Dr Alan Stewart
Senior Lecturer in Molecular Medicine
Tel: 01334 463546
Current work is focused within three main areas as described below:
Circulatory fatty acid and zinc dynamics
Histidine-rich glycoprotein (HRG) is a plasma adaptor protein that regulates a number of biological processes in the blood, most notably coagulation. Clinically, elevated levels of HRG are linked to thrombosis. Zn2+ ions can stimulate HRG-complex formation. However, under normal conditions the majority of Zn2+ in the blood associates with human serum albumin (HSA). Crystallographic and mutagenesis studies reveal that Zn2+-binding to albumin occurs at a high-affinity site conserved across mammalian species (Stewart et al., PNAS, 2003; 100: 3701-3706; Handing et al., Chem. Sci., 2016; 7: 6635-6648). In collaboration with Dr Claudia Blindauer & Prof Peter Sadler we have demonstrated that high levels of free fatty acids disrupt the major Zn2+-binding site on HSA to increase the proportion of Zn2+ associated with HRG ( Lu et al., JACS, 2012; 134: 1454-1457; Kassaar et al., J. Thromb. Haemost. 2015; 13: 101-110). We speculate that this mechanism potentiates an increased risk of thrombosis in individuals with elevated fatty acid levels such as those associated with cancer, obesity and diabetes. Our primary aim is to establish whether plasma fatty acid levels regulate the Zn2+-dependent activities of HRG. If so, maintenance of plasma Zn2+ (or fatty acid) levels may provide a therapeutic strategy for managing thrombotic complications in high-risk individuals.
Functional and biochemical characterisation of histidine-rich glycoprotein
Histidine-rich glycoprotein (HRG) is a plasma protein that regulates angiogenesis, coagulation and immune function in vertebrates. In plasma HRG binds to and regulates the function of a diverse variety of targets that include fibrinogen, plasminogen, thrombospondin, IgG, complement factors and heparin as well as cell surface molecules such as Fcγ receptors and heparan sulphate. The protein possesses two N-terminal domains (N1 and N2), a central histidine-rich region (HRR) flanked by two proline rich regions (PRR1 and PRR2) and a C-terminal domain (C). HRG binds divalent metal cations at the HRR. In particular, Zn2+ is known to bind this region and modulate HRG activity by altering the protein’s affinity for other targets. We are currently examining the role of Zn2+ in regulating HRG functioning and aim to structurally charactrise the molecule. Recently, in collaboration with Prof Jim Naismith, we crystallised the N2 domain of serum-purified HRG, which provided a first structural snapshot of HRG (Kassaar et al., Blood 2014; 123: 1948-1955). The structure revealed the N2 domain to possess a cystatin-like fold. A native N-linked glycosylation site was identified at Asn184. Moreover, the structure reveals the presence of an S-glutathionyl adduct at Cys185, which has implications for angiogenic regulation.
Role of plasma proteins in sub-retinal epithelial deposit formation
A major feature of the ageing retina is the thickening of Bruch’s membrane and the formation of sub-retinal pigment epithelial (RPE) deposits. These sub-RPE deposits can block metabolic exchange between the choroidal blood circulation and the retina leading to sensory retinal degeneration and eventually to age-related macular degeneration (AMD). Recently, the presence of small (0.5-20 μm diameter) hydroxyapatite (HAP) spherules were identified within sub-RPE deposits isolated from human cadaver eyes (Thompson et al., PNAS, 2015; 112: 1565-1570). Furthermore, protein aggregates containing the AMD-associated plasma proteins complement factor H (CFH) and histidine-rich glycoprotein (HRG) were found to form on the surface of the HAP spherules. These new data led us to the idea that the spherules provide nucleation sites for sub-RPE deposit formation, where the initiation, growth and retention of deposits are controlled by the binding of proteins present in the sub-RPE space to the spherules. We are currently examining the contribution of plasma protein-HAP binding in sub-RPE deposit formation.
Selected Recent Publications (Full list is available here)
Reilly-O’Donnell, B, Robertson, GB, Karumbi, A, McIntyre, C, Bal, W, Nishi, M, Takeshima, H, Stewart, AJ & Pitt, SJ 2017, ‘Dysregulated Zn2+ homeostasis impairs cardiac type-2 ryanodine receptor and mitsugumin 23 functions, leading to sarcoplasmic reticulum Ca2+ leakage‘ Journal of Biological Chemistry, vol 292, no. 32, pp. 13361-13373.
Handing, KB, Shabalin, IG, Kassaar, O, Khazaipoul, S, Blindauer, CA, Stewart, AJ, Chruszcz, M & Minor, W 2016, ‘Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins‘ Chemical Science, vol 7, no. 11, pp. 6635-6648.
Blindauer, CA, Khazaipoul, S, Yu, R & Stewart, AJ 2016, ‘Fatty acid-mediated inhibition of metal binding to the multi-metal site on serum albumin: implications for cardiovascular disease‘ Current Topics in Medicinal Chemistry, vol 16, no. 27, pp. 3021-3032.
Woodier, J, Rainbow, R , Stewart, AJ & Pitt, SJ 2015, ‘ Intracellular zinc modulates cardiac ryanodine receptor-mediated calcium release ‘ Journal of Biological Chemistry, vol 290, no. 28, pp. 17599-17610.
Kassaar, O , Schwarz-Linek, U , Blindauer, CA & Stewart, AJ 2015, ‘ Plasma free fatty acid levels influence Zn 2+ -dependent histidine-rich glycoprotein-heparin interactions via an allosteric switch on serum albumin ‘ Journal of Thrombosis and Haemostasis, vol 13, no. 1, pp. 101-110.
Kassaar, O, McMahon, SA, Thompson, R , Botting, CH , Naismith, JH & Stewart, AJ 2014, ‘ Crystal structure of histidine-rich glycoprotein N2 domain reveals redox activity at an interdomain disulfide bridge: implications for angiogenic regulation ‘ Blood, vol 123, no. 12, pp. 1948-1955.
Barnett, JP, Blindauer, CA, Kassaar, O , Khazaipoul, S , Martin, EM, Sadler, PJ & Stewart, AJ 2013, ‘ Allosteric modulation of zinc speciation by fatty acids ‘ Biochimica et Biophysica Acta – General Subjects, vol 1830, no. 12, pp. 5456-5464.
Popovics, P, Gray, A, Arastoo, M, Finelli, DK, Tan, AJL & Stewart, AJ 2013, ‘ Phospholipase C-η2 is required for retinoic acid-stimulated neurite growth ‘ Journal of Neurochemistry, vol 124, no. 5, pp. 632-644.
Majorek, KA, Porebski, PJ, Dayal, A, Zimmerman, MD, Jablonska, K , Stewart, AJ , Chruszcz, M & Minor, W 2012, ‘ Structural and immunologic characterization of bovine, horse, and rabbit serum albumins ‘ Molecular Immunology, vol 52, no. 3-4, pp. 174-182.
Lu, J , Stewart, AJ , Sadler, PJ, Pinheiro, TJT & Blindauer, CA 2012, ‘ Allosteric inhibition of cobalt binding to albumin by fatty acids: Implications for the detection of myocardial ischemia ‘ Journal of Medicinal Chemistry, vol 55, no. 9, pp. 4425-4430.
Lu, J , Stewart, AJ , Sleep, D, Sadler, PJ, Pinheiro, TJT & Blindauer, CA 2012, ‘ A molecular mechanism for modulating plasma Zn speciation by fatty acids ‘ Journal of the American Chemical Society, vol 134, no. 3, pp. 1454-1457.