Dr Alan Stewart
Reader 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 other proteins (Kassaar et al., J. Thromb. Haemost. 2015; 13: 101-110; Coverdale et al. Metallomics 2019; 11: 1805-1819). Our work also suggests 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 (Sobczak et al., Chem. Sci. 2021; 12: 4079-4093). A primary aim going forward is to establish which proteins in plasma “pick up” Zn2+ displaced from HSA and to determine the functional/pathological consequences of these interactions.
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 characterise the molecule. Previously, 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. More recently, we characterised the metal binding sites on the molecule together with Dr Bela Bode’s group (Ackermann et al., JACS 2023; 145: 8064-8072). We revealed that HRG acts like a sponge for Cu2+ (and other divalent metal ions, with Zn2+ known to play an important regulatory role), using a set of high-affinity binding sites involving histidine coordination of the metal ions and a much larger number of lower-affinity binding sites not involving histidine residues. We further conclude that the predicted disordered PRR1-HRR-PRR2 region of HRG allows for a
gradual and flexible adaptation of structural features dependent on the metal ion loading. We are currently working to elucidate the structure and biochemical properties of HRG and its associated complexes in collaboration with various groups.
Quantitative Cellular and Plasma Proteomics
Recently in collaboration with Dr Sally Shirran (St Andrews Mass Spectrometry Facility) we have established a platform in St Andrews for cellular and plasma quantitative proteomics using a technique called Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS). Using this approach we can identify and relatively quantify several hundreds of plasma proteins in a single drop of blood. Recently we utilised this method to identify hydroxyapatite-binding plasma proteins in blood samples taken from genotyped individuals with age-related macular degeneration (Arya et al., Exp. Eye Res. 2018; 172: 21-29). In addition we have employed this approach to examine cellular proteomic changes. for example, we measured changes in monocyte-derived dendritic cell protein abundance during LPS-induced maturation, where we were able to detect and quantify >4400 proteins at different stages of the process enabing pathway analysis (Arya et al., Sci. Rep. 2019; 9: 4343). We are currently using this platform to identify prognostic and diagnostic markers of diseases including cancer. My group also have an interest in developing new “speciomic” approaches to examine metal-protein interactions in complex mixtures. Current work in this area is being carried out in collaboration with Profs. Claudia Blindauer (University of Warwick) and Marco Arruda (University of Campinas, Brazil; Arruda et al., J. Proteomics 2022; 263: 104615). We are interested in collaboratively exploring further applications of these technologies to address medically-relevant problems.
Selected Recent Publications (Full list is available here)
Ackermann, K, Khazaipoul, S, Wort, JL, Sobczak, AIS, El Mkami, H, Stewart, AJ & Bode, BE 2023, ‘Investigating native metal ion binding sites in mammalian histidine-rich glycoprotein. ‘ Journal of the American Chemical Society, vol 145, 8064-8072.
Arruda, MAZ, de Jesus, JR, Blindauer, CA & Stewart, AJ 2022, ‘Speciomics as a concept involving chemical speciation and omics. ‘ Journal of Proteomics, vol 263, 104615.
Coverdale, JPC, van den Burgh, HA, Khazaipoul, S, Bridgewater, HE, Stewart, AJ & Blindauer, CA 2022, ‘Albumin-mediated extracellular zinc speciation drives cellular zinc uptake‘ Chemical Communications, vol 58, 7384-7387.
Sobczak, AIS, Katundu, KG, Phoenix, F, Khazaipoul, S, Yu, R, Lampiao, F, Stefanowicz, F, Blindauer, CA, Pitt, SJ, Smith, TK, Ajjan, RA & Stewart, AJ 2021, ‘Albumin-mediated alteration of plasma zinc speciation by fatty acids modulates blood clotting in type-2 diabetes‘ Chemical Science, vol 12, 4079-4093.
Wort, JL, Ackermann, K, Giannoulis, A, Stewart, AJ, Norman, DG & Bode BE 2019, ‘Submicromolar pulse dipolar EPR spectroscopy reveals increasing CuII-labelling of double-histidine motifs with lower temperature‘ Angewandte Chemie International Edition, vol 58, 11681-11685.
Arya, S, Wiatrek-Moumoulidis, D, Synowsky, SA, Shirran, SL, Botting, CH, Powis, SJ & Stewart, AJ 2019, ‘Quantitative proteomic changes in LPS-activated monocyte-derived dendritic cells: A SWATH-MS study‘ Scientific Reports, vol 9, 4343.
Bergen, AA, Arya, S, Koster, C., Pilgrim, MG, Wiatrek-Moumoulidis, D, van der Spek, P, Hauck, SM, Boon, CJF, Emri, E, Stewart, AJ & Lengyel I 2019, ‘On the origin of proteins in human drusen: The meet, greet and stick hypothesis‘ Progress in Retinal and Eye Research, vol 70, 55-84.
Martin, EM, Kondrat, FDL, Stewart, AJ, Scrivens, JH, Sadler, PJ & Blindauer, CA 2018, ‘Native electrospray mass spectrometry approaches to probe the interaction between zinc and an anti-angiogenic peptide from histidine-rich glycoprotein‘ Scientific Reports, vol 8, 8646.
Sobczak, AIS, Pitt, SJ & Stewart, AJ 2018, ‘Glycosaminoglycan neutralization in coagulation control‘ Arteriosclerosis, Thrombosis, and Vascular Biology, vol 38, no. 6, pp. 1258-1270.
Arya, S, Emri, E, Synowsky, SA, Shirran, SL, Barzegar-Befroei, N, Peto, T, Botting, CH, Lengyel, I & Stewart, AJ 2018, ‘Quantitative analysis of hydroxyapatite-binding plasma proteins in genotyped individuals with late-stage age-related macular degeneration‘ Experimental Eye Research, vol 172, pp. 21-29.
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.
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.