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Download from the ETH Zurich Research Collection Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2019 Substrate Identification and Functional Analysis of ฀/฀ Hydrolase Fold Epoxide Hydrolases Hew, Bettina DOI: https://doi.org/10.3929/ethz-b-000350557 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-186648 Dissertation Published Version Originally published at: Hew, Bettina. Substrate Identification and Functional Analysis of ฀/฀ Hydrolase Fold Epoxide Hydrolases. 2019, ETH Zurich. DOI: https://doi.org/10.3929/ethz-b-000350557 Research Collection Doctoral Thesis Substrate Identification and Functional Analysis of α/β Hydrolase Fold Epoxide Hydrolases Author(s): Hew, Bettina Publication Date: 2019 Permanent Link: https://doi.org/10.3929/ethz-b-000350557 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH NO 25662 SUBSTRATE IDENTIFICATION AND FUNCTIONAL ANALYSIS OF α/β HYDROLASE FOLD EPOXIDE HYDROLASES A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) presented by BETTINA HEW MSc ETH in Pharmaceutical Sciences, ETH Zurich born on 30.07.1987 citizen of Klosters-Serneus GR accepted on the recommendation of Prof. Dr. Hanns Ulrich Zeilhofer Prof. Dr. Ursula Quitterer und Prof. Dr. Michael Arand 2019 i Preamble PREAMBLE The main project of the present work; “Deciphering the (patho)physiologic role of epoxide hydrolases by ex vivo cartography of their substrate landscape”, was a joined project of me together with Monika Dengler. Originally, it was planned to result in two independent projects after a joined part at the beginning, which did not work out. Thus, the research questions, methods and results were the same for both of us. We worked out the technical details, performed the experiments and analyzed the data with equal contributions. I particularly had the lead in AAV production and cell culture work, while M. Dengler had the lead in LC-MS/MS method development and analysis. In the two corresponding, individually written PhD dissertations the experimental parts were divided accordingly. For the sake of completeness missing sections were summarized and referred to the respective other work. In contrast, the side project dealt with in this work; “The CYP blocker MS-PPOH is an inhibitor of sEH”, was my individual project. i Summary SUMMARY Epoxide hydrolases (EHs) are enzymes that convert epoxides to their corresponding vicinal diols. In mammals, they play a central role in the detoxification of genotoxic epoxides and have an important function in the regulation of physiological processes by hydrolyzing signaling epoxides. The best characterized EHs are the microsomal EH (mEH) and the soluble EH (sEH). mEH is generally considered to be primarily important in detoxification and only recently gained attention in signaling molecule metabolism. sEH, on the other hand, has long been recognized as the key enzyme in the hydrolysis of signaling fatty acid epoxides (FAEs), such as epoxyeicosatrienoic acids (EETs). The most recently discovered family member, EH3, was also shown to hydrolyze FAEs, thus a role in mammalian physiology seems likely. EH4 and mesoderm specific transcript (MEST) are also predicted to be mammalian EHs based on their primary structure, but their EH activity is not yet proven. Bacteria also possess EHs, sometimes with pathophysiologic consequences for host organisms as reported for the CFTR inhibitory factor (Cif), secreted by P. aeruginosa. However, the exact mechanism by which Cif fulfills its pathologic function is still unknown. The knowledge about the aforementioned EHs is typically based on in vitro analysis of their substrate preference and does not allow direct conclusions on their real role in mammalian physiology. Thus, the general aim of the main project of this work was to identify the major (patho)physiologic roles of the six EHs via the identification of their preferred in vivo substrates. For this purpose, a novel unbiased in vivo substrate trapping approach, taking advantage of the characteristic molecular mechanism of EHs, was developed and applied. EH variants able to trap their substrate in a covalent ester-bond due to introduction of a point mutation, were expressed in target tissues of mice using adeno-associated virus (AAV) infection. The resulting enzyme-substrate complex was subsequently retrieved from tissue lysates by His-tag based affinity enrichment and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the bound substrates. A LC-MS/MS method to analyze bound substrates was successfully developed. The method could be confirmed by in vitro trapping experiments with sEH trapping mutant and its known substrate 14,15- EET. Moreover, successful AAV-mediated trapping EH expression was detected in different target organs of mice by Western blot and immunohistochemistry (IHC) stainings. However, in a comprehensive series of experiments, it was not possible to identify or verify any substrates with this novel trapping approach. The primary issues were the expression of non-functional trapping mutants unable to bind their substrate (i.e. mEH, MEST, Cif) and the selective loss of the peptides during sample ii Summary preparation for the LC-MS/MS analysis (i.e. sEH, EH3, EH4). Another shortcoming was that the selected AAV serotypes did not lead to transgene expression in the relevant organs for certain EHs (i.e. Cif, EH3). In addition, transgene expression for some EH trapping mutants was low and thus hindered their analysis (i.e. EH3, EH4). Finally, further optimization is required for the success of our developed trapping method. Moreover, trapping attempts in vitro could be a valuable alternative for Cif and EH3 substrate identification. Unexpectedly, mice expressing the mEH trapping mutant in the brain developed a striking progressive trembling phenotype. This phenotype was not observed in any other group of AAV-infected mice. A potential underlying cause is a loss of dopaminergic neurons in the brain motor system of these mice, producing a pathology similar to Parkinson’s disease. However, the exact mechanism remains unclear and needs further examination. In a smaller side project pursued in parallel to the main project, a classic in vitro inhibitor screening was performed. The cytochrome P450 (CYP) inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)- benzenehexanamide (MS-PPOH), which is generally used in vitro and in vivo to prevent EET formation, was examined for its potential to inhibit mEH and sEH activity. While it did not inhibit mEH, MS-PPOH inhibition of sEH was almost two orders of magnitude stronger compared to CYPs. This would lead to erroneous conclusions concerning the physiological EET functions when sEH inhibition is not considered in experiments using MS-PPOH. Nonetheless, additional studies to examine the consequences of these findings in vivo would be of major importance. In the present work, novel insight into EH physiology has been obtained and a method has been established which has the potential to enable unbiased ex vivo substrate identification for EHs in the near future. iii Zusammenfassung ZUSAMMENFASSUNG Epoxidhydrolasen (EHs) sind Enzyme, die Epoxide zu ihren entsprechenden Diolen hydrolysieren. Sie spielen eine zentrale Rolle in der Entgiftung genotoxischer Epoxide und haben eine wichtige Funktion in der Regulation physiologischer Prozesse, indem sie Signalmoleküle hydrolysieren. Die bestcharakterisierten Säugetier-EHs sind die mikrosomale EH (mEH) und die lösliche EH (sEH). Die mEH wird hauptsächlich im Fremdstoffmetabolismus angesiedelt und erlangte erst vor Kurzem Aufmerksamkeit für ihre Funktion im Signalmolekülstoffwechsel. Die sEH hingegen ist das Schlüsselenzym im Umsatz von Fettsäureepoxiden (FAEs), wie Epoxyeicosatriensäuren (EETs). Auch für die erst kürzlich entdeckte EH3 wurde gezeigt, dass sie FAEs hydrolysiert, was eine physiologische Aufgabe nahelegt. Basierend auf ihren Primärstrukturen, sind auch EH4 und mesoderm specific transcript (MEST) potentielle Säugetier-EHs. Ihre EH Aktivität ist jedoch noch nicht erwiesen. Bakterien besitzen auch EHs, die pathophysiologische Konsequenzen für den Wirt mit sich ziehen können. Ein Beispiel dafür ist CFTR inhibitory factor (Cif), ein Virulenzfaktor sekretiert von P. aeruginosa. Der genaue Mechanismus, mit welchem Cif seine pathologische Funktion erfüllt, ist aber noch ungeklärt. Das Wissen über die genannten EHs basiert typischerweise auf in vitro Untersuchen ihrer Substratpräferenz und erlaubt darum keine direkten Rückschlüsse auf ihre eigentliche physiologische Aufgabe. Das Ziel des Hauptprojekts dieser Arbeit war deshalb die Aufklärung der jeweiligen (patho)physiologischen Hauptaufgaben der sechs EHs, mittels Identifizierung ihrer bevorzugten in vivo Substrate. Dazu wurde eine neuartige, unvoreingenommene in vivo Substrat-Fangmethode entwickelt und angewendet. EH Varianten, die aufgrund einer Punktmutation ihr Substrat in einer kovalenten Esterbindung irreversibel festhalten (Trapping-Mutanten), wurden über Adeno-assoziierte Viren (AAV) in Zielorganen von Mäusen exprimiert. Die Enzym-Substrat Komplexe wurden anschliessend via His- Tag Affinitätschromatographie aus dem Gewebe angereichert und mit Flüssigchromatographie- Tandem Massenspektrometrie (LC-MS/MS) analysiert,
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