Thesis
Probing the biology of zinc alpha2-glycoprotein
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2025
- Thesis identifier
- T17382
- Person Identifier (Local)
- 202085806
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Zinc Alpha 2-Glycoprotein (AZGP1;ZAG) is a ~40-kDa single-chain polypeptide protein thought to contribute to the regulation of weight and body fat through lipid and glucose metabolism. In healthy individuals, ZAG exerts a homeostatic effect by inducing lipolysis of adipose tissue to help reduce fat storage and overall weight. ZAG is upregulated in various carcinomas; cancer patients with upregulated ZAG lose weight rapidly and there is a clear link between ZAG and cancer cachexia. The crystal structure of ZAG revealed an MHC-Class Ilike protein fold which has been proposed to act as a potential lipid binding site that could be important for ZAG’s function. The work in this thesis aimed to produce an in silico docking approach to identify ligand(s) which may bind this groove, and to understand the key interactions between them and ZAG. Using PLANTS software and the protein visualisation software UCSF Chimera, we identified candidate ligands for binding in ZAGs pocket that have equalled or outscored previous identified ligands which can now be incorporated into competition binding assays to test their affinity for ZAG. In parallel, we developed approaches to produce recombinant ZAG using bacterial and mammalian expression systems, confirmed their structure using fluorescence spectroscopy and outline initial work to show that the recombinant protein is functional and can promote lipolysis in fat cells. Because a key target of ZAG action is adipocytes, methods were developed to utilise Stimulated Raman Scattering (SRS) to provide a workflow that allows for a label-free, high throughput, single cell analysis to investigate heterogeneric metabolic activity of adipocytes. Specifically, the approach described allows measurement of lipid droplet numbers, size and quantifying glucose metabolism using glucose-d7. We have demonstrated the validity of our method in 3T3-L1 adipocytes incubated in culture for different periods as a model of adipocyte hypertrophy. Lastly, we adapted a mitochondrial isolation assay to separate cytosolic and peridroplet mitochondria from 3T3-L1 adipocytes. In sum, this study provided tools and insight to help progress the knowledge and understanding of ZAG biology, signalling mechanisms and for the investigation of both phenotypic and metabolic adipocyte heterogeneity.
- Advisor / supervisor
- Gould, Gwyn W.
- Resource Type
- DOI
- Date Created
- 2024
- Funder
Relations
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PDF of thesis T17382 | 2025-06-12 | Public | Download |