Publications by authors named "James P Tam"

113 Publications

Identification and characterization of a wolfberry carboxypeptidase inhibitor from Lycium barbarum.

Food Chem 2021 Feb 19;351:129338. Epub 2021 Feb 19.

School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore. Electronic address:

Hyperstable cysteine-rich peptides (CRPs) represent an underexplored superfamily of bioactives in functional foods. An example is wolfberry of the Lycium barbarum family. Previously, we discovered a CRP, designated α-lybatide, from L. barbarum bark. Herein, we report the discovery of β-lybatide, a novel carboxypeptidase inhibitor belonging to a different CRP family from the wolfberry plant. Proteomic and transcriptomic analyses showed that β-lybatide contains 36 amino acids with six cysteine residues. NMR spectroscopy revealed that β-lybatide displays a knottin-like structure that renders it highly resistant to thermal, chemical and enzymatic degradation, conditions important for keeping its structural integrity in gastrointestinal tract. Biochemical assays showed that β-lybatide is a potent carboxypeptidase inhibitor which could contribute to the wolfberry biological activities. Bioinformatics analysis revealed an additional 49 β-lybatide-like plant carboxypeptidase inhibitors. Together, our results show that β-lybatide is the first and the smallest plant-derived hyperstable carboxypeptidase inhibitor discovered from a functional food.
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http://dx.doi.org/10.1016/j.foodchem.2021.129338DOI Listing
February 2021

Construction of FeO@α-glucosidase magnetic nanoparticles for ligand fishing of α-glucosidase inhibitors from a natural tonic Epimedii Folium.

Int J Biol Macromol 2020 Dec 10;165(Pt A):1361-1372. Epub 2020 Oct 10.

School of Pharmacy, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China; School of Traditional Chinese Pharmacy, China Pharmaceutical University, 639 Longmian Avenue, Nanjing 211198, China. Electronic address:

Inhibition of α-glucosidase activity is an effective way for treatment of type 2 diabetes mellitus. Epimedii Folium is an important source of α-glucosidase inhibitors (AGIs), however bioactive compounds and pharmacological mechanisms remained unclear. In this study, a novel strategy was established, which harnessed α-glucosidase functionalized magnetic beads to fish out potential AGIs, followed by UPLC-MS/MS analysis for their identification. Furthermore, molecular docking was employed to predict binding patterns between the AGIs and the enzyme, and IC values was estimated as well. After response surface methodology optimization, the highest activity of FeO@α-glucosidase has been achieved when 1.17 mg/mL of α-glucosidase was immobilized in phosphate buffer (pH 6.81) for 4.22 h. Moreover, eight flavonoids were fished out from the extract of Epimedii Folium, and then identified to be epimedin A, epimedin B, epimedin C, icariin, sagittatoside A, sagittatoside B, 2"-O-rhamnosyl icariside II and baohuoside I. All of them were further confirmed to be AGIs through in vitro inhibitory assay and molecular docking. Among those, baohuoside I and sagittatoside B possessed stronger inhibitory activity than acarbose. The approach has a significant prospect in conveniently screening bioactive compounds that target various receptors, which provided an efficient platform for new drug development from natural products.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.10.018DOI Listing
December 2020

Tagging Transferrin Receptor with a Disulfide FRET Probe To Gauge the Redox State in Endosomal Compartments.

Anal Chem 2020 09 3;92(18):12460-12466. Epub 2020 Aug 3.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.

Although the basic process of receptor-mediated endocytosis (RME) is well established, certain specific aspects, like the endosomal redox state, remain less characterized. Previous studies used chemically labeled ligands or antibodies with a FRET (fluorescence resonance energy transfer) probe to gauge the redox activity of the endocytic pathway with a limitation being their inability to track the apo receptor. New tools that allow direct labeling of a cell surface receptor with synthetic probes would aid in the study of its endocytic pathway and function. Herein, we use a peptide ligase, butelase 1, to label the human transferrin receptor 1 (TfR1) in established human cell lines with a designer disulfide FRET probe. This strategy enables us to obtain real-time live cell imaging of redox states in TfR1-mediated endocytosis, attesting a reducing environment in the endosomal compartments and the dynamics of TfR1 trafficking. A better understanding of endocytosis of different cell surface receptors has implications in designing strategies that hijack this natural process for intracellular drug delivery.
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http://dx.doi.org/10.1021/acs.analchem.0c02264DOI Listing
September 2020

Correction to Immobilized Peptide Asparaginyl Ligases Enhance Stability and Facilitate Macrocyclization and Site-Specific Ligation.

J Org Chem 2020 Mar 5;85(6):4581. Epub 2020 Mar 5.

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http://dx.doi.org/10.1021/acs.joc.0c00321DOI Listing
March 2020

Cyclization of a G4-specific peptide enhances its stability and G-quadruplex binding affinity.

Chem Commun (Camb) 2020 Jan;56(7):1082-1084

School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore637371.

G-quadruplexes (G4) are non-canonical nucleic acid structures with important implications in biology. Based on an α-helical fragment of the RHAU helicase that displays high specificity for parallel-stranded G-quadrplexes, herein we demonstrate its head-to-tail cyclization by a high-efficiency ligase. The cyclic peptide exhibits superior stability and binding affinity to a G-quadruplex, and can serve as an excellent investigational tool for chemical biology applications.
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http://dx.doi.org/10.1039/c9cc06748eDOI Listing
January 2020

Immobilized Peptide Asparaginyl Ligases Enhance Stability and Facilitate Macrocyclization and Site-Specific Ligation.

J Org Chem 2020 02 3;85(3):1504-1512. Epub 2020 Jan 3.

School of Biological Sciences , Nanyang Technological University , 637551 Singapore.

The recently discovered peptide asparaginyl ligases (PALs) from cyclotide-producing plants are efficient and versatile tools for protein and peptide engineering. Here, we report immobilization of two glycosylated PALs, butelase-1 and VyPAL2, using three different attachment methods and their applications for peptide engineering. We compared immobilization indirectly via noncovalent affinity capture using NeutrAvidin or concanavalin A agarose beads or directly via covalent coupling of free amines on the enzyme surface with the -hydroxysuccinimide (NHS) ester attached on agarose beads. The catalytic efficiency of immobilized PALs correlated with the distance between the biocatalysts and the solid supports, and in turn, the mobility of enzymes and the accessibility of substrates. Compared to their soluble counterparts, the site separations of immobilized PALs retain higher activity after prolonged storage and confer reusability for over 100 runs with less than 10% activity loss. We also showed that the cyclization and ligation of peptides and proteins with varying shapes and sizes can be accelerated by providing higher concentration of reusable immobilized PALs. These advantages could be exploited for large-scale industrial applications and nanodevices.
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http://dx.doi.org/10.1021/acs.joc.9b02524DOI Listing
February 2020

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

J Biol Chem 2019 12 14;294(51):19604-19615. Epub 2019 Nov 14.

School of Biological Sciences, Nanyang Technological University, Singapore 637551

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 (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 -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.
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http://dx.doi.org/10.1074/jbc.RA119.010796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926453PMC
December 2019

Eco-efficient biphasic enzymatic hydrolysis for the green production of rare baohuoside I.

Enzyme Microb Technol 2019 Dec 17;131:109431. Epub 2019 Sep 17.

School of Pharmacy, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, People's Republic of China; School of Traditional Chinese Pharmacy, China Pharmaceutical University, 639 Longmian Avenue, Nanjing 211198, People's Republic of China. Electronic address:

Baohuoside I, a rare secondary glycoside from Epimedii Folium, has anti-osteoporosis and other pharmacological effects. In addition, it exhibits improved bioactivities compared with its original glycoside icariin. Conventional methods for the production of baohuoside I, such as acid hydrolysis, exhibit very low efficiency with serious pollution and substantial byproducts. The aim of this study was to develop an eco-efficient biphasic enzymatic hydrolysis system for the green production of rare baohuoside I. β-glucanase was selected to hydrolyze icariin and showed better performance than β-glucosidase. The biphasic system was constructed using HAc-NaAc buffer (pH 4.5) containing β-glucanase/icariin (C = 100 mg/mL; 1:1, w/w) and propyl acetate (1:2, v/v), and the hydrolysis was performed at 60℃ for 6 h. The hydrolysis capacity of icariin in the system was largely increased from 0.5 mg/mL to 100 mg/mL. The conversion ratio of icariin was greater than 99% in a 100-fold scale-up pilot test, demonstrating its strong potential for industrial applications. Furthermore, a process flow and production plant were designed to produce 1 ton of baohuoside I annually, and the profit would be approximately 4.5-fold its total cost. Biphasic enzymatic hydrolysis is an eco-efficient technology to produce baohuoside I and represents a promising method in the pharmaceutical industry.
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http://dx.doi.org/10.1016/j.enzmictec.2019.109431DOI Listing
December 2019

Prooxidant modifications in the cryptome of beef jerky, the deleterious post-digestion composition of processed meat snacks.

Food Res Int 2019 11 19;125:108569. Epub 2019 Jul 19.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore. Electronic address:

Snacking has traditionally been associated with consumption of foods rich in fats and carbohydrates. However, new dietary trends switched to consumption of protein-rich foods. This study investigates the impact of food processing on the cryptome of one of the most widely consumed meat snacks, beef jerky. We have performed discovery-driven proteome-wide analyses, which identified a significantly elevated presence of reactive prooxidant post-translational modifications in jerky. We also found that these protein decorations impact an important subset of in-silico predicted DNA binding cryptides. Furthermore, we observed cell-dependent reduction in cell viability after prolonged treatments with endogenous-like jerky digests. Collectively these findings uncover the presence of prooxidant modifications in processed dried beef snacks and associate their presence with cytotoxicity. Thus, the findings reported here can pave the way for future studies aimed to establish appropriate dietary recommendations on snacking trends.
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http://dx.doi.org/10.1016/j.foodres.2019.108569DOI Listing
November 2019

Cysteine-Rich Peptide Fingerprinting as a General Method for Herbal Analysis to Differentiate Radix Astragali and Radix Hedysarum.

Front Plant Sci 2019 31;10:973. Epub 2019 Jul 31.

School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

Species misidentification and adulteration are major concerns in authenticating herbal medicines. Radix Astragali (RA), the roots of , is a traditional herbal medicine used for treating diabetes. However, it is often substituted by Radix Hedysarum (RH), the roots of from the same plant family Fabaceae, which possesses different bioactivities. Current authentication methods, focusing on the chemical composition differences of herbal medicines based on small molecules, have limitations when these chemical markers are found in many species. Herein, we describe a rapid and general method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), coupled with multivariate analyses to differentiate herbal medicines. We used cysteine-rich peptide (CRP) fingerprinting, a method that exploits an underexplored chemical space between 2 to 6 kDa and which is populated by highly stable CRPs. To show the generality of the method, we screened 100 medicinal plant extracts and showed that CRP fingerprints are unique chemical markers. In addition, CRP fingerprinting was many-fold faster than the conventional authentication method using ultra-performance liquid chromatography (UPLC). Multivariate analyses showed that it has comparable classification accuracy as UPLC fingerprinting. Together, our findings revealed that CRP fingerprinting coupled with multivariate analyses is a rapid and general method for authentication and quality control for natural products in medicinal plants.
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http://dx.doi.org/10.3389/fpls.2019.00973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684776PMC
July 2019

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

Molecules 2019 Jun 30;24(13). Epub 2019 Jun 30.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.

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 . 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.
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http://dx.doi.org/10.3390/molecules24132417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6651024PMC
June 2019

Butelase 1-Mediated Ligation of Peptides and Proteins.

Methods Mol Biol 2019 ;2012:83-109

School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

Structurally, butelase 1 is a cysteine protease of the asparaginyl endoprotease (AEP) family, but functionally, it displays intense Asn/Asp-specific (Asx) ligase activity and is virtually devoid of protease activity. Butelase 1 recognizes specifically a C-terminal Asx-containing tripeptide motif, Asx-His-Val, to form an Asx-Xaa peptide bond (Xaa = any amino acid), either intramolecularly or intermolecularly, resulting in cyclic peptides or site-specific modified peptides/proteins, respectively. Our work in the past 4 years has validated that butelase 1 is a potent and versatile tool for peptide and protein modification. Here we describe our protocols using butelase 1 for efficient and site-specific peptide and protein ligation, N-terminal labeling, preparation of thioesters, and bioconjugation of dendrimers. Additionally, we provide an example using butelase 1 for protein cyclization in combination with genetic code expansion in order to incorporate unnatural building blocks.
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http://dx.doi.org/10.1007/978-1-4939-9546-2_6DOI Listing
March 2020

Structural determinants for peptide-bond formation by asparaginyl ligases.

Proc Natl Acad Sci U S A 2019 06 23;116(24):11737-11746. Epub 2019 May 23.

School of Biological Sciences, Nanyang Technological University, 637551 Singapore;

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants () and () of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the -ester intermediate. Recombinantly expressed PAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on PAL1-3, AEP1, and AEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of PAL3 and converted the protease AEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.
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http://dx.doi.org/10.1073/pnas.1818568116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6576118PMC
June 2019

Pulsed SILAC-based proteomic analysis unveils hypoxia- and serum starvation-induced protein synthesis with PHD finger protein 14 (PHF14) as a hypoxia sensitive epigenetic regulator in cell cycle progression.

Oncotarget 2019 Mar 15;10(22):2136-2150. Epub 2019 Mar 15.

Division of Structural Biology and Biochemistry School of Biological Sciences, Nanyang Technological University, Singapore 637551.

Hypoxia is an environmental cue that is associated with multiple tumorigenic processes such as immunosuppression, angiogenesis, cancer invasion, metastasis, drug resistance, and poor clinical outcomes. When facing hypoxic stress, cells initiate several adaptive responses such as cell cycle arrest to reduce excessive oxygen consumption and co-activation of oncogenic factors. In order to identify the critical novel proteins for hypoxia responses, we used pulsed-SILAC method to trace the active cellular translation events in A431 cells. Proteomic discovery data and biochemical assays showed that cancer cells selectively activate key glycolytic enzymes and novel ER-stress markers, while protein synthesis is severely suppressed. Interestingly, deprivation of oxygen affected the expression of various epigenetic regulators such as histone demethylases and NuRD (nucleosome remodeling and deacetylase) complex in A431 cells. In addition, we identified PHF14 (the plant homeodomain finger-14) as a novel hypoxia-sensitive epigenetic regulator that plays a key role in cell cycle progress and protein synthesis. Hypoxia-mediated inhibition of PHF14 was associated with increase of key cell cycle inhibitors, p14, p15, and p16, which are responsible for G1-S phase transition and decrease of AKT-mTOR-4E-BP1/pS6K signaling pathway, a master regulator of protein synthesis, in response to environmental cues. Analysis of TCGA colon cancer (n=461) and skin cancer (n=470) datasets revealed a positive correlation between PHF14 expression and protein translation initiation factors, eIF4E, eIF4B, and RPS6. Significance of PHF14 gene was further demonstrated by mouse xenograft model using PHF14 KD cell lines.
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http://dx.doi.org/10.18632/oncotarget.26669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481330PMC
March 2019

Potentides: New Cysteine-Rich Peptides with Unusual Disulfide Connectivity from Potentilla anserina.

Chembiochem 2019 08 24;20(15):1995-2004. Epub 2019 Jun 24.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore.

Cysteine-rich peptides (CRPs), which are disulfide-constrained peptides with 3 to 5 disulfide bonds and molecular weights of 2 to 6 kDa, are generally hyperstable and resistant to thermal, chemical, and enzymatic degradation. Herein, the discovery and characterization of a novel suite of CRPs, collectively named potentides pA1-pA16 from the root of the medicinal herb Potentilla anserina L, are described. Through a combination of proteomic and transcriptomic methods, it is shown that 35-residue potentide pA3, which is the most abundant member of potentides, exhibits high stability against heat, acidic, and proteolytic degradation. Transcriptomic analysis revealed that potentide precursor sequences contained four tandem repeats in the mature domain, which is the first report on tandem repeats being found in the Rosaceae family. Disulfide mapping showed that potentide pA3 displayed a novel disulfide connectivity of C1-C3, C2-C6, and C4-C5; a cystine motif that has not been reported in plant CRPs. Transcriptomic data mining and a neighbor-joining clustering analysis revealed 56 potentide homologues and their distribution in the families of Rosaceae and Ranunculaceae in angiosperm. Altogether, these results reveal a new plant CRP structure with an unusual cystine connectivity. Additionally, this study expands the families and structure diversity of CRPs as potentially active peptide pharmaceuticals.
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http://dx.doi.org/10.1002/cbic.201900127DOI Listing
August 2019

Hypoxia-induced tumor exosomes promote M2-like macrophage polarization of infiltrating myeloid cells and microRNA-mediated metabolic shift.

Oncogene 2019 06 12;38(26):5158-5173. Epub 2019 Mar 12.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore.

Developing tumors rapidly outgrow their oxygen supply and are subject to hypoxia, which stimulates hypersecretion of tumor-derived exosomes that promote angiogenesis, metastasis, and immunosuppression, but the molecular mediators of these pathological effects remain poorly defined. Using quantitative proteomics, we identified that exosomes produced by hypoxic tumor cells are highly enriched in immunomodulatory proteins and chemokines including CSF-1, CCL2, FTH, FTL, and TGFβ. Modeling exosome effects on tumor-infiltrating immune cells, we observed a potent ability of these hypoxia-induced vesicles to influence macrophage recruitment and promote M2-like polarization both in vitro and in vivo. In addition, hypoxic, but not normoxic, tumor exosomes enhanced oxidative phosphorylation in bone marrow-derived macrophages via transfer of let-7a miRNA, resulting in suppression of the insulin-Akt-mTOR signaling pathway. Together, these data demonstrate that hypoxia promotes tumor secretion of biomolecule-loaded exosomes that can modify the immunometabolic profile of infiltrating monocyte-macrophages to better evade host immunity and enhance tumor progression.
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http://dx.doi.org/10.1038/s41388-019-0782-xDOI Listing
June 2019

Ligase-Controlled Cyclo-oligomerization of Peptides.

Org Lett 2019 04 14;21(7):2029-2032. Epub 2019 Feb 14.

School of Biological Sciences , Nanyang Technological University , 60 Nanyang Drive , Singapore 637551.

A biomimetic one-step ligase-catalyzed cyclo-oligomerization mediated by butelase 1, an Asn/Asp-specific ligase, is introduced that is time-, concentration-, length-, and sequence-dependent. This reaction yields cyclic mono-, di-, tri-, and tetramers from peptide precursors containing 3-15 amino acids ended with Asn and a His-Val tail. The cyclomonomers were favored when the peptide lengths were >9 amino acids. A turn-forming Pro residue at the P2 position favored the formation of higher-order cyclo-oligomers.
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http://dx.doi.org/10.1021/acs.orglett.9b00151DOI Listing
April 2019

Astratides: Insulin-Modulating, Insecticidal, and Antifungal Cysteine-Rich Peptides from Astragalus membranaceus.

J Nat Prod 2019 02 13;82(2):194-204. Epub 2019 Feb 13.

School of Biological Sciences , Nanyang Technological University , 60 Nanyang Drive , Singapore 637551.

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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http://dx.doi.org/10.1021/acs.jnatprod.8b00521DOI Listing
February 2019

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

J Biol Chem 2019 03 23;294(11):4000-4011. Epub 2019 Jan 23.

From the School of Biological Sciences, Nanyang Technological University, 637551 Singapore

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 , 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.
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http://dx.doi.org/10.1074/jbc.RA118.006693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422099PMC
March 2019

Convenient preparation of sagittatoside B, a rare bioactive secondary flavonol glycoside, by recyclable and integrated biphase enzymatic hydrolysis.

Enzyme Microb Technol 2019 Feb 3;121:51-58. Epub 2018 Dec 3.

School of Pharmacy, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China; School of Traditional Chinese Pharmacy, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, 211198, China. Electronic address:

Sagittatoside B, a rare secondary flavonol glycoside in Epimedii Folium, has much better in vivo bioactivities than its original glycoside epimedin B. Its preparation methods, such as acidic hydrolysis, are of low efficiency, and byproducts are generated. The objective of this study was to establish a novel catalysis system for convenient preparation of this compound based on recyclable and integrated biphase enzymatic hydrolysis. β-glucanase was selected from five commercial enzymes based on the best catalysis performance. After optimization of the conditions, the biphase system was constructed with propyl acetate and HAc-NaAc buffer (pH 4.5) (1:1, v/v) containing β-glucanase/epimedin B (1:2, w/w), and the hydrolysis was performed at 60 °C for 1 h. Consequently, epimedin B was completely hydrolyzed to sagittatoside B, and 95.7% of the product was transferred into the organic phase. Moreover, a high conversion ratio of 94.0% was achieved, even after the enzyme solution was used for six cycles. Additionally, the procedure was much simplified compared with conventional enzymatic hydrolysis. The newly proposed strategy is an efficient and promising approach for the preparation of sagittatoside B in industrial applications.
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http://dx.doi.org/10.1016/j.enzmictec.2018.12.002DOI Listing
February 2019

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

Sci Rep 2018 11 1;8(1):16201. Epub 2018 Nov 1.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore, Singapore.

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.
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http://dx.doi.org/10.1038/s41598-018-33894-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212409PMC
November 2018

Vascular Bed Molecular Profiling by Differential Systemic Decellularization In Vivo.

Arterioscler Thromb Vasc Biol 2018 10;38(10):2396-2409

From the School of Biological Sciences, Nanyang Technological University, Singapore (A.S., X.G.-P., J.E.P., J.P.T., S.K.S.).

Objective- Vascular endothelial dysfunction is a key component of several major human diseases, but the molecular basis of this complex disorder has been difficult to determine in vivo. Previous attempts to identify key mediators of vascular endothelial dysfunction in experimental models have been limited by the lack of suitable methods for system-wide analyses of vascular bed biology. Here, we aimed to develop a novel method for investigating vascular endothelial dysfunction pathogenesis that enables system-wide analyses of molecular interactions between endothelial glycocalyx, endothelial cells, and smooth muscle cells in murine. Approach and Results- We developed a new technique using whole-body differential perfusion with increasing concentrations of detergent buffer to selectively solubilize distinct layers of vascular bed tissue in rodents. When combined with proteomics techniques, our novel approach of differential systemic decellularization in vivo enabled quantitative profiling of vascular beds throughout the body. Initial perfusion with phosphate buffer was used to obtain the endothelial glycocalyx, followed by subsequent extraction of endothelial cell components, and finally by smooth muscle cell constituents with increasing concentrations of detergent. Differential systemic decellularization in vivo has also been successfully applied to characterize molecular events in the vascular bed pathology of lipopolysaccharide-challenged mice. Conclusions- Together, these data indicate that differential systemic decellularization in vivo permits system-wide molecular characterization of vascular bed proteomes in rodent models and can be used to advance our current understanding of vascular endothelial dysfunction pathogenesis and progression in a wide range of disease settings.
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http://dx.doi.org/10.1161/ATVBAHA.118.311552DOI Listing
October 2018

One-Pot Dual Labeling of IgG 1 and Preparation of C-to-C Fusion Proteins Through a Combination of Sortase A and Butelase 1.

Bioconjug Chem 2018 10 21;29(10):3245-3249. Epub 2018 Sep 21.

Program in Cellular and Molecular Medicine , Boston Children's Hospital, Harvard Medical School , Boston , Massachussets 02115 , United States.

Site-specific chemical modification of proteins can assist in the study of their function. Furthermore, these methods are essential to develop biologicals for diagnostic and therapeutic use. Standard protein engineering protocols and recombinant expression enable the production of proteins with short peptide tags recognized by enzymes capable of site-specific modification. We report here the application of two enzymes of orthogonal specificity, sortase A and butelase 1, to prepare non-natural C-to-C fusion proteins. Using these enzymes, we further demonstrate site-selective installation of different chemical moieties at two sites in a full-size antibody molecule.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429940PMC
October 2018

Molecular diversity and function of jasmintides from Jasminum sambac.

BMC Plant Biol 2018 Jul 11;18(1):144. Epub 2018 Jul 11.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore.

Background: Jasmintides jS1 and jS2 from Jasminum sambac were previously identified as a novel family of cysteine-rich peptides (CRPs) with an unusual disulfide connectivity. However, very little else is known about jasmintides, particularly their molecular diversity and functions. Here, we report the discovery and characterization of a novel suite of jasmintides from J. sambac using transcriptomic, peptidomic, structural and functional tools.

Results: Transcriptomic analysis of leaves, flowers and roots revealed 14 unique jasmintide precursors, all of which possess a three-domain architecture comprising a signal peptide, a pro-domain and a mature jasmintide domain. Peptidomic analysis, using fractionated mixtures of jasmintides and chemical derivatization of cysteine to pseudolysine, trypsin digestion and MS/MS sequencing, revealed an additional 86 jasmintides, some of which were post-translationally modified. NMR analysis showed that jasmintide jS3 has three anti-parallel β-strands with a three-disulfide connectivity of CysI-CysV, CysII-CysIV and CysIII-CysVI, which is similar to jasmintide jS1. Jasmintide jS3 was able to withstand thermal, acidic and enzymatic degradation and, importantly, exhibited antifeedant activity against mealworm Tenebrio molitor.

Conclusion: Together, this study expands the existing library of jasmintides and furthers our understanding of the molecular diversity and cystine framework of CRPs as scaffolds and tools for engineering peptides targeting pests.
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http://dx.doi.org/10.1186/s12870-018-1361-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042386PMC
July 2018

Immobilization and Intracellular Delivery of Circular Proteins by Modifying a Genetically Incorporated Unnatural Amino Acid.

Bioconjug Chem 2018 07 14;29(7):2170-2175. Epub 2018 Jun 14.

School of Biological Sciences , Nanyang Technological University , 60 Nanyang Drive , 637551 , Singapore.

Backbone-cyclic proteins are of great scientific and therapeutic interest owing to their higher stability over their linear counterparts. Modification of such cyclic proteins at a selected site would further enhance their versatility. Here we report a chemoenzymatic strategy to engineer site-selectively modified cyclic proteins by combining butelase-mediated macrocyclization with the genetic code expansion methodology. Using this strategy, we prepared a cyclic protein which was modified with biotin or a cell-penetrating peptide at a genetically incorporated noncanonical amino acid, making the cyclization-stabilized protein further amenable for site-specific immobilization and intracellular delivery. Our results point to a new avenue to engineering novel cyclic proteins with improved physicochemical and pharmacological properties for potential applications in biotechnology and medicine.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00244DOI Listing
July 2018

Identification and application of self-binding zipper-like sequences in SARS-CoV spike protein.

Int J Biochem Cell Biol 2018 08 22;101:103-112. Epub 2018 May 22.

School of Biological Sciences, Nanyang Technological University, Singapore. Electronic address:

Self-binding peptides containing zipper-like sequences, such as the Leu/Ile zipper sequence within the coiled coil regions of proteins and the cross-β spine steric zippers within the amyloid-like fibrils, could bind to the protein-of-origin through homophilic sequence-specific zipper motifs. These self-binding sequences represent opportunities for the development of biochemical tools and/or therapeutics. Here, we report on the identification of a putative self-binding β-zipper-forming peptide within the severe acute respiratory syndrome-associated coronavirus spike (S) protein and its application in viral detection. Peptide array scanning of overlapping peptides covering the entire length of S protein identified 34 putative self-binding peptides of six clusters, five of which contained octapeptide core consensus sequences. The Cluster I consensus octapeptide sequence GINITNFR was predicted by the Eisenberg's 3D profile method to have high amyloid-like fibrillation potential through steric β-zipper formation. Peptide C6 containing the Cluster I consensus sequence was shown to oligomerize and form amyloid-like fibrils. Taking advantage of this, C6 was further applied to detect the S protein expression in vitro by fluorescence staining. Meanwhile, the coiled-coil-forming Leu/Ile heptad repeat sequences within the S protein were under-represented during peptide array scanning, in agreement with that long peptide lengths were required to attain high helix-mediated interaction avidity. The data suggest that short β-zipper-like self-binding peptides within the S protein could be identified through combining the peptide scanning and predictive methods, and could be exploited as biochemical detection reagents for viral infection.
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http://dx.doi.org/10.1016/j.biocel.2018.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108413PMC
August 2018

A novel PCR-based technology for rapid and non-sequencing authentication of Bombyx batryticatus using species-specific primers.

Nat Prod Res 2019 May 23;33(9):1251-1256. Epub 2018 Apr 23.

a School of Pharmacy , Jiangsu University , Zhenjiang , China.

A novel PCR technology was developed to detect short DNA fragments using species-specific primers for rapid and non-sequencing authentication of Bombyx batryticatus based on differences in the mitochondrial genome. Three specifically designed primer reactions were established to target species for the reliable identification of their commercial products. They were confirmed to have a high inter-species specificity and intra-species stability. The limit of detection was estimated as 1 ng of genomes for Beauveria bassiana and 100 pg for Bombyx mori and Metarhizium anisopliae. Furthermore, validation results demonstrated that raw materials and their processed products can be conveniently authenticated with good sensitivity and precision using this newly proposed approach. In particular, when counterfeits were assayed, these primer sets performed well, whereas COI barcoding technology did not. These could also assist in the discrimination and identification of adulterates of other animal-derived medicines in their pulverized and processed forms and even in complexes.
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http://dx.doi.org/10.1080/14786419.2018.1466127DOI Listing
May 2019

Peptidomic Identification of Cysteine-Rich Peptides from Plants.

Methods Mol Biol 2018 ;1719:379-393

School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

Plant cysteine-rich peptides (CRPs) constitute a majority of plant-derived peptides with high molecular diversity. This protocol describes a rapid and efficient peptidomic approach to identify a whole spectrum of CRPs in a plant extract and decipher their molecular diversity and bioprocessing mechanism. Cyclotides from C. ternatea are used as the model CRPs to demonstrate our methodology. Cyclotides exist naturally in both cyclic and linear forms, although the linear forms (acyclotide) are generally present at much lower concentrations. Both cyclotides and acyclotides require linearization of their backbone prior to fragmentation and sequencing. A novel and practical three-step chemoenzymatic treatment was developed to linearize and distinguish both forms: (1) N-terminal acetylation that pre-labels the acyclotides; (2) conversion of Cys into pseudo-Lys through aziridine-mediated S-alkylation to reduce disulfide bonds and to increase the net charge of peptides; and (3) opening of cyclic backbones by the novel asparaginyl endopeptidase butelase 2 that cleaves at the native bioprocessing site. The treated peptides are subsequently analyzed by liquid chromatography coupled to mass spectrometry using electron transfer dissociation fragmentation and sequences are identified by matching the MS/MS spectra directly with the transcriptomic database.
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http://dx.doi.org/10.1007/978-1-4939-7537-2_26DOI Listing
January 2019

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

Front Plant Sci 2017 22;8:2162. Epub 2017 Dec 22.

School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

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 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 , and 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 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.
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http://dx.doi.org/10.3389/fpls.2017.02162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743680PMC
December 2017

β-Ginkgotides: Hyperdisulfide-constrained peptides from Ginkgo biloba.

Sci Rep 2017 07 21;7(1):6140. Epub 2017 Jul 21.

School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Singapore.

Hyperdisulfide-constrained peptides are distinguished by their high stability and diverse functions. Thus far, these peptides have been reported from animals only but their occurrence in plants are rare. Here, we report the discovery, synthesis and characterization of a hyperdisulfide-constrained peptides family of approximately 2 kDa, β-ginkgotides (β-gB1 and β-gB2) from Ginkgo biloba. Proteomic analysis showed β-ginkgotides contain 18‒20 amino acids, of which 16 residues form a conserved six-cysteine core with a highly clustered cysteine spacing of C‒CC‒C‒CC, an arrangement that has not been reported in cysteine-rich peptides. Disulfide mapping revealed a novel disulfide connectivity of CysI‒IV, CysII‒VI and CysIII‒V. Oxidative folding of synthetic β-gB1 to the native form was obtained in 70% yield. The synthetic β-gB1 displays a compact structure with no regular secondary structural elements, as determined by NMR spectroscopy. Transcriptomic analysis showed precursor βgb1 has a four-domain architecture and revealed an additional 76 β-ginkgotide-like peptides in 59 different gymnosperms, but none in angiosperms. Phylogenetic clustering analysis demonstrated β-ginkgotides belong to a new cysteine-rich peptide family. β-Ginkgotide is resistant to thermal, chemical and proteolytic degradation. Together, β-ginkgotides represent the first-in-class hyperdisulfide-constrained peptide family from plants with a novel scaffold that could be useful for engineering metabolically stable peptidyl therapeutics.
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http://dx.doi.org/10.1038/s41598-017-06598-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522442PMC
July 2017