Protein quality control and antibiotics: the role of the small heat shock protein YocM and the disaggregase ClpC in B. subtilis

Publikation: Qualifikations-/Studienabschlussarbeit › Dissertation

Autorschaft

  • Ingo Hantke

Organisationseinheiten

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Details

OriginalspracheEnglisch
QualifikationDoctor rerum naturalium
Gradverleihende Hochschule
Betreut von
  • Kürşad Turgay, Betreuer*in
Datum der Verleihung des Grades16 Sept. 2019
ErscheinungsortHannover
PublikationsstatusVeröffentlicht - 2019

Abstract

Alle lebenden Zellen müssen Fluktuationen von abiotischen Umweltfaktoren (z.B. Temperatur, Salzgehalt etc.) und daraus resultierendem proteotoxischen Stress entgegen-wirken. Ein funktionales Proteom wird dabei durch die Aktivität verschiedener Chaperone und Proteasen gewährleistet. Dieses Proteinqualitätskontrollsystem (PQK) ist hoch konserviert. Einen Teil bilden kleine Hitzeschockproteine (sHsp), die Proteine vor Entfaltung schützen und deren Rückfaltung in Kooperation mit Chaperonsystemen begünstigen können. In dieser Arbeit wurde YocM als erstes Stress relevantes sHsp in B. subtilis identifiziert und dessen protektive Rolle während eines Salzschocks charakterisiert. Zudem wurde ein YocM-mCherry Fusionsprotein als in vivo-Aggregatmarker etabliert. Mittels dieses Markers wurde McsB als entscheidendes Adapterprotein für die vom Hsp100/Clp Protein ClpC vermittelte Disaggregation von Proteinaggregaten unter Hitzestress identifiziert und gleichzeitig dessen Proteinargininkinase als essentiell für ebenjene Aktivität erkannt. Dabei stellte sich in der Stressantwort von B. subtilis generell die Disaggregation und Rückfaltung im Vergleich zu der Degradation von Proteinaggregaten als bedeutsamer heraus. Kürzlich wurden einige Antibiotika charakterisiert, welche auf ClpC als zentralen Punkt in der PQK abzielen (z.B. Cyclomarin). In dieser Arbeit konnte gezeigt werden, dass der Austausch einer Aminosäure in ClpC (F436A) zu vermehrter Bildung von Proteinaggregaten sowie zu erheblich gestörtem Wachstum und sogar Zelltod führt. Diese durch ClpC vermittelte Toxizität untermauert die Eignung von ClpC als Ziel für Antibiotika. Im Folgenden wurde ein target-based screen in B. subtilis etabliert und als proof of concept validiert. Neue Antibiotika, die auf ClpC und die PQK abzielen, können so schnell selektiert und anschließend auf deren Effekt auf Gram-positive Pathogene weiter getestet werden.

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Protein quality control and antibiotics: the role of the small heat shock protein YocM and the disaggregase ClpC in B. subtilis. / Hantke, Ingo.
Hannover, 2019. 194 S.

Publikation: Qualifikations-/Studienabschlussarbeit › Dissertation

Hantke, I 2019, 'Protein quality control and antibiotics: the role of the small heat shock protein YocM and the disaggregase ClpC in B. subtilis', Doctor rerum naturalium, Gottfried Wilhelm Leibniz Universität Hannover, Hannover. https://doi.org/10.15488/5491
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abstract = "All living cells have to deal with fluctuations of abiotic factors, such as temperature and salt content, which can cause different types of proteotoxic stress. A functional proteome depends on an intricate protein quality control network (PQC) of chaperones and proteases, which is highly conserved in all domains of life. As one part of the PQC system, small heat shock proteins (sHsp) protect proteins from unfolding and facilitate refolding in cooperation with molecular chaperons. During this work, YocM was identified as the first stress-related sHsp of B. subtilis, ensuring survival of cells during salt shock. Furthermore, a YocM-mCherry fusion protein was established as a protein aggregate marker. Thereby, McsB was identified as the main adaptor protein for disaggregation of subcellular protein aggregates by the Hsp100/Clp protein ClpC during heat stress, which was dependent on its protein arginine kinase activity. In general, protein disaggregation instead of degradation was observed to be the predominant process regarding protein aggregate removal in stress response in B. subtilis. ClpC as a central player of PQC and stress response was recently identified as a target for various antibiotic compounds (e.g. cyclomarin). During this work it was demonstrated that a clpC F436A mutation led to severe formation of protein aggregates in vivo and substantially impaired survival of B subtilis, which was dependent on the presence of McsB. As this deregulation of ClpC by exchange of only one amino acid residue displayed such a toxic phenotype, ClpC was confirmed to be an adequate target for antibiotics. Consequently, a ClpC-target based screen was successfully established and validated in B. subtilis as proof of concept to discover and characterize novel antibiotics compounds that address the PQC system in Gram-positive pathogenic bacteria.",
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T2 - the role of the small heat shock protein YocM and the disaggregase ClpC in B. subtilis

AU - Hantke, Ingo

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