Kallikrein-related peptidase 4 activation of protease-activated receptor family members and association with prostate cancer

Two areas of particular importance in prostate cancer progression are primary tumour development and metastasis. These processes involve a number of physiological events, the mediators of which are still being discovered and characterised. Serine proteases have been shown to play a major role in cancer invasion and metastasis. The recently discovered phenomenon of their activation of a receptor family known as the protease activated receptors (PARs) has extended their physiological role to that of signaling molecule. Several serine proteases are expressed by malignant prostate cancer cells, including members of the kallikreinrelated peptidase (KLK) serine protease family, and increasingly these are being shown to be associated with prostate cancer progression. KLK4 is highly expressed in the prostate and expression levels increase during prostate cancer progression. Critically, recent studies have implicated KLK4 in processes associated with cancer. For example, the ectopic over-expression of KLK4 in prostate cancer cell lines results in an increased ability of these cells to form colonies, proliferate and migrate. In addition, it has been demonstrated that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone forming cells). The ability of KLK4 to influence cellular behaviour is believed to be through the selective cleavage of specific substrates. Identification of relevant in vivo substrates of KLK4 is critical to understanding the pathophysiological roles of this enzyme. Significantly, recent reports have demonstrated that several members of the KLK family are able to activate PARs. The PARs are relatively new members of the seven transmembrane domain containing G protein coupled receptor (GPCR) family. PARs are activated through proteolytic cleavage of their N-terminus by serine proteases, the resulting nascent N-terminal binds intramolecularly to initiate receptor activation. PARs are involved in a number of patho-physiological processes, including vascular repair and inflammation, and a growing body of evidence suggests roles in cancer. While expression of PAR family members has been documented in several types of cancers, including prostate, the role of these GPCRs in prostate cancer development and progression is yet to be examined. Interestingly, several studies have suggested potential roles in cellular invasion through the induction of cytoskeletal reorganisation and expression of basement membrane-degrading enzymes. Accordingly, this program of research focussed on the activation of the PARs by the prostate cancer associated enzyme KLK4, cellular processing of activated PARs and the expression pattern of receptor and agonist in prostate cancer. For these studies KLK4 was purified from the conditioned media of stably transfected Sf9 insect cells expressing a construct containing the complete human KLK4 coding sequence in frame with a V5 epitope and poly-histidine encoding sequences. The first aspect of this study was the further characterisation of this recombinant zymogen form of KLK4. The recombinant KLK4 zymogen was demonstrated to be activatable by the metalloendopeptidase thermolysin and amino terminal sequencing indicated that thermolysin activated KLK4 had the predicted N-terminus of mature active KLK4 (31IINED). Critically, removal of the pro-region successfully generated a catalytically active enzyme, with comparable activity to a previously published recombinant KLK4 produced from S2 insect cells. The second aspect of this study was the activation of the PARs by KLK4 and the initiation of signal transduction. This study demonstrated that KLK4 can activate PAR-1 and PAR-2 to mobilise intracellular Ca2+, but failed to activate PAR-4. Further, KLK4 activated PAR-1 and PAR-2 over distinct concentration ranges, with KLK4 activation and mobilisation of Ca2+ demonstrating higher efficacy through PAR-2. Thus, the remainder of this study focussed on PAR-2. KLK4 was demonstrated to directly cleave a synthetic peptide that mimicked the PAR-2 Nterminal activation sequence. Further, KLK4 mediated Ca2+ mobilisation through PAR-2 was accompanied by the initiation of the extra-cellular regulated kinase (ERK) cascade. The specificity of intracellular signaling mediated through PAR-2 by KLK4 activation was demonstrated by siRNA mediated protein depletion, with a reduction in PAR-2 protein levels correlating to a reduction in KLK4 mediated Ca2+mobilisation and ERK phosphorylation. The third aspect of this study examined cellular processing of KLK4 activated PAR- 2 in a prostate cancer cell line. PAR-2 was demonstrated to be expressed by five prostate derived cell lines including the prostate cancer cell line PC-3. It was also demonstrated by flow cytometry and confocal microscopy analyses that activation of PC-3 cell surface PAR-2 by KLK4 leads to internalisation of this receptor in a time dependent manner. Critically, in vivo relevance of the interaction between KLK4 and PAR-2 was established by the observation of the co-expression of receptor and agonist in primary prostate cancer and prostate cancer bone lesion samples by immunohistochemical analysis. Based on the results of this study a number of exciting future studies have been proposed, including, delineating differences in KLK4 cellular signaling via PAR-1 and PAR-2 and the role of PAR-1 and PAR-2 activation by KLK4 in prostate cancer cells and bone cells in prostate cancer progression.

Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis

DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.

In vitro and in vivo studies on protease-activated receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) that is activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. Cleavage of this receptor exposes a neoepitope, termed the tethered ligand (TL), which binds intramolecularly within the receptor to stimulate signal transduction via coupled G proteins. PAR2-mediated signal transduction is also experimentally stimulated by hexapeptides (agonist peptides; APs) that are homologous to the TL sequence. Due to the irreversible nature of PAR2 proteolysis, downstream signal transduction is tightly regulated. Following activation, PAR2 is rapidly uncoupled from downstream signalling by the post-translational modifications phosphorylation and ubiquination which facilitate interactions with â- arrestin. This scaffolding protein couples PAR2 to the internalisation machinery initiating its desensitisation and trafficking through the early and late endosomes followed by receptor degradation. PAR2 is widely expressed in mammalian tissues with key roles for this receptor in cardiovascular, respiratory, nervous and musculoskeletal systems. This receptor has also been linked to pathological states with aberrant expression and signalling noted in several cancers. In prostate cancer, PAR2 signalling induces migration and proliferation of tumour derived cell lines, while elevated receptor expression has been noted in malignant tissues. Importantly, a role for this receptor has also been suggested in prostate cancer bone metastasis as coexpression of PAR2 and a proteolytic activator has been demonstrated by immunohistochemical analysis. Based on these data, the primary focus of this project has been on two aspects of PAR2 biology. The first is characterisation of cellular mechanisms that regulate PAR2 signalling and trafficking. The second aspect is the role of this receptor in prostate cancer bone metastasis. In addition, to permit these studies, it was first necessary to evaluate the specificity of the commercially available anti-PAR2 antibodies SAM11, C17, N19 and H99. The evaluation of the four commercially available antibodies was assessed using four techniques: immunoprecipitation; Western blot analysis; immunofluorescence; and flow cytometry. These approaches demonstrated that three of the antibodies efficiently detect ectopically expressed PAR2 by each of these techniques. A significant finding from this study was that N19 was the only antibody able to specifically detect N-glycosylated endogenous PAR2 by Western blot analysis. This analysis was performed on lysates from prostate cancer derived cell lines and tissue derived from wildtype and PAR2 knockout mice. Importantly, further evaluation demonstrated that this antibody also efficiently detects endogenous PAR2 at the cell surface by flow cytometry. The anti-PAR2 antibody N19 was used to explore the in vitro role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Significantly, use of the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling experiments using two approaches which showed that PAR2 stably expressed by CHO cells is palmitoylated and that palmitoylation occurs on cysteine 361. Another key finding from this study is that palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ~9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. Importantly, this study also identified that palmitoylation of this receptor within the Golgi apparatus is required for efficient agonist-induced rab11amediated trafficking of PAR2 to the cell surface. Interestingly, palmitoylation is also required for receptor desensitization, as agonist-induced â-arrestin recruitment and receptor degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. Collectively, these data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor. This project also evaluated PAR2 residues involved in ligand docking. Although the extracellular loop (ECL)2 of PAR2 is known to be required for agonist-induced signal transduction, the binding pocket for receptor agonists remains to be determined. In silico homology modelling, based on a crystal structure for the prototypical GPCR rhodopsin, and ligand docking were performed to identify PAR2 transmembrane (TM) amino acids potentially involved in agonist binding. These methods identified 12 candidate residues that were mutated to examine the binding site of the PAR2 TL, revealed by trypsin cleavage, as well as of the soluble ligands 2f-LIGRLO-NH2 and GB110, which are both structurally based on the AP SLIGRLNH2. Ligand binding was evaluated from the impact of the mutated residues on PAR2-mediated calcium mobilisation. An important finding from these experiments was that mutation of residues Y156 and Y326 significantly reduced 2f-LIGRLO-NH2 and GB110 agonist activity. L307 was also important for GB110 activity. Intriguingly, mutation of PAR2 residues did not alter trypsin-induced signalling to the same extent as for the soluble agonists. The reason for this difference remains to be further examined by in silico and in vitro experimentation and, potentially, crystal structure studies. However, these findings identified the importance of TM domains in PAR2 ligand docking and will enhance the design of both PAR2 agonists and potentially agents to inhibit signalling (antagonists). The potential importance of PAR2 in prostate cancer bone metastasis was examined using a mouse model. In patients, prostate cancer bone metastases cause bone growth by disrupting bone homeostasis. In an attempt to mimic prostate cancer growth in bone, PAR2 responsive 22Rv1 prostate cancer cells, which form mixed osteoblastic and osteolytic lesions, were injected into the proximal aspect of mouse tibiae. A role for PAR2 was assessed by treating these mice with the recently developed PAR2 antagonist GB88. As controls, animals bearing intra-tibial tumours were also treated with vehicle (olive oil) or the prostate cancer chemotherapeutic docetaxel. The effect of these treatments on bone was examined radiographically and by micro-CT. Consistent with previous studies, 22Rv1 tumours caused osteoblastic periosteal spicule formation and concurrent osteolytic bone loss. Significantly, blockade of PAR2 signalling reduced the osteoblastic and osteolytic phenotype of 22Rv1 tumours in bone. No bone defects were detected in mice treated with docetaxel. These qualitative data will be followed in the future by quantitative micro-CT analysis as well as histology and histomorphometry analysis of already collected tissues. Nonetheless, these preliminary experiments highlight a potential role for PAR2 in prostate cancer growth in bone. In summary, in vitro studies have defined mechanisms regulating PAR2 activation, downstream signalling and trafficking and in vivo studies point to a potential role for this receptor in prostate cancer bone metastasis. The outcomes of this project are that a greater understanding of the biology of PAR2 may lead to the development of strategies to modulate the function of this receptor in disease.

Hypoxia-inducible factor-1α in non small cell lung cancer: Relation to growth factor, protease and apoptosis pathways

Hypoxia-inducible factor (HIF)-1α is the regulatory subunit of HIF-1 that is stabilized under hypoxic conditions. Under different circumstances, HIF-1α may promote both tumorigenesis and apoptosis. There is conflicting data on the importance of HIF-1α as a prognostic factor. This study evaluated HIF-1α expression in 172 consecutive patients with stage I-IIIA non small cell lung cancer (NSCLC) using standard immunohistochemical techniques. The extent of HIF-1α nuclear immunostaining was determined using light microscopy and the results were analyzed using the median (5%) as a low cut-point and 60% as a high positive cut-point. Using the low cut-point, positive associations were found with epidermal growth factor receptor (EGFR; p = 0.01), matrix metalloproteinase (MMP)-9 (p = 0.003), membranous (p < 0.001) and perinuclear (p = 0.004) carbonic anhydrase (CA) IX, pS3 (p = 0.008), T-stage (p = 0.042), tumor necrosis (TN; p < 0.001) and squamous histology (p < 0.001). No significant association was found with Bcl-2 or either N- or overall TMN stage or prognosis. When the high positive cut-point was used, HIF-1α was associated with a poor prognosis (p = 0.034). In conclusion, the associations with EGFR, MMP-9, p53 and CA IX suggest that these factors may either regulate or be regulated by HIF-1α. The association with TN and squamous-type histology, which is relatively more necrotic than other NSCLC types, reflects the role of hypoxia in the regulation of HIF-1α. The prognostic data may reflect a change in the behavior of HIF-1α in increasingly hypoxic environments. © 2004 Wiley-Liss, Inc.

The M(r) 43K major capsid protein of rice ragged stunt oryzavirus is a post-translationally processed product of a M(r) 67 348 polypeptide encoded by genome segment 8

The nucleotide sequence of DNA complementary to rice ragged stunt oryzavirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was determined. RRSV S8 is 1 914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1 810 which is capable of encoding a protein of M(r) 67 348. The N-terminal amino acid sequence of a ~43K virion polypeptide matched to that inferred for an internal region of the S8 coding sequence. These data suggest that the 43K protein is encoded by S8 and is derived by a proteolytic cleavage. Predicted polypeptide sizes from this possible cleavage of S8 protein are 26K and 42K. Polyclonal antibodies raised against a maltose binding protein (MBP)-S8 fusion polypeptide (expressed in Escherichia coli) recognised four RRSV particle associated polypeptides of M(r) 67K, 46K, 43K and 26K and all except the 26K polypeptide were also highly immunoreactive to polyclonal antibodies raised against purified RRSV particles. Cleavage of the MBP-S8 fusion polypeptide with protease Factor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K. Antibodies to purified RRSV particles reacted strongly with the intact fusion protein and the 46K cleavage product but weakly to the 26K product. Furthermore, in vitro transcription and translation of the S8 coding region revealed a post-translational self cleavage of the 67K polypeptide to 46K and 26K products. These data indicate that S8 encodes a structural polypeptide, the majority of which is auto- catalytically cleaved to 26K and 46K proteins. The data also suggest that the 26K protein is the self cleaving protease and that the 46K product is further processed or undergoes stable conformational changes to a ~43K major capsid protein.

Molecular and functional characterizations of a Kunitz-type serine protease inhibitor FcKuSPI of the shrimp Fenneropenaeus chinensis

Serine proteinase inhibitors play important and diverse roles in biological processes such as coagulation, defense mechanisms, and immune responses. Here, we identified and characterized a Kunitz-type proteinase inhibitor, designated FcKuSPI, of the BPTI/Kunitz family of serine proteinase inhibitors from the hemocyte cDNA library of the shrimp Fenneropenaeus chinensis. The deduced amino acid sequence of FcKuSPI comprises 80 residues with a putative signal peptide of 15 amino acids. The predicted molecular weight of the mature peptide is 7.66 kDa and its predicted isoelectric point is 8.84. FcKuSPI includes a Kunitz domain containing six conserved cysteine residues that are predicted to form three disulfide bonds. FcKuSPI shares 44e53% homology with BPTI/Kunitz family members from other species. FcKuSPI mRNAwas expressed highly in the hemocytes and moderately in muscle in healthy shrimp. Recombinant FcKuSPI protein demonstrated anti-protease activity against trypsin and anticoagulant activity against citrated human plasma in a dose-dependent manner in in vitro assays.

Guidelines on building design and operation to minimize air quality impacts from major roads in transit oriented development

Poor air quality has a huge detrimental effect, both economic and on the quality of life, in Australia. Transit oriented design (TOD), which aims to minimise urban sprawl and lower dependency on vehicles, leads to an increasing number of buildings close to transport corridors. This project aims at providing guidelines that are appropriate to include within City Plan to inform future planning along road corridors, and provide recommendations on when mitigation measures should be utilised.

A comparative analysis of major ERP lifecycle implementation, management and support issues in Queensland Government

This paper reports on a study of ERP lifecycle major issues from the perspectives of individuals with substantial and diverse involvement with SAP Financials in Queensland Government. A survey was conducted of 117 ERP system project participants in five closely related state government agencies. A modified Delphi technique identified, rationalized and weighed perceived major issues in ongoing ERP life cycle implementation, management and support. The five agencies each implemented SAP Financials simultaneously using a common implementation partner. The three survey rounds of the Delphi technique, together with coding and synthesizing procedures, resulted in a set of 10 major issue categories with 38 sub-issues. Relative scores of issue importance are compared across government agencies, roles (client vs implementation partner) and organizational levels (strategic, technical and operational). Study findings confirm the importance of this finer partitioning of the data, and distinctions identified reflect the circumstances of ERP lifecycle implementation, management and support among the stakeholder groups. The study findings should also be of interest to stakeholders who seek to better understand the issues surrounding ERP systems and to better realise the benefits of ERP.