Site-specific incorporation of 3-nitrotyrosine in proteins generated via genetic code expansion in E.coli

Gerding, Hanne R.

Site-specific incorporation of 3-nitrotyrosine in proteins generated via genetic code expansion in E.coli

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Date

2018

Publication type

Dissertation

Publication status

Published

Abstract

Tyrosine nitration is an oxidative post-translational protein modification relevant to various pathophysiological processes. It is potentially also involved in the redox regulation of normal cellular functions. The scientific literature provides little information on residue specificity and efficiency of nitration. In initial experiments of this thesis, we further showed that the commonly used chemical tyrosine nitration methods also lead to unwanted modifications at other amino acid residues. The multiple side reactions make it arduous to establish direct and quantitative relationships between extents of tyrosine nitration on specific sites in a protein and biological responses. Therefore, in this thesis, a novel strategy was explored, allowing incorporation of 3-nitrotyrosine (3NT) during ribosomal protein synthesis, based on a genetic code modification. It was used to generate recombinant proteins with defined 3NT-sites, in the absence of other post-translational modifications. We studied the generation and stability of the 3NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (ASYN) was selected as exemplary protein, relevant in Parkinson’s disease (PD). A procedure was established to obtain pure tyrosine-modified ASYN in mg amounts. However, a rapid (t1/2 = 0.4 h) reduction of 3NT to 3-aminotyrosine (3AT) was observed. When screening for potential mechanisms, we found that 3NT can be reduced enzymatically to 3AT, depending on the spatial orientation of 3NT within the protein. A genetic screen of E.coli proteins, involved in the observed 3NT reduction, revealed the contribution of several, possibly redundant pathways. Green fluorescent protein was studied as an alternative model protein. These data confirm 3NT reduction as a broadly-relevant pathway in E.coli. In conclusion, incorporation of 3NT as a genetically-encoded non-natural amino acid allows for generation of recombinant proteins with specific nitration sites, opening up new possibilities for investigating the role of tyrosine nitration in regulating protein structure and function in physiological and pathophysiological conditions. The potential reduction of the 3NT moiety by E.coli, however, requires attention to the design of the purification strategy for obtaining purified nitrated protein. The findings of this thesis open for new research questions: (i) which role does the reduction of 3NT in E.coli have as a defence mechanism under inflammatory conditions? (ii) which role does 3AT play as a potential end product of potential 3NT reduction in mammalian tissue? This novel post-translational modification could be relevant in diseases related to inflammation and oxidative stress.

Summary in another language

Die Nitrierung von Tyrosinresten ist eine oxidative post-translationale Modifikation, relevant in verschiedenen pathophysiologischen Prozessen. Sie spielt möglicherweise auch eine Rolle in der Redox-Regulation normaler zellulärer Funktionen. Die wissenschaftliche Literatur gibt leider nur wenig Auskunft über die Spezifität der nitrierten Tyrosinreste und die Effizienz der Nitrierung. In ersten Experimenten zu Beginn dieser Arbeit konnten wir zudem zeigen, dass die gebräuchlichen chemischen Tyrosin-Nitrierungsverfahren auch eine Vielzahl von Modifikationen an anderen Aminosäuren erzeugen. Diese Vielzahl von Nebenreaktionen macht es sehr mühselig, direkte und quantitative Zusammenhänge zwischen dem Grad der Nitrierung an bestimmten Tyrosinresten eines Proteins und einer biologischen Reaktion zu etablieren. Deswegen wurde in der vorliegenden Arbeit eine neue Strategie erforscht, basierend auf einer Modifizierung des genetischen Codes, die den Einbau von Nitrotyrosin während der ribosomalen Proteinsynthese erlaubt. Diese Strategie wurde genutzt, um rekombinante Proteine mit definierten nitrierten Tyrosinresten zu generieren ohne die gleichzeitige Modifikation anderer Aminosäuren des Proteins. Wir konnten damit die Generierung und die Stabilität der Nitrotyrosin-Gruppe in rekombinanten Proteinen untersuchen, die in E.coli hergestellt wurden. Nitriertes Alpha-Synuclein (ASYN), das in der Parkinson-Krankheit eine Rolle spielt, wurde als exemplarisches Protein gewählt. Ein Verfahren wurde etabliert, um Milligramm-Mengen von reinem Tyrosin-modifizierten ASYN herzustellen. Allerdings wurde dabei eine schnelle (t1/2= 0.4 h) Reduktion des Nitrotyrosins zu Aminotyrosin beobachtet. Auf der Suche nach potentiellen Mechanismen konnten wir feststellen, dass Nitrotyrosin abhängig von seiner Position im Protein enzymatisch zu Aminotyrosin reduziert werden kann. Ein genetischer Screen von E.coli-Proteinen, die in die beobachte Reduktion des Nitrotyrosins involviert sein könnten, zeigte, dass mehrere, eventuell redundante Reaktionswege beteiligt sind. Grün fluoreszierendes Protein wurde als zusätzliches alternatives Modell-Protein untersucht. Diese Daten bestätigen, dass die Reduktion von Nitrotyrosin ein allgemein relevanter Prozess in E.coli zu sein scheint. Schlussfolgernd erlaubt der Einbau von Nitrotyrosin als genetisch-codierte Aminosäure die Herstellung rekombinanter Proteine mit spezifischen Nitrierungsstellen und somit neue Möglichkeiten, die Rolle von Tyrosin-Nitrierung in der Regulation von Proteinstruktur und -funktion unter physiologischen und pathophysiologischen Bedingungen zu untersuchen. Die mögliche Reduktion der Nitrotyrosin-Gruppe durch E.coli macht es jedoch notwendig, größere Aufmerksamkeit auf die Aufreinigungsstrategie zu legen, um reine nitrierte Proteine zu erhalten. Die Ergebnisse dieser Arbeit eröffnen neue wissenschaftliche Fragen: (i) welche Rolle spielt die Reduktion des Nitrotyrosins in E.coli als möglicher Abwehrmechanismus unter Entzündungsbedingungen? (ii) welche Rolle spielt Aminotyrosin als potentielles Endprodukt der möglichen Reduktion von Nitrotyrosin in Säugetiergewebe? Diese neue post-translationale Modifikation könnte in Krankheiten relevant sein, die in Zusammenhang mit Entzündung und oxidativem Stress stehen.

Subject (DDC)

570 Biosciences, Biology

Keywords

alpha-synuclein, Parkinson's disease, nitration, E.coli, unnatural amino acids, genetic encoding

Cite This

ISO 690

GERDING, Hanne R., 2018. Site-specific incorporation of 3-nitrotyrosine in proteins generated via genetic code expansion in E.coli [Dissertation]. Konstanz: University of Konstanz

BibTex

@phdthesis{Gerding2018Sites-45156,
  year={2018},
  title={Site-specific incorporation of 3-nitrotyrosine in proteins generated via genetic code expansion in E.coli},
  author={Gerding, Hanne R.},
  address={Konstanz},
  school={Universität Konstanz}
}

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Examination date of dissertation

December 10, 2018

University note

Konstanz, Univ., Doctoral dissertation, 2018