Characterization of phenolic compound-modified alpha-2-macroglobulin in vitro

Author: Emma-Jean Hillman

Hillman, Emma-Jean, 2023 Characterization of phenolic compound-modified alpha-2-macroglobulin in vitro, Flinders University, College of Medicine and Public Health

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Alpha-2-macroglobulin (α2M) belongs to the alpha-macroglobulin family of secreted proteins and is constitutively abundant in human biological fluids. α2M is a highly multifunctional protein with ancient ties to innate immunity. The functions of α2M are dependent on its conformation, which can be native, transformed, or dimeric. Each conformation is functionally distinct, all playing a crucial role in the regulation of inflammatory and immune responses. For example, during inflammation, dissociation of the native α2M tetramer into dimers is induced by hypochlorite and significantly enhances the holdase-type chaperone activity of α2M. Where the α2M and its cargo are rapidly cleared by autophagy mediated via low-density lipoprotein receptor protein 1 (LRP-1). However, since hypochlorite is localized to the site of inflammation, the potential benefits of this protein may be limited. Consequently, if a drug or drug-like compound could induce the dimeric conformation of α2M, it could present a promising new therapeutic avenue.

Preliminary investigations by the Wyatt laboratory identified that phenolic compounds such as rosmarinic acid (RA), caffeic acid (CA) and salvianolic acid β (Saβ), induce α2M to dissociate into dimer-like molecules.

Considering the superior chaperone activity of dimeric α2M compared to native and transformed α2M tetramers, this study undertook a series of experiments to characterise the structure and function of phenolic compound-treated α2M in order to generate proof-of-principle data, to demonstrate whether phenolic compounds could be used to target the chaperone activity of α2M. Based on the assumption that native α2M treated with RA, CA or Saβ , will have the same structure and function as hypochlorite treated α2M, this study aimed to characterise functions in holdase-type chaperone activity and cell surface receptor binding of phenolic compound-modified α2M. The evaluation of holdase-type chaperone activity involved conducting a Thioflavin T (ThT) assay with the Alzheimer’s disease-related amyloid beta peptide, Aβ1-42. Assessment of cell surface receptor binding was accomplished through flow cytometry, utilizing SH SY5Y neuroblastoma cells pre-treated with biotinylated phenolic compound-modified α2M and streptavidin-conjugated fluorophore, STR488. The structural characterization of phenolic compound-modified α2M included native gel electrophoresis and a bis-ANS assay to analyse migration patterns and hydrophobicity, respectively. The proof-of-principle data generated in this study supported the conclusion that although phenolic compound-modified α2M enhanced the ability of α2M to stabiles Aβ1-42, this effect does not occur via a canonical holdase chaperone action. The results also suggest that phenolic compounds may induce a slightly transformed conformation in α2M, which is electrophoretically fast but shields the hydrophobic regions. When considering these findings alongside Western blot analysis of ThT endpoint samples and the cell surface binding assay, it seems to support the idea that treatment of α2M with phenolic compounds may be exposing the internal thiol ester. This exposure may be important in the stabilisation of Aβ1-42 via covalent binding and exposure of the LRP-1 binding domain. But additional studies are required to confirm the model proposed for phenolic compound-induced modification of α2M.

A greater understanding to the functions of phenolic compound-modified α2M has the potential to lay the groundwork for developing treatments for conditions characterized by protein misfolding and inflammation.

Keywords: Alpha-2-macroglobulin, α2M, phenolic compound-modified α2M,

Subject: Medical Biotechnology thesis

Thesis type: Masters
Completed: 2023
School: College of Medicine and Public Health
Supervisor: Amy Wyatt