Professor Steve Bottomley

Telephone: +61 3 9902 9362 Facsimile: +61 3 9905 2726 Office:  Room 244, Building 77, Clayton Email: steve.bottomley@med.monash.edu.au

Professor Stephen Bottomley is currently a Senior Research Fellow of the NH&MRC. He obtained his PhD at the University of Southampton in 1994 working with Prof. Michael Gore. He worked as a post-doctoral scientist in the laboratory of Prof. Stuart Stone at Cambridge University before moving to Monash University in 1996. His research is currently supported by a program grant from the NH&MRC and discovery grants from the Australian Research Council. He heads the recently formed Protein Production Unit, which has established high-throughput cloning, expression and purification technologies at Monash University.

The Bottomley laboratory investigates the molecular basis of two reactions; protein folding and misfolding, with the ultimate aim of preventing a range of devastating diseases.

Most proteins have no trouble folding quickly and efficiently to their native state. However, an increasing number of diseases such as emphysema, Alzheimer's and Huntington's disease are associated with the failure of proteins to fold correctly.

We study two protein families involved in misfolding disorders: the serine proteinase inhibitor (serpin) and polyglutamine family of proteins. Variants of the serpin, antitrypsin, misfold and accumulate in the endoplasmic reticulum of hepatocyte cells, leading to a plasma deficiency and hepatocyte damage due to the accumulation of aggregated protein, leading to cirrhosis and emphysema. At least eight neurodegenerative disorders are caused by an expanded polyglutamine tract in the various disease proteins, including Huntington's disease and spinocerebellar ataxia type 3. Polyglutamine expansion causes the disease protein to misfold and aggregate, ultimately leading to neuronal death.

Cellataxin pic to be inserted here

We apply a multi-disciplinary approach to define the molecular basis of disease, including the techniques of protein engineering, DNA cloning and mutagenesis, fluorescence and circular dichroism (CD) spectroscopy, rapid kinetics, NMR and X-ray crystallography and cell biology. All of our projects benefit greatly from their collaborative nature with other research groups within Monash and elsewhere.

Mapping the Folding Pathway of the Serpin Superfamily:

The ability of proteins to rapidly fold from a structureless state to their native conformation is remarkable; however the process is poorly understood especially in the case of large proteins. The aim of this project is to investigate the folding pathway of the archetypal serpin antitrypsin. We have identified residues that form the folding nucleus and identified one intermediate involved in the folding pathway. Using a range of approaches, the aim is to characterise the interactions involved in stabilising the folding nucleus and intermediate state. This project involves inserting new amino acids into the structure to act as probes for the formation of local structures during folding. Using this approach in combination with rapid fluorescence and CD techniques we aim to characterise critical structures in the folding pathway of antitrypsin.

Additional studies involve determining the role of chaperones in the folding of antitrypsin, in particular we aim to compare the conformational changes involved in the in vitro and chaperone assisted folding pathways.

Serpin Misfolding and Disease

The aim of this project is to investigate the conformational changes involved in the misfolding and polymerisation of antitrypsin, which in vivo results in emphysema and liver disease. Our previous data has shown that mutations cause antitrypsin to adopt an intermediate conformation with a high propensity to aggregate. The aim of the present study is to determine the conformation of this intermediate using biophysical techniques such as fluorescence and CD spectroscopy in combination with site-directed mutagenesis. Through a combination of protein chemistry and spectroscopic techniques we also aim to determine the kinetics and mechanism of antitrypsin aggregation, this information will be invaluable in the quest for aggregation inhibitors. Additional studies will focus on the use of chemical chaperones as polymerisation inhibitors, their effectiveness and mode of action.

Polyglutamine Proteins and Disease

The polyglutamine family of proteins causes neurological disease through protein aggregation. The aim of this project is to investigate the mechanism of conformational change and fibrillogenesis using ataxin-3. Using biophysical techniques we will compare the structure of ataxin-3 with 20 glutamine repeats, which does not aggregate, with ataxin-3 with 57 repeats which rapidly forms fibrils. In addition we are using NMR and X-ray crystallography to determine the three dimensional structure of ataxin-3.

An additional project involves the cloning and expression of a fragment of the androgen receptor, which also possess an expanded glutamine tract. Once cloned and expressed the aim of the work is to characterise the fragments structure and conformational changes involved in fibril formation.

Structural changes involved in Serpin function

Members of the serpin superfamily control many fundamental biological processes where proteolysis is required such as coagulation, inflammation and apoptosis. When antitrypsin inhibits its proteinase it undergoes a dramatic conformational change that inactivates the proteinase. There is also increasing evidence to suggest that the proteinase itself undergoes a dramatic conformational change. Using mutagenesis we have produced a fluorescently silent antitrypsin molecule with which we can explore conformational changes within the proteinase. Through a combination of protein engineering and biophysical techniques the aim of this project is to elucidate the structural changes of the proteinase during inhibition by the serpin and therefore understand the fundamental process of proteinase inhibition.

Recent Publications

2009

Saunders HM, Bottomley SP. Multi-domain misfolding: understanding the aggregation pathway of polyglutamine proteins. Protein Eng Des Sel. 2009 Aug;22(8):447-51. Epub 2009 Jul 9.

Levina V, Dai W, Knaupp AS, Kaiserman D, Pearce MC, Cabrita LD, Bird PI, Bottomley SP. Expression, purification and characterization of recombinant Z alpha(1)-Antitrypsin-The most common cause of alpha(1)-Antitrypsin deficiency. Protein Expr Purif. 2009 Jun 23.

2008

Whisstock JC, Bottomley SP. Structural biology: Serpins' mystery solved. Nature. 2008 Oct 30;455(7217):1189-90.

Robertson AL, Horne J, Ellisdon AM, Thomas B, Scanlon MJ, Bottomley SP. The structural impact of a polyglutamine tract is location-dependent. Biophys J. 2008 Dec 15;95(12):5922-30.

Knaupp AS, Bottomley SP. Serpin polymerization and its role in disease--the molecular basis of alpha1-antitrypsin deficiency. IUBMB Life. 2009 Jan;61(1):1-5. Review.

Pearce MC, Morton CJ, Feil SC, Hansen G, Adams JJ, Parker MW, Bottomley SP. Preventing serpin aggregation: the molecular mechanism of citrate action upon antitrypsin unfolding. Protein Sci. 2008 Dec;17(12):2127-33.

2007

Cabrita LD, Gilis D, Robertson AL, Dehouck Y, Rooman M, Bottomley SP. Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 2007 Nov;16(11):2360-7.

Cabrita LD, Irving JA, Pearce MC, Whisstock JC, Bottomley SP. Aeropin from the extremophile Pyrobaculum aerophilum bypasses the serpin misfolding trap. J Biol Chem. 2007 Sep 14;282(37):26802-9.

Pearce MC, Cabrita LD, Ellisdon AM, Bottomley SP. The loss of tryptophan 194 in antichymotrypsin lowers the kinetic barrier to misfolding. FEBS J. 2007 Jul;274(14):3622-32.

Ellisdon AM, Pearce MC, Bottomley SP. Mechanisms of ataxin-3 misfolding and fibril formation: kinetic analysis of a disease-associated polyglutamine protein. J Mol Biol. 2007 Apr 27;368(2):595-605.

Powers GA, Pham CL, Pearce MC, Howlett GJ, Bottomley SP. Serpin acceleration of amyloid fibril formation: a role for accessory proteins. J Mol Biol. 2007 Feb 16;366(2):666-76.