LIR Motif-Containing Hyperdisulfide β-Ginkgotide is Cytoprotective, Adaptogenic, and Scaffold-Ready

Abstract

Grafting a bioactive peptide onto a disulfide-rich scaffold is a promising approach to improve its structure and metabolic stability. The ginkgo plant-derived β-ginkgotide β-gB1 is a highly unusual molecule: Small, hyperdisulfide, and found only in selected ancient plants. It also contains a conserved 16-amino-acid core with three interlocking disulfides, as well as a six-amino-acid inter-cysteine loop 2 suitable for grafting peptide epitopes. However, very little is known about this recently-discovered family of molecules. Here, we report the biophysical and functional characterizations of the β-ginkgotide β-gB1 from G. biloba. A circular dichroism spectroscopy analysis at 90 °C and proteolytic treatments of β-gB1 supported that it is hyperstable. Data mining revealed that the β-gB1 loop 2 contains the canonical LC3 interacting region (LIR) motif crucial for selective autophagy. Cell-based assays and pull-down experiments showed that β-gB1 is an adaptogen, able to maintain cellular homeostasis through induced autophagosomes formation and to protect cells by targeting intracellular proteins from stress-mediated damage against hypoxia and the hypoxia-reoxygenation of induced cell death. This is the first report of an LIR-containing peptide natural product. Together, our results suggest that the plant-derived β-ginkgotide is cytoprotective, capable of targeting intracellular proteins, and holds promise as a hyperdisulfide scaffold for engineering peptidyl therapeutics with enhanced structural and metabolic stability.

Identification and characterization of a wolfberry carboxypeptidase inhibitor from Lycium barbarum

“…The hyperstability of β-lybatide can be attributed to its compact cystine-knot structure, which is known to confer the high metabolic stability in CRPs, particularly against endopeptidases (Loo et al, 2016;Nguyen et al, 2014;Tam et al, 2018;Wong et al, 2016). These results are in agreement with our previous studies as well as those by others, in which CRPs of 2 to 5 kDa with three to five disulfides are known to be hyperstable and able to resist heat, acid and enzymatic degradation due to their compact and multiple disulfide-constrained structures (Dutta, Huang, To, & Tam, 2019;Tam, Wang, Wong, & Tan, 2015;Wong et al, 2017a;Wong, Tan, Xiao, & Tam, 2017b).…”

Discovery and characterization of hevein-like peptides in functional food and medicinal plants

“…Similarly, this result agrees with β-ginkgotide isolated from G. biloba, which contained an LIR-like motif (DEYGCI) and induced cytoprotective effects in both cardiomyocytes and neuronal cells [398]. Additionally, eT1 promotes cell migration, which is a hallmark of wound healing.…”

“…To examine the effects of eleutide eT1 in hypoxia stress, the rat cardio-myoblast H9c2 cells were employed and the procedure was followed according to Dutta et al [398]. The cells were cultured with or without eT1 and incubated in a normoxic and hypoxic environment for 48 h before evaluating the cell viability via MTT assay.…”

Plant-derived cell-penetrating microprotein α-astratide aM1 targets Akt signaling and alleviates insulin resistance

“…This example provides a precedent to develop plant-derived highly disulfided peptides for oral administration. Furthermore, we have recently reported that such cystine-dense peptides with MW 2–4 kDa appeared much more common than previously thought [ 12 , 14 , 15 , 18 , 19 ]. Noteworthy examples include roseltides [ 15 , 18 , 19 ], β-ginkgotides [ 14 ], and ginsentides [ 12 , 17 ], which exhibit diverse bioactivities with potential therapeutic applications.…”

“…Furthermore, we have recently reported that such cystine-dense peptides with MW 2–4 kDa appeared much more common than previously thought [ 12 , 14 , 15 , 18 , 19 ]. Noteworthy examples include roseltides [ 15 , 18 , 19 ], β-ginkgotides [ 14 ], and ginsentides [ 12 , 17 ], which exhibit diverse bioactivities with potential therapeutic applications. These cell-penetrating peptides offer certain advantages in targeting intracellular proteins and the potential for oral administration.…”