Astratides: Insulin-Modulating, Insecticidal, and Antifungal Cysteine

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Astratides: Insulin-Modulating, Insecticidal, and Antifungal Cysteine-Rich Peptides from Astragalus membranaceus Jiayi Huang, Ka H. Wong, Stephanie V. Tay, Aida Serra, Siu Kuan Sze, and James P. Tam* School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551

J. Nat. Prod. Downloaded from pubs.acs.org by MACQUARIE UNIV on 02/13/19. For personal use only.

S Supporting Information *

ABSTRACT: Astragalus membranaceus root, Huang Qi in Chinese, is a popular medicinal herb traditionally used to regulate blood glucose. Herein, the identification and characterization of two families of cysteine-rich peptides (CRPs), designated α- and β-astratides, from A. membranaceus roots are reported. Proteomic analysis showed that α-astratide aM1 and β-astratide bM1 belong to two distinct CRP families. The six-cysteine-containing and proline-rich α-astratide aM1 displayed high sequence identity to Pea Albumin 1 Subunit b (PA1b), while the eight-cysteine-containing β-astratide bM1 showed sequence similarity to plant defensins. An antifungal assay revealed that bM1 possessed potent antifungal activity. In contrast, aM1 showed a cytotoxic effect against insect Sf9 cells. More importantly, aM1 decreased insulin secretion in mouse pancreatic β cells, suggesting it could interfere in glucose homeostasis, which accounts for the adaptogenic property of A. membranaceus. Phylogenetic clustering analysis suggested that the proline-rich aM1 is a putative prolyl oligopeptidase inhibitor and belongs to a novel subfamily of PA1b-like peptides, while bM1 belongs to a new subfamily of plant defensins. Together, the study reveals that astratides are multifunctional CRPs in plants, which expand the existing library of PA1b-like peptides and plant defensins and further our understanding of their roles in host-defense system and leads as peptidyl therapeutics.

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ATPase (V-ATPase) in theinsect midgut.11 In plants, it has been found that the addition of PA1b homologue from soybean enhanced cell proliferation, suggesting that the PA1blike peptide can act as a peptidic plant hormone and function in signal transduction system.12 In mammals, PA1b and its counterpart leginsulin, have been shown to interfere with glucose metabolism and insulin secretion.10,13−15 Defensins, a well-studied CRP family, are widely expressed in plants, animals, and fungi for host defense.16 The term “plant defensins” was introduced in 1995 to describe proteins formerly known as γ-thionins, when the antifungal peptides discovered from radish seeds (Rs-AFP1 and Rs-AFP2) were found to be more similar to insect and mammalian defensins than to plant thionins.17 Plant defensins are generally cationic peptides with 45−54 amino acids (aa) and display a wide range of biological functions including antifungal, antibacterial, and trypsin inhibitory activities.18 Astragalus membranaceus, a perennial plant belonging to the Fabaceae family, is native to China and northern Asia. Its root, commonly known as Huang Qi in Chinese, is one of the popular health-promoting herbs in China. Documented as early as 300 A.D. in the Shennong’s Classic of Materia Medica, A. membranaceus roots are used to treat diabetes, restore

edicinal plants play an important role in traditional medicines in treating and managing human diseases. Currently, approximately 45% of all clinically approved drugs are plant-derived, small molecules with molecular weight 97% purity (Figure S1, Supporting Information). The extracted yield of aM1 and bM1 were approximately 0.5 and 2 mg per kg of dried plant material, respectively. The number of cysteines present in α-astratide aM1 was determined as six, based on the mass shift of 348 Da after SB

DOI: 10.1021/acs.jnatprod.8b00521 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. Comparison of the Primary Sequences and Physiochemical Properties of α-Astratide aM1 and Other Reported PA1blike Peptides

1

Mass (Da) = reported mass 2Charge: the total charge is the sum of positive (lysine, arginine, and histidine residues) and negative (glutamate and aspartate residues) charges present in the sequence 3Approach: the primary sequence was obtained by transcriptomic (T) and/or proteomic (P) approach. The Cys are shown with the letter “C” and indicated by the brackets. Assignment of isobaric amino acids such as Leu/Ile were confirmed by transcriptomic method.

With five prolines accounting for 14% of the sequence, aM1 are considered proline-rich. Other families of CRPs are also proline-rich, including cystine knot α-amylase inhibitors and αginkgotides.28,29 In addition, it is reported that cyclotide kalata B1 and psysol 2 displayed an inhibitory effect against human prolyl oligopeptidase (POP), a serine-type protease related to psychiatric and neurodegenerative diseases.30 Inhibition of POP may be useful for treating cognitive deficits associated with central nerve system disorders and neurodegenerative diseases.31 Interestingly, kalata B1 and psysol 2, together with other active cyclotides that have been identified in the POP inhibitory fractions of plant extracts, contain three prolines in loops 3, 5, and 6.30 A similar pattern can be found in aM1, as it contains three prolines in loop 2 and two prolines in loops 5 and 6, which suggests that aM1 may act as a potential POP inhibitor. Unlike α-astratide aM1, β-astratide bM1 displays a 50% sequence similarity to plant defensins (Table S1, Supporting Information). For example, β-astratide bM1 showed 43% −55% sequence similarity with the plant defensins RS-AFP1 from Raphanus sativus, NaD1 from Nicotiana alata, VrD1 from Vigna radiate, Lc-def from Lens culinaris, NsD7 from Nicotiana suaveolens, and AhPDF1 from Arabidopsis halleri. Both bM1 and other reported plant defensins are positively charged, containing 45 to 51 amino acids and eight cysteines. Based on the sequence similarity between bM1 and plant defensins AhPDF1,32 it is highly likely that they share a similar disulfide connectivity as Cys I−VIII, Cys II−V, Cys III−VI, and Cys IV−VII (Table S1, Supporting Information) and a βαβ structural fold with cis oriented disulfides from the C-terminal β-strand (Figure S6, Supporting Information).33 Biosynthesis of Astratides. A transcriptomic search revealed the full-length precursor sequence am1, which encodes for α-astratide aM1. Both OneKP and tBLASTn search using aM1 as a query were performed to determine the distribution of the other 19 PA1b-like peptides from the plant

families Fabaceae, Amaranthaceae, and Convolvulaceae (Figure 2A). Similar to PA1b, aM1 and other PA1b-like peptides comprise a five-domain architecture, which include an endoplasmic reticulum signal peptide, a mature PA1b-like peptide, a hinge domain, a PA1a-like domain, and a C-terminal tail. The presence of a signal peptide suggests that astratide is a secretory peptide.34 The N-terminal cleavage site was a highly conserved Ala, whereas the C-terminal processing of the mature peptides was a highly conserved Gly residue. The length of all PA1b-like peptides is highly conserved, with 37 amino acid residues. In contrast, the lengths of the C-terminal domains varied from 8 to 28 amino acid residues, with the shortest C-tail found in aM1. A transcriptomic search revealed that the precursor sequence of β-astratide bM1 comprised a two-domain architecture. A comparison of bM1 with other plant defensins showed that they shared a similar two-domain precursor arrangement, comprising a signal peptide and a mature peptide (Figure 2C). The preprotein precursor PA1 is known to be bioprocessed to yield two peptides, PA1a (C-terminal fragment) and PA1b (N-terminal fragment),7 and in theory, the two are equimolar. However, PA1a was not detected by proteomic methods. As such, PA1a has been speculated to assist in the correct folding of PA1b.35 Similar to PA1b, the release of the mature astratide aM1 involves the cleavage of a signal peptide by signal peptidase and the removal of the hinge domain and C-terminal tail by endopeptidase. Subsequently, the mature aM1 was transported to the Golgi apparatus for post-translational modifications and packed into vesicles for secretion.28 Cliotides extracted from Clitoria ternatea, belonging to the cyclotide family, display chimera characteristics in their precursors. Half of their precursor comes from cyclotide and the other from albumin-1, with the cyclotide domain displacing the PA1b mature domain. A major factor in the biosynthesis to form a cyclic peptide is due to the lack of C-terminal Asn, which together with asparaginyl endopeptidase can form a C



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Figure 2. Gene alignment and biosynthesis pathway of Astratides. (A) Precursor sequences alignment of α-astratide aM1 and PA1b-like peptides. The precursors of PA1b-like peptides are divided into five domains, including signal peptide, mature PA1b-like peptide domain, a hinge domain, a PA1a-like peptide domain, and a C-terminal tail. Signal peptide was removed from the full precursor sequences by SPase. The hinge domain and Cterminal tail is then cleaved by endopeptidase to release the mature peptides. (B) Sequence analysis of all PA1b-like peptides. The conservation are indicated by the overall height of the stack, whereas the frequency of the amino acids on positions were revealed by the height of symbols within the stack. Color yellow, red, blue, and green are used for indicating cysteine, negatively charged (D and E), positively charged (H, K, R), and aromatic acid residues (F, W, Y), respectively. (C) Gene alignment and biosynthesis pathway of β-astratide bM1 and other defensin-like peptides. The precursors of bM1 and other defensin-like peptides are divided into two domains, including signal peptide, mature peptide. Signal peptide was removed from the full precursor sequences by SPase. (D) Sequence analysis of defensin-like peptides. Accession numbers of all the sequences are as follows: am1, ac1, cp1, cp2, cp3, and bm1 (OneKp: HJMP-2011398, XSSD-2076575, AHRN-2020593, AHRN-2005129, AHRN-2005130, and HJMP-2064542), pa1b, psaa1b012, psaa1b014, psaa1b015, leginsulin1, leginsulin2, pv1, pc1, pa1, vu1, cac1, vf1, lc1, mt1, gs1, ctc1, ctc2, ctc3, mt2, gu1, ms1,ps1, vf2, vrd1, lc-def, and Gamma-thionin1 (Genbank: CAE00468.1, CAB82859.1, AAA33638.1, CAE00468.1, CAE00463.1, CAA11040.1, GW907308.1, CA909171.1, HO790552.1, GH619272.1, GW353628.1, FL506347.1, AHG94969.1, CAE00461.1, CAA09880.2, AEK26402.1, JF931989.1, JF931990.1, BG452703.1, FS281873.1, CO516397.1, CD859908.1, FL506862.1, AAR08912.1, and BAB19054.1) * Represents the stop codon.

domain architecture and a minor group with a three-domain architecture.37 The three-domain plant defensin precursor has an extra acidic C-terminal tail, which was reported to be associated with the vacuolar sorting mechanism.38 Evolution and Origin of Astratides. Transcriptomic analysis revealed most PA1b-like peptides were distributed in the Fabaceae family, with only cP1, cP2, and cP3 in the family Convolvulaceae and aC1 in Amaranthaceae. Genetic divergence within the plant phyla results from mutations in the mature peptide, which leads to functional diversification.39 A phylogenetic tree was constructed using the mature peptide sequences of astratide aM1, cliotides, and other PA1b-like peptides to study their evolutionary relationship (Figure 4A). In the Fabaceae family, the phylogenetic tree revealed the relationship of two major clusters of CRPs, cliotides and PA1blike peptides. It is hypothesized that the occurrence of chimeric structures of cliotides in the Fabaceae is the result of horizontal gene transfer between plant nuclear genomes or convergent evolution from PA1b-like peptides to cyclotides.36 To narrow the range, PA1b-like peptides in the Fabaceae family were separated into tribes, on the basis of a limited range of floral

reactive thioester bond that leads head-to-tail ligation to afford the cyclized structure.36 A distinguishing feature of the six-cysteine-containing αastratide aM1 is its five-domain architecture compared to other 6C-CRPs, which generally have two to four domains (Figure 3). α-Astratide aM1 has a relatively long full precursor sequence (128 aa) compared with cystine knot α-amylase inhibitors (93 aa), 6C-hevein-like peptides (84 aa), carboxypeptidase inhibitors (90 aa), jasmintides (100 aa), and βginkgotides (90 aa). Furthermore, aM1 is similar to 6C-heveinlike peptides and thionins, the pro-peptide domain was absent in their precursors. However, unlike thionins, which have a long full precursor sequence (132 aa), aM1 have a short C-tail (8 aa). Understanding the astratide precursor arrangement could provide insight into their biosynthesis and will be beneficial in developing a bacterial expression system and transgenic crops in the future. In contrast, bM1 adopts a two-domain defensin-like precursor, which comprises a signal peptide and a mature peptide. Two groups of precursors have been identified in plant defensins, containing a majority group with a twoD



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Figure 3. Summary of precursor sequences of α-astratide aM1 and other known CRPs with six cysteine residues. The number on top of each domain represents the average number of amino acids.

characteristics, with a greater emphasis on petal morphology and stamen arrangement.40 The aM1 diverged from leginsulin, PA1b and its isoforms from P. sativum, suggesting that there might be a close evolutionary relationship between aM1, PA1b, and leginsulin. Furthermore, in the Fabaceae family, most of the PA1b-like peptides belong to either the tribe of pea (Fabeae) or the tribe of soybean (Phaseoleae). aM1, which belongs to the tribe galegeae, suggests the diversity of PA1blike peptides may represent a novel direction for discovering PA1b-like peptides.41 Similarly, a phylogenetic tree was generated to show the relationship between bM1 and other plant defensins using a neighbor-joining clustering algorithm (Figure 4B). Based on the number of cysteines, plant defensins are divided into 8Cand 10C-plant defensins. Recently, Anderson et al. further classified defensins on the basis of their cysteine motifs. The commonly distributed cysteine motif in 8C-plant defensins is C-X10-C-X5-C-X3-C-X [9−10]-C-X [6−8]-C-X-C-X3-C.33 Compared to other reported two-domain plant defensins, a distinct characteristic of bM1 is that it contains a unique CXCXC motif at the C-terminus. Previously, fabatin-2 from Vicia faba of the Fabaceae family was the only reported plant defensin with this unique motif.42 In addition, a transcriptomic search showed that it is a unique motif that can only be found in plants of the Fabaceae family. In the phylogenetic tree, two major clusters, designated Cluster 1 and 2, were observed. Cluster 1 represents bM1 and the other plant defensins that contain the CXCXC motif at the C-terminal from Fabaceae family, whereas Cluster 2 refers to plant defensins from other plant families. These clues suggested that bM1 might belong to a new subfamily of plant defensins. Insecticidal Activity of α-Astratide aM1 on Sf9 Cells. To determine the insecticidal effect of aM1, a cytotoxicity assay on insect Sf9 cells was performed, in which the cells were incubated with different concentrations of aM1. The mortality rate was determined by an MTT assay. After a 24-h incubation with aM1, the mortality rate of the Sf9 cells increased in a

dose-dependent manner with an average lethal dose (LD50) of 5.9 μM (Supplementary Figure S7). The 0.1% DMSO was incubated with Sf9 cells as a negative control, with no significant cytotoxic effect, and 10% Triton-X 100 was used as a positive control, causing more than 99% cell death after 24 h treatment. More importantly, aM1 is nontoxic to CHO-K1 cells at concentrations up to 200 μM (Figure S7, Supporting Information), suggesting that aM1 is insect-cell-specific. Bright-field microscopy was employed to monitor the cytotoxic effect of aM1 on Sf9 cells. Sf9 cells were incubated with 5 μM of aM1 at 27 °C for 15 h, and the images were recorded at different time points. At 0 h, Sf9 cells showed a normal, spherical shape. At later time points, the cell membrane started to lose its integrity, leading to cell death (Figure S8A, Supporting Information). In addition, the membranolytic effect of aM1 on Sf9 cells was shown by confocal microscopy. The cell membrane of Sf9 cells was labeled with PKH26, while the nucleic acid was labeled with Hoechst. The labeled Sf9 cells were incubated with astratide aM1, which was labeled with Alexa488 fluorescent dye for 24 h at 27 °C. The results showed that incubation with aM1 destroyed the cell membrane of Sf9 cells and caused their death (Figure S8C, Supporting Information). In contrast, control Sf9 cells showed no morphological changes during the incubation (Figures S8B, S8D, Supporting Information). Until now, only a few cystine-knot peptides have been shown to possess insecticidal activity. They include cyclotides,43 the amaranthus α-amylase inhibitor (AAI) from A. hypocondriacus, and PA1b.9,44 However, the mechanisms are different. For cyclotides, it has been postulated that activity is related to membrane binding,45 while AAI relies on specific inhibition of the α-amylase of insect larvae. More diversely, PA1b has been shown to have insecticidal activity through a membrane protein-based receptor. It was reported that the insect Sf9 cells were found to contain a high-affinity binding site to PA1b, due to the presence of four hydrophobic residues, E



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Figure 4. Phylogenetic tree analysis. The precursor sequences were aligned by MUSCLE. Neighbor-joining clustering algorithm was used to construct a phylogenetic tree with bootstrap test of 1000 replicates in MEGA6. The phylogenetic tree was displayed using iTOL v3. (A) Phylogenetic tree of aM1, PA1b-like peptides, and cliotides. All the PA1b-like peptides are clustered separately due to the (a) different plant families, (b) different CRP families, and (c) different tribes under the same family. (B) Phylogenetic tree of bM1 and other defensin-like peptides. Neighbor-joining clustering algorithm was employed to analyze the aligned mature domain of defensins. All the defensins are clustered separately due to the (a) different plant families and (b) different C-terminal motif. Uniprot accession number: cT1 (G1CWH0), cT2 (G1CWH1), cT3 (C0HKG1), LCR74 (Q9FFP8), LCR71 (P82781), LCR66 (Q9C947), J1-1 (Q43413), J1-2 (O65740), LCR72 (Q9ZUL8), VrD1 (Q6T418), LCR69 (Q39182), LCR68 (Q9ZUL7), LCR70 (Q41914), PPT (Q40901), LCR75 (P82784), LCR76 (P82785), MtDef4 (G7L736), SD2 (P82659), NaD1 (Q8GTM0), NP-THN1 (O24115), Lc-def (B3F051), VrD2 (Q8W434), MsDef1 (Q9FPM3), LCR78 (P82787), RS-AFP1 (P69241), Rs-Apf2 (P30230), At-AFP1 (P30224), AhPDF1 (Q29SA6), AFP3 (Q39313), RS-AFP4 (O24331), LCR77 (Q9FI23), and Rs-AFP At2g26010 (O80995). GenBank accession number: pS1 (CAB82859.1), pS2 (AAA33638.1), PsDef 1 (EF455616.1), and Gamma-thionin 1(BAB19054.1).

structure homology. The predicted structure of α-astratide aM1 shows a similar disulfide arrangement and secondary structure (Figure S5, Supporting Information) to PA1b, with a TM-align score of 0.61793 (Table S2, Supporting Informa-

Phe10, Ile23, Val25, and Leu27, and the charged amino acid, Arg21.35 The insecticidal activity of aM1 could be explained by a mechanism similar to PA1b, due to their sequence and F



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tion). The structure of aM1 comprises two antiparallel β sheets, adopting the cystine-knot linkage but lacks the α-helical turn compared to PA1b. In PA1b, there is clearly a hydrophobic face formed by the residues of the hydrophobic loop 5: Val25, Leu27, Val28, and Ile29, together with the opposite residue Phe10. The hydrophilic face, situated at the other pole of the molecule, is contributed by Ser2, Asn4, Thr17, Ser18, Asn34, and Ser36.10 This similar amphipathic structure found in aM1 compared with PA1b (Figure S5B, Supporting Information) may favor its interaction with the membrane. For example, the insecticidal activity is based on the interaction of PA1b with a membrane-based receptor in insect cells.9 Effect of α-Astratide aM1 on Insulin Secretion. Different concentrations of aM1 were coincubated with mouse pancreatic β-TC cells for 24 h. GW9508 is a compound that dose-dependently potentiated glucose-stimulated insulin secretion in pancreatic β cells,46 and thus, 10 μM of GW9508 was used as a positive control with mouse pancreatic β-TC cells for 24 h. The insulin released by pancreatic β cells was measured by ELISA after incubation. Figure 5 shows that aM1

resulting in a reduced ability to respond to glucose stimuli and subsequent degeneration.47 By reducing the insulin secretion, the beta cell function can be restored and the insulin secretory response to beta cell stimulation would be increased. This proposed mechanism was supported by an effective antidiabetic drug pioglitazone, which reduces insulin secretion. The reduction of beta cell metabolism caused by decreased insulin secretion helps to prevent loss of beta cells due to exhaustion or to reduce insulin resistance.48 As a result, the decreased insulin secretion exerted by aM1 may follow a similar mechanism as pioglitazone. However, details of the putative mechanism of aM1 in regulating blood glucose need further studies. Thermal, Acidic, Enzymatic, and Serum Stability of Astratides. Several tests were performed on astratides aM1 and bM1 to demonstrate their stability against thermal, acid, proteolytic, and serum-mediated degradation. Results showed that 82% of aM1 remained intact after incubation in boiling water for 2 h (Figure S9A, Supporting Information). It also displayed a high tolerance to acidic conditions for 2 h, with 92% of the peptide remaining (Figure S9B, Supporting Information). Furthermore, aM1 was resistant to digestion with the endopeptidase pepsin and exopeptidase aminopeptidase I for up to 6 h, with >88% of the peptide remaining (Figures S9C, S9D, Supporting Information). In addition, more than 90% of aM1 remain intact after incubating with human serum for 36 h (Figure S9E, Supporting Information). β-Astratide bM1 was similarly stable against thermal, enzymatic, acidic and human serum degradation (Figure S10, Supporting Information). These stability features suggest that both α- and β-astratides are comparable to hevein-like and cystine-knot peptides such as α-amylase inhibitors.28,29,49 The ability of astratides to withstand harsh treatment suggests that they could be relevant as active compounds in A. membranaceus, which has the potential to serve as an orally active therapeutic. Antifungal Activity of β-Astratide bM1. A disc diffusion assay was employed to test the inhibitory effect of bM1 on four phytopathogenic fungal strains. The fungi strains were allowed to grow for 24−72 h at 30 °C until a radical colony was formed. Different concentrations of bM1 (1, 5, and 10 mg/ mL) were placed on discs at the end of growing mycelia. The antifungal activity of bM1 was indicated by the formation of crescent-shape inhibition zones (Figure 6A). A dose−response curve was generated, from which the calculated IC50 value was in the range of 2.7−130.3 μg/mL after 24 h incubation at 30 °C, depending on the fungal strain tested (Figure 6B). Brightfield microscopy revealed the morphological changes of fungal spores with bM1. It also resulted in shorter and highly brunched hyphae, vacuolar granulation, and swollen hyphal tips, as well as restarted budding hyphae compared to the untreated fungi (Figure 6C). Collectively, these results suggest that bM1 inhibits fungal growth in a dose-dependent manner. Plant defensins such as AlfAFP and Ms-Def1 from Medicago sativa seeds, Rs-AFPs purified from Raphanus sativus radish seeds, and Ct-AMP1 from Clitoria ternatea are reported to have a broad antifungal spectrum with an IC50 = 0.3−100 μg/mL.18 At present, there are three proposed mechanisms by which plant defensins may inhibit fungal growth. Proposed mechanisms include receptor-mediated internalization, membrane translocation, and membrane permeabilization.50 It has been demonstrated that defensins can specifically interact with host membrane compounds. Thus, upon interaction with their

Figure 5. Effect of aM1 on insulin secretion level in β-TC cells. 0.01, 0.1, 1, 10, 100 μM of aM1, and 10 μM of GW9508 were incubated with β-TC cells for 24 h. 1, 10, and 100 μM of aM1 showed significant reduction in the insulin secretion level in mouse pancreatic β-TC cells as compared with 0.1% DMSO. 0.01, 0.1 μM of aM1, and 10 μM of GW9508 increased the insulin secretion level as compared with control. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

has no significant effect on insulin secretion up to 1 μM. However, at concentrations >1 μM, aM1 decreased insulin secretion of pancreatic β cells in a dose-dependent manner. The insulin concentration decreased from 1225.20 μIU/mL to 811.14 μIU/mL with increasing concentrations of aM1 (1− 100 μM). Previously, it was reported that >5 μg/g of PA1b increased blood glucose in healthy C57BL/6 and type II diabetic mice, while no effect was observed at the 2.5 μg/g concentration.13 In addition, PA1b was shown to have a hyperglycemic effect in mice upon binding to VDAC-1, an ion channel protein on the pancreatic β cell membrane.14 Therefore, it can be speculated that aM1 could bind and subsequently block the ion channel function to reduce the cation influx, leading to reduced insulin exocytosis. This effect confirmed that, despite its role in the insect world, aM1 can interfere with mammalian physiology in regulating glucose homeostasis. The balance between the release and action of insulin is important for maintaining glucose homeostasis, while dysfunctional insulin secretion is a pivotal component of type 2 diabetes. It has been proposed that relative insulin hypersecretion would cause the beta cells to become exhausted, G



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Figure 6. Inhibitory activity of bM1 toward four phytopathogenic fungal strains. (A) Formation of arc-shaped inhibition zone of fungal mycelia indicates the susceptibility of F. oxysporum, R. solani, C. lunata, and A. alternate to different concentration of bM1. (B) Dose−response curves generated from the microbroth dilution assay were used to calculate the IC50 values. (C) Bright-filed microscopy of the mycelium growth of A. alternate with different concentrations of bM1. Fungi treated with bM1 showed distinct morphological changes.

biosynthesis pathway, and evolutionary relationship and suggesting the role of CRPs as an active compound in plants.

target, plant defensins either stay at the cell surface and induce cell death or are internalized by the fungal wall and interact with intracellular targets.51 The mechanism of action of βastratide bM1 remains to be elucidated and further experiments to clarify this issue are warranted. This study reported the discovery of two CRP families, the α-astratides and β-astratides, from the roots of A. membranaceus. The α-astratide aM1 was characterized and shown to be a 6C-CRP and PA1b-like peptide, while β-astratide bM1 is an 8C-CRP and plant defensin. Both astratides showed high stability against heat, acid, enzyme, and human serum degradation. Transcriptomic data showed that the aM1 precursor sequence comprised a signal peptide domain, a mature peptide domain, a hinge domain, a PA1a-like domain, and a C-terminal domain, which differentiated it from other 6C-CRPs with a three- to four-domain architecture. In addition, neighbor-joining clustering analysis showed that astratide aM1 is a novel PA1b-like peptide from a new tribe of the Fabaceae family. Functional assays revealed that aM1 was cytotoxic to Sf9 cells and reduced insulin secretion in normal mouse pancreatic β cells. In contrast, β-astratides displayed similar cysteine spacing as a defensin and a typical two-domain defensin-like precursor. Phylogenetic analysis revealed that bM1 belongs to a new subfamily of plant defensins and has strong antifungal activity against four phytopathogenic fungi strains. Overall, our discovery expands the existing library of PA1b-like peptides and plant defensins, providing insight into their sequence diversity, structure,

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EXPERIMENTAL METHODS

General Experimental Procedures. High-performance liquid chromatography (HPLC) and ultraperformance liquid chromatography (UPLC) were performed on Shimadzu systems (Shimadzu, CA, U.S.A.). Preparative, semipreparative, and analytical reversed-phase (RP) HPLC were performed on C18 columns (Phenomenex, CA, U.S.A.) (particle size: 5 μm; pore size: 300 Å) with dimensions of 250 × 22 mm, 250 × 10 mm, and 250 × 4.6 mm, respectively. An ABI 4800 matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF/TOF) system (Applied Biosystems, MA, U.S.A.) was used for mass spectrometry (MS) analysis of crude extracts and HPLC fractions. Absorbance was acquired using an Infinite 200 PRO microplate reader (Tecan Group Ltd., Maennedorf, Switzerland). Screening of Astragalus membranaceus. Dried A. membranaceus roots were purchased from a local herb distributor (Hung Soon Medical Trading Ltd., Singapore). The roots were authenticated by Dr. Yan Zhao, registered TCM physician from Nanyang Technological University, Singapore and a voucher specimen was stored at the Nanyang Herbarium, Nanyang Technological University, Singapore with accession number AMR-20171010. Dried roots (2 g) of A. membranaceus were homogenized in 2 mL of water and centrifuged at 10 000g for 10 min. The supernatant was subjected to a C18 solid-phase extraction (SPE) column. The SPE column was washed with 20% CH3CN and eluted with 80% CH3CN. The eluted fraction was subjected to MALDI-TOF MS to scan the mass range from 2−6 kDa. H



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Large-Scale Extraction. Dried A. membranaceus roots (1 kg) were ground into a powder and extracted twice with water in a ratio of 1:5 kg/L. After centrifugation (10 000g, 20 min, 4 °C), the supernatant was filtered through 1-μm and 0.45-μm pore filters, followed by the addition of 4536.05 g of (NH4)2SO4 and mixing for 1 h. The mixture was centrifuged, and the supernatant was decanted. The pellet was dissolved in 500 μL of 10% CH3CN and purified using a C18 flash column (Grace Davison, MD, U.S.A.) by washing with water and eluting with 20, 40, 60, and 80% ethanol. The eluted fraction was subjected to MALDI-TOF MS. Fractions that showed a positive signal in the target range from 2−6 kDa were combined and lyophilized to be purified by multiple rounds of preparative and analytical RP-HPLC. In preparative RP-HPLC, a linear gradient at a flow rate of 5 mL/min was employed using buffer A (0.1% TFA in milli-Q water) and buffer B (0.1% TFA in 100% CH3CN) as follows: 5−5% B from 0.01−20 min, 5−40% B from 20−75 min, 40−60% B from 75−120 min, and 60−100% B from 120−130 min. For analytical RP-HPLC, a linear gradient at a flow rate of 0.4 mL/min was employed using buffer A and buffer B as follows: 5−5% B from 0.01− 30 min, 5−35% B from 30−65 min, 35−50% B from 65−130 min, and 50−100% B from 130−140 min. Sequence Determination of Astratides. Lyophilized peptide (20 μg) was dissolved in 50 mM NH4HCO3 buffer (pH 8.0) with 50 mM dithiothreitol and subsequently incubated at 37 °C for 1 h. The sample was alkylated with 100 mM iodoacetamide at room temperature for 1 h. The S-reduced and S-alkylated sample was desalted using a C18 Zip-tip and lyophilized. The sample was redissolved in 0.1% formic acid before LC-MS/MS analysis using a Dionex UltiMate 3000 UHPLC system coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Inc., Bremen, Germany) with a nanoelectrospray ion source (BrukerMichrom Inc., Auburn, U.S.A.). The mobile phase was water with 0.1% formic acid as eluent A and 90% CH3CN as eluent B, with a flow rate of 0.3 μL/min. Peptide separations were performed with a 60 min gradient as follows: 3% of buffer B for 1 min, 3−35% of buffer B over 47 min, 35−50% buffer B over 4 min, 50−80% buffer B over 6 s, and 80% buffer B for 1 min. Later, the system was reverted to the initial conditions over 6 s and was maintained for 6.5 min. Data were acquired using alternating Full FT-MS (350−3000 m/z, resolution 60.000, with 1 μscan per spectrum) and an FT-MS/MS scan applying 65, 80, and 95 ms ETD activation times (110−2000 m/ z, resolution 30.000, with 2 μscan averaged per MS/MS spectrum). The isolation window for precursor selection was varied in different runs, and the three most intense ions with charge >2+ were isolated with a 2 Da mass isolation window and fragmented. The lower threshold for targeting precursor ions in the MS scans was 5000 counts. A source voltage of 1.5 kV with a 250 °C capillary temperature was set, and the automatic gain control for Full MS and MS/MS was set to 1 × 106. Data were processed using PEAKS studio version 7.5 (Bioinformatics Solutions, Waterloo, ON, Canada), applying a 10 ppm MS and 0.05 Da MS/MS tolerance. Data Mining and Bioinformatics Analysis. The database search was performed using GenBank and OneKP.52,53 The astratide precursor sequences were obtained by translating the expressed sequence tag (EST) of the A. membranaceus roots from OneKP, and a basic local alignment search tool for translated nucleotide (BLASTn) was used to search for sequence homologues of astratide for expressed nucleotides in the EST database. The EST sequence results were translated using the ExPaSy translation tool.54 The accession numbers of astratide precursors were as follows: aM1 (HJMP-2011398) and bM1 (HJMP-2064542). The open reading frame was defined as the region between the specified start (ATG) and stop (TAA, TAG, and TGA) codons. The cleavage site of the signal peptide in the precursor sequence was determined by SignalP 4.0.55 The isoelectric point was predicted using the ProtParam tool.56 The alignment of the primary amino acid and precursor sequences was performed using BioEdit.57 Identity and similarity were compared using EMBOSS Water Pairwise Sequence Alignment.58 The sequence logos were generated using WebLogo 3.59 The aligned precursor sequences were analyzed using a neighbor-joining clustering algorithm by MEGA 6.0. 60 The

phylogenetic tree was constructed using a bootstrap test of 1000 replications, and the Poisson correction method was employed for substitution modeling. The phylogenetic tree was displayed using iTOL v3.61 Structure Prediction of α-Astratide aM1. The predicted 3D structure of aM1 and bM1 were obtained from I-TASSER.62 The solution structure of PA1b was obtained from the Protein Data Bank (PDB). A cartoon representation and electrostatic surface of the PA1b (PDB: 1P8B) and predicted aM1 were created using PyMOL.63 Structural similarity was calculated using the TM-align algorithm.64 Thermal and Acid Stability Assays. Purified astratides (200 μM) were incubated in a water bath at 100 °C or with HCl (pH 2) for 2 h. Then, 65 μL of the treated sample was aliquoted and quenched in an ice bath for 10 min or by adding 0.8 μL of 1 M NaOH at different time points (0, 30, 60, 90, and 120 min). Later, 20 μL of samples from each time point were analyzed by RP-HPLC to determine the amount of astratides. Each experiment was performed in triplicate. Proteolytic Enzyme Stability Assays. Purified astratides (200 μM) were incubated at 37 °C for 6 h with 4 mg/mL pepsin or 20 U/ mL aminopeptidase I in HCl (pH 2) or 20 mM tricine and 0.05% bovine serum albumin (pH 8.0). At each time point (0, 2, 4, and 6 h), 65 μL of the treated sample was aliquoted and quenched by adding 0.8 μL of 1 M NaOH or 0.4 μL of 1 M HCl. Later, 20 μL of samples from each time point were analyzed by RP-HPLC to determine the amount of astratides. Each experiment was performed in triplicate. Human Serum Stability Assay. Purified astratides (0.2 mg) were incubated with 25% human male serum AB type in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C for 48 h. At specified intervals (0, 12, 24, and 48 h), aliquots were collected and quenched with 95% EtOH. Samples were kept at 4 °C for 15 min and were centrifuged at 13 000 rpm for 10 min. Later, 20 μL of samples from each time point was analyzed by RP-HPLC to determine the amount of astratides. Each experiment was performed in triplicate. Cell Culture. Ovarian cells from Spodoptera f rugiperda Sf9 cells (ATCC no. CRL-1711) were grown at 26 °C in Sf-900 III SFM culture medium (Sigma-Aldrich, St. Louis, U.S.A.) supplemented with 2% fetal bovine serum (FBS) and with 1% (v/v) antibiotics (100 U/ mL penicillin and 100 ng/mL streptomycin). CHO-K1 cells from Chinese hamster ovary (ATCC no. CCL-61) were grown at 37 °C with 5% CO2 in DMEM supplemented with 10% (v/v) FBS, and 1% penicillin-streptomycin. Mouse pancreatic β cell line beta-TC-6 cells (ATCC no. CRL-11506) were grown at 37 °C with 5% CO2 in ATCC-formulated DMEM high-glucose medium, supplemented with 15% FBS and 1% penicillin−streptomycin. Viability Assays. Sf9 cells were split every 3 days and seeded in 96-well plates 24 h with a starting density of 1 × 105 cell/mL. Then, 100 μL of Sf9 cells and CHO-K1 cells was exposed to various different concentrations of peptide (0.01, 0.1, 1, 5, 10, 25, 50, 100, 140, and 200 μM) as well as the negative control (0.1% DMSO) for another 24 h. Ten percent of Triton-X 100 was used as a positive control as described in previous studies.5,65,66 Cell viability was determined by a colorimetric assay based on the ability of viable cells to reduce 3-(4,5-dime-thylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT). Cells were loaded with 10 μL of MTT solution (0.5 mg/mL) and incubated at 26 °C for 4 h before treatment with DMSO for 1 h. Cell homogenates were used to measure absorbance at 550 nm using a microplate reader (Dynatech Laboratories Inc., Virginia, U.S.A.). Sf9 cells were incubated with 5 μM of aM1 at 26 °C for 15 h. At various time points (0, 1, 4, 8, and 15 h), cells were observed under a Zeiss Live Cell Observer (Zeiss, Oberkochen, Germany). The images were recorded and analyzed using axiovision rel 4.8 software (Zeiss, Oberkochen, Germany). Cell Membrane Labeling Assay. The Sf9 cells were labeled with PKH26 (Sigma-Aldrich, St. Louis, U.S.A.) according to the manufacturer’s recommendations with modifications. Sf9 cells (60,000 cells per coverslip per well in a 12-well plate) were washed with PBS three times and incubated with 4 μM of PKH26 in PBS for 4 min. An equal volume of FBS was added to stop the reaction. I



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Confocal Microscopy. A 10 μM sample of aM1 was added to the Sf9 cells immediately after cell membrane labeling with PKH26 for 24 h at 26 °C. The medium was removed after incubation and washed with PBS twice. The fluorescence image of samples was subjected to a Zeiss 710 Confocal Microscope (Zeiss, Oberkochen, Germany). Images were processed and analyzed with ZEN image software (Zeiss, Oberkochen, Germany). Insulin Secretion Assay. Beta-TC-6 cells were split every 7 days and seeded in a 96-well plate for 24 h with a starting density of 5000 cell/mL. Various concentrations of aM1 (0.01, 0.1, 1, 10, and 100 μM) as well as positive control of 10 μM of GW9508 (Sigma-Aldrich, St. Louis, U.S.A.) and negative control of 0.1% DMSO were added to 100 μL of beta-TC-6 cells for 24 h. An insulin Mouse ELISA Kit (Thermo Fisher Scientific, Massachusetts, U.S.A.) was used to measure insulin secretion level. Antifungal Assay. A radical disc diffusion assay was employed to evaluate the antifungal activities of bM1 as previously described.29,39 Four phytopathogenic fungal strains were obtained from the China Center of Industrial Culture Collection (CICC), namely, Fusarium oxysporum (CICC 2532), Alternaria alternata (CICC 2465), Rhizoctonia solani (CICC 40259), and Curvularia lunata (CICC 40301). The susceptibility of these four fungal strains to bM1 (0.02 to 0.2 mg) was tested using a disc diffusion assay.39 Potato dextrose agar was used to grow the four fungal strains at 30 °C, and the mycelia were harvested by punching a hole in the growing fungi and transferring to a new agar plate. The plate was incubated at 30 °C for 2−3 days until sufficient formation of a radical mycelial colony. Then, 6 mm discs impregnated with 20 μL of different concentrations of bM1 were placed equidistant (1 cm) from the colony end and incubated for 24 h at 30 °C. Deionized water was used for a negative control. A microbroth dilution assay was employed to determine the IC50 levels of bM1 against these four fungal strains as previously described.34,39 Fungal spores were obtained from a confluent agar plate. The spores were seeded in half-strength potato dextrose broth at a density of 2.5 × 103 cells/mL. The spore solution was mixed with β-astratide bM1 (20 μL) at various concentrations in a 96-well plate and incubated at 30 °C for 24 h. Then, 100 μL of MeOH was added to fix the fungi for 30 min, and 1% methylene blue stain in 0.01 M borate buffer was added. The plate was incubated at room temperature for 30 min. Excess dye was washed away with water, and HCl (50 mM) in 50% EtOH was added to redissolve the stain after the plates were dried. Absorbance was read at 640 nm using an Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland).

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Author Contributions

J.P.T., J.Y.H., and K.H.W. conceived and designed the experiments. J.Y.H., K.H.W., S.V.T., and A.S. performed the experiments, analyzed the data and wrote the manuscript. J.P.T. and S.K.S revised the manuscript. All authors read and approved the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was in part by the Competitive Research Grant by supported by National Research Foundation in Singapore (NRF-CRP8-2011-05), a Nanyang Technological University internal funding−Synzyme and Natural Products Center (SYNC) and the AcRF Tier 3 funding (MOE2016-T3-1003).We would like to thank Dr Yan Zhao from School of Biological Sciences, Nanyang Technological University, Singapore for the authentication of plant sample Astragalus membranaceus.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00521.

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REFERENCES

(1) Schmidt, B. M.; Ribnicky, D. M.; Lipsky, P. E.; Raskin, I. Nat. Chem. Biol. 2007, 3, 360−366. (2) Bhardwaj, G.; Mulligan, V. K.; Bahl, C. D.; Gilmore, J. M.; Harvey, P. J.; Cheneval, O.; Buchko, G. W.; Pulavarti, S. V.; Kaas, Q.; Eletsky, A.; et al. Nature 2016, 538, 329−335. (3) Wong, K. H.; Tan, W. L.; Xiao, T.; Tam, J. P. Sci. Rep. 2017, 7, 6140−6152. (4) Tan, W. L.; Wong, K. H.; Lei, J.; Sakai, N.; Tan, H. W.; Hilgenfeld, R.; Tam, J. P. Sci. Rep. 2017, 7, 5194−5204. (5) Tam, J. P.; Nguyen, G. K. T.; Loo, S.; Wang, S.; Yang, D.; Kam, A. Sci. Rep. 2018, 8, 16201−16215. (6) Qi, S.; Huang, H.; Huang, J.; Wang, Q.; Wei, Q. Food Control 2015, 50, 195−201. (7) Higgins, T.; Chandler, P.; Randall, P.; Spencer, D.; Beach, L.; Blagrove, R.; Kortt, A.; Inglis, A. J. Biol. Chem. 1986, 261, 11124− 11130. (8) Da Silva, P.; Strzepa, A.; Jouvensal, L.; Rahioui, I.; Gressent, F.; Delmas, A. F. Biopolymers 2009, 92, 436−444. (9) Da Silva, P.; Rahioui, I.; Laugier, C.; Jouvensal, L.; Meudal, H.; Chouabe, C.; Delmas, A. F.; Gressent, F. J. Biol. Chem. 2010, 285, 32689−32694. (10) Gressent, F.; Da Silva, P.; Eyraud, V.; Karaki, L.; Royer, C. Toxins 2011, 3, 1502−1517. (11) Eyraud, V.; Balmand, S.; Karaki, L.; Rahioui, I.; Sivignon, C.; Delmas, A. F.; Royer, C.; Rahbe, Y.; Da Silva, P.; Gressent, F. Sci. Rep. 2017, 7, 4902−4911. (12) Yamazaki, T.; Takaoka, M.; Katoh, E.; Hanada, K.; Sakita, M.; Sakata, K.; Nishiuchi, Y.; Hirano, H. Eur. J. Biochem. 2003, 270, 1269−1276. (13) Dun, X. P.; Li, F. F.; Wang, J. H.; Chen, Z. W. Peptides 2008, 29, 891−897. (14) Dun, X. P.; Wang, J. H.; Chen, L.; Lu, J.; Li, F. F.; Zhao, Y. Y.; Cederlund, E.; Bryzgalova, G.; Efendic, S.; Jornvall, H.; Chen, Z. W.; Bergman, T. FEBS J. 2007, 274, 751−759. (15) Lu, J.; Zeng, Y.; Hou, W.; Zhang, S.; Li, L.; Luo, X.; Xi, W.; Chen, Z.; Xiang, M. J. Nutr. Biochem. 2012, 23, 1449−1457. (16) Shafee, T. M.; Lay, F. T.; Phan, T. K.; Anderson, M. A.; Hulett, M. D. Cell. Mol. Life Sci. 2017, 74, 663−682. (17) Broekaert, W. F.; Terras, F.; Cammue, B.; Osborn, R. W. Plant Physiol. 1995, 108, 1353−1358. (18) Tam, J. P.; Wang, S.; Wong, K. H.; Tan, W. L. Pharmaceuticals 2015, 8, 711−757. (19) Fu, J.; Wang, Z.; Huang, L.; Zheng, S.; Wang, D.; Chen, S.; Zhang, H.; Yang, S. Phytother. Res. 2014, 28, 1275−1283. (20) Rios, J.; Waterman, P. Phytother. Res. 1997, 11, 411−418.

UPLC profiles of aM1 and bM1; MALDI-TOF MS spectra of aM1; LC-ESI-LTQ-Orbitrap MS/MS analysis of aM1 and bM1; 3D structure prediction of aM1 and bM1; cell viability data on Sf9 cells and CHO-K1 cells; microscopic images; stability assays of aM1 and bM1; large-scale extraction flowchart; sequence comparison; TM alignment scores (PDF)

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*Tel: 65-6316 2863. Fax: 65-6515 1632. E-mail: jptam@ntu. edu.sg. ORCID

Siu Kuan Sze: 0000-0002-5652-1687 James P. Tam: 0000-0003-4433-198X J



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(21) Wang, Z.; Wang, J.; Chan, P. J. Evidence-Based Complementary Altern. Med. 2013, 2013, 343594−343611. (22) Agyemang, K.; Han, L.; Liu, E.; Zhang, Y.; Wang, T.; Gao, X. J. Evidence-Based Complementary Altern. Med. 2013, 2013, 654643− 654652. (23) Gruber, C. W.; Elliott, A. G.; Ireland, D. C.; Delprete, P. G.; Dessein, S.; Göransson, U.; Trabi, M.; Wang, C. K.; Kinghorn, A. B.; Robbrecht, E.; Craik, D. J. Plant Cell 2008, 20, 2471−2483. (24) Louis, S.; Delobel, B.; Gressent, F.; Rahioui, I.; Quillien, L.; Vallier, A.; Rahbé, Y. Plant Sci. 2004, 167, 705−714. (25) Watanabe, Y.; Barbashov, S. F.; Komatsu, S.; Hemmings, A. M.; Miyagi, M.; Tsunasawa, S.; Hirano, H. Eur. J. Biochem. 1994, 224, 167−172. (26) Jianzhong, T. Chin. J. Appl. Environ. Biol. 1999, 5, 259−263. (27) Taylor, W. G.; Sutherland, D. H.; Zhang, H.; Hegedus, D. D. Phytochem. Lett. 2015, 12, 105−112. (28) Nguyen, P. Q.; Luu, T. T.; Bai, Y.; Nguyen, G. K.; Pervushin, K.; Tam, J. P. J. Nat. Prod. 2015, 78, 695−704. (29) Wong, K. H.; Tan, W. L.; Serra, A.; Xiao, T.; Sze, S. K.; Yang, D.; Tam, J. P. Front. Plant Sci. 2016, 7, 1639−1653. (30) Hellinger, R.; Koehbach, J.; Puigpinós, A.; Clark, R. J.; Tarragó, T.; Giralt, E.; Gruber, C. W. J. Nat. Prod. 2015, 78, 1073−1082. (31) Männistö, P. T.; Vanäläinen, J.; Jalkanen, A.; García-Horsman, J. A. Drug News Perspect. 2007, 20, 293−305. (32) Meindre, F.; Lelièvre, D.; Loth, K.; Mith, O.; Aucagne, V.; Berthomieu, P.; Marquès, L.; Delmas, A. s. F.; Landon, C. l.; Paquet, F. o. Biochemistry 2014, 53, 7745−7754. (33) Shafee, T. M.; Lay, F. T.; Hulett, M. D.; Anderson, M. A. Mol. Biol. Evol. 2016, 33, 2345−2356. (34) Wong, K. H.; Tan, W. L.; Kini, S. G.; Xiao, T.; Serra, A.; Sze, S. K.; Tam, J. P. Front. Plant Sci. 2017, 8, 1100−1113. (35) Eyraud, V.; Karaki, L.; Rahioui, I.; Sivignon, C.; Da Silva, P.; Rahbé, Y.; Royer, C.; Gressent, F. PLoS One 2013, 8, e81619− e81627. (36) Nguyen, G. K.; Wang, S.; Qiu, Y.; Hemu, X.; Lian, Y.; Tam, J. P. Nat. Chem. Biol. 2014, 10, 732−738. (37) Finkina, E. I.; Shramova, E. I.; Tagaev, A. A.; Ovchinnikova, T. V. Biochem. Biophys. Res. Commun. 2008, 371, 860−865. (38) Lay, F. T.; Brugliera, F.; Anderson, M. A. Plant Physiol. 2003, 131, 1283−1293. (39) Kini, S. G.; Wong, K. H.; Tan, W. L.; Xiao, T.; Tam, J. P. BMC Plant Biol. 2017, 17, 68−80. (40) El-Gazzar, A.; El-Ghani, M.; El-Husseini, N.; Khattab, A. Not. Sci. Biol. 2013, 5, 499−507. (41) Doyle, J. J.; Luckow, M. A. Plant Physiol. 2003, 131, 900−910. (42) Zhang, Y.; Lewis, K. FEMS Microbiol. Lett. 1997, 149, 59−64. (43) Jennings, C.; West, J.; Waine, C.; Craik, D.; Anderson, M. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 10614−10619. (44) Chagolla-Lopez, A.; Blanco-Labra, A.; Patthy, A.; Sánchez, R.; Pongor, S. J. Biol. Chem. 1994, 269, 23675−23680. (45) Daly, N. L.; Craik, D. J. Curr. Opin. Chem. Biol. 2011, 15, 362− 368. (46) Briscoe, C. P.; Peat, A. J.; McKeown, S. C.; Corbett, D. F.; Goetz, A. S.; Littleton, T. R.; McCoy, D. C.; Kenakin, T. P.; Andrews, J. L.; Ammala, C.; et al. Br. J. Pharmacol. 2006, 148, 619−628. (47) Leahy, J. Diabetes Rev. 1996, 4, 298−319. (48) Lamontagne, J.; Jalbert-Arsenault, É .; Pepin, É .; Peyot, M.-L.; Ruderman, N. B.; Nolan, C. J.; Joly, E.; Madiraju, S. M.; Poitout, V.; Prentki, M. Diabetes 2013, 62, 2122−2129. (49) Kini, S. G.; Nguyen, P. Q.; Weissbach, S.; Mallagaray, A.; Shin, J.; Yoon, H. S.; Tam, J. P. Biochemistry 2015, 54, 6639−6649. (50) Nicolas, P. FEBS J. 2009, 276, 6483−6496. (51) Vriens, K.; Cammue, B.; Thevissen, K. Molecules 2014, 19, 12280−12303. (52) Benson, D. A.; Cavanaugh, M.; Clark, K.; Karsch-Mizrachi, I.; Lipman, D. J.; Ostell, J.; Sayers, E. W. Nucleic Acids Res. 2012, 41, D36−D42. (53) Matasci, N.; Hung, L.-H.; Yan, Z.; Carpenter, E. J.; Wickett, N. J.; Mirarab, S.; Nguyen, N.; Warnow, T.; Ayyampalayam, S.; Barker,

M.; Burleigh, J. G.; Gitzendanner, M. A.; Wafula, E.; Der, J. P.; dePamphilis, C. W.; Roure, B.; Philippe, H.; Ruhfel, B. R.; Miles, N. W.; Graham, S. W.; Mathews, S.; Surek, B.; Melkonian, M.; Soltis, D. E.; Soltis, P. S.; Rothfels, C.; Pokorny, L.; Shaw, J. A.; DeGironimo, L.; Stevenson, D. W.; Villarreal, J. C.; Chen, T.; Kutchan, T. M.; Rolf, M.; Baucom, R. S.; Deyholos, M. K.; Samudrala, R.; Tian, Z.; Wu, X.; Sun, X.; Zhang, Y.; Wang, J.; Leebens-Mack, J.; Wong, G. K.-S. GigaScience 2014, 3, 17−26. (54) Gasteiger, E.; Gattiker, A.; Hoogland, C.; Ivanyi, I.; Appel, R. D.; Bairoch, A. Nucleic Acids Res. 2003, 31, 3784−3788. (55) Petersen, T. N.; Brunak, S.; von Heijne, G.; Nielsen, H. Nat. Methods 2011, 8, 785−786. (56) Gasteiger, E.; Hoogland, C.; Gattiker, A.; Wilkins, M. R.; Appel, R. D.; Bairoch, A. Protein identification and analysis tools on the ExPASy server. In The proteomics protocols handbook; Walker, J. M., Ed.; Springer, 2005; pp 571−607. (57) Hall, T. GERF Bull. Biosci. 2011, 2, 60−61. (58) Rice, P.; Longden, I.; Bleasby, A. Trends Genet. 2000, 16, 276− 277. (59) Crooks, G. E.; Hon, G.; Chandonia, J.-M.; Brenner, S. E. Genome Res. 2004, 14, 1188−1190. (60) Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. Mol. Biol. Evol. 2013, 30, 2725−2729. (61) Letunic, I.; Bork, P. Nucleic Acids Res. 2016, 44, W242−W245. (62) Roy, A.; Kucukural, A.; Zhang, Y. Nat. Protoc. 2010, 5, 725− 738. (63) DeLano, W. L. CCP4 Newsletter On Protein Crystallography 2002, 40, 82−92. (64) Zhang, Y.; Skolnick, J. Nucleic Acids Res. 2005, 33, 2302−2309. (65) Castro-Garza, J.; Barrios-Garcia, H. B.; Cruz-Vega, D. E.; SaidFernandez, S.; Carranza-Rosales, P.; Molina-Torres, C. A.; VeraCabrera, L. J. Med. Microbiol. 2007, 56, 733−737. (66) Riaz, M.; Rasool, N.; Bukhari, I. H.; Shahid, M.; Zubair, M.; Rizwan, K.; Rashid, U. Molecules 2012, 17, 14275−14287.

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