Richard Kelwick

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defects in certain family members give rise to inherited genetic disorders, while the aberrant expression or function of others is associated with arthritis, cancer and cardiovascular disease. In particular, ADAMTS4 and 5 have emerged as therapeutic targets in arthritis. Multiple ADAMTSs from different sub-groupings exert either positive or negative effects on tumorigenesis and metastasis, with both metalloproteinase-dependent and -independent actions known to occur. The basic ADAMTS structure comprises a metalloproteinase catalytic domain and a carboxy-terminal ancillary domain, the latter determining substrate specificity and the localization of the protease and its interaction partners; ancillary domains probably also have independent biological functions. Focusing primarily on the aggrecanases and proteoglycanases, this review provides a perspective on the evolution of the ADAMTS family, their links with developmental and disease mechanisms, and key questions for the future.

Developments in the tools and methodologies of synthetic biology.

Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices, or systems. However, biological systems are generally complex and unpredictable, and are therefore, intrinsically difficult to engineer. In order to address these fundamental challenges, synthetic biology is aiming to unify a “body of knowledge” from several foundational scientific fields, within the context of a set of engineering principles. This shift in perspective is enabling synthetic biologists to address complexity, such that robust biological systems can be designed, assembled, and tested as part of a biological design cycle. The design cycle takes a forward-design approach in which a biological system is specified, modeled, analyzed, assembled, and its functionality tested. At each stage of the design cycle, an expanding repertoire of tools is being developed. In this review, we highlight several of these tools in terms of their applications and benefits to the synthetic biology community.

The roles of ADAMTS metalloproteinases in tumorigenesis and metastasis.

The human ADAMTS (a disintegrin and metalloproteinase with thrombospondin-like motifs) family of 19 secreted, multidomain proteolytic enzymes is involved in a wide range of biological processes including ECM assembly and degradation, hemostasis, organogenesis and the regulation of angiogenesis. Defects in certain family members give rise to inherited human genetic diseases, while aberrant expression of other ADAMTSs has been linked to the pathogenesis of arthritis and cancer. Several ADAMTSs act as tumor or metastasis suppressors whose functions are lost either by mutation or epigenetic silencing during tumor progression. This review looks in depth at the involvement of ADAMTSs as positive and negative mediators in cancer growth and spread.

Metalloproteinase-Dependent and -Independent processes contribute to inhibition of breast cancer cell migration, angiogenesis and liver metastasis by a disintegrin and metalloproteinase with thrombospondin motifs-15

The ADAMTS proteinases are a family of secreted, matrix‐associated enzymes that have diverse roles in the regulation of tissue organization and vascular homeostasis. Several of the 19 human family members have been identified as having either tumor promoting or suppressing roles. We previously demonstrated that decreased ADAMTS15 expression correlated with a worse clinical outcome in mammary carcinoma (e.g., Porter et al., Int J Cancer 2006;118:1241–7). We have explored the effects of A Disintegrin and Metalloproteinase with Thrombospondin motifs‐15 (ADAMTS‐15) on the behavior of MDA‐MB‐231 and MCF‐7 breast cancer cells by stable expression of either a wild‐type (wt) or metalloproteinase‐inactive (E362A) protein. No effects on mammary cancer cell proliferation or apoptosis were observed for either form of ADAMTS‐15. However, both forms reduced cell migration on fibronectin or laminin matrices, though motility on a Type I collagen matrix was unimpaired. Knockdown of syndecan‐4 attenuated the inhibitory effects of ADAMTS‐15 on cell migration. In contrast to its effects on cell migration, wt ADAMTS‐15 but not the E362A inactive mutant inhibited endothelial tubulogenesis in 3D collagen gels and angiogenesis in the aortic ring assay. In experimental metastasis assays in nude mice, MDA‐MB‐231 cells expressing either form of ADAMTS‐15 showed reduced spread to the liver, though lung colonization was enhanced for cells expressing wt ADAMTS‐15. These studies indicate that extracellular ADAMTS‐15 has multiple actions on tumor pathophysiology. Via modulation of cell‐ECM interactions, which likely involve syndecan‐4, it attenuates mammary cancer cell migration independent of its metalloproteinase activity; however, its antiangiogenic action requires catalytic functionality, and its effects on metastasis in vivo are tissue niche‐dependent.

Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements

Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type Pgrac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.

A protease-based biosensor for the detection of schistosome cercariae.

Parasitic diseases affect millions of people worldwide, causing debilitating illnesses and death. Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings. A common signature of parasitic diseases is the release of specific proteases by the parasites at multiple stages during their life cycles. To this end, we engineered several modular Escherichia coli and Bacillus subtilis whole-cell-based biosensors which incorporate an interchangeable protease recognition motif into their designs. Herein, we describe how several of our engineered biosensors have been applied to detect the presence and activity of elastase, an enzyme released by the cercarial larvae stage of Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide. Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access.

Promoting microbiology education through the iGEM synthetic biology competition

Synthetic biology has developed rapidly in the 21st century. It covers a range of scientific disciplines that incorporate principles from engineering to take advantage of and improve biological systems, often applied to specific problems. Methods important in this subject area include the systematic design and testing of biological systems and, here, we describe how synthetic biology projects frequently develop microbiology skills and education. Synthetic biology research has huge potential in biotechnology and medicine, which brings important ethical and moral issues to address, offering learning opportunities about the wider impact of microbiological research. Synthetic biology projects have developed into wide-ranging training and educational experiences through iGEM, the International Genetically Engineered Machines competition. Elements of the competition are judged against specific criteria and teams can win medals and prizes across several categories. Collaboration is an important element of iGEM, and all DNA constructs synthesized by iGEM teams are made available to all researchers through the Registry for Standard Biological Parts. An overview of microbiological developments in the iGEM competition is provided. This review is targeted at educators that focus on microbiology and synthetic biology, but will also be of value to undergraduate and postgraduate students with an interest in this exciting subject area.

A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples

Synthetic biology designed cell-free biosensors are a promising new tool for the detection of clinically relevant biomarkers in infectious diseases. Here, we report that a modular DNA-encoded biosensor in cell-free protein expression systems can be used to measure a bacterial biomarker of Pseudomonas aeruginosa infection from human sputum samples. By optimizing the cell-free system and sample extraction, we demonstrate that the quorum sensing molecule 3-oxo-C12-HSL in sputum samples from cystic fibrosis lungs can be quantitatively measured at nanomolar levels using our cell-free biosensor system, and is comparable to LC-MS measurements of the same samples. This study further illustrates the potential of modular cell-free biosensors as rapid, low-cost detection assays that can inform clinical practice.

ADAMTS15 metalloproteinase inhibits breast cancer cell migration

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) family are a group of 19 extracellular, secreted proteases whose known functions include processing of procollagen molecules, cleavage of extracellular matrix proteoglycans and anti-angiogenesis. Our previous studies have shown that ADAMTS15 is a novel predictor of good prognosis in breast cancer; patients whose tumours had high levels of ADAMTS15 expression had an increased relapse-free survival compared with those with lower levels [1]. ADAMTS15 has also emerged as a candidate cancer gene from cancer genome sequencing, and its tumour suppressive function has recently been documented in colorectal cancer [2]. Our study has focused on the cellular effects of overexpression in MCF7 and MDA-MB-231 breast cancer cell lines of full-length wild-type ADAMTS15 and an E-A mutant that lacks metalloproteinase activity. We have generated stable transfectants carrying either an inducible (lentivirus tet-off system) or constitutive vector system. The effects on cell adhesion, migration and proliferation have subsequently been analysed. Proliferation (MTT assay) and adhesion to various matrix components (including collagen, fibronectin and laminin) was not altered with the addition of ADAMTS15. However, ectopic expression (inducible and constitutive) of either full-length ADAMTS15 or the catalytically dead mutant significantly reduced migration in both cell lines. Wild-type ADAMTS15 also enhanced the aggregation of MCF7 cells. These data suggest that ADAMTS15 may exert tumour suppressive effects via modulation of the interactions of breast carcinoma cells with their environment independent of its metalloproteinase activity.

A forward-design approach to increase the production of poly-3-hydroxybutyrate in genetically engineered Escherichia coli.

Biopolymers, such as poly-3-hydroxybutyrate (P(3HB)) are produced as a carbon store in an array of organisms and exhibit characteristics which are similar to oil-derived plastics, yet have the added advantages of biodegradability and biocompatibility. Despite these advantages, P(3HB) production is currently more expensive than the production of oil-derived plastics, and therefore, more efficient P(3HB) production processes would be desirable. In this study, we describe the model-guided design and experimental validation of several engineered P(3HB) producing operons. In particular, we describe the characterization of a hybrid phaCAB operon that consists of a dual promoter (native and J23104) and RBS (native and B0034) design. P(3HB) production at 24 h was around six-fold higher in hybrid phaCAB engineered Escherichia coli in comparison to E. coli engineered with the native phaCAB operon from Ralstonia eutropha H16. Additionally, we describe the utilization of non-recyclable waste as a low-cost carbon source for the production of P(3HB).