Protein Discovery Centre

Jeff Gorman Staff
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Lab Head: Professor Jeff Gorman

The QIMR Protein Discovery Centre (QPDC) was established at QIMR in early 2006. The QPDC aims to discover the identities of proteins involved in and/or affected by physiological and disease processes and the ways in which these proteins function and interact. This may involve a range of techniques such as sequencing proteins isolated from polyacrylamide gels through to identifying the ways in which the proteins are modified (post-translational modifications) in order to affect their specific functions. Our expertise is applicable to all biological systems in which proteins participate. Projects of particular interest within the centre involve viruses that cause serious respiratory diseases of children and signal-activated transcription factors involved in cancer progression. However, the generic nature of our expertise allows us to engage in collaborations with colleagues who seek integration of our expertise into their studies of other biological systems.

The PDC is equipped with an array of the latest high-performance mass spectrometers which are complemented with a range of other state-of-the-art ancillary equipment required for contemporary protein chemistry and proteomics. This platform is broadly applicable to defining the chemical features of purified proteins, interactions between proteins at the molecular and cellular levels and the dynamics of the protein repertoires of cells in response to disease states and other stimuli. The combination of expertise and infrastructure within the QPDC is broadly applicable to:

o Probing protein folding and interactions
o Mapping of protein networks and pathways in cells by the analysis of post-translational modifications
o Characterisation of the proteomes of organisms and organelles

Our work has the potential to produce important leads for development of therapeutic agents to treat viral infections and other important medical conditions.

Projects

Interactions and structures of proteins in assembled virus particles.
Viruses consist of nucleic acid genomes packaged within protein-containing coats. Coat proteins function to ensure efficient attachment to target host cells and transmission of the viral genomes into these cells in which the viruses replicate. Proteins also contribute to structural integrity of the viral particles and the viral genome as well as functioning in viral replication. The process of assembly (morphogenesis) of the virus particles, stabilities of the assembled particles and functional activities of the viral proteins are governed by protein-protein interactions.

This project aims to develop and apply methods to better understand the interactions between viral proteins during morphogenesis and in the assembled particles.

The principal viruses to be studied are from the Paramyxoviridae family. This family contains many important pathogens of humans and animals, including mumps, measles, parainfluenza viruses, respiratory syncytial virus (RSV) and Newcastle disease virus (NDV) of poultry.

The innocuous avirulent Queensland or V4 strain of NDV is the principal model virus for these studies. This avirulent strain does not cause disease in any species and can be grown and purified in sufficient quantities to enable detailed biochemical characterisation. Our group has considerable experience and expertise in preparation and analysis of proteins from this virus. Importantly, there are precedents for using avian viruses as models to understand and control related viruses that affect humans, eg. influenza. Thus, it is possible that any findings may be used in development of agents and strategies to control human pathogens and the economically significant NDV.

Like all members of the Paramyxoviridae, NDV has major structural coat proteins for attachment (haemagglutinin-neuraminidase or HN) to host cell membrane receptors and fusion (F) with host cell membranes. Other viral proteins are important for maintenance of the integrity of the membrane coat of the virus (membrane protein M) and the viral nucleocapsid (nucleocapsid protein NP) through association with the negative strand RNA of the genome.

Three proteomics methods are being developed for the study of interactions between these proteins in conjunction with mass spectrometric analysis. The first method is chemical crosslinking of the viral proteins. Viruses can be exposed to different crosslinkers under different conditions. Crosslinked complexes are isolated and digested with proteolytic enzymes in the presence of H218O. Crosslinked peptides are identified by virtue of their crosslink-specific isotope profiles and sequenced by tandem mass spectrometry. This will allow identification of the points of close contact (interaction) between the viral proteins and the impact of environmental factors and functional events on these interactions. Thus, an image of the network and dynamics of interactions between viral proteins in assembled virus particles will be established at a chemical level.

Another method for studying these interactions is deuterium exchange. The amide protons of peptide bonds undergo rapid exchange with water from the solvent unless solvent access is restricted by close contact with a closely juxtaposed piece of polypeptide such as an interacting protein. Mass spectrometry can be used to determine the kinetics of proton exchange using D2O as a solvent and hence probe the stabilities of interactions between regions of proteins. Data from this method will further refine the image of protein interaction networks in paramyxoviral particles obtained from chemical crosslinking studies.

The third method involves mapping the sensitivity of amino acid side chains on viral proteins to modification by hydroxyl radicals. In this method, side chains are assessed for shielding from modification due to sequestration into the internal environment of the protein or an interaction site with a partner protein.

Viral proteins undergo structural changes and changes in their interactions in response to various stimuli, such as attachment to host cell receptors. These methods are also being used to probe the dynamics of change in paramyxoviral protein structures and interactions.

Interactions of viral proteins with host cell proteins during infection and assembly.
There is a growing appreciation that viral proteins can interact in a variety of ways with cellular proteins during the viral replicative cycle. The interaction of viral attachment proteins with host cell receptors to initiate infection is well known, but there is evidence of interaction at other steps in the cycle, such as during RNA synthesis, viral assembly, and antagonism of host cell defences. Frequently, these interactions are essential for efficient viral replication and can have a major impact on pathogenesis. Relatively little is known about this area, in part because it has not been amenable to investigation by traditional biochemical approaches. However, application of improved techniques of mass spectrometry and proteomic-related techniques have enabled the analysis of proteins present in low quantities or in complex mixtures, and provide new impetus for examining the effect of viral infection on the host proteome and possible physical interaction between viral and cellular proteins.

This project aims to identify cellular proteins that interact with proteins of replicating RSV and to define the structural details and biological significance of these interactions. Similar analytical strategies to those described above to define interactions of viral proteins in assembled virus particles are used to define the interactions with cellular proteins during replication. The global impact of virus infection on the host proteome will also be investigated.

Respiratory syncytial virus (RSV) is the major focus of these studies because it is a serious respiratory pathogen for infants, young children, immunocompromised individuals and the elderly. An important aspect of this project is access to expertise in RSV molecular virology and reverse genetics through collaboration with expert virologists throughout the world.

Our collaborators are using recombinant plasmids and viruses to produce RSV proteins in transfected and infected cells, respectively. These proteins have specific sequence tags to enable affinity capture of complexes with cellular proteins. Our role is to identify the proteins in these complexes and their specific sites of interaction using mass spectrometry.

Regulation of signal-activated transcription factors by post-translational modifications and protein-protein interactions.
Transcription factors with a general basic helix-loop-helix/Per Arnt-Sim (bHLH/PAS) domain architecture act within the cell nucleus to coordinate transcription of specific genes. Heterodimerisation with the aryl hydrocarbon receptor nuclear translocator (Arnt) is essential to form DNA binding complexes. Other proteins are recruited to form the active transcription complexes. Ligand binding or other cellular signals can modulate nuclear translocation of the transcription factors and their ability to form active complexes.

In collaboration with Dr Murray Whitelaw's group from the University of Adelaide, we have demonstrated that the transcriptional potency of hypoxia-inducible factor (HIF)-transcriptional complexes is inhibited by post-translational hydroxylation of a specific asparagine residue of HIF. Oxygen serves as the substrate for the hydroxylase (Factor that Inhibits HIF, or FIH) which hydroxylates the regulatory asparagine of HIF. This normoxic hydroxylation inhibits the recruitment of the transcriptional co-activator p300/CBP to the DNA-bound complex, thereby repressing transcription. The purpose of this regulation is to prevent transcription of genes whose products are only required to respond to low cellular oxygen levels (hypoxia).

Other uncharacterised proteins are also recruited to the transcription complexes to influence the transcriptional activity of HIF. One of our current aims is to identify these unknown proteins and to determine their functional roles in the hypoxic regulation of transcription.

Phosphorylation is believed to be the impetus for translocation of another bHLH-PAS transcription factor, the ligand-activated xenobiotic receptor, otherwise known as the Dioxin receptor (DR), in response to the binding of ligands such as Dioxin. DR is also constitutively phosphorylated. We are working to differentiate between constitutive and ligand-activated phosphorylation and to identify other regulatory post-translational modifications of DR.

Characterisation of proteins secreted (secretomes) by parasitic organisms
Parasites secrete proteins at the sites of attachment to their hosts to facilitate feeding from the host and to block host defences. Some of the secreted proteins may achieve beneficial effects for the parasite by blocking blood clotting and others may dampen the host immune response. Some of these properties are similar to activities that are sometimes desirable for therapeutic intervention to treat human diseases, such as thrombosis or allergic responses. Thus, we are collaborating with Dr Alex Loukas and Dr James McCarthy of QIMR to characterise proteins secreted by various parasites as a way of initiating the development of therapeutic agents.

Other parasites can secrete compounds that actually lead to highly detrimental effects on their hosts. One such parasite is Opisthorcis viverini which causes bile duct cancer of infected individuals in South East Asia. Various lines of evidence indicate that the cancer causing agent is a protein. Consequently, we are collaborating with Dr Alex Loukas, in an endeavour to identify the cancer causing component of this parasite so as to be able to better understand and treat the disease it causes.

Characterisation of the proteomes of neurosecretory granules
Coordinated secretion of neurotransmitters from neural cells involves a complex series of molecular events. For example neurosecretory granules have to be able to dock at specific intracellular sites on membranes within nerve cells in order to secrete their contents. We are collaborating with Dr Fred Meunier from the University of Queensland to gain insights into the mechanism of interactions between proteins from neurosecretory granules and phosphatidyl inositides and the roles these interactions play in docking of neurosecretory granules with cell membranes.

Interactions of Cpn10 with cellular receptors
Cpn10 has been identified as a protein with potential therapeutic uses for modulating human diseases. Rational exploitation of this small protein in disease modulation will benefit from gaining an understanding of its mode(s) of action. Accordingly the QIMR Protein Discovery Centre is collaborating with CBio as an industry partner to characterise the interactions of Cpn10 with cellular receptors. This project involves 3 streams of research. Chemical crosslinking is being used to identify cellular receptors that Cpn10 exploits to bind to cells. Hhydroxyl radical footprinting is being used to study the structure, folding and interactions of mammalian Cpn10. Finally, we are analysing post-translational modifications of cellular proteins induced by exposure of cells to Cpn10 as a means of identifying cellular pathways activated and/or repressed by the protein.

Facilitation
QPDC also provides access to mass spectrometry infrastructure and expertise to other QIMR scientists on both collaborative and non-collaborative bases. We are also open to proposals from external parties from both academia and industry.

Staff

Labhead:	Professor Jeff Gorman
Post Docs:	Dr Brett Hamilton Dr Marcus Hastie (Visiting Scientist from UQ) Dr Madeleine Headlam Dr Jason Mulvenna Dr Tristan Wallis
Research Assistant:	Ms Una Cuffe
PhD Scholar:	Mr Keyur Dave

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Funding

NHMRC
ARC
QIMR

External Collaborators

Interactions of viral proteins with host cell proteins during infection and assembly - Dr Peter Collins, Respiratory Virus Laboratory, National Institutes of Allergic and Infectious Diseases, National Institutes of Health (USA), Prof Mark Peeples, Department of Pediatrics, The Ohio State University Center for Vaccines and Immunity, Columbus Children's Research Institute, Columbus, Ohio (USA) and Dr Kirsten Spann, Respiratory Virus Research Unit Head, Clinical Medical Virology Centre University of Queensland and Sir Albert Sakzewski Virus Research Centre, Herston, Queensland, Australia

Mechanism of interaction of the RSV attachment protein with cell membranes - Prof Craig Gerard, Department of Pediatrics, Harvard Medical School, Division of Respiratory Diseases, Children's Hospital, Boston MA (USA).

Post-translational regulation of signal-activated transcription factors - A/Prof Murray Whitelaw and Dr Daniel Peet, University of Adelaide, South Australia.

Neurosecretion - Dr Fred Meunier, The University of Queensland, St Lucia, Queensland, Australia.

Interactions of Cpn10 with cell membranes - Dr Dean Naylor, CBio Ltd, Eight Mile Plains, Queensland, Australia and Prof Ross Smith, The University of Queensland, St Lucia, Queensland, Australia.

Key Publications

(since 2000) Gorman, J.J. and Folk, J.E. (1980). Structural Features of Glutamine Substrates for Human Plasma Factor XIIIa (Activated Blood Coagulation Factor XIII). J. Biol. Chem. 255 419-427.

Gorman, J.J. and Folk, J.E. (1980). Transglutaminase Amine Substrates for Photochemical Labelling and Cleavable Cross-Linking of Proteins. J. Biol. Chem. 255: 1175-1180.

Moseley, J.M., Findlay, D.M., Martin, T.J. and Gorman, J.J. (1982). Covalent Cross-Linking of a Photoactive Derivative of Calcitonin to Human Breast Cancer Cell Receptors. J. Biol. Chem. 257: 5846-5851.

Gorman, J.J., Nestorowicz, A., Corino, G.L., Mitchell, S.J. and Selleck, P.W. (1988). Characterisation of the Sites of Proteolytic Activation of Newcastle Disease Virus Membrane Glycoprotein Precursors. J. Biol. Chem. 263: 12522-12531.

Gorman, J.J., Corino, G.L. and Selleck, P.W. (1990). Comparison of the Positions and Efficiency of Cleavage-Activation of Fusion Protein Precursors of Virulent and Avirulent Strains of Newcastle Disease Virus: Insights into the Specificities of Activating Proteases. Virology. 177: 339-351.

Gorman, J. J., Ferguson, B. L. Speelman, D. and Mills, J. (1997) Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein Science. 6, 1308-1315.

Pitt, J. J., Da Silva, E. and Gorman, J. J. (2000) Determination of the Disulfide Bond Arrangement of Newcastle Disease Virus Hemagglutinin Neuraminidase: Correlation with a -Sheet propeller Structural Fold Predicted For Paramyxoviridae Attachment Proteins. J. Biol. Chem. 275, 6469-6478.

Chen, L., Gorman, J. J., McKimm-Breshkin, J., Lawrence, L. J., Tulloch, P. A., Smith, B. J., Colman, P. M. and lawrence, M. C. (2001) Structure of the Fusion Glycoprotein of Newcastle Disease Virus. Structure. 9, 255-266.

Gorman, J. J., McKimm-Breshkin, J., Norton, R. S. and Barnham K. J. (2001) Antiviral Activity and Structural Features of the Non-Glycosylated Central Subdomain of Human Respiratory Virus Attachment (G) Glycoprotein. J. Biol. Chem. 276, 38988-38994.

Lando, D., Peet, D. J., Whelan, D. A., Gorman, J. J. and Whitelaw, M. L. (2002) Asn Hydroxylation of the HIF Transactivation Domain: A Hypoxic Switch. Science. 295, 858-861.

Gorman, J.J., Wallis, T. P. and Pitt, J. J. (2002) Determination of Disulfide Bond Arrangements of Proteins by Mass Spectrometry. Mass Spec. Rev. 21, 183-216.

Lando, D., Peet, D. J., Gorman, J. J., Whelan, D. A., Whitelaw, M. L. and Bruick, R. K. (2002) Identification of the asparagine hydroxylase responsible for regulating the transcriptional activity of HIF. Genes Devel. 16, 1466-1471.

Wallis, T. P., Huang, C.-Y., Nimkar, S. B., Young, P. R. and Gorman J. J. (2004) Determination of the Disulfide Bond Arrangement of Dengue Virus NS1 Protein. J. Biol. Chem. 279, 20729-20741.

Purcell, A. W. and Gorman, J. J. (2004) Immunoproteomics: Mass spectrometry based methods to study the targets of the immune response. Molecular and Cellular Proteomics 3, 193-208.

Gorman, J. J., Wallis, T. P., Whelan, D. A., Shaw, J. and Both, G. W. (2005) LH3, a structural "homologue" of the mast adenoviral E1B 55kDa protein is a structural protein of Atadenoviruses. Virol. 342, 159-66.