The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We sought to determine if there was antibody deposition in SAH livers and whether antibodies extracted from SAH livers were cross-reactive against both bacterial antigens and human proteins. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissue from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. Employing human proteome arrays, we profiled the antibodies extracted from explanted SAH, alcoholic cirrhosis (AC), nonalcoholic steatohepatitis (NASH), primary biliary cholangitis (PBC), autoimmune hepatitis (AIH), hepatitis B virus (HBV), hepatitis C virus (HCV) and HD livers and found that antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins as autoantigens. The use of an E. coli K12 proteome array revealed the presence of unique anti- E. coli antibodies in SAH, AC or PBC livers. Further, both Ig and E. coli captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion and focal adhesion (IgG). Except IgM from PBC livers, no common autoantigen was recognized by Ig and E. coli captured Ig from AC, HBV, HCV, NASH or AIH suggesting no cross-reacting anti- E. coli autoantibodies. The presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in the liver may participate in the pathogenesis of SAH.
Heritage Science In their Research Article on page 19144, Hwan-Ching Tai et al. use ICP-MS to identify the unique mineral recipe used by Antonio Stradivari to treat his spruce wood, the material for the violin top plate.
Cellular proteins are constantly damaged by reactive oxygen species generated by cellular respiration. Because of its metal-chelating property, the histidine residue is easily oxidized in the presence of Cu/Fe ions and H2O2 via metal-catalyzed oxidation, usually converted to 2-oxohistidine. We hypothesized that cells may have evolved antioxidant defenses against the generation of 2-oxohistidine residues on proteins, and therefore there would be cellular proteins which specifically interact with this oxidized side chain. Using two chemically synthesized peptide probes containing 2-oxohistidine, high-throughput interactome screening was conducted using the E. coli K12 proteome microarray containing >4200 proteins. Ten interacting proteins were identified, and successfully validated using a third peptide probe, fluorescence polarization assays, as well as binding constant measurements. We discovered that 9 out of 10 identified proteins seemed to be involved in redox-related cellular functions. We also built the functional interaction network to reveal their interacting proteins. The network showed that our interacting proteins were enriched in oxido-reduction processes, ion binding, and carbon metabolism. A consensus motif was identified among these 10 bacterial interacting proteins based on bioinformatic analysis, which also appeared to be present on human S100A1 protein. Besides, we found that the consensus binding motif among our identified proteins, including bacteria and human, were located within α-helices and faced the outside of proteins. The combination of chemically engineered peptide probes with proteome microarrays proves to be an efficient discovery platform for protein interactomes of unusual post-translational modifications, and sensitive enough to detect even the insertion of a single oxygen atom in this case. Cellular proteins are constantly damaged by reactive oxygen species generated by cellular respiration. Because of its metal-chelating property, the histidine residue is easily oxidized in the presence of Cu/Fe ions and H2O2 via metal-catalyzed oxidation, usually converted to 2-oxohistidine. We hypothesized that cells may have evolved antioxidant defenses against the generation of 2-oxohistidine residues on proteins, and therefore there would be cellular proteins which specifically interact with this oxidized side chain. Using two chemically synthesized peptide probes containing 2-oxohistidine, high-throughput interactome screening was conducted using the E. coli K12 proteome microarray containing >4200 proteins. Ten interacting proteins were identified, and successfully validated using a third peptide probe, fluorescence polarization assays, as well as binding constant measurements. We discovered that 9 out of 10 identified proteins seemed to be involved in redox-related cellular functions. We also built the functional interaction network to reveal their interacting proteins. The network showed that our interacting proteins were enriched in oxido-reduction processes, ion binding, and carbon metabolism. A consensus motif was identified among these 10 bacterial interacting proteins based on bioinformatic analysis, which also appeared to be present on human S100A1 protein. Besides, we found that the consensus binding motif among our identified proteins, including bacteria and human, were located within α-helices and faced the outside of proteins. The combination of chemically engineered peptide probes with proteome microarrays proves to be an efficient discovery platform for protein interactomes of unusual post-translational modifications, and sensitive enough to detect even the insertion of a single oxygen atom in this case. The complexity of the proteome arises in a large part because of the hundreds of post-translational modifications (PTMs) 1The abbreviations used are:PTMpost-translational modificationMCOmetal-catalyzed oxidationRAGEreceptors for advanced glycation end-productsAβamyloid betaADAlzheimer's diseaseGOGene OntologyKEGGKyoto Encyclopedia of Genes and GenomesBSAbovine serum albuminTBSTTris-buffered saline with Tween 20Kddissociation constantAG peptideAGAQVAHGNEVAG, SE peptideSEAGVNHGSAGQAIA peptideIAVENVHAQGLAOxo-AG peptide, AG peptide with 2-oxohistidine residueOxo-SE peptideSE peptide with 2-oxohistidine residueOxo-IA peptideIA peptide with 2-oxohistidine residueNADPHdihydronicotinamide-adenine dinucleotide phosphate. already discovered. Many PTMs are enzyme-catalyzed, such as phosphorylation, glycosylation, or ubiquitination (1.Wold F. In vivo chemical modification of proteins (post-translational modification).Annu. Rev. 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For instance, 14-3-3 family protein can recognize protein phosphorylation motifs (5.Morrison D.K. The 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development.Trends Cell Biol. 2009; 19: 16-23Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar) and various lectins can recognize protein glycosylation (6.Kilpatrick D.C. Animal lectins: a historical introduction and overview.Biochim. Biophys. Acta. 2002; 1572: 187-197Crossref PubMed Scopus (373) Google Scholar). However, recognition factors may also exist for nonenzymatic PTMs, such as receptor for advanced glycation end-products (RAGE) (7.Sparvero L.J. Asafu-Adjei D. Kang R. Tang D. Amin N. Im J. Rutledge R. Lin B. Amoscato A.A. Zeh H.J. Lotze M.T. RAGE (Receptor for Advanced Glycation Endproducts), RAGE ligands, and their role in cancer and inflammation.J. Transl. Med. 2009; 7: 17Crossref PubMed Scopus (446) Google Scholar). In this study we seek to uncover cellular binding factors for 2-oxohistidine, the oxidized product of histidine, which is an important but little-understood nonenzymatic PTM. post-translational modification metal-catalyzed oxidation receptors for advanced glycation end-products amyloid beta Alzheimer's disease Gene Ontology Kyoto Encyclopedia of Genes and Genomes bovine serum albumin Tris-buffered saline with Tween 20 dissociation constant AGAQVAHGNEVAG, SE peptide IA peptide Oxo-AG peptide, AG peptide with 2-oxohistidine residue SE peptide with 2-oxohistidine residue IA peptide with 2-oxohistidine residue dihydronicotinamide-adenine dinucleotide phosphate. The generation of ROS is an unavoidable consequence of cellular respiration, which leads to the oxidation of proteins, lipids, and nucleic acids (4.Davies M.J. The oxidative environment and protein damage.Biochim. Biophys. Acta. 2005; 1703: 93-109Crossref PubMed Scopus (1097) Google Scholar, 8.Muller F.L. Lustgarten M.S. Jang Y. 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The reactions of proteins with ROS may lead to almost 100 types of side chain modifications (12.Shacter E. Quantification and significance of protein oxidation in biological samples.Drug Metab. Rev. 2000; 32: 307-326Crossref PubMed Scopus (658) Google Scholar, 13.Xu G. Chance M.R. Hydroxyl radical-mediated modification of proteins as probes for structural proteomics.Chem. Rev. 2007; 107: 3514-3543Crossref PubMed Scopus (539) Google Scholar). Histidine is highly susceptible to ROS damage, because it has strong metal chelation affinity and often constitutes the binding site for metal ions (14.Tainer J.A. Roberts V.A. Getzoff E.D. Metal-binding sites in proteins.Curr. Opin. Biotechnol. 1991; 2: 582-591Crossref PubMed Scopus (120) Google Scholar, 15.Regan L. The design of metal-binding sites in proteins.Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 257-287Crossref PubMed Scopus (145) Google Scholar). The presence of H2O2 and redox-active metals (Cu and Fe) can lead to metal-catalyzed oxidation (MCO, also called Fenton-type chemistry), which converts histidine side chain to 2-oxohistidine (16.Uchida K. Kawakishi S. Identification of oxidized histidine generated at the active site of Cu, Zn-superoxide dismutase exposed to H2O2. Selective generation of 2-oxo-histidine at the histidine 118.J. Biol. Chem. 1994; 269: 2405-2410Abstract Full Text PDF PubMed Google Scholar, 17.Lewisch S.A. Levine R.L. Determination of 2-oxohistidine by amino acid analysis.Anal. Biochem. 1995; 231: 440-446Crossref PubMed Scopus (56) Google Scholar). The conversion of histidine to 2-oxohistidine alters its charge state, hydrogen bonding property, and metal chelation affinity, and hence may have serious impacts on protein structure and function. The net reaction is oxygen insertion (+16 Da), which makes it an irreversible PTM. It is unclear if cells simply tolerate such damages on histidines or employ active mechanisms to recognize them and use them as redox sensors or as damage markers for promoting protein degradation. The only known biological function of 2-oxohistidine is to serve as a redox sensor on bacterial transcription factor PerR (18.Traore D.A. El Ghazouani A. Jacquamet L. Borel F. Ferrer J.L. Lascoux D. Ravanat J.L. Jaquinod M. Blondin G. Caux-Thang C. Duarte V. Latour J.M. Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein.Nat. Chem. Biol. 2009; 5: 53-59Crossref PubMed Scopus (91) Google Scholar), whereas other studies have used 2-oxohistidine as a stable marker of protein damage during oxidative stress (12.Shacter E. Quantification and significance of protein oxidation in biological samples.Drug Metab. Rev. 2000; 32: 307-326Crossref PubMed Scopus (658) Google Scholar, 19.Davies M.J. Fu S. Wang H. Dean R.T. Stable markers of oxidant damage to proteins and their application in the study of human disease.Free Radic. Biol. Med. 1999; 27: 1151-1163Crossref PubMed Scopus (416) Google Scholar). Judging by the potential biological significance of 2-oxohistidine modification, we hypothesized that there may be cellular factors to recognize it. Previous research on 2-oxohistidine had been impeded by the difficulty in generating this side chain with reasonable yields. Recently, we managed to greatly improve the yield of 2-oxohistidine conversion by optimizing MCO reaction conditions using the copper/ascorbate system (20.Huang C.F. Liu Y.H. Tai H.C. Synthesis of peptides containing 2-oxohistidine residues and their characterization by liquid chromatography-tandem mass spectrometry.J. Pept. Sci. 2015; 21: 114-119Crossref PubMed Scopus (1) Google Scholar), allowing us to synthesize and purify peptide probes containing 100% 2-oxohistidine for this study. Here, we used 2-oxohistidine-containing peptides to mimic the oxidative conversion of histidine residues on native proteins. Then, we utilized the Escherichia coli (E. coli) K12 proteome chip to identify 2-oxohistidine-interacting proteins via high-throughput screening, and the interactors turned out to be largely involved redox-related metabolism. From the bacterial interactors we predicted a consensus binding motif, which could be validated across different species and correctly predicted S100A1 as a human binding factor for 2-oxohistidine. Thus, recognition of 2-oxohistidine appears to be an evolutionarily conserved capacity from bacteria to human. The high throughput protein expression, protein purification, and protein printing were modified from the previous study (21.Chen C.S. Korobkova E. Chen H. Zhu J. Jian X. Tao S.C. He C. Zhu H. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli.Nat. Methods. 2008; 5: 69-74Crossref PubMed Scopus (101) Google Scholar). Briefly, we expressed and purified E. coli K12 proteins in 96-well plate format and subsequently printed the proteome microarray. All purified proteins were spotted in duplicate on each aldehyde slide (BaiO, Shanghai, China) by SmartArrayer 136 (CapitalBio, Beijing, China) at 4 °C. After printing proteins, the proteome microarray chips were kept at 4 °C for protein immobilization on the slides for 12 h. The chips were stored at −80 °C before probing with samples. Solutions containing 1 mm peptide, 5 mm Cu2+ and 200 mm sodium ascorbate were exposed to air with gentle shaking at 37 °C for 24 h (AG and SE peptide) or 6 h (IA peptide). The oxidation reaction was quenched with 20 mm EDTA and analyzed by reverse-phase high-performance liquid chromatography (HPLC) (10–30% acetonitrile and 0.1% TFA in water, C18 column from Dr. Maisch, Ammerbuch, Germany) to determine the reaction yield. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of crude reaction mixtures and HPLC fractions, 10 μl samples were acidified with 2 μl 10% TFA and desalted with ZipTip (Millipore, Billerica, MA) following manufacturer's protocols. Oxidized peptides were purified by semi-preparative HPLC (C18 column, Dr. Maisch). LC-MS/MS experiments were conducted under previously reported conditions (20.Huang C.F. Liu Y.H. Tai H.C. Synthesis of peptides containing 2-oxohistidine residues and their characterization by liquid chromatography-tandem mass spectrometry.J. Pept. Sci. 2015; 21: 114-119Crossref PubMed Scopus (1) Google Scholar). Oxidized and nonoxidized peptides were dissolved in 50 mm sodium borate buffer at pH 7.5 and analyzed by HPLC to determine peptide concentration by 210 nm absorbance. DyLight-conjugated NHS esters (Thermo, Waltham, MA) were dissolved in anhydrous DMF to 10 mg/ml and added to peptide solutions for 1 h incubation at room temperature, at the following fluorophore/peptide ratios: DyLight 650/AG = 3:1, DyLight 650/SE = 5:1, DyLight 650/oxo-IA = 1.5:1; DyLight 550/oxo-AG = 5:1, DyLight 550/oxo-SE = 7:1, DyLight 550/IA = 3:1. Labeled peptides were analyzed and purified by HPLC as described above. Labeled products were verified by LC-MS/MS, and quantified by absorbance measurements based on known fluorophore properties. The chips were first blocked with 3% bovine serum albumin (BSA) (Sigma, St. Louis, MO) for 5 min. DyLight 550-conjugated 2-oxohistidine peptide and DyLight 650-conjugated nonoxidized peptide (10 μm each) were probed together onto the chip with LifterSlips (Thermo) at room temperature for 45 min. Finally, the chips were washed by Tris-buffered saline-Tween 20 (TBST) on an orbital shaker three times and 5 min each time. The chip was dried by centrifugation and then scanned with a LuxScan microarray scanner (CapitalBio). Signal intensities, defined as foreground median subtracted by background median, were acquired and analyzed using GenePix Pro 6.0 software. Then, we used quantile normalization to normalize the signal intensity from both 2-oxohistidine containing probes and nonoxidized probes. To identify positive 2-oxohistidine interacting proteins, four cutoff criteria were set: (1) The signal from experimental group was >1.5 standard deviations above the mean for all experimental groups. (2) To identify large signal differences between experimental groups and negative controls, the delta, defined as signal difference between experimental group and control group, was >1.5 standard deviations above the mean for all deltas. (3) To exclude the nonspecific binding to 2-oxohistidine residue, the signal from the negative control was >1.5 standard deviations below the mean for all control groups. (4) To remove irreproducible hits among triplicate chip assays, the student's t test p values between experimental groups and negative controls were less than 0.05. The R programming language (22.Team R.C. R: A Language and Environment for Statistical Computing.R Foundation for Statistical Computing. 2015; Google Scholar) was used to display heat map. The data was presented by the signal intensity of foreground subtracted by background. The gplots package (23.Warnes G.R. Bolker B. Bonebakker L. Gentleman R. Huber W. Liaw A. Lumley T. Maechler M. Magnusson A. Moeller S. gplots: Various R programming tools for plotting data. 2009; (R package Version 2)Google Scholar) was used for classifying 2-oxohistidine containing peptides and nonoxidized peptides in hierarchy. The identified proteins were used for functional interaction analyses by using EcID (24.Andres Leon E. Ezkurdia I. Garcia B. Valencia A. Juan D. EcID. A database for the inference of functional interactions in E. coli.Nucleic Acids Res. 2009; 37: D629-D635Crossref PubMed Scopus (25) Google Scholar) and Cytoscape (25.Shannon P. Markiel A. Ozier O. Baliga N.S. Wang J.T. Ramage D. Amin N. Schwikowski B. Ideker T. Cytoscape: a software environment for integrated models of biomolecular interaction networks.Genome Res. 2003; 13: 2498-2504Crossref PubMed Scopus (25321) Google Scholar). Briefly, the files of EcID entities and EcID pairs were downloaded from EcID database. Before mapping identified proteins to their EcID entities and EcID pairs, we removed the pairs that were based on the prediction mode, such as phylogenetic profiles, gene neighborhood, mirror tree, in silico two-hybrid, or context mirror. After mapping, we used Cytoscape to generate the functional interaction network, and visualized the identified proteins and their interacting proteins. Subsequently, we used AmiGO 2 (26.Carbon S. Ireland A. Mungall C.J. Shu S. Marshall B. 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Identified proteins and S100A1 (Abnova, Taipei, Taiwan) were printed on aldehyde chips in a multiple-well format. After printing, the chips were immobilized at 4 °C for 12 h and then stored at −80 °C. The printed chips were blocked at room temperature for 5 min with 3% BSA. Two-fold serial-diluted DyLight 550-conjugated 2-oxohistidine peptides, DyLight 650-conjugated nonoxidized peptides, and quenched fluorescent dyes were added into the chip wells individually with Multi-Well Microarray Hybridization Cassettes (Arrayit, Sunnyvale, CA), and incubated at room temperature for 45 min. The fluorescent dyes, NHS esters of DyLight 550 and DyLight 650, were already quenched by Tris-HCl (Bionovas, Toronto, Canada). To check whether calcium affects interaction between S100A1 and 2-oxohistidine, 1 mm CaCl2 was added in the assay buffer. After several washes with TBST, the chips were dried by centrifugation and then scanned with a microarray scanner. 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To identify proteins which may bind specifically to 2-oxohistidine residue, we devised an experimental strategy illustrated in Fig. 1. First, we fabricated the E. coli K12 proteome chip, generated the 2-oxohistidine containing peptides, and probed these peptides with E. coli K12 proteome chips. After identifying the positive hits, we used fluorescence polarization assays to validate the interactions and measured the binding affinity by dose-response measurements. Then, we surveyed the consensus motif among these identified proteins and applied to human proteome to look for possible human 2-oxohistidine interacting proteins. Finally, we used the functional interaction network to find out the possible interacting proteins and used GO and KEGG to figure out related processes and pathways (Fig. 1). To synthesize peptide probes, histidine residues were placed in the middle of 12-mer or 13-mer peptides to eliminate possible charge effects at N terminus and C terminus, creating a context similar to proteins. Easily oxidized amino acids, such as methionine, cysteine, tyrosine, tryptophan, phenylalanine, lysine, and arginine, were avoided. Three peptides containing a single histidine residue and random selections of other residues, namely AGAQVAHGNEVAG (AG), SEAGVNHGSAGQA (SE), and IAVENVHGGLA (IA), were used for chip assays. We carried out MCO reactions using the copper/ascorbate/air system shown in Fig. 2 to convert them to 2-oxohistidine containing peptides (Oxo-AG, Oxo-SE, Oxo-IA). The HPLC yield of peptides Oxo-AG and Oxo-SE were around 10%, and for Oxo-IA peptide around 20% (Fig. 2). The site of oxidative modification was confirmed by LC-MS/MS for all peptides (supplemental Fig. S1). To investigate 2-oxohistidine interacting proteins, AGAQVAH*GNEVAG (Oxo-AG peptide) and SEAGVNH*GSAGQA (Oxo-SE peptide) were conjugated to DyLight 550 fluo
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Antimicrobial peptides (AMPs) have potential antifungal activities; however, their intracellular protein targets are poorly reported. Proteome microarray is an effective tool with high-throughput and rapid platform that systematically identifies the protein targets. In this study, we have used yeast proteome microarrays for systematical identification of the yeast protein targets of Lactoferricin B (Lfcin B) and Histatin-5. A total of 140 and 137 protein targets were identified from the triplicate yeast proteome microarray assays for Lfcin B and Histatin-5, respectively. The Gene Ontology (GO) enrichment analysis showed that Lfcin B targeted more enrichment categories than Histatin-5 did in all GO biological processes, molecular functions, and cellular components. This might be one of the reasons that Lfcin B has a lower minimum inhibitory concentration (MIC) than Histatin-5. Moreover, pairwise essential proteins that have lethal effects on yeast were analyzed through synthetic lethality. A total of 11 synthetic lethal pairs were identified within the protein targets of Lfcin B. However, only three synthetic lethal pairs were identified within the protein targets of Histatin-5. The higher number of synthetic lethal pairs identified within the protein targets of Lfcin B might also be the reason for Lfcin B to have lower MIC than Histatin-5. Furthermore, two synthetic lethal pairs were identified between the unique protein targets of Lfcin B and Histatin-5. Both the identified synthetic lethal pairs proteins are part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) protein complex that regulates gene expression via histone modification. Identification of synthetic lethal pairs between Lfcin B and Histatin-5 and their involvement in the same protein complex indicated synergistic combination between Lfcin B and Histatin-5. This hypothesis was experimentally confirmed by growth inhibition assay.
The acid-hydrolyzed fragments of Ganoderma lucidum polysaccharides (GLPS) obtained by Smith degradation were separated by size-exclusion chromatography into two major water-soluble fractions: peptidoglycans (GLPS-SF1) and oligosaccharides (GLPS-SF2). Both fractions induced CD69 in human peripheral blood mononuclear cells (hPB-MNCs), and they displayed distinct immunomodulating properties. GLPS-SF1, with a molecular weight of around 20 kDa, were heterogeneous peptidoglycans composed of glucose/mannose (4:1) that exhibited biological activities with Th1 cytokines IL-12, IL-2, TNF-α, and IFN-γ in hPB-MNCs and stimulated macrophage cytokine expression via Toll-like receptor 4 (TLR4) signaling. For GLPS-SF2, with a molecular weight of around several kilodaltons, its sugar sequence was elucidated by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy as [-α-1,4-Glc-(β-1,4-GlcA)(3)-](n). This oligosaccharide displayed specific immune property with low monocyte induction, greatly stimulated cell activation and proliferation of NK and T cells. This oligosaccharide isolated from G. lucidum polysaccharides with internal glucuronic acids/glucose repeat unit in a 3:1 ratio may be responsible for the active stimulation of NK and T cells.
The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We sought to determine if there was antibody deposition in SAH livers and whether antibodies extracted from SAH livers were cross-reactive against both bacterial antigens and human proteins. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissue from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. Employing human proteome arrays, we profiled the antibodies extracted from explanted SAH, alcoholic cirrhosis (AC), nonalcoholic steatohepatitis (NASH), primary biliary cholangitis (PBC), autoimmune hepatitis (AIH), hepatitis B virus (HBV), hepatitis C virus (HCV) and HD livers and found that antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins as autoantigens. The use of an E. coli K12 proteome array revealed the presence of unique anti- E. coli antibodies in SAH, AC or PBC livers. Further, both Ig and E. coli captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion and focal adhesion (IgG). Except IgM from PBC livers, no common autoantigen was recognized by Ig and E. coli captured Ig from AC, HBV, HCV, NASH or AIH suggesting no cross-reacting anti- E. coli autoantibodies. The presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in the liver may participate in the pathogenesis of SAH.
Antimicrobial peptides have been considered well‐deserving candidates to fight the battle against microorganisms due to their broad‐spectrum antimicrobial activities. Several studies have suggested that membrane disruption is the basic mechanism of AMPs that leads to killing or inhibiting microorganisms. Also, AMPs have been reported to interact with macromolecules inside the microbial cells such as nucleic acids (DNA/RNA), protein synthesis, essential enzymes, membrane septum formation and cell wall synthesis. Proteins are associated with many intracellular mechanisms of cells, thus protein targets may be specifically involved in mechanisms of action of AMPs. AMPs like pyrrhocoricin, drosocin, apidecin and Bac 7 are documented to have protein targets, DnaK and GroEL. Moreover, the intracellular targeting AMPs are reported to influence more than one protein targets inside the cell, suggesting for the multiple modes of actions. This complex mechanism of intracellular targeting AMPs makes them more difficult for the development of resistance. Herein, we have summarized the current status of AMPs in terms of their mode of actions, entry to cytoplasm and inhibition of macromolecules. To reveal the mechanism of action, we have focused on AMPs with intracellular protein targets. We have also included the use of high‐throughput proteome microarray to determine the unidentified AMP protein targets in this review.
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