N. Taylor

University of Melbourne, Biochemistry and Molecular Biology, Alumna

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Glucose transporter 1 expression identifies a population of cycling CD4+CD8+ human thymocytes with high CXCR4-induced chemotaxis

by N. Taylor and Marc Sitbon

Proceedings of the National Academy of Sciences, 2005

GLUT1, the major glucose transporter in peripheral T lymphocytes, is induced upon T cell receptor... more GLUT1, the major glucose transporter in peripheral T lymphocytes, is induced upon T cell receptor activation. However, the role of GLUT1 during human thymocyte differentiation remains to be evaluated. Our identification of GLUT1 as the human T lymphotrophic virus (HTLV) receptor has enabled us to use tagged HTLVreceptor-binding domain fusion proteins to specifically monitor surface GLUT1 expression. Here, we identify a unique subset of CD4 ؉ CD8 ؉ double-positive (DP) thymocytes, based on their GLUT1 surface expression. Whereas these cells express variable levels of CD8, they express uniformly high levels of CD4. Glucose uptake was 7-fold higher in CD4 hi DP thymocytes than in CD4 lo DP thymocytes (P ‫؍‬ 0.0002). Further analyses indicated that these GLUT1 ؉ thymocytes are early post-␤-selection, as demonstrated by low levels of T cell receptor (TCR)␣␤ and CD3. This population of immature GLUT1 ؉ DP cells is rapidly cycling and can be further distinguished by specific expression of the transferrin receptor. Importantly, the CXCR4 chemokine receptor is expressed at 15-fold higher levels on GLUT1 ؉ DP thymocytes, as compared with the DP GLUT1 ؊ subset, and the former cells show enhanced chemotaxis to the CXCR4 ligand CXCL12. Thus, during human thymopoiesis, GLUT1 is up-regulated after ␤-selection, and these immature DP cells constitute a population with distinct metabolic and chemotactic properties.

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Substrate-mediated Stabilization of a Tetrameric Drug Target Reveals Achilles Heel in Anthrax

by Renwick Dobson, Craig Hutton, and N. Taylor

Journal of Biological Chemistry, 2010

Bacillus anthracis is a Gram-positive spore-forming bacterium that causes anthrax. With the incre... more Bacillus anthracis is a Gram-positive spore-forming bacterium that causes anthrax. With the increased threat of anthrax in biowarfare, there is an urgent need to characterize new antimicrobial targets from B. anthracis. One such target is dihydrodipicolinate synthase (DHDPS), which catalyzes the committed step in the pathway yielding meso-diaminopimelate and lysine. In this study, we employed CD spectroscopy to demonstrate that the thermostability of DHDPS from B. anthracis (Ba-DHDPS) is significantly enhanced in the presence of the substrate, pyruvate. Analytical ultracentrifugation studies show that the tetramer-dimer dissociation constant of the enzyme is 3-fold tighter in the presence of pyruvate compared with the apo form. To examine the significance of this substrate-mediated stabilization phenomenon, a dimeric mutant of Ba-DHDPS (L170E/ G191E) was generated and shown to have markedly reduced activity compared with the wild-type tetramer. This demonstrates that the substrate, pyruvate, stabilizes the active form of the enzyme. We next determined the high resolution (2.15 Å ) crystal structure of Ba-DHDPS in complex with pyruvate (3HIJ) and compared this to the apo structure (1XL9). Structural analyses show that there is a significant (91 Å 2 ) increase in buried surface area at the tetramerization interface of the pyruvatebound structure. This study describes a new mechanism for stabilization of the active oligomeric form of an antibiotic target from B. anthracis and reveals an "Achilles heel" that can be exploited in structure-based drug design.

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Expression, purification, crystallization and preliminary X-ray analysis of maleylacetate reductase from Burkholderia sp. strain SJ98

by N. Taylor and Paula Clancy

Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2009

The soluble domain (residues 483-913) of PPA0092, a putative copper-containing nitrite reductase ... more The soluble domain (residues 483-913) of PPA0092, a putative copper-containing nitrite reductase from Propionibacterium acnes KPA171202, has been overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 2.4 A. The crystal belonged to space group P2(1)3, with unit-cell parameters a = b = c = 108.63 A. Preliminary diffraction data show that one molecule is present in the asymmetric unit; this corresponds to a V(M) of 2.1 A(3) Da(-1).

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$Research paper thumbnail of Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate$

Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate

by Renwick Dobson, Craig Hutton, and N. Taylor

Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2009

Printed in Denmark ± all rights reserved Mycobacterium tuberculosis (H37Rv), the causative agent ... more Printed in Denmark ± all rights reserved Mycobacterium tuberculosis (H37Rv), the causative agent of the dreaded disease tuberculosis, contains three thioredoxins and a single thioredoxin reductase. Thioredoxin reductase is a member of the pyridine-nucleotide disul®de oxidoreductase family of¯avoenzymes. The thioredoxin reductase gene with a His tag at the C-terminus was expressed in Escherichia coli and puri®ed. The dimeric (70 kDa) protein was incubated with 10 mM DTT for 30 min and then crystallized using the hanging-drop vapour-diffusion method in the presence of 15% PEG 3350 and phosphate±citrate buffer pH 5 at room temperature (298 K). A diffraction data set complete to 3 A Ê resolution has been collected under cryoconditions and the space group was determined to be P4 1 2 1 2, with unit-cell parameters a = 107.4, c = 118.2 A Ê . Matthews coef®cient calculations revealed the presence of two monomers in the asymmetric unit.

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