaria replication in the bloodstream
Schematic picture of a merozoite
A particular focus of our research is the identification of antibodies that inhibit the replication and growth of P. falciparum in the bloodstream. This type of antibody is thought to be important both in acquired immunity and as mediators of immunity generated by vaccination. However, the role of these antibodies in protection from clinical disease has not been established and the primary targets of inhibitory antibodies remain unclear.
We are examining the acquisition and targets of antibodies that inhibit P. falciparum invasion of red blood cells and the potential role of invasion-inhibitory antibodies in protective immunity. To achieve these goals, we are combining novel approaches using transgenic parasite lines in functional assays with unique samples from well defined clinical cohort studies in Kenya and Papua New Guinea. In recent studies (Persson et al 2008), we have identified two erythrocyte invasion ligand families, known as the EBA and PfRH proteins, as important targets of inhibitory antibodies. P. falciparum can switch between the use of different members of the EBA and PfRH invasion ligands, which facilitates evasion of immune responses. A malaria vaccine may need to target multiple ligands in order to generate an immune response that counters malaria's immune evasion strategy.
Malaria in Pregnancy
Red blood cells infected with P. falciparum can stick to molecules on the surface of endothelial cells in various organs. During pregnancy, large number of parasitized RBCs can accumulate in the blood spaces of the placenta resulting in severe consequences for mother and baby. Inside red blood cells, parasites produce proteins that are transported to the cell surface. A protein called PfEMP1 enables parasites to adhere to particular receptors in the vascular beds of various organs such as the brain and placenta. These PfEMP1 proteins are very diverse, encoded by a family of genes termed var, and allow parasites to undergo antigenic variation to evade the immune response. We are presently examining how immunity develops despite the parasites evasion strategies, and what immune responses should be induced by a possible vaccine.
