Arabidopsis thaliana phytocystatin 6 forms functional oligomer and amyloid fibril states
Cystatins not only encode a high functional variability because of their ability to inhibit different classes of proteases but also because of their propensity to form oligomers and amyloid fibrils. Phytocystatins, essential regulators of protease activity in plants, specifically inhibit papain-like cysteine proteases (PLCPs) and legumains through two distinct cystatin domains. Mammalian cystatins can form amyloid fibrils, however, the potential for amyloid fibril formation of phytocystatins remains unknown. In this study, we demonstrate that Arabidopsis thaliana phytocystatin 6 (AtCYT6) exists as a mixture of monomeric, dimeric, and oligomeric forms in solution. Non-covalent oligomerization was facilitated by the N-terminal cystatin domain, while covalent dimerization occurred through disulfide bond formation in the interdomain linker. The non-covalent dimeric form of AtCYT6 retained activity against its target proteases, papain, and legumain, albeit with reduced inhibitory potency. Additionally, we observed the formation of amyloid fibrils by AtCYT6 under acidic pH conditions and upon heating. The amyloidogenic potential could be attributed to AtCYT6s N-terminal domain (AtCYT6-NTD). Importantly, AtCYT6 amyloid fibrils harbored inhibitory activities against both papain and legumain. These findings shed light on the oligomerization and amyloidogenic behavior of AtCYT6, expanding our understanding of phytocystatin biology and its potential functional implications in plant protease regulation.