Professor Robert Pike
Deputy Dean (Academic Planning)
Faculty of Medicine, Nursing and Health Sciences
Telephone: +61-3-9902 9300
Facsimile: +61-3-9902 9500
Office: Bld 77, Room 204
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.
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.
The Pike Laboratory 2013
Left to Right:
Selected Peer-Reviewed Publications
Lourbakos A, Yuan Y, Jenkins AL, Travis J, Andrade-Gordon P, Santulli R, Potempa J & Pike RN. Activation of protease activated receptors by gingipains from Porphyromonas gingivalis leads to platelet aggregation: a new trait in microbial pathogenicity. (2001) Blood 97, 3790-3797.
Lourbakos A, Potempa J, Travis J, D'Andrea MR, Andrade-Gordon P, Santulli R, Mackie EJ & Pike RN. An arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion. (2001) Infect. Immun. 69, 5121-5130.
Irving JA, Shushanov SS, Pike RN, Popova E, Bromme D, Coetzer THT, Bottomley SP, Boulynko IA, Grigoryev SA & Whisstock JC. Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation. (2002) J. Biol. Chem. 277, 13192-13201.
Pagel CN, DeNiese MR, Abraham LA, Chinni C, Song S-J, Pike RN & Mackie EJ. Inhibition of osteoblast apoptosis by thrombin. (2003) Bone 33, 733-43.
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. J. Bone Min. Res. 19, 507-516.
Song S-J, Campbell TM, Pagel CN, Pike RN & Mackie EJ. (2005) The Role of Protease-Activated Receptor-1 in Bone Healing. American J Pathol. 166, 857-868.
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. J. Biol. Chem. 280, 39510-39514.
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 J. 25, 3144-3155.
Bromfield KM, Quinsey NS, Duggan PJ & Pike RN. (2006) Approaches to selective peptidic inhibitors of factor Xa. Chem. Biol. Drug Des. 68, 11-19.
Fenalti G, Law RHP, Buckle AM, Langendorf C, Tuck K, Rosado CJ, Faux NG, Mahmood K, Hampe CS, Banga P, Wilce M, Schmidberger J, Rossjohn J, El-Kabbani O, Pike RN, Smith AI, Mackay IR, Rowley MJ & Whisstock JC. (2007) GABA Production by Glutamic Acid Decarboxylase is Regulated by a Dynamic Catalytic Loop. Nature Struct. Mol. Biol. 14, 280-286.
Kerr FK, Thomas AR, Wijeyewickrema LC, Whisstock JC, Boyd SE, Kaiserman D, Matthews AY, Bird PI, Thielens NM, Rossi V & Pike RN. (2007) Elucidation of the substrate specificity of the MASP-2 protease of the lectin complement pathway and identification of the enzyme as a major physiological target of the serpin, C1-inhibitor. Mol. Immunol. 45, 670-677.
Rosado CJ, Buckle AM, Law RHP, 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, Reid H, Hartland EL, Voskoboinik I, Trapani JA, Bird PI, Dunstone MA & Whisstock JC. (2007) A common fold mediates vertebrate defense and bacterial attack. Science 317, 1548-1551.
Ong PC, McGowan S, Pearce MC, Irving JA, Kan W-T, Grigoryev SA, Turk B, Silverman GA, Brix K, Bottomley SP, Whisstock JC & Pike RN. (2007) DNA accelerates the inhibition of human cathepsin V by serpins. J. Biol. Chem. 282, 36980-36986.
Fitzpatrick RE, Aprico A, Wijeyewickrema LC, Pagel CN, Wong DM, Potempa J, Mackie EJ & Pike RN. (2009) High molecular weight gingipains from Porphyromonas gingivalis induce cytokine responses from human macrophage-like cells via a non-proteolytic mechanism. J. Innate Immun. 1, 109-117.
Beckham SA, Piedrafita D, Phillips CI, Samarawickrema N, Law RHP, Smooker PM, Quinsey NS, Irving JA, Greenwood D, Verhelst SHL, Bogyo M, Turk B, Coetzer TH, Wijeyewickrema LC, Spithill TW & Pike RN. (2009) A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites. Int. J. Biochem. Cell Biol. 41, 1601-1612.
Murray-Rust TA, Kerr FK, Thomas AR, Wu T, Yongqing T, Ong PC, Quinsey NS, Whisstock JC, Wagenaar-Bos I, Freeman C & Pike RN. (2009) Modulation of the proteolytic activity of the complement protease C1s by polyanions: implications for polyanion-mediated acceleration of interaction between C1s and SERPING1. Biochem. J. 422, 295-303.
Beckham SA, Boyd SE, Reynolds S, Willis C, Johnstone M, Mika A, Simerská P, Wijeyewickrema LC, Smith AI, Kemp DJ, Pike RN and Fischer K. (2009) Characterisation of a serine protease homologous to house dust mite group 3 allergens from the scabies mite Sarcoptes scabiei. J. Biol. Chem. 284, 34413-34422.
Bergström FC, Reynolds S, Johnstone M, Pike RN, Buckle AM, Kemp DJ, Fischer K & Blom AM. (2009) Scabies mite inactivated serine protease paralogues inhibit the human complement system. J. Immunol. 182, 7809-7817.
Wong DM, Tam V, Lam R, Walsh KA, Tatarczuch L, Pagel CN, Reynolds EC, O'Brien-Simpson NM, Mackie EJ & Pike RN. (2010) Protease-activated receptor-2 has pivotal roles in cellular mechanisms involved in experimental periodontitis. Infect. Immun. 78, 629-638.
Ng NM, Pike RN & Boyd SE. (2009) Subsite Cooperativity in Protease Specificity. Biol. Chem. 390, 401-407.
Kennan RM, Wong W, Dhungye OP, Han X, Wong D, Parker D, Rosado CJ, Law RHP, McGowan S, Reeve SB, Levina V, Powers GA, Pike RN, Bottomley SP, Smith AI, Marsh I, Whittington RJ, Whisstock JC, Porter CJ & Rood JI. (2010) The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates. PLOS Pathogens 6, e1001210.
Duncan RC, Bergström F, Coetzer TH, Blom AM, Wijeyewickrema LC & Pike RN. (2011) Multiple domains of MASP-2, an initiating complement protease, are required for interaction with its substrate C4. Mol. Immunol. 49, 593-600.
Wong W, Wijeyewickrema LC, Kennan RM, Reeve SB, Steer DL, Reboul C, Smith AI, Pike RN, Rood JI, Whisstock JC & Porter CJ. (2011) The S1 pocket of a bacterial derived subtilisin-like protease underpins effective tissue destruction. J. Biol. Chem. 286, 42180-42187.
Ng NM, Pierce JD, Webb GI, Ratnikov BI, Wijeyewickrema LC, Duncan RC, Robertson AL, Bottomley SP, Boyd SE & Pike RN. (2011) Discovery of amino acid motifs for thrombin cleavage and validation using a model substrate. Biochemistry 50, 10499-10507.
Hsieh YSY, Taleski D, Wilkinson BL, Wijeyewickrema LC, Adams TE, Pike RN & Payne RJ. (2011) Role of O-Glycosylation and Tyrosine Sulfation on Leech-Derived Peptides for Binding and Inhibitory Activity against Thrombin. Chemical Commun. 48, 1547-1549.
Georgy SR, Pagel CN, Ghasem-Zadeh A, Zebaze RMD, Pike RN, Sims NA & Mackie EJ. (2012) Proteinase-activated receptor-2 is required for normal osteoblast and osteoclast differentiation during skeletal growth and repair. Bone 50, 704-712.
Norbury LJ, Hung A, Beckham S, Pike RN, Spithill TW, Craik CS, Choe Y, Fecondo JV & Smooker PM. (2012) Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1-P4 specificity reveals an unusual specificity. Biochimie 94, 1119-1127.
Law RH, Caradoc-Davies T, Cowieson N, Horvath AJ, Quek AJ, Encarnacao JA, Steer D, Cowan A, Zhang Q, Lu BG, Pike RN, Smith AI, Coughlin PB & Whisstock JC. (2012) The X-ray crystal structure of full-length human plasminogen. Cell Reports 1, 185-190.
Duncan RC, Mohlin F, Taleski D, Coetzer TH, Huntington JA, Payne RJ, Blom AM, Pike RN & Wijeyewickrema LC. (2012) Identification of a catalytic exosite for complement component C4 on the serine protease domain of C1s. Journal of Immunology 189, 2365-2373. [joint last and corresponding author]
Mika A, Reynolds SL, Mohlin FC, Halilovic V, Willis C, Pickering DA, Swe PM, Wijeyewickrema LC, Pike RN, Blom AM, Kemp DJ, Fischer K. (2012) Novel Scabies mite serpins inhibit the three pathways of the human complement system. PLoS One 7, e40489.
Song J, Hao Tan H, Perry AJ, Akutsu T, Webb GI, Whisstock JC & Pike RN. (2012) PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites. PLoS One 7, e50300.
Langendorf CG, Tuck KL, Key TL, Fenalti G, Pike RN, Rosado CJ, Wong AS, Buckle AM, Law RH, Whisstock JC. (2012) Structural characterization on the mechanism of auto-inactivation for Human Glutamic Acid Decarboxylase. Bioscience Reports (in press).
Dunstan RA, Heinz E, Wijeyewickrema LC, Pike RN, Purcell AW, Evans TJ, Praszkier J, Robins-Browne RM, Strugnell RA, Korotkov KV & Lithgow T. (2012) Assembly of the Type II Secretion System such as found in Vibrio cholerae depends on the novel pilotin AspS. PLoS Pathogens (in press). (Accepted 21/11/12)