Proteomic Investigation of the Antibacterial Mechanism of trans-Cinnamaldehyde against Escherichia coli.

Trans-Cinnamaldehyde (TC) is a widely used food additive, known for its sterilization, disinfection, and antiseptic properties. However, its antibacterial mechanism is not completely understood. In this study, quantitative proteomics was performed to investigate differentially expressed proteins (DEPs) in Escherichia coli in response to TC treatment. Bioinformatics analysis suggested aldehyde toxicity, acid stress, oxidative stress, interference of carbohydrate metabolism, energy metabolism, and protein translation as the bactericidal mechanism. E. coli BW25113ΔyqhD, ΔgldA, ΔbetB, ΔtktB, ΔgadA, ΔgadB, ΔgadC, and Δrmf were used to investigate the functions of DEPs through biochemical methods. The present study revealed that TC exerts its antibacterial effects by inducing the toxicity of its aldehyde group producing acid stress. These findings will contribute to the application of TC in the antibacterial field.

A hidden human proteome encoded by 'non-coding' genes.

It has been a long debate whether the 98% 'non-coding' fraction of human genome can encode functional proteins besides short peptides. With full-length translating mRNA sequencing and ribosome profiling, we found that up to 3330 long non-coding RNAs (lncRNAs) were bound to ribosomes with active translation elongation. With shotgun proteomics, 308 lncRNA-encoded new proteins were detected. A total of 207 unique peptides of these new proteins were verified by multiple reaction monitoring (MRM) and/or parallel reaction monitoring (PRM); and 10 new proteins were verified by immunoblotting. We found that these new proteins deviated from the canonical proteins with various physical and chemical properties, and emerged mostly in primates during evolution. We further deduced the protein functions by the assays of translation efficiency, RNA folding and intracellular localizations. As the new protein UBAP1-AST6 is localized in the nucleoli and is preferentially expressed by lung cancer cell lines, we biologically verified that it has a function associated with cell proliferation. In sum, we experimentally evidenced a hidden human functional proteome encoded by purported lncRNAs, suggesting a resource for annotating new human proteins.

Quantitative Mitochondrial Proteomics Reveals ANXA7 as a Crucial Factor in Mitophagy.

Mitochondria are involved in many crucial cellular processes. Maintaining healthy mitochondria is essential for cellular homeostasis. Parkin-dependent mitophagy plays an important role in selectively eliminating damaged mitochondria in mammalian cells. However, mechanisms of Parkin-dependent mitophagy remain elusive. In this research, we performed data-independent acquisition-based quantitative mitochondrial proteomics to study the proteomic alterations of carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced Parkin-mediated mitophagy. We identified 222 differentially expressed proteins, with 76 upregulations and 146 downregulations, which were potentially involved in mitophagy. We then demonstrated that annexin A7 (ANXA7), a calcium-dependent phospholipid-binding protein, can translocate to impaired mitochondria upon CCCP treatment, where it played a pivotal part in the process of Parkin-dependent mitophagy via interacting with BASP1. As a mitochondrial uncoupling agent, CCCP indirectly regulated ANXA7 and BASP1 to induce Parkin-dependent mitophagy.

2H-Azirine-Based Reagents for Chemoselective Bioconjugation at Carboxyl Residues Inside Live Cells.

Protein modification by chemical reagents has played an essential role in the treatment of human diseases. However, the reagents currently used are limited to the covalent modification of cysteine and lysine residues. It is thus desirable to develop novel methods that can covalently modify other residues. Despite the fact that the carboxyl residues are crucial for maintaining the protein function, few selective labeling reactions are currently available. Here, we describe a novel reactive probe, 3-phenyl-2H-azirine, that enables chemoselective modification of carboxyl groups in proteins under both in vitro and in situ conditions with excellent efficiency. Furthermore, proteome-wide profiling of reactive carboxyl residues was performed with a quantitative chemoproteomic platform.

Proteomic Analysis Reveals that Odoroside A Triggers G2/M Arrest and Apoptosis in Colorectal Carcinoma Through ROS-p53 Pathway.

Odoroside A (OA) is an active ingredient extracted from the leaves of Nerium oleander Linn. (Apocynaceae). This study aims to examine the anticancer bioactivity of OA against CRC cells and to investigate the action mechanisms involved. As a result, OA can significantly inhibit cellular ability and induce apoptosis of CRC cells in a concentration-dependent manner without any obvious cytotoxicity in normal colorectal epithelial cells. Then, quantitative proteomics combined with bioinformatics is adopted to investigate the alterations of proteins and signaling pathways in response to OA treatment. As suggested by the proteomic analysis, flow cytometry and Western blotting analyses validate that exposure of CRC cells to OA causes cell cycle arrest and apoptosis, accompanied with the activation of the ROS/p53 signaling pathway. This observation demonstrates that OA, as a natural product, can induce oxidative stress to suppress tumor cell growth, implicating a novel therapeutic agent against CRC without obvious side effects.

Crenolanib-Derived Probes Suitable for Cell- and Tissue-Based Protein Profiling and Single-Cell Imaging.

Crenolanib (CP-868,596), a potent inhibitor of FLT3 and PDGFRα/ß, is currently under phase III clinical investigation for the treatment of acute myeloid leukemia. However, the protein targets of Crenolanib in cancer cells remain obscure, which results in difficulties in understanding the mechanism of actions and side effects. To alleviate this issue, in this study, a photoaffinity probe and two fluorescent probes were created based on Crenolanib, followed by competitive protein profiling and bioimaging studies, with the aim of characterizing the cellular targets. A series of unknown protein hits, such as MAPK1, SHMT2, SLC25A11, and HIGD1A, were successfully identified by means of pull-down/LC-MS/MS; these might provide valuable clues for understanding drug action and potential toxicities. Moreover, the fluorescent probes are suitable for imaging drug distribution at the single-cell level.

Detergent-Insoluble Proteome Analysis Revealed Aberrantly Aggregated Proteins in Human Preeclampsia Placentas.

Preeclampsia (PE) is a placenta disease, featured by hypertension, proteinuria, and other multiorgan dysfunctions, and its etiology is unclear. We and others have shown that intensive endoplasmic reticulum (ER) stress and unfolded protein response (UPR) occur in the PE placenta. In this study, we isolated detergent-insoluble proteins (DIPs) from human placenta tissues, which were enriched with protein aggregates, to characterize the placenta UPR in PE. With data-independent acquisition (DIA) mass spectrometry, we identified 2066 DIPs across all normal (n = 10) and PE (n = 10) placenta samples, among which 110 and 108 DIPs were significantly up- and down-regulated in PE, respectively. Per clustering analysis, differential DIPs could generally distinguish PE from normal placentas. We verified the MS quantitation of endoglin and vimentin by immunoblotting. In addition, we observed that PE placenta tissues have remarkably more endoglin in the cytoplasm. Furthermore, we found that DIPs were evenly distributed across different chromosomes and could be enriched in diversified gene ontology terms, while differential DIPs avoided to distribute on X-chromosome. Significantly up-regulated DIPs in PE were focused on the top functions of lipid metabolism, while 23 of these DIPs could form the top network regulating cellular movement, development, growth, and proliferation. Our results implicate that human PE placentas have disease-relevant differential DIPs, which reflect aberrantly aggregated proteins of placental tissues. The mass spectrometry proteomics data have been deposited to ProteomeXchange consortium with the data set identifier PXD006654, and iProX database (accession number: IPX0000948000).

ACK1 promotes gastric cancer epithelial-mesenchymal transition and metastasis through AKT-POU2F1-ECD signalling.

Amplification of the activated Cdc42-associated kinase 1 (ACK1) gene is frequent in gastric cancer (GC). However, little is known about the clinical roles and molecular mechanisms of ACK1 abnormalities in GC. Here, we found that the ACK1 protein level and ACK1 phosphorylation at Tyr 284 were frequently elevated in GC and associated with poor patient survival. Ectopic ACK1 expression in GC cells induced epithelial-mesenchymal transition (EMT) and promoted migration and invasion in vitro, and metastasis in vivo; the depletion of ACK1 induced the opposite effects. We utilized SILAC quantitative proteomics to discover that the level of the cell cycle-related protein ecdysoneless homologue (ECD) was markedly altered by ACK1. Overexpression of ECD promoted EMT, migration, and invasion in GC, similar to the effects of ACK1 overexpression. Silencing of ECD completely blocked the augmentation of ACK1 overexpression-induced EMT, migration, and invasion. Mechanistically, ACK1 phosphorylated AKT at Thr 308 and Ser 473 and activated the AKT pathway to up-regulate the transcription factor POU2F1, which directly bound to the promoter region of its novel target gene ECD and thus regulated ECD expression in GC cells. Furthermore, the phosphorylation levels of AKT at Thr 308 and Ser 473 and POU2F1 and ECD levels were positively associated with ACK1 levels in clinical GC specimens. Collectively, we have demonstrated that ACK1 promotes EMT, migration, and invasion by activating AKT-POU2F1-ECD signalling in GC cells. ACK1 may be employed as a new prognostic factor and therapeutic target for GC.

Finding Missing Proteins from the Epigenetically Manipulated Human Cell with Stringent Quality Criteria.

The chromosome-centric human proteome project (C-HPP) has made great progress of finding protein evidence (PE) for missing proteins (PE2-4 proteins defined by the neXtProt), which now becomes an increasingly challenging field. As a majority of samples tested in this field were from adult tissues/cells, the developmental stage specific or relevant proteins could be missed due to biological source availability. We posit that epigenetic interventions may help to partially bypass such a limitation by stimulating the expression of the "silenced" genes in adult cells, leading to the increased chance of finding missing proteins. In this study, we established in vitro human cell models to modify the histone acetylation, demethylation, and methylation with near physiological conditions. With mRNA-seq analysis, we found that histone modifications resulted in overall increases of expressed genes in an even distribution manner across different chromosomes. We identified 64 PE2-4 and six PE5 proteins by MaxQuant (FDR < 1% at both protein and peptide levels) and 44 PE2-4 and 7 PE5 proteins by Mascot (FDR < 1% at peptide level) searches, respectively. However, only 24 PE2-4 and five PE5 proteins in Mascot, and 12 PE2-4 and one PE5 proteins in MaxQuant searches could, respectively, pass our stringently manual spectrum inspections. Collectively, 27 PE2-4 and five PE5 proteins were identified from the epigenetically modified cells; among them, 19 PE2-4 and three PE5 proteins passed FDR < 1% at both peptide and protein levels. Gene ontology analyses revealed that the PE2-4 proteins were significantly involved in development and spermatogenesis, although their chemical-physical features had no statistical difference from the background. In addition, we presented an example of suspicious PE5 peptide spectrum matched with unusual AA substitutions related to post-translational modification. In conclusion, the epigenetically manipulated cell models should be a useful tool for finding missing proteins in C-HPP. The mass spectrometry data have been deposited to the iProx database (accession number: IPX00020200).

Translating mRNAs strongly correlate to proteins in a multivariate manner and their translation ratios are phenotype specific.

As a well-known phenomenon, total mRNAs poorly correlate to proteins in their abundances as reported. Recent findings calculated with bivariate models suggested even poorer such correlation, whereas focusing on the translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) subset. In this study, we analysed the relative abundances of mRNAs, RNC-mRNAs and proteins on genome-wide scale, comparing human lung cancer A549 and H1299 cells with normal human bronchial epithelial (HBE) cells, respectively. As discovered, a strong correlation between RNC-mRNAs and proteins in their relative abundances could be established through a multivariate linear model by integrating the mRNA length as a key factor. The R(2) reached 0.94 and 0.97 in A549 versus HBE and H1299 versus HBE comparisons, respectively. This correlation highlighted that the mRNA length significantly contributes to the translational modulation, especially to the translational initiation, favoured by its correlation with the mRNA translation ratio (TR) as observed. We found TR is highly phenotype specific, which was substantiated by both pathway analysis and biased TRs of the splice variants of BDP1 gene, which is a key transcription factor of transfer RNAs. These findings revealed, for the first time, the intrinsic and genome-wide translation modulations at translatomic level in human cells at steady-state, which are tightly correlated to the protein abundance and functionally relevant to cellular phenotypes.

Two synonymous single-nucleotide polymorphisms promoting fluoroquinolone resistance of Escherichia coli in the environment.

Single-nucleotide polymorphism (SNP) is one of the core mechanisms that respond to antibiotic resistance of Escherichia coli (E. coli), which is a major issue in environmental pollution. A specific type of SNPs, synonymous SNPs, have been generally considered as the "silent" SNPs since they do not change the encoded amino acid. However, the impact of synonymous SNPs on mRNA splicing, nucleo-cytoplasmic export, stability, and translation was gradually discovered in the last decades. Figuring out the mechanism of synonymous SNPs in regulating antibiotic resistance is critical to improve antimicrobial therapy strategies in clinics and biological treatment strategies of antibiotic-resistant E. coli-polluted materials. With our newly designed antibiotic resistant SNPs prediction algorithm, Multilocus Sequence Type based Identification for Phenotype-single nucleotide polymorphism Analysis (MIPHA), and in vivo validation, we identified 2 important synonymous SNPs 522 G>A and 972 C>T, located at hisD gene, which was previously predicted as a fluoroquinolone resistance-related gene without a detailed mechanism in the E. coli samples with environmental backgrounds. We first discovered that hisD causes gyrA mutation via the upregulation of sbmC and its downstream gene umuD. Moreover, those 2 synonymous SNPs of hisD cause its own translational slowdown and further reduce the expression levels of sbmC and its downstream gene umuD, making the fluoroquinolone resistance determining region of gyrA remains unmutated, ultimately causing the bacteria to lose their ability to resist drugs. This study provided valuable insight into the role of synonymous SNPs in mediating antibiotic resistance of bacteria and a new perspective for the treatment of environmental pollution caused by drug-resistant bacteria.

Qualitative and quantitative expression status of the human chromosome 20 genes in cancer tissues and the representative cell lines.

Under the guidance of the Chromosome-centric Human Proteome Project (C-HPP), (1, 2) we conducted a systematic survey of the expression status of genes located at human chromosome 20 (Chr.20) in three cancer tissues, gastric, colon, and liver carcinoma, and their representative cell lines. We have globally profiled proteomes in these samples with combined technology of LC-MS/MS and acquired the corresponding mRNA information upon RNA-seq and RNAchip. In total, 323 unique proteins were identified, covering 60% of the coding genes (323/547) in Chr.20. With regards to qualitative information of proteomics, we overall evaluated the correlation of the identified Chr.20 proteins with target genes of transcription factors or of microRNA, conserved genes and cancer-related genes. As for quantitative information, the expression abundances of Chr.20 genes were found to be almost consistent in both tissues and cell lines of mRNA in all individual chromosome regions, whereas those of Chr.20 proteins in cells are different from tissues, especially in the region of 20q13.33. Furthermore, the abundances of Chr.20 proteins were hierarchically evaluated according to tissue- or cancer-related distribution. The analysis revealed several cancer-related proteins in Chr.20 are tissue- or cell-type dependent. With integration of all the acquired data, for the first time we established a solid database of the Chr.20 proteome.

Identification and analysis of glycogen synthase kinase 3 beta1 interactome.

Glycogen synthase kinase-3 beta (GSK3ß) was initially identified as a key protein in glucose metabolism. GSK3ß might be involved in cell growth, motility and apoptosis. Systematic identification of GSK3ß-associated proteins is crucial for the exhaustive understanding of its functions. Using GST pull-down experiment and LCMS/MS analysis coupled to bioinformatics tools, we have identified 114 proteins that interacted with GSK3ß1 in hepatocellular carcinoma HepG2 cells. Most of the identified proteins are implicated in metabolic process, whereas other proteins are important for cell proliferation or migration, and have been associated with cancer development and metastasis. Several representative proteins, such as hnRNPK, PCNA, Ezrin and STAT1, have been confirmed to interact with GSK-3ß1 by co-immunoprecipitation in HepG2 cells. Further studies of these interactions may discover the precise roles and the underlying mechanisms of GSK-3ß1 in tumour growth and metastasis.

Inhibition of nuclear deacetylase Sirtuin-1 induces mitochondrial acetylation and calcium overload leading to cell death.

Sirtuin-1 (SIRT1) is a critical nuclear deacetylase that participates in a wide range of biological processes. We hereby employed quantitative acetyl-proteomics to globally reveal the landscape of SIRT1-dependent acetylation in colorectal cancer (CRC) cells stimulated by specific SIRT1 inhibitor Inauhzin (INZ). We strikingly observed that SIRT1 inhibition enhances protein acetylation levels, with the multisite-acetylated proteins (acetyl sites >4/protein) mainly enriched in mitochondria. INZ treatment increases mitochondrial fission and depolarization in CRC cells. The acetylation of mitochondrial proteins promoted by SIRT1 inhibition prevents the recruitment of ubiquitin and LC3 for mitophagic degradation. We then found that, SIRT1 inhibition increases the acetylation of mitochondrial calcium uniporter (MCU) at residue K332, resulting in mitochondrial Ca2+ overload and depolarization, and ultimately CRC apoptosis. Arginine substitution of the K332 (K332R) dramatically decreases the mitochondrial Ca2+ influx, mitochondrial membrane potential loss and ROS burst induced by INZ. This finding uncovers a non-canonical role of SIRT1 in regulating mitochondrial function and implicates a possible way for anticancer intervention through SIRT1 inhibition.

Crizotinib Shows Antibacterial Activity against Gram-Positive Bacteria by Reducing ATP Production and Targeting the CTP Synthase PyrG.

Infections caused by drug-resistant bacteria are a serious threat to public health worldwide, and the discovery of novel antibacterial compounds is urgently needed. Here, we screened an FDA-approved small-molecule library and found that crizotinib possesses good antimicrobial efficacy against Gram-positive bacteria. Crizotinib was found to increase the survival rate of mice infected with bacteria and decrease pulmonary inflammation activity in an animal model. Furthermore, it showed synergy with clindamycin and gentamicin. Importantly, the Gram-positive bacteria showed a low tendency to develop resistance to crizotinib. Mechanistically, quantitative proteomics and biochemical validation experiments indicated that crizotinib exerted its antibacterial effects by reducing ATP production and pyrimidine metabolism. A drug affinity responsive target stability study suggested crizotinib targets the CTP synthase PyrG, which subsequently disturbs pyrimidine metabolism and eventually reduces DNA synthesis. Subsequent molecular dynamics analysis showed that crizotinib binding occurs in close proximity to the ATP binding pocket of PyrG and causes loss of function of this CTP synthase. Crizotinib is a promising antimicrobial agent and provides a novel choice for the development of treatment for Gram-positive infections. IMPORTANCE Infections caused by drug-resistant bacteria are a serious problem worldwide. Therefore, there is an urgent need to find novel drugs with good antibacterial activity against multidrug-resistant bacteria. In this study, we found that a repurposed drug, crizotinib, exhibits excellent antibacterial activity against drug-resistant bacteria both in vivo and in vitro via suppressing ATP production and pyrimidine metabolism. Crizotinib was found to disturb pyrimidine metabolism by targeting the CTP synthase PyrG, thus reducing DNA synthesis. This unique mechanism of action may explain the decreased development of resistance by Staphylococcus aureus to crizotinib. This study provides a potential option for the treatment of drug-resistant bacterial infections in the future.

Potential Role of Lysine Acetylation in Antibiotic Resistance of Escherichia coli.

Antibiotic resistance is increasingly becoming a challenge to public health. The regulation of bacterial metabolism by post-translational modifications (PTMs) has been widely studied. However, the mechanism underlying the regulation of acetylation in bacterial resistance to antibiotics is still unknown. Here, we performed a quantitative analysis of the acetylated proteome of a wild-type (WT) Escherichia coli (E. coli) sensitive strain and ampicillin- (Re-Amp), kanamycin- (Re-Kan), and polymyxin B-resistant (Re-Pol) strains. Based on bioinformatics analysis combined with biochemical validations, we found a common regulatory mechanism between the different resistant strains. Our results showed that protein acetylation negatively regulates bacterial metabolism to regulate antibiotic resistance and positively regulates bacterial motility. Further analyses revealed that key enzymes in various metabolic pathways were differentially acetylated. In particular, pyruvate kinase (PykF), a glycolytic enzyme that regulates bacterial metabolism, and its acetylated form were highly expressed in the three resistant strains and were identified as reversibly acetylated by the deacetylase CobB and the acetyl-transferase PatZ (peptidyl-lysine N-acetyltransferase). Results showed that PykF also could be acetylated by nonenzymatic acetyl phosphatase (AcP) in vitro. Furthermore, the deacetylation of Lys413 in PykF increased PykF enzymatic activity by changing the conformation of its ATP binding site, resulting in an increase in energy production which, in turn, increased the sensitivity of drug-resistant strains to antibiotics. This study provides novel insights for understanding bacterial resistance and lays the foundation for future research on the regulation of acetylation in antibiotic-resistant strains. IMPORTANCE The misuse of antibiotics has resulted in the emergence of many antibiotic-resistant strains which seriously threaten human health. Protein post-translational modifications, especially acetylation, tightly control bacterial metabolism. However, the comprehensive mechanism underlying the regulation of acetylation in bacterial resistance remains unexplored. Here, acetylation was found to positively regulate bacterial motility and negatively regulate energy metabolism, which was common in all antibiotic-resistant strains. Moreover, the acetylation and deacetylation process of PykF was uncovered, and deacetylation of the Lys 413 in PykF was found to contribute to bacterial sensitivity to antibiotics. This study provides a new direction for research on the development of bacterial resistance through post-translational modifications and a theoretical basis for developing antibacterial drugs.

Targeting PFKL with penfluridol inhibits glycolysis and suppresses esophageal cancer tumorigenesis in an AMPK/FOXO3a/BIM-dependent manner.

As one of the hallmarks of cancer, metabolic reprogramming leads to cancer progression, and targeting glycolytic enzymes could be useful strategies for cancer therapy. By screening a small molecule library consisting of 1320 FDA-approved drugs, we found that penfluridol, an antipsychotic drug used to treat schizophrenia, could inhibit glycolysis and induce apoptosis in esophageal squamous cell carcinoma (ESCC). Gene profiling and Ingenuity Pathway Analysis suggested the important role of AMPK in action mechanism of penfluridol. By using drug affinity responsive target stability (DARTS) technology and proteomics, we identified phosphofructokinase, liver type (PFKL), a key enzyme in glycolysis, as a direct target of penfluridol. Penfluridol could not exhibit its anticancer property in PFKL-deficient cancer cells, illustrating that PFKL is essential for the bioactivity of penfluridol. High PFKL expression is correlated with advanced stages and poor survival of ESCC patients, and silencing of PFKL significantly suppressed tumor growth. Mechanistically, direct binding of penfluridol and PFKL inhibits glucose consumption, lactate and ATP production, leads to nuclear translocation of FOXO3a and subsequent transcriptional activation of BIM in an AMPK-dependent manner. Taken together, PFKL is a potential prognostic biomarker and therapeutic target in ESCC, and penfluridol may be a new therapeutic option for management of this lethal disease.

Phosphoproteome analysis of the pathogenic bacterium Helicobacter pylori reveals over-representation of tyrosine phosphorylation and multiply phosphorylated proteins.

Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is a major regulatory post-translational modification in the bacteria. To reveal the phosphorylation state in the Gram-negative pathogenic bacterium Helicobacter pylori, we carried out a global and site-specific phosphoproteomic analysis based on TiO(2) -phosphopeptide enrichment and high-accuracy LC-MS/MS determination. Eighty-two phosphopeptides from 67 proteins were identified with 126 phosphorylation sites, among which 79 class I sites were determined to have a distribution of 42.8:38.7:18.5% for the Ser/Thr/Tyr phosphorylation, respectively. The H. pylori phosphoproteome is characterized by comparably big size, high ratio of Tyr phosphorylation, high abundance of multiple phosphorylation sites in individual phosphopeptides and over-representation of membrane proteins. An interaction network covering 28 phosphoproteins was constructed with a total of 163 proteins centering on the major H. pylori virulence factor VacA, indicating that protein phosphorylation in H. pylori may be delicately controlled to regulate many aspects of the metabolic pathways and bacterial virulence.

Putative copper- and zinc-binding motifs in Streptococcus pneumoniae identified by immobilized metal affinity chromatography and mass spectrometry.

The aim of metalloproteomics is to identify and characterize putative metal-binding proteins and metal-binding motifs. In this study, we performed a systematical metalloproteomic analysis on Streptococcus pneumoniae through the combined use of efficient immobilized metal affinity chromatography enrichment and high-accuracy linear ion trap-Orbitrap MS to identify metal-binding proteins and metal-binding peptides. In total, 232 and 166 putative metal-binding proteins were respectively isolated by Cu- and Zn-immobilized metal affinity chromatography columns, in which 133 proteins were present in both preparations. The putative metalloproteins are mainly involved in protein, nucleotide and carbon metabolisms, oxidation and cell cycle regulation. Based on the sequence of the putative Cu- and Zn-binding peptides, putative Cu-binding motifs were identified: H(X)mH (m=0-11), C(X)(2) C, C(X)nH (n=2-4, 6, 9), H(X)iM (i=0-10) and M(X)tM (t=8 or 12), while putative Zn-binding motifs were identified as follows: H(X)mH (m=1-12), H(X)iM (i=0-12), M(X)tM (t=0, 3 and 4), C(X)nH (n=1, 2, 7, 10 and 11). Equilibrium dialysis and inductively coupled plasma-MS experiments confirmed that the artificially synthesized peptides harboring differential identified metal-binding motifs interacted directly with the metal ions. The metalloproteomic study presented here suggests that the comparably large size and diverse functions of the S. pneumoniae metalloproteome may play important roles in various biological processes and thus contribute to the bacterial pathologies.

Proteomic analysis of excretory secretory products from Clonorchis sinensis adult worms: molecular characterization and serological reactivity of a excretory-secretory antigen-fructose-1,6-bisphosphatase.

Clonorchis sinensis is a food-borne zoonotic parasite that resides in bile ducts and causes clonorchiasis, which may result in cholelithiasis, cholecystitis, hepatic fibrosis, and liver tumors. Although total excretory secretory products (ESP) of C. sinensis adults induce hepatic fibrosis in vivo in rats, the causative mechanism is not well understood. To study components of the ESP, C. sinensis culture medium was collected and analyzed using shotgun LC-MS/MS. We identified a total of 110 proteins, including glycometabolic enzymes (such as fructose-1,6-bisphosphatase (FBPase) and enolase), detoxification enzymes (such as glutamate dehydrogenase, dihydrolipoamide dehydrogenase and cathepsin B endopeptidase), and a number of RAB family proteins. To identify a potential causative agent for hepatic fibrosis, we expressed and purified a recombinant FBPase, a 1,041-bp gene product that encodes a 41.7-kDa protein with prototypical FBPase domains and that can form a tetramer with a molecular mass of 166.8 kDa. In addition, we found that FBPase is an antigen present in the ESP and in circulation. Immunofluorescence showed that FBPase localizes to the intestinal cecum and vitellarium in C. sinensis adults. Our results describe the components of the excretory secretory products from C. sinensis adult worms and suggest that FBPase may be an important antigen present in the ESP of C. sinensis and may lay the foundation for additional studies on the development of clonorchiasis-associated hepatic fibrosis.