Abstract
Members of the CAP superfamily (Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-Related 1 proteins) are characterized by the presence of a structurally conserved CAP domain. The common structure-function relationship of this domain is still poorly understood. In this study, we unravel specific molecular mechanisms modulating the quaternary structure of the mammalian CAP protein GAPR-1 (Golgi-Associated plant Pathogenesis-Related protein 1). Copper ions are shown to induce a distinct amyloid-like aggregation pathway of GAPR-1 in the presence of heparin. This involves an immediate shift from native multimers to monomers which are prone to form amyloid-like fibrils. The Cu2+-induced aggregation pathway is independent of a conserved metal-binding site and involves the formation of disulfide bonds during the nucleation process. The elongation process occurs independently of the presence of Cu2+ ions, and amyloid-like aggregation can proceed under oxidative conditions. In contrast, the Zn2+-dependent aggregation pathway was found to be independent of cysteines and was reversible upon removal of Zn2+ ions. Together, our results provide insight into the regulation of the quaternary structure of GAPR-1 by metal ions and redox homeostasis with potential implications for regulatory mechanisms of other CAP proteins.
Highlights
Members of the CAP superfamily (Cysteine-rich secretory proteins, Antigen 5, and PathogenesisRelated 1 proteins) are characterized by the presence of a structurally conserved CAP domain
In order to determine whether the modulation of GAPR-1 amyloidogenicity is specific for zinc, GAPR-1 was incubated with various metal ions in the presence of heparin and Thioflavin T (ThT) fluorescence was monitored over time
We have shown here that formation of GAPR-1 amyloid-like structures can occur through distinct molecular pathways dependent on zinc/copper ion binding and redox conditions, providing novel insights into the possible regulatory mechanisms controlling GAPR-1 oligomerization and aggregation
Summary
Members of the CAP superfamily (Cysteine-rich secretory proteins, Antigen 5, and PathogenesisRelated 1 proteins) are characterized by the presence of a structurally conserved CAP domain. Upon interaction with phosphatidylinositol- and cholesterol-containing liposomes, GAPR-1 forms oligomers and amyloid-like structures[20] This indicates that structural dynamics play an important role in fluorescence intensity after 20 h incubation is shown for the histidine mutants relative to that of WT GAPR-1 for each metal ion, respectively. Copper ions exert their effect independently of the conserved putative metal binding site, but through oxidation of the two cysteine residues in the formation of disulfide-linked oligomeric intermediates. This infers a critical role for redox and metal homeostasis in the regulation of specific high-molecular weight assemblies of GAPR-1
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