Broad-spectrum ginsentides are principal bioactives in unraveling the cure-all effects of ginseng.

Stress and illness connection is complex and involves multiple physiological systems. Panax ginsengs, reputed for their broad-spectrum "cure-all" effect, are widely prescribed to treat stress and related illnesses. However, the identity of ginseng's "cure-all" medicinal compounds that relieve stress remains unresolved. Here, we identify ginsentides as the principal bioactives that coordinate multiple systems to restore homeostasis in response to stress. Ginsentides are disulfide-rich, cell-penetrating and proteolytic-stable microproteins. Using affinity-enrichment mass spectrometry target identification together with in vitro, ex vivo and in vivo validations, we show that highly purified or synthetic ginsentides promote vasorelaxation by producing nitric oxide through endothelial cells via intracellular PI3K/Akt signaling pathway, alleviate α1-adrenergic receptor overactivity by reversing phenylephrine-induced constriction of aorta, decrease monocyte adhesion to endothelial cells via CD166/ESAM/CD40 and inhibit P2Y12 receptors to reduce platelet aggregation. Orally administered ginsentides were effective in animal models to reduce ADP-induced platelet aggregation, to prevent collagen and adrenaline-induced pulmonary thrombosis as well as anti-stress behavior of tail suspension and forced swimming tests in mice. Together, these results strongly suggest that ginsentides are the principal panacea compounds of ginsengs because of their ability to target multiple extra- and intra-cellular proteins to reverse stress-induced damages.

Ultrafast Biomimetic Oxidative Folding of Cysteine-rich Peptides and Microproteins in Organic Solvents.

Disulfides in peptides and proteins are essential for maintaining a properly folded structure. Their oxidative folding is invariably performed in an aqueous-buffered solution. However, this process is often slow and can lead to misfolded products. Here, we report a novel concept and strategy that is bio-inspired to mimic protein disulfide isomerase (PDI) by accelerating disulfide exchange rates many thousand-fold. The proposed strategy termed organic oxidative folding is performed under organic solvents to yield correctly folded cysteine-rich microproteins instantaneously without observable misfolded or dead-end products. Compared to conventional aqueous oxidative folding strategies, enormously large rate accelerations up to 113,200-fold were observed. The feasibility and generality of the organic oxidative folding strategy was successfully demonstrated on 15 cysteine-rich microproteins of different hydrophobicity, lengths (14 to 58 residues), and numbers of disulfides (2 to 5 disulfides), producing the native products in a second and in high yield.

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

Insulin-resistant diabetes is a common metabolic disease with serious complications. Treatments directly addressing the underlying molecular mechanisms involving insulin resistance would be desirable. Our laboratory recently identified a proteolytic-resistant cystine-dense microprotein from huáng qí (Astragalus membranaceus) called α-astratide aM1, which shares high sequence homology to leginsulins. Here we show that aM1 is a cell-penetrating insulin mimetic, enters cells by endocytosis, and activates the PI3K/Akt signaling pathway independent of the insulin receptor leading to translocation of glucose transporter GLUT4 to the cell surface to promote glucose uptake. We also showed that aM1 alters gene expression, suppresses lipid synthesis and uptake, and inhibits intracellular lipid accumulation in myotubes and adipocytes. By reducing intracellular lipid accumulation and preventing lipid-induced, PKCθ-mediated degradation of IRS1/2, aM1 restores glucose uptake to overcome insulin resistance. These findings highlight the potential of aM1 as a lead for developing orally bioavailable insulin mimetics to expand options for treating diabetes.

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.

Ginsentide TP1 Protects Hypoxia-Induced Dysfunction and ER Stress-Linked Apoptosis.

Hypoxia-induced vascular endothelial dysfunction (VED) is a significant contributor to several severe human diseases, including heart disease, stroke, dementia, and cancer. However, current treatment options for VED are limited due to the lack of understanding of the underlying disease mechanisms and therapeutic leads. We recently discovered a heat-stable microprotein in ginseng, called ginsentide TP1, that has been shown to reduce vascular dysfunction in cardiovascular disease models. In this study, we use a combination of functional assays and quantitative pulsed SILAC proteomics to identify new proteins synthesized in hypoxia and to show that ginsentide TP1 provides protection for human endothelial cells against hypoxia and ER stress. Consistent with the reported findings, we also found that hypoxia activates various pathways related to endothelium activation and monocyte adhesion, which in turn, impairs nitric oxide (NO) synthase activity, reduces the bioavailability of NO, and increases the production of reactive oxygen species that contribute to VED. Additionally, hypoxia triggers endoplasmic reticulum stress and initiates apoptotic signaling pathways associated with cardiovascular pathology. Treatment with ginsentide TP1 reduced surface adhesion molecule expression, prevented activation of the endothelium and leukocyte adhesion, restored protein hemostasis, and reduced ER stress to protect against hypoxia-induced cell death. Ginsentide TP1 also restored NO signaling and bioavailability, reduced oxidative stress, and protected endothelial cells from endothelium dysfunction. In conclusion, this study shows that the molecular pathogenesis of VED induced by hypoxia can be mitigated by treatment with ginsentide TP1, which could be one of the key bioactive compounds responsible for the "cure-all" effect of ginseng. This research may lead to the development of new therapies for cardiovascular disorders.

Hyperstable EGF-like bleogen derived from cactus accelerates corneal healing in rats.

Corneal scarring reduces corneal transparency, compromises vision, and is a major cause of vision loss worldwide. Epidermal growth factor (EGF), which is the prototypic member of the EGF receptor (EGFR) agonists, is present in tears to provide repair and regeneration. Recently, we discovered bleogen pB1 in the cactus plant Pereskia bleo and showed that it is a non-canonical and hyperstable EGFR agonist with EGF-like wound healing properties for diabetic rats. Here, we apply bleogen pB1 to accelerate corneal wound healing in rats. To assess the corneal healing effects of bleogen pB1, we induced an acute alkali burn to the right eye of male Wistar rats. After five consecutive ophthalmic applications, fluorescein staining and opacity scores of the bleogen pB1-treated, and the positive control EGF-treated groups improved significantly compared to the saline control group. Immunohistochemical analyses revealed that infiltrated CD68+ macrophages and the expression of the myofibroblast marker alpha smooth muscle actin (α-SMA) were significantly decreased in the bleogen pB1- and the EGF-treated groups. By employing a differential gene expression analysis of bleogen pB1- and EGF-treated keratinocytes through RNA-seq, we demonstrated that bleogen pB1 or EGF treatments can affect the expression of genes associated with inflammatory responses and extracellular matrix remodeling. Taken together, our results indicate that the plant-derived EGFR agonist bleogen pB1 can produce similar effects to those of EGF in accelerating corneal wound healing as well as in reducing persistent inflammation and myofibroblast accumulation in the cornea.

Hololectin Interdomain Linker Determines Asparaginyl Endopeptidase-Mediated Maturation of Antifungal Hevein-Like Peptides in Oats.

Heveins and hevein-containing (hev-) lectins play important roles in stress and pathogenic responses in plants but cause health concerns in humans. Hev-hololectins contain multiple modular hev-peptide domains and are abundantly present in cereals and pseudocereals. However, it is unclear why some cereal hev-hololectins are presented as different forms of proteolytically processed proteoforms. Here we show the precursor architectures of hev-hololectins lead to different processing mechanisms to give either hololectins or hevein-like peptides. We used mass spectrometry and datamining to screen hev-peptides from common cereals, and identified from the oat plant Avena sativa nine novel hevein-like peptides, avenatide aV1-aV9. Bioinformatic analysis revealed that asparaginyl endopeptidase (AEP) can be responsible for the maturation of the highly homologous avenatides from five oat hev-hololectin precursors, each containing four tandemly repeating, hev-like avenatide domains connected by AEP-susceptible linkers with 13-16 residues in length. Further analysis of cereal hev-hololectins showed that the linker lengths provide a distinguishing feature between their cleavable and non-cleavable precursors, with the cleavables having considerably longer linkers (>13 amino acids) than the non-cleavables (<6 amino acids). A detailed study of avenatide aV1 revealed that it contains eight cysteine residues which form a structurally compact, metabolic-resistant cystine-knotted framework with a well-defined chitin-binding site. Antimicrobial assays showed that avenatide aV1 is anti-fungal and inhibits the growth of phyto-pathogenic fungi. Together, our findings of cleavable and non-cleavable hololectins found in cereals expand our knowledge to their biosynthesis and provide insights for hololectin-related health concerns in human.

Anti-Fungal Hevein-like Peptides Biosynthesized from Quinoa Cleavable Hololectins.

Chitin-binding hevein-like peptides (CB-HLPs) belong to a family of cysteine-rich peptides that play important roles in plant stress and defense mechanisms. CB-HLPs are ribosomally synthesized peptides that are known to be bioprocessed from the following two types of three-domain CB-HLP precursor architectures: cargo-carrying and non-cargo-carrying. Here, we report the identification and characterization of chenotides biosynthesized from the third type of precursors, which are cleavable hololectins of the quinoa (Chenopodium quinoa) family. Chenotides are 6-Cys-CB-HLPs of 29-31 amino acids, which have a third type of precursor architecture that encompasses a canonical chitin-binding domain that is involved in chitin binding and anti-fungal activities. Microbroth dilution assays and microscopic analyses showed that chenotides are effective against phyto-pathogenic fungi in the micromolar range. Structure determination revealed that chenotides are cystine knotted and highly compact, which could confer resistance against heat and proteolytic degradation. Importantly, chenotides are connected by a novel 18-residue Gly/Ala-rich linker that is a target for bioprocessing by cathepsin-like endopeptidases. Taken together, our findings reveal that chenotides are a new family of CB-HLPs from quinoa that are synthesized as a single multi-modular unit and bioprocessed to yield individual mature CB-HLPs. Importantly, such precursors constitute a new family of cleavable hololectins. This unusual feature could increase the biosynthetic efficiency of anti-fungal CB-HLPs, to provide an evolutionary advantage for plant survival and reproduction.

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.

Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization.

Butelase-1, an asparaginyl endopeptidase or legumain, is the prototypical and fastest known Asn/Asp-specific peptide ligase. It is highly useful for engineering and macrocyclization of peptides and proteins. However, certain biochemical properties and applications of naturally occurring and recombinant butelase-1 remain unexplored. Here we report methods to increase the yield of natural and bacterial expressed recombinant butelase-1 and how they can be used to improve the stability and activity of two important industrial enzymes, lipase and phytase, by end-to-end circularization. First, the yield of natural butelase-1 was increased 3-fold to 15 mg kg-1 by determining its highest distribution which is found in young tissues, such as shoots. The yield of recombinantly-produced soluble butelase-1 was improved by promoting cytoplasmic disulfide folding, codon changes, and truncation of the N-terminal pro-domain. Natural and recombinant butelase-1 displayed similar ligase activity, physical stability, and salt tolerance. Furthermore, the processing and glycosylation sites of natural and recombinant butelase-1 were determined by proteomic analysis. Storage conditions for both forms of butelase-1, frozen or lyophilized, were also optimized. Cyclization of lipase and phytase mediated by either soluble or immobilized butelase-1 was highly efficient and simple, and resulted in increased thermal stability and enhanced enzymatic activity. Overall, improved production of butelase-1 can be exploited to improve the biocatalytic efficacy of lipase and phytase by end-to-end cyclization. In turn, ligase-improved enzymes could be a general and environmentally friendly strategy for producing more stable and efficient industrial enzymes.

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.

Bleogens: Cactus-Derived Anti-Candida Cysteine-Rich Peptides with Three Different Precursor Arrangements.

Cysteine-rich peptides (CRPs) play important host-defense roles in plants. However, information concerning CRPs in the Cactaceae (cactus) family is limited, with only a single cactus-derived CRP described to date. Here, we report the identification of 15 novel CRPs with three different precursor architectures, bleogens pB1-15 from Pereskia bleo of the Cactaceae family. By combining proteomic and transcriptomic methods, we showed that the prototype, bleogen pB1, contained 36 amino acid residues, a six-cysteine motif typical of the six-cysteine-hevein-like peptide (6C-HLP) family, and a type I two-domain precursor consisting of an endoplasmic reticulum (ER) and a mature domain. In contrast, the precursors of the other 14 bleogens contained a type II three-domain architecture with a propeptide domain inserted between the ER and the mature bleogen domain. Four of these 14 bleogens display a third type of architecture with a tandemly repeating bleogen domain. A search of the Onekp database revealed that <1% plant species possess three different precursor architectures for the biosynthesis of 6C-HLPs, including Lophophora williamsii, Pereskia aculeate, Portulaca cryptopetala, Portulaca oleracea, Portulaca suffruticosa, and Talinum sp. NMR analysis confirmed that bleogen pB1 has cystine-knot disulfide connectivity as well as a two-beta-sheet and a four-loop structural fold that is similar to other 6C-HLPs. Sequence analysis, structural studies, and in silico modeling revealed that bleogen pB1 has a cation-polar-cation motif, a signature heparin-binding motif that was confirmed by heparin affinity chromatography. Cell-based assays showed that bleogen pB1 is non-toxic to mammalian cells but functions as an anti-Candida peptide. Taken together, our findings provide insight into the occurrence, functions and precursor architectures of CRPs in the cactus family.

Identification and Characterization of Roseltide, a Knottin-type Neutrophil Elastase Inhibitor Derived from Hibiscus sabdariffa.

Plant knottins are of therapeutic interest due to their high metabolic stability and inhibitory activity against proteinases involved in human diseases. The only knottin-type proteinase inhibitor against porcine pancreatic elastase was first identified from the squash family in 1989. Here, we report the identification and characterization of a knottin-type human neutrophil elastase inhibitor from Hibiscus sabdariffa of the Malvaceae family. Combining proteomic and transcriptomic methods, we identified a panel of novel cysteine-rich peptides, roseltides (rT1-rT8), which range from 27 to 39 residues with six conserved cysteine residues. The 27-residue roseltide rT1 contains a cysteine spacing and amino acid sequence that is different from the squash knottin-type elastase inhibitor. NMR analysis demonstrated that roseltide rT1 adopts a cystine-knot fold. Transcriptome analyses suggested that roseltides are bioprocessed by asparagine endopeptidases from a three-domain precursor. The cystine-knot structure of roseltide rT1 confers its high resistance against degradation by endopeptidases, 0.2 N HCl, and human serum. Roseltide rT1 was shown to inhibit human neutrophil elastase using enzymatic and pull-down assays. Additionally, roseltide rT1 ameliorates neutrophil elastase-stimulated cAMP accumulation in vitro. Taken together, our findings demonstrate that roseltide rT1 is a novel knottin-type neutrophil elastase inhibitor with therapeutic potential for neutrophil elastase associated diseases.

Butelase 1: A Versatile Ligase for Peptide and Protein Macrocyclization.

Macrocyclization is a valuable tool for drug design and protein engineering. Although various methods have been developed to prepare macrocycles, a general and efficient strategy is needed. Here we report a highly efficient method using butelase 1 to macrocyclize peptides and proteins ranging in sizes from 26 to >200 residues. We achieved cyclizations that are 20,000 times faster than sortase A, the most widely used ligase for protein cyclization. The reactions completed within minutes with up to 95% yields.