Publications & Prizes

Biochemistry Department Honour Board

Awards given to Department Members

Publications on this page begin in 2012. Some staff have a record of earlier publications on their personal pages.

  • Antifreeze proteins in the Antarctic springtail, Gressittacantha terranova
  • T. C. Hawes, C J Marshall, and D A Wharton, Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology 2011 vol. 181 (6) pp. 713-719
  • Published:2 August 2011
  • Antarctic springtails are exemplars of extreme low temperature adaptation in terrestrial arthropods. This paper represents the first examination of such adaptation in the springtail, Gressittacantha terranova. Acclimatization state was measured in field-fresh samples over a 22-day period at the beginning of the austral summer. No evidence of temperature tracking was observed. Mean temperature of crystallization (T c) for all samples was -20.67 ± 0.32°C and the lowest T c recorded was -32.62°C. Ice affinity purification was used to collect antifreeze proteins (AFPs) from springtail homogenate. The purified ice fraction demonstrated both thermal hysteresis activity and recrystallisation inhibition. Growth-melt observations revealed that ice crystals grow normal to the c-axis (basal plane). Reverse-phased HPLC produce one clearly resolved peak (P1) and one compound peak (P2). Mass spectrometry identified the molecular mass of P1 as 8,599 Da. The P1 protein was also the most prominent in P2, although additional peptides of 6-7 KDa were also prominent. The main AFP of the Antarctic springtail, G. terranova has been isolated, although like other AFP-expressing arthropods, it shows evidence of expressing a family of AFPs. © 2011 Springer-Verlag.

  • Smac mimetics activate the E3 ligase activity of cIAP1 protein by promoting RING domain dimerization
  • R Feltham, B Bettjeman, R Budhidarmo, P D Mace, S Shirley, S.M. Condon, S.K. Chunduru, M.A. McKinlay, D L Vaux, J Silke, and C L Day, The Journal of Biological Chemistry 2011 vol. 286 (19) pp. 17015-17028
  • Published:31 May 2011
  • The crystal structure of alanine racemase from Streptococcus pneumoniae, a target for structure-based drug design
  • Im H, Sharpe ML, Strych U, Davlieva M, Krause KL, BMC Microbiology (2011) 11:116
  • Published:25 May 2011
  • Background: Streptococcus pneumoniae is a globally important pathogen. The Gram-positive diplococcus is a leading cause of pneumonia, otitis media, bacteremia, and meningitis, and antibiotic resistant strains have become increasingly common over recent years. Alanine racemase is a ubiquitous enzyme among bacteria and provides the essential cell wall precursor, D-alanine. Since it is absent in humans, this enzyme is an attractive target for the development of drugs against S. pneumoniae and other bacterial pathogens. Results: Here we report the crystal structure of alanine racemase from S. pneumoniae (Alr SP). Crystals diffracted to a resolution of 2.0 and belong to the space group P3 121 with the unit cell parameters a = b = 119.97 , c = 118.10 , = = 90° and = 120°. Structural comparisons show that Alr SPshares both an overall fold and key active site residues with other bacterial alanine racemases. The active site cavity is similar to other Gram positive alanine racemases, featuring a restricted but conserved entryway. Conclusions: We have solved the structure of Alr SP, an essential step towards the development of an accurate pharmacophore model of the enzyme, and an important contribution towards our on-going alanine racemase structure-based drug design project. We have identified three regions on the enzyme that could be targeted for inhibitor design, the active site, the dimer interface, and the active site entryway. © 2011 Im et al; licensee BioMed Central Ltd.

  • New classes of alanine racemase inhibitors identified by high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis
  • K G Anthony, U Strych, K R Yeung, C S Shoen, O Perez, K L Krause, M H Cynamon, P A Aristoff, and R A Koski, PLoS ONE 2011 vol. 6 (5)
  • Published:8 May 2011
  • Background: In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target. Methodology/Principal Findings: To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells. Conclusions/Significance: This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents. © 2011 Anthony et al.

  • Noncanonical Translation Initiation of the Arabidopsis Flowering Time and Alternative Polyadenylation Regulator FCA
  • G G Simpson, R E Laurie, P P Dijkwel, V Quesada, P A Stockwell, C Dean, and R C Macknight, THE PLANT CELL ONLINE 2010 vol. 22 (11) pp. 3764-3777
  • Published:1 November 2010
  • Discovery and characterization of a distinctive exo-1,3/1,4?-glucanase from the marine bacterium Pseudoalteromonas sp. strain BB1
  • Y Nakatani, I L Lamont, and J.F. Cutfield, Applied and environmental microbiology 2010 vol. 76 (20) pp. 6760-6768
  • Published:10 August 2010
  • Marine bacteria residing on local red, green, and brown seaweeds were screened for exo-1,3-β-glucanase (ExoP) activity. Of the 90 bacterial species isolated from 32 seaweeds, only one, a Pseudoalteromonas sp., was found to display such activity. It was isolated from a Durvillaea sp., a brown kelp known to contain significant amounts of the storage polysaccharide laminarin (1,3-β-D-glucan with some 1,6-β branching). Four chromatographic steps were utilized to purify the enzyme (ExoP). Chymotryptic digestion provided peptide sequences for primer design and subsequent gene cloning. The exoP gene coded for 840 amino acids and was located just 50 bp downstream from a putative lichenase (endo-1,3-1,4-β-glucanase) gene, suggesting possible cotranscription of these genes. Sequence comparisons revealed ExoP to be clustered within a group of bacterial glycosidases with high similarity to a group of glycoside hydrolase (GH3) plant enzymes, of which the barley exo-1,3/1,4-β-glucanase (ExoI) is the best characterized. The major difference between the bacterial and plant proteins is an extra 200- to 220-amino-acid extension at the C terminus of the former. This additional sequence does not correlate with any known functional domain, but ExoP was not active against laminarin when this region was removed. Production of recombinant ExoP allowed substrate specificity studies to be performed. The enzyme was found to possess similar levels of exoglucanase activity against both 1,4-β linkages and 1,3-β linkages, and so ExoP is designated an exo-1,3/1,4-β-exoglucanase, the first such bacterial enzyme to be characterized. This broader specificity could allow the enzyme to assist in digesting both cell wall cellulose and cytoplasmic laminarin.

  • Impact of different milk fat globule membrane preparations on protein composition, xanthine oxidase activity, and redox potential.
  • Zahra Haddadian, Graham T Eyres, Alan Carne, David W Everett, and Phil Bremer., International Dairy Journal 2017 vol. 64 pp. 14-21.
  • Published:
  • © 2016 Elsevier LtdThe protein composition, redox potential (Eh), and xanthine oxidase (XO) content and activity were determined for anhydrous milk fat emulsions containing milk fat globule membrane (MFGM) fractions derived from either buttermilk or commercial sources of bovine α-serum, β-serum, and buttermilk powder (BMP). Caseins were the dominant proteins in the MFGM fractions isolated from BMP and buttermilk, whereas fractions from α- and β-serum contained higher amounts of membrane proteins. The XO content and activity was >70-fold and >700-fold higher in α- and β-serum samples, respectively, compared with the BMP fraction. The Eh values of the recombined emulsions were highest for α-serum (196 mV), and β-serum (169 mV), followed by BMP (131 mV). These positive values contrasted with the highly negative Eh of the buttermilk emulsion (−580 mV). This study demonstrates how milk-processing methods can alter the composition and functionality of the MFGM. 

  • Infiltration-RNAseq: transcriptome profiling of Agrobacterium-mediated infiltration of transcription factors to discover gene function and expression networks in plants.
  • Donna M Bond, Nick W Albert, Robyn H Lee, Gareth B Gillard, Chris M Brown, Roger P Hellens, and Richard C Macknight., Plant Methods 2016 vol. 12 (1) p. 41
  • Published:
  • BACKGROUND:Transcription factors (TFs) coordinate precise gene expression patterns that give rise to distinct phenotypic outputs. The identification of genes and transcriptional networks regulated by a TF often requires stable transformation and expression changes in plant cells. However, the production of stable transformants can be slow and laborious with no guarantee of success. Furthermore, transgenic plants overexpressing a TF of interest can present pleiotropic phenotypes and/or result in a high number of indirect gene expression changes. Therefore, fast, efficient, high-throughput methods for assaying TF function are needed.

    RESULTS:Agroinfiltration is a simple plant biology method that allows transient gene expression. It is a rapid and powerful tool for the functional characterisation of TF genes in planta. High throughput RNA sequencing is now a widely used method for analysing gene expression profiles (transcriptomes). By coupling TF agroinfiltration with RNA sequencing (named here as Infiltration-RNAseq), gene expression networks and gene function can be identified within a few weeks rather than many months. As a proof of concept, we agroinfiltrated Medicago truncatula leaves with M. truncatula LEGUME ANTHOCYANIN PRODUCITION 1 (MtLAP1), a MYB transcription factor involved in the regulation of the anthocyanin pathway, and assessed the resulting transcriptome. Leaves infiltrated with MtLAP1 turned red indicating the production of anthocyanin pigment. Consistent with this, genes encoding enzymes in the anthocyanin biosynthetic pathway, and known transcriptional activators and repressors of the anthocyanin biosynthetic pathway, were upregulated. A novel observation was the induction of a R3-MYB transcriptional repressor that likely provides transcriptional feedback inhibition to prevent the deleterious effects of excess anthocyanins on photosynthesis.

    CONCLUSIONS:Infiltration-RNAseq is a fast and convenient method for profiling TF-mediated gene expression changes. We utilised this method to identify TF-mediated transcriptional changes and TF target genes in M. truncatula and Nicotiana benthamiana. This included the identification of target genes of a TF not normally expressed in leaves, and targets of TFs from other plant species. Infiltration-RNAseq can be easily adapted to other plant species where agroinfiltration protocols have been optimised. The ability to identify downstream genes, including positive and negative transcriptional regulators, will result in a greater understanding of TF function.

  • Effects of pH, temperature and pulsed electric fields on the turbidity and protein aggregation of ovomucin-depleted egg white.
  • Ya-Fei Liu, Indrawati Oey, Phil Bremer, Alan Carne, and Pat Silcock., Food Research International 2017 vol. 91 pp. 161-170.
  • Published:
  • The effect of either pulsed electric fields (PEF) or thermal processing on protein aggregation of ovomucin-depleted egg white (OdEW) solutions at different pH was assessed by solution turbidity and SDS-PAGE. Heating to 60 °C for 10 min caused marked protein aggregation of OdEW at pH 5, 7, and 9. At constant electric field strength (E = 1.4–1.8 kV/cm), PEF processing under high specific energy input (Wspec = 260–700 kJ/kg) induced some protein aggregation at pH 5 and 7, but not at either pH 4 or 9. Similar effects of pH on protein aggregation were observed upon PEF processing at varied E (from 0.7 to 1.7 kV/cm) but with constant Wspec (713 kJ/kg). Analysis by SDS-PAGE revealed that proteins in the OdEW solution at pH 5 were most susceptible to both PEF- and heat-induced protein aggregation and lysozyme was only involved in the formation of insoluble aggregates under PEF. The present study shows that PEF treatment has considerable potential for minimizing protein aggregation in the processing of heat-labile egg white proteins. Retaining the OdEW proteins in solution during processing has potential industry application, for example, protein fortification of drinks with OdEW, where minimizing solution turbidity would be advantageous.

     

  • A PAM50-based Chemo-Endocrine Score for Hormone Receptor-Positive Breast Cancer with an Intermediate Risk of Relapse.
  • Aleix Prat, Ana Lluch, Arran K Turnbull, Anita K Dunbier, Lourdes Calvo, Joan Albanell, Juan de la Haba-Rodríguez, Angels Arcusa, Ignacio Chacón, Pedro Sánchez-Rovira, Arrate Plazaola, Montse Muñoz, Laia Paré, Joel S Parker, Nuria Ribelles, Begona Jimenez, Abdul Aziz Bin Aiderus, Rosalía Caballero, Barbara Adamo, Mitch Dowsett, Eva M Carrasco, Miguel Martín, J Michael Dixon, Charles M Perou, and Emilio Alba., Clin Cancer Res 2016.
  • Published:
  • PURPOSE:Hormone receptor-positive (HR+) breast cancer is clinically and biologically heterogeneous and subgroups with different prognostic and treatment sensitivities need to be identified.

    EXPERIMENTAL DESIGN:Research-based PAM50 subtyping and expression of additional genes was performed on 63 patients with HR+/HER2- disease randomized to neoadjuvant multi-agent chemotherapy versus endocrine therapy in a phase II trial. The biology associated with treatment response was used to derive a PAM50-based Chemo-Endocrine Score (CES). CES's predictive ability was evaluated in 4 independent neoadjuvant datasets (n=675) and 4 adjuvant datasets (n=1,505). The association of CES, intrinsic biology and PAM50 risk of relapse (ROR) was explored across 6,007 tumors.

    RESULTS:Genes associated with endocrine sensitivity were also found associated with chemotherapy resistance. In the chemotherapy test/validation datasets, CES was independently associated with pathological complete response, even after adjusting for intrinsic subtype. pCR rates of the CES endocrine sensitive (CES-E), uncertain (CES-U) and chemotherapy sensitive (CES-C) groups in both datasets combined were 25%, 11% and 2%, respectively. In the endocrine test/validation datasets, CES was independently associated with response. Compared to ROR, >90% of ROR-low and ROR-high tumors were identified as CES-E and CES-C, respectively; however, each CES-group represented >25% of ROR-intermediate disease. In terms of survival outcome, CES-C was associated with poor relapse-free survival in patients with ROR-intermediate disease treated with adjuvant endocrine therapy-only or no adjuvant systemic therapy, but not in patients treated with (neo)adjuvant chemotherapy.

    CONCLUSIONS:CES is a genomic signature capable of estimating chemo-endocrine sensitivity in HR+ breast cancer beyond intrinsic subtype and risk of relapse.