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PEPTIDE BIOLOGY & PROTEOMICS LABORATORIES

Scientific Staff:

Ian Smith

ian.smith@med.monash.edu.au

 

Sanjaya Kuruppu

sanjaya.kuruppu@med.monash.edu.au

 

Ed Nice

ed.nice@med.monash.edu.au

 

Rui Zeng

rui.zeng@med.monash.edu.au

 

David Steer

david.steer@med.monash.edu.au


Professional &
Technical:



Shane Reeve



shane.reeve@med.monash.edu.au

Iresha Hanchapola

iresha.hanchapola@med.monash.edu.au

 

Josie Lawrence

josie.lawrence@med.monash.edu.au

Background: The vascular endothelial cells which line blood vessels express many proteases at the cell surface whose activity can directly affect blood pressure and vascular function. These enzymes can inactivate vasodilator peptides, generate vasoconstrictor peptides or convert inactive peptides into active vasoconstrictors. Manipulation of peptidase activities by specific inhibitors may allow us to control cardiovascular function and treat cardiovascular disease. The major aim of our research program is to apply advanced proteomic, structural, cellular and molecular biological technologies in a truly multidisciplinary approach to better understand the role that vascular peptides and peptidases play in the regulation of cardiovascular function. The laboratory is well-equipped with state of the art proteomic technologies (ToF ToF Mass Spectrometry, peptide sequencing, HPLC protein and peptide purification, 1 & 2D gels and gel analysis) as well as the necessary technologies to characterise peptidase function (automated plate readers for high throughput screening, recombinant enzyme expression, tissue culture etc.).

We are particularly interested in two membrane peptidases 1) Endothelin Converting Enzyme (ECE) and 2) Angiotensin Converting Enzyme 2 (ACE2), both of which act on circulating peptides to directly regulate blood pressure and/or vascular function.

Endothelin Converting Enzyme (ECE): Endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. Indeed, endothelin was until very recently considered the most potent natural vasoconstrictor and is second only to the newly discovered urotensin II. The generation of endothelin in the vasculature is crucially dependent on the local presence and activity of the endothelin converting enzyme or ECE. Our research on ECE has two main aspects: first to understand the regulatory mechanisms underlying the transport of the enzyme to and from the cell surface where it acts to generate endothelin; and second, in collaboration with the Structural Biology Laboratories at Monash (Dr James Whisstock), determine the X-ray structure of ECE to help facilitate the rational design of ECE specific inhibitors.

Angiotensin Converting Enzyme 2 (ACE2): Recent studies published over the last few years have not only changed the way we think about the role of angiotensin and angiotensin converting enzyme (ACE) in regulating vascular tone but more importantly, have shown that the system is far more complex than first thought. This new research has uncovered new players in the system such as the ACE homologue ACE2, as well as new roles for what were previously thought of as inactive peptide congeners of angiotensin (eg. angiotensin 1-7 and angiotensin IV). ACE2 is abundant in human heart, kidney, testes and intestine and has been implicated in heart and kidney function. ACE2 has also been identified as a receptor to the severe acute respiratory syndrome (SARS) coronavirus. We have shown that expression of ACE2 is increased in both heart disease and liver failure (hepatitis and cirrhosis). Interestingly, we have recently shown that ACE2 can be both constitutively and actively shed from the cell surface and have developed a method to measure soluble ACE2 activity. We have a number of projects aimed at trying to understand the physiological role of ACE2 in health and disease, characterizing the mechanisms by which ACE2 is shed from the cell surface and understanding the physiological significance of shedding, the mechanisms by which ACE2 is selectively targeted to different cell surfaces and finally, the design of ACE2 specific inhibitors.

Understanding the regulation, structure and function of these two peptidases along with our knowledge and ability to characterize peptide/peptidase interactions will allow us to design, synthesise and characterise specific peptidase inhibitors, not only as research tools to further probe their physiological function, but also as potential therapeutic agents to treat cardiovascular disease.

Research Project Areas:

  1. Regulation of Endothelin Converting Enzyme Expression at the Cell Surface
  2. Design and Analysis of Specific ECE Inhibitors
  3. Subcellular Trafficking of ACE2
  4. Regulation of ACE2 Expression in Health and Disease
  5. Secretion of ACE2 in vitro and in vivo

A Selection of recent publications, relevant to the above projects:

Chandana B H, Warner F J, Lubel J S, Dean R G, Zhiyuan J, Lew R A, Smith A I, Burrell L M, Angus P W. Upregulation of hepatic angiotensin-converting enzyme 2 (ACE2) and angiotensin-(1-7) levels in experimental biliary fibrosis. J Hepatol. 2007 Sep;47(3):387-95. Epub 2007 Apr 2.

1Rosado CJ, Buckle AM, Law RH, Butcher RE, Kan WT, Bird CH, Ung K, Browne KA, Baran K, Bashtannyk-Puhalovich TA, Faux NG, Wong W, Porter CJ, Pike RN, Ellisdon AM, Pearce MC, Bottomley SP, Emsley J, Smith AI, Rossjohn J, Hartland EL, Voskoboinik I, Trapani JA, Bird PI, Dunstone MA, Whisstock JC. A common fold mediates vertebrate defense and bacterial attack. Science. 2007 Sep 14;317(5844):1548-51. Epub 2007 Aug 23

1M.-I. Aguilar, A.W. Purcell, R. Devi, R. Lew, J. Rossjohn, A.I. Smith and P. Perlmutter (2007) " Beta-Amino acid-containing hybrid peptides - new opportunities in peptidomimetics", Org Biomol Chem. 2007 Sep 21;5(18):2884-90. Epub 2007 Aug 3

Kuruppu S, Reeve S, Smith A I. Characterisation of endothelin converting enzyme-1 shedding from endothelial cells. FEBS Letters, 2007 Volume 581, Issue 23, Pages 4501-4506

Jokubaitis V J Sinka S, Driessen R, Whitty G , Haylock D N, Bertoncello I, Smith A I, Péault B, Tavian M and Simmons P J. Angiotensin-converting enzyme (CD143) marks hematopoietic stem cells in human embryonic, fetal and adult hematopoietic tissues. Blood 2008 Apr 15;111(8):4055-6

R A. Lew, FJ. Warner, I Hanchapola, M A. Yarski, J. Manohar, L M. Burrell, A. I Smith. ACE2 catalytic activity in plasma is masked by an endogenous inhibitor. Experimental Physiology 2008 May;93(5):685-93

L Burchill, E Velkoska, R G Dean, R A Lew, A. I Smith, V Levidiotis, and L Burrell. Acute kidney injury in the rat causes cardiac remodelling and increases ACE2 expression. Experimental Physiology 93 5 2008)

Lai Z-W, Hanchapola I & Smith A I, Quenched fluorescent peptide substrates: tools for the discovery of novel biomarkers. Chemistry Today 2008 vol 26 n 2 / March-April 2008 29-31

Lai, R A. Lew, M A. Yarski, F-T Mu, R K. Andrews and A. I Smith. The identification of a calmodulin-binding domain within the cytoplasmic tail of angiotensin-converting enzyme-2.Endocrinology 2009 May;150(5):2376-81

Velkoska E, Warner FJ, Cole TJ, Smith AI, Morris MJ. Metabolic effects of low dose angiotensin converting enzyme inhibitor in dietary obesity in the rat. Nutrition, Metabolism & Cardiovascular Diseases 2009 Apr 8. [Epub ahead of print]

Clayton D, Hanchapola I, Perlmutter P, Smith AI, Aguilar MI. The active site specificity of angiotensin II converting enzyme 2 investigated through single and multiple residue changes and beta-amino acid substrate analogs. Adv Exp Med Biol. 2009;611:559-60

Smith AI, Warner FJ, Lew RA, Yarski M, McGrath B, Burrell LM. Quenched fluorescent peptide substrates as tools for the discovery of novel cardiovascular disease biomarkers. Adv Exp Med Biol. 2009;611:419-22