Charakterisierung des Redoxproteoms von Hefe (Saccharomyces cerevisiae) nach Allicin-induziertem oxidativem Stress

Albrecht, Frank; Slusarenko, Alan (Thesis advisor); Jacob, Claus (Thesis advisor)

Aachen (2018, 2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2018

Abstract

In 1944 Cavallito & Bailey showed that allicin was the major antimicrobial compound in garlic juice. Allicin is a reactive sulfur species (RSS) which oxidises thiols by S-thioallylation. It occurs as a defence molecule in some species of the genus Allium, e.g. garlic (Allium sativum) and ramsons (Allium ursinum). The present study characterises the effects of a sublethal dose of allicin on the Baker’s Yeast (Saccharomyces cerevisiae) Proteome. Synthetic allicin was used and for this purpose a new procedure was developed and optimised, achieving >98 % pure Allicin with a yield of 98 % after only 15 minutes reaction time and a final yield of 92 % after purification. The OxICATmethod, coupled with LC-MS/MS and MaxQuant software, was used to identify specific cysteine-containing peptides from yeast proteins. Approx. 10,000 peptides per experiment were identified in five independent experiments. To streamline the analysis of the results and identify peptides oxidised by allicin a spreadsheet-based computer analysis tool was developed. In total 1808 peptide pairs (oxidised/non-oxidised) were found. The procedure revealed the specific cysteine which was oxidised and the degree of oxidation in the population for 448 individual proteins. Thirteen candidate proteins were selected for detailed characterisation by the criteria that they were detected in at least three experiments and were ≥10 % more oxidised. The candidate proteins were investigated by molecular and biochemical methods to assess their biological relevance in the context ofallicin’s effects. After allicin treatment, alcohol dehydroganse-1 (ADH1) was 20 % oxidised at cysteines 277 and 278, which are outside the catalytic centre. ADH1 activity was inhibited noncompetitively in vitro by allicin, which would be expected for allosteric inhibition. Sitedirected mutagenesis was used to prepare C277S,C278S and C277,278S exchange mutants and these were investigated for growth phenotype, altered resistance to allicin and ADH activity. The mutants showed various degrees of enhanced allicin-resistance compared to the wild type. The C277, 278S double mutant had lost ADH activity but grew normally, whereas an ADH1 deletion mutant grew with the characteristic petite yeast colony morphology. This suggests an important novel role for the ADH1 protein in S. cerevisiae that is independent of its ADH activity, i.e. a ‘moonlighting’ role which enables the cells to grow and divide efficiently. This interesting effect should be a topic for further investigation.

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