ISSN 1443-0193 Australian Biochemist The Magazine of the Australian Society for Biochemistry and Molecular Biology Inc. Volume 47 AUGUST 2016 No.2

SHOWCASE ON RESEARCH Protein Misfolding and Proteostasis

THIS ISSUE INCLUDES

Showcase on Research Regular Departments  A Short History of Amyloid  SDS (Students) Page  Molecular Chaperones:  The Cutting Edge Guardians of the Proteome  Off the Beaten Track  When Proteostasis Goes Bad:  Intellectual Property Protein Aggregation in the Cell  Our Sustaining Members  Extracellular Chaperones and  Forthcoming Meetings Proteostasis  Directory

INSIDE ComBio2016 International Speaker Profiles Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 1 ‘OSE’ Fill-in Puzzle

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Australian Biochemist – Editor Chu Kong Liew, Editorial Officer Liana Friedman © 2016 Australian Society for Biochemistry and Molecular Biology Inc. All rights reserved.

Page 2 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 SHOWCASE ON RESEARCH EDITORIAL Molecular Origami: the Importance of Managing Protein Folding In my humble opinion, the most important biological transcription, RNA processing and transport, translation, molecule is the protein. After water, it is by far the most protein folding, protein transport and protein degradation abundant molecule in the human body, making up must be tightly regulated. Together these processes make approximately 20% of our body weight. This means that, up the proteostasis network. on average, a human of 75 kg contains around 15 kg of The proteostasis network regulates proteome protein, which translates to an astounding 1.5 x 1028 protein homeostasis by maintaining the delicate balance between molecules (on par with the estimated number of stars in the production and disposal of proteins. In our second the universe). This protein content is not a static entity, article, Dezerae Cox, Rebecca San Gil, Anthea Rote and it has been estimated that about 400 grams of protein is Heath Ecroyd describe the function of arguably the most synthesised and degraded each day in the human body. important contributors to the proteostasis network, the Making even a single protein is not an easy task; one chaperone proteins. Degradation of proteins by autophagy only has to look at any structure in the protein data or the ubiquitin-proteasome system (UPS) and active bank to appreciate the exquisite beauty and complexity compartmentalisation of misfolded proteins into specific of a natively folded protein structure. Importantly, the regions in the cell (eg. the aggresome) also contribute to biological function of proteins is most often critically proteome quality control. The careful regulation of these dependent on them reaching their folded state. Correct processes is critical for protection against the toxicity protein folding is not always achieved. Single point associated with mutant, misfolded and/or damaged mutations can destabilise, and thus prevent, proper folding proteins associated with human disease. In their of a protein and certain environmental conditions, such as contribution, Mona Radwan, Rebecca Wood, Xiaojing macromolecular crowding, inappropriate ionic strength, Sui and Danny Hatters consider protein aggregation in oxidative stress and extremes of pH and temperature are the cell and its role in the toxicity associated with this known to promote the formation of misfolded states. If left process. Last, while the vast majority of research into unchecked, misfolded proteins can aggregate into insoluble protein misfolding has centered on what happens inside protein deposits. Many disease states are associated with the cell, in our last article, Amy Wyatt explains why the abnormal protein deposits comprised of aggregated proteostasis network is also crucial outside the cell. protein, including an insoluble fibrillar aggregate known The study of protein misfolding is an exciting as amyloid. In our first article, one of the pioneers in this and dynamic field which has moved from original field, Margie Sunde, describes the early discoveries in observations of single proteins to that of the misfolding protein misfolding and the formation and structure of of large subsections of the proteome. Whilst this area amyloid associated with human disease, before outlining of research has obvious importance to age-related the intriguing world of functional amyloid. neurodegenerative diseases in which protein aggregates The term protein homeostasis or proteostasis refers are a hallmark of the disorders (eg. Parkinson’s disease), to the maintenance of the proteome in a conformation, there is emerging recognition of the importance of the concentration and in a location that is required for their maintenance of proteostasis in a range of conditions correct function. Given that a single cell has to handle including type 2 diabetes, cataract, pre-eclampsia and around 200 million protein molecules made from up to many forms of cancer. Indeed, given the astounding 20,000 protein encoding genes, it is an understatement number of protein molecules that need to be kept in check to say that proteostasis is important in the normal in the human body, it is remarkable that the proteostasis housekeeping of a cell. In order to produce a properly network manages to protect cells from diseases associated functioning (non-aggregating) proteome, the processes of with proteome stress at all. Justin Yerbury Illawarra Health and Medical Research Institute, University of Wollongong, NSW 2522 [email protected]

Protein Misfolding and Proteostasis Cover Illustration Total internal reflection fluorescence Guest Editors: Justin Yerbury and Heath Ecroyd microscopy of amyloid fibrils 4 A Short History of Amyloid: from Abnormal Aggregation to formed from a-synuclein, imaged Functional Assembly using Thioflavin T (blue) and Nile Margie Sunde Red (green) staining. The small heat 8 Molecular Chaperones: Guardians of the Proteome shock protein Hsp27 (red) binds to Dezerae Cox, Rebecca San Gil, Anthea Rote and Heath Ecroyd these amyloid fibrils. 11 When Proteostasis Goes Bad: Protein Aggregation in the Cell Image courtesy of Mathew Horrocks, Mona Radwan, Rebecca Wood, Xiaojing Sui and Danny Hatters Dezerae Cox and Caitlin Johnston 14 Extracellular Chaperones and Proteostasis (Illawarra Health and Medical Research Amy Wyatt Institute, University of Wollongong).

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 3 SHOWCASE ON RESEARCH

A Short History of Amyloid: from Abnormal Aggregation to Functional Assembly Margie Sunde* Discipline of Pharmacology, School of Medical Sciences, University of Sydney, NSW 2006 *Corresponding author: [email protected]

Amyloid fibrils are long protein fibrils, usually straight the way in diagnosis and treatment of systemic amyloid and unbranching (Fig. 1A), which have an underlying disease. At amyloid meetings, clinicians presented ordered β-sheet structure in which the β strands run at alongside basic scientists such as Jeff Kelly, Ron Wetzel, right angles to the fibril long axis. This structured core Dan Kirschner, Peter Lansbury and Paul Fraser, who gives rise to a cross-β X-ray fibre diffraction pattern, with were starting to apply biophysical methods to study dominant reflections at ~4.7 Å on the meridian and ~10 Å disease-associated variant proteins in order to understand on the equator of the pattern, and to diagnostic staining why proteins with a stable globular structure would self- with the dye Congo red (Fig. 1B). This simple description assemble into an insoluble, fibrillar form (3). Colin Blake, of amyloid structure and character has held even as our as a pioneer protein crystallographer, had solved the three understanding of the roles of amyloid fibrils in biology dimensional structures of both human lysozyme and and disease has changed dramatically in recent years. transthyretin and he was keen to understand why and Extracellular, fibrillar amyloid deposits associated with how variants of these two proteins formed amyloid fibrils human disease were first described by the pathologist and human disease. Louise Serpell was a DPhil student Virchow in 1854 (1). The observation of apple-green when I joined the laboratory and she initiated electron birefringence from Congo red-stained deposits in tissue microscopy studies of transthyretin fibrils isolated from sections was considered pathognomonic for amyloidosis patients and also analysed detailed X-ray fibre diffraction and different amyloid diseases were characterised by patterns that she collected from Val30Met transthyretin the nature of the component protein and the location of amyloid fibrils, known to cause familial amyloidotic the deposits. When I joined Colin Blake’s group in the polyneuropathy (4). In collaboration with David Booth Laboratory of Molecular Biophysics in Oxford in late and Vittorio Bellotti, who were working with Mark 1993, to start postdoctoral work on familial lysozyme Pepys in London, we started to study the process of fibril amyloidosis, the study of disease-associated amyloid formation by two variant human lysozymes, identified fibrils dominated the field (2). Clinician scientists such as as amyloidogenic in two families (5). Along the way we Mark Pepys, Joel Buxbaum and Giampaolo Merlini led came to understand that all disease-associated fibrils had

Fig. 1. A. Negatively stained transmission electron micrograph showing amyloid fibrils with typical long, relatively straight and unbranching morphology. These fibrils were formed from an amyloidogenic peptide derived from a fungal hydrophobin protein. B. X-ray fibre diffraction pattern collected from the fibrils shown in part A, showing the strong inter-strand spacing at ~4.7 Å on the meridian, parallel to the fibril long axis, and the weaker and more diffuse inter-sheet spacing at ~10 Å on the equator of the pattern. Inset shows apple-green birefringence observed when amyloid is stained by Congo red. C. Schematic representation of disease-associated fibril formation, where misfolding of the protein or polypeptide allows an amyloidogenic region (coloured green) to take part in intermolecular interactions, through hydrogen bonding, and to generate the β-sheet core of the amyloid fibrils. APP = amyloid precursor protein

Page 4 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 SHOWCASE ON A Short History of Amyloid RESEARCH the common core cross-β structure, regardless of their first detailed information about the packing of amyloid native protein conformation and also recognised that protofibrils and protofilaments on a larger scale andin disease-associated amyloid formation is essentially a fibrils formed from full-length amyloidogenic proteins protein misfolding process (Fig. 1C) (6). (15,16). As early as 1996, discussion between Colin Blake and The understanding of protein (mis)folding intermediates Dan Kirschner at a Ciba Foundation Amyloid meeting and their interactions with chaperones grew with the in Portugal highlighted the fact that there were no increased understanding of the cellular machinery that known animal protein structures that displayed a cross-β acts to maintain proteostasis. Whereas disease-associated structure. In β-keratin and most insect silks, the β-strands amyloid deposition had always been characterised in within the component β-sheets lie parallel to the fibril long clinical samples as an extracellular phenomenon, there axis. However, Dan Kirschner drew attention to the fact was a growing recognition of intracellular amyloid- that the silk egg stalks produced by the lacewing Chrysopa structured aggregates. Sue Lindquist’s group connected had a cross β-structure, described by Geddes in 1968 (7). yeast prions (structurally unrelated to mammalian This wasn’t recognised at the time for what it was, the Prion protein; PrP) and amyloids and it was shown that first identification of a functional amyloid, but the work chaperones affected the maintenance of the prion state by Geddes and colleagues did underlie the development and amyloid formation (17). From 2000 onwards there of models of human disease-associated amyloid fibrils(8). has been a growing recognition that there is widespread With the realisation that disease-associated fibrils are application of the amyloid fibril structure for functional often an undesirable end-point of protein misfolding, Chris purposes in microorganisms. In addition to the Sup35 Dobson, Sheena Radford and Carol Robinson brought and Ure2p yeast prions, the fibrils formed from the fungal protein-folding methodology and an understanding of protein HET-s were shown to have an amyloid structure, chaperones to studies of fibril formation (5). It was at this with a highly-ordered β-solenoid structure (18). The Het-s time that Iñaki Guijarro, working with Chris Dobson in an element functions in the heterokaryon incompatibility effort to use NMR to study the folding pathway of an SH3 system that leads to cell death. Fibrillar structures on domain, found that it formed a gel in the bottom of his the surface of Streptomyces coelicor spores and spores NMR tube when incubated at low pH for extended times. from filamentous fungi such as Neurospora crassa, long We realised that the gel contained amyloid fibrils with a recognised and described by mycologists, were shown to cross-β structure, formed by misfolding of a domain never have a robust amyloid nature, with the added property previously known to be associated with disease (9). This that they formed monolayers that were amphipathic led to the idea that the amyloid fold is a generic protein and served to waterproof aerial bacterial and fungal fold, with (perhaps almost) all polypeptide sequences structures (Fig. 2A) (19,20). It was demonstrated that being able to form extended structures and to take part in bacterial curli fibres have an amyloid structure and that the backbone, interchain hydrogen bonding (H-bonding) some of the natural adhesives produced by algae contain that forms the basis of the cross-β structure of amyloid amyloid structures, adding mechanical strength to the fibrils (8). substances (21). The list of natural, functional amyloids in Now amyloids were everywhere, produced from microorganisms is likely to grow as there is recognition everything, under all conditions and in large quantities that the amyloid fold is applied in multiple settings and for and these in vitro studies led to the recognition that multiple purposes: it can provide a stable superstructure oligomeric species, formed when misfolded monomers (eg. lacewing egg stalk, curli fibrils), allow controlled self- associated on the pathway to the formation of the long, assembly into a macromolecular form that generates or straight, unbranching fibrils recognised as amyloid fibrils, displays additional structural or functional properties (eg. were cytotoxic (10,11). Peptide fibril forming assays and amphipathic monolayer coatings on fungal spores) and mutational studies with model proteins were used to can allow sequestration of peptides and proteins in stored, develop algorithms that could be used to predict and inactive or insoluble forms (eg. yeast prions or phenol- identify amyloidogenic sequences within proteins (12). soluble modulins in Staphylococcus aureus) (22). Seeding was recognised as a key feature in the formation The first report of a mammalian functional amyloid of prion amyloid deposits in the transmissible spongiform came from Douglas Fowler and Jeff Kelly in 2005, when encephalopathies and Stan Prusiner’s work on prions they published their finding that cytotoxic melanin led to him being awarded the Nobel Prize (13). High biosynthetic intermediates are sequestered within resolution work from David Eisenberg’s group, using melanosomes as a result of binding to PMel amyloid microcrystals grown from short amyloidogenic peptides, fibrils (23). In 2009, it was reported that certain peptide underpinned our understanding of the cross-β structure hormones were stored in an amyloid form (24). Not only of amyloid fibrils but highlighted the specific side-chain did this show that unique amyloid fibrils could play inter-digitation and unique β-sheet interfaces that may important functional roles within mammalian systems, it explain the homogeneous nature of most amyloid fibrils demonstrated that the amyloid forming machinery was (14). This implied that the amyloid cross-β fold is more under tight control, presumably necessary to prevent than a generic structure involving only H-bonding the unwanted consequences and toxicity observed in between amide and carbonyl groups of the polypeptide amyloid-associated diseases. The list of recognized backbone. Cryo-electron microscopy and solid state NMR mammalian, functional amyloids continues to grow. studies led by Helen Saibil and Rob Tycko gave us the In 2012, Hao Wu’s laboratory demonstrated that large

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 5 SHOWCASE ON A Short History of Amyloid RESEARCH necroptosis-associated signalling complexes involving Recently, my laboratory has found evidence that some the kinases RIPK1 and RIPK3 are stabilised by hetero- viral RHIM-containing proteins are able to form amyloid oligomeric amyloid interactions formed by common RIP fibrils Fig.( 2B, unpublished). While mammalian cells use homotypic interaction motifs (RHIMs) (25). Sequence functional amyloid structures to signal for cell death in analysis indicates that these RHIMs are similar to the response to viral infection, certain viruses express proteins sequences that stabilize HET-s prion structures and which that can form functional amyloid that may interfere with signal for heterokaryon incompatibility in fungi (26). the host response and allow latent infection. The formation Many RNA-binding proteins, known to have (yeast) of hetero-oligomeric amyloid fibrils may be a distinctive prion-like domains, have been shown to form aggregated feature of functional or biologically active amyloid, where structures within cells and in some cases these are linked different active domains are brought together through to diseases such as amyotrophic lateral sclerosis. In vitro association of similar amyloidogenic motifs (Fig. 2C). studies indicate that many of these proteins can form Disease-associated amyloid fibrils isolated from human amyloid-like structures and some may form amyloid in patients typically contain only a single protein component vivo (27,28). These amyloids appear to be less stable than (30), although recent evidence indicates that Aβ and IAPP most microbial amyloids and also less stable than disease- amyloid may be found together in vivo and that IAPP may associated fibrils found in humans. This may reflect seed Aβ amyloid formation (31). High-resolution studies their ability to undergo phase transitions and to allow of the amyloidogenic core of these hetero-amyloid fibrils the sequestering of proteins in functional but dynamic will be required to determine how side-chain differences structures such as ribonucleoprotein granules (29). and sequence-specific recognition are both accommodated.

Fig. 2. A. Negatively stained transmission electron micrograph showing that amyloid fibrils formed by a hydrophobin protein associate laterally to form a stable protein layer that provides a functional, amphipathic coating on the surface of fungal spores in contact with air. B. Viral proteins containing RHIM sequences can form amyloid fibrils. Here a fluorescent protein domain, substituting for the viral ribonucleotide reductase domain, is attached to the RHIM sequence from the M45 protein from murine cytomegalovirus. The fusion protein assembles spontaneously into amyloid fibrils, with the fluorescent partner domain displayed along the length of the assembled fibrils. C. Schematic representation of two routes to the formation of functional amyloid fibrils. Left panel: Exposure or generation of an amyloidogenic motif (coloured green) by controlled conformational change or proteolysis can lead to fibril assembly. Right panel: Amyloidogenic motifs (coloured green) may be attached to separate, functional domains and assembly of the motif into the β-sheet amyloid structure can drive formation of fibrils decorated with the active domains.

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Amyloid deposits are not always undesirable protein Newcombe, E.A., Hosoki, K., Goshima, N., Kawaguchi, aggregates and not all undesirable protein aggregates T., Hatters, D., Trinkle-Mulcahy, L., Hirose, T., Bond, are amyloid. A major challenge is to understand the C.S., and Fox, A.H. (2015) J. Cell Biol. 210, 529-539 mechanisms that control appropriate protein self- 28. Hervas, R., Li, L., Majumdar, A., et al. (2016) PLoS Biol. assembly for functional purposes and which allow 14, e1002361 turnover of biologically active amyloid. 29. Wu, H., and Fuxreiter, M. (2016) Cell 165, 1055-1066 ­­­ 30. Tuttle, M.D., Comellas, G., Nieuwkoop, A.J., et al. References (2016) Nat. Struct. Mol. Biol. 23, 409-415 1. Virchow, R. (1854) Vichows Arch. 6, 415-426 31. Oskarsson, M.E., Paulsson, J.F., Schultz, S.W., 2. Pepys, M.B. (2006) Annu. Rev. Med. 57, 223-241 Ingelsson, M., Westermark, P., and Westermark, G.T. 3. Kelly, J.W. (1997) Structure 5, 595-600 (2015) Am. J. Pathol. 185, 834-846 4. 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Molecular Chaperones: Guardians of the Proteome Dezerae Cox, Rebecca San Gil, Anthea Rote and Heath Ecroyd* Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522 *Corresponding author: [email protected]

Chaperones can be defined as proteins that interact with, Protein Folding and Molecular Chaperones stabilise, and/or aid in the folding of another protein, Anfinsen’s dogma of protein folding states that the without becoming a component of its final conformation. amino acid sequence contains all the information Cells have evolved a complex network of molecular required for a protein to fold into its native conformation chaperones that act to stabilise proteins during folding (2). Whilst the folding of small proteins/domains can and unfolding, and hence minimise the occurrence of occur spontaneously in vitro, protein folding in vivo protein misfolding and aggregation. Moreover, transient typically requires the assistance of molecular chaperones, interactions between chaperones, co-chaperones and their particularly for large and/or multidomain proteins (3-6). client proteins ensure that efficient folding of nascent and As such, molecular chaperones are a central component of existing polypeptides occurs on a biologically relevant the proteostasis network in that they maintain a stable and timescale. Molecular chaperones are arguably the most functional proteome. A recent and comprehensive analysis important components of the proteostasis network since of the human ‘chaperome’ identified 332 chaperone genes they are the first line of defence against protein misfolding (7). Chaperones are often broadly classified as having either and aggregation in cells (1). foldase or holdase type activity. Models of their chaperone

Fig. 1. The heat shock protein chaperone network highlighting the interconnected and dynamic nature of the interactions between Hsp members. The six major Hsp families are shown, ie. Hsp100, Hsp90, Hsp60, Hsp70, Hsp40 and small Hsps. Taken from the heat shock protein internet resource webpage: http://pdslab.biochem.iisc.ernet.in/hspir/

Page 8 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 SHOWCASE ON Molecular Chaperones: RESEARCH Guardians of the Proteome action posit that, during periods of cellular stress, holdase maintenance of cellular proteostasis and viability under chaperones prevent protein aggregation by binding to conditions of stress. HSF1-mediated transcription is exposed hydrophobic regions on partially folded proteins, attenuated by an auto-regulatory mechanism, whereby sequestering them into chaperone–client protein complexes HSF1-induced Hsps inhibit HSF1 trimer activity (11). This (8,9). When energy levels permit, holdase chaperones pass negative feedback loop provides an important mechanism this aggregation-prone cargo over to foldases which use by which cells can regulate the activation, duration and ATP to facilitate refolding. Alternatively, aggregation- strength of a heat shock response via the presence and prone proteins bound to chaperones can be targeted for concentration of Hsps in the cell. degradation via the proteasome or autophagy. Thus, when studying the chaperones, it is important to consider Protein Aggregation – a Failure of Molecular the interactive and dynamic nature of the chaperone/co- Chaperones? chaperone network (Fig. 1). The finding that protein aggregation and protein Whilst significant progress has been made in better aggregates are the hallmarks of various diseases has led understanding the role chaperones play in maintaining to an enormous amount of effort (and research funding) proteostasis, the precise molecular mechanisms by which to identify the culprit protein or peptide responsible for they prevent protein aggregation and refold proteins forming the aggregates associated with each disorder. remains elusive. For instance, due to the transient and However, equally important to consider is that these heterogeneous nature of chaperone and co-chaperone aggregates are a signature of a molecular chaperone interactions with client proteins, the exact stoichiometries, network that has failed to prevent this protein forming the order in which they occur and the kinetic processes aggregates in the first place. The failure of chaperones involved (ie. on/off rates of substrate binding) are yet to prevent protein aggregation associated with disease to be established for many of these interactions. The is often interpreted as the aggregation-prone proteins application of high-resolution techniques to study ‘escaping’ the molecular chaperones, or the chaperones dynamic protein interactions promises to uncover more becoming ‘overwhelmed’ by the burden (ie. amount) detailed information on precisely how chaperones and co- of misfolded protein. However, specific factors or chaperones work together to stabilise and refold proteins. mechanisms that account for proteins escaping chaperones are yet to be identified. Cells Feel Stressed Too In terms of how aggregation-prone proteins can One of the pivotal roles of molecular chaperones is overwhelm the chaperone network, the most likely to stabilise proteins during periods of cellular stress explanation is that at some point, insufficient levels of (increased temperature, changes in pH and/or redox the chaperones are present, thus enabling destabilised state) as these conditions favour protein unfolding, proteins to aggregate. Transcriptome analyses have misfolding and aggregation. Cells respond to acute stress indicated that the expression of a subset of chaperones by dramatically up-regulating the expression of heat that decline with age (7). This decline in the levels of some shock proteins (Hsps) via induction of the heat shock chaperones may therefore be the tipping point that enables response (10). Of the 332 chaperone genes in humans, 147 aggregates to form in cells. It may also help explain why correspond to Hsps (7), which are subclassified based on many of the diseases associated with protein aggregation their monomeric molecular mass into the Hsp100 (Clp are age-related. Once aggregation occurs it is probable proteins), Hsp90, Hsp70, Hsp60 (chaperonins), Hsp40 that a further collapse in proteostasis follows because (J-proteins) and small Hsp (sHsps) families. The Hsps are chaperones and other proteostasis network components evolutionarily-conserved and its members have diverse (eg., proteasome subunits) become incorporated into cellular functions. They are endogenously expressed in the inclusions. Recent work investigating the cellular some cells for ‘house-keeping’ roles (e.g. Hsp27, a sHsp, aggregation of huntingtin exon 1 has shown that there is is important in stabilising actin for the maintenance of no induction of a heat shock response in cells caused by the cytoskeleton). However, under conditions of cellular the production of aggregates (12). In other words, cells stress when the levels of many, but not all, Hsps are do not perceive the aggregation of huntingtin as a ‘stress’ dramatically upregulated (see below), they function and therefore do not activate the signalling pathway that to stabilise the cytoskeleton, prevent the aggregation would lead to increased levels of the Hsps. It is of interest of destabilised proteins, regulate stress responses, and to establish whether the aggregation of other proteins mitigate apoptotic signalling. induces a stress response (and hence higher levels of During periods of stress, the upregulation of Hsp Hsps) in cells. Since higher levels of chaperones are likely levels is controlled by heat shock transcription factor to protect cells from further aggregation, activation of 1 (HSF1), which becomes activated (via concurrent HSF1 (and restoration of cellular proteostasis) is being hyperphosphorylation and trimerisation), resulting in investigated as a possible therapeutic approach to treat it binding to heat shock elements (HSEs) in promoter diseases associated with protein aggregation. regions of target genes. Stress-inducible mRNA transcripts are then synthesised and translated, many Who Will Guard the Guardians Themselves? of which are Hsps (increased levels of Hsp70 are often Whilst an insufficient level of chaperones may prompt used as markers of induction of a heat shock response) the pathogenesis of some diseases, the opposite is true in (10). The heat shock response is therefore pivotal for the cancer. Cancerous cells typically express very high levels

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 9 Molecular Chaperones: SHOWCASE ON Guardians of the Proteome RESEARCH of Hsps and their anti-apoptotic activities promote cell diseases. We are interested in developing new tools to growth and survival (13,14). In rapidly dividing cancer study the molecular mechanisms and cellular functions cells genomic instability leads to proteomic instability of Hsps in an effort to uncover just how these molecular through the accumulation of mutated proteins. This chaperones ensure our cells maintain a healthy proteome. higher burden of mutated proteins makes cancer cells highly dependent on the cellular systems that maintain References proteostasis, in turn making Hsps an attractive target 1. Yerbury, J.J., Ooi, L., Dillin, A., Saunders, D.N., Hatters, for the development of anti-cancer therapies. As such, a D.M., Beart, P.M., Cashman, N.R., Wilson, M.R., and number of anti-cancer drugs target Hsp activity (eg. 17- Ecroyd, H. (2016) J. Neurochem. 137, 489-505 AAG which targets Hsp90). Other approaches aim to 2. Anfinsen, C.B. (1973) Science 181, 223-230 decrease the levels of Hsps in cells. A recent clinical trial 3. Kim, Y.E., Hipp, M.S., Bracher, A., Hayer-Hartl, M., and of an antisense oligonucleotide (ASO; OGX-427) designed Hartl, F.U. (2013) Annu. Rev. Biochem. 82, 323-355 to specifically inhibit the expression of Hsp27 for the 4. Voisine, C., Pedersen, J.S., and Morimoto, R.I. (2010) treatment of prostate, bladder, breast and lung cancer has Neurobiol. Dis. 40, 12-20 shown promising results (15). Future cancer therapies 5. Barral, J.M., Broadley, S.A., Schaffar, G., and Hartl, F.U. may, therefore, include those which aim to suppress the (2004) Semin. Cell Dev. Biol. 15, 17-29 action of Hsps and/or other arms of the proteostasis 6. Balchcin, D., Hayer-Hartl, M., and Hartl, F.U. (2016) network. Science 353, aac4354 The critical role of chaperones in proteostasis is also 7. Brehme, M., Voisine, C., Rolland, T., et al. (2014) Cell evidenced by the association of mutations in Hsps with Rep. 9, 1135-1150 various diseases (16). For example, at least 15 distinct 8. Hartl, F.U., Bracher, A., and Hayer-Hartl, M. (2011) mutations in Hsp27 have been identified as being Nature 475, 324-332 causative of Charcot-Marie-Tooth disease type 2 and/or 9. Hendrick, J.P., and Hartl, F.U. (1993) Annu. Rev. distal hereditary motor neuropathy; most of these have an Biochem. 62, 349-384 autosomal dominant inheritance pattern. Disease-related 10. Velichko, A.K., Markova, E.N., Petrova, N.V., Razin, mutations have also been identified in Hsp60, Hsp40 S.V., and Kantidze, O.L. (2013) Cell. Mol. Life Sci. 70, and other sHsp members. Intriguingly however, no 4229-4241 disease-related mutations have been identified in Hsp70 11. Anckar, J., and Sistonen, L. (2011) Annu. Rev. Biochem. or Hsp90 family members. This may be due to functional 80, 1089-1115 redundancy between family members (although this 12. Bersuker, K., Hipp, M.S., Calamini, B., Morimoto, R.I., would also be expected in the Hsp40s and sHsps) or and Kopito, R.R. (2013) J. Biol. Chem. 288, 23633-23638 because mutations in these Hsps are embryonic lethal. 13. Lianos, G.D., Alexiou, G.A., Mangano, A., Mangano, A., Rausei, S., Boni, L., Dionigi, G., and Roukos, D.H. (2015) Conclusions Cancer Lett. 360, 114-118 Maintenance of the proteome in a folded and functional 14. Jego, G., Hazoume, A., Seigneuric, R., and Garrido, C. state requires the coordinated and interconnected (2010) Cancer Lett. 332, 275-285 interactions of the chaperone network. However, the 15. Chi, K.N., Yu, E.Y., Jacobs, C., Bazov, J., dynamic and complex nature of the interactions Hsps Kollmannsberger, C., Higano, C.S., Mukherjee, S.D., have with themselves and their client proteins presents a Gleave, M.E., Stewart, P.S., and Hotte, S.J. (2016) Ann. barrier to studying them. Yet we need to persist; the Hsps Oncol. 27, 1116-1122 play a critical role in governing the proteostasis capacity 16. Kakkar, V., Meister-Broekema, M., Minoia, M., Carra, S., of cells and their (mal)function is linked with diverse and Kampinga, H.H. (2014) Dis. Model Mech. 7, 421-434

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When Proteostasis Goes Bad: Protein Aggregation in the Cell Mona Radwan, Rebecca Wood, Xiaojing Sui and Danny Hatters* Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC 3010 *Corresponding author: [email protected] Protein aggregation is a hallmark of the major Nuts and Bolts of Proteostasis neurodegenerative diseases including Alzheimer’s, To maintain proteostasis, the cell employs a proteostasis Parkinson’s, Huntington’s and amyotrophic lateral network (PN) of quality control machinery that shield sclerosis (ALS; also known as motor neuron disease) and is proteins from non-native interactions, act as folding a symptom of a breakdown in the management of proteome checkpoints, and govern protein synthesis, turnover and foldedness (or proteostasis) (1). Indeed it is remarkable that trafficking (shown conceptually in Fig. 1). During protein under normal conditions cells can keep their proteome in a synthesis, hydrophobic regions of nascent polypeptide highly crowded and confined space without uncontrollable chains are shielded from non-specific interactions by aggregation. Proteins pose a particular challenge relative ribosome-associated chaperones including the nascent to other classes of biomolecules because upon synthesis, chain associated complex, Hsp70 chaperones and Hsp40 they must typically follow a complex folding pathway to co-chaperones. More than two-thirds of the proteome, reach their functional conformation (native state). Non- especially membrane proteins and other complex native conformations, including the unfolded nascent multidomain proteins, require additional substantial chain, are highly prone to aberrant interactions, leading to guidance to fold (2). Early steps of this guidance include aggregation. Here we review recent advances in knowledge hsp70 family members acting as triage points connecting of proteostasis, approaches to monitor proteostasis and the to other branches of the PN, such as the calnexin cycle. impact that protein aggregation has on biology. We also At the triage, proteins can be engaged with downstream include a discussion of the outstanding challenges. chaperones including Hsp90s and chaperonins, and

Fig. 1. Proteostasis governs the life of a protein from synthesis to degradation. Shown is a simplified diagram of key steps in the life of a protein and how this is guided by the proteostasis network (in green). Management of protein folding is normally regulated by a network of about 800 proteins in humans. When proteostasis fails, proteins can misassemble into aggregates (red). The failure of proteostasis correlates with a gain of proteotoxicity; some of the postulated mechanisms covered in this review are indicated.

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 11 When Proteostasis Goes Bad: SHOWCASE ON Protein Aggregation in the Cell RESEARCH in the case of being irreparably misfolded, targeted for Mechanisms of Misfolded Protein Sequestration – degradation by the ubiquitin proteasome system (UPS) or Cell Driven Inclusion Formation autophagy. Hsp70 family proteins are also important in While protein oligomers or nascent proteins may preventing misfolding of nascent chains during translation themselves be toxic, there is also evidence that cells actively and ensuring optimum translation rates. Elements of the PN cluster together misfolded proteins for sequestration. also play critical roles targeting and translocating proteins The most compelling evidence for this model is from to cellular organelles including the nucleus, mitochondria a key study showing that in cells expressing mutant and the endoplasmic reticulum (ER), which contain their Huntingtin exon 1, cells that formed inclusions had own subnetworks of quality control machinery and greater rates of survival than cells than cells that did not checkpoints for folding. (13). The mechanisms for this effect with Huntingtin exon 1 remain to be determined, however, similar ‘active’ Failure of Proteostasis and Protein Aggregation mechanisms of inclusion building have been proposed Unfolded states are permissive to entering misfolding with other misfolded proteins. The original model was that pathways that result in aggregation, which provides of the ‘aggresome’, which described a dynein-mediated a rival low energy state to the native fold (3). For this retrograde transport mechanism of misfolded DF508 reason, aggregation is often observed as symptomatic of Cystic Fibrosis Transmembrane conductance regulator problems in maintaining proteostasis (1). For example, protein into the microtubule organising centre (14). The proteins accumulate into punctate aggregate structures aggresome model however, is problematic in that it does inside neurons, known as inclusions, as a hallmark of not appropriately define the diversity of processes that neurodegenerative diseases including Alzheimer’s disease, sequester aggregating proteins into a centralsed location. ALS and Huntington’s disease (1). Proteins may also Recent studies have partly addressed this deficiency by naturally aggregate during the normal ageing process, with unearthing two distinct aggresome-like compartments the PN remodeling to accommodate the changes – a process for aggregating proteins. One is the juxtanuclear quality that may be dysregulated in neurodegenerative disease (4). control (JUNQ), which comprises reversibly-aggregated Mutations can also change the folding stability of individual proteins and the other is the insoluble protein deposit proteins, which may modify baseline proteostasis capacity (IPOD), which comprises irreversibly-aggregated proteins and influence disease risk (5). (Fig. 1) (9). Different disease-associated aggregating One of the great challenges of current research efforts proteins seem to preferentially partition into either JUNQ is in understanding how protein aggregation relates to or IPOD exclusively, and that these compartments may toxicity. One of the longest standing theorems is that correlate with different mechanisms of aggregation (15). soluble protein oligomers, as precursors to a larger Huntingtin exon 1 accumulates in IPOD-like structures. amyloid state, are directly proteotoxic and that this seems By contrast, polyalanine, which aggregates via soluble to be independent of the protein sequence (6). However, a-helical clusters, and SOD1 mutants, which also cluster the mechanisms for toxicity, and the relevance to disease into soluble oligomers prior to aggregation, accumulate remain to be robustly validated (6). Possible routes of in JUNQ-like structures (16). FUS and TDP-43 partitioned toxicity include physical disruption of membranes or into both structures and another unidentified structure other cellular structures, or interference with synaptic (17). These data indicate that aggregation into foci arises structure and plasticity (7). Larger aggregates seem to be via diverse processes, which are in part consistent with the less toxic, and this may arise from a lower concentration of JUNQ and IPOD models, but which also seem to involve effective reactive ‘ends’ of fibrils or cellular mechanisms a further uncharacterised layer of complexity. Hence, to sequester the dispersed oligomers together (8,9). critical questions remain as to what factors drive proteins One of the more interesting recent hypotheses for to different compartments, how the elements of different proteotoxicity of protein aggregates arises from the models fit together, how the additional inclusion types observation that markers of stress granule abnormalities relate to the JUNQ and IPOD structures, and whether there appear in pathology (notably ALS) (10). Indeed several RNA are further inclusion types that remain to be discovered. granule proteins, most notably trans-activation element (TAR) DNA binding protein-43 (TDP-43), FUS and hnRNP An ‘Omics View of Proteostasis family proteins cause ALS when mutated. Stress granules, ‘Omics technologies have begun to transform our and related structures, P-bodies (Fig. 1), are condensed understanding of what happens to the proteome under foci of mRNA and ribonucleoproteins that form under proteostasis stress. Of note is a recent study showing that translational stresses (10). They act as sites for temporal the nuclear pore is fundamentally damaged in cells that translational repression and quality control of mRNA- display extensive cytoplasmic aggregates of unrelated, ribonucleoprotein. Recent studies have suggested many aggregation-prone proteins (18). Components of the RNA granule proteins contain predicted prion-like domains import and/or export machinery coaggregate with disease- that mediate liquid:liquid protein phase separation and/ associated mutant proteins, including polyglutamine- or ‘functional’ amyloid scaffolding (11). The presence of containing huntingtin protein, mutant TDP-43 and the ‘functional amyloid’ indicates a necessity for these structures C9orf72-associated polydipeptides (18). Indeed, the to be rigorously tamed to avoid pathological aggregation progressive loss of proteostasis may account for the into amyloid fibrils. Indeed, mutations or conditions that tip observation of nuclear transport becoming increasingly the balance to pathological aggregation have been proposed ‘leaky’ with ageing (19). as a basis for these neurodegenerative diseases (12). ‘Omics, along with computational approaches, has also

Page 12 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 SHOWCASE ON When Proteostasis Goes Bad: RESEARCH Protein Aggregation in the Cell enabled insight to which proteins in the proteome become transferring this approach to eukaryotic cells remains an more vulnerable to aggregation under proteostasis stress. ongoing challenge because of a vastly more complicated Indeed in different organisms and cell culture conditions, proteostasis networks. hundreds of proteins aggregate under stress (20,21). More importantly, distinct stress types tend to induce the The Path Ahead same proteins to aggregate by reducing their aggregation The true potential of proteostasis measurement will be threshold, indicating the presence of a metastable realised when computational models can be integrated sub-proteome (22). This includes many regulators with powerful tools for measuring proteostasis activity. of proteostasis, such as chaperones, and proteasome Further work, which our lab is actively engaged in, will be subunits (23). Others have shown that in C. elegans, required to develop extremely well-characterised sensors highly abundant proteins become supersaturated with that are able probe the diverse arms of the proteostasis respect to their solubility and most extensively contribute network without disrupting it, and yield quantitative to aggregate loads during ageing, despite their otherwise information that can feed into network models that enable low propensity to aggregate (24). A supersaturation score, information-rich analysis. which combines experimental and bioinformatics data, In conclusion, research into mechanisms of proteostasis has been used to explain which proteins coaggregate with and their relationship to protein aggregation in disease amyloid plaques, neurofibrillary tangles, Lewy bodies has progressed substantially in the last few years. We and artificialb -proteins, as well as proteins that aggregate see several key questions as immediate challenges to be in C. elegans during ageing (25). addressed. An important question is how generalisable While many proteins are likely to be aggregating are the mechanisms of toxicity caused by aggregation of ‘inappropriately’ under stress, there is also evidence for an different proteins? Are the mechanisms of proteotoxicity adaptive aggregation response as well. For example, under non-specific? And are there certain machinery hotspots, heat stress, many proteins form transiently aggregated such as the nuclear pore, that need to be ‘hit’ to trigger complexes that remain functionally competent, and these disease? Another gap is defining precisely which proteins are not degraded upon recovery (20). Such structures aggregate in the proteome under proteostasis collapse and were suggested to aid in the management of the stressed probing whether this explains the changes seen in disease. proteome (20). Given that TDP-43 mislocalisation is observed as marker of all forms of sporadic ALS, is TDP-43 simply a bellwether for Quantification of Proteostasis a broader subproteome highly prone to aggregation under A major area of development in the study of proteostasis stress? A large part of our focus is on building new tools involves building quantitative tools to mechanistically and approaches that can get at such problems including probe the PN. One approach to do this relies on metastable more quantitative measures of proteostasis. reporter proteins that engage with the PN and read out the effectiveness of suppressing aggregation as an indicator of References proteostasis efficacy. This includes temperature-sensitive 1. Schneider, K., and Bertolotti, A. (2015) J. Cell Sci. 128, endogenous proteins, which have been used to identify 3861-3869 novel regulators of the PN in C. elegans (26) and to probe 2. Braselmann, E., Chaney, J.L., and Clark, P.L. (2013) the collapse of proteostasis when disease-associated Trends Biochem. Sci. 38, 337-344 proteins are expressed. More advanced strategies involved 3. Jahn, T.R., and Radford, S.E. (2005) FEBS J. 272, 5962- designer ectopic reporters including destabilised mutants 5970 of firefly luciferase, which is a known chaperone substrate 4. Brehme, M., Voisine, C., Rolland, T., Wachi, S., Soper, that requires Hsp70 and Hsp90 to fold (27), and the de novo James H., Zhu, Y., Orton, K., Villella, A., Garza, D., designed enzyme retroaldolase as a sensor of proteome stress Vidal, M., Ge, H., and Morimoto, R.I. (2014) Cell Rep. 9, (28). These reporters offer the advantage of not interfering 1135-1150 with normal biological pathways and have been used in the 5. Gidalevitz, T., Krupinski, T., Garcia, S., and Morimoto, studies cited above to probe changes in proteostasis induced R.I. (2009) PLoS Genet. 5, e1000399 by drugs, disease proteins, and ageing. 6. Fändrich, M. (2012) J. Mol. Biol. 421, 427-440 Another strategy for gaining insight into proteostasis 7. Haass, C., and Selkoe, D.J. (2007) Nat. Rev. Mol. Cell Biol. has been the systems approach of computationally 8, 101-112 modelling the entire PN. This strategy offers mechanistic 8. Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., insight into the connections between different modules Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, of the PN in a way that makes it possible to make C.M., and Stefani, M. (2002) Nature 416, 507–511 sense of experimental data and make predictions that 9. Kaganovich, D., Kopito, R., and Frydman, J. (2008) informatively guide experiments. FoldEco provides Nature 454, 1088-1095 a comprehensive computational model of the PN in 10. Ramaswami, M., Taylor, J.P., and Parker, R. (2013) Cell Escherichia coli (29). This platform provides the capacity 154, 727-736 to model, and make predictions about, the kinetics of 11. Molliex, A., Temirov, J., Lee, J., Coughlin, M., Kanagaraj, chaperone-protein interactions as well as synthesis, A.P., Kim, H.J., Mittag, T., and Taylor, J.P. (2015) Cell 163, aggregation and degradation, in a fully integrated 123-133 system. It can also be ‘fitted’ to experimental data for a References continued on page 7 mechanistic understanding of proteostasis (30). However, Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 13 SHOWCASE ON RESEARCH Extracellular Chaperones and Proteostasis Amy Wyatt* Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, NSW 2522 *Corresponding author: [email protected]

Introduction oxidative stress or (iv) stimulate inflammatory responses. Disease-associated extracellular protein deposition was Of course, not all misfolded protein assemblies will have first documented over a century ago. Remarkably, it the same biological consequences, however, it appears that has only been in the last decade that attention has been molecules with a high degree of surface hydrophobicity given to identifying and characterising the elements are inherently cytotoxic and immunostimulatory (7,8). The of the proteostasis network that exist outside cells (i.e. ability of extracellular chaperones to selectively bind to extracellular proteostasis). The pioneering work in this regions of exposed hydrophobicity on misfolded proteins field has been led by Senior Professor Mark Wilson supports the concept that they are the front line defenders (University of Wollongong), who originally proposed against the pathogenesis of protein misfolding disorders. that protein folding quality control systems must exist New lines of evidence support the notion that cell-to-cell beyond the boundary of the cell membrane in order to transmission of pathogenic proteins (similar to that which protect the proteins that are secreted into the extracellular is known to occur in prion diseases) could be responsible milieu (1,2). Proteins are a major component of biological for the progression of degenerative protein misfolding fluids including blood plasma, cerebral spinal fluid (CSF), disorders. This includes elegant studies, including many synovial fluid and interstitial fluid. Compared to proteins carried out in Australia, that demonstrate the prion-like that are retained within the cytosol, secreted proteins are cell-to-cell propagation of misfolded superoxide dismutase subjected to relatively harsh conditions in the extracellular 1 (SOD1; implicated in amyotrophic lateral sclerosis) (9). At milieu. Structural modifications have evolved to increase least five different mechanisms for the transfer of misfolded the stability of extracellular proteins (eg. disulphide proteins between cells have been proposed including (i) bonds, glycosylation). Despite these modifications, direct penetration of the plasma membrane; (ii) uptake by many abundant extracellular proteins are susceptible macropinocytosis; (iii) receptor-mediated endocytosis; (iv) to misfolding in the presence of physiological stressors. transport via membranous exosomes or (v) transfer through For example, albumin, fibrinogen, and ceruloplasmin nanotubes that directly connect adjacent cells (reviewed can be induced to misfold by shear stress conditions in (10)). Although transfer via exosomes or nanotubes that are comparable to those encountered within the would shield misfolded proteins from extracellular fluids, bloodstream (3). Also, biological oxidants potently induce transfer of misfolded proteins via any of the three other the misfolding and aggregation of albumin, fibrinogen mechanisms would involve them being directly exposed to and apolipoprotein B-100, which has direct implications on their functions and is implicated in disease (4-6). Table 1. Some examples of extracellular protein deposition in disease. Extracellular Protein Deposition in Human Disorders The deposition of misfolded extracellular proteins is Disorder Major deposit components implicated in the pathology of a large number of debilitating disorders (Table 1). Depending on the peptide or protein(s) Alzheimer’s disease Amyloid beta (Aβ) involved, the deposits formed can be ordered (most Pre-eclampsia Many proteins present commonly, amyloid) or amorphous. Some diseases are the including Aβ, ceruloplasmin, direct result of genetic mutations that destabilise proteins, immunoglobulin light chain, such as familial amyloidosis. Other diseases appear to SERPINA1 and albumin result from complex (and in many cases poorly defined) Macular degeneration Many proteins present mechanisms, which are likely to involve a combination of including albumin, vitronectin, genetic, epigenetic and environmental factors. matrix metalloproteinase The role of misfolded proteins in the pathogenesis of inhibitor 3 and complement many other disorders remains enigmatic. The deposition components of large quantities of insoluble proteins (kilograms in Type 2 diabetes Amylin some instances) in tissues is undoubtedly responsible for the disruption of organs in some conditions (eg. systemic Systemic amyloidosis Immunoglobulin light chain a amyloidosis). On the other hand, the results of many Atherosclerosis Apolipoproteins, Aβ and 1- studies support the conclusion that soluble assemblies (or antitrypsin oligomers) formed prior to end-point insoluble aggregates AA amyloidosis Serum amyloid A can be toxic. Some theories regarding the toxicity of Spongiform Prion protein soluble misfolded protein assemblies include that they encephalopathies (i) disrupt membranes; (ii) induce apoptosis; (iii) enhance Page 14 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 SHOWCASE ON Extracellular Chaperones RESEARCH and Proteostasis extracellular fluids. Therefore, it is feasible that components misfolded client proteins are very rapidly taken up by the of the extracellular proteostasis network also contribute to liver in rats and degraded following scavenger receptor- controlling disorders typically characterised by intracellular mediated endocytosis (18). Furthermore, aged clusterin misfolded protein deposits. knockout mice develop glomerular neuropathy (typified by the deposition of immunoglobulins and complement Extracellular Chaperones components), which directly implicates clusterin in the Decades of research have characterised, in detail, the disposal of pathological protein deposits in vivo (19). central roles that molecular chaperones play in monitoring Taken together, the results of these in vitro and in vivo intracellular protein folding and facilitating the refolding studies support the conclusion that the role of clusterin in or degradation of misfolded proteins. Comparatively, we extracellular proteostasis is to selectively target misfolded are now only beginning to understand the corresponding proteins for intracellular disposal (Fig. 1). systems that function outside of cells. A growing number Genome-wide association studies have shown that some of secreted proteins have been proposed as extracellular polymorphisms in clusterin are strong genetic risk factors chaperones (Table 2; reviewed in more detail in (2)). With for Alzheimer’s disease (20-22)), however, the precise role the exception of serum amyloid P component (SAP), of clusterin in the disease remains unresolved. Several which reportedly has refolding activity when present at a studies suggest that clusterin could play a protective high molar excess in vitro (11), all proposed extracellular role within the brain by sequestering the Aβ peptide and chaperones stabilise misfolded proteins in an ATP- facilitating its clearance (2,23,24). On the other hand, it independent manner similar to intracellular small heat has been shown that knocking out clusterin reduces Aβ shock proteins (sHsps). The following section highlights fibril deposition and neurotoxicity in a mouse model of some of our own work involving clusterin and alpha-2- Alzheimer’s disease (25). The data from a subsequent macroglobulin (α2M), two extracellular chaperones that double knockout study suggests that clusterin and apoE can target misfolded proteins for receptor-mediated could work synergistically to inhibit amyloid deposition disposal. (26), but further studies are needed to fully elucidate this relationship. Table 2. Proposed extracellular chaperones (reviewed in more detail in (2)). a 2M a M is a multifunctional protein that is best known Protein 2 for its role as a broad-spectrum protease inhibitor. The

quaternary structure of a M comprises four identical Clusterin 2 subunits that are paired by disulfide bonds. These a 2M disulfide-linked dimers then non-covalently associate a Haptoglobin into a 720 kDa tetramer. 2M sterically traps proteases Serum amyloid P component (SAP) via reaction with an intramolecular thioester bond. This reaction results in a major conformational change that Caseins a exposes a cryptic binding site on 2M for the low density Apolipoprotein E lipoprotein receptor-related protein (LRP-1). Albumin a It has been demonstrated that 2M inhibits amyloid Secreted protein acidic and rich in cysteine (SPARC) formation and protect cells against Aβ toxicity in vitro and Fibrinogen-420 in vivo (2,24,27,28). At physiological levels, hypochlorite (an oxidant produced during inflammation) induces the a 1-acid glycoprotein a dissociation of the native 2M tetramer into stable dimers a 1-antitrypsin that are no longer able to trap proteases, but can bind Lipocalin-type prostaglandin D synthase (L-PGDS)/ to a range of pro-inflammatory cytokines and growth β-trace factors and deliver them to LRP-1 (29). Recently, we have a demonstrated that the dissociation of native 2M into dimers markedly enhances its small heat shock protein Clusterin (sHsp)-like chaperone activity (30). This is significant Clusterin, also known as apolipoprotein J, is a highly given that hypochlorite is known to potently induce conserved protein with near ubiquitous expression in protein misfolding and the accumulation of hypochlorite- mammals. A large number of alternative functions have damaged proteins is implicated in the pathology of a been proposed for clusterin (eg. complement inhibition, number of disorders (reviewed in (31)). The results of our lipid transport, regulating apoptosis), nevertheless, studies support the conclusion that during inflammation, a clusterin (i) potently inhibits fibrillary and amorphous hypochlorite-induced dissociation of 2M is a mechanism protein aggregation in vitro (reviewed in (2)), (ii) is that reduces collateral damage to the host by disposing upregulated in response to heat and oxidative stress (12,13) of misfolded proteins (Fig. 1). Consistent with having a and (iii) is found colocalised with misfolded proteins in a a role in extracellular proteostasis in vivo, 2M is found variety of disorders (14-17). It has also been demonstrated colocalised with misfolded protein deposits in many that blood-borne complexes formed between clusterin and diseases (32-34).

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 15 Extracellular Chaperones SHOWCASE ON and Proteostasis RESEARCH

a Fig. 1. A model for the roles of 2M and clusterin in the disposal of misfolded proteins. (1) Extensive intracellular protein folding quality control processes ensure that proteins that are destined for a extracellular fluids are secreted in their native conformation. 2M and clusterin are two secreted proteins that have been identified as extracellular chaperones. (2) Secreted proteins are susceptible to stress-induced misfolding, which is heightened during inflammation. a 2M is rapidly induced to dissociate into chaperone active dimers by reaction with hypochlorite (a product of inflammation). The levels of clusterin in extracellular fluids are upregulated in response to heat or oxidative stress. a (3) Misfolded extracellular proteins form soluble oligomers that bind to and are stabilised by 2M dimers or clusterin. Similarly, misfolded intracellular proteins are targets for extracellular chaperones if they are released a into extracellular fluids. 2M-misfolded protein complexes bind to LRP-1 on the surface of cells, which facilitates their clearance via endocytosis. Concomitantly, clusterin-misfolded protein complexes bind to scavenger receptors, which also mediate their disposal.

a Extracellular Degradation of Misfolded Proteins protease- 2M complexes (36). Degradation of fibrinogen a Many different proteases are present in extracellular by protease- 2M complexes is initiated at the A-alpha fluids, however, components of the ubiquitin-proteasome chain, which is highly flexible due to the presence of system, which can specifically target misfolded proteins intrinsically disordered regions. It is tempting to speculate a for degradation, are only present at trace levels. Moreover, that protease- 2M complexes could therefore selectively the ubiquitin-proteasome system requires ATP to function, target misfolded proteins for degradation based on their which is also scarce in biological fluids. Nevertheless, it exposed hydrophobicity and flexibility. is possible that specific extracellular proteolytic systems that target misfolded proteins do exist. In this regard, the Concluding Remarks and Future Directions tissue plasminogen activator (tPA) system is of interest It is clear that in multicellular organisms there is a given that it has been shown that that misfolded proteins requirement for proteostasis systems that function outside are cofactors for tPA and substrates of the fibrinolytic the cell. Befitting this, extracellular proteostasis research system in vitro (35). is now a vibrant field, particularly within Australia. The a Although 2M is commonly referred to as a protease results of our work support the conclusion that extracellular a inhibitor, proteases trapped by 2M remain active. chaperones minimise the toxic effects of misfolded proteins Therefore, substrates that are accessible to proteases within by targeting them for receptor-mediated disposal (Fig. 1), a the steric cage formed by 2M, such as small amyloidogenic and we aim to fully characterise the mechanisms involved. proteins and peptides, are readily degraded. Although it is It is feasible that chaperone-mediated clearance is a generally accepted that size is a major factor that restricts mechanism that (i) limits the accumulation of misfolded a the access of substrates to proteases trapped by 2M, it is extracellular proteins and (ii) prevents pathological cell-to- astonishing that fibrinogen (640 kDa) can be degraded by cell transfer of intracellular misfolded proteins. Therefore,

Page 16 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 SHOWCASE ON Extracellular Chaperones RESEARCH and Proteostasis a better understanding of the functions of the extracellular 18. Wyatt, A. R., Yerbury, J. J., Berghofer, P., Greguric, I., proteostasis network has the potential to inform the Katsifis, A., Dobson, C. M., and Wilson, M. R. (2011) design of novel therapeutic strategies for a broad range of Cell. Mol. Life Sci. 68, 3919-3931 debilitating and costly human disorders. 19. Rosenberg, M. E., Girton, R., Finkel, D., Chmielewski, D., Barrie, A., 3rd, Witte, D. P., Zhu, G., Bissler, J. J., References Harmony, J. A., and Aronow, B. J. (2002) Mol. Cell. 1. Yerbury, J.J., Stewart, E.M., Wyatt, A.R., and Wilson, Biol. 22, 1893-1902 M.R. (2005) EMBO Rep. 6, 1131-1136 20. Harold, D., Abraham, R., Hollingworth, P., et al. 2. Wyatt, A.R., Yerbury, J.J., Ecroyd, H., and Wilson, (2009) Nat. Genet. 41, 1088-1093 M.R. (2013) Annu. Rev. Biochem. 82, 295-322 21. Lambert, J.C., Heath, S., Even, G., et al. (2009) Nat. 3. Wyatt, A.R., and Wilson, M.R. (2010) J. Biol. Chem. 285, Genet. 41, 1094-1099 3532-3539 22. Wijsman, E.M., Pankratz, N.D., Choi, Y., Rothstein, 4. Bruschi, M., Candiano, G., Santucci, L., and Ghiggeri, J.H., Faber, K.M., Cheng, R., Lee, J.H., Bird, T.D., G.M. (2013) Biochim. Biophys. Acta 1830, 5473-5479 Bennett, D.A., Diaz-Arrastia, R., Goate, A.M., Farlow, 5. Becatti, M., Marcucci, R., Bruschi, G., Taddei, N., Bani, M., Ghetti, B., Sweet, R.A., Foroud, T.M., and Mayeux, D., Gori, A.M., Giusti, B., Gensini, G.F., Abbate, R., R. (2011) PLoS Genet. 7, e1001308 and Fiorillo, C. (2014) Arterioscler. Thromb. Vasc. Biol. 23. Bell, R.D., Sagare, A.P., Friedman, A.E., Bedi, G.S., 34, 1355-1361 Holtzman, D.M., Deane, R., and Zlokovic, B.V. (2007) 6. Ursini, F., Davies, K.J.A., Maiorino, M., Parasassi, T., J. Cereb. Blood Flow Metab. 27, 909-918 and Sevanian, A. (2002) Trends Mol. Med. 8, 370-374 24. Cascella, R., Conti, S., Tatini, F., Evangelisti, E., 7. Campioni, S., Mannini, B., Zampagni, M., Pensalfini, Scartabelli, T., Casamenti, F., Wilson, M.R., Chiti, F., A., Parrini, C., Evangelisti, E., Relini, A., Stefani, M., and Cecchi, C. (2013) Biochim. Biophys. Acta 1832, 1217- Dobson, C.M., Cecchi, C., and Chiti, F. (2010) Nat. 1226 Chem. Biol. 6, 140-147 25. DeMattos, R.B., O’Dell M, A., Parsadanian, M., Taylor, 8. Seong, S.Y., and Matzinger, P. (2004) Nat. Rev. J.W., Harmony, J. A., Bales, K.R., Paul, S.M., Aronow, Immunol. 4, 469-478 B.J., and Holtzman, D.M. (2002) Proc. Natl. Acad. Sci. 9. Grad, L.I., Yerbury, J.J., Turner, B.J., Guest, W.C., USA 99, 10843-10848 Pokrishevsky, E., O’Neill, M. A., Yanai, A., Silverman, 26. DeMattos, R.B., Cirrito, J.R., Parsadanian, M., May, J.M., Zeineddine, R., Corcoran, L., Kumita, J.R., P.C., O’Dell, M.A., Taylor, J.W., Harmony, J.A., Luheshi, L.M., Yousefi, M., Coleman, B.M., Hill, A.F., Aronow, B.J., Bales, K.R., Paul, S.M., and Holtzman, Plotkin, S.S., Mackenzie, I.R., and Cashman, N.R. D.M. (2004) Neuron 41, 193-202 (2014) Proc. Natl. Acad. Sci. USA 111, 3620-3625 27. Yerbury, J.J., Kumita, J.R., Meehan, S., Dobson, C.M., 10. Guo, J.L., and Lee, V.M.Y. (2014) Nat. Med. 20, 130-138 and Wilson, M.R. (2009) J. Biol. Chem. 284, 4246-4254 11. Coker, A.R., Purvis, A., Baker, D., Pepys, M.B., and 28. Yerbury, J.J., and Wilson, M.R. (2010) Cell Stress Wood, S.P. (2000) FEBS Lett. 473, 199-202 Chaperones 15, 115-121 12. Clark, A.M., and Griswold, M.D. (1997) J. Androl. 18, 29. Wu, S.M., Patel, D.D., and Pizzo, S.V. (1998) J. 257-263 Immunol. 161, 4356-4365 13. Kwon, H.S., Kim, T.B., Lee, Y.S., Jeong, S.H., Bae, Y.J., 30. Wyatt, A.R., Kumita, J.R., Mifsud, R.W., Gooden, Moon, K.A., Bang, B.R., Moon, H.B., and Cho, Y.S. C.A., Wilson, M.R., and Dobson, C.M. (2014) Proc. (2014) Ann. Allergy Asthma Immunol. 112, 217-221 Natl. Acad. Sci. USA 111, E2081-2090 14. Calero, M., Rostagno, A., Matsubara, E., Zlokovic, B., 31. Davies, M. J. (2011) J. Clin. Biochem. Nutr. 48, 8-19 Frangione, B., and Ghiso, J. (2000) Microsc. Res. Tech. 32. Van Gool, D., De Strooper, B., Van Leuven, F., Triau, 50, 305-315 E., and Dom, R. (1993) Neurobiol. Aging 14, 233-237 15. Freixes, M., Puig, B., Rodriguez, A., Torrejon- 33. Adler, V., and Kryukov, V. (2007) Neurochem. J. 1, 43- Escribano, B., Blanco, R., and Ferrer, I. (2004) Acta 52 Neuropathol. 108, 295-301 34. Hollander, W., Colombo, M.A., Kirkpatrick, B., and 16. Wang, H., Ma, J., Tan, Y., Wang, Z., Sheng, C., Chen, Paddock, J. (1979) Atherosclerosis 34, 391-405 S., and Ding, J. (2010) J. Alzheimers Dis. 21, 597-610 35. Machovich, R., and Owen, W.G. (1997) Arch. Biochem. 17. Ishikawa, Y., Akasaka, Y., Ishii, T., Komiyama, K., Biophys. 344, 343-349 Masuda, S., Asuwa, N., Choi-Miura, N. H., and 36. Harpel, P.C., and Mosesson, M.W. (1973) J. Clin. Tomita, M. (1998) Arterioscler. Thromb. Vasc. Biol. 18, Invest. 52, 2175-2184 665-672

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 17 Short Discussions SDS Page for Students Page

Conference Survival Guide – The Dos and Don’ts

One of the best perks of doing research is being able to attend conferences around the world, especially if they are held in idyllic locations. As a postgraduate student or early career researcher, you are often encouraged to attend conferences to receive feedback on your work, share ideas with peers, practice your science communication skills and promote your research. It may seem daunting having to present in front of a group of experts in your field and you may have a number of excuses why you should not attend one – not enough time, not enough data or that you don’t know anyone. But think of all the new people you can meet, new ideas you may generate and how much fun you can have at the conference dinner, where plenary speakers let their hair down and party like it’s 1999. Here are a few tips on how to get the most from your conference attendance:

1. Choosing the right conference Do: • Attend a conference where you have the opportunity to present your work, either as a poster or an oral presentation. That way you feel like an active Sightseeing in Berlin, Germany, after attending the 40th FEBS participant rather than just a spectator. Congress in July 2015. I was very fortunate to receive funding • Choose a conference that is more focused on your to attend the meeting from the ASBMB Fred Collins Award. area of research. This might make networking less daunting. Don’t: • Location, location, location! Choosing a destination • Expect your supervisor to cover all the costs. Your such as Barcelona or LA for a conference is always a supervisor may have some funding set aside to allow good excuse to escape the Australian winter. students to attend conferences but this should not be Don’t: an expectation. • Choose a conference that nobody has ever heard about before. There is a worrying, increasing trend of fake 3. Registration Process conferences and you don’t want to be short-changed. Do: • Choose a conference based on location alone or only • Register before the early bird registration closes because it is a convenient time of the year to go. (typically three months before the conference kicks off). Booking in advance not only keeps your 2. Apply for Funding registration price lower, but will also keep your travel Conferences can be expensive so it is always a good idea and accommodation costs lower. to seek for funding support. • Have your abstract ready far enough in advance to Do: allow your supervisor and collaborators to review it • Apply for departmental or university travel well before the deadline. fellowships. Keep an eye on the application deadlines Don’t: so you don’t miss the opportunity to apply. • Miss the early bird deadline. A lot of the time the early • Become a member of a professional society like the bird deadline coincides with the oral presentation Australian Society for Biochemistry and Molecular deadline so you don’t want to miss the opportunity to Biology (ASBMB), which has fellowships to assist be considered for an oral presentation by not making recipients to attend overseas conferences. Bear in mind the deadline. that for a lot of the awards you need to be a member of • Write your abstract at the last minute. Being considered the society for a period of time before being eligible to for a presentation usually comes down to a well- apply. written abstract.

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4. Conference Preparation 6. Network, Network, Network! Do: Do: • Keep up with the literature, so you have an idea what • Sell yourself. You may be finishing your PhD or to expect. looking for a new postdoc position. Meeting members • Read the conference abstracts and select any talks or of other labs may help you make a more informed poster presentations you want to attend. Typically the decision about your next career move. Listen to what final schedule for a conference will be posted about a research other people are doing and think about what month in advance. Pick out presentations that sound you would like to learn/do in your next position. interesting and create a schedule for each day. • Have informal discussions with people who attended • Read about the plenary speakers. You may have the your presentation. They may come up with useful opportunity to speak to them and you would sound feedback and it is a good opportunity to strengthen very impressive if you could bring up some of their your connections. published work or it may help you ask better questions. • Have a chat to speakers; they may be your future • Prepare, prepare, freak out, prepare for your employer. presentation. It is always a good idea to have a • Get involved in social activities. Although it may be rehearsal in front of your lab group and have them tempting to just hang out with people you already think of possible questions you may get. know, try to interact with new people. Go to dinner or Don’t: a drink with your new contacts outside the conference • Plan a busy schedule. A conference program may venue. Even if you are shy by nature, try your best to be overwhelming, particularly if you attend a large meet and talk to as many people as possible, whether conference with plenary lectures, parallel sessions and they be students, postdocs or professors. You’ll likely multiple poster sessions. Try to find a balance between find that most people are quite friendly and willing to attending interesting presentations and leaving some engage. free time in your schedule to rest and network. Don’t: • Overstress about your presentation. Rehearse but • Spend all your free time getting free stuff from the don’t obsess. When you become so stressed that you industry booths. Although it may seem like a good can’t stop thinking about it every day leading up to the idea getting free pens, USB sticks etc, remember that conference, it is time to stop. most of these end up in the back of your wardrobe and only get found again when you are moving. 5. Attending a Session • Burn yourself out. Although it may not seem like it, Enthusiasm is usually at its highest at the start of the listening to endless talks and networking can be very conference but that can quickly go away when you attend tiring. Make the time to relax. an uninteresting session. Do: When you return from your conference, before going • Take a pen and notepad with you at all times. back to your everyday routine, spend some time going • Plan to stay for all presentations in a session. Some through the program and have a look at your notes. Have presentations may be easier to understand than others a think about how you can put some of the ideas you got but you never know what you may learn. into action. It is also important to solidify the contacts you • Think of questions to ask. Even if you do not have the made by sending a follow up email. courage to ask them, it may help you stay alert. You I hope these tips can make your next conference may also have the opportunity to ask the questions experience more enjoyable. Remember to just relax and later during a break. have fun. You will be surrounded by people who are just Don’t: as excited about science as you are. Lastly, take the time • Fall asleep. If you are feeling tired before the start of to do some sightseeing in your free time. You will most a session, sit near an exit so you can quietly sneak out likely be in an exciting location so do a bit of travelling if when tiredness gets the best out of you. you can. Mixing work with pleasure is one of the pros of being a research scientist after all!

The Student’s Page is coordinated by Dr Tatiana Soares da Costa, who is an NHMRC Early Career Fellow at the La Trobe Institute for Molecular Science ([email protected]).

Send us your articles! If you’ve recently (in the past 12 months) published an interesting article, please feel free to send its details onto the Editor ([email protected]) as we are always looking for new articles to profile in our ‘Cutting Edge’feature.

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 19 International ComBio2016 Plenary Speakers Brisbane 3 to 7 October 2016

Andrea Ballabio, Telethon Institute of Genetics and Medicine, Italy Andrea Ballabio is the founder and director of the Telethon Institute of Genetics and Medicine (TIGEM) in Naples, Italy. He is Professor of Medical Genetics at the University of Naples Federico II and Visiting Professor at both Baylor College of Medicine and University of Oxford. His research interests are focused on biological mechanisms and innovative therapies for genetic diseases. His recent discovery of a gene network that regulates lysosomal biogenesis and autophagy expanded our view of the lysosome from a degradation and recycling center to a signaling hub that controls cell homeostasis. He was President of the European Society of Human Genetics, Council member of EMBO and recipient of an ERC Advanced Investigator Grant. He has received numerous national and international awards for research and culture, among them the 2007 Award of the European Society of Human Genetics, the Knighthood of the Italian Republic by the President of Italy in 2007 and the 2016 Louis-Jeantet Prize for Medicine. Xiaofeng Cao, Centre for Genome Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Science, China Professor Xiaofeng Cao obtained her BSc and MSc degree in Biochemistry from Peking University and China Agricultural University, respectively. She received her PhD in Molecular Biology from Peking University. Professor Cao had postdoctoral training with Professor Steve Jacobsen at UCLA, where she initiated her studies on plant epigenetics. In 2003, Dr Cao was awarded a Young Talented Investigator Award in China and took up a professorship at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing. The Cao lab investigates the epigenetic control of plant development, genome stability and stress response. Her lab uses rice as a model to study plant adaptation to high temperature. Professor Cao has received many awards including the China Young Female Scientists Award (2010), the National Outstanding Scientist Award (2010) and the DuPont Young Professor Award (2008). She is an editor for several leading journals including The Plant Cell and Current Opinion in Plant Biology, and is Associate Editor-in-Chief for Science China Life Sciences. In 2015, she was elected an Academician of the Chinese Academy of Science. Jennifer Elisseeff, Translational Tissue Engineering Center, John Hopkins University, Maryland, USA Dr Elisseeff received a bachelors degree in Chemistry from Carnegie Mellon University and a PhD in Medical Engineering from the Harvard-MIT Division of Health Sciences and Technology. She was then a Fellow at the National Institute of Dental and Craniofacial Research. In 2001, she became an Assistant Professor in the Department of Biomedical Engineering at Johns Hopkins University. In 2004, she cofounded Cartilix, Inc., a startup that translated adhesive and biomaterial technologies for treating orthopedic disease. In 2009, she also founded Aegeria Soft Tissue and Tissue Repair, focused on soft tissue regeneration and wound healing. Dr Elisseeff is now the Jules Stein Professor at the Wilmer Eye Institute and directs the recently established Translational Tissue Engineering Center at Johns Hopkins. She serves on the Scientific Advisory Boards of Bausch and Lomb, Kythera Biopharmaceutical, and Cellular Bioengineering Inc. Dr Elisseeff has received awards including the Carnegie Mellon Young Alumni Award, Arthritis Investigator Award (Arthritis Foundation), the Yasuda Award (Society of Physical Regulation in Medicine and Biology), and was named by Technology Review magazine as a top innovator under 35 in 2002 and top 10 technologies to change the future. She has published over 120 articles, book chapters and patent applications and given over 130 national and international invited lectures. Page 20 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 International ComBio2016 Plenary Speakers Brisbane 3 to 7 October 2016

Harsha Gowda, Institute of Bioinformatics, India Harsha Gowda is a faculty scientist at the Institute of Bioinformatics, Bangalore, India and Associate Director of the YU-IOB Center for Systems Biology and Molecular Medicine at Yenepoya University. He did his PhD at the Institute of Bioinformatics in Dr Akhilesh Pandey’s laboratory at Johns Hopkins University, USA, on proteomic profiling of pancreatic cancers. He did his postdoctoral work in Dr Gary Siuzdak’s laboratory at the Scripps Center for Metabolomics and Mass Spectrometry, USA. His research group employs cutting-edge technologies in genomics, proteomics and metabolomics to investigate molecular mechanisms that drive cancers and to identify biomarkers and therapeutic targets. In addition, he is using proteogenomics methods to identify novel protein coding regions in the human genome. He is a recipient of a Wellcome Trust-DBT Fellowship awarded to promising young researchers in India and the Sir CV Raman young scientist award by the Karnataka state government. He is a reviewer for several international journals and an Editorial Board member of the Journal of Proteomics and Journal of Proteins and Proteomics. He is a member of the Indian Association for Cancer Research and an elected executive council member of the Proteomics Society of India.

Dave Jackson, Cold Spring Harbor Laboratory, New York, USA Dave Jackson is a Professor of Plant Genetics at Cold Spring Harbor Labs, NY. He obtained his PhD at the John Innes Institute, Norwich, UK, working with Cathie Martin and Keith Roberts, and did his postdoc with Sarah Hake at the Plant Gene Expression Center, Berkeley, CA. He has been at CSHL since 1997, where his lab studies genes and signals that regulate plant growth and architecture. Recent examples of his research include discovery of a heterotrimeric G protein subunit that controls stem cell proliferation. This protein interacts with a completely different class of receptors than in animals, which helps explain how signaling from diverse receptors is achieved in plants. They also demonstrated that a weak mutation in one of the developmental genes enhances seed production in maize, which could lead to yield increases. The lab has also characterised networks of gene expression, using next-gen profiling and chromatin immunoprecipitation methods that have revealed new hypotheses in networks controlling inflorescence development.

Shigeru Kondo, Faculty of Frontier Bioscience, Osaka University, Japan Shigeru Kondo is a Professor of Molecular Biology at Osaka University. He started his scientific career as an immunologist (1983) in the graduate school of medicine atKyoto University, where he learned the basic technology of molecular biology. After getting his PhD, he was interested in animal morphogenesis and joined the laboratory of Professor Walter Gehring at Basel University as a postdoc to learn developmental biology (1991). During his stay in Basel, he met Professor Hans Meinhardt who impressed him with the beauty of mathematical models. He returned to Kyoto University in 1993 to work in immunology because he could not find a job as a developmental biologist. He worked in experimental immunology during the daytime. However, in the evening and on the weekend, he took care of a tropical angelfish in his apartment. During the growth of the fish, the stripe pattern dynamically changed in the way that the Turing’s reaction-diffusion equation predicted, which turned out to be proof of the Turing pattern. The paper was published in Nature (1995), and his boss fired him due to his secret research. What happened after that, you can find out at his talk.

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Xia Li, College of Plant Science and Technology, Huazhong Agricultural University, China Xia Li received her PhD from Purdue University in the United States, and then joined Vector Tobacco Inc (USA) as a research scientist from 2001 to 2004. In 2004, she was selected as one of the Hundred Talents of the Chinese Academy of Sciences (CAS) and became a Principal Investigator in the Institute of Genetics and Developmental Biology, CAS. In 2015, she moved to the College of Plant Science and Technology, Huazhong Agricultural University, as an outstanding professor. Her work is focused on the molecular genetics of legumes- rhizobia symbiosis. Additionally, her lab is interested in molecular mechanisms underlying plant response to abiotic stresses, with a focus on the ABA signaling pathway and plastic development.

Patrick Lupardus, Department of Structural Biology, Genentech, California, USA Dr Patrick Lupardus is a Senior Scientist at Genentech in South San Francisco, California. He received his bachelor’s degree from the University of Wisconsin-Madison in 1999, followed by his doctorate in Molecular Pharmacology from Stanford University in 2005. He then moved on to postdoctoral training in Structural Biology at Stanford and received a Damon Runyon Cancer Research Foundation fellowship for his work on structural characterisation of JAK kinases and cytokine receptors in 2007. In 2010, he joined the Department of Structural Biology at Genentech. His group utilises biochemistry, structural biology, biophysical methods, and computational techniques to investigate protein–protein and protein–small molecule interactions of therapeutic interest. In addition, his lab has a basic research focus revolving around the structural biology receptor-coupled kinase signalling cascades involved in innate and adaptive immunity.

Gene Myers. Max-Planck Institute of Molecular Biology and Genetics, Dresden, Germany Gene Myers is a computer scientist and biotechnologist known for the BLAST search engine and the sequencing of the human genome, for which he advocated whole genome shotgun sequencing. He developed an assembler to do so at Celera Genomics, and assembled high- quality reconstructions of the fruit fly, human, mouse, and mosquito genomes in rapid succession. In the 80s, Myers invented algorithms for sequence comparison and searches including suffix arrays that enable the Burroughs-Wheeler transform used in today’s compact indices for genomic search. In the 90s, Myers created and perfected the string graph approach to DNA sequencing used at Celera. From 2005 to today, he has focused on the construction of novel microscopes and software for building single cell expression atlases across developmental epochs. Myers has been a professor at the University of Arizona and UC Berkeley, a vice president at Celera Genomics, a group leader with HHMI, and currently is a director of the Max-Planck Society. He is a member of the National Academy of Engineering, USA, the National Academy of Germany, and won the ACM Kannellakis Prize in 2002.

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Andrew Oates, The National Institute for Medical Research, London, UK After undergraduate studies at the University of Adelaide, Andrew received his PhD at the Ludwig Institute for Cancer Research and the University of Melbourne working with Andrew Wilks. His postdoctoral time was at Princeton University and the University of Chicago in the lab of Robert Ho, where his studies on the segmentation clock in zebrafish began in 1998. In 2003 he moved to Germany and started his group at the Max Planck Institute for Molecular Cell Biology and Genetics in Dresden. In 2012 he accepted a position at University College London as Professor of vertebrate developmental genetics and moved his group to the MRC- National Institute for Medical Research at Mill Hill in London. Since April 2015, he has been a member of the Francis Crick Institute in London.

Lacey Samuels, Department of Botany, University of British Columbia, Canada Lacey Samuels is a Professor at the University of British Columbia in Vancouver, BC, Canada. She is also the Institutional Leader of the UBC Centre for the Integration of Research, Teaching and Learning. She has a BSc in Honours Neurobiology from McGill University in Montreal, and a PhD in Botany from the UBC Vancouver. She did postdoctoral studies at the University of Colorado, Boulder, USA and became an Assistant Professor at UBC in 2000. Research in the Samuels laboratory focuses on biosynthesis of plant cell walls, both cell wall polysaccharides as well as specialised cell wall components, such as lipidic waxes on the plant surface and lignin in wood. The approach is to integrate advanced microscopy techniques with molecular biology and biochemistry to put cell wall biosynthesis and secretion into a cellular context.

Joseph Thornton, Department of Human Genetics & Department of Ecology and Evolution, University of Chicago, USA Joe Thornton is a Professor in the Departments of Human Genetics and Ecology and Evolution at the University of Chicago, Illinois, USA. After studying English literature at Yale University, he worked for a decade as an environmental activist. He then pursued graduate and postdoctoral training in molecular biology, phylogenetics and evolution at Columbia University and the American Museum of Natural History, both in New York, USA, where he began to study the molecular mechanisms by which nuclear receptor proteins diversified in sequence, structure and function. His laboratory has played a key role in establishing a ‘functional synthesis’ in molecular biology and evolution. His lab has also developed ancestral protein resurrection as a strategy for characterising the mechanisms of protein evolution. He has received the Hans Falk Award from the National Institute of Environmental Health Sciences, the US Presidential Early Career Award for Scientists and Engineers, a Howard Hughes Medical Institute Early Career Scientist Award, and a Guggenheim Foundation Fellowship.

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Shubha Tole, Tata Institute for Fundamental Research, India Professor Shubha Tole obtained her BSc in Life Sciences and Biochemistry from St Xavier’s College, Mumbai in 1987. Her MSc and PhD are from Caltech, USA. She worked at the University of Chicago as a postdoctoral fellow, and then joined the Tata Institute in Mumbai, India as a faculty member in 1999. Her research interests focus on the mechanisms that shape the development of the brain. Tole’s lab discovered an ‘organiser’ in the developing brain, a signalling centre that induces the formation of the hippocampus. They created embryos with multiple signaling centers and discovered that each induces a hippocampus in adjacent neuroepithelium. Tole has received the Infosys prize for Life Sciences (2014), the SS Bhatnagar Award (2010), the Research Award for Innovation in Neurosciences (2008) from the Society for Neuroscience and the Wellcome Trust Senior International Fellowship (1999). She has offered workshops on Ethics in Science at national and international forums She also teaches science communication, actively engages in public outreach via workshops in schools and colleges, and encourages her students to do the same. Jennifer Van Eyk, Cedars-Sinai, California, USA Dr Jennifer Van Eyk is a Professor of Medicine at Cedars-Sinai Medical Center, Director of Basic Science Research in the Barbra Streisand Woman’s Heart Center and Director of the new Advance Clinical Biosystems Institute where she recently moved to from Johns Hopkins University. The Van Eyk laboratory’s central philosophy is that compelling biological and clinical questions drive innovation through development, optimisation and adaption of proteomic technologies, functional analysis, and large-scale data handling. The primary research focuses i) on understanding the molecular mechanism underlying acute and chronic disease and treatment therapies and ii) in the development of clinically robust circulating biomarkers focusing primarily on cardiovascular disease and women’s health.

Rajeev Varshney, International Crops Research Institute for Semi Arid Tropics, India Rajeev Varshney is a Global Research Program Director of the Genetic Gains Research Program for crop development at ICRISAT. This program encompasses different disciplines including Genebank, Pre-Breeding, Genomics & Trait Discovery, Cell & Molecular Biology and Genetic Engineering, Forward and Integrated Breeding, and Seed Systems. Rajeev recently won the Shanti Swarup Bhatnagar Award – Biological Sciences, the most coveted and prestigious award from the Indian Government. He is internationally recognised for his contribution to genome sequencing of pigeonpea, chickpea, peanut, pearl millet, sesame, mungbean and adzuki bean and the development of molecular breeding products in chickpea, pigeonpea and peanut. Rajeev has published over 300 publications in journals such as Nature, Nature Biotechnology, Nature Communications, PNAS, etc. Rajeev is a Fellow of various societies, including the Crop Science Society of America (CSSA), the Indian National Science Academy (INSA), the National Academy of Sciences, India (NASI), and the National Academy of Agricultural Sciences, India (NAAS). Rajeev has received several prestigious awards, including the Research Excellence India Citation Award 2015, the Illumina Agricultural Greater Good Initiative Award, and the NASI-Scopus Young Scientist Award. Rajeev has been an invited speaker at several conferences including the G-8 Conference on Open Data for Agriculture, the FAO conference on Agricultural Biotechnologies, and brainstorming session on digital agriculture chaired by Bill Gates.

Page 24 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 EPPENDORF EDMAN AWARD REPORT Networks Social and Posttranslational Benjamin Schulz reports on his Eppendorf Edman Award travels As a recipient of the 2015 with pirates in centuries past. The braver souls enjoyed a Eppendorf Edman Award relaxed swim in the warm waters. from the ASBMB, I was Unscheduled excitement struck on the final day of the fortunate enough to be able meeting, with the arrival of Typhoon Linfa. The tropical to attend the 2015 Gordon storm was expected to make landfall directly at Hong Research Conference (GRC) on Kong, prompting an emergency ‘Typhoon Number 8 Posttranslational Modification Signal’ to be called. This meant that all public transport Networks. Held at the Hong Kong University of Science and non-essential services were to be closed for the and Technology, on the eastern outskirts of Hong Kong, duration of the storm. The last session of the conference the meeting promised to showcase research linking was rushed through literally in double time, but more proteomics, posttranslational modifications of proteins, unfortunately the conference banquet was also cut short and cellular regulatory networks. to allow the staff to travel home before public transport Although Gordon Research Conferences have a long and stopped. Luckily there was still enough time to enjoy some distinguished history, the Hong Kong site is a relatively delicious jellyfish and dumplings before a spontaneous new GRC location. I certainly enjoyed attending a major post-banquet gathering waiting for the typhoon to hit. international conference without jetlag! The location also Fortunately, the typhoon made landfall to the east of increased attendance from Hong Kong and mainland Hong Kong and was downgraded early in the evening. China, which made for an internationally diverse meeting. I spent the day after the conference seeing the sites of The conference organisers did a stellar job of showcasing Hong Kong with some new friends from the conference. exciting research from a variety of excellent researchers. We enjoyed a Yum Cha lunch with plenty of amazing Invited talks were spread evenly amongst women and dumplings, caught the bus up Victoria Peak, enjoyed a men, and young and established researchers from Europe, surprisingly wild bushwalk around the peak, and came North America and Asia. back down on the precipitously steep funicular Peak Tram. A key feature of GRCs is that much of the research The conference highlighted to me the amazing diversity presented is unpublished, and that conference proceedings and biological importance of posttranslational protein are to be treated as private communications. Nonetheless, modifications. However, while the Posttranslational some of the work presented was already published. An Modification Networks conference was strong on overarching theme of the meeting was the development posttranslational modifications, the networks actually of new approaches in mass spectrometry to identify and discussed were professional and social. I certainly quantify posttranslational modifications on proteins. hope these new collaborations will bring us closer While pushing technology is always exciting, it was to understanding the roles of networks of protein made even more so at this meeting by being directly used posttranslational modifications in biological systems. to ask fundamentally interesting questions in biology. Ben Schulz is an NHMRC Career Development Fellow Presentations that particularly stood out to me were and Senior Lecturer in the School of Chemistry and from: Pedro Beltrao (EMBL, EBI, UK) who presented his Molecular Biosciences, University of Queensland. recent work suggesting that many phosphorylation sites Left: View are evolutionary noise, rather than being functionally of Hong important; Feng Shao (National Institute of Biological Kong from Sciences, China) who described diverse newly-identified Victoria modifications by bacterial pathogens that covalently Peak. modify proteins of the host cell; and Barbara Panning (UCSF, USA) who told us how stem cells use glycosylation to count to two. The opportunities for social and professional networking are clearly a highlight of any conference. GRCs have relatively few attendees and are held as far as possible from exciting locations – by design. This traps conference attendees and forces social interactions. All attendees including invited and plenary speakers also stay for the entire length of the conference, so it was relatively easy to find and meet with people. I certainly enjoyed many Right: relaxed discussions over breakfast, lunch, or drinks, which Boat cruise sparked research questions and potential collaborations. from the Organised activities were also held, with a boat cruise Hong Kong one afternoon taking conference attendees around the University of bay to the east of the campus. We anchored in one of the Science and small protected bays that made Hong Kong so popular Technology. Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 25 Off the Beaten Track

Written by former researchers who have now established careers outside of research, Off the Beaten Track is intended to give the readers insights into the range of alternative careers available to them. Authors describe the paths they have taken to arrive at their present career and provide a detailed description of exactly what the job entails on a day-to-day basis. You Can Trust Me, I’m a Regulatory Scientist Michael Dornbusch, Assistant Secretary, Evaluation Branch, Office of the Gene Technology Regulator, Australian Government Department of Health

Let me begin by saying that I never really imagined that I that control the sale and use of chemicals, medicines would be in the type of job I have now. When I was a child and food, activities that impact on the environment and I was fascinated by dinosaurs, archaeology and human even some types of research. The reason they do this is evolution. As I went through school I imagined my career to protect people and the environment from the possible in science or architecture. I was accepted into university harmful effects of these products/activities. These to study both. I decided to go to the same university as agencies employ scientists to conduct risk analysis using my best friend and that meant I would study science. sound science for regulatory decision making – regulatory During my undergraduate degree, I enjoyed studying scientists. The regulatory scientists provide the technical biochemistry and microbiology, and I was interested inputs into decisions about whether or not the products in recombinant DNA technology. I considered doing or activities outlined above should be approved and what honours, but decided that I wanted to see what science risk management measures, if any, might be required to jobs were really like. ensure that people and the environment are protected. I worked for a time as a research technician in a veterinary Regulatory scientists often have PhD-level research school and in human pathology laboratories. Looking training and postdoctoral experience. Searching literature, back, I am glad I decided to do this. These jobs gave me a critically analysing scientific information, identifying gaps better understanding of bench science and research and I in the information, drawing valid conclusions using a was introduced to such things as standards, accreditation weight of evidence approach, writing concise, accurate and and the regulation of the work of science. readable reports and giving oral presentations are critical After a few years, I decided to pursue a career in for regulatory scientists. In addition to practising those research. I undertook a PhD in an area that could be skills learnt during my research training, I have learnt new described as veterinary biochemical toxicology. Although things on the job – the art of risk analysis, administering it was hard work, and I didn’t quite realise it at the time, and complying with legislation, government processes I had developed extremely useful skills that would turn and many others. Apart from doing assessment work out to be critical for my future career. (A bit more about at the desk, regulatory staff also attend and present at that later). conferences and other meetings and travel to inspect field After my PhD, I was offered a role in a project aiming trials of GM crops and visit a range of laboratories and to commercialise a novel treatment for gastric ulcers. The role was a mix of research and development work, with one of my tasks being the preparation of information for future regulatory submissions. It was at this time that I realised that there were potential careers outside of the usual postdoctoral researcher/academic career path. I saw a job ad for a scientist to work in a government regulatory agency and applied. After working in a research type environment, my first job in government was quite a change. Over the last 20 years, I have worked in the regulation of pesticides, veterinary medicines, human complementary medicines, industrial chemicals and most recently, gene technology. I have made the change from research scientist to regulatory scientist. So what is regulatory science and how is research training relevant to working in government? A number of government agencies administer laws OGTR staff inspecting a high-level containment facility. Page 26 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 Off the Beaten Track containment facilities. As many of us tend to, I have now and presenting information on Australia’s regulatory moved into a management/leadership role and spend system. We have also received a number of international ever more time on such things as strategic and operational delegations to our office. It’s interesting and varied work planning and management and stakeholder engagement. – trust me, I’m a regulatory scientist! However, I still have an active role in regulatory science. In my job heading up the part of the Office of the Gene Technology Regulator (OGTR) that does all of the risk assessments and processes applications for licences and certifications etc, one of my key responsibilities is to build and maintain the scientific, technical, and risk analysis capabilities of the organisation. It is an exciting time to be in this type of role. There is a lot of really interesting research and development work going on around the world and in Australia. Genetic modification techniques are continually and rapidly evolving and being applied in such areas as crops, vaccines and other therapeutics, bioenergy, and bioremediation. Australia’s regulatory system for gene technology and our approach to risk analysis are very highly regarded overseas. As a consequence I (and other staff in the office) have recently been visiting a number of different countries, helping to build risk analysis capabilities for GMOs Michael (back left) with visiting Indian government officials.

ELECTION OF COUNCIL 2017 Nominations are called for the following positions on the Council of the Australian Society for Biochemistry and Molecular Biology Inc for 2017: Secretary, Treasurer, Editor, Secretary for Sustaining Members and State Representatives for ACT, NSW, Qld, SA, Tas, Vic and WA.

The Council for the President M. Ryan period 1 January 2016 President Elect L. Tilley to 31 December 2016 Secretary B. Forbes# is composed of the Treasurer T. Piva# following members: Editor C.K. Liew* Education Representative J. Macaulay# Secretary for Sustaining Members S. Jay# Representatives for: ACT Y.P. Mabbitt# NSW K. Michie# Vic D. Stojanovski* * Retiring member, not Qld D. Ng# eligible for re-election SA S. Polyak# # Eligible for re-election Tas A. Holloway* WA N. Taylor#

Nomination forms are available on the ASBMB website. Nominations for all vacant positions must be signed and seconded by members of the Society. The nominations must be signed by the nominee to indicate his/her willingness to stand. If more than one nomination is received for any position, a ballot will be held to determine the successful candidate. All members may vote for all positions except those of State Representatives where election is by members in the State concerned.

NOMINATIONS MUST REACH THE SECRETARY 19 SEPTEMBER 2016 (14 DAYS BEFORE THE ANNUAL GENERAL MEETING TO BE HELD 3 OCTOBER 2016)

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 27 ASBMB Awards 2017

SOCIETY Medals, Awards and Fellowships NOW OPEN

Nomination or application forms for all 2017 Medals, Awards and Fellowships can be obtained directly from the ASBMB website: http://www.asbmb.org.au/awards.html

Nominations or applications must be submitted no later than 31 October 2016. Nominations or applications must be emailed to the Secretary of the Society, Briony Forbes: [email protected] Please note that hard copies are not required.

All applicants (excluding those for the Boomerang Award) must be current members of ASBMB. The requirement for the number of years of prior membership varies between awards. All recipients will receive complimentary registration for the 2017 ComBio meeting.

u NOMINATIONS FOR MEDALS AND AWARDS U

The Lemberg Medal is awarded to a distinguished ASBMB member who will present the Lemberg Lecture at the ComBio meeting. The Medal is presented in memory of Emeritus Professor M.R. Lemberg who was the Society’s first President and Honorary Member. The award will be made to an individual who has demonstrated excellence in biochemistry and molecular biology and who has made significant contributions to the scientific community. An honorarium is provided by ASBMB.

The Merck Research Medal is awarded to an outstanding ASBMB member with less than 15 years postdoctoral experience at the nominated deadline. The successful candidate will present the Merck Medal Lecture at the ComBio meeting. An honorarium is provided through the courtesy of Merck.

The Beckman Coulter Discovery Science Award is awarded to an ASBMB member for distinguished contributions to the field of biochemistry and molecular biology. The nominee should demonstrate involvement in research innovation, technology transfer, and communication. The Award is intended as a Travelling Lectureship to enable the awardee to present his/her work at a number of centres within Australia and New Zealand. The awardee will also present a Symposium talk at the ComBio meeting. The award carries an honorarium to cover the travelling expenses, provided through the courtesy of Beckman Coulter.

Page 28 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 ASBMB Awards 2017

u APPLICATIONS FOR TRAVEL AWARDS AND FELLOWSHIPS U

The Eppendorf Edman Award is awarded to an ASBMB member with no more than 7 years postdoctoral experience, in recognition of their outstanding research work. The Award provides funds to assist the recipient to attend an overseas conference in a field associated with biochemistry or molecular biology or to visit briefly a research laboratory in Australia or elsewhere to access specialised equipment or to learn new research techniques. The contribution to travel expenses is provided through the courtesy of Eppendorf South Pacific.

The Shimadzu Education Award rewards outstanding achievement in education in biochemistry or molecular biology, especially innovation and creativity in education, with a view to fostering leadership in this important area of the Society’s objectives. The Award will enable the recipient to participate in an international conference with a significant focus on education, or to spend a period of time at another institution for the purposes of undertaking developments in education in biochemistry and molecular biology. The recipient will present a lecture within the Education Symposium at the ComBio meeting. The contribution to travel expenses is provided through the courtesy of Shimadzu.

The Boomerang Award is awarded to an outstanding expatriate Australian biochemist or molecular biologist to allow them to return to Australia to present their work in a symposium at the ComBio meeting and to give seminars at universities or research institutes. This will provide the awardee with exposure in Australia and will facilitate interactions with local researchers. The Award makes a significant contribution to the cost of a return airfare and accommodation for ComBio, and towards domestic travel expenses to visit at least one other Australian city. Applicants must have been a member of a recognised Australian scientific society for at least 2 years, and be no more than 7 years since the award of their PhD. The contribution to travel expenses is provided by ASBMB.

The Awards Committee will also award several ASBMB Fellowships to postgraduate students who are no more than 2 years prior to the completion of their PhD degree or recently graduated postdoctoral researchers no more than 2 years subsequent to the award of their PhD degree. The contribution to travel expenses is provided by ASBMB. The most outstanding ASBMB Fellowship applicant may receive the Fred Collins Award. These travel grants are awarded to early career researchers, normally resident in Australia, in recognition of their outstanding work in an area of biochemistry and molecular biology. The Fellowships provide funds to assist the recipient to attend an overseas conference in a field associated with biochemistry or molecular biology, or to visit briefly a research laboratory in Australia or elsewhere to access specialised equipment or to learn new research techniques.

u APPLICATIONS FOR OTHER AWARDS U

The Bioplatforms Australia Award is awarded to a biochemist or molecular biologist with no more than 7 years postdoctoral experience working in the field of genomics, transcriptomics, proteomics, metabolomics or relevant bioinformatics. The award is based on recognition of outstanding research and the potential to carry out independent research. Preference is given to those setting up an independent laboratory for the first time. The Award provides $10,000 worth of access to the services provided by nodes of Bioplatforms Australia, provided through the courtesy of Bioplatforms Australia. The recipient will give a talk at the ComBio meeting.

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 29 The Cutting Edge The Cutting Edge profiles recent, interesting publications by ASBMB members. These short summaries showcase some of the latest research being carried out by our members by presenting the work in a brief but accessible manner. A dynamic intron retention program in the mammalian megakaryocyte and erythrocyte lineages. Blood (2016) 127, e24-e34

Christopher R. Edwards1, William Ritchie2,3,4, Justin J.-L. Wong2,4, Ulf Schmitz2,4, Robert Middleton2,3,4, Xiuli An5, Narla Mohandas6, John E.J. Rasko2,4,7 and Gerd A. Blobel1,8* 1Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, USA 2Gene and Stem Cell Therapy Program, Centenary Institute, University of Sydney, Camperdown, Australia 3Bioinformatics Lab, Centenary Institute, Camperdown, Australia 4Sydney Medical School, University of Sydney, Camperdown, Australia 5Laboratory of Membrane Biology, New York Blood Center, New York, USA 6Red Cell Physiology Laboratory, New York Blood Center, New York, USA 7Cell and Molecular Therapies, Royal Prince Alfred Hospital, Camperdown, Australia 8Perelman School of Medicine, The University of Pennsylvania, Philadelphia, USA *Corresponding author: [email protected] Names in bold denote members of ASBMB Summary In Depth Alternative splicing is a key biological process that Intron retention is not a new term in the ‘dictionary’ of promotes protein diversity, which is essential for cell/ molecular biologists. However, studies into the functions tissue specific functions. Over 95% of all multi-exonic of intron retention are few because intron-retaining human genes undergo alternative splicing in a tissue and transcripts that usually harbour premature-termination differentiation-stage-specific manner. Of all modes of codons (PTCs) are typically degraded by NMD and alternative splicing (including alternative 5′ and 3′ splice thus difficult to detect. We were first alerted to intron site selection, exon skipping, alternative promoters, and retention after observing sequencing reads mapping to mutually exclusive exons) intron retention has been the intronic regions in extremely deep RNA-seq data (over least studied and understood. Intron retention occurs 100 million reads per sample) generated from poly-A when an intron is transcribed into pre-mRNA, but is not enriched RNA libraries (1). We further developed a novel excised by the splicing machinery. In most cases studied, bioinformatics algorithm called IRFinder to improve the intron-retaining mRNA is exported into the cytoplasm detection of ‘true’ intron retention events. We achieved following addition of the 5′ cap and poly-A tail. In 2013, this by systematically recovering reads that do not map we reported in Cell that many normal intron-retaining uniquely to the reference genome due to the presence of transcripts from normal white blood cells are recognised repetitive sequences in introns. We also filter for intron- by the nonsense-mediated decay (NMD) machinery derived long and short non-coding RNA species (1). Intron and degraded, thereby preventing their translation into retention levels predominantly increased in the cytoplasm proteins (Fig. 1). Myeloid cells from normal human during granulocyte differentiation. It affects genes that are and mouse blood take advantage of this mechanism to enriched for functions relevant to granulocytes including reduce the level of, for example, a nuclear lamina protein, nuclear lamina and periphery. Importantly, we were Lmnb1. This process ensures a physiologically normal able to measure a marked increase in intron retention differentiation of promyelocytes into granulocytes, of affected genes by 10–100 fold following inhibition of characterised by the nuclei that become ring-shaped or NMD using chemical or RNA interference approaches mutilobular in terminal granulocytes. Indeed, reduced (1). This confirms that intron-retention is a mechanism granulocyte number and abnormal nuclear morphology for controlling gene expression mediated via NMD. Our were observed in mice when we introduced an ‘intronless’ efforts and those recently undertaken by others have led version of Lmnb1 transcripts into mouse bone marrow. to a resurgence of studies to dissect the roles of intron In 2014, a team from ANU showed the importance of retention in normal biology and cancer (1,2,3). A notable intron retention in normal mouse lymphoid cells. In discovery reported in Nature Genetics identified intron collaboration with Gerd Blobel’s group at the University retention as an important mechanism that inactivates a of Pennsylvania, we have now shown a dynamic intron myriad of classical tumour suppressor genes including retention program associated with the differentiation of p53, PTEN and CDKN2A in diverse solid cancers (3). megakaryocyte and erythrocyte precursors. More recently, intron retention has been shown to regulate gene expression during human erythroblast differentiation (4). The genes affected by intron retention in erythroblasts

Page 30 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 The Cutting Edge exhibit little overlap with those that we described in nucleus splicing granulocytes (1) and that of other haemopoietic cells. This indicates a tissue and differentiation-stage specific intron pre-mRNA regulation of intron retention. As opposed to granulocytes, PTC intron retention levels predominantly decreased during exons erythroblast differentiation. Our collaboration with Gerd Blobel’s group at the University of Pennsylvania confirmed observations by Pimentel et al. (5). Both studies demonstrate PTC AAAAA that intron-retaining genes are enriched for functions related to RNA processing and binding in erythroblasts.

Intron-retaining transcripts are enriched for PTCs and are associated with reduced gene expression, however they cytoplasm are not always subject to NMD. Degradation of intron- retaining transcripts may also occur via NMD-independent PTC mechanism(s), which remain to be determined. We ribosome NMD retained further examined intron retention changes during mouse intron erythroblast differentiation and demonstrated their conservation between human and mouse. Degraded mature mRNA Our main focus in this current study was to examine IR Fig. 1. Degradation of intron retaining transcript containing in megakaryocytes and erythroblasts and their common a premature termination codon (PTC) via nonsense mediated precursor cells (MEPs) in order to compare changes during decay (NMD) at the pioneer round of translation. lineage specification in human and mouse haemopoiesis. Similar to erythroblast differentiation, there is a decrease References in intron retention events when MEPs differentiate into 1. Wong, J.J.-L., Ritchie, W., Ebner, O.A., et al. (2013) Cell megakaryocytes. Nearly 600 genes exhibited differential 154, 583–595 intron retention in either lineage, many of which are 2. Cho, V., Mei, Y., Sanny, A., et al. (2014) Genome Biol. megakaryocyte or erythroid-specific. 15, R26 In summary, recent reports published by our group and 3. Jung, H., Lee, D., Lee, J., et al. (2015) Nat. Genet. 47, Pimentel et al. provide further evidence that intron retention 1242–1248 is widespread and is regulated during physiologically 4. Pimentel, H., Parra, M., Gee, S.L., et al. (2016) Nucleic normal cell differentiation and lineage specification. It is thus Acids Res. 44, 838–851 appropriate to recognise intron retention as an important 5. Edwards, C.R., Ritchie, W., Wong, J.J.-L., et al. (2016) contributor to tissue-specific control of gene expression. Blood 127, e24–34 Justin Wong and John Rasko

ANNUAL GENERAL MEETING of the AUSTRALIAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY Inc.

The 60th Annual General Meeting of the Australian Society for Biochemistry and Molecular Biology Inc. will be held in conjunction with the Annual Conference of the Society, in this instance at ComBio2016. The venue will be the Brisbane Convention and Exhibition Centre, Brisbane, on Thursday, 6 October at 1815 hours.

AGENDA 1. Apologies 2. Confirmation of the Minutes of Annual General Meeting No. 59 3. Results of Council Elections 4. President’s Report 5. Treasurer’s Report 6. Fees for 2017 7. Notification of Changes to the By-Laws of the Constitution 8. Changes to the Constitution 9. Any Other Business Briony Forbes Secretary, ASBMB

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 31 Science meets Parliament 2016 Briony Forbes and Suresh Mathivanan report on SmP2016

Science Meets Parliament is an annual event organised of Epidemiology and Public Health, ANU) and Dr Subho by Science & Technology Australia (STA). It brings Banerjee (Deputy Secretary, Department of Education and approximately 200 scientists from different backgrounds and Training) enlightened us with their thoughts and strategies societies together in Canberra over the course of two days, for bringing scientific advances into policy. Professor Banks with the aim of helping develop skills of communication highlighted the need to incorporate the intention and with politicians, policymakers and the members of the structure to work towards changing policy right from the media. This year, Associate Professor Briony Forbes beginning of undertaking scientific investigations. Professor (Flinders University) and Dr Suresh Mathivanan (La Trobe Graham Durant AM (Director, Questacon) shared his University) attended on the behalf of ASBMB. passion for inspiring engagement of the general public, and particularly young Australians, in science. He stressed the importance of science communication and education. The day was capped off with a workshop requiring audience participation and was conducted by Dr Rod Lamberts and Dr Will Grant (Australian National Centre for Public Awareness of Science, ANU). Here, we learned how to pitch our message to our politician whom we would meet the next day. There was a knockout competition to be the best to spruik our ‘science in 60 seconds’. This was a lot of fun and also allowed us to network with others. Finally we made our way to Parliament house for further networking and dinner with politicians and speeches from Christopher Pyne MP (Minister for Industry, Innovation and Science) and Bill Shorten MP (Leader of the Opposition) both of whom outlined their visions for innovation and science. Suresh Mathivanan and Briony Forbes at Parliament House. Day 2 Day 1 The next day, we worked our way back to Parliament As a short break from grant writing, we made our way house and through the crowds of visitors. There was much early on Tuesday March 1 to Canberra. This first day anticipation as each of us was allocated to a group of three focused on professional development and was held at the or four to meet with our allocated parliamentarians. While Hotel Realm. waiting, we heard from several politicians about their The day started with a welcome from Emeritus Professor visions for innovation. Those of us who were lucky enough Jim Piper AM (President, STA) and Catriona Jackson (STA to have meetings at other times could make their way to the CEO) followed by a welcome speech from the Nobel Laureate Brian Schmidt (ANU), who provided an overview of what to expect and key ways to get our message as scientists to politicians and the general public. Over the years, Brian has had significant interactions with politicians and policy makers regarding scientific matters and hence was able to precisely educate us on how to effectively communicate our science. More importantly, he enlightened us on some of the ways in which a scientist ‘should not’ interact with politicians and policy makers. Among the highlights of the day was a lively discussion between Paul Bongiorno AM (Contributing Editor for Ten News and columnist for The Saturday Paper) and Alison Carabine (Political Editor, ABC Radio National Breakfast) chaired by Kylie Walker, (Australian Academy of Science) that provided insight into how the media interacts with politicians and From left: Briony Forbes, Tony Zappia MP (Member for Makin), how scientists might make their message engaging for Lisa Akison (University of Queensland), Prasad Paradkar politicians and the media. Professor Emily Banks (Professor (CSIRO) and Chris Embry (SafeWork SA).

Page 32 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 Science meets Parliament 2016 National Press Club to hear the first address by the incoming Later in the afternoon, we managed to sneak into question Chief Scientist for Australia, Dr Alan Finkel AO. Briony at time, which was particularly entertaining and included this time was meeting with Tony Zappia MP (Member for some lively heckling from the public gallery at one stage! It Makin) who listened to descriptions of scientific activities was amazing to see parliament in action first hand. ranging from insulin peptide development, malaria We would like to thank the ASBMB for providing the research, the effects of alcohol on foetal development and funding to enable us to attend Science meets Parliament. the handling of dangerous explosive substances! Mr Zappia We are grateful to Catriona Jackson and the rest of STA for provided us with a great insight into the life of politics and organising such a comprehensive and informative two days. decision making in Canberra.

Biochemical Education: an ASBMB Special Interest Group

This year, the Biochemical Education SIG is under At ComBio 2016, the Education SIG will have a full new management. Our previous Chair, Janet Macaulay day program on Tuesday 4 October. This is a new and (Monash University), has been elected as the Chair of the improved presence for Education at ComBio and our four Committee for Education and Training for IUBMB. The SIG session chairs are lining up a top-notch set of speakers members thank Janet for her stewardship and give her our for the program. Amber Willems-Jones (University of hearty congratulations – we know she will enjoy her new Melbourne) and Terry Piva (RMIT University) are chairing role! You might like to take a look at Janet starring here: Session 1 on effective assessment and inquiry-based http://iubmb.org/about-iubmb/executive-committee/ learning in a practical setting. Nirma Samarawickrema current-members/ (Monash University) and Greg Blatch (Victoria University) Susan Rowland (University of Queensland) has taken are developing Session 2, which will address workplace over as the SIG Chair and is appreciating the helpful advice readiness, critical thinking, and students as partners. The from many experienced leaders in the Education space, SIG will also host a lunch for its members on day 1 of the especially Janet, Graham Parslow and Sally Jay. Susan is meeting. also a Member of the Education Committee of FAOBMB, We hope to see you at ComBio 2016! so the SIG members have front-line access to information Susan Rowland, Chair, ASBMB Education SIG about any and all Education initiatives from the FAOBMB and the IUBMB. As a teaching-focused academic, Susan has an interest in helping SIG members publish their Scholarship of Teaching and Learning work through a community of practice. This year, the SIG will have its first writing retreat; eight members of the SIG are spending three days before ComBio working on project ideas, examining data and drafting manuscripts. We hope to produce a series of papers for submission to BAMBED (Biochemistry and Molecular Biology Education) in the future. We will keep working on them throughout the year. We also hope that we will be able to have the retreat every year to coincide with ComBio. If you are a member of the Education SIG, you will be invited to join us. Janet Macaulay receives the 2013 Susan Rowland. FAOBMB Education Award from Professor Andrew Wang.

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 33 L for Lectin, L for Laura, L for Life

From Scottish PhD student to postdoctoral fellow in Australia, Laura McCaughey shows us that not letting the fear of failure stop you from giving things a go can sometimes lead to great results.

the Wellcome trust PhD program is very competitive and I felt I didn’t know ANY biology. It is at this point that I have to get a bit soppy and thank Laura McCaughey. my husband for making me go to the interview for the Wellcome Trust PhD program, as I was going through a Early Years complete crisis of confidence by this point. This was only Among the many great perks of being Scottish, Scottish heightened by the fact that the night before the interview, students are entitled to free university education, as all of the candidates were invited to dinner. This sounded long as they attend a Scottish University. For me, this like a nice idea, until the talk at the table traversed the made the prospect of university appear attainable from general awkward chitchat about the weather and turned a young age, without having to worry too much about towards science, and more specifically, biology. Having the financial consequences. Going through high school I only had a total of one series of lectures on biology over really enjoyed maths and science and I had a few ideas a whole five-year chemistry degree I had no idea what of what I wanted to do in the future. At the time of people were talking about (at this stage in my career I applying for university I was a massive fan of the TV likely thought that protein was just something that you show CSI (Crime Scene Investigation), which ultimately ate). After the meal I went home and was adamant I led me to undertake an integrated Masters degree in wasn’t going for the interview; there was no point, I wasn’t Forensic and Analytical Chemistry at the University of going to get it anyway. Luckily my husband marched me Strathclyde, Glasgow, UK. Although I really enjoyed to the interview and gave me a pep talk along the way. the course, and the forensics lecturers at Strathclyde In the end, the interview went great, I was offered the University were excellent, I felt that the TV show had PhD, and have loved biology ever since. The main advice misled me (who could have guessed?). The reality of I would now give to anyone in a similar position is first of being a forensic scientist wasn’t as glamorous or exciting all, believe in yourself, second realise that you never know as I had thought, or probably hoped, it to be (if only I what interviewers are looking for so don’t try and second had known that there would be no on-scene shoot outs guess them, and third just be yourself. or solving a crime in two days). After five years of study and deciding that forensic science was not for me, it left PhD Years me with the big question, ‘Where to next?’ I could not have found a better group of people to do my PhD with than those in Dr Dan Walker’s lab at the My First Taste of Biology University of Glasgow. The Walker group, including My final year Masters project was carried out in the myself, consisted of five PhD students, all of whom started lab of Professor Duncan Graham at the University of and completed their PhDs at the same time. There were Strathclyde. The main focus of the research in Professor no postdocs or technical staff, just five eager and excitable Graham’s lab is the creation of functionalised metallic PhD students. We all had different projects and developed nanoparticles that can be used for a variety of different purposes, including the diagnosis and treatment of disease. The thought of doing research that had direct links to benefiting human health really excited me, and so I started looking for PhDs that had health-related outcomes. It was at this point that I came across the Wellcome Trust four-year PhD program in Molecular Functions of Disease at the University of Glasgow. The format of this program seemed to be ideally suited to my situation. Having never studied biology, I had no idea what type of biology I wanted to focus on during my PhD. However, this program would give me the opportunity to undertake three 12-week projects on a range of biological topics before choosing one for my PhD project. Ideal! There was only one rather major hurdle left to negotiate; Halloween: scary what a PhD can do to your health! Page 34 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 different skills sets, which resulted in great collaborations across the projects over the three years. As a team we encouraged, challenged (annoyed) and inspired each other, and our outputs reflected our great working dynamic. Moreover, we were a very social group and I have made great friends for life. Dan was a great supervisor, always there if you needed his help (much to the annoyance of the person who shared his office I’m sure) but happy to let you explore your own ideas and make the project your own. I think this was a really critical learning point that helped me secure a fellowship straight out of my PhD. My project was to identify and characterise pyocins (protein antibiotics produced by Pseudomonas aeruginosa for intraspecies competition) and to test their therapeutic Current UTS lab group – Cynthia Whitchurch’s lab. potential in an animal model of disease. During this project I learned a great deal about molecular biology, teaching into my workload. microbiology, protein biochemistry and structural My fellowship is a four-year, Wellcome trust-funded and biophysical protein characterisation (mainly fellowship with appointments in both the Biochemistry through making and correcting mistakes!). My greatest department at the University of Oxford, working with achievement during this time was taking one of these Professor Colin Kleanthous, and the ithree institute protein antibiotics, pyocin L1, from discovery, through (infection, immunity and innovation) at the University characterisation to demonstrating its excellent activity in of Technology Sydney (UTS), with Associate Professor vivo. I like to tell people that the ‘L’ in pyocin L1 stands for Cynthia Whitchurch. My project is based around pyocin Laura. Unfortunately though, it actually stands for lectin, L1, the protein antibiotic that I discovered during my as it is a lectin-like bacteriocin. PhD (pyocin Laura1), and trying to identify the molecular target through which it and other lectin-like bacteriocins exert their cytotoxicity. I started my fellowship at UTS in February 2015 and I love living and working in Sydney. Australia is a great country, not just because of the weather (although being Scottish this is a massive factor, even if at times my skin doesn’t agree), but the lifestyle and laidback attitude is great for the soul. I am really enjoying my time at UTS. The people are all welcoming and friendly, the research is exciting and the institute couldn’t be under better leadership than that of Professor Liz Harry; she has great vision and really makes every member of the institute feel valued and heard. I have really tried to throw myself Celebrating a year’s hard work at into the academic community in Australia, becoming a the Walker lab Christmas party. member of several societies (including the Australian Society for Biochemistry and Molecular Biology) and PhD–fellowship Transition and My Research attending several great national conferences, including the If I’m being honest, I found the transition from PhD 41st Lorne Conference on Protein Structure and Function. student to fellowship recipient much harder than I I was honoured to receive a Young Investigator Award expected. This may be due in part, or more likely almost at this conference, with the opportunity to present my completely, to the fact I moved half way around the world research at such an internationally renowned conference at the same time as starting in a new lab, meeting new both terrifying and exciting me in equal measure. I was people and learning new procedures and work practises. also very honoured to be awarded the Sydney Protein However, the main issue I struggled with (well actually Group’s Thompson Prize for my research in November still struggle with) is the additional administrative side of last year. The competition was a great showcase of managing my own grant. I almost always have a feeling the protein research being undertaken by early career of guilt when I am not in the lab doing research, as this researchers in Sydney and I was excited to be a part of is what my fellowship is paying me to do. However, it. I would strongly recommend, especially to ECRs, to I need to get my head around the idea that planning, apply for prizes and awards, even if you don’t think you ordering, writing, managing the finances and becoming will win. As I mentioned before, you never know what an integrated member of the department (by volunteering interviewers/judges are looking for so back yourself. It to help with projects such as organising seminars) are all shows your supervisors and institution that you think you part of research life. Looking back, I don’t think I fully are good enough to get the award, and in turn they will appreciated the luxury of solely doing lab work during my also think you are good enough. Also, once you win the PhD. However, I plan to take full advantage of focussing first it becomes much easier to win others (also the prize on my research over the next few years before I integrate money is a great added bonus!). Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 35 My Ever-present Imposter Syndrome numerous events and lectures. To overcome this fear, and Imposter syndrome is the feeling that you are not good my ever-present imposter syndrome, I decided quite early enough to do what you have been asked or that you got on in my PhD to become the Yes Woman in all matters to where you are through luck. Almost all researchers relating to public engagement. This was after reading experience this at some point in their career, whether they the book The Yes Man by Danny Wallace, a very funny admit it or not. My imposter syndrome stems from doing and motivational story, well worth a read. Thankfully, chemistry as an undergraduate degree and then moving unlike the character in the book, only good has come to into the field of biology. I feel like I missed out on a lot me through being the Yes Woman. I have challenged of the basics and so get nervous whenever people ask me myself, participated in events that I never thought I could questions about a topic that is not directly related to my do, improved my confidence and presentation skills and research. I always think that I should know what they are made a lot of excellent connections along the way. The talking about and that the other person standing next to me greatest and probably most unexpected outcome of my is probably a walking encyclopaedia on the topic. This is public engagement activities is how much they impact on silly, because science is such a broad topic, with thousands my research and broaden my knowledge of my general of papers being published every week, so it is impossible research area. Not only am I educating the public, the to keep up with it all, even within your field. I am actively public are educating me by asking questions that I would trying to overcome this as I progress through my career. never have thought of previously; like ‘can you get My tactics involve being the Yes Woman and doing things new antibiotics from crocodile blood?’ I would strongly like writing this article, accepting praise when it’s due and encourage all researchers, especially those who think they reminding myself that I put a lot of hard work into my wouldn’t enjoy public engagement or wouldn’t be good at research. Deep down I know I deserve to be where I am it, to give it a go. Say yes and see where it takes you. (probably!). I don’t know if I will ever completely silence my imposter syndrome and be 100% confident; so as a Women in STEM back up I will go with the mantra ‘you’ve got to fake it I feel fortunate that from an early age I was encouraged to to make it’ (although this should be applied carefully in study maths and science. At my high school, the advanced research, and NEVER in relation to experimental data!). higher (equivalent to Extension 2 in the Australian Higher School Certificate program) maths, chemistry and physics classes all had an equal portion of males and females. Below: Winning the Again, my undergraduate chemistry degree appeared to Young Investigator have an equal gender balance, if not slightly more females. Award at the Lorne However, as I progress through my research career, I can Conference on see that there are fewer and fewer women in the more Protein Structure and senior academic positions. As a female scientist hoping to Function 2016. pursue a career in academia, I find this discouraging. With very few female role models to look up to and get advice from, I am led to feel that the attainability of an academic career has question marks surrounding it. I am encouraged though by programs such as Athena Swan and Science in Australia Gender Equity (SAGE), where gender equity and diversity is discussed and people/practices are called out for bias, be it conscious or unconscious. I think that over the next few years, the topic of women in science is going to come to the fore, and hopefully we will start to see a move towards gender equality in the high-ranking Above: Talking science roles. antibiotic resistance at the Natural History Future Ambitions Museum in London. In the near future, I am looking forward to spending some time at the University of Oxford. Having visited the Department of Biochemistry before departing Public Engagement and Being the Yes Woman the UK, it has everything that you would imagine of Like a lot of researchers, I got involved in public such a leading institution: history, ceremony, prestige, engagement for entirely selfish reasons, primarily because excellent researchers and state-of-the-art technology (and it was a glaring hole in my CV. However, I quickly fell in importantly, significantly better weather than Scotland). love with the idea of educating and influencing the public In the long term, I hope to continue working on my own on the problem of antibiotic resistance, a topic that I find research ideas and would like to run my own research exciting and can talk about for hours (much to some of my group. I am excited about my future in academia. I love family and colleagues’ annoyance). Although I loved the research and I want to continue doing research that has idea of public engagement, public speaking is something direct links to benefiting human health, which was what that still terrifies me, even though I have participated in inspired me to pursue this career path all those years ago. Page 36 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 Queen’s Birthday Honours for ASBMB Members

Professor Doug Hilton was Dr TJ Higgins was awarded an awarded an Officer of the Order of Officer of the Order of Australia Australia (AO) for ‘distinguished (AO) for ‘distinguished service service to medical research and to agricultural biotechnology as a education, particularly in the field biologist and researcher, particularly of haematology, as a molecular in the area of plant nutritional value biologist and author, to gender and resistance to pests and disease, equity, and as a mentor of young and to professional scientific scientists’. organisations’. Doug is the sixth Director of the TJ did agricultural science at the Walter and Eliza Hall Institute, National University of Ireland Head of the Department of Medical and completed a PhD in Plant Biology in the Faculty of Medicine, Dentistry and Health Physiology in 1971 at the University of California, Davis. Sciences at the University of Melbourne, and the current He was a Postdoctoral Fellow in the Research School of President of the Association of Australian Medical Biological Sciences at ANU and then moved to CSIRO Research Institutes (AAMRI). He is best known for his Plant Industry from which he recently retired as Deputy discoveries in the area of cytokine signalling, and his Chief of the Division. He is a Professor of Science and advocacy for health and medical research. The Hilton Technology at the Queensland University of Technology. lab aims to understand which of the 30,000 genes are TJ’s research interests have been in the biochemistry important in the production and function of blood cells, and molecular biology of legume seed proteins and in and how this information can be used to better prevent, the improvement of their amino acid composition for diagnose and treat blood cell diseases such as leukaemia, animal and human nutrition. This led on to an interest in arthritis and asthma. Professor Hilton has been awarded protecting food legumes from insect pests which is now numerous prizes for his research into how blood cells moving toward application in West Africa. His long term communicate and has led major collaborations with colleagues in this work have been Don Spencer, Hart industry to translate his discoveries from the bench to the Schroeder and Linda Tabe. bedside. He is an inventor of more than 20 patent families, He has been active in outreach to the wider community most of which have been licensed, and is a co-founder of about gene technology and GMOs and was given the Seed the biotechnology company MuriGen. of Light Award by the Grains Research and Development As Institute Director, Doug introduced mentoring Corporation. programs for staff at WEHI, and formed the Gender Other awards include the Goldacre Medal (awarded by Equity in Science Committee to help alleviate problems Australian Society of Plant Physiology), the Rivett Medal faced by women scientists who are combining research (awarded by CSIRO Officers’ Association), the Centenary with family responsibilities. His work in these areas Medal (awarded by the Australian Government) and the have been recognised by the award of the Eureka Prize RN Rutherford Award (awarded by Australian Society of for Outstanding Mentor of Young Researchers and he Plant Scientists). was invited to become a Victorian Male Champion of TJ has been a member of the ASBMB since the early 70s Change in 2015. This year Doug was invited to join two and was awarded the Pharmacia-LKB Biotechnology major committees: the NHMRC Structural Review Expert Medal in 1993. He was a founding member International Advisory Group and the Australian Medical Research Society for Plant Molecular Biology and served as a Advisory Board (MRFF Committee). He is also honoured Director in the 80s and again in the 90s. He has served to be the 2016 recipient of the John Curtin Medal for on the NSW Agricultural Advisory Council on Gene Excellence in Medical Research. Technology and the NSW Ministerial Scientific Advisory Council on Agriculture Fisheries & Food and the Australian Government Department of Agriculture, Fisheries & Forestry – Eminent Scientists Group. He is currently a director of the Agricultural Biotechnology Council of Australia and was Chairman of the Board of Gene Shears Company. He also served on the Board of the Cotton Research and Development Corporation. TJ is a Fellow of the Academy of Technological Sciences and Engineering and a Fellow of the Australian Academy of Science. He served as Chair of the Australian Academy of Science’s National Committee for Plant and Animal Sciences and was a member of the Academy’s Council. He is now Vice-President (Biology) of the Australian Academy of Science.

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 37 Gene Patenting in Australia – What’s the Story?

A series of regular articles coding for the BRCA1 gene, and variations thereof. on intellectual property, In a highly-anticipated decision, the High Court held written by Sarah Hennebry, that Myriad’s claims to the isolated BRCA1 gene should Patent Attorney, Freehills be revoked because the claims related to subject matter Patent Attorneys. Here Sarah which was not considered patent eligible under Australian reports on Myriad Genetics’ law (ie, was not ‘patentable subject matter’). attempt to patent the BRAC1 gene and what this means Does Anyone Really Care? for gene patenting. The claims considered by the High Court were only directed to isolated nucleic acids; the use of such nucleic acids in methods of diagnosis in the Myriad patent was In October last year, the Australian High Court not considered (nor were these claims challenged). This considered a landmark case relating to the patentability means that Myriad would still have the right to enforce of genes. A lot of emotion surrounded the case, but now its claims directed to the use of the nucleic acid in a that the dust has settled, it is timely to revisit the case diagnostic method, if the patent had not expired before and consider the potential impact of the decision on the commencement of the case. Australian researchers. Given that the real commercial utility of Myriad’s patent lay in the use of a diagnostic method, the question arises Synopsis as to whether it really matters that the High Court decided that the isolated nucleic acid claims of Myriad’s patent In Australia, if you make something new in the course were invalid. of your research, for example, a new antibody, then the A key factor in the High Court’s departure from the nucleic acid molecule that encodes that new antibody decisions of the lower courts, was the way in which the will likely be eligible for patent protection. claims at issue were interpreted. Specifically, while the Similarly, if you modify a known nucleic acid sequence lower courts understood the claims to be directed to a to give it a new function (for example, making a construct chemical compound per se, the High Court understood encoding a fusion protein), then that new vector construct that the substance of the patent claim was no more than will likely also be patent eligible in Australia. ‘mere information’. However, if you discover a new use of a nucleic acid Given this interpretation by the High Court, it is perhaps molecule that has a sequence found in nature (eg, an not that surprising that the relevant claims were found not siRNA molecule), then you may only be able to obtain to be patent eligible. For example, it seems unlikely that protection for the use of that molecule in a new method, any one today would actively pursue patent protection for rather than protection for the molecule per se. mere data. In addition to being ‘patent eligible’, any patent claim must also be new and inventive before an Australian Response from the Australian Patent Office patent can be granted on that claim. The Australian Patent Office is the government body The scope of what can now be claimed in Australian responsible for examining patent applications to determine patent applications has been narrowed, although whether they meet the requirements for patentability guidelines issued by the Australian Patent Office have under Australian law. provided some degree of certainty as to what claims are In the wake of the High Court decision, the Australian likely to be allowed during the examination process. It Patent Office outlined what it considers to be patent is still possible to patent genes in Australia, but only in eligible. The Patent Office has adopted a broad certain circumstances; your Patent Attorney can advise interpretation of the decision, such that the implications you on what is required and what are the best steps for of the High Court decision are further reaching than many protecting your valuable innovations. observers initially predicted. The Patent Office has adopted a blanket exclusion to The Detail the patentability of isolated nucleic acids which replicate The High Court Decision naturally occurring DNA and RNA sequences. Last year, the High Court of Australia considered the Synthetic nucleic acids (including cDNA), probes, validity of certain claims in a patent owned by Myriad primers and isolated interfering/inhibitory nucleic acids Genetics Inc. are also excluded where they ‘merely replicate the genetic The patent at issue was directed to mutations in the information of a naturally occurring organism’. breast cancer gene BRCA1, associated with an increased What this means in practice is that if a claim to an isolated risk of breast cancer. The patent also related to diagnostic nucleic acid molecule merely replicates the sequence methods for detecting breast cancer, based on the presence found in nature, the Patent Office will likely reject the of a mutated BRCA1 gene. The Court only considered the claim on the basis that nothing has been ‘made’ by human validity of the claims directed to an ‘isolated nucleic acid’ intervention.

Page 38 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 Gene Patenting in Australia – What’s the Story?

However, if the sequence is different, then the claim may In 2013, the US Supreme Court considered the US be patent eligible: for example, if the sequence contains counterpart to the patent considered by the Australian a non-naturally occurring mutation, is coupled to a High Court and found that Myriad’s claims to the isolated promoter sequence which would not normally be coupled BRCA1 gene were not patentable. While the situation in to the sequence, or if the sequence relates to an entirely Australia and the US is now similar, the legal basis for this new molecule (for example, a nucleic acid encoding a new difference is not the same in the two jurisdictions. antibody or new fusion protein). An important distinction between the US and Australian Importantly, even if the subject matter of the claim is considered approach is what has become known as the ‘product of to be patent eligible, the claim still needs to pass the other tests nature’ exception to patentable subject matter in the US. In for patentability: is it new? is it inventive? another US Supreme Court decision, (Mayo v Prometheus) the Court set forth a framework for distinguishing patents What Does ‘Made’ Mean? that claim laws of nature, natural phenomena, and abstract Previous tests for determining patent eligibility in ideas from those that claim patent eligible applications of Australia required consideration of whether a patent those concepts. This has had a significant impact on the claim related to an ‘artificially created state of affairs patentability of diagnostic methods in the US and is a having economic utility’. This, in part, explains why the complex and evolving story – and perhaps best suited to word ‘isolated’ has been adopted in patent claims to a future article! denote artificiality and to distinguish nucleic acid claims While last year’s High Court decision initiated much as products of human action from the naturally occurring emotional discussion about the patentability of genes, it DNA sequence. is unclear whether the decision has had any significant The High Court’s decision sought to make a distinction impact on the approach taken by biotechnology companies between ‘artificiality’ and ‘made’ and held that the (particularly given the similarities between what is now substance of the claim must have been created or ‘made’ allowable in Australia and the US). Further, while the by human action. ‘Made’ is intended to include created or Patent Office has outlined how it interprets the High modified, but more than mere replication. Court’s decision, the decision has yet to be considered and applied by the lower courts. It will be interesting to observe future developments in this area, and whether The Situation Outside of Australia any future Court decisions impact on the examination In Europe, patenting of a gene sequence ‘isolated’ from guidelines issued by the Patent Office. In the meantime, the human body is permitted, even if the structure of if you have any questions relating to what aspects of the gene is identical to that of a natural element. Other your inventions can be patented, you should direct these requirements for patentability must also be met, including queries to your Patent Attorney who can best advise as ‘industrial application’ (can the invention be used in some to the most appropriate approach for protecting your kind of industry?). innovation, whether in Australia or overseas.

Australian Society for Biochemistry and Molecular Biology Inc PUBLICATION SCHEDULE FOR AUSTRALIAN BIOCHEMIST, volume 47, 2016

Issue ASBMB Content Copy Deadline Issue Date

April 2016 47 (1) Profiles of medal, award and fellowship winners Monday 8th February Monday 28th March Nominations for Executive/Council

August 2016 47 (2) Nominations for medals, awards and fellowships Monday 13th June Monday 25th July Notice of AGM/proposed constitutional changes

December 2016 47 (3) Annual Reports/finances Monday 10th October Monday 29th November ComBio2016 reports

The Program for ComBio2016 (Brisbane) will be placed on the ASBMB webpage (www.asbmb.org.au). The Proceedings of the Australian Society for Biochemistry and Molecular Biology is published in conjunction with the Annual Conference of the Society. The electronic version of the Proceedings (Volume 48) will be made available online.

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 39 The Custom Science NZSBMB Award for Research Excellence 2015 Resistance is Not Futile: Bacterial ‘Innate’ and ‘Adaptive’ Immune Systems Peter Fineran Department of Microbiology & Immunology, University of Otago, New Zealand, [email protected]

What Has Phage Research Ever Done For Us? been converted into a ‘benchtop centrifuge’ for spinning Bacteriophages, or phages, are viruses that specifically down PCR tubes. Diversions aside, phages further revealed infect bacteria and their name literally translates as the architecture of the gene in the classic phage T4 rII gene ‘bacteria eaters’. The year 2016 marks 100 years since their mapping experiments by Seymour Benzer (11). We still independent discovery by Frederick Twort in 1915 and teach these experiments to our students at Otago, not only Félix d’Herelle in 1917 (1) – well, it is the average to keep for the historical interest, but as elegant logical approaches both Twort and d’Herelle supporters happy! Therefore, it to tackle scientific problems. is an appropriate time to write about our current phage Phages also provided many of the tools used in molecular research and reflect briefly on the past. Phages are the biology research. T4 ligase and restriction enzymes are two most abundant and genetically diverse biological entities of the most well-known, but many cloning vectors were on the planet. They outnumber bacteria by an order also derived from phages, as were some high-fidelity DNA of magnitude (estimated at >1030 phages) and infect sequencing methods. It is perhaps unsurprising then, that ~1025 bacteria per second, influencing many important the first genomes to be sequenced were phages. Phage- processes such as global nutrient cycles and the evolution based tools were then applied to the genome projects of of bacterial pathogens. The result is that they participate other organisms, including Escherichia coli and humans. in an ongoing evolutionary ‘arms race’, with phages and I believe that it is salient to remember that the work on bacteria trying to ‘outwit’ each other (2). As such, despite restriction enzymes originated from observations in the going unseen, phages have a major influence on our daily early 1950s on non-heritable variations in phage resistance lives and are also being seriously considered as specific when grown on different E. coli strains (12,13). These antibacterial agents (3). For example, in recent years we restriction-modification systems endow bacteria with a have been isolating and characterising phages that infect form of defence against phages that is akin to our innate Pseudomonas syringae pv. actinidiae (commonly known as immune systems. CRISPR-Cas research also highlights the Psa), which is the causative agent of kiwifruit canker, with serendipitous nature of studying phage resistance – who the aim of developing a phage-based biocontrol strategy could have known that trying to understand these bacterial (4,5). Phage research also continues to transform society adaptive immune systems would result in a revolution in through biotechnological spin-offs – as exemplified by tools for genetic manipulation. Both restriction enzymes the incredible rise of CRISPR-Cas9 gene editing, gene and CRISPR-Cas are billion dollar biotech successes and we regulation and related technologies (6,7,8). should keep in mind that these outcomes were built firmly The impact of phage research on molecular biology is on high quality curiosity-driven science. These are just some not recent. In fact, it is widely accepted that the study of the amazing ways that 100 years of phage research has and application of phage biology was responsible for influenced science, but I refer anyone interested to other the origin of molecular biology. The simple nature of articles (14,1). phages led to their exploitation as model systems to tackle It was during my undergraduate degree that I first complex fundamental questions in biology – such as developed an interest in phages, when I performed my ‘what is the nature of the gene’. As molecular biologists Honours research project with Jack Heinemann at the and biochemists, most of us are aware of this early University of Canterbury. I now head a research team based research, since we should have read it in our textbooks. at the University of Otago that investigates the interactions For example, Salvador Luria and Max Delbrück provided between phages, other mobile genetic elements and bacteria. evidence that mutations pre-existed in the absence The current major focus of my group is phage resistance, of selection, in support of Darwin’s theory of natural in particular the innate (toxin-antitoxin/abortive infection) selection (9). Alfred Hershey and Martha Chase later and adaptive (CRISPR-Cas) immune systems. Below I will showed that DNA (not protein) was the genetic material discuss some of our work in these areas. by using the simple, yet elegant, blender experiment (10) – demonstrating that sophisticated equipment is not always Bacterial Innate Immunity – Abortive a requisite to perform science of high impact. Kitchenware Infection and Toxin–antitoxin Systems often provides important lab equipment when on a tight To mitigate the ever-present risk from phage attack, budget – one of my first lab equipment purchases was a bacteria have evolved multiple defence strategies (2). Akin $50 pie warmer (complete with pie smell) through Trademe to our innate immune systems, the first line of defence is from Southland! It is still running many years later. I also provided by cell surface modifications, restriction of foreign worked in a lab with a hand powered salad dryer that had DNA by restriction-modification systems and abortive

Page 40 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 Resistance is Not Futile: Bacterial ‘Innate’ and ‘Adaptive’ Immune Systems infection. Abortive infection systems cause ‘altruistic cell a catch 22. I had just read the seminal paper in Science by suicide’ of phage-infected bacteria, which reduces the phage Rodolphe Barrangou and colleagues about CRISPR-Cas spread through the population. In 2006 I began studying a (clustered regularly interspaced short palindromic repeats new abortive infection system as a postdoctoral fellow in the – CRISPR associated proteins) systems that could provide lab of George Salmond at the University of Cambridge. At adaptive immunity in Streptococcus thermophilus, a common that time, the mechanism of most abortive infection systems bacterium used for fermentations in the dairy industry (24). was unknown. We showed that this system, which we As I was working on the ToxIN phage immunity system at named ToxIN, functioned as a toxin-antitoxin module (15). that time, I thought these sounded like incredible systems. Toxin–antitoxin systems are widespread and are composed So, I decided to start a new line of research investigating of a toxin, which targets an essential cellular process, and CRISPR-Cas when I started my group and managed to get a an antitoxin that inhibits the toxin (16). Their physiological CRISPR-Cas research theme established in New Zealand in functions were hotly debated but our work clearly showed 2008. However, since I had no track record in this new area, that toxin–antitoxin systems can provide phage resistance for a few years major funding was elusive and I continued (17,15). There were two Types of toxin–antitoxin systems, working on my other more established research areas. For RNA–RNA (Type I) and protein–protein (Type II) known the first few years from 2008, almost nothing was known at that time. Our discovery led to an entirely new class, the about CRISPR-Cas systems and we eagerly read any paper Type III toxin–antitoxin systems, which uses a protein– that was published in this area. Now, I typically get five RNA toxin-antitoxin mechanism (18,15). The toxin (ToxN) to ten new CRISPR paper alerts every day – such is the is an RNase that binds and cleaves cellular and phage exponential growth of this area! RNAs, leading to phage and host cell arrest (19,15). This What are these systems and how do they work? The activity is counteracted by the toxin-inhibitor (ToxI), a short CRISPR-Cas small RNA-mediated resistance systems repetitive non-coding RNA, which binds to ToxN and provide protection against foreign genetic material, such as forms a heterohexameric complex entirely consisting of phages and plasmids and are found in ~50% of bacteria and RNA–protein interactions (19,20). So, how are these systems ~85% of archaea (25,26). CRISPR arrays contain short repeats triggered by phage infection? We showed that upon phage separated by similar sized spacer sequences that provide infection, the levels of ToxI decrease, which leaves ToxN the genetic ‘memory’ of past infections. Briefly, immunity available to target RNA (21,22). In a surprising evolutionary involves three phases (Fig. 1): 1. During adaptation short twist, some phages overcome this innate immunity by one invader-derived sequences are added as new spacers to the of two strategies. Firstly, some phages hijack the toxI gene CRISPR array(s) – much like immunisation (27). 2. Next, the and place it in their own genome. In the second strategy, expression of CRISPRs from a leader sequence upstream phages repeat and expand a short segment of their own of the array results in an RNA that is processed into small genome to produce a small RNA the acts as a molecular guide crRNAs by Cas proteins. 3. Finally, Cas protein(s) and mimic of the ToxI RNA. In future infections these phages guide crRNAs form complexes that recognise and degrade express the anti-dote RNAs, which mop up any ToxN in the complementary nucleic acids (termed protospacers) during cell and enable phage replication (21,22). interference. It is this relative ease in which a protein (with Our work on ToxIN also demonstrated a direct link nuclease activity) can be directed to a specific nucleotide between toxin–antitoxin and abortive infection systems (15) sequence by a small RNA (the crRNA) that has resulted in – providing insight into the mode of action of some abortive the widespread application of CRISPR-Cas systems in gene infection systems. Taking this concept further, we have editing. There is incredible diversity in CRISPR systems, now identified, and begun characterising, other abortive with two major classes of system that are subdivided infection systems that function via novel toxin-antitoxin into (at least 6) types and some of these types are further mechanism(s). For example, we demonstrated that the AbiE categorised into multiple subtypes (25). Cas9 belongs to the phage resistance system functions through a toxin–antitoxin Class 2, type II systems and is currently the most widely mechanism (23). The toxin has a mechanism not previously used CRISPR-Cas biotechnological tool (6,8), due to editing observed in bacterial toxins and is a nucleotidyl transferase functions being encoded in a single protein that is guided that utilises GTP to modify a currently unidentified cellular by an RNA (28). More recently, type V proteins have been target to elicit growth inhibition. The antitoxin is an unusual discovered and may offer some benefits or complementary autoregulatory DNA-binding protein with a distinct improvements over Cas9 (29,30). Other types of CRISPR- C-terminal domain that alone is sufficient for antitoxicity Cas system (e.g. types I and III) have more complicated (23). There is still a paucity of information about how abortive multiprotein complexes required for invader recognition infection systems are triggered by phages, despite their and interference. My laboratory is using plant-pathogenic successful commercial use for protecting bacterial cultures bacteria of the Pectobacterium (formerly Erwinia (31) and from phage spoilage during cheese and yoghurt production. Serratia (32) genera as models to investigate these adaptive immune systems. Below I will highlight some of our work Bacterial Adaptive Immunity – CRISPR-Cas to understand the aspects of these immune systems during Systems adaptation, expression and interference, and in the CRISPR- In 2007, I was planning what I wanted my lab to research. Cas evasion strategies employed by phages. Like many postdocs at this stage in their career, I knew Adaptation: In the last few years we have been it was important to choose an area in which I had both investigating adaptation, which was poorly-understood experience and a track-record, but that would give me until recently (33,27,34). There are two proteins conserved independence from my former supervisor – somewhat of across all CRISPR-Cas systems that are involved in

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Fig. 1. Mechanism of CRISPR- Cas immunity as shown for the system present in Pectobacterium atrosepticum (type I-F). Immunity is provided in three major stages termed adaptation, expression and interference. When phages escape CRISPR-Cas immunity, the adaptive immune system can respond using the priming process. For details, see the text. Figure courtesy of Ron Dy. adaptation – the acquisition of invader DNA and its Cas system. addition to the CRISPR array ‘memory bank’ (35). Our Once the CRISPR arrays are transcribed, they need to be group was the first to demonstrate that these proteins (Cas1 processed into the functional guide crRNAs. In late 2008, and Cas2) interact to form a complex (36). More recently, in papers from the labs of John van der Oost, and Michael and collaboration with Kurt Krause’s group at Otago, we have Rebecca Terns were published on the E. coli and Pyrococcus elucidated a high-resolution crystal structure of the Cas1 furiosus CRISPR-Cas systems respectively and the proteins dimer from Pectobacterium atrosepticum (37). This structure responsible for CRISPR processing and crRNA generation gave us insight into the DNA binding by this complex were identified (39,40). We were working in a different and we are now studying the generation of new immune type of CRISPR-Cas system, so a critical question was memories both in vitro and in vivo during adaptation. how the mature crRNAs were generated in Pectobacterium Expression: We have also begun to make significant atrosepticum. We subsequently identified that Csy4 (aka advances in our understanding of how CRISPR-Cas systems Cas6f) was the endoribonuclease involved in the generation are controlled – i.e. when are the cas genes and CRISPR arrays of the small interfering RNAs (crRNAs) in vivo (41). turned on and off? We have demonstrated that the metabolic Interference: We also set out to test whether the Cas state of the cell is sensed through the global DNA-binding proteins in Pectobacterium formed complexes, and if so, transcriptional regulator, CRP-cAMP (38). CRP-cAMP which protein–protein interactions contributed to their activates the adaptive immune system during unfavourable formation. This work led us to identify two different protein metabolic times, potentially such as those triggered by complexes. The first of these consisted of the Csy proteins phage infection. The altered cas gene transcription affects and together with the crRNA form the interference complex both the level of existing immunity during interference in Pectobacterium. The second complex involved Cas1 and the subsequent degree of new immunity developed and a Cas2-3 fusion protein. Since Cas1 and Cas2 engage through both adaptation and interference by the CRISPR- in adaptation and Cas3 is required for interference, we

Page 42 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 Resistance is Not Futile: Bacterial ‘Innate’ and ‘Adaptive’ Immune Systems proposed that this complex engages in both adaptation and Cas immunity, some phages have evolved proteins that interference in this particular CRISPR-Cas type (36). specifically inhibit the activity of bacterial CRISPR-Cas An important step in understanding CRISPR-Cas systems. These proteins are termed anti-CRISPRs and are immunity is defining the targets of crRNAs encoded by part of the phage arsenal that gives them the upper hand CRISPR arrays. Working with Chris Brown at Otago, a tool against some CRISPR-Cas systems (53,54). These proteins called CRISPRTarget was developed that enabled us to were discovered in Pseudomonas phages by Alan Davidson’s identify the viruses and plasmids to which bacteria are likely laboratory at Toronto University. We have been fortunate to be immune (42,43). Together, we have also developed to collaborate recently with Alan’s group to test the tools for predicting CRISPR arrays and their direction of promiscuity of these proteins. By using our Pectobacterium transcription (44). Surprisingly, one of the pre-existing strain, we have tested whether anti-CRISPRs derived from spacers in Pectobacterium atrosepticum has a perfect match Pseudomonas phages and a variety of other mobile genetic to its own genome in a pathogenicity island – an apparent elements are able to inhibit this particular CRISPR-Cas case of ‘autoimmunity’. We showed that this CRISPR-Cas- system. A number of the proteins are capable of blocking or mediated ‘autoimmunity’ can result in a toxic ‘suicide’ (45). reducing activity of the Pectobacterium CRISPR-Cas system, These autoimmune effects can be tolerated in surviving cells demonstrating that, these proteins are not only widespread, that have undergone major genomic changes, including the but they function against diverse CRISPR-Cas systems deletion of this large pathogenicity island. In collaboration (55). These anti-CRISPR proteins provide another amazing with Andrew Pitman at Plant and Food Research, who example of the evolutionary flexibility of phages and is was studying this particular pathogenicity island, we have reminiscent of the acquisition of antitoxins genes by phages, recently shown that CRISPR-Cas-mediated deletion of the as mentioned earlier (22). island has altered the virulence of Pectobacterium in potato plants (46). These ‘self-inflicted’ genomic changes indicated Final Remarks a possible role of CRISPR-Cas that had not previously been It has been, and will continue to be, an exciting time to appreciated: one that can result in significant, and rapid, work on bacterial immune systems with many mysteries genomic changes (47). Indeed, it is now becoming apparent about CRISPR-Cas systems and their functions awaiting that CRISPR-Cas systems can have functions additional discovery. I think it is relevant to end on a note about to their role in defence (48). Our study of ‘autoimmunity’ funding, before I thank those who kindly supported this also led to the demonstration that systems other than Cas9 work! Restriction enzymes and CRISPR-Cas provide yet can be harnessed for bacterial genome editing to generate another perfect example where fundamental research point mutations or large (e.g. 100 kb) deletions in bacterial to understand a process as apparently esoteric as phage chromosomes (49,45). resistance can uncover not only critical knowledge about important life-processes, but can lead to biotechnological How Do Phages Avoid CRISPR-Cas revolutions with far-reaching impacts. Maintaining a sufficient base of investigator-led fully funded research Immunity? grants that support excellent fundamental research is Priming: In the phage–bacterium arms race, phages can essential for the health of the science and technology sectors escape CRISPR-Cas immunity with point mutations that in any country, including New Zealand. I have been very disrupt interference – eg mutations that impede the ability of fortunate to obtain grants from the Marsden Fund and the crRNA to base-pair with the target viral DNA (50). This a Rutherford Discovery Fellowship – these funds were was seen as a weakness of CRISPR-Cas immunity, but recent particularly significant in making possible the research that studies by our groups and others, revealed that a process, I have described here. termed priming, enables rapid adaptation to these escapees (51,50,52). Upon reinfection with an escape phage, the original interference-deficient spacer accelerates the incorporation of Acknowledgements new functional spacers, which elicit interference (51,50,52). Most importantly I would like to thank all of the fantastic Multiple new spacers can be incorporated though priming, lab members and collaborators who have contributed providing increased resistance and reducing the probability massively to the work highlighted in this essay. I have been of further escape – indeed, in Pectobacterium we observed incredibly lucky to work with so many talented students, up to nine new spacers in a single bacterium (52). While I technicians, postdocs and collaborators. Thanks to Ron Dy was on research and study leave in the Netherlands with who provided the figure used in the article and to Raymond Stan Brouns, we discovered that priming occurs even when Staals for providing critical feedback. I am very grateful to the invader has >10 escape mutations (50). This suggests the multitude of funding agencies that have supported my that these systems provide a robust protection against the research over the years: the Marsden Fund, Royal Society of rapidly evolving viral and plasmid populations, and even New Zealand (RSNZ), a Rutherford Discovery Fellowship possibly to invaders not previously encountered (50). We (RSNZ), Zespri International Ltd, the Tertiary Education have subsequently proposed, and are currently testing, Commission, the Bio-protection Centre of Research a model for how the Cas protein machinery translocates Excellence and the University of Otago, Division of Health along the invader DNA during this rapid priming mode of Sciences, Otago School of Medical Research and Department adaptation (52). of Microbiology and Immunology. Finally, I would like to thank Renwick Dobson for nominating me for this prize and Anti-CRISPRs: In addition to their ability to obtain point mutations that lead to initial avoidance of CRISPR- Custom Science for generously sponsoring this award.

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Pawluk, A., Staals, R.H., Taylor, C., Watson, B.N., Viruses 4, 2291-2311 Saha, S., Fineran, P.C., Maxwell, K.L., and Davidson, 27. Fineran, P.C., and Charpentier, E. (2012) Virology 434, A.R. (2016). Nat. Microbiol. in press 202-209 This article was also published in the Autumn 2016 issue 28. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, of Southern Blot: the newsletter of the New Zealand Society for J.A., and Charpentier, E. (2012) Science 337, 816-821 Biochemistry and Molecular Biology. Page 44 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 OUR SUSTAINING MEMBERS

Disclaimer The Australian Biochemist is published by the Australian Society for Biochemistry and Molecular Biology Inc. The opinions expressed in this magazine do not necessarily represent the views of the Australian Celase® GMP OmicsLink™ shRNA Clones Society for Biochemistry and Celase® GMP is a proprietary OmicsLink™ shRNA clone collections Molecular Biology Inc. enzyme containing a unique blend of from GeneCopoeia include lentiviral and collagenase, neutral protease and buffer non-viral vector-based shRNA constructs salts that are produced using avian and against genome-wide human, mouse DAINTREE mammalian tissue-free raw materials, and rat genes. shRNA of varying lengths aseptically processed, sterile filtered and (19 to 29 bases) were designed using a scientific highly purified under GMP guidelines. proprietary algorithm to make shRNA AUSTRALIA expression constructs that have high Manufactured by Cytori Therapeutics, A new addition to the DAINTREE knockdown efficiency with minimal off- this product line is ideal for cell isolation range of electrophoresis hardware, target effect. studies for laboratories aiming to power supplies, transilluminators, facilitate a smooth transition from bench Guaranteed shRNA knockdown software, and integrated gel imaging and animal research to downstream A set of four expression constructs is systems. clinical applications. offered against every target gene with the The Bio-1000F is an innovative, user- guarantee that at least one of the four will A single, sterile, ready-to-use enzyme friendly, and cost-effective device that have a knockdown effect of 70% or more containing both collagenase and neutral integrates image capture, gel preview, as determined by qRT-PCR. protease is ideal for a wide range of and gel extraction essential for routine adipose stem cell, biomedical and All cell types covered nucleic acid gel electrophoresis. With the bioprocessing applications. Lentiviral and non-viral vector options combination of a high-sensitivity charge- allows transfection or transduction of Not all research applications require coupled device (CCD) system and blue- shRNA into difficult-to-transfect cells as the use of a GMP grade enzyme in light emitting diode (LED) illuminators, well as conventional dividing cell lines. early phase studies. However, the the Bio-1000F is compatible with all ethidium bromide (EtBr)-alternative recent FDA guidance issued for tissue Multiple delivery formats and cell products specifically cites that shRNA are delivered as 4 individual fluorescent stains, and provides GMP grade reagents should be utilized constructs of 5 µg purified plasmid. publication-quality images up to 0.04 in drug-type validated processes. Lentiviral particles for shRNA also ng per band, significantly enhancing Subsequently, both regenerative available from GeneCopoeia. fluorescent signal expression over other medicine researchers and clinicians are gel documentation systems that depend Markers and reporters now looking for GMP quality products, on ultraviolet and blue-LED light sources. Vectors with mCherry or eGFP reporter which provide a smoother regulatory Thanks to its removable filter plate and gene for monitoring transfection or approval process. intuitive MiBioFluo software interface, transduction efficiencies. Stable cell users can visualize banding patterns The Cytori Celase® Enzyme is selection with puromycin marker. and conduct gel extraction directly on currently used in US FDA approved Fully sequenced expression cassettes the Bio-1000F without moving between clinical trials. Expression cassettes of all shRNA the transilluminator and gel imager. Worthington is excited to offer a clones are fully sequenced including the The Bio-1000F’s compact design enables foundational and versatile enzyme promoter, sense and antisense target it to fit in crowded laboratory spaces. for advancing adipose-based research sequences, hairpin, termination and other The Bio-1000F features an integrated, programs from pre-clinical to clinical linker sequences. environment-friendly, and ultrasensitive levels, while eliminating the need to gel imager for researchers dedicated For further information please contact perform costly and time consuming to improving laboratory safety and the United Bioresearch, GeneCopoeia’s bridging studies. efficiency of gel electrophoresis. Australian partner. ScimaR For information please contact United Bioresearch Products Email [email protected] Murdoch Macaskill Kirrily Smith Freecall 1800 639 364 Daintree Scientific Australia Phone (02) 4575 0309 Phone 03 9842 3386 Phone (03) 6376 3335 [email protected] Fax 03 9842 3407 Email [email protected] www.genecopoeia.com www.daintreescientific.com.au

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The range offers extremely low and the Royal Society of Biology and halogenated and other organic will be a reviews journal featuring compounds to prevent sample invited articles. The journal will focus contamination, ideal for research on new and growing fields, as well as and analytical laboratories that test interdisciplinary themes, drawing from samples for the presence of pesticides or all areas of life science. Each issue will insecticides. cover one emerging topic of interest and will be guest edited by a subject-matter Produced from consistent quality raw expert. materials under ISO 9001 conditions: SWATHXtend • Filtered at 0.2 μm APAF is a world leader in the use • Specified to the required low of SWATH-mass spectrometry for residue levels necessary proteomics. To improve the data analysis • Bottled under nitrogen workflow to discover even greater • Fitted with caps that have PTFE proteome coverage, our bioinformatics liners to prevent contamination team has developed a new open source • Packed in BDH Prolabo standard software module, SWATHXtend. neck size 2.5 L glass bottles SWATHXtend is designed to merge VWR International Pty Ltd locally generated MS data with spectral Tel: 1300 727 696 membraPure Amino Acid libraries from external sources such Fax: 1300 135 123 Analyzer as large repositories in SWATHAtlas. Email: [email protected] This extends the number of proteins Web: au.vwr.com Scientex has been appointed Distributor quantified using SWATH-MS. for membraPure (Germany) for its range SWATHXtend takes the hassle out of of Amino Acid Analyzers (AAA). The manual data analysis, providing the new generation ARACUS makes analysis user with statistical analysis to discover of amino acids more modern, fast and changes in protein levels between simple. It uses the classic means of sample sets. routine analysis in amino acids by post- Read about SWATHXtend, recently column derivatization with ninhydrin, Biochemical Society Launching also known as the “gold standard”. published in Molecular and Cellular Two New Journals Proteomics and access the software A new procedure was developed The Biochemical Society is excited to via the APAF website (http:// which allows for the analysis of announce two new journals, Neuronal www.proteome.org.au/Services/ physiological and hydrolysate amino Signaling and Emerging Topics in Life Bioinformatics/SWATHXtend) acids with the same buffer system and Sciences, strengthening the Society’s column. commitment to promoting and sharing knowledge. The ARACUS has a compact design consisting of three individual units: the Neuronal Signaling will be an online- main unit, the Autosampler and the only, fully Open Access journal eluent rack EluBox. This modular design publishing research and reviews on VWR Chemicals BDH Prolabo allows the instrument to be used in all aspects of signaling within and PESTINORM® Solvents for research, quality control and in clinical between neurons. Grounded in the Pesticide Residue Analysis laboratories. The Autosampler provides fields of cellular signaling pathways, continuous analysis of 96 samples (2 x PESTINORM® Solvents from the biochemistry and molecular biology, this 48 vials) and samples injected without VWR Chemical range combine the journal will also serve the neurosciences, sample loss. Washing routine of the best quality and price competitive covering a range of subjects from injection valve and syringe pump characteristics, ensuring we meet your molecular mechanisms of neuro- guarantees zero cross-contamination. demands now and in the future. pathologies and neuro-degeneration to Detection of trace organic substances signaling in consciousness and memory. The software aminoPeak is very user in food and the environment requires We welcome Professor Aideen Sullivan friendly, self-explanatory and meets the the use of highly purified solvents (@asneuro on Twitter) as the journal’s latest GLP criteria. for all stages of analysis, starting with first Editor-in-Chief. For further information contact:- sample preparation. Emphasizing our dedication to Scientex Pty Ltd PESTINORM® solvents are connecting and collaborating for the Tel 03 9899 6100 guaranteed specifically for use in future of the life sciences, we are Fax 03 9899 6122 pesticide residue analysis and designed particularly glad that one of the new Email [email protected] to meet the day-to-day requirements of titles Emerging Topics in Life Sciences, is www.scientex.com.au quality control laboratories. co-owned by the Biochemical Society Page 46 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 OUR SUSTAINING MEMBERS

Abcam Enables Faster Drug and Biomarker Discovery with 95% QE Back Illuminated Automated Tissue Immunoassay Research Suite Scientific CMOS Camera Microarrayers Now Available from AXT Matched antibody pair kits provide The new Prime 95B Back Illuminated assays with reproducible, sensitive and AXT is pleased to announce that we Scientific CMOS (sCMOS) camera high-throughput detection have just added automated Tissue from Photometrics with 95% Quantum Microarray (TMA) systems from Matched antibody pairs are central Efficiency (QE) is an ideal choice Integrated Systems Engineering to enzyme-linked immunoabsorbant for Super-Resolution Microscopy, (ISENET) to our product portfolio. assays (ELISAs), which are widely used Confocal Microscopy, Single Molecule These products fit nicely with existing to study secreted biomarkers, and are Fluorescence and Light Sheet product lines for biospecimen storage fundamental to research in areas such Microscopy. from TTP Labtech (automated as immunology and cancer. Abcam The Photometrics Prime 95B sCMOS cryo-storage systems) and the optimises its antibody pairs to avert camera is the first sCMOS camera to OpenSpecimen (biobanking sample- the reproducibility and scalability offer 95% Quantum Efficiency (QE) management database). challenges previously faced by researchers, which can cause variability and Back Illumination (BI) in the ISENET is a leading designer, in results. Abcam’s matched antibody same camera and it now outperforms manufacturer and supplier of automated pairs use recombinant monoclonal EMCCD cameras. TMAs. Since entering this market, they antibody technology, such as the have developed an enviable reputation The Prime 95B’s sensor converts patented RabMAb® technology, to in the TMA community and have up to 95% of incident photons provide improved consistency over systems in laboratories all over the into a measurable signal. The back polyclonal antibodies. The pairs are world. illuminated sensor brings light into the screened for sensitive performance and pixel photodiode from behind, avoiding ISENET’s TMA solutions create arrays rigorously tested in a variety of complex structures that reflect or absorb light. of large numbers of representative core sample types including plasma, serum Combined with large 11μm pixels, the samples from your bank of existing and cell lysates. Prime 95B camera can deliver over 300% pathology samples for further detailed more signal than other sCMOS cameras analysis. ISENET’s range of TMAs These optimised pairs are already at 100X magnification. devices spans from fully automated part of Abcam’s SimpleStep ELISA® systems, through to semi-automated kits, and will now be available as The extreme sensitivity not only allows models. stand-alone matched antibody pair fainter signals to be detected, it provides kits. The new stand-alone high volume Using a TMA from ISENET will allow the flexibility to increase frame rates, or format provides greater flexibility to you to more quickly prepare accurate turn down the excitation intensity to researchers and facilitates cost-effective and reliable tissue micro-arrays for reduce cellular photo-damage. drug discovery, by enabling scale up your area of medical research. This will from small pilot studies to large-scale Key features include 95% QE, 16 bit help you to analyse more samples more high-throughput assay platforms. dynamic range, large11μm x 11μm Pixel quickly, producing statistically relevant Area, 1.3e- Read Noise (rms), 41fps results, translating research, into clinical To find out more, visit: http://www. @ 16-bit / 82fps @ 12-bit, regulated trials and ultimately commercial abcam.com/antibody_pair air cooling to -10 deg C, single cable treatments. connection rather than a dual camera Abcam For more information on ISENET’s link, large field of view, C Mount Email: [email protected] range of TMA systems, please visit interface, multiple expose out triggering Tel: 1800 024 968 www.axt.com.au. and SMART streaming. AXT Pty Ltd SciTech Pty Ltd www.axt.com.au (03) 9480 4999 [email protected] [email protected] 02 9450 1359 www.scitech.com.au

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 47 OUR SUSTAINING MEMBERS exceptional speed (40 cycles in 40 minutes) produces high quality data suitable for both single users and core facilities. Thermal cyclers complete the range from PCRmax with innovative, reliable, fast, LATEX Bead Conjugation Kits easy to use single 96 or 384 well block instruments, as well as a four block (96 Innova Biosciences has introduced well/384 well/combo) work stations. a range of LATEX Bead Conjugation Kits. These simple to use, one-step kits For more information or a for covalently conjugating antibodies, LabGear Australia has a suite of demonstration, please contact LabGear proteins and peptides (or any other complimentary products for today’s Australia on 1800 LABGEAR or email biomolecule with an amine group) to Life Scientist, from quantification to [email protected] specially treated latex beads without the amplification and imaging. IMPLEN is need for extensive optimisation. a leading supplier of innovative ultra- low volume nanophotometers that The latex conjugation reaction has been includes cuvette options. Using sample developed using Innova Biosicences compression and true path technologies, expertise in simple and quick one- IMPLEN has created a family of step antibody conjugations such as Nanophotometers which accurately our InnovaCoat® GOLD kits (covalent measure the concentration of nano conjugation to gold nanoparticle) volume nucleic acids and proteins with and Lightning-Link® kits (covalent unmatched reproducibility. The choice conjugation of antibodies, proteins and of four models covers the full workflow Analytical Ultracentrifugation peptides to enzymes and fluorophores) of a research or clinical laboratory Sample Characterisation to produce a kit unlike any latex bead with flexible unit control by PC, built- Beckman Coulter delivered the first conjugation product available. in touchscreen or smartphone/tablet AUC sample characterization tool to Quick and easy to use: connectivity. Not requiring calibration the scientific community powering • 30 seconds to set up the one-step for the life of the instrument ensures discoveries. The tradition continues into conjugation reaction lower ongoing costs and better reliability. the 21st century with the new Optima • 3 minutes hands-on time and On-board vortexing and the option of AUC system. battery power for up to eight hours offers 35 minutes total time until the This latest offering is the most robust further reliability and flexibility to the conjugates are ready to use technology for providing protein IMPLEN range. Specially treated latex is resistant to molecular weight in basic protein research aggregation: Syngene imaging systems are and quantification of aggregation • High yields of functional conjugates recognised world-wide as high levels for academic and biopharma can be made without the need for quality, high performance instruments research. Analytical ultracentrifugation harsh resuspension methods like for the image capture and analysis is the most versatile, rigorous and sonication and vortexing of fluorescent gels, stain-free gels, accurate means for determining the chemiluminescent western blots, molecular weight, hydrodynamic and Only two buffers to test for optimal bioluminescence and protein samples. thermodynamic properties of a protein activity: The versatile and powerful G:BOX or other macromolecule. Currently, • No extensive pH optimisations as range are designed to perform to the there is no other technique capable of is typically required for traditional highest level for specific applications. providing the same range of information passive conjugation methods The PXi range of high resolution multi- with a comparable level of precision and Choice of bead colour and kit sizes: application systems are compact, easy accuracy. to use and offer full automation in • Available with red, blue or black Providing over 65 years of global the capture of chemiluminescent and 400nm latex beads leadership in centrifugation, Beckman fluorescent blots, visible gels, blots and • Two kit sizes available: Mini kits Coulter Life Sciences designs, small-format 2D gels. The NuGenius are ideal for antibody screening or manufactures, sells, and services a system is a new generation low- ‘proof of principle’ experiments, complete line of centrifuge systems. By cost integrated system for DNA and and Midi kits are 10 times the size offering unique rotors and innovative protein analysis and gel documentation of the Mini kits. Bulk material is also bottles, tubes and accessories, coupled featuring an integrated 7-inch touch available for further scale up with advanced centrifugation software, screen and a built in processor. BioNovus Life Sciences Beckman Coulter delivers intelligent Ph: (02) 9484-0931 PCRmax Eco 48 qPCR instrument is centrifugation solutions to laboratory Email: [email protected] MIQE compliant with HRM functionality science. as standard utilising four colours for Web: www.bionovuslifesciences.com.au Learn more at info.beckmancoulter. easy multiplexing. Its industry leading com/OptimaAUC +/-0.1°C temperature uniformity and Page 48 AUSTRALIAN BIOCHEMIST Vol 47 No 2 August 2016 OUR SUSTAINING MEMBERS

Cell Signaling Technology has Genesearch is sponsoring Charles developed PTMScan® Technology, Farnsworth from Cell Signaling which uses validated PTM- and Motif- Technology, on a technical tour in specific antibodies to enrich PTM- Australia Aug 15-26. Charles will be containing peptides prior to LC-MS/ running workshops and speaking at PTMScan® Technology: MS analysis. PTMScan allows the a number of venues about PTMScan Antibody Based Proteomics identification and quantification of technology and its applications. Please Post-translational modifications hundreds to thousands of even low let us know if you are interested in (PTMs), such as phosphorylation, abundance PTM sites. PTMScan can be attending a seminar, or workshop acetylation, methylation, or used to: 1) determine novel PTM sites, or having small group talk on PTM ubiquitination, are critical regulators 2) identify and validate drug targets, proteomics. 3) discover biomarkers, 4) elucidate of protein activity and function. Contact: [email protected] Proteomic methods that profile PTMs off-target drug effects and 5) explore provide insight into both normal and the mechanism of action of drugs or disease biology that is not feasible at a chemical modulators. genetic level. FORTHCOMING MEETINGS

ComBio2016 Registrations can be made at: 25th FAOBMB Conference 3–7 October 2016 http://www.asbmb.org.au/ 5–7 December 2016 Brisbane Convention and Exhibition combio2016/registration.html Manila, Philippines Centre, Brisbane The theme of the Conference is The program will feature 15 plenary Biochemistry and Molecular Biology in presentations from some of the best ComBio2016 will be held at the Health and Wellness. international scientists (please see the Brisbane Convention Centre, located Further information: last page of this magazine for details), in the heart of Brisbane’s unique www.psbmb.org plus a number of society speciality Southbank Precinct – a 17 hectare Email: [email protected] lectures. Several poster sessions are riverside oasis renowned for its also planned. The scientific program of abundance of creative, cultural, the conference will include the themes: entertainment and lifestyle activities. In the tradition of ComBio’s, this • Plant Cell and Developmental conference will be scientifically 26th FAOBMB Biology and Genetics rewarding with the range and depth • Plant Physiology and Ecology Conference demanded by the modern bioscientist 6–9 December 2017 • Developmental, Stem Cell and and undoubtedly is one of the Kobe, Japan Regenerative Biology highlights of the annual Australasian The 26th FOABMB Conference will be • Proteins and Proteomics scientific calendar. held as a combined meeting with the • Genomes and Bioinformatics Japanese Biochemical Society and the • Cell Biology The Program Committee are pleased Molecular Biology Society of Japan. • Cell Signalling to advise that the poster abstract • Biochemistry and Metabolism Further information: deadlines have been extended as • Education and Career Development Email: [email protected] follows: • Education and Career Development Poster abstracts will be included in There will be the three core ComBio Late Posters if received by Monday, societies participating (ASBMB, ASPS 15 August and details published in the and ANZSCDB). conference program, conference App and on the website (but will not be programmed within themes). Further information: www.asbmb.org.au/combio2016 Poster abstracts will be detailed as On Site Posters on the program revisions Scientific Program: board at the conference if received Joe Rothnagel by Monday, 26 September. The [email protected] poster presentation details will not be published in the conference program, Registration/Exhibition conference App or on the website. Sally Jay [email protected]

Vol 47 No 2 August 2016 AUSTRALIAN BIOCHEMIST Page 49 DIRECTORY COUNCIL FOR 2016 STATE SPECIAL INTEREST PRESIDENT REPRESENTATIVES GROUPS Professor Michael Ryan Department of Biochemistry and AUSTRALIAN CAPITAL TERRITORY ADELAIDE PROTEIN GROUP Molecular Biology Dr Peter Mabbitt Contact: Dr Christopher McDevitt Monash University ANU College of Physical and Mathematical Research Centre for Infectious Diseases CLAYTON VIC 3800 Sciences University of Adelaide Ph (03) 9902 4909 Australian National University ADELAIDE SA 5005 Email: [email protected] CANBERRA ACT 0200 Ph (08) 8313 0413 Email: [email protected] Email: [email protected] PRESIDENT ELECT Professor Leann Tilley NEW SOUTH WALES AUSTRALIAN YEAST GROUP Department of Biochemistry and Dr Katharine Michie Chair: Dr Alan Munn Molecular Biology School of Physics Griffith University Gold Coast University of Melbourne University of New South Wales PMB 50, Gold Coast Mail Centre PARKVILLE VIC 3010 SYDNEY NSW 2052 SOUTHPORT QLD 9726 Ph (03) 8344 2227 Ph (02) 9385 4587 Ph (07) 07 5552 9307 Email: [email protected] Email: [email protected] Email: [email protected] TREASURER QUEENSLAND BIOCHEMICAL EDUCATION Associate Professor Terrence Piva Dr Dominic Chi Hiung Ng Chair: Associate Professor Susan Rowland School of Medical Sciences School of Biomedical Sciences School of Chemistry and Molecular Biosciences RMIT University, PO Box 71 University of Queensland University of Queensland BUNDOORA VIC 3083 ST LUCIA QUEENSLAND 4072 ST LUCIA QLD 4072 Ph (03) 9925 6503 Ph (07) 3365 3077 Ph: (07) 3365 4615 Email: [email protected] Email: [email protected] Email: [email protected] SECRETARY SOUTH AUSTRALIA MELBOURNE PROTEIN GROUP Associate Professor Briony Forbes Dr Steven Polyak President: Dr Douglas Fairlie Medicinal Biochemistry School of Molecular and Biomedical Science Olivia Newton John Cancer Research Institute Flinders University University of Adelaide Austin Hospital BEDFORD PARK SA 5042 ADELAIDE SA 5005 HEIDELBERG VIC 3084 Ph (08) 8204 4221 Ph (08) 8313 6042 Email: [email protected] Email: [email protected] Email: [email protected] METABOLISM AND MOLECULAR EDITOR TASMANIA MEDICINE GROUP Dr Chu Kong Liew Dr Adele Holloway Chair: Dr Nigel Turner Email: [email protected] School of Medicine, University of Tasmania School of Medical Science HOBART TAS 7008 University of New South Wales EDUCATION REPRESENTATIVE Ph (03) 6226 2670 KENSINGTON NSW 2052 Associate Professor Janet Macaulay Email: [email protected] Department of Biochemistry and Ph (02) 9385 2548 Email: [email protected] Molecular Biology, Monash University VICTORIA CLAYTON VIC 3800 Dr Diana Stojanovski QUEENSLAND PROTEIN GROUP Ph (03) 9905 3730 Biochemistry and Molecular Biology Chair: Dr Brett Collins Email: [email protected] Bio21 Institute Institute for Molecular Bioscience University of Melbourne University of QLD, ST LUCIA QLD 4072 FAOBMB REPRESENTATIVE PARKVILLE VIC 3010 Ph (07) 3346 2043 Professor Paul Gleeson Ph (03) 9035 3197 Email: [email protected] Department of Biochemistry and Email: [email protected] Molecular Biology RNA NETWORK AUSTRALASIA University of Melbourne WESTERN AUSTRALIA Chair: Dr Archa Fox PARKVILLE VIC 3010 Associate Professor Nicolas Taylor Harry Perkins Institute of Medical Research Ph (03) 8344 2354 ARC Centre of Excellence in Plant Energy 6 Verdun Street Email: [email protected] Biology NEDLANDS WA 6009 University of Western Australia Ph (08) 6151 0762 SECRETARY FOR CRAWLEY WA 6009 Email: [email protected] SUSTAINING MEMBERS Ph (08) 6488 7005 Sally Jay Email: [email protected] SYDNEY PROTEIN GROUP c/- ASBMB National Office Chair: Dr Liza Cubeddu PO Box 2331 School of Science and Health, University of KENT TOWN SA 5071 Western Sydney, PENRITH NSW 2751 Ph (08) 8362 0009 Ph (02) 4620 3343 Fax (08) 8362 0009 Email: [email protected] Email: [email protected] ASBMB NATIONAL OFFICE PO Box 2331 KENT TOWN SA 5071 FOLLOW US ON: Ph (08) 8362 0009 Fax (08) 8362 0009 Email: [email protected] http://www.asbmb.org.au COPY DEADLINE FOR NEXT ISSUE: Monday, 10 October 2016

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