MSc Opportunity

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Monday 1 August 2022

MSc(Res) Opportunity at the School of Medicine, University of St Andrews

Project Outline

Probing insulin complexation using fluorescent approaches

Supervisor(s): Dr Alan Stewart (University of St Andrews) & Prof Carlos Penedo (University of St Andrews).

An opportunity for a funded Masters of Science (by research) degree is available under the supervision of Dr Alan Stewart. You should have a 2:1 degree or above in a relevant subject.

 

Deadline: Position will remain open until filled.

Start date: September-October 2022

Fees: home tuition fees and lab costs will be covered but there is no provision for living expenses.

 

Project description

Zinc plays central roles in controlling numerous processes in the circulation. The concentration of free/labile Zn2+ in plasma is controlled by human serum albumin (HSA), influencing both its quantity and distribution (also called “speciation”). Zn2+ speciation plays a major role in regulating insulin signalling. Insulin is stored within granular vesicles inside pancreatic β-cells in an inactive Zn2+-bound hexameric form (Zn2Ins6) [1-3]. The concentration of Zn2+ is high within β-cell secretory vesicles (ca. 20 mM; [4]) due to the action of ZnT8 zinc transporters, which shuttle cytosolic Zn2+ into the secretory vesicle [5]. Upon its immediate release, insulin in the Zn2Ins6 form only becomes active once Zn2+ disengages from the complex, a necessary step in activation. A role for zinc in insulin clearance by the liver has also been suggested [6]; hence Zn2+ availability impacts the levels of total circulating and active insulin. There are indications that HSA affects insulin pharmacodynamics through regulation of hexamer to monomer conversion. Radioimmunoassays and insulin tolerance tests in rats showed that albumin promoted the dissociation of inactive Zn2Ins6 into active monomer [7]. This demonstrates that the Zn2+-binding properties of HSA aid dissociation of insulin into active subunits – a process not fully understood nor appreciated on a quantitative level, but central to blood glucose control.

The Stewart group has shown that fatty acid binding to HSA perturbs its ability to bind Zn2+ [8]. Furthermore, others have shown that glycation reduces the affinity of HSA-Zn2+ binding [9]. Given that both elevations in fatty acid and increased HSA glycation are associated with diabetes, it is likely that defective Zn2+ buffering/binding by HSA (caused by high concentrations of fatty acids or glycation) alter insulin pharmacokinetics in a manner that contributes to insulin resistance.  Insulin complexation has been studied using various methods including ITC [10], and EPR [11]. However, these methods unsuitable to study HSA-mediated insulin decomplexation under physiological conditions due to their low sensitivity (i.e. insulin concentrations in plasma are in the pM/nM range, whilst HSA is ~600 µM, causing dynamic range and sensitivity issues). To overcome this, we have developed fluorescently labelled insulins that form FRET complexes enabling examination of complexation at the pM scale. Further work is required to understand the signals these generate in the context of multimerization.

Aims / Approach: 1. Characterise FRET signals derived from fluorescently-labelled insulin mixtures. For this, we will use native MS-based approaches to characterize insulin complexes and their labelling positions, followed by single-molecule FRET to characterize structural states. Compounds known to stabilise hexamers in certain conformations (e.g., phenol, KSCN and Cl [3]) will be used in combination with different concentrations of zinc.

  1. Assess the impact of HSA on decomplexation dynamics at physiological concentrations. Here we will examine the impact of HSA addition to pre-formed “FRET active” insulin complexes. This will allow the effect of HSA to be assessed at a quantitative level. If time allows, we will also examine glycated and fatty acid-loaded HSA forms.

Impact of the work: In addition to the interdisciplinary biomedical research experience offered, this work will enhance our understanding of insulin pharmacokinetics in both health and disease. This work will increase our toolkit and allow assessment of clinical samples and examination of insulin analogues used clinically to treat diabetes and related conditions.

For further details on the project, please contact Dr Stewart: ajs21@st-andrews.ac.uk.

 

How to apply

Informal application enquiries can be made to pgmed@st-andrews.ac.uk. Full applications should be made via the University’s online portal.

Applications should include:

A covering letter

A full curriculum vitae

Supporting document including a list of refereed publications and key conference contributions (if applicable)

 

 

School of Medicine, University of St Andrews

The successful applicants will join a thriving research laboratory and will have the opportunity to work in an exciting and collaborative research environment, equipped to the highest international standards. The School of Medicine is located at the core of a highly interactive and vibrant research campus at the forefront of cell signalling and molecular medicine research with a focus on understanding and combating disease. The School is committed to equal opportunities and values all applicants. The School currently has Athena SWAN bronze accreditation.

 

Relevant References

  1. 1. Jeffrey PD & Coates JH (1966) Biochemistry 5:489-498. 2. Carpenter FH (1966) Am J Med. 40:750-758. 3. Hassiepan U et al. (1999) Biophys J. 77:1638. 4. Foster MC et al. (1993) Biophys J. 64:525-532. 5. Kawasaki E (2012) J. 59:531-537. 6. Fukunaka & Fujitani (2018) Int J Mol Sci 19:476. 7. Pertusa JAG et al. (2017) PLoS One 12:e0187547. 8. Sobczak AIS et al. (2021) Chem Sci 12: 4079-4093. 9. Jacobs MJ et al. (2020) Metallomics 12:1036-1043. 10. Carpenter M.C. & Wilcox D.E. (2014) Biochemistry 53:1296-1301. 11. Steinhoff H.J. et al. (1997) Biophys J 73:3287-3298.

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