- Caringbah, New South Wales, Australia
Bret Church
The University of Sydney, Bachelor of Pharmacy, Faculty Member
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The solute carrier organic anion transporting polypeptide 1A2 (OATP1A2, SLCO1A2) is implicated in the cellular influx of a number of drugs. We identified five novel single nucleotide polymorphisms (SNPs) in coding exons of the SLCO1A2... more
The solute carrier organic anion transporting polypeptide 1A2 (OATP1A2, SLCO1A2) is implicated in the cellular influx of a number of drugs. We identified five novel single nucleotide polymorphisms (SNPs) in coding exons of the SLCO1A2 gene in a cohort of subjects: G550A, G553A, G673A, A775C, and G862A, that encoded the OATP1A2 variants E184K, D185N, V255I, T259P, and D288N, respectively. The function and expression of these variant transporters were assessed in HEK-293 cells. We found that the novel variants, E184K, D185N, T259P, and D288N, were associated with impaired estrone-3-sulfate, imatinib, and methotrexate transport (∼20-50% of wild-type control); function was retained by OATP1A2-V255I. From biotinylation assays, the decreased function of these variants was due, at least in part, to impaired plasma membrane expression. The four loss-of-function variants were studied further using mutagenesis to produce variants that encode residues with different charges or steric properties. From immunoblotting, the replacement of negatively charged residues at amino acid positions 184 and 185 impaired membrane expression, while either a positive or negative charge at residue 288 supported the correct membrane targeting of OATP1A2. Replacement of T259 with bulky residues disrupted transporter stability. From molecular models, E184, D185, and D288 were located near several charged residues such that intramolecular ionic interactions may stabilize the transporter structure. Individuals who carry these novel SNPs in the SLCO1A2 gene may be at risk from impaired efficacy or enhanced toxicity during treatment with drugs that are substrates for OATP1A2.
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Schizophrenia is a complex neuropsychiatric disorder with limited treatment options and highly debilitating symptoms, leading to poor personal, social, and occupational outcomes for an afflicted individual. Our current understanding of... more
Schizophrenia is a complex neuropsychiatric disorder with limited treatment options and highly debilitating symptoms, leading to poor personal, social, and occupational outcomes for an afflicted individual. Our current understanding of schizophrenia suggests that dopaminergic and glutamatergic systems have a significant role in the pathogenesis of the disease. Kynurenic acid, an endogenous glutamate antagonist, is found in elevated concentrations in the prefrontal cortex and cerebrospinal fluid of patients with schizophrenia, and this affects neurotransmitter release in a similar manner to previously observed psychotomimetic agents, such as phencyclidine, underlining the molecular basis to its link in schizophrenia pathophysiology. Kynurenic acid is a breakdown product of tryptophan degradation, through a transamination process mediated by kynurenine aminotransferase (KAT) enzymes. There are four KAT homologues reported, all of which are pyridoxal-5'-phosphate-dependent enzymes....
ABSTRACT In Some Cyanobacteria, Poor Nutrient Conditions Induce A Reduction Of The Phycobilisome Light-Harvesting System And An Up-Regulation Of A Chlorophyll Antenna Protein, Isia, Which Can Form An 18-Mer Ring Around Photosystem I (Psi)... more
ABSTRACT In Some Cyanobacteria, Poor Nutrient Conditions Induce A Reduction Of The Phycobilisome Light-Harvesting System And An Up-Regulation Of A Chlorophyll Antenna Protein, Isia, Which Can Form An 18-Mer Ring Around Photosystem I (Psi) Trimers. We Have Used The Recent High-Resolution Structure Of Photosystem Ii (Psii) To Obtain A New Model Of The Molecular Structure Of Isia, Which Evolved From Cp43. This Model Was Further Refined By Analysis Of Sequence Variation Among Cp43, Isia, And The Nterminal Part Of Psaa, To Produce A Model In Which There Are 16 Chlorophylls (Chls) Per Isia Subunit, Two More Than Previously Predicted. The Structure Of The Isia-Ps I Supercomplex Was Then Examined By Comparing The Potential Of Excitation Transfer, Which Is Done By Optimizing The Chl-Chl Distances.
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Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activities of certain chemokines and neuropeptides. In addition, human DPPIV, also known as the T-cell activation antigen CD26, binds adenosine... more
Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activities of certain chemokines and neuropeptides. In addition, human DPPIV, also known as the T-cell activation antigen CD26, binds adenosine deaminase (ADA) to the T-cell surface, thus protecting the T-cell from adenosine-mediated inhibition of proliferation. Mutations were engineered into DPPIV (five point, 16 single point and six deletion mutations) to examine the binding of ADA and 19 monoclonal antibodies. Deletions of C-terminal residues from the 738-residue extracellular portion of DPPIV showed that the 214 residues C-terminal to Ser552 were not required for ADA binding and that peptidase activity could be ablated by deletion of 20 residues from the C-terminus. Point mutations at either of two locations, Leu294 and Val341, ablated ADA binding. Binding by six anti-DPPIV antibodies that inhibited ADA binding was found to require Leu340 to Arg343 and Thr440/Lys441 but not the 214 residues C-terminal to Ser552. The 13 other antibodies studied bound to a truncated DPPIV consisting of amino acids 1-356. Therefore, the binding sites on DPPIV of ADA and antibodies that inhibit ADA binding are discontinuous and overlapping. Moreover, the 47 and 97 residue spacing of amino acids in these binding sites concords with their location on a beta propeller fold consisting of repeated beta sheets of about 50 amino acids.
Research Interests: Humans, Mutagenesis, Animals, Ligand Binding, Monoclonal Antibodies, and 12 moreEuropean, T lymphocytes, Protein Secondary Structure Prediction, Rats, Transfection, Amino Acid Sequence, Amino Acid Substitution Rates, Recombinant Proteins, Adenosine Deaminase, Epitopes, Ligands, and Biochemistry and cell biology
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BAMLET (Bovine Alpha-lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET-like complexes, a novel class of protein-based anti-cancer complexes that incorporate oleic acid and deliver it to cancer cells. Small angle... more
BAMLET (Bovine Alpha-lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET-like complexes, a novel class of protein-based anti-cancer complexes that incorporate oleic acid and deliver it to cancer cells. Small angle X-ray scattering (SAXS) was performed on the complex at pH 12, examining the high pH structure as a function of oleic acid added. The SAXS data for BAMLET species prepared with a range of oleic acid concentrations indicate extended, irregular, partially unfolded protein conformations that vary with the oleic acid concentration. Increases in oleic acid concentration correlate with increasing radius of gyration without an increase in maximum particle dimension, indicating decreasing protein density. The models for the highest oleic acid content BAMLET indicate an unusual coiled elongated structure that contrasts with apo-α-lactalbumin at pH 12, which is an elongated globular molecule, suggesting that oleic acid inhibits the folding or collapse of the protein component of BAMLET to the globular form. Circular dichroism of BAMLET and apo-α-lactalbumin was performed and the results suggest that α-lactalbumin and BAMLET unfold in a continuum of increasing degree of unfolded states. Taken together, these results support a model in which BAMLET retains oleic acid by non-specific association in the core of partially unfolded protein, and represent a new type of lipoprotein structure.
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ABSTRACT Homology modeling methods have been used to construct models of two proteins--the histidine-containing phosphocarrier protein (HPr) from Mycoplasma capricolum and human eosinophil-derived neurotoxin (EDN). Comparison of the... more
ABSTRACT Homology modeling methods have been used to construct models of two proteins--the histidine-containing phosphocarrier protein (HPr) from Mycoplasma capricolum and human eosinophil-derived neurotoxin (EDN). Comparison of the models with the subsequently determined X-ray crystal structures indicates that the core regions of both proteins are reasonably well reproduced, although the template structures are closer to the X-ray structures in these regions--possible enhancements are discussed. The conformations of most of the side chains in the core of HPr are well reproduced in the modeled structure. As expected, the conformations of surface side chains in this protein differ significantly from the X-ray structure. The loop regions of EDN were incorrectly modeled--reasons for this and possible enhancements are discussed.
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Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino... more
Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino acid positions necessary for EF-G functions, a series of mutations were constructed in the EF-G structural gene (fusA) of Escherichia coli, specifically at positions flanking the effector domain. A mutated allele was isolated in which the wild-type sequence from codons 29 to 47 ("EFG2947") was replaced with a sequence encoding 28 amino acids from ribosomal protein S7. This mutated gene was unable to complement a fusAts strain when supplied in trans at the nonpermissive temperature. In vitro biochemical analysis demonstrated that nucleotide crosslinking was unaffected in EFG2947, while ribosome binding appeared to be completely abolished. A series of point mutations created within this region, encoding L30A, Y32A, H37A, and K38A were shown to give rise to fully functional proteins, suggesting that side chains of these individual residues are not essential for EF-G function. A sixth mutant, E41A, was found to inefficiently rescue growth in a fusAts background, and was also unable to bind ribosomes normally in vitro. In contrast E41Q could restore growth at the nonpermissive temperature. These results can be explained within the context of a three-dimensional model for the effector region of EF-G. This model indicates that the effector domain contains a negative potential field that may be important for ribosome binding.
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The development of high-throughput genome sequencing and protein structure determination techniques have provided researchers with a wealth of biological data. Integrated analysis of such data is difficult due to the disparate nature of... more
The development of high-throughput genome sequencing and protein structure determination techniques have provided researchers with a wealth of biological data. Integrated analysis of such data is difficult due to the disparate nature of the repositories used to store this biological data and of the software used for its analysis. This paper presents a framework based upon the use of semi-structured database management systems that would provide an integrated interface for the collection, storage and retrieval of biological data from existing repositories and of biological information generated by existing analysis programs. A simple implementation that integrates information from databases and analytical programs is presented as a proof of concept.
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Galanin and its newly discovered relative galanin-like peptide (GALP) are neuropeptides that are implicated in the neuroendocrine regulation of body weight and reproduction. GALP encompasses within its sequence the first 13 residues of... more
Galanin and its newly discovered relative galanin-like peptide (GALP) are neuropeptides that are implicated in the neuroendocrine regulation of body weight and reproduction. GALP encompasses within its sequence the first 13 residues of galanin, known to be crucial to binding and activation of galanin receptor (GalR) subtypes. Using 2D-NMR and circular dichroism spectroscopy we demonstrated that GALP does not adopt a preferred conformation in pure water alone. However, it shows characteristics of transient turn-like structures in two distinct regions of its sequence, 11-23 and 41-49. These transient ordered structures, nascent helices, probably form stable helical structures upon addition of the helix-inducing solvent, trifluoroethanol, as determined by circular dichroism studies. Secondary structure prediction methods also predict the presence of two helical regions in the sequence of GALP overlapping reasonably with those regions identified as nascent helical structures by experimental methods.
Research Interests: Water, Structural Biology, NMR Spectroscopy, Magnetic Resonance Spectroscopy, Structural, and 10 moreCircular Dichroism, Animals, Protein Secondary Structure Prediction, Swine, Circular Dichroism Spectroscopy, Trifluoroethanol, Body Weight, Protein Conformation, Experimental Method, and Biochemistry and cell biology
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Kinks have been observed to provide important functional and structural features for membrane proteins. Despite their ubiquity in membrane proteins, and their perceived importance, no protein modeling methods explicitly considers kinks.... more
Kinks have been observed to provide important functional and structural features for membrane proteins. Despite their ubiquity in membrane proteins, and their perceived importance, no protein modeling methods explicitly considers kinks. In spite of the limited data for transmembrane proteins, we were able to develop a knowledge-based modeling method for introducing kinks, which we demonstrate can be exploited in modeling approaches to improve the quality of models. The work entailed a thorough analysis of the available high resolution membrane protein structures, concomitantly demonstrating the complexity of the structural considerations for kink prediction. Furthermore, our results indicate that there are systematic and significant differences in the sequence as well as the structural environment between kinked and nonkinked transmembrane helices. To the best of our knowledge, we are reporting a method for modeling kinks for the first time.
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Major advances have been made in the prediction of soluble protein structures, led by the knowledge-based modeling methods that extract useful structural trends from known protein structures and incorporate them into scoring functions.... more
Major advances have been made in the prediction of soluble protein structures, led by the knowledge-based modeling methods that extract useful structural trends from known protein structures and incorporate them into scoring functions. The same cannot be reported for the class of transmembrane proteins, primarily due to the lack of high-resolution structural data for transmembrane proteins, which render many of the knowledge-based method unreliable or invalid. We have developed a method that harnesses the vast structural knowledge available in soluble protein data for use in the modeling of transmembrane proteins. At the core of the method, a set of transmembrane protein decoy sets that allow us to filter and train features recognized from soluble proteins for transmembrane protein modeling into a set of scoring functions. We have demonstrated that structures of soluble proteins can provide significant insight into transmembrane protein structures. A complementary novel two-stage modeling/selection process that mimics the two-stage helical membrane protein folding was developed. Combined with the scoring function, the method was successfully applied to model 5 transmembrane proteins. The root mean square deviations of the predicted models ranged from 5.0 to 8.8 Å to the native structures.
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Spontaneously hypertensive rats (SHR) are the most extensively used animal model for genetic hypertension, increased stroke damage, and insulin resistance syndromes; however, the identification of target genes has proved difficult. SHR... more
Spontaneously hypertensive rats (SHR) are the most extensively used animal model for genetic hypertension, increased stroke damage, and insulin resistance syndromes; however, the identification of target genes has proved difficult. SHR show elevated sympathetic nerve activity, and stimulation of the central blood pressure control centers with glutamate or nicotine results in exaggerated blood pressure responses, effects that appear to be genetically determined. Kynurenic acid, a competitive glutamate antagonist and a non-competitive nicotinic antagonist, can be synthesized in the brain by the enzyme kynurenine aminotransferase-1 (KAT-1). We have previously shown that KAT-1 activity is significantly reduced in SHR compared with normotensive Wistar Kyoto rats (WKY). Here we show that KAT-1 contains a missense mutation, E61G, in all the strains of SHR examined but not in any of the WKY or outbred strains. Previous studies on F2 rats from a cross of stroke-prone SHR and WKY have shown a suggestive level of linkage between elevated blood pressure and the KAT-1 locus on chromosome 3. In addition, the mutant enzyme expressed in Escherichia coli displays altered kinetics. This mutation may explain the enhanced sensitivity to glutamate and nicotine seen in SHR that may be related to an underlying mechanism of hypertension and increased sensitivity to stroke.
Research Interests: Kinetics, Biological Chemistry, Western blotting, Biological Sciences, Brain, and 10 moreMutation, Escherichia coli, Animals, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, CHEMICAL SCIENCES, Protein Secondary Structure Prediction, Rats, Wistar Rats, Protein Conformation, and Transaminases
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Research Interests: Immunology, Macrophages, Gene expression, Cell line, Humans, and 11 moreImmunology & Cell Biology, Protein Secondary Structure Prediction, Transfection, Protein isoforms, Cell communication, Amino Acid Substitution Rates, Structure activity Relationship, Molecular Interactions, Biochemistry and cell biology, Dimerization, and Tyrosine Kinase
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Research Interests: Immunology, Cytokines, Membrane Proteins, Apoptosis, Liver diseases, and 13 moreCell Differentiation, Humans, Liver Cirrhosis, Animals, Hepatitis, Liver Transplantation, Rats, Liver Disease, Immune Tolerance, Structure activity Relationship, Adenosine Deaminase, Gene Expression Regulation, and Gene expression profiling
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Phage display represents an important approach in the development pipeline for producing peptides and peptidomimetics therapeutics. Using randomly generated DNA sequences and molecular biology techniques, large diverse peptide libraries... more
Phage display represents an important approach in the development pipeline for producing peptides and peptidomimetics therapeutics. Using randomly generated DNA sequences and molecular biology techniques, large diverse peptide libraries can be displayed on the phage surface. The phage library can be incubated with a target of interest and the phage which bind can be isolated and sequenced to reveal the displayed peptides' primary structure. In this review, we focus on the 'mechanics' of the phage display process, whilst highlighting many diverse and subtle ways it has been used to further the drug-development process, including the potential for the phage particle itself to be used as a drug carrier targeted to a particular pathogen or cell type in the body.
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Research Interests: Algorithms, Protein Structure Prediction, Biological Sciences, Crystal structure, Computer Simulation, and 14 moreMathematical Sciences, G protein-coupled receptors, Structured data, BMC Bioinformatics, Protein Sequence Analysis, High Resolution, Very high throughput, Three Dimensional, Amino Acid Profile, Protein Conformation, Amino Acid Sequence, In Silico, Membrane Protein, and Physicochemical Properties
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Herein we report 6-ethoxy-6-oxo-5-(2-phenylhydrazono) hexanoic acid and 3-(2-carboxyethyl)-1H-indole-2-carboxylic acid derivatives as synthetically accessible leads for human kynurenine aminotransferase-I (KAT-I) inhibitors. In total, 12... more
Herein we report 6-ethoxy-6-oxo-5-(2-phenylhydrazono) hexanoic acid and 3-(2-carboxyethyl)-1H-indole-2-carboxylic acid derivatives as synthetically accessible leads for human kynurenine aminotransferase-I (KAT-I) inhibitors. In total, 12 compounds were synthesized and their biological activities were determined using the HPLC-UV based KAT-I inhibition assay. Of the 12 compounds synthesized, 10 were found to inhibit human KAT-I and the most active compound was found to be 5-(2-(4-chlorophenyl) hydrazono)-6-ethoxy-6-oxohexanoic acid (9a) with an IC(50) of 19.8 μM.
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Proteins are the workhorses of biomolecules and their function is affected by their structure and their structural rearrangements during ligand entry, ligand binding and protein-protein interactions. Hence, the knowledge of protein... more
Proteins are the workhorses of biomolecules and their function is affected by their structure and their structural rearrangements during ligand entry, ligand binding and protein-protein interactions. Hence, the knowledge of protein structure and, importantly, the dynamic behaviour of the structure are critical for understanding how the protein performs its function. The predictions of the structure and the dynamic behaviour can be performed by combinations of structure modelling and molecular dynamics simulations. The simulations also need to be sensitive to the constraints of the environment in which the protein resides. Standard computational methods now exist in this field to support the experimental effort of solving protein structures. This review presents a comprehensive overview of the basis of the calculations and the well-established computational methods used to generate and understand protein structure and function and the study of their dynamic behaviour with the reference to lung-related targets.