Untargeted Metabolomics Analysis Reveals a Link between ETHE1-Mediated Disruptive Redox State and Altered Metabolic Regulation

Defects in the gene encoding the persulfide dioxygenase ETHE1 are known to cause the severe inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of known clinical characteristics, the molecular mechanisms underlying the ETHE1 deficiency are still obscure. Herein, to further analyze the molecular phenotype of the disease, we applied an untargeted metabolomics approach on cultivated fibroblasts of EE patients for pinpointing alterations in metabolite levels. Metabolites, as direct signatures of biochemical functions, can decipher biochemical pathways involved in the cellular phenotype of patient cells. Using liquid chromatography–mass spectrometry-based untargeted metabolomics, we identified 18 metabolites that have altered levels in fibroblasts from EE patients. Our data demonstrate disrupted redox state in EE patient cells, which is reflected by significantly decreased level of reduced glutathione. Furthermore, the down-regulation of several intermediate metabolites such as the redox cofactors NAD⁺ and NADH as well as Krebs cycle intermediates revealed clear alteration in metabolic regulation. Pantothenic acid and several amino acids exhibited decreased levels, whereas the β-citrylglutamate with a putative role in brain development had an increased level in the EE patient cells. These observations indicate the severe impact of ETHE1 deficiency on cellular physiology and redox state, meanwhile suggesting targets for experimental studies on novel treatment options for the devastating metabolic disorder

Cytokeratins.

<p>All identified cytokeratins (Hair- and hair follicle keratins excluded) were grouped according to Moll <i>et al</i>.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104103#pone.0104103-Moll1" target="_blank">[38]</a>. Letters above the standard deviation bars indicate the tissues compared to which differences were found in the pairwise comparisons between the four keratinizing tissues (results from comparisons including the middle ear mucosa are not shown). a: Group A proteins; b: Group B proteins; b(t): Triplicate values in group B. Gene names shown below the columns. Tymp Membr: Tympanic membrane; Chol: Cholesteatoma.</p

Minimum fold changes of proteins meeting the group A and B criteria for fold change.

<p>Only comparisons including cholesteatoma are shown. Neck: Neck of cholesteatoma; Tymp: Tympanic membrane; EACS: external auditory canal skin; Mucosa: Middle ear mucosa.</p

Top 20 Up-regulated proteins in cholesteatoma.

<p>Up-regulated proteins in cholesteatoma sorted by 1: The number of tissues compared to which cholesteatoma showed higher levels of the protein; 2: Protein group (fold change criteria A then B); 3: Fold change. C: Cholesteatoma; N: Neck of cholesteatoma; T: Tympanic membrane; S: External auditory canal skin; a: Protein group A; b: Protein group B; b(t): Triplicates in protein group B.</p

Network of differential level proteins in cholesteatoma with associations to connective tissue.

<p>A. The top scoring, automatically-synthesized network of related proteins in IPA: Connective Tissue Development and Function, Embryonic Development, Organ Developmentive tissue.d proteins: Group B proteins.d EACS.holesteatoma a STRING. All proteins, except for MMP9, showed the same expression direction comparing cholesteatoma with tympanic membrane and EACS, respectively. Protein level differences meeting the group A or B criteria were detected in at least one of the two comparisons. 41 interactions (7.95 expected) were identified between the 23 proteins, network <i>p</i> value = 1.11e-16. B. Some of the top scoring significant associations of the network with: GO Biological Processes, GO Cellular Components, and KEGG Pathways; ordered by <i>p</i> value.</p

Activation direction of biological functions in cholesteatoma.

<p>Statistical predictions on the direction of activation of biological functions associated with cholesteatoma (see experimental procedures). Biological functions with z - scores of the predictions above the numerical value 1.645 (90% significance level) are shown. In a first step, biological functions were statistically associated with cholesteatoma based on the up- and down-regulated proteins that could be assigned to these functions. Subsequently, predictions on the activation direction were calculated from the composition of up- and down-regulations among these proteins. The listed results were found in comparisons between cholesteatoma sack (C), neck of cholesteatoma (N), tympanic membrane (T), and external auditory canal skin (S). *The proteins had to show the same direction of expression in both tissues and meet the fold change criteria in at least one of the tissues.</p

Overview of levels of 11 related proteins involved in inflammation, response to bacteria, and/or protein degradation.

<p>Letters above the standard deviation bars indicate the tissues compared to which differences were found in the pairwise comparisons between the four keratinizing tissues. Left: Leukocyte-associated proteins. Middle: Inhibitor of enzyme activity. Right other protein degrading and/or immune-response related proteins. a: Group A proteins, b: Group B proteins, b(t): Triplicate values in group B. Gene names below the columns. Tymp Membr: Tympanic membrane; Chol: Cholesteatoma.</p

Canonical pathways associated with the proteins meeting the fold-change criteria.

<p>A. The top scoring (lowest <i>p</i>-values) canonical pathways associated with the differentially expressed proteins found in the pairwise comparisons between the four keratinizing tissues. *The second highest score "Methylglyoxal Degradation III" was found in the comparison between tympanic membrane and EACS. All others were found in the comparison between cholesteatoma and EACS. Horizontal blue line indicates <i>p</i> value  = 0.05. B. <i>p</i> values and the involved differential-level proteins of three selected pathways from the comparison between cholesteatoma and EACS. All proteins showed lower levels (green color) in cholesteatoma compared with EACS. Underlined proteins: Group B proteins.</p

Simple overview of workflow.

<p>EACS: external auditory canal skin; Tymp: tympanic membrane; Neck: neck of cholesteatoma; Chol: cholesteatoma; Muc: middle ear mucosa; ME: Middle ear; LC: Liquid Chromatography; MS: Mass Spectrometry.</p

Synthesized network of immune response-related up-regulated proteins in cholesteatoma and neck of cholesteatoma.

<p>A. All proteins showed higher protein levels in cholesteatoma and neck of cholesteatoma compared to the external auditory canal skin. The proteins met the fold change criteria in at least one of the two tissue comparisons. B. Examples of significant associations of the network with: GO Biological Processes, GO Cellular Components and KEGG Pathways; ordered by <i>p</i> value. The networks were generated in STRING.</p