International Protease Network

Department of Biochemistry and Molecular Biology
Monash University
Melbourne, Vic 3800
Email: phil.bird@monash.edu.au
Tel: +61-3-9902-9365
Fax: +61-3-9902-9500
Homepage: http://www.med.monash.edu.au/biochem/staff/bird.html

Research

We have proposed that intracellular serpins protect cells against protease-induced damage and apoptosis. PI-9 (SERPINB9) is a specific granzyme B inhibitor produced by cytotoxic and dendritic cells that prevents their inadvertent destruction by ectopic granzyme B. It efficiently protects against granzyme B - mediated apoptosis, without blocking death receptor-mediated apoptosis. To further understand the role of PI-9 we are studying its localization within cytotoxic cells, its expression outside the immune system, and analysing knockout mice. We have recently shown that PI-9 shuttles between the nucleus and cytoplasm using an unconventional mechanism, and have also demonstrated that it is expressed in non-immune tissue such as placenta and testis. How and when granzyme B is released from cytotoxic lymphocytes and enters target cells, and how perforin releases it into the cytoplasm, is under investigation. We have identified regions on granzyme B required for its entry into cells, and have shown that it has an extracellular matrix-remodelling function, suggesting that its role extends beyond initiating apoptosis in compromised cells. We are also interested in the design of granzyme B substrates and inhibitors. The substrates can be used to monitor granzyme B activation, while the inhibitors may prevent apoptosis. We generate large quantities of recombinant granzyme B in Pichia pastoris and E.coli for these studies, and have successfully produced other granzymes for related work.

Collaborations

Prof Joe Trapani, Peter MacCallum Cancer Institute
Prof Rob Pike, Monash University
Prof James Whisstock, Monash University
Prof Steve Bottomley, Monash University
Prof Ian Smith, Monash University
Dr Ashley Buckle, Monash University
Dr Nigel Waterhouse
Dr Jamie Simpson, Monash Institute of Pharmaceutical Sciences
Dr Martin Scanlon, Monash Institute of Pharmaceutical Sciences
Dr Stephen Turner, University of Melbourne

Publications

Bird CH, Sutton VR, Sun J, Hirst CE, Novak A, Kumar S, Trapani JA and Bird PI (1998). Selective regulation of apoptosis: the cytotoxic lymphocyte serpin PI-9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway. Mol. Cell. Biol. 18, 6387-6398.

Scott FL, Hirst CE, Sun J, Bird CH, Bottomley SP and Bird PI (1999). The intracellular serpin proteinase inhibitor 6 (PI-6) is expressed in monocytes and granulocytes and is a potent inhibitor of the azurophilic granule protease, cathepsin G. Blood 93, 2089-2097.

Bird CH, Blink EJ, Hirst CE, Buzza MS, Steele PM, Sun J, Jans DA and Bird PI (2001). Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a non-conventional nuclear import pathway and the export factor Crm1. Mol Cell Biol 21, 5396-5407.

Hirst CE, Buzza MS, Bird CH, Warren HS, Cameron PU, Zhang M, Ashton-Rickardt PG and Bird PI (2003). The intracellular granzyme B inhibitor PI-9 is upregulated during accessory cell maturation and effector cell degranulation, and its overexpression enhances CTL potency. J Immunol 170, 805-815.

Buzza MS, Zamurs L, Sun J, Bird CH, Smith AI, Trapani JA, Froelich CJ, Nice EC, and Bird PI. (2005). Extracellular matrix remodeling by human granzyme B via cleavage of vitronectin, fibronectin and laminin. J Biol Chem. 280, 23549-58
Bird CH, Sun J, Ung K, Karambalis D, Whisstock JC, Trapani JA and Bird PI. (2005). Cationic sites on granzyme B contribute to cytotoxicity by promoting its uptake into target cells. Mol Cell Biol 25, 7854-7867.

Kaiserman D and Bird PI (2005). Analysis of vertebrate genomes suggests a new mechanism of clade B serpin evolution. BMC Genomics 6, 167.

Kaiserman D, Bird CH, Sun J, Matthews A, Ung K, Whisstock JC, Thompson PE, Trapani JA, and Bird PI (2006). The major human and mouse granzymes are structurally and functionally divergent. J Cell Biol 175, 619-630.

Rosado 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 (2007). A common fold mediates vertebrate defense and bacterial attack. Science 317, 1548-51.

Buzza MS, Dyson JM, Choi H, Gardiner EE, Andrews RK, Kaiserman D, Mitchell CA, Berndt MC, Dong J-F and Bird PI. (2008). Anti-hemostatic activity of human granzyme B mediated by cleavage of von Willebrand factor and fibrinogen. J Biol Chem 283, 22498-504

Kaiserman D, Buckle AM, Van Damme P, Irving JA, Law RH, Matthews AY, Bashtannyk-Puhalovich T, Langendorf C, Thompson P, Vandekerckhove J, Gevaert K, Whisstock JC, and Bird PI. (2009). Structure of granzyme C reveals an unusual mechanism of protease autoinhibition. Proc Natl Acad Sci U S A.106, 5587-92.