Professor Robert Pike

Head, Department of Biochemistry and Molecular Biology

Telephone:           +61-3-9902 9300 Facsimile:             +61-3-9902 9435 Office:                  Bld 77, Room G31 Email: rob.pike@med.monash.edu.au

Proteases, inhibitors and receptors:relationship to disease states.

Proteases are involved in a diverse range of biological processes. Their control by inhibitors is critical to the maintenance of health. Proteases also cleave cell surface receptors leading to activation of second messenger systems and cellular responses such as differentiation and proliferation. Micro-organisms use proteases to alter normal biological mechanisms in the host and thus facilitate pathogenesis.

Project Areas

Regulation and control of the Complement system in immunity
The complement system is vital in preventing disease caused by infections. It is also strongly implicated to be involved in a number of diseases where there is excess inflammation occurring. An example of this is the situation following a heart attack, where a condition called reperfusion injury results after the circulation has been restored. The reperfusion injury results from excessive inflammation caused by overactivity of the complement system in particular, resulting in reocclusion of the circulation and another heart attack. This strongly implies that control of the complement pathway in this context would prevent heart disease.

Model of the MASP-2 complement protease.
One of the CCP domains is shown in green and the active serine protease is in pink. The CCP domain is thought to participate in the interaction with substrate proteins such as the C4 complement protein.

We are studying both the classical and mannose-binding lectin (MBL) pathways of complement activation. These pathways involve the sequential activation of proteins by a cascade of proteases. A particular focus in the laboratory at present, is the initiating proteases of the two pathways, C1r, C1s and the MBL-associated serine proteases (MASPs). There are three of the latter enzymes and their respective roles in complement activation are not clear, although it appears that MASP-2 carries out the bulk of the direct complement activating activity.

The major studies in the laboratory are aimed at examining how these proteases interact with their target substrates, the complement C2 and C4 proteins and their regulatory inhibitor, C1-inhibitor. We are using phage display technology (in association with A/Prof Phil Bird) to map the substrate specificity of the proteases. Based on these results, we plan to develop specific protein and peptide inhibitors of the different proteases in order to map their respective roles in complement activation. We also plan to investigate the roles of the other domains of the proteases in the interaction with substrates and inhibitors.

Dissection of catalytic and adhesin activities of proteases from Porphyromonas gingivalis

The anaerobic bacterium, Porphyromonas gingivalis, is a causative agent of periodontal (gum) disease. This disease is the primary cause of tooth loss in the Western world and is also associated with heart disease and other more systemic disease. The bacterium secretes potent cysteine proteases called gingipains. These proteases are vital for the virulence of the bacterium. The proteases are made up of a catalytic subunit and several other protein subunits which have been characterized as adhesins. The adhesins bind to other bacteria, a process which is vital for colonization of the oral cavity, and also bind to host cells and proteins. It would seem most likely that the adhesin and protease activities of this protease are vital to its role in the virulence of P. gingivalis. We are seeking to understand the determinants of both binding and protease specificity and we are using phage display technology to reveal this.

Crystal structure of a gingipain.
The active site of the catalytic domain is shown at the top of the red colloured domain, the green coloured domain is the other segment of the catalytic domain, while the gold coloured domain is an adhesin domain.

Selected recent publications from the Pike laboratory

Ally N, Whisstock JC, Sieprawska-Lupa M, Potempa J, Le Bonniec BF, Travis J and Pike RN. (2003) Characterisation of the specificity of Arginine-Specific gingipains from Porphyromonas gingivalis reveals active site differences between different forms of the enzymes. Biochemistry 42, 11693-11700.

O’Brien G, Quinsey NS, Whisstock JC & Pike RN. (2003) The importance of the prime subsites of the C1s protease of the classical complement pathway for recognition of substrates. Biochemistry 42, 14939-14945.

Smith RA, Ransjo M, Tatarczuch L, Song S-J, Pagel CN, Morrison JR, Pike RN & Mackie EJ. (2004) Activation of Protease-Activated Receptor-2 Leads to Inhibition of Osteoclast Differentiation. Journal of Bone & Mineral Research 19, 507-516.

Sztukowska M, Sroka A, Bugno M, Banbula A, Takahashi Y, Pike RN, Genco CA, Travis J, & Potempa J. (2004) The C-terminal domains of the gingipain K polyprotein are necessary for assembly of the active enzyme and expression of associated activities. Molecular Microbiology 54, 1393-1408.

Song S-J, Campbell TM, Pagel CN, Pike RN & Mackie EJ. (2005) The Role of Protease-Activated Receptor-1 in Bone Healing. American Journal of Pathology 166, 857-868.

Boyd SE, Pike RN, M., Rudy GB, Whisstock JC & Garcia de la Banda M. PoPS: A Computational Tool for Modelling and Predicting Protease Specificity. (2005) Journal of Bioinformatics and Computational Biology 3, 551-585.

Horvath AJ, Irving JA, Rossjohn J, Law RH, Bottomley SP, Quinsey NS, Pike RN, Coughlin PB, Whisstock JC. (2005) The murine orthologue of human antichymotrypsin: A structural paradigm for clade A3 serpins. Journal of Biological Chemistry 280, 43168-43178.

Kerr FK, O’Brien G, Quinsey NS, Whisstock JC, Boyd S, Garcia de la Banda M, Kaiserman D, Matthews AY, Bird PI & Pike RN. (2005) Elucidation of the substrate specificity of the C1s protease of the classical complement pathway. Journal of Biological Chemistry 280, 39510-39514.

Beckham SA, Law RH-P, Smooker PM, Quinsey NS, Caffrey CR, McKerrow JH, Pike RN and Spithill TW. (2006) Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite. Biological Chemistry (in press). [joint last author]

McGowan S, Buckle AM, Irving JA, Ong PC, Bashtannyk-Puhalovich TA, KanW-T, Henderson KN, Bulynko YA, Popova EY, Smith AI, Bottomley SP, Rossjohn J, Grigoryev SA, Pike RN & Whisstock JC. (2006) X-ray Crystal Structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation. EMBO Journal 25, 3144-3155. [joint last author]