Publications by authors named "Robert N Pike"

99 Publications

Mapping the binding site of C1-inhibitor for polyanion cofactors.

Mol Immunol 2020 10 24;126:8-13. Epub 2020 Jul 24.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia; ARC Centre of Excellence in Advanced Molecular Imaging, La Trobe University, Melbourne, Victoria 3086, Australia. Electronic address:

The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules.
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http://dx.doi.org/10.1016/j.molimm.2020.06.018DOI Listing
October 2020

Protease-associated import systems are widespread in Gram-negative bacteria.

PLoS Genet 2019 10 15;15(10):e1008435. Epub 2019 Oct 15.

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Australia.

Bacteria have evolved sophisticated uptake machineries in order to obtain the nutrients required for growth. Gram-negative plant pathogens of the genus Pectobacterium obtain iron from the protein ferredoxin, which is produced by their plant hosts. This iron-piracy is mediated by the ferredoxin uptake system (Fus), a gene cluster encoding proteins that transport ferredoxin into the bacterial cell and process it proteolytically. In this work we show that gene clusters related to the Fus are widespread in bacterial species. Through structural and biochemical characterisation of the distantly related Fus homologues YddB and PqqL from Escherichia coli, we show that these proteins are analogous to components of the Fus from Pectobacterium. The membrane protein YddB shares common structural features with the outer membrane ferredoxin transporter FusA, including a large extracellular substrate binding site. PqqL is an active protease with an analogous periplasmic localisation and iron-dependent expression to the ferredoxin processing protease FusC. Structural analysis demonstrates that PqqL and FusC share specific features that distinguish them from other members of the M16 protease family. Taken together, these data provide evidence that protease associated import systems analogous to the Fus are widespread in Gram-negative bacteria.
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http://dx.doi.org/10.1371/journal.pgen.1008435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6793856PMC
October 2019

Determination of the crystal structure and substrate specificity of ananain.

Biochimie 2019 Nov 12;166:194-202. Epub 2019 Jul 12.

Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia; ARC Centre of Excellence for Advanced Molecular Imaging. Melbourne, Australia. Electronic address:

Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. Despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference and inhibitory profile compared to stem bromelain. A tripeptidyl substrate library (REPLi) was used to further characterize the substrate specificity of ananain and identified an optimal substrate for cleavage by ananain. The optimal tripeptide, PLQ, yielded a high k/K value of 1.7 x 106 Ms, with cleavage confirmed to occur after the Gln residue. Crystal structures of unbound ananain and an inhibitory complex of ananain and E-64, solved at 1.73 and 1.98 Å, respectively, revealed a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in the specificity studies. A further illustration of the atomic interactions between E-64 and ananain explains the high inhibitory efficiency of E-64 toward ananain. These data reveal the first in depth structural and functional data for ananain and provide a basis for further study of the natural properties of the enzyme.
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http://dx.doi.org/10.1016/j.biochi.2019.07.011DOI Listing
November 2019

Twenty years of bioinformatics research for protease-specific substrate and cleavage site prediction: a comprehensive revisit and benchmarking of existing methods.

Brief Bioinform 2019 11;20(6):2150-2166

Biomedicine Discovery Institute and Department of Biochemistry & Molecular Biology, Monash University, Melbourne, VIC 3800, Australia.

The roles of proteolytic cleavage have been intensively investigated and discussed during the past two decades. This irreversible chemical process has been frequently reported to influence a number of crucial biological processes (BPs), such as cell cycle, protein regulation and inflammation. A number of advanced studies have been published aiming at deciphering the mechanisms of proteolytic cleavage. Given its significance and the large number of functionally enriched substrates targeted by specific proteases, many computational approaches have been established for accurate prediction of protease-specific substrates and their cleavage sites. Consequently, there is an urgent need to systematically assess the state-of-the-art computational approaches for protease-specific cleavage site prediction to further advance the existing methodologies and to improve the prediction performance. With this goal in mind, in this article, we carefully evaluated a total of 19 computational methods (including 8 scoring function-based methods and 11 machine learning-based methods) in terms of their underlying algorithm, calculated features, performance evaluation and software usability. Then, extensive independent tests were performed to assess the robustness and scalability of the reviewed methods using our carefully prepared independent test data sets with 3641 cleavage sites (specific to 10 proteases). The comparative experimental results demonstrate that PROSPERous is the most accurate generic method for predicting eight protease-specific cleavage sites, while GPS-CCD and LabCaS outperformed other predictors for calpain-specific cleavage sites. Based on our review, we then outlined some potential ways to improve the prediction performance and ease the computational burden by applying ensemble learning, deep learning, positive unlabeled learning and parallel and distributed computing techniques. We anticipate that our study will serve as a practical and useful guide for interested readers to further advance next-generation bioinformatics tools for protease-specific cleavage site prediction.
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http://dx.doi.org/10.1093/bib/bby077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954447PMC
November 2019

Keratinocyte-specific ablation of protease-activated receptor 2 prevents gingival inflammation and bone loss in a mouse model of periodontal disease.

Cell Microbiol 2018 Nov 31;20(11):e12891. Epub 2018 Jul 31.

Department of Veterinary Biosciences, Melbourne Veterinary School, University of Melbourne, Parkville, Victoria, Australia.

Chronic periodontitis is characterised by gingival inflammation and alveolar bone loss. A major aetiological agent is Porphyromonas gingivalis, which secretes proteases that activate protease-activated receptor 2 (PAR ). PAR expressed on oral keratinocytes is activated by proteases released by P. gingivalis, inducing secretion of interleukin 6 (IL-6), and global knockout of PAR prevents bone loss and inflammation in a periodontal disease model in mice. To test the hypothesis that PAR expressed on gingival keratinocytes is required for periodontal disease pathology, keratinocyte-specific PAR -null mice were generated using K14-Cre targeted deletion of the PAR gene (F2rl1). These mice were subjected to a model of periodontitis involving placement of a ligature around a tooth, combined with P. gingivalis infection ("Lig + Inf"). The intervention caused a significant 44% decrease in alveolar bone volume (assessed by microcomputed tomography) in wildtype (K14-Cre:F2rl1 ), but not littermate keratinocyte-specific PAR -null (K14-Cre:F2rl1 ) mice. Keratinocyte-specific ablation of PAR prevented the significant Lig + Inf-induced increase (2.8-fold) in the number of osteoclasts in alveolar bone and the significant up-regulation (2.4-4-fold) of the inflammatory markers IL-6, IL-1β, interferon-γ, myeloperoxidase, and CD11b in gingival tissue. These data suggest that PAR expressed on oral epithelial cells is a critical regulator of periodontitis-induced bone loss and will help in designing novel therapies with which to treat the disease.
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http://dx.doi.org/10.1111/cmi.12891DOI Listing
November 2018

Molecular basis for the folding of β-helical autotransporter passenger domains.

Nat Commun 2018 04 11;9(1):1395. Epub 2018 Apr 11.

Research School of Biology, Australian National University, Canberra, ACT, 0200, Australia.

Bacterial autotransporters comprise a C-terminal β-barrel domain, which must be correctly folded and inserted into the outer membrane to facilitate translocation of the N-terminal passenger domain to the cell exterior. Once at the surface, the passenger domains of most autotransporters are folded into an elongated β-helix. In a cellular context, key molecules catalyze the assembly of the autotransporter β-barrel domain. However, how the passenger domain folds into its functional form is poorly understood. Here we use mutational analysis on the autotransporter Pet to show that the β-hairpin structure of the fifth extracellular loop of the β-barrel domain has a crucial role for passenger domain folding into a β-helix. Bioinformatics and structural analyses, and mutagenesis of a homologous autotransporter, suggest that this function is conserved among autotransporter proteins with β-helical passenger domains. We propose that the autotransporter β-barrel domain is a folding vector that nucleates folding of the passenger domain.
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http://dx.doi.org/10.1038/s41467-018-03593-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895577PMC
April 2018

PROSPERous: high-throughput prediction of substrate cleavage sites for 90 proteases with improved accuracy.

Bioinformatics 2018 02;34(4):684-687

ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, VIC 3800, Australia.

Summary: Proteases are enzymes that specifically cleave the peptide backbone of their target proteins. As an important type of irreversible post-translational modification, protein cleavage underlies many key physiological processes. When dysregulated, proteases' actions are associated with numerous diseases. Many proteases are highly specific, cleaving only those target substrates that present certain particular amino acid sequence patterns. Therefore, tools that successfully identify potential target substrates for proteases may also identify previously unknown, physiologically relevant cleavage sites, thus providing insights into biological processes and guiding hypothesis-driven experiments aimed at verifying protease-substrate interaction. In this work, we present PROSPERous, a tool for rapid in silico prediction of protease-specific cleavage sites in substrate sequences. Our tool is based on logistic regression models and uses different scoring functions and their pairwise combinations to subsequently predict potential cleavage sites. PROSPERous represents a state-of-the-art tool that enables fast, accurate and high-throughput prediction of substrate cleavage sites for 90 proteases.

Availability And Implementation: http://prosperous.erc.monash.edu/.

Contact: [email protected] or [email protected] or [email protected]

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btx670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5860617PMC
February 2018

The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech.

J Immunol 2017 12 23;199(11):3883-3891. Epub 2017 Oct 23.

Department of Biochemistry and Genetics and Australian Research Council Centre of Excellence in Advanced Molecular Imaging, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia;

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.
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http://dx.doi.org/10.4049/jimmunol.1700158DOI Listing
December 2017

A T cell-specific knockout reveals an important role for protease-activated receptor 2 in lymphocyte development.

Int J Biochem Cell Biol 2017 11 22;92:95-103. Epub 2017 Sep 22.

Department of Veterinary Biosciences, Melbourne Veterinary School, University of Melbourne, Parkville, VIC 3010, Australia. Electronic address:

Activation of protease-activated receptor-2 (PAR) expressed by T cells has been linked to the bone loss associated with periodontitis. We generated PAR conditional-null mice and crossed these with mice expressing Cre recombinase under control of the Lck proximal promoter, to produce T cell-specific PAR-null mice in order to further study the cellular mechanism involved in periodontitis. Here we report that efficient deletion of PAR in thymocytes isolated from T cell-specific PAR-null mice resulted in thymic and splenic hypoplasia and a reduction in the cells of the cortex and a loss of distinction between the cortex and the medulla of the thymus. FACS analysis confirmed significant reductions in CD4 and CD8 double negative (DN3 and DN4) sub-populations, as well as double positive and single positive T cells, in T cell-specific PAR-null mice compared to Cre expressing PAR wild-type mice. The proportion of annexin V positive and propidium iodide negative cells was increased in CD4 and CD8 double negative, double positive and single positive T cells from T cell-specific PAR-null mice. No change in the proportion of Ki67 positive cells was observed in sections of thymus from T cell-specific PAR-null mice, suggesting that the depletion of T cell sub-populations in T cell-specific PAR-null mice resulted from increased apoptosis rather than reduced proliferation. Together, these results demonstrate that PAR plays an important and previously unrecognised anti-apoptotic role in T cell development and suggest that the PAR conditional-null mouse will be an important resource for determining tissue and cell specific effects of PAR.
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http://dx.doi.org/10.1016/j.biocel.2017.09.015DOI Listing
November 2017

Polyphosphate is a novel cofactor for regulation of complement by a serpin, C1 inhibitor.

Blood 2016 09 23;128(13):1766-76. Epub 2016 Jun 23.

Centre for Blood Research, Department of Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada;

The complement system plays a key role in innate immunity, inflammation, and coagulation. The system is delicately balanced by negative regulatory mechanisms that modulate the host response to pathogen invasion and injury. The serpin, C1-esterase inhibitor (C1-INH), is the only known plasma inhibitor of C1s, the initiating serine protease of the classical pathway of complement. Like other serpin-protease partners, C1-INH interaction with C1s is accelerated by polyanions such as heparin. Polyphosphate (polyP) is a naturally occurring polyanion with effects on coagulation and complement. We recently found that polyP binds to C1-INH, prompting us to consider whether polyP acts as a cofactor for C1-INH interactions with its target proteases. We show that polyP dampens C1s-mediated activation of the classical pathway in a polymer length- and concentration-dependent manner by accelerating C1-INH neutralization of C1s cleavage of C4 and C2. PolyP significantly increases the rate of interaction between C1s and C1-INH, to an extent comparable to heparin, with an exosite on the serine protease domain of the enzyme playing a major role in this interaction. In a serum-based cell culture system, polyP significantly suppressed C4d deposition on endothelial cells, generated via the classical and lectin pathways. Moreover, polyP and C1-INH colocalize in activated platelets, suggesting that their interactions are physiologically relevant. In summary, like heparin, polyP is a naturally occurring cofactor for the C1s:C1-INH interaction and thus an important regulator of complement activation. The findings may provide novel insights into mechanisms underlying inflammatory diseases and the development of new therapies.
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http://dx.doi.org/10.1182/blood-2016-02-699561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043130PMC
September 2016

Structural basis for substrate specificity of Helicobacter pylori M17 aminopeptidase.

Biochimie 2016 Feb 1;121:60-71. Epub 2015 Dec 1.

Infection and Immunity Program, Monash Biomedical Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia. Electronic address:

The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases.
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http://dx.doi.org/10.1016/j.biochi.2015.11.021DOI Listing
February 2016

Protein unfolding is essential for cleavage within the α-helix of a model protein substrate by the serine protease, thrombin.

Biochimie 2016 Mar 25;122:227-34. Epub 2015 Sep 25.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia.

Proteolysis has a critical role in transmitting information within a biological system and therefore an important element of biology is to determine the subset of proteins amenable to proteolysis. Until recently, it has been thought that proteases cleave native protein substrates only within solvent exposed loops, but recent evidence indicates that cleavage sites located within α-helices can also be cleaved by proteases, despite the conformation of this secondary structure being generally incompatible with binding into an active site of a protease. In this study, we address the mechanism by which a serine endopeptidase, thrombin, recognizes and cleaves a target sequence located within an α-helix. Thrombin was able to cleave a model substrate, protein G, within its α-helix when a suitable cleavage sequence for the enzyme was introduced into this region. However, structural data for the complex revealed that thrombin was not perturbing the structure of the α-helix, thus it was not destabilizing the helix in order to allow it to fit within its active site. This indicated that thrombin was only cleaving within the α-helix when it was in an unfolded state. In support of this, the introduction of destabilizing mutations within the protein increased the efficiency of cleavage by the enzyme. Our data suggest that a folded α-helix cannot be proteolytically cleaved by thrombin, but the species targeted are the unfolded conformations of the native state ensemble.
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http://dx.doi.org/10.1016/j.biochi.2015.09.021DOI Listing
March 2016

The protease cathepsin L regulates Th17 cell differentiation.

J Autoimmun 2015 Dec 3;65:56-63. Epub 2015 Sep 3.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA; Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Previously we reported that IL-17(+) T cells, primarily IL-17(+) γδ cells, are increased in mice lacking the protease inhibitor serpinB1 (serpinb1(-/-) mice). Here we show that serpinB1-deficient CD4 cells exhibit a cell-autonomous and selective deficiency in suppressing T helper 17 (Th17) cell differentiation. This suggested an opposing role for one or more protease in promoting Th17 differentiation. We found that several SerpinB1-inhibitable cysteine cathepsins are induced in Th17 cells, most prominently cathepsin L (catL); this was verified by peptidase assays, active site labeling and Western blots. Moreover, Th17 differentiation was suppressed by both broad cathepsin inhibitors and catL selective inhibitors. CatL is present in Th17 cells as single chain (SC)- and two-chain (TC)-forms. Inhibiting asparagine endopeptidase (AEP) blocked conversion of SC-catL to TC-catL and increased generation of serpinb1(-/-) Th17 cells, but not wild-type Th17 cells. These findings suggest that SC-catL is biologically active in promoting Th17 generation and is counter-regulated by serpinB1 and secondarily by AEP. Thus, in addition to regulation by cytokines and transcription factors, differentiation of CD4 cells to Th17 cells is actively regulated by a catL-serpinB1-AEP module. Targeting this protease regulatory module could be an approach to treating Th17 cell-driven autoimmune disorders.
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http://dx.doi.org/10.1016/j.jaut.2015.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679515PMC
December 2015

Investigation of the mechanism of interaction between Mannose-binding lectin-associated serine protease-2 and complement C4.

Mol Immunol 2015 Oct 27;67(2 Pt B):287-93. Epub 2015 Jun 27.

Department of Biochemistry & Genetics, La Trobe Institute of Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia.

The interaction between mannose-binding lectin [MBL]-associated serine protease-2 (MASP-2) and its first substrate, C4 is crucial to the lectin pathway of complement, which is vital for innate host immunity, but also involved in a number of inflammatory diseases. Recent data suggests that two areas outside of the active site of MASP-2 (so-called exosites) are crucial for efficient cleavage of C4: one at the junction of the two complement control protein (CCP) domains of the enzyme and the second on the serine protease (SP) domain. Here, we have further investigated the roles of each of these exosites in the binding and cleavage of C4. We have found that both exosites are required for high affinity binding and efficient cleavage of the substrate protein. Within the SP domain exosite, we have shown here that two arginine residues are most important for high affinity binding and efficient cleavage of C4. Finally, we show that the CCP domain exosite appears to play the major role in the initial interaction with C4, whilst the SP domain exosite plays the major role in a secondary conformational change between the two proteins required to form a high affinity complex. This data has provided new insights into the binding and cleavage of C4 by MASP-2, which may be useful in the design of molecules that modulate this important interaction required to activate the lectin pathway of complement.
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http://dx.doi.org/10.1016/j.molimm.2015.06.011DOI Listing
October 2015

Scabies mite inactive serine proteases are potent inhibitors of the human complement lectin pathway.

PLoS Negl Trop Dis 2014 May 22;8(5):e2872. Epub 2014 May 22.

Infectious Diseases Division, QIMR Berghofer Medical Research Institute, Brisbane, Australia.

Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics.
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http://dx.doi.org/10.1371/journal.pntd.0002872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031079PMC
May 2014

Total synthesis of homogeneous variants of hirudin P6: a post-translationally modified anti-thrombotic leech-derived protein.

Angew Chem Int Ed Engl 2014 Apr 11;53(15):3947-51. Epub 2014 Mar 11.

School of Chemistry, The University of Sydney, Sydney, NSW 2006 (Australia) http://sydney.edu.au/science/chemistry/∼payne/index.

Hirudin P6 is a leech-derived anti-thrombotic protein which possesses two post-translational modifications, O-glycosylation and tyrosine sulfation. In this study we report the ligation-based synthesis of a library of hirudin P6 proteins possessing homogeneous glycosylation and sulfation modifications. The nature of the modifications incorporated was shown to have a drastic effect on inhibition against both the fibrinogenolytic and amidolytic activities of thrombin and thus highlights a potential means for attenuating the biological activity of the protein.
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http://dx.doi.org/10.1002/anie.201310777DOI Listing
April 2014

A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis.

J Exp Med 2013 Nov 4;210(12):2569-82. Epub 2013 Nov 4.

Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.

Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine-containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01(+) individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4(+) T cells in the peripheral blood of HLA-DRB1*04:01(+) RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.
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http://dx.doi.org/10.1084/jem.20131241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832918PMC
November 2013

The molecular switches controlling the interaction between complement proteases of the classical and lectin pathways and their substrates.

Curr Opin Struct Biol 2013 Dec 7;23(6):820-7. Epub 2013 Aug 7.

Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. Electronic address:

Complement represents a major bridge between the innate and adaptive immune systems of the body. It plays a vital role in host defences against pathogens, but has also been implicated in numerous inflammatory diseases. The system has been the subject of intensive research in recent times with a number of key structural insights into the functioning of the system. Here, we will give an overview of the activation of each pathway, following which recent developments in our understanding of the mechanisms governing the interaction between enzymes and substrates in the classical and lectin pathways in particular will be discussed.
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http://dx.doi.org/10.1016/j.sbi.2013.07.016DOI Listing
December 2013

The x-ray crystal structure of mannose-binding lectin-associated serine proteinase-3 reveals the structural basis for enzyme inactivity associated with the Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome.

J Biol Chem 2013 Aug 21;288(31):22399-407. Epub 2013 Jun 21.

Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

The mannose-binding lectin associated-protease-3 (MASP-3) is a member of the lectin pathway of the complement system, a key component of human innate and active immunity. Mutations in MASP-3 have recently been found to be associated with Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome, a severe developmental disorder manifested by cleft palate, intellectual disability, and skeletal abnormalities. However, the molecular basis for MASP-3 function remains to be understood. Here we characterize the substrate specificity of MASP-3 by screening against a combinatorial peptide substrate library. Through this approach, we successfully identified a peptide substrate that was 20-fold more efficiently cleaved than any other identified to date. Furthermore, we demonstrated that mutant forms of the enzyme associated with 3MC syndrome were completely inactive against this substrate. To address the structural basis for this defect, we determined the 2.6-Å structure of the zymogen form of the G666E mutant of MASP-3. These data reveal that the mutation disrupts the active site and perturbs the position of the catalytic serine residue. Together, these insights into the function of MASP-3 reveal how a mutation in this enzyme causes it to be inactive and thus contribute to the 3MC syndrome.
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http://dx.doi.org/10.1074/jbc.M113.483875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829330PMC
August 2013

A molecular switch governs the interaction between the human complement protease C1s and its substrate, complement C4.

J Biol Chem 2013 May 16;288(22):15821-9. Epub 2013 Apr 16.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Victoria 3800, Australia.

The complement system is an ancient innate immune defense pathway that plays a front line role in eliminating microbial pathogens. Recognition of foreign targets by antibodies drives sequential activation of two serine proteases, C1r and C1s, which reside within the complement Component 1 (C1) complex. Active C1s propagates the immune response through its ability to bind and cleave the effector molecule complement Component 4 (C4). Currently, the precise structural and biochemical basis for the control of the interaction between C1s and C4 is unclear. Here, using surface plasmon resonance, we show that the transition of the C1s zymogen to the active form is essential for C1s binding to C4. To understand this, we determined the crystal structure of a zymogen C1s construct (comprising two complement control protein (CCP) domains and the serine protease (SP) domain). These data reveal that two loops (492-499 and 573-580) in the zymogen serine protease domain adopt a conformation that would be predicted to sterically abrogate C4 binding. The transition from zymogen to active C1s repositions both loops such that they would be able to interact with sulfotyrosine residues on C4. The structure also shows the junction of the CCP1 and CCP2 domains of C1s for the first time, yielding valuable information about the exosite for C4 binding located at this position. Together, these data provide a structural explanation for the control of the interaction with C1s and C4 and, furthermore, point to alternative strategies for developing therapeutic approaches for controlling activation of the complement cascade.
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http://dx.doi.org/10.1074/jbc.M113.464545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668739PMC
May 2013

Molecular determinants of the substrate specificity of the complement-initiating protease, C1r.

J Biol Chem 2013 May 15;288(22):15571-80. Epub 2013 Apr 15.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

The serine protease, C1r, initiates activation of the classical pathway of complement, which is a crucial innate defense mechanism against pathogens and altered-self cells. C1r both autoactivates and subsequently cleaves and activates C1s. Because complement is implicated in many inflammatory diseases, an understanding of the interaction between C1r and its target substrates is required for the design of effective inhibitors of complement activation. Examination of the active site specificity of C1r using phage library technology revealed clear specificity for Gln at P2 and Ile at P1', which are found in these positions in physiological substrates of C1r. Removal of one or both of the Gln at P2 and Ile at P1' in the C1s substrate reduced the rate of C1r activation. Substituting a Gln residue into the P2 of the activation site of MASP-3, a protein with similar domain structure to C1s that is not normally cleaved by C1r, enabled efficient activation of this enzyme. Molecular dynamics simulations and structural modeling of the interaction of the C1s activation peptide with the active site of C1r revealed the molecular mechanisms that particularly underpin the specificity of the enzyme for the P2 Gln residue. The complement control protein domains of C1r also made important contributions to efficient activation of C1s by this enzyme, indicating that exosite interactions were also important. These data show that C1r specificity is well suited to its cleavage targets and that efficient cleavage of C1s is achieved through both active site and exosite contributions.
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http://dx.doi.org/10.1074/jbc.M113.451757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668718PMC
May 2013

Assembly of the type II secretion system such as found in Vibrio cholerae depends on the novel Pilotin AspS.

PLoS Pathog 2013 Jan 10;9(1):e1003117. Epub 2013 Jan 10.

Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS.
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http://dx.doi.org/10.1371/journal.ppat.1003117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542185PMC
January 2013

PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

PLoS One 2012 29;7(11):e50300. Epub 2012 Nov 29.

Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.

The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s). Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database) with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate sequence using machine learning techniques. It is freely available at http://lightning.med.monash.edu.au/PROSPER/.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050300PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510211PMC
June 2013

Structural characterization of the mechanism through which human glutamic acid decarboxylase auto-activates.

Biosci Rep 2013 Jan 11;33(1):137-44. Epub 2013 Jan 11.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, VIC 3800, Australia.

Imbalances in GABA (γ-aminobutyric acid) homoeostasis underlie psychiatric and movement disorders. The ability of the 65 kDa isoform of GAD (glutamic acid decarboxylase), GAD65, to control synaptic GABA levels is influenced through its capacity to auto-inactivate. In contrast, the GAD67 isoform is constitutively active. Previous structural insights suggest that flexibility in the GAD65 catalytic loop drives enzyme inactivation. To test this idea, we constructed a panel of GAD65/67 chimaeras and compared the ability of these molecules to auto-inactivate. Together, our data reveal the important finding that the C-terminal domain of GAD plays a key role in controlling GAD65 auto-inactivation. In support of these findings, we determined the X-ray crystal structure of a GAD65/67 chimaera that reveals that the conformation of the catalytic loop is intimately linked to the C-terminal domain.
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http://dx.doi.org/10.1042/BSR20120111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546353PMC
January 2013

Identification of a catalytic exosite for complement component C4 on the serine protease domain of C1s.

J Immunol 2012 Sep 1;189(5):2365-73. Epub 2012 Aug 1.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

The classical pathway of complement is crucial to the immune system, but it also contributes to inflammatory diseases when dysregulated. Binding of the C1 complex to ligands activates the pathway by inducing autoactivation of associated C1r, after which C1r activates C1s. C1s cleaves complement component C4 and then C2 to cause full activation of the system. The interaction between C1s and C4 involves active site and exosite-mediated events, but the molecular details are unknown. In this study, we identified four positively charged amino acids on the serine protease domain that appear to form a catalytic exosite that is required for efficient cleavage of C4. These residues are coincidentally involved in coordinating a sulfate ion in the crystal structure of the protease. Together with other evidence, this pointed to the involvement of sulfate ions in the interaction with the C4 substrate, and we showed that the protease interacts with a peptide from C4 containing three sulfotyrosine residues. We present a molecular model for the interaction between C1s and C4 that provides support for the above data and poses questions for future research into this aspect of complement activation.
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http://dx.doi.org/10.4049/jimmunol.1201085DOI Listing
September 2012

The X-ray crystal structure of full-length human plasminogen.

Cell Rep 2012 Mar 8;1(3):185-90. Epub 2012 Mar 8.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, VIC 3800 Australia.

Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change.
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http://dx.doi.org/10.1016/j.celrep.2012.02.012DOI Listing
March 2012

Novel scabies mite serpins inhibit the three pathways of the human complement system.

PLoS One 2012 11;7(7):e40489. Epub 2012 Jul 11.

Infectious Diseases Program, Biology Department, Queensland Institute of Medical Research, Brisbane, Queensland, Australia.

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040489PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3394726PMC
April 2013

Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1-P4 specificity reveals an unusual preference.

Biochimie 2012 May 20;94(5):1119-27. Epub 2012 Jan 20.

School of Applied Sciences, RMIT University, Bundoora, Victoria 3083, Australia.

Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S(2) subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola. An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P(2) proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P(2) proline, further highlighting the importance of Y69 for FhCatL2 P(2) proline acceptance. Finally, the P(1)-P(4) specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P(2,) which is unique amongst Fasciola cathepsins examined to date.
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http://dx.doi.org/10.1016/j.biochi.2012.01.011DOI Listing
May 2012

Effect of O-glycosylation and tyrosine sulfation of leech-derived peptides on binding and inhibitory activity against thrombin.

Chem Commun (Camb) 2012 Feb 23;48(10):1547-9. Epub 2011 Nov 23.

School of Chemistry, The University of Sydney, NSW 2006, Australia.

Synthesis of sulfated and unsulfated (glyco)peptide fragments of Hirudin P6 (a potent anticoagulant from the leech Hirudinaria manillensis) is described. The effect of O-glycosylation and tyrosine sulfation on thrombin binding and peptidolytic activity was investigated, together with the inhibition of fibrinogen cleavage.
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http://dx.doi.org/10.1039/c1cc14773kDOI Listing
February 2012

Predicting serpin/protease interactions.

Methods Enzymol 2011 ;501:237-73

Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia.

Proteases are tightly regulated by specific inhibitors, such as serpins, which are able to undergo considerable and irreversible conformational changes in order to trap their targets. There has been a considerable effort to investigate serpin structure and functions in the past few decades; however, the specific interactions between proteases and serpins remain elusive. In this chapter, we describe detailed experimental protocols to determine and characterize the extended substrate specificity of proteases based on a substrate phage display technique. We also describe how to employ a bioinformatics system to analyze the substrate specificity data obtained from this technique and predict the potential inhibitory serpin partners of a protease (in this case, the immune protease, granzyme B) in a step-by-step manner. The method described here could also be applied to other proteases for more generalized substrate specificity analysis and substrate discovery.
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http://dx.doi.org/10.1016/B978-0-12-385950-1.00012-2DOI Listing
March 2012
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