Dr Michelle Dunstone - Researcher Profile

Michelle Dunstone

Address

Department of Microbiology
Faculty of Medicine, Nursing & Health Sciences, Clayton

Contact Details

Tel: +61 3 990 29269

Email: Michelle.Dunstone@monash.edu


Biography

X-ray vision reveals protein gateway to cell life and death

Understanding the life and death of cells is crucial information for scientists around the world working to treat illness and disease. Through the process of X-ray crystallography, Dr Michelle Dunstone has been able to make an important contribution to this field of science. She has been able to capture images that allow scientists to visualise the behaviour of “hole punching” proteins, called pore forming toxins. These toxins are used by our immune system to combat disease and also used by bacteria to cause disease.

As a molecular biologist, Michelle has been studying the structure of several toxic proteins that create holes in the cell surface, resulting in cell death. She has captured world acclaim by imaging how these proteins work through X-ray crystallography.

The success of this technique relies largely on the skill of the researcher in preparing samples – purifying and crystallising proteins. This is an important prerequisite to using advanced imaging technology to visualise the protein molecules and to identify their biological purpose.

This understanding will allow future researchers to consider how these proteins can be used or manipulated to achieve desired outcomes, such as promoting the growth of beneficial cells or killing disease.

Michelle says the proteins she studies create pores in the cell membrane and are used by organisms in all kingdoms of life, from bacteria to humans. They come in two varieties. There are life-givers in humans, allowing the immune system to kill disease, and there are life-takers used by disease-causing bacteria.

Her research targets a class of proteins that behaves like toxins, called MACPF toxin. They are used by the human immune system to kill invading bacteria, for example. But bacteria also use them as part of their infection strategy.

Michelle has been able to crystallise and image the free-floating form of several MACPF toxin specimens. It turns out these proteins used by the immune system are similar in their three-dimensional structure to those used by bacteria, which indicates they may all use the same mechanism to punch holes. By studying these structures we can come to understand how they do it.

“When the proteins come across a target cell membrane with the right composition then these toxins change shape and bind to the membrane,” Michelle says. “As more individual proteins do this, they form a doughnut-shaped ring in the membrane, which triggers the formation of a pore that punctures the membrane.”

When she is not at the synchrotron bombarding her crystallised samples with high-energy beams, Michelle has another passion. Fascinated by the antiquity of the proteins she images, she trawls the world’s genome sequence database, trying to understand the evolution of the hole-punching proteins.

“These proteins are present in everything from bacteria all the way through to humans. It seems that an ancestor may have existed in the organism that gave rise to both modern bacteria and mammals,” she says. “That places the origin of this molecule deep in the past, before there was divergence among bacteria. So it was present in living cells very early in evolution.”

She calls this activity “genome gazing” along the evolutionary tree of life. She keeps finding new members of the toxin family in all sorts of different organisms. In the process, she has started to see her proteins in a different light.

“It is not always obvious that the organism needs the protein for a pore-forming activity,” she says. “That implies they may have some other role. What we would postulate is that a common activity across the family is that they can recognise lipid membranes – see them chemically – and insert into the recognised subtype. But we need to identify more family members before we can assign a universal role.”

Keywords

3-D protein structures, X-ray crystallography, imaging biological molecules, cell-lysing toxins, biochemistry, microbiology, X-ray crystallography, toxin biology

Qualifications

PHD
Institution: The University of Melbourne
Year awarded: 2006
BACHELOR OF SCIENCE (HONS)
Institution: Monash University
Year awarded: 1997

Publications

Book Chapters

Dunstone, M.A., Whisstock, J.C., 2011, Crystallography of serpins and serpin complexes, in Methods in Enzymology, Volume 501: Serpin Structure and Evolution, eds James C Whisstock and Phillip I Bird, Academic Press, USA, pp. 63-87.

Journal Articles

Dunstone, M.A., Tweten, R.K., 2012, Packing a punch: the mechanism of pore formation by cholesterol dependent cytolysins and membrane attack complex/perforin-like proteins, Current Opinion in Structural Biology [P], vol 22, issue 3, Elsevier Ltd. * Current Opinion Journals, UK, pp. 342-349.

D'Angelo, M., Dunstone, M.A., Whisstock, J.C., Trapani, J., Bird, P.I., 2012, Perforin evolved from a gene duplication of MPEG1, followed by a complex pattern of gene gain and loss within Euteleostomi, BMC Evolutionary Biology [P], vol 12, issue Art. ID: 59, BioMed Central Ltd, UK, pp. 1-12.

Reboul, C.F., Mahmood, K., Whisstock, J.C., Dunstone, M.A., 2012, Predicting giant transmembrane ß-barrel architecture, Bioinformatics [P], vol 28, issue 10, Oxford University Press, UK, pp. 1299-1302.

Hatfaludi, T.Z., Al-Hasani, K., Gong, L., Boyce, J.D., Ford, M., Wilkie, I.W., Quinsey, N.S., Dunstone, M.A., Hoke, D.E., Adler, B., 2012, Screening of 71 P. multocida proteins for protective efficacy in a fowl cholera infection model and characterization of the protective antigen PlpE, PLoS ONE [P], vol 7, issue 7 (Art. ID: e39973), Public Library of Science, USA, pp. 1-11.

Theodossis, A., Guillonneau, C., Welland, A.D., Ely, L.K., Clements, C.S., Williamson, N.A., Webb, A.I., Wilce, J.A., Mulder, R.J., Dunstone, M.A., Doherty, P.C., McCluskey, J., Purcell, A.W., Turner, S.J., Rossjohn, J., 2010, Constraints within major histocompatibility complex class I restricted peptides: presentation and consequences for T-cell recognition, Proceedings Of The National Academy Of Sciences Of The United States Of America [P], vol 107, issue 12, National Academy of Sciences, USA, pp. 5534-5539.

Voskoboinik, I., Dunstone, M.A., Baran, K., Whisstock, J., Trapani, J.A., 2010, Perforin: structure, function, and role in human immunopathology, Immunological Reviews [P], vol 235, issue 1, Wiley-Blackwell Publishing Inc, USA, pp. 35-54.

Law, R.H.P., Lukoyanova, N., Voskoboinik, I., Caradoc-Davies, T.T., Baran, K., Dunstone, M.A., D'Angelo, M., Orlova, E.V., Coulibaly, F.J., Verschoor, S., Browne, K.A., Ciccone, A., Kuiper, M.J., Bird, P.I., Trapani, J.A., Saibil, H.R., Whisstock, J.C., 2010, The structural basis for membrane binding and pore formation by lymphocyte perforin, Nature [P], vol 468, issue 7322, Nature Publishing Group, UK, pp. 447-451.

Kondos, S., Hatfaludi, T.Z., Voskoboinik, I., Trapani, J.A., Law, R.H., Whisstock, J.C., Dunstone, M.A., 2010, The structure and function of mammalian membrane-attack complex/perforin-like proteins, Tissue Antigens [P], vol 76, issue 5, Wiley-Blackwell Publishing Inc, USA, pp. 341-351.

Newton, H.J., Pearson, J.S., Badea, L., Kelly, M., Lucas, M., Holloway, G., Wagstaff, K.M., Dunstone, M.A., Sloan, J., Whisstock, J.C., Kaper, J.B., Robins-Browne, R.M., Jans, D.A., Frankel, G., Phillips, A.D., Coulson, B.S., Hartland, E.L., 2010, The type III effectors NleE and NleB from enteropathogenic E. coli and OspZ from Shigella block nuclear translocation of NF-kappaB p65, Plos Pathogens [P], vol 6, issue 5 (e1000898), Public Library of Science, USA, pp. 1-16.

Beddoe, T.C., Chen, Z., Clements, C.S., Ely, L.K., Bushell, S.R., Vivian, J.P., Kjer-Nielsen, L., Pang, S.S., Dunstone, M.A., Liu, Y.C., Macdonald, W.A., Perugini, M.A., Wilce, M.C.J., Burrows, S.R., Purcell, A.W., Tiganis, T., Bottomley, S.P., McCluskey, J., Rossjohn, J., 2009, Antigen ligation triggers a conformational change within the constant domain of the alphabeta T cell receptor, Immunity [P], vol 30, issue 6, Cell Press, USA, pp. 777-788.

Chia, J., Yeo, K., Whisstock, J., Dunstone, M.A., Trapani, J.A., Voskoboinik, I., 2009, Temperature sensitivity of human perforin mutants unmasks subtotal loss of cytotoxicity, delayed FHL, and a predisposition to cancer, Proceedings Of The National Academy Of Sciences O..., vol 106, issue 24, National Academy of Sciences, USA, pp. 9809-9814.

Baran, K., Dunstone, M.A., Chia, J., Ciccone, A., Browne, K.A., Clarke, C.J.P., Lukoyanova, N., Saibil, H., Whisstock, J., Voskoboinik, I., Trapani, J.A., 2009, The molecular basis for perforin oligomerization and transmembrane pore assembly, Immunity [P], vol 30, issue 5, Cell Press, USA, pp. 684-695.

Hatfaludi, T.Z., Al-Hasani, K., Dunstone, M.A., Boyce, J.D., Adler, B., 2008, Characterization of TolC efflux pump proteins from Pasteurella multocida, Antimicrobial Agents and Chemotherapy, vol 52, issue 11, American Society for Microbiology, United States, pp. 4166-4171.

Sansom, F.M., Riedmaier, P., Newton, H., Dunstone, M.A., Muller, C.E., Stephan, H., Byres, E., Beddoe, T.C., Rossjohn, J., Cowan, P.J., d'Apice, A.J.F., Robson, S.C., Hartland, E.L., 2008, Enzymatic properties of an ecto-nucleoside triphosphate diphosphohydrolase from Legionella pneumophila: Substrate specificity and requirement for virulence, Journal of Biological Chemistry, vol 283, issue 19, American Society for Biochemistry and Molecular Biology, Inc., United States, pp. 12909-12918.

La Gruta, N.L., Thomas, P.G., Webb, A.I., Dunstone, M.A., Cukalac, T., Doherty, P.C., Purcell, A.W., Rossjohn, J., Turner, S.J., 2008, Epitope-specific TCRbeta repertoire diversity imparts no functional advantage on the CD8+ T cell response to cognate viral peptides, Proceedings of the National Academy of Sciences, vol 105, issue 6, National Academy of Sciences, United States, pp. 2034-2039.

Butler, N.S., Theodossis, A., Webb, A.I., Nastovska, R., Ramarathinam, S.H., Dunstone, M.A., Rossjohn, J., Purcell, A.W., Perlman, S., 2008, Prevention of cytotoxic T cell escape using a heteroclitic subdominant viral T cell determinant, PL o S Pathogens (online), vol 4, issue 10, Public Library of Science, United States, pp. 1-13.

Butler, N.S., Theodossis, A., Webb, A.I., Dunstone, M.A., Nastovska, R., Ramarathinam, S.H., Rossjohn, J., Purcell, A.W., Perlman, S., 2008, Structural and biological basis of CTL escape in coronavirus-infected mice, Journal of Immunology, vol 180, issue 6, American Association of Immunologists, United States, pp. 3926-3937.

Rosado, C.J., Kondos, S., Bull, T.E., Kuiper, M., Law, R.H.P., Buckle, A.M., Voskoboinik, I., Bird, P.I., Trapani, J.A., Whisstock, J., Dunstone, M.A., 2008, The MACPF/CDC family of pore-forming toxins, Cellular Microbiology, vol 10, issue 9, Wiley-Blackwell Publishing Ltd., United Kingdom, pp. 1765-1774.

Rosado, C.J., Buckle, A.M., Law, R.H.P., Butcher, R.E., Kan, W., Bird, C.H., Ung, K., Browne, K.A., Baran, K., Bashtannyk-Puhalovich, T.A., Faux, N.G., Wong, W., Porter, C.J., Pike, R.N., Ellisdon, A.M., Pearce, M.C., Bottomley, S.P., Emsley, J., Smith, A.I., Rossjohn, J., Hartland, E.L., Voskoboinik, I., Trapani, J.A., Bird, P.I., Dunstone, M.A., Whisstock, J., 2007, A common fold mediates vertebrate defense and bacterial attack, Science, vol 317, issue 5844, American Association of Advancement in Science, Washington, DC, USA, pp. 1548-1551.

Clements, C.S., Dunstone, M.A., Macdonald, W.A., McCluskey, J., Rossjohn, J., 2006, Specificity on a knife-edge: The alphabeta T cell receptor, Current Opinion in Structural Biology, vol 16, issue 6, Current Biology Ltd, London UK, pp. 787-795.

Clements, C.S., Kjer-Nielsen, L., Kostenko, L., Hoare, H.L., Dunstone, M.A., Moses, E.K., Freed, K., Brooks, A.G., Rossjohn, J., McCluskey, J., 2005, Crystal structure of HLA-G: A nonclassical MHC class I molecule expressed at the fetal-maternal interface, Proceedings of the National Academy of Sciences of the United States of America, vol 102, issue 9, National Academy of Sciences, Washington USA, pp. 3360-3365.

Turner, S.J., Kedzierska, K., Komodromou, H., La Gruta, N.L., Dunstone, M.A., Webb, A.I., Webby, R., Walden, H., Xie, W., McCluskey, J., Purcell, A.W., Rossjohn, J., Doherty, P.C., 2005, Lack of prominent peptide-major histocompatibility complex features limits repertoire diversity in virus-specific CD8+ T cell populations, Nature Immunology, vol 6, issue 4, Nature Publishing Group, New York USA, pp. 382-389.

Webb, A.I., Dunstone, M.A., Williamson, N.A., Price, J.D., de Kauwe, A., Chen, W., Oakley, A.J., Perlmutter, P., McCluskey, J., Aguilar, M., Rossjohn, J., Purcell, A.W., 2005, T cell determinants incorporating beta-amino acid residues are protease resistant and remain immunogenic in vivo, Journal of Immunology, vol 175, issue 6, American Association of Immunologists, Bethesda USA, pp. 3810-3818.

Kjer-Nielsen, L., Dunstone, M.A., Kostenko, L., Ely, L.K., Beddoe, T.C., Mifsud, N.A., Purcell, A.W., Brooks, A.G., McCluskey, J., Rossjohn, J., 2004, Crystal structure of the human T cell receptor CD3 heterodimer complexed to the therapeutic mAb OKT3, Proceeedings of the National Academy of Sciences of the United Stated of America, vol 101, issue 20, National Academy of Sciences, Washington USA, pp. 7675-7680.

Webb, A.I., Dunstone, M.A., Chen, W., Aguilar, M.I., Chen, Q., Jackson, H., Chang, L., Kjer-Nielsen, L., Beddoe, T.C., McCluskey, J., Rossjohn, J., Purcell, A.W., 2004, Functional and structural characteristics of NY-ESO-1-related HLA A2-restricted epitopes and the design of a novel immunogenic analogue, The Journal of Biological Chemistry, vol 279, issue 22, American Society for Biochemistry and Molecular Biology Inc., Bethesda USA, pp. 23438-23446.

Dunstone, M.A., Kjer-Nielsen, L., Kostenko, L., Purcell, A.W., Brooks, A.G., Rossjohn, J., McCluskey, J., 2004, The production and purification of the human T-cell receptors, the CD3and CD3heterodimers: Complex formation and crystallization with OKT3, a therapeutic monoclonal antibody, Acta Crystallographica Section D - Biological Crystallography, vol 60, issue 8, Blackwell Munksgaard, Copenhagen Denmark, pp. 1425-1428.

Webb, A.I., Borg, N.A., Dunstone, M.A., Kjer-Nielsen, L., Beddoe, T.C., McCluskey, J., Carbone, F.R., Bottomley, S.P., Aguilar, M.I., Purcell, A.W., Rossjohn, J., 2004, The structure of H-2Kb and Kbm8 complexed to a herpes simplex virus determinant: evidence for a conformational switch that governs T cell repertoire selection and viral resistance, The Journal of Immunology, vol 173, issue 1, American Association of Immunologists, Bethesda USA, pp. 402-409.

Dunstone, M.A., Dai, W., Whisstock, J.C., Rossjohn, J., Pike, R.N., Feil, S.C., Le Bonniec, B., Parker, M.W., Bottomley, S.P., 2000, Cleaved antitrypsin polymers at atominc resolution, Protein Science, vol 9, Cambridge University Press, New York NY USA, pp. 417-420.

Conference Proceedings

Webb, A.I., Dunstone, M.A., Aguilar, M.I., Rossjohn, J., Purcell, A.W., 2005, Engineering protease resistance into T cell epitopes improves bioavailability of vaccine components, Tissue Antigens, 29 November 2005 - 3rd December 2005, Blackwell Publishing, Oxford England, p. 586.

La Gruta, N.L., Webb, A.I., Dunstone, M.A., Doherty, P.C., Purcell, A.W., Rossjohn, J., Turner, S.J., 2005, The relationship between peptide-MHC class I structures and the TCR repertoire diversity of influenza a virus specific CD8+T cells, Tissue Antigens, 29 November 2005 - 3rd December 2005, Blackwell Publishing, Oxford England, pp. 570-571.

Prescott, M., Bateson, M., Boyle, G.M., Lourbakos, A., Dunstone, M.A., Nagley, P., Devenish, R.J., 1998, Protein engineering of subunits of the Fa stalk sector of yeast mitochondrial ATP synthase, Proceedings, 23rd Annual Lorne Conference on Protein Structure and Function, Lorne 8-12 February 1998, Lorne Protein Conference Inc, Melbourne Vic Australia, p. C-94.

Dunstone, M.A., Prescott, M., Nagley, P., Devenish, R.J., 1998, Targeting GFP to the yeast mitochondrion as a functional fusion with an ATP synthase subunit, Proceedings, 23rd Annual Lorne Conference on Protein Structure and Function, Lorne 8-12 February 1998, Lorne Protein Conference Inc, Melbourne Vic Australia, p. C-57.

Other

Rossjohn, J., Beddoe, T.C., Dunstone, M.A., Ely, L.K., McCluskey, J., Kjer-Nielsen, L., Kostenko, L., Purcell, A.W., 2004, Crystal structure of CD3ϵgammaad;/OKT3 complex, Australia.

Postgraduate Research Supervisions

Current Supervision

Program of Study:
(DOCTORATE BY RESEARCH).
Thesis Title:
Structural and Biophysical studies of MACPF pore-forming toxins, pleurotolysin.
Supervisors:
Dunstone, M (Main), Law, H (Associate), Whisstock, J (Associate).