Alan Stewart

Dr Alan Stewart

Reader in Molecular Medicine

ajs21@st-andrews.ac.uk

Tel: 01334 463546

 

Research summary
Current work is focused within three main areas as described below:

fatty switch

The fatty-acid/zinc switch on serum albumin. Overlay of crystal structures of human serum albumin with (grey; PDB: 1BJ5) and without myristic acid bound (green; PDB: 1AO6) showing the location of the major zinc-binding site and the movement of zinc-coordinating residues His247 and Asp249 relative to His67 and Asn99 between the two structures.

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.

HRG N2

X-ray crystal structure of the N2 domain of HRG. PDB: 4CCV.

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. 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. We are currently working to elucidate the structure and biochemical properties of HRG and its associated complexes in collaboration with Prof Ramzi Ajjan (Univeristy of Leeds).

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 and are interested in collaboratively exploring further applications of this technology to address other medically-relevant problems.

 

Selected Recent Publications (Full list is available here)

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 diabetesChemical 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 temperatureAngewandte 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 studyScientific 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 hypothesisProgress 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 glycoproteinScientific Reports, vol 8, 8646.

Sobczak, AIS, Pitt, SJ & Stewart, AJ 2018, ‘Glycosaminoglycan neutralization in coagulation controlArteriosclerosis, 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 degenerationExperimental 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+ leakageJournal 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 albuminsChemical 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 diseaseCurrent 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.