Ginsentide-like Coffeetides Isolated from Coffee Waste Are Cell-Penetrating and Metal-Binding Microproteins.

Coffee processing generates a huge amount of waste that contains many natural products. Here, we report the discovery of a panel of novel cell-penetrating and metal ion-binding microproteins designated coffeetide cC1a-c and cL1-6 from the husk of two popular coffee plants, Coffea canephora and Coffea liberica, respectively. Combining sequence determination and a database search, we show that the prototypic coffeetide cC1a is a 37-residue, eight-cysteine microprotein with a hevein-like cysteine motif, but without a chitin-binding domain. NMR determination of cC1a reveals a compact structure that confers its resistance to heat and proteolytic degradation. Disulfide mapping together with chemical synthesis reveals that cC1a has a ginsentide-like, and not a hevein-like, disulfide connectivity. In addition, transcriptomic analysis showed that the 98-residue micrcoproten-like coffeetide precursor contains a three-domain arrangement, like ginsentide precursors. Molecular modeling, together with experimental validation, revealed a Mg2+ and Fe3+ binding pocket at the N-terminus formed by three glutamic acids. Importantly, cC1a is amphipathic with a continuous stretch of 19 apolar amino acids, which enables its cell penetration to target intracellular proteins, despite being highly negatively charged. Our findings suggest that coffee by-products could provide a source of ginsentide-like bioactive peptides that have the potential to target intracellular proteins.

Discovery of Hyperstable Noncanonical Plant-Derived Epidermal Growth Factor Receptor Agonist and Analogs.

Here, we report the discovery of the first plant-derived and noncanonical epidermal growth factor receptor (EGFR) agonist, the 36-residue bleogen pB1 from Pereskia bleo of the Cactaceae family. We show that bleogen pB1 is a low-affinity EGFR agonist using a suite of chemical, biochemical, cellular, and animal experiments which include incisor eruption and wound-healing mouse models. A focused positional scanning pB1 library of Ala- and d-amino acid scans yielded a high-affinity pB1 analog, [K29k]pB1, with a 60-fold-improved EGFR affinity and mitogenicity. We show that the potency of [K29k]pB1 and the epidermal growth factor (EGF) is comparable in a diabetic mouse wound-healing model. We also show that both bleogen pB1 and [K29k]pB1 are hyperstable, being >100-fold more stable than EGF against proteolytic degradation. Overall, our discovery of a noncanonical proteolytic-resistant EGFR agonist scaffold could open new avenues for developing wound healing and skin regeneration therapeutics and biomaterials.

Roseltide rT7 is a disulfide-rich, anionic, and cell-penetrating peptide that inhibits proteasomal degradation.

Disulfide-rich plant peptides with molecular masses of 2-6 kDa represent an expanding class of peptidyl-type natural products with diverse functions. They are structurally compact, hyperstable, and underexplored as cell-penetrating agents that inhibit intracellular functions. Here, we report the discovery of an anionic, 34-residue peptide, the disulfide-rich roseltide rT7 from Hibiscus sabdariffa (of the Malvaceae family) that penetrates cells and inhibits their proteasomal activities. Combined proteomics and NMR spectroscopy revealed that roseltide rT7 is a cystine-knotted, six-cysteine hevein-like cysteine-rich peptide. A pair-wise comparison indicated that roseltide rT7 is >100-fold more stable against protease degradation than its S-alkylated analog. Confocal microscopy studies and cell-based assays disclosed that after roseltide rT7 penetrates cells, it causes accumulation of ubiquitinated proteins, inhibits human 20S proteasomes, reduces tumor necrosis factor-induced IκBα degradation, and decreases expression levels of intercellular adhesion molecule-1. Structure-activity studies revealed that roseltide rT7 uses a canonical substrate-binding mechanism for proteasomal inhibition enabled by an IIML motif embedded in its proline-rich and exceptionally long intercysteine loop 4. Taken together, our results provide mechanistic insights into a novel disulfide-rich, anionic, and cell-penetrating peptide, representing a potential lead for further development as a proteasomal inhibitor in anti-cancer or anti-inflammatory therapies.

Plant-derived mitochondria-targeting cysteine-rich peptide modulates cellular bioenergetics.

Mitochondria are attractive therapeutic targets for developing agents to delay age-related frailty and diseases. However, few promising leads have been identified from natural products. Previously, we identified roseltide rT1, a hyperstable 27-residue cysteine-rich peptide from Hibiscus sabdariffa, as a knottin-type neutrophil elastase inhibitor. Here, we show that roseltide rT1 is also a cell-penetrating, mitochondria-targeting peptide that increases ATP production. Results from flow cytometry, live-cell imaging, pulldown assays, and genetically-modified cell lines supported that roseltide rT1 enters cells via glycosaminoglycan-dependent endocytosis, and enters the mitochondria through TOM20, a mitochondrial protein import receptor. We further showed that roseltide rT1 increases cellular ATP production via mitochondrial membrane hyperpolarization. Using biotinylated roseltide rT1 for target identification and proteomic analysis, we showed that human mitochondrial membrane ATP synthase subunit O is an intramitochondrial target. Collectively, these data support our discovery that roseltide rT1 is a first-in-class mitochondria-targeting, cysteine-rich peptide with potentials to be developed into tools to further our understanding of mitochrondria-related diseases.

Ginsentides: Cysteine and Glycine-rich Peptides from the Ginseng Family with Unusual Disulfide Connectivity.

Ginseng, a popular and valuable traditional medicine, has been used for centuries to maintain health and treat disease. Here we report the discovery and characterization of ginsentides, a novel family of cysteine and glycine-rich peptides derived from the three most widely-used ginseng species: Panax ginseng, Panax quinquefolius, and Panax notoginseng. Using proteomic and transcriptomic methods, we identified 14 ginsentides, TP1-TP14 which consist of 31-33 amino acids and whose expression profiles are species- and tissues-dependent. Ginsentides have an eight-cysteine motif typical of the eight-cysteine-hevein-like peptides (8C-HLP) commonly found in medicinal herbs, but lack a chitin-binding domain. Transcriptomic analysis showed that the three-domain biosynthetic precursors of ginsentides differ from known 8C-HLP precursors in architecture and the absence of a C-terminal protein-cargo domain. A database search revealed an additional 50 ginsentide-like precursors from both gymnosperms and angiosperms. Disulfide mapping and structure determination of the ginsentide TP1 revealed a novel disulfide connectivity that differs from the 8C-HLPs. The structure of ginsentide TP1 is highly compact, with the N- and C-termini topologically fixed by disulfide bonds to form a pseudocyclic structure that confers resistance to heat, proteolysis, and acid and serum-mediated degradation. Together, our results expand the chemical space of natural products found in ginseng and highlight the occurrence, distribution, disulfide connectivity, and precursor architectures of cysteine- and glycine-rich ginsentides as a class of novel non-chitin-binding, non-cargo-carrying 8C-HLPs.