Substrate Specificity of Clostridial Glucosylating Toxins and Their Function on Colonocytes Analyzed by Proteomics Techniques

Zeiser, Johannes; Gerhard, Ralf; Just, Ingo; Pich, Andreas

doi:10.1021/pr300973q

Cited by 51 publications

(65 citation statements)

References 70 publications

(161 reference statements)

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“…Statistical tests are indicated with horizontal lines as described in the legend to S10). In another high-throughput analysis of the host cell response to TcdA, Zeiser et al analyzed the changes in the proteome of Caco2 cells 24 h after TcdA exposure (47). By mapping transcripts to proteins, we found that the proteomics data were poorly correlated with transcriptional changes in HCT8 cells and our in vivo data (r 2 Ͻ 0.01 for all comparisons).…”

Section: Discussionmentioning

confidence: 71%

In VivoPhysiological and Transcriptional Profiling Reveals Host Responses to Clostridium difficile Toxin A and Toxin B

et al. 2013

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Toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile cause gross pathological changes (e.g., inflammation, secretion, and diarrhea) in the infected host, yet the molecular and cellular pathways leading to observed host responses are poorly understood. To address this gap, we evaluated the effects of single doses of TcdA and/or TcdB injected into the ceca of mice, and several endpoints were analyzed, including tissue pathology, neutrophil infiltration, epithelial-layer gene expression, chemokine levels, and blood cell counts, 2, 6, and 16 h after injection. In addition to confirming TcdA's gross pathological effects, we found that both TcdA and TcdB resulted in neutrophil infiltration. Bioinformatics analyses identified altered expression of genes associated with the metabolism of lipids, fatty acids, and detoxification; small GTPase activity; and immune function and inflammation. Further analysis revealed transient expression of several chemokines (e.g., Cxcl1 and Cxcl2). Antibody neutralization of CXCL1 and CXCL2 did not affect TcdA-induced local pathology or neutrophil infiltration, but it did decrease the peripheral blood neutrophil count. Additionally, low serum levels of CXCL1 and CXCL2 corresponded with greater survival. Although TcdA induced more pronounced transcriptional changes than TcdB and the upregulated chemokine expression was unique to TcdA, the overall transcriptional responses to TcdA and TcdB were strongly correlated, supporting differences primarily in timing and potency rather than differences in the type of intracellular host response. In addition, the transcriptional data revealed novel toxin effects (e.g., altered expression of GTPase-associated and metabolic genes) underlying observed physiological responses to C. difficile toxins.

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Section: Discussionmentioning

confidence: 71%

In VivoPhysiological and Transcriptional Profiling Reveals Host Responses to Clostridium difficile Toxin A and Toxin B

et al. 2013

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“…Analysis-These analyses were performed as described previously (40). Reversed-phase chromatography of peptide samples was conducted with a nano-flow ultra-high-pressure liquid chromatography system (RSLC, Thermo Fisher Scientific).…”

Section: Lc-ms/ms and Automated Ms Datamentioning

confidence: 99%

Cleavage of E-Cadherin and β-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells

Konze

Diepen²,

Schröder

et al. 2014

Molecular & Cellular Proteomics

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The envisioned clinical and industrial use of human pluripotent stem cells and their derivatives has given major momentum to the establishment of suspension culture protocols that enable the mass production of cells. Understanding molecular changes accompanying the transfer from adherent to suspension culture is of utmost importance because this information can have a direct effect on the development of optimized culture conditions. In this study we assessed the gene expression of human embryonic stem cells and induced pluripotent stem cells grown in surface-adherent culture (two-dimensional) versus free-floating suspension culture spheroids (three-dimensional). We combined a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture with deep-sequencing-based transcriptomics. Cells in three-dimensional culture showed reduced expression of proteins forming structural components of cell-cell and cell-extracellular matrix junctions. However, fully unexpected, we found up-regulation of secreted inhibitors of the canonical Wnt signaling pathway and, concomitantly, a reduction in the level of active ␤-catenin and in the expression of Wnt target genes. In Western blot analyses the cysteine protease calpain was shown to cleave E-cadherin and ␤-catenin under three-dimensional culture conditions. Our data al- Human embryonic and induced pluripotent stem cells (hESCs and hiPSCs, respectively) 1 hold the potential for indefinite self-renewal and differentiation into all somatic cell types (1, 2). Beyond their application as models for studying mechanisms of pluripotency, these cells have been considered as a potent source for cell therapies and in vitro assays in pharmacology and toxicology, raising the need for largescale cell production under defined conditions (3). Conventional, surface adherent, two-dimensional culture is not suited to generate billions of human pluripotent stem cells (hPSCs) and their respective progenies required for clinical applications (3). To overcome these limits, three-dimensional culture From the ‡Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany; §REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; ¶Institute for

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“…'HeLa Whole Cell Lysate' was used as positive control lysate, while detection of the abundantly expressed loading control vinculin was applied [20,21] to calculate relative ALDH2 expression in blood samples. Detailed information regarding the generation of protein lysates from frozen whole blood samples, relative ALDH2 protein quantification and validation of precise detection of ALDH2 by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) as described earlier [22] , can be found in the online supplementary material.…”

Section: Western Blot Analysis Of Relative Aldh2 Protein Quantificatimentioning

confidence: 99%

Impaired Regulation of ALDH2 Protein Expression Revealing a Yet Unknown Epigenetic Impact of rs886205 on Specific Methylation of a Negative Regulatory Promoter Region in Alcohol-Dependent Patients

Nassab

Rhein²,

Hagemeier³

et al. 2015

Eur Addict Res

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Acetaldehyde, the carcinogenic metabolite of ethanol known to provoke aversive symptoms of alcohol consumption, is predominantly eliminated by aldehyde dehydrogenase 2 (ALDH2). Reduced ALDH2 activity correlates with low alcohol tolerance and low risk for alcohol dependence. The ALDH2 promoter polymorphism rs886205 (A>G) is associated with decreased promoter activity, but a molecular mechanism and allele-dependent ALDH2 protein expression has not been described yet. On the basis of allele-dependent epigenetic effects, we analyzed the rs886205 genotype, methylation rates of cytosine-phosphatidyl-guanine (CpG)-sites within a regulatory promoter region and ALDH2 protein levels in 82 alcohol-dependent patients during a 2-week withdrawal and compared them to 34 matched controls. Patients without the G-allele of rs886205 showed higher methylation of the promoter region than controls and readily adapted epigenetically as well as on protein level during withdrawal, while patients with the G-allele displayed retarded methylation readjustment and no change in ALDH2 protein levels. Our data provide novel insights into an unknown genetic-epigenetic interaction, revealing impaired ALDH2 protein expression in patients with the G-allele of rs886205. Additionally, we checked for an association between rs886205 and protection against alcohol dependence and found a trend association between the G-allele and protection against alcohol dependence that needs replication in a larger Caucasian cohort.

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Substrate Specificity of Clostridial Glucosylating Toxins and Their Function on Colonocytes Analyzed by Proteomics Techniques

Cited by 51 publications

References 70 publications

In VivoPhysiological and Transcriptional Profiling Reveals Host Responses to Clostridium difficile Toxin A and Toxin B

In VivoPhysiological and Transcriptional Profiling Reveals Host Responses to Clostridium difficile Toxin A and Toxin B

Cleavage of E-Cadherin and β-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells

Impaired Regulation of ALDH2 Protein Expression Revealing a Yet Unknown Epigenetic Impact of rs886205 on Specific Methylation of a Negative Regulatory Promoter Region in Alcohol-Dependent Patients

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