James P. Tam

Nanyang Technological University, Tumasik, Singapore

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Publications (207)387.61 Total impact

  • James P Tam · Shujing Wang · Ka H Wong · Wei Liang Tan ·
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    ABSTRACT: Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms.
    Pharmaceuticals 11/2015; 8(4):711-757. DOI:10.3390/ph8040711
  • Giang K T Nguyen · Yuan Cao · Wei Wang · Chuan Fa Liu · James P Tam ·
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    ABSTRACT: An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields.
    Angewandte Chemie International Edition 11/2015; DOI:10.1002/anie.201506810 · 11.26 Impact Factor
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    ABSTRACT: Cysteine-rich peptides (CRPs) are natural products with privileged peptidyl structures that represent a potentially rich source of bioactive compounds. Here, the discovery and characterization of a novel plant CRP family, jasmintides from Jasminum sambac of the Oleaceae family, are described. Two 27-amino acid jasmintides (jS1 and jS2) were identified at the gene and protein levels. Disulfide bond mapping of jS1 by mass spectrometry and its confirmation by NMR spectroscopy revealed disulfide bond connectivity of C-1-C-5, C-2-C-4, and C-3-C-6, a cystine motif that has not been reported in plant CRPs. Structural determination showed that jS1 displays a well-defined structure framed by three short antiparallel β-sheets. Genomic analysis showed that jasmintides share a three-domain precursor arrangement with a C-terminal mature domain preceded by a long pro-domain of 46 residues and an intron cleavage site between the signal sequence and pro-domain. The compact cysteine-rich structure together with an N-terminal pyroglutamic acid residue confers jasmintides high resistance to heat and enzymatic degradation, including exopeptidase treatment. Collectively, these results reveal a new plant CRP structure with an unusual cystine connectivity, which could be useful as a scaffold for designing peptide drugs.
    Journal of Natural Products 11/2015; DOI:10.1021/acs.jnatprod.5b00762 · 3.80 Impact Factor
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    ABSTRACT: Cystine knot α-amylase inhibitors are cysteine-rich, proline-rich peptides found in the Amaranthaceae and Apocynaceae plant species. They are characterized by a pseudocyclic backbone with 2-4 prolines and three disulfides arranged in a knotted motif. Similar to other knottins, cystine knot α-amylase inhibitors are highly resistant to degradation by heat and protease treatments. Thus far, only the α-amylase inhibition activity has been described for members of this family. Here, we show that cystine knot α-amylase inhibitors named alstotides discovered from the Alstonia scholaris plant of the Apocynaceae family display antiviral activity. The alstotides (As1-As4) were characterized by both proteomic and genomic methods. All four alsotides are novel, heat- and enzyme-stable and contain 30 residues. NMR determination of As1 and As4 structures reveals their conserved structural fold and the presence of one or more cis proline bonds, characteristics shared by other cystine knot α-amylase inhibitors. Genomic analysis showed that they contain a three-domain precursor, an arrangement common to other knottins. We also showed that alstotides are antiviral and cell-permeable to inhibit the early phase of infectious bronchitis virus and Dengue infection, in addition to their ability to inhibit α-amylase. Taken together, our results expand membership of cystine knot α-amylase inhibitors in the Apocynaceae family and their bioactivity, functional promiscuity which could be exploited as leads in developing therapeutics.
    Journal of Biological Chemistry 11/2015; DOI:10.1074/jbc.M115.654855 · 4.57 Impact Factor
  • Geeta Kumari · Aida Serra · Shin Joon · James P Tam ·

    10th Annual Peptide Therapeutic Symposium; 10/2015
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    ABSTRACT: Hevein-like peptides are a family of cysteine-rich peptides (CRPs) and play a defensive role in plants against insects and fungal pathogens. In this study, we report isolation and characterization of six hevein-like peptides, aSG1-G3 and aSR1-R3, collectively named altides from green and red varieties of Alternanthera sessilis, a perennial herb belonging to the Amaranthaceae family. Proteomic analysis of altides revealed they contain six cysteines (6C), seven glycines, four prolines, and a conserved chitin-binding domain (SXYGY/SXFGY). Thus far, only four 6C-hevein-like peptides have been isolated and characterized; hence, our study expands the existing library of these peptides. Nuclear magnetic resonance (NMR) study of altides showed its three disulfide bonds were arranged in a cystine knot motif. As a consequence of this disulfide arrangement, they are stable against thermal and enzymatic degradation. Gene cloning studies revealed altides contain a three-domain precursor with an endoplasmic reticulum signal peptide followed by a mature CRP domain and a short C-terminal tail. This indicates that the biosynthesis of altides is through the secretory pathway. 1H-NMR titration experiments showed that the 29-30 amino-acid-long altides bind to β-(1-4)-linked N-acetylglucosamine (GlcNAc) oligomers with dissociation constants in the micromolar range. Aromatic residues in the chitin-binding domain of altides were involved in the binding interaction. To our knowledge, aSR1 is the smallest hevein-like peptide with dissociation constant towards (GlcNAc)3 comparable to hevein and other hevein-like peptides. Together, our study expands the existing library of 6C-hevein-like peptides and provides insights into their structure, biosynthesis, and interaction with GlcNAc oligomers.
    Biochemistry 10/2015; DOI:10.1021/acs.biochem.5b00872 · 3.02 Impact Factor
  • Yuan Cao · Giang K T Nguyen · James P Tam · Chuan-Fa Liu ·
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    ABSTRACT: Protein thioesters are versatile intermediates for various ligation schemes. Currently, protein thioesters are prepared by intein-mediated technology. Using a recently discovered peptide ligase, butelase 1, we developed a complementary method to prepare protein thioesters in good yield. We successfully combined it with native chemical ligation and sortase-mediated ligation in tandem for protein C-terminal labeling and dual-terminal labeling through exploiting the orthogonality of these three ligation methods.
    Chemical Communications 10/2015; DOI:10.1039/c5cc07227a · 6.83 Impact Factor
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    Si Min Zhang · Alenka Jejcic · James P Tam · Anders Vahlne ·
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    ABSTRACT: The membrane proximal external region (MPER) is a highly conserved membrane-active region located at the juxtamembrane positions within class I viral fusion glycoproteins and essential for membrane fusion events during viral entry. The MPER in the human immunodeficiency virus type I (HIV-1) envelope protein (Env) interacts with the lipid bilayers through a cluster of tryptophan (Trp) residues and a C-terminal cholesterol-interacting motif. The inclusion of the MPER N-terminal sequence contributes to the membrane reactivity and anti-viral efficacy of the first two anti-HIV peptidyl fusion inhibitors T20 and T1249. As a type I transmembrane protein, Env also interacts with the cellular membranes during its biosynthesis and trafficking. Here we investigated the roles of MPER membrane-active sequences during both viral entry and assembly, specifically, their roles in the design of peptidyl fusion inhibitors and the biosynthesis of viral structural proteins. We found that elimination of the membrane-active elements in MPER peptides, namely, penta Trp→alanine (Ala) substitutions and the disruption of the C-terminal cholesterol-interacting motif through deletion inhibited the anti-viral effect against the pseudotyped HIV-1. Furthermore, as compared to C-terminal dimerization, N-terminal dimerization of MPER peptides and N-terminal extension with five helix-forming residues enhanced their anti-viral efficacy substantially. The secondary structure study revealed that the penta-Trp→Ala substitutions also increased the helical content in the MPER sequence, which prompted us to study the biological relevance of such mutations in pre-fusion Env. We observed that Ala mutations of Trp664, Trp668 and Trp670 in MPER moderately lowered the intracellular and intraviral contents of Env while significantly elevating the content of another viral structural protein, p55/Gag and its derivative p24/capsid. The data suggest a role of the gp41 MPER in the membrane-reactive events during both viral entry and budding, and provide insights into the future development of anti-viral therapeutics.
    PLoS ONE 08/2015; 10(7):e0134851. DOI:10.1371/journal.pone.0134851 · 3.23 Impact Factor
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    ABSTRACT: Gelatinous Chinese medicines made from mammalian skin or horn or reptile shell are a very important type of animal-derived Chinese medicine. They have been extensively used either as both hemopoietic and hemostatic agents to treat vertigo, palpitation, hematuria, and insomnia in traditional Chinese medicine clinics; consumed as a popular tonic for weaker persons such as the elderly or women after giving birth; or further manufactured to health supplements for certain populations. However, they cannot be discriminated from each other by only using the routine approach in the Chinese Pharmacopoeia, as it lacks enough specificity and, consequently, and the requirements can be met even by adding assayed ingredients. In this study, our efforts to differentiate three gelatinous Chinese medicines, Asini Corii Colla, Cervi Cornus Colla, and Testudinis Carapacis ET Plastri Colla, are presented, and a novel strategy based on enzymatic digestion followed by nano-flow liquid chromatography in tandem with orbitrap mass spectrum detector analysis is proposed herein. Fourteen diagnostic fragments identified from the digests of these medicines were exclusively selected for their discrimination. By taking advantage of the favorable features of this strategy, it is feasible and convenient to identify enzymatic-digested peptides originated from signature proteins in each medicine, which thus could be employed as potential biomarkers for their form of raw medicinal material, and the pulverized and the complex especially, that being the direct basis for authentication purpose.
    International Journal of Nanomedicine 08/2015; 10:4947. DOI:10.2147/IJN.S82291 · 4.38 Impact Factor
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    Bamaprasad Dutta · Yan Ren · Sai Kiang Lim · James P Tam · Siu Kwan Sze ·

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    ABSTRACT: Cystine knot α-amylase inhibitors belong to a knottin family of peptidyl inhibitors of 30-32 residues and contain two to four prolines. Thus far, only four members of the group of cystine knot α-amylase inhibitors have been characterized. Herein, the discovery and characterization of five cystine knot α-amylase inhibitors, allotides C1-C5 (Ac1-Ac5) (1-5), from the medicinal plant Allamanda cathartica are reported using both proteomic and genomic methods. Proteomic analysis showed that 1-5 are 30 amino acids in length with three or four proline residues. NMR determination of 4 revealed that it has two cis- and one trans-proline residues and adopts two equally populated conformations in solution. Determination of disulfide connectivity of 2 by differential S-reduction and S-alkylation provided clues of its unfolding process. Genomic analysis showed that allotide precursors contain a three-domain arrangement commonly found in plant cystine knot peptides with conserved residues flanking the processing sites of the mature allotide domain. This work expands the number of known cystine knot α-amylase inhibitors and furthers the understanding of both the structural and biological diversity of this type of knottin family.
    Journal of Natural Products 04/2015; 78(4). DOI:10.1021/np500866c · 3.80 Impact Factor
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    Ying Liao · Si Min Zhang · Tuan Ling Neo · James P Tam ·
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    ABSTRACT: The spike (S) protein of severe acute respiratory syndrome-associated CoV (SARS-CoV) mediates membrane fusion and viral entry. These events involve structural rearrangements, including heteromerization between two heptad repeats (HR1 and HR2) to form a trimer-of dimer as a six-helix-bundle (6-HB), a quaternary protein structure which brings two distant clusters of hydrophobic sequences to proximity, the internal fusion peptide (IFP) preceding the HR1 and the highly conserved tryptophan (Trp)-rich membrane proximal external region (MPER) following the HR2. Here, we show that MPER can undergo self-oligomerization and heteromerization with IFP, events that are Trp-dependent. To delineate the roles of Trp residues of MPER in forming these quaternary structures and interacting with membranes, we employed a panel of synthetic peptides: MPER peptide (M-wt) and its alanine (Ala) or phenylalanine (Phe) analogs. Ala substitutions of Trp inhibited its association to cellular membranes. Chemical cross-linking experiments showed that M-wt can self-interact to form oligomers and cross-interact with IFP23, a synthetic IFP peptide, to form a hetero-hexamer. In comparison, little high-order oligomer was formed between M-wt and fusion peptide. The specific interaction between M-wt and IFP23 was confirmed by immunofluorescence staining experiments. In aqueous solutions, both M-wt and IFP23 displayed random secondary structures which became helical in hydrophobic solvents. Triple-Ala substitutions of Trp in M-wt, but not the corresponding triple-Phe analog, disrupted oligomerization of M-wt and hetero-oligomerization of M-wt with IFP23. Overall, our results show that Trp residues of MPER play a key role in maintaining the structure and functions of MPER, enabling it to interact with IFP to form a MPER-IFP heteromer, a putative quaternary structure extending from the 6-HB and function in membrane fusion. Finally, we showed that a MPER peptide could serve as an inhibitor in the entry process.
    Biochemistry 02/2015; 54(9). DOI:10.1021/bi501352u · 3.02 Impact Factor
  • Piliang Hao · Yan Ren · Arnab Datta · James P Tam · Siu Kwan Sze ·
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    ABSTRACT: Nonenzymatic deamidation occurs readily under the condition of trypsin digestion, resulting in the identification of many artificial deamidation sites. To evaluate the effect of trypsin digestion buffers on artificial deamidation, we compared the commonly used buffers of Tris-HCl (pH 8), ammonium bicarbonate (ABB) and triethylammonium bicarbonate (TEAB), and ammonium acetate (pH 6) which was reported to reduce Asn deamidation. iTRAQ quantification on rat kidney tissue digested in these four buffers indicates that artificial Asn deamidation is produced in the order of ammonium acetate<Tris-HCl<ABB<TEAB, and Gln deamidation has no significant differences in all tested buffers. Label-free experiments show the same trend, while protein and unique peptide identification are comparable during using these four buffers. To explain the differences of these four buffers in producing artificial Asn deamidation, we determined the half-time of Asn deamidation in these buffers using synthetic peptides containing -Asn-Gly- sequences. It is 51.4±6.0 days in 50 mM of ammonium acetate (pH 6) at 37ºC, which is about 23, 104 and 137 times of that in Tris-HCl, ABB and TEAB buffers, respectively. In conclusion, ammonium acetate (pH 6) is more suitable than other tested buffers for characterizing endogenous deamidation and N-glycosylation.
    Journal of Proteome Research 12/2014; 14(2). DOI:10.1021/pr500903b · 4.25 Impact Factor
  • Xinya Hemu · Yibo Qiu · James P. Tam ·
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    ABSTRACT: Cyclic cysteine peptides are peptide macrocycles endowed with enhanced metabolic stability and potentially, with membrane permeability. They have attracted attention in drug design and interest in their synthesis. The chemical approach for macrocyclization through transpeptidation bears striking similarity to the biological approach using an intein. Both use a similar design of thioester precursors and an amide-to-amide transpeptidation scheme, employing a series of acyl shifts to break and make amide bonds. Here we describe the synthesis of two cyclic cysteine peptides, hedyotide B1 and sunflower trypsin inhibitor-1, highlighting the similarities between the intein-based and chemical amide-to-amide schemes. In our intein-based and chemical schemes, we employed an intein Mxe or a thioethylbutylamido linkage at the C-terminus of their linear precursors, respectively. Our results demonstrated that the chemical approach provides a useful alternative to the intein approach with high efficiency.
    Tetrahedron 10/2014; 70(42):7707–7713. DOI:10.1016/j.tet.2014.05.112 · 2.64 Impact Factor
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    Bamaprasad Dutta · Yan Ren · Sai Kiang Lim · James P Tam · Siu Kwan Sze ·
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    ABSTRACT: In contrast to the intensely studied genetic and epigenetic changes that induce host cell transformation to initiate tumor development, those that promote malignant progression of cancer remain poorly defined. As emerging evidence suggests that the hypoxic tumor microenvironment could re-model chromatin-associated proteome (chromatome) to induce epigenetic changes and alter gene expression in cancer cells, we hypothesized that hypoxia-driven evolution of the chromatome promotes malignant changes and development of therapy resistance in tumor cells. To test this hypothesis, chromatins were isolated from tumor cells treated with varying conditions of normoxia, hypoxia and re-oxygenation, partially digested with DNase I and analyzed for changes in euchromatin- and heterochromatin-associated proteins using quantitative iTRAQ-based quantitative proteomic approach. We identified a total of 1446 proteins at a high level of confidence, including 819 proteins that were observed to change their chromatin association topology under hypoxic conditions. These hypoxia-sensitive proteins included key mediators of chromatin organization, transcriptional regulation and DNA repair. Furthermore, our proteomic and functional experiments revealed a novel role for the chromatin organizer protein HP1BP3 in mediating chromatin condensation during hypoxia, leading to increased tumor cell viability, radio-resistance, chemo-resistance and self-renewal. Taking together, our findings indicate that HP1BP3 is a key mediator of tumor progression and cancer cell acquisition of therapy-resistant traits, and may thus represent a novel therapeutic target in a range of human malignancies.
    Molecular &amp Cellular Proteomics 08/2014; 13(12). DOI:10.1074/mcp.M114.038232 · 6.56 Impact Factor
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    ABSTRACT: Proteases are ubiquitous in nature, whereas naturally occurring peptide ligases, enzymes catalyzing the reverse reactions of proteases, are rare occurrences. Here we describe the discovery of butelase 1, to our knowledge the first asparagine/aspartate (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 shares 71% sequence identity and the same catalytic triad with legumain proteases but does not hydrolyze the protease substrate of legumain. Instead, butelase 1 cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s(-1) and catalytic efficiencies as high as 542,000 M(-1) s(-1), butelase 1 is the fastest peptide ligase known. Notably, butelase 1 also displays broad specificity for the N-terminal amino acids of the peptide substrate, thus providing a new tool for C terminus-specific intermolecular peptide ligations.
    Nature Chemical Biology 07/2014; 10(9). DOI:10.1038/nchembio.1586 · 13.00 Impact Factor
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    ABSTRACT: Obesity and type-2 diabetes are chronic metabolic diseases that could be benefited by the use of α-amylase inhibitors to manage starch intake. The pseudocyclics, wrightides Wr-AI1 to Wr-AI3, isolated from an Apocynaceae plant show promising potentials for further development as orally active α-amylase inhibitors. These linear peptides retain the stability known for cystine knot peptides in harsh treatment. They are resistant to treatment by heat, endopeptidase or exopeptidase, characteristics of cyclic cystine knot peptides. Our NMR and crystallography analysis also showed that wrightides, currently the smallest proteinaceous α -amylase inhibitors reported, contain the backbone-twisting cis proline which is preceded by a non-aromatic residue rather than a conventional aromatic residue. Modeled structure and molecular dynamics study of Wr-AI1 in complex with yellow meal worm α-amylase suggested that despite similar structure and cystine knot fold, members of knottin-type α-amylase inhibitors may bind to insect α-amylase via a different set of interactions. Finally, we showed that the precursors of pseudocyclic cystine knot α-amylase inhibitors and their biosynthesis in plants follow secretory protein synthesis pathway. Together, our work provides insights for the use of the pseudocyclic α-amylase inhibitors as useful leads for developing orally active peptidyl bioactives as well as an alternative scaffold to cyclic peptides for engineering metabolic-stable human α-amylase inhibitors.This article is protected by copyright. All rights reserved.
    FEBS Journal 07/2014; 281(19). DOI:10.1111/febs.12939 · 4.00 Impact Factor
  • Yibo Qiu · Xinya Hemu · Ding Xiang Liu · James P. Tam ·
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    ABSTRACT: A selective bi-directional peptide bond cleavage mediated by N-methylcysteine (MeCys) in Xaa-MeCys-Yaa peptides (Xaa and Yaa, non-cysteine residues) leading to thioesters and thiolactones is described. Rate and product analyses showed that an Nα-amide bond cleavage occurred at the Xaa-MeCys bond by an N–S acyl shift to generate an Xaa-S-(MeCys-Yaa) thioester at pH 1–5, whereas under strongly acidic conditions of H0 = –5, the MeCys-Yaa bond underwent a Cα-amide bond cleavage via an oxazolone intermediate, which was trapped by thiocresol (TC) as an Xaa-MeCys-TC thioester. This thioester was then transformed into an Xaa-MeCys-β-thiolactone at pH 4–5. Replacing MeCys by a Cys residue did not result in significant bi-directional peptide bond cleavage, which suggests that N-methylation in a MeCys residue is important for the N–S acyl shift reaction and formation of oxazolone. The isomerization of amides and thioesters was successfully used to prepare cyclic peptides.
    European Journal of Organic Chemistry 07/2014; 2014(20). DOI:10.1002/ejoc.201402261 · 3.07 Impact Factor
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    ABSTRACT: The chromatin-associated proteome (chromatome) regulates cellular gene expression by restricting access of transcriptional machinery to template DNA, and dynamic re-modeling of chromatin structure is required to regulate critical cell functions including growth and replication, DNA repair and recombination, and oncogenic transformation in progression to cancer. Central to the control of these processes is efficient regulation of the host cell cycle, which is maintained by rapid changes in chromatin conformation during normal cycle progression. A global overview of chromatin protein organization is therefore essential to fully understand cell cycle regulation, but the influence of the chromatome and chromatin binding topology on host cell cycle progression remains poorly defined. Here we used partial MNase digestion together with iTRAQ-based high-throughput quantitative proteomics to quantify chromatin-associated proteins during interphase progression. We identified a total of 481 proteins with high confidence that were involved in chromatin-dependent events including transcriptional regulation, chromatin re-organization, and DNA replication and repair, while the quantitative data revealed the temporal interactions of these proteins with chromatin during interphase progression. When combined with biochemical and functional assays, these data revealed a strikingly dynamic association of protein HP1BP3 with the chromatin complex during different stages of interphase, and uncovered a novel regulatory role for this molecule in transcriptional regulation. We report that HP1BP3 protein maintains heterochromatin integrity during G1-S progression and regulates the duration of G1 phase to critically influence cell proliferative capacity.
    Molecular &amp Cellular Proteomics 05/2014; 13(9). DOI:10.1074/mcp.M113.034975 · 6.56 Impact Factor
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    ABSTRACT: Unlabelled: Secretion of bioactive mediators regulates cell interactions with the microenvironment in tissue homeostasis and wound healing processes. We assessed the cardiomyocyte secretory response to hypoxia with the aim of identifying key mediators of tissue pathology and repair after ischemic heart attack. We profiled the secretome of rat H9C2 cardiomyoblast cells subjected to 16h hypoxia followed by 24h re-oxygenation using iTRAQ and label-free quantitative proteomics. A total of 860 and 2007 proteins were identified in the iTRAQ and label-free experiments respectively. Among these proteins, 1363 were identified as being secreted proteins, including mediators of critical cellular functions that were modulated by hypoxia/re-oxygenation stress (SerpinH1, Ppia, Attractin, EMC1, Postn, Thbs1, Timp1, Stip1, Robo2, Fat1). Further analysis indicated that hypoxia is associated with angiogenesis, inflammation, and remodeling of the extracellular matrix (ECM), whereas subsequent re-oxygenation was instead associated with modified secretion of proteins involved in suppression of inflammation, ECM modification, and decreased output of anti-apoptosis proteins. These data indicate that hypoxia and subsequent re-oxygenation modify the cardiomyocyte secretome in order to mitigate cellular injury and promote healing. The identified changes in cardiomyocyte secretome advance our current understanding of cardiac biology in ischemia/reperfusion injury and may lead to the identification of novel prognostic biomarker. Biological significance: Cardiovascular diseases (CVDs) are the leading cause of death globally. Myocardial infarction (MI) resulting from ischemic heart disease represents a substantial component of CVD-associated mortality, and is associated with obstruction of blood flow to the myocardium. Restoration of blood flow through the occluded coronary artery is the current most effective therapy to limit infarct size and preserve cardiac function after acute myocardial infarction. However, this treatment does not prevent subsequent development of heart failure in some patients. Reperfusion following ischemia causes additional cell death and increase in infarct size, a phenomenon called myocardial ischemia/reperfusion (I/R) injury. In order to advance our current understanding of cardiac biology in ischemia/reperfusion injury, we assessed the cardiomyocyte secretory response to hypoxia with the aim of identifying key mediators of tissue pathology and repair after ischemic heart attack. We profiled the secretome of rat H9C2 cardiomyoblast cells subjected to 16h hypoxia followed by 24h re-oxygenation using LC-MS/MS-based iTRAQ and label-free quantitative proteomics approaches. We identified many secreted proteins as mediators of critical cellular functions that were modulated by hypoxia and re-oxygenation stress. Further analysis of these modulated secretory proteins indicated that hypoxia is associated with angiogenesis, inflammation, and remodeling of the extracellular matrix (ECM), whereas subsequent re-oxygenation/reperfusion was instead associated with modified secretion of proteins involved in suppression of inflammation, ECM modification, and decreased output of anti-apoptosis proteins. These data indicate that hypoxia and subsequent re-oxygenation modify the cardiomyocyte secretome in order to mitigate cellular injury and promote healing. The identified changes in cardiomyocyte secretome may lead to the identification of novel prognostic biomarkers secreted from injured heart tissues into the circulation of patients with cardiovascular disease.
    Journal of proteomics 01/2014; 98. DOI:10.1016/j.jprot.2013.12.025 · 3.89 Impact Factor

Publication Stats

9k Citations
387.61 Total Impact Points


  • 2005-2015
    • Nanyang Technological University
      • School of Biological Sciences
      Tumasik, Singapore
  • 1994-2010
    • Vanderbilt University
      • Department of Pathology, Microbiology and Immunology
      Nashville, Michigan, United States
  • 2005-2009
    • The Scripps Research Institute
      • Department of Chemistry
      لا هویا, California, United States
  • 1977-2009
    • The Rockefeller University
      • Laboratory of Mass Spectrometry and Gaseous Ion Chemistry
      New York City, New York, United States
  • 1989
    • Weill Cornell Medical College
      • Department of Medicine
      New York, New York, United States
    • IT University of Copenhagen
      København, Capital Region, Denmark
  • 1984
    • NCI-Frederick
      Фредерик, Maryland, United States