A*STAR-Dundee Partnership (ADP) Research Projects
1. The role of PIP2 in cell movement Bob Robinson (Institute of Molecular & Cell Biology, Singapore) and Nicholas Leslie (Division of Molecular Physiology, College of Life Sciences)
2. Curing Cancer with Computers: virtual drug design from the inhibition of growth factor signalling to translation
Chandra Verma (Bioinformatics Institute, Singapore) and Ruth Brenk (Division of Biological Chemistry and Molecular Microbiology, College of Life Sciences)
3. Defects in the skin barrier and their role in eczema and psoriasis
E. Birgit Lane (Institute of Medical Biology, Singapore) and W.H. Irwin McLean, (Division of Molecular Medicine, College of Life Sciences and Medicine, Dentistry & Nursing)
4. Relationship(s) between two the tumour suppressors p53 and APC in cancer
David Lane (Institute of Molecular & Cell Biology, Singapore) and Inke Nathke (Division of Cell & Developmental Biology, College of Life Sciences)
5. Specificity and regulation of interactions between 14-3-3 proteins and actin binding proteins
Ed Manser (Institute of Molecular & Cell Biology, Singapore) and Carol MacKintosh (MRC Protein Phosphorylation Unit, College of Life Sciences)
6. Lowe syndrome protein (OCRL-1) and endosome-to-TGN trafficking
Wanjin Hong (Institute of Molecular & Cell Biology, Singapore) and John Lucocq (Division of Cell Biology & Immunology, College of Life Sciences,
7. The role of the cell cycle during vertebrate somite segmentation
Yun-Jin JIANG (Institute of Molecular & Cell Biology, Singapore) and Miguel Maroto (Division of Cell & Developmental Biology)
8. Novel cellular targets of Human Papilloma Virus – implications in disease
Francoise Thierry (Institute of Medical Biology, Singapore) and Sam Crouch (Division of Molecular Medicine, College of Life Science and Medicine, Dentistry & Nursing)
9. Characterisation of the Wnt and GSK3 signalling and their role in cancer
David Virshup (Institute of Medical Biology, Singapore) and Dario Alessi (MRC Protein Phosphorylation Unit, College of Life Sciences)
10. Mutations in type VII collagen and their role in wound healing and cancer
E. Birgit Lane (Institute of Medical Biology, Singapore) and Andrew South, (Division of Medical Sciences, Medicine, College of Medicine, Dentistry & Nursing)
11. Analysis into the function and regulation of Kirrel in the paraxial mesoderm of the chick embryo
Mike Jones (Institute of Medical Biology, Immunos, Singapore ) and Kim Dale (Division of Cell & Developmental Biology, College of Life Sciences)
12. Function of the proto-oncogene ect2 in cell motility and cell signalling
Ed Manser, (Institute of Molecular & Cell Biology, A*STAR, Singapore) and Arno Muller, Division of Cell & Developmental Biology, College of Life Sciences)
13. Exploring and exploiting conserved synthetic lethal interactions of DNA double strand break repair factors.
David Lane (Institute of Molecular & Cell Biology, Singapore) and Anton Gartner, (Wellcome Trust Centre for Regulation and Expression)
14. Mathematical/Systems biology, Computational Biology/Bioinformatics
Vladimir Kuznetsov (Bioinformatics Institute, Singapore) and Mark Chaplain (Division of Mathematics)
15. The role of Aurora A in the cell cycle and transformation
Ed Manser (Institute of Molecular & Cell Biology, Singapore) and Ron Hay (College of Life Sciences)
16. The role of the Cdc7 kinase in DNA replication and in the DNA damage response
Philipp Kaldis (Institute of Molecular & Cell Biology, Singapore) and Julian Blow (Wellcome Trust Centre for Regulation and Expression, College of Life Sciences)
DETAILS OF PROJECTS
(1) The Role of PIP2 in Cell Movement
Supervisors: Dr Bob Robinson (Institute of Molecular & Cell Biology, Singapore) and Dr Nicholas Leslie (College of Life Sciences, University of Dundee)
Actin polymerization provides the force that drives cellular movement. Concerted polymerization, in a particular direction, is required in order for a cell to push forward its leading edge. There are a host of actin-binding proteins that regulate the spatial and temporal patterning of this actin polymerization. The list of actin-interacting proteins now exceeds 150 classes of proteins. Actin-filament uncapping and actin-filament nucleation are the two known mechanisms through which a cell can produce the explosive actin filament elongation that is required for cell movement. However, filament capping (to prevent non-productive polymerization), filament crosslinking (to provide a strong base against which to push), and actin filament recycling (to provide an inexhaustible supply of actin monomers) are also vital components of this system.
Phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid, is a major regulator of many of these actin-binding proteins, generally favouring the transition to polymeric actin. PIP2 is, however, just one of several related phosphatidylinositol signalling molecules and the precursor for the second messengers diacylglycerol, inositol 1,4,5-trisphosphate, and phosphatidylinositol 3,4,5-trisphosphate (PIP3). The first phase of this project is a cell biology approach to investigate how a single phospholipid species fulfils such a range of independent functions. This will involve quantitative and spatial analysis of PIP2 in motile cells and under conditions where PIP2 levels are manipulated by pharmacological and molecular biological means. The effects of sequestering PIP2 in vivo, using specific lipid binding protein domains, on the distribution and functionality of the actin network will also be analysed. A second aim of this phase is to identify the protein partners of PIP2 that regulate cell movement, using affinity isolation techniques. This portion of research will be carried out in the first 2 years of the PhD in Dundee under the supervision of Pete Downes, who is a world renowned leader in the phosphatidylinositol signalling field.
www.dundee.ac.uk/biocentre/SLSBDIV6cpd.htm
The second half of the PhD, years 3 and 4, will be carried out in Singapore. In this phase the molecular basis of the interactions of PIP2 with actin-binding proteins and actin will be studied in vitro. Proteins that were identified in the first phase as potential PIP2-controlled actin regulators will be characterized in biochemical assays to determine these relationships. PIP2-protein complexes will be subjected to protein crystallographic studies in order to unravel the specificity of these proteins for PIP2 over PIP3 or inositol 1,4,5-trisphosphate. Bob Robinson is a PI at IMCB specializing in the structural basis of cell movement.
http://www.imcb.a-star.e.sg/research/research_group/
This project is intended to enhance our understanding of the balance between cell proliferation, cell growth and apoptosis through inositol signalling and PIP2 initiated movement. These questions are particularly pertinent to tumour growth and metastasis. To apply or to obtain more information on this PhD opportunity, please contact Pete or Bob directly:
Nick Leslie
(2) Curing Cancer with Computers: virtual drug design from the inhibition of growth factor signalling to translation
Supervisors: Chandra Verma (Bioinformatics Institute, Singapore) and Ruth Brenk (College of Life Sciences, University of Dundee)
Introduction: Translation in eukaryotic systems begins with the recognition/binding of 5’ mRNA by the eukaryotic initiation factor 4E (eIF4E)1,2. eIF4G then binds eIF4E thereby enabling the formation of a larger translational assembly which then anchors to the mRNA. Formation of this complex is crucial for recruiting the ribosome to the initiation codon and subsequent translation. Translation is suppressed when 4E binding protein (4E-BP) binds to the same site as eIF4G, preventing the formation of the translation initiation complex (see figure below). This pathway is of great medical interest as translational control appears to be a major route of action of rapamycin the immunosuppressive drug and now a promising anti-cancer agent. mTOR, the kinase inhibited by rapamycin, phosphorylates the BP1 proteins and blocks their binding to eIF4E, thus allowing eIF4E to bind eIF4G and initiate translation of capped mRNA's. The BP1 and eIF4G proteins share a common peptide motif that binds to the peptide binding pocket on eIF4e.
Project: The project will characterize the interactions of the mRNA cap and the 4E-BP in detail using simulation and bioinformatics tools. This will then be used to design ligands/inhibitory-peptides for both pockets. High resolution crystal structures3 and assays for measuring the interactions are available in the laboratory of Prof Sir David Lane. The project will complement efforts in the Lane lab at optimizing protein aptamers for eIF4E inhibition4 that can be used as proof of concept in genetic animal model systems for the anti-tumour affect of eIF4E inhibition.
1. Richter JD, Sonenberg N (2005) Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature. 433:477-80.
a. Curing Cancer with Computers: virtual drug design from the inhibition of growth factor signalling to translation
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2. Gingras AC, Raught B, Sonenberg N. (1999) eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem.68:913-63.
3. Quiocho FA, Hu G, Gershon PD. (2000) Structural basis of mRNA cap recognition by proteins. Curr Opin Struct Biol. 10:78-86
4. Herbert TP, Fahraeus R, Prescott A, Lane DP, Proud CG (2000) Rapid induction of apoptosis mediated by peptides that bind initiation factor eIF4E. Curr Biol. 10:793-6.
(3) Defects in the skin barrier and their role in eczema and psoriasis
Supervisors: E. Birgitte Lane (Institute of Medical Biology, Singapore) and W.H. Irwin McLean (Division of Molecular Medicine, University of Dundee)
This project is aimed at identifying the cause of one of the most common groups of diseases in the developed world. Atopic disease (including atopic dermatitis (eczema), allergy and asthma) is becoming increasingly common (Holgate 1999) and now affects ~20% of the population in the developed world. Predisposition to atopic disease is highly heritable (Van Eerdewegh et al. 2002). Similarly, psoriasis affects about 4% of population and has as strong genetic component. Attempts to identify the genes involved have mostly focused on immunological mechanisms, although a defective epithelial barrier has been suggested (Cookson and Moffatt 2002). Both atopic dermatitis and psoriasis show genetic linkage to the “epidermal differentiation locus” on chromosome 1q21, where many genes encoding epidermal proteins are found. One of these is filaggrin (FLG), a key protein that facilitates terminal differentiation of the epidermis and also formation of the skin barrier to water loss – an extremely important function of the epidermis.
Prof Birgit Lane (Singapore) and Prof Irwin McLean (Dundee) have collaborated for many years in studies of genetic skin disorders. In a recent important breakthrough, Prof McLean’s group in Dundee showed that two independent loss-of-function genetic variants in the gene for filaggrin (FLG) are the cause of ichthyosis vulgaris, the most common inherited disorder of keratinisation (Smith et al., Nature Genetics 2006). Because many ichthyosis patients also have eczema, the group also looked at these filaggrin variants in atopic disease (eczema) and showed that filaggrin mutations are also very strong predisposing factors for eczema (Palmer et al. 2006). There was also a highly significant association with the subtype of asthma that occurs in association with atopic dermatitis (eczema). This work has established a key role for impaired skin barrier function in the development of atopic disease. This project will extend these studies into other clinically important areas, building on synergistic resources between Singapore and Dundee.
The two FLG variants, R510X and 2282del4, are carried by ~9% of people of European origin, but it is not yet known whether these same variants are important in Asian populations. Singaporean patients with ichthyosis vulgaris will therefore also be screened initially for filaggrin mutations. Other closely-related genes may also be involved in these and related disorders. Filaggrin is one of a cluster of 7 large “fused S100” genes that lie within the 1q21 locus (http://genome.ucsc.edu). The other genes are filaggrin-2 (FLG2), trichohyalin (THH), trichohyalin-like 1 (THHL1), cornulin (CRNN), repetin (no gene symbol yet assigned) and hornerin (HRNR). All these proteins are involved in terminal differentiation of the epidermis and skin barrier function. About 40% of the UK paediatric atopic dermatitis patients do not carry the prevalent filaggrin mutations and may have defects in the related fused S100 genes. Pilot studies on UK patients suggest that the filaggrin variants linked to atopic dermatitis may be in inverse phase linkage with psoriasis, as individuals generally develop either atopic dermatitis or psoriasis and seldom, if ever, develop both. One interpretation is that filaggrin variants lead to atopic dermatitis and variants in a neighbouring gene cause psoriasis. Thus, the remaining fused S100 proteins are prime candidates for psoriasis.
Skin and DNA samples from patients with these disorders will be collected for this study in collaboration with Dr Jean Ho of the National Skin Centre. Biopsies from UK and Singapore patients with atopic dermatitits and psoriasis showing no filaggrin mutations will be carefully analysed for any changes in expression of other fused S100 proteins by immunohistochemistry, immunoblotting and quantitative RT-PCR. Prof McLean’s lab have developed long-range PCR methods for analysis of the filaggrin gene (Smith et al. 2006) which are being adapted for mutation detection strategies for the remaining 6 genes. Also, no antibodies currently exist against the THHL1 protein and limited reagents are available to the others; there is a particular need for reagents against additional epitopes on filaggin-2, cornulin, repetin and hornerin. Using the expertise for antibody generation in Prof Lane’s lab antibodies will be raised against the six proteins and used for immunohistochemistry and protein biochemistry analysis of biopsies. This will lead to identification of target genes for analysis, and to identification of further predisposing genes for atopic dermatitis, psoriasis and other diseases precipitated by skin barrier defects. Depending on the nature of any fused S100 variants identified, expression studies in organotypic keratinocyte cultures will be performed to assess the functional consequences of these genetic defects, which may lead to development of therapeutic strategies for these widespread disorders.
References:
Cookson WO, Moffatt MF (2002) The genetics of atopic dermatitis. Curr Opin Allergy Clin Immunol 2:383-387
Holgate ST (1999) The epidemic of allergy and asthma. Nature 402:B2-4
Palmer CNA, Irvine AD, Terron-Kwiatkowski A, Zhao Y, Liao H, Lee SP, Goudie DR, Sandilands A, Campbell LE, Smith FJD, O'Regan GM, Watson RM, Cecil JE, Bale SJ, Compton JG, DiGiovanna JJ, Fleckman P, Lewis-Jones S, Arseculeratne G, Sergeant A, Munro CS, El Houate B, McElreavey K, Halkjaer LB, H. B, Mukhopadhyay S, McLean WHI (2006) Common loss-of-function variants of the epithelial barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nature Genetics, in press.
Smith FJD, Irvine AD, Terron-Kwiatkowski A, Sandilands A, Campbell LE, Zhao Y, Liao H, Evans AT, Goudie DR, Lewis-Jones S, Arseculeratne G, Munro CS, Sergeant A, O'Regan G, Bale SJ, Compton JG, Digiovanna JJ, Presland RB, Fleckman P, McLean WHI (2006) Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris. Nature Genetics (e-pub ahead of print).