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.

Diversity of dicotyledenous-infecting geminiviruses and their associated DNA molecules in Southern Africa, including the South-west Indian Ocean Islands

The family Geminiviridae comprises a group of plant-infecting circular ssDNA viruses that severely constrain agricultural production throughout the temperate regions of the world, and are a particularly serious threat to food security in sub-Saharan Africa. While geminiviruses exhibit considerable diversity in terms of their nucleotide sequences, genome structures, host ranges and insect vectors, the best characterised and economically most important of these viruses are those in the genus Begomovirus. Whereas begomoviruses are generally considered to be either monopartite (one ssDNA component) or bipartite (two circular ssDNA components called DNA-A and DNA-B), many apparently monopartite begomoviruses are associated with additional subviral ssDNA satellite components, called alpha- (DNA-αs) or betasatellites (DNA-βs). Additionally, subgenomic molecules, also known as defective interfering (DIs) DNAs that are usually derived from the parent helper virus through deletions of parts of its genome, are also associated with bipartite and monopartite begomoviruses. The past three decades have witnessed the emergence and diversification of various new begomoviral species and associated DI DNAs, in southern Africa, East Africa, and proximal Indian Ocean islands, which today threaten important vegetable and commercial crops such as, tobacco, cassava, tomato, sweet potato, and beans. This review aims to describe what is known about these viruses and their impacts on sustainable production in this sensitive region of the world. © 2012 by the authors licensee MDPI, Basel, Switzerland.

Genetic polymorphisms in the human tissue kallikrein (KLK) locus and their implication in various malignant and non-malignant diseases

The Kallikrein (KLK) gene locus encodes a family of serine proteases and is the largest contiguous cluster of protease-encoding genes attributed an evolutionary age of 330 million years. The KLK locus has been implicated as a high susceptibility risk loci in numerous cancer studies through the last decade. The KLK3 gene already has established clinical relevance as a biomarker in prostate cancer prognosis through its encoded protein, prostate-specific antigen. Data mined through genome-wide association studies (GWAS) and next-generation sequencing point to many important candidate single nucleotide polymorphisms (SNPs) in KLK3 and other KLK genes. SNPs in the KLK locus have been found to be associated with several diseases including cancer, hypertension, cardiovascular disease and atopic dermatitis. Moreover, introducing a model incorporating SNPs to improve the efficiency of prostate-specific antigen in detecting malignant states of prostate cancer has been recently suggested. Establishing the functional relevance of these newly-discovered SNPs, and their interactions with each other, through in silico investigations followed by experimental validation, can accelerate the discovery of diagnostic and prognostic biomarkers. In this review, we discuss the various genetic association studies on the KLK loci identified either through candidate gene association studies or at the GWAS and post-GWAS front to aid researchers in streamlining their search for the most significant, relevant and therapeutically promising candidate KLK gene and/or SNP for future investigations.

Structure of the immature retroviral capsid at 8 Å resolution by cryo-electron microscopy

The assembly of retroviruses such as HIV-1 is driven by oligomerization of their major structural protein, Gag. Gag is a multidomain polyprotein including three conserved folded domains: MA (matrix), CA (capsid) and NC (nucleocapsid)(1). Assembly of an infectious virion proceeds in two stages(2). In the first stage, Gag oligomerization into a hexameric protein lattice leads to the formation of an incomplete, roughly spherical protein shell that buds through the plasma membrane of the infected cell to release an enveloped immature virus particle. In the second stage, cleavage of Gag by the viral protease leads to rearrangement of the particle interior, converting the non-infectious immature virus particle into a mature infectious virion. The immature Gag shell acts as the pivotal intermediate in assembly and is a potential target for anti-retroviral drugs both in inhibiting virus assembly and in disrupting virus maturation(3). However, detailed structural information on the immature Gag shell has not previously been available. For this reason it is unclear what protein conformations and interfaces mediate the interactions between domains and therefore the assembly of retrovirus particles, and what structural transitions are associated with retrovirus maturation. Here we solve the structure of the immature retroviral Gag shell from Mason-Pfizer monkey virus by combining cryo-electron microscopy and tomography. The 8-angstrom resolution structure permits the derivation of a pseudo-atomic model of CA in the immature retrovirus, which defines the protein interfaces mediating retrovirus assembly. We show that transition of an immature retrovirus into its mature infectious form involves marked rotations and translations of CA domains, that the roles of the amino-terminal and carboxy-terminal domains of CA in assembling the immature and mature hexameric lattices are exchanged, and that the CA interactions that stabilize the immature and mature viruses are almost completely distinct.

Feasibility of agent-based modelling of articular cartilage including a conceptual representation of its structure

Articular cartilage is a complex structure with an architecture in which fluid-swollen proteoglycans constrained within a 3D network of collagen fibrils. Because of the complexity of the cartilage structure, the relationship between its mechanical behaviours at the macroscale level and its components at the micro-scale level are not completely understood. The research objective in this thesis is to create a new model of articular cartilage that can be used to simulate and obtain insight into the micro-macro-interaction and mechanisms underlying its mechanical responses during physiological function. The new model of articular cartilage has two characteristics, namely: i) not use fibre-reinforced composite material idealization ii) Provide a framework for that it does probing the micro mechanism of the fluid-solid interaction underlying the deformation of articular cartilage using simple rules of repartition instead of constitutive / physical laws and intuitive curve-fitting. Even though there are various microstructural and mechanical behaviours that can be studied, the scope of this thesis is limited to osmotic pressure formation and distribution and their influence on cartilage fluid diffusion and percolation, which in turn governs the deformation of the compression-loaded tissue. The study can be divided into two stages. In the first stage, the distributions and concentrations of proteoglycans, collagen and water were investigated using histological protocols. Based on this, the structure of cartilage was conceptualised as microscopic osmotic units that consist of these constituents that were distributed according to histological results. These units were repeated three-dimensionally to form the structural model of articular cartilage. In the second stage, cellular automata were incorporated into the resulting matrix (lattice) to simulate the osmotic pressure of the fluid and the movement of water within and out of the matrix; following the osmotic pressure gradient in accordance with the chosen rule of repartition of the pressure. The outcome of this study is the new model of articular cartilage that can be used to simulate and study the micromechanical behaviours of cartilage under different conditions of health and loading. These behaviours are illuminated at the microscale level using the socalled neighbourhood rules developed in the thesis in accordance with the typical requirements of cellular automata modelling. Using these rules and relevant Boundary Conditions to simulate pressure distribution and related fluid motion produced significant results that provided the following insight into the relationships between osmotic pressure gradient and associated fluid micromovement, and the deformation of the matrix. For example, it could be concluded that: 1. It is possible to model articular cartilage with the agent-based model of cellular automata and the Margolus neighbourhood rule. 2. The concept of 3D inter connected osmotic units is a viable structural model for the extracellular matrix of articular cartilage. 3. Different rules of osmotic pressure advection lead to different patterns of deformation in the cartilage matrix, enabling an insight into how this micromechanism influences macromechanical deformation. 4. When features such as transition coefficient were changed, permeability (representing change) is altered due to the change in concentrations of collagen, proteoglycans (i.e. degenerative conditions), the deformation process is impacted. 5. The boundary conditions also influence the relationship between osmotic pressure gradient and fluid movement at the micro-scale level. The outcomes are important to cartilage research since we can use these to study the microscale damage in the cartilage matrix. From this, we are able to monitor related diseases and their progression leading to potential insight into drug-cartilage interaction for treatment. This innovative model is an incremental progress on attempts at creating further computational modelling approaches to cartilage research and other fluid-saturated tissues and material systems.

The role of HtrA as a chaperone and protease in bacterial pathogenesis

HtrA (High Temperature Requirement A) is a critical stress response protease and chaperone for many bacteria. HtrA is a multitasking protein which can degrade unfolded proteins, conduct specific proteolysis of some substrates for correct assembly, interact with substrates to ensure correct folding, assembly or localisation, and chaperone unfolded proteins. These functions are critical for the virulence of a number of bacterial pathogens, in some cases not simply due to the broad activities of HtrA in protection against the protein stress conditions which occur during virulence. But also due to the role of HtrA in either specific proteolysis or assembly of key protein substrates which function directly in virulence. Remarkably, these activities are all conducted without any requirement for ATP. The biochemical mechanism of HtrA relies both on the chymotryptic serine protease active site as well as the presence of two PDZ (protein binding) domains. The mechanism is a unique combination of activation by substrate motifs to alter the confirmation of the active site, and assembly into a multimeric complex which has enhanced degradation and may also act as a protective cage for proteins which are not degraded. The role of this protease in the pathogenesis of a number of bacteria and the details of its distinctive biochemical activation and assembly mechanisms are discussed in this chapter.

The role of HtrA as a chaperone and protease in bacterial pathogenisis

HtrA (High Temperature Requirement A) is a critical stress response protease and chaperone for many bacteria. HtrA is a multitasking protein which can degrade unfolded proteins, conduct specific proteolysis of some substrates for correct assembly, interact with substrates to ensure correct folding, assembly or localisation, and chaperone unfolded proteins. These functions are critical for the virulence of a number of bacterial pathogens, in some cases not simply due to the broad activities of HtrA in protection against the protein stress conditions which occur during virulence. But also due to the role of HtrA in either specific proteolysis or assembly of key protein substrates which function directly in virulence. Remarkably, these activities are all conducted without any requirement for ATP. The biochemical mechanism of HtrA relies both on the chymotryptic serine protease active site as well as the presence of two PDZ (protein binding) domains. The mechanism is a unique combination of activation by substrate motifs to alter the confirmation of the active site, and assembly into a multimeric complex which has enhanced degradation and may also act as a protective cage for proteins which are not degraded. The role of this protease in the pathogenesis of a number of bacteria and the details of its distinctive biochemical activation and assembly mechanisms are discussed in this chapter.

Insights into the nature of the coupling interactions between uracil corrosion inhibitors and copper : a DFT and molecular dynamics study

In this study, the nature of the coupling interactions between copper and uracil as well as its several derivatives has been systematically investigated employing the atoms in molecules (AIM) theory and energy decomposition analyses. The whole interaction process has been investigated through the analyses of the radial distribution functions of the Cu⋯X (X = S and O) contact on the basis of the ab initio molecular dynamics. No direct relationship between the adsorption strengths and inhibition efficiencies of the inhibitors has been observed. Additionally, the possibility of the methyl-substituted dithiouracil species to act as copper corrosion inhibitors has been tested.

Simplified method for including spatial correlations in mean-field approximations

Biological systems involving proliferation, migration and death are observed across all scales. For example, they govern cellular processes such as wound-healing, as well as the population dynamics of groups of organisms. In this paper, we provide a simplified method for correcting mean-field approximations of volume-excluding birth-death-movement processes on a regular lattice. An initially uniform distribution of agents on the lattice may give rise to spatial heterogeneity, depending on the relative rates of proliferation, migration and death. Many frameworks chosen to model these systems neglect spatial correlations, which can lead to inaccurate predictions of their behaviour. For example, the logistic model is frequently chosen, which is the mean-field approximation in this case. This mean-field description can be corrected by including a system of ordinary differential equations for pair-wise correlations between lattice site occupancies at various lattice distances. In this work we discuss difficulties with this method and provide a simplication, in the form of a partial differential equation description for the evolution of pair-wise spatial correlations over time. We test our simplified model against the more complex corrected mean-field model, finding excellent agreement. We show how our model successfully predicts system behaviour in regions where the mean-field approximation shows large discrepancies. Additionally, we investigate regions of parameter space where migration is reduced relative to proliferation, which has not been examined in detail before, and our method is successful at correcting the deviations observed in the mean-field model in these parameter regimes.

Effects of soluble dietary cellulose on specific growth rate, survival and digestive enzyme activities in three freshwater crayfish (Cherax) species

Three native freshwater crayfish Cherax species are farmed in Australia namely; Redclaw (Cherax quadricarinatus), Marron (C. tenuimanus), and Yabby (C. destructor). Lack of appropriate data on specific nutrient requirements for each of these species, however, has constrained development of specific formulated diets and hence current use of over-formulated feeds or expensive marine shrimp feeds, limit their profitability. A number of studies have investigated nutritional requirements in redclaw that have focused on replacing expensive fish meal in formulated feeds with non-protein, less expensive substitutes including plant based ingredients. Confirmation that freshwater crayfish possess endogenous cellulase genes, suggests their potential ability to utilize complex carbohydrates like cellulose as nutrient sources in their diet. To date, studies have been limited to only C. quadricarinatus and C. destructor and no studies have compared the relative ability of each species to utilize soluble cellulose in their diets. Individual feeding trials of late-juveniles of each species were conducted separately in an automated recirculating culture system over 12 week cycles. Animals were fed either a test diet (TD) that contained 20% soluble cellulose or a reference diet (RD) substituted with the same amount of corn starch. Water temperature, conductivity and pH were maintained at constant and optimum levels for each species. Animals were fed at 3% of their body weight twice daily and wet body weight was recorded bi-weekly. At the end of experiment, all animals were harvested, measured and midgut gland extracts assayed for alpha-amylase, total protease and cellulase activity levels. After the trial period, redclaw fed with RD showed significantly higher (p<0.05) specific growth rate (SGR) compare with animals fed the TD while SGR of marron and yabby fed the two diets were not significantly different (p<0.05). Cellulase expression levels in redclaw were not significantly different between diets. Marron and yabby showed significantly higher cellulase activity when fed the RD. Amylase and protease activity in all three species were significantly higher in the animals fed with RD (Table 1). These results indicate that test animals of all species can utilize starch better than dietary soluble cellulose in their diet and inclusion of 20% soluble cellulose in diets does not appear to have any significant negative effect on their growth rate but survival was impacted in C. quadricarinatus while not in C. tenuimanus or C. destructor.

In vitro functional characterisation of IGF-I : VN-induced breast cancer progression

Members of the insulin-like growth factor (IGF) family have been shown to play critical roles in normal growth and development, as well as in tumour biology. The IGF system is complex and the biological effects of the IGFs are determined by their diverse interactions between many molecules, including their interactions with extracellular matrix (ECM) proteins. Recent studies have demonstrated that IGFs associate with the ECM protein vitronectin (VN) through IGF-binding proteins (IGFBP) and that this interaction modulates IGF-stimulated biological functions, namely cell migration and cell survival through the cooperative involvement of the type-I IGF receptor (IGF-1R) and VN-binding integrins. Since IGFs play important roles in the transformation and progression of breast cancer and VN has been found to be over-expressed at the leading edge of breast tumours, this project aimed to describe the effects of IGF-I:VN interactions on breast cell function. This was undertaken to dissect the molecular mechanisms underlying IGF-I:VN-induced responses and to design inhibitors to block the effects of such interactions. The studies described herein demonstrate that the increase in migration of MCF-7 breast cancer cells in response to the IGF-I:IGFBP-5:VN complex is accompanied by differential expression of genes known to be involved in migration, invasion and/or survival, including Tissue-factor (TF), Stratifin (SFN), Ephrin-B2, Sharp-2 and PAI-1. This „migration gene signature‟ was confirmed using real-time PCR analysis. Substitution of the native IGF-I within the IGF-I:IGFBP:VN complex with the IGF-I analogue, \[L24]\[A31]-IGF-I, which has a reduced affinity for the IGF-1R, failed to stimulate cell migration and interestingly, also failed to induce the differential gene expression. This supports the involvement of the IGF-1R in mediating these changes in gene expression. Furthermore, lentiviral shRNA-mediated stable knockdown of TF and SFN completely abrogated the increased cell migration induced by IGF-I:IGFBP:VN complexes in MCF-7 cells. Indeed, when these cells were grown in 3D Matrigel™ cultures a decrease in the overall size of the 3D spheroids in response to the IGF-I:IGFBP:VN complexes was observed compared to the parental MCF-7 cells. This suggests that TF and SFN have a role in complex-stimulated cell survival. Moreover, signalling studies performed on cells with the reduced expression of either TF or SFN had a decreased IGF-1R activation, suggesting the involvement of signalling pathways downstream of IGF-1R in TF- and/or SFN-mediated cell migration and cell survival. Taken together, these studies provide evidence for a common mechanism activated downstream of the IGF-1R that induces the expression of the „migration gene signature‟ in response to the IGF-I:IGFBP:VN complex that confers breast cancer cells the propensity to migrate and survive. Given the functional significance of the interdependence of ECM and growth factor (GF) interactions in stimulating processes key to breast cancer progression, this project aimed at developing strategies to prevent such growth factor:ECM interactions in an effort to inhibit the downstream functional effects. This may result in the reduction in the levels of ECM-bound IGF-I present in close proximity to the cells, thereby leading to a reduction in the stimulation of IGF-1R present on the cell surface. Indeed, the inhibition of IGF-I-mediated effects through the disruption of its association with ECM would not alter the physiological levels of IGF-I and potentially only exert effects in situations where abnormal over expression of ECM proteins are found; namely carcinomas and hyperproliferative diseases. In summary, this PhD project has identified novel, innovative and realistic strategies that can be used in vitro to inhibit the functions exerted by the IGF-I:IGFBP:VN multiprotein complexes critical for cancer progression, with a potential to be translated into in vivo investigations. Furthermore, TF and SFN were found to mediate IGF-I:IGFBP:VN-induced effects, thereby revealing their potential to be used as therapeutic targets or as predictive biomarkers for the efficacy of IGF-1R targeting therapies in breast cancer patients. In addition to its therapeutic and clinical scope, this PhD project has significantly contributed to the understanding of the role of the IGF system in breast tumour biology by providing valuable new information on the mechanistic events underpinning IGF-I:VN-mediated effects on breast cell functions. Furthermore, this is the first instance where favourable binding sites for IGF-II, IGFBP-3 and IGFBP-5 on VN have been identified. Taken together, this study has functionally characterised the interactions between IGF-I and VN and through innovative strategies has provided a platform for the development of novel therapies targeting these interactions and their downstream effects.

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.

Mutations in cardiac T-Box Factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy

The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up.

Security ceremonies : including humans in cryptographic protocols

Whether by using electronic banking, by using credit cards, or by synchronising a mobile telephone via Bluetooth to an in-car system, humans are a critical part in many cryptographic protocols daily. We reduced the gap that exists between the theory and the reality of the security of these cryptographic protocols involving humans, by creating tools and techniques for proofs and implementations of human-followable security. After three human research studies, we present a model for capturing human recognition; we provide a tool for generating values called Computer-HUman Recognisable Nonces (CHURNs); and we provide a model for capturing human perceptible freshness.

Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to Chlamydia trachomatis

The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human C. trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydia CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targetted for anti-microbial therapy for intracellular pathogens.