School of Biomedical Sciences - Research Publications
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ItemStructural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition(SPRINGER, 2013-03)Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.
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ItemFrom Knock-Out Phenotype to Three-Dimensional Structure of a Promising Antibiotic Target from Streptococcus pneumoniae(PUBLIC LIBRARY SCIENCE, 2013-12-13)Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a k(cat) = 22 s(-1), K(M)(PYR) = 2.55 ± 0.05 mM and K(M)(ASA) = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (T(M)(app)) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (T(M)(app) = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a K(D)(4→2) = 1.7 nM, which is considerably tighter than its E. coli ortholog (K(D)(4→2) = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å(2)) compared to its E. coli counterpart (500 Å(2)). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target.
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ItemPlant cell shape: modulators and measurements(FRONTIERS MEDIA SA, 2013-11-19)Plant cell shape, seen as an integrative output, is of considerable interest in various fields, such as cell wall research, cytoskeleton dynamics and biomechanics. In this review we summarize the current state of knowledge on cell shape formation in plants focusing on shape of simple cylindrical cells, as well as in complex multipolar cells such as leaf pavement cells and trichomes. We summarize established concepts as well as recent additions to the understanding of how cells construct cell walls of a given shape and the underlying processes. These processes include cell wall synthesis, activity of the actin and microtubule cytoskeletons, in particular their regulation by microtubule associated proteins, actin-related proteins, GTP'ases and their effectors, as well as the recently-elucidated roles of plant hormone signaling and vesicular membrane trafficking. We discuss some of the challenges in cell shape research with a particular emphasis on quantitative imaging and statistical analysis of shape in 2D and 3D, as well as novel developments in this area. Finally, we review recent examples of the use of novel imaging techniques and how they have contributed to our understanding of cell shape formation.
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ItemSkeletal Muscle Insulin Resistance Associated with Cholesterol-Induced Activation of Macrophages Is Prevented by High Density Lipoprotein(PUBLIC LIBRARY SCIENCE, 2013-02-21)Emerging evidence suggests that high density lipoprotein (HDL) may modulate glucose metabolism through multiple mechanisms including pancreatic insulin secretion as well as insulin-independent glucose uptake into muscle. We hypothesized that HDL may also increase skeletal muscle insulin sensitivity via cholesterol removal and anti-inflammatory actions in macrophages associated with excess adiposity and ectopic lipid deposition.
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ItemEstimation and partitioning of polygenic variation captured by common SNPs for Alzheimer's disease, multiple sclerosis and endometriosis(OXFORD UNIV PRESS, 2013-02-15)Common diseases such as endometriosis (ED), Alzheimer's disease (AD) and multiple sclerosis (MS) account for a significant proportion of the health care burden in many countries. Genome-wide association studies (GWASs) for these diseases have identified a number of individual genetic variants contributing to the risk of those diseases. However, the effect size for most variants is small and collectively the known variants explain only a small proportion of the estimated heritability. We used a linear mixed model to fit all single nucleotide polymorphisms (SNPs) simultaneously, and estimated genetic variances on the liability scale using SNPs from GWASs in unrelated individuals for these three diseases. For each of the three diseases, case and control samples were not all genotyped in the same laboratory. We demonstrate that a careful analysis can obtain robust estimates, but also that insufficient quality control (QC) of SNPs can lead to spurious results and that too stringent QC is likely to remove real genetic signals. Our estimates show that common SNPs on commercially available genotyping chips capture significant variation contributing to liability for all three diseases. The estimated proportion of total variation tagged by all SNPs was 0.26 (SE 0.04) for ED, 0.24 (SE 0.03) for AD and 0.30 (SE 0.03) for MS. Further, we partitioned the genetic variance explained into five categories by a minor allele frequency (MAF), by chromosomes and gene annotation. We provide strong evidence that a substantial proportion of variation in liability is explained by common SNPs, and thereby give insights into the genetic architecture of the diseases.
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ItemRapid Decline in Episodic Memory in Healthy Older Adults with High Amyloid-β(IOS PRESS, 2013)High levels of amyloid-β (Aβ) have been associated with greater rates of decline in episodic memory over 18 months in healthy older adults. Serial assessments over shorter time intervals may facilitate earlier detection of Aβ-related memory decline in healthy older adults. In forty-four healthy older adults enrolled in the Australian Imaging, Biomarkers and Lifestyle Rate of Change Sub-Study, we compared rates of change in cognition over six months in healthy older adults with high and low levels of Aβ. High Aβ was associated with greater decline in episodic memory measures over 6 months in healthy older adults.
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ItemTargeting antigen to bone marrow stromal cell-2 expressed by conventional and plasmacytoid dendritic cells elicits efficient antigen presentation(WILEY-BLACKWELL, 2013-03)Bone marrow stromal cell-2 (BST-2) has major roles in viral tethering and modulation of interferon production. Here we investigate BST-2 as a receptor for the delivery of antigen to dendritic cells (DCs). We show that BST-2 is expressed by a panel of mouse and human DC subsets, particularly under inflammatory conditions. The outcome of delivering antigen to BST-2 expressed by steady state and activated plasmacytoid DC (pDC) or conventional CD8(+) and CD8(-) DCs was determined. T-cell responses were measured for both MHC class I (MHCI) and MHC class II (MHCII) antigen presentation pathways in vitro. Delivering antigen via BST-2 was compared with that via receptors DEC205 or Siglec-H. We show that despite a higher antigen load and faster receptor internalisation, when antigen is delivered to steady state or activated pDC via BST-2, BST-2-targeted activated conventional DCs present antigen more efficiently. Relative to DEC205, BST-2 was inferior in its capacity to deliver antigen to the MHCI cross-presentation pathway. In contrast, BST-2 was superior to Siglec-H at initiating either MHCI or MHCII antigen presentation. In summary, BST-2 is a useful receptor to target with antigen, given its broad expression pattern and ability to access both MHCI and MHCII presentation pathways with relative efficiency.
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ItemHepatitis B virus-like particles access major histocompatibility class I and II antigen presentation pathways in primary dendritic cells(ELSEVIER SCI LTD, 2013-04-26)Virus-like particles (VLPs) represent high density displays of viral proteins that efficiently trigger immunity. VLPs composed of the small hepatitis B virus envelope protein (HBsAgS) are useful vaccine platforms that induce humoral and cellular immune responses. Notably, however, some studies suggest HBsAgS VLPs impair dendritic cell (DC) function. Here we investigated HBsAgS VLP interaction with DC subsets and antigen access to major histocompatibility complex (MHC) class I and II antigen presentation pathways in primary DCs. HBsAgS VLPs impaired plasmacytoid DC (pDC) interferon alpha (IFNα) production in response to CpG in vitro, but did not alter conventional DC (cDC) or pDC phenotype when administered in vivo. To assess cellular immune responses, HBsAgS VLPs were generated containing the ovalbumin (OVA) model epitopes OVA(257-264) and OVA(323-339) to access MHCI and MHCII antigen presentation pathways, respectively; both in vitro and following immunisation in vivo. HBsAgS VLP-OVA(257-264) elicited CTL responses in vivo that were not enhanced by inclusion of an additional MHCII helper epitope. HBsAgS VLP-OVA(257-264) administered in vivo was cross-presented by CD8(+) DCs, but not CD8(-) DCs. Therefore, HBsAgS VLPs can deliver antigen to both MHCI and MHCII antigen presentation pathways in primary DCs and promote cytotoxic and helper T cell priming despite their suppressive effect on pDCs.
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ItemRapid Deletion and Inactivation of CTLs upon Recognition of a Number of Target Cells over a Critical Threshold(AMER ASSOC IMMUNOLOGISTS, 2013-10-01)Initiation of CTL responses against foreign pathogens also primes anti-self CTLs. Mechanisms of CTL inactivation inhibit anti-self CTLs to prevent tissue damage. These mechanisms are exploited by pathogens and tumors to evade the immune response, and present a major hurdle to adoptive CTL therapies. It is unclear whether CTL inactivation is Ag specific and, if so, which APCs are involved. Potential candidates include the target cells themselves, dendritic cells, myeloid-derived suppressor cells, and macrophages. In this study, we show that lymphoma-specific CTLs are rapidly deleted in an Ag-specific manner after adoptive transfer into lymphoma-bearing mice, and the surviving CTLs are functionally impaired. The only APCs responsible were the target cells directly presenting Ag, notwithstanding the presence of myeloid-derived suppressor cells, and CD8(+) dendritic cells cross-presenting tumor Ag. The capacity to inactivate CTLs critically depended on the number of tumor/target cells; small numbers of targets were readily killed, but a large number caused quick deletion and functional inactivation of the CTLs. Application of mild, noninflammatory, and nonlymphoablative chemotherapy to specifically reduce tumor burden before CTL injection prevented CTL deletion and inactivation and allowed eradication of tumor. Our results advocate the use of adoptive CTL therapy soon after mild chemotherapy. They also suggest a simple mechanism for Ag-specific impairment of anti-self CTLs in the face of an active anti-foreign CTL response.