TU Darmstadt / ULB / TUbiblio

Studies on the interaction of the inhibitor of apoptosis protein Survivin with DNA-dependent protein kinase to modulate DNA double-strand break repair

Weipert, Fabian (2018):
Studies on the interaction of the inhibitor of apoptosis protein Survivin with DNA-dependent protein kinase to modulate DNA double-strand break repair.
Darmstadt, Technische Universität, [Online-Edition: http://tuprints.ulb.tu-darmstadt.de/7473],
[Ph.D. Thesis]

Abstract

During the last years, the ability of tumour cells to evade apoptosis was considered to be an important mechanism to develop resistance against tumour therapies. In this context, members of the inhibitor of apoptosis protein (IAP) family gained increasing attention. Survivin, the smallest member of the IAP family, is a functionally unique protein that is involved in a variety of molecular mechanisms and cellular networks including cell proliferation, regulation of apoptosis and metastasis formation. Furthermore, an overexpression of Survivin in the tumour tissue was correlated with tumour progression as well as a decreased survival of the patients. Besides inhibition of apoptosis and its role as a member of the chromosomal passenger complex, Survivin was also found being accumulated in the nucleus after irradiation. That accumulation was linked to a modulation of DNA double-strand break repair due to its interaction with DNA repair factors such as the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). The aim of this thesis was to gain further insight on the molecular mechanisms facilitating a Survivin-mediated regulation of DNA repair by characterising the interaction between Survivin and DNA-PKcs, a major enzyme in the non-homologous end-joining (NHEJ) DNA double-strand break repair pathway, in more detail. Docking of Survivin wild type (wt) and a X-linked IAP (XIAP) binding site deletion mutant (ΔXIAP) of Survivin to DNA-PKcs was evaluated in colorectal cancer SW480 and glioblastoma LN-229 cells via immunoprecipitation experiments. These experiments indicated that recombinant Survivin (wt) was able to co-immunoprecipitate with DNA-PKcs in both lines while the ΔXIAP mutant of Survivin did not show complexation to DNA-PKcs. In case of the Aurora-B kinase it has been reported that Survivin stimulates Aurora-B kinase activity by binding to the catalytic domain. In analogy, an interaction of Survivin with the kinase domain of DNA-PKcs (PI3K) was analysed by different methods, including GST pulldown assay, NanoLuc Binary Technology (NanoBiT®) complementation assay and flow cytometry-based Förster resonance energy transfer (FRET). All of these methods confirmed an interaction between Survivin and the PI3K domain of DNA-PKcs, indicating that Survivin is binding directly to the kinase domain but not to other domains like the HEAT1 and FATC domain. Additionally, functional analysis, such as autophosphorylation of serine 2056 of DNA-PKcs, revealed a decreased DNA-PK activity after Survivin knockdown in both SW480 and LN-229 cells. Finally, attenuation of endogenous Survivin in the ΔXIAP mutant of Survivin resulted in a decreased DNA-PK activity measured by SignaTECT kinase assay, while recombinant Survivin (wt) rescued DNA-PK activity following irradiation with 4 Gy. In conclusion these findings for the first time indicate that Survivin not only interacts with DNA-PKcs but directly binds to its kinase domain. Thus, it modulates DNA-PKcs kinase activity and as a consequence repair of radiation induced DNA double-strand breaks. These results add a further facet to the plethora of functions exerted by the nodal protein Survivin in the cellular radiation response in cancer cells.

Item Type: Ph.D. Thesis
Erschienen: 2018
Creators: Weipert, Fabian
Title: Studies on the interaction of the inhibitor of apoptosis protein Survivin with DNA-dependent protein kinase to modulate DNA double-strand break repair
Language: English
Abstract:

During the last years, the ability of tumour cells to evade apoptosis was considered to be an important mechanism to develop resistance against tumour therapies. In this context, members of the inhibitor of apoptosis protein (IAP) family gained increasing attention. Survivin, the smallest member of the IAP family, is a functionally unique protein that is involved in a variety of molecular mechanisms and cellular networks including cell proliferation, regulation of apoptosis and metastasis formation. Furthermore, an overexpression of Survivin in the tumour tissue was correlated with tumour progression as well as a decreased survival of the patients. Besides inhibition of apoptosis and its role as a member of the chromosomal passenger complex, Survivin was also found being accumulated in the nucleus after irradiation. That accumulation was linked to a modulation of DNA double-strand break repair due to its interaction with DNA repair factors such as the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). The aim of this thesis was to gain further insight on the molecular mechanisms facilitating a Survivin-mediated regulation of DNA repair by characterising the interaction between Survivin and DNA-PKcs, a major enzyme in the non-homologous end-joining (NHEJ) DNA double-strand break repair pathway, in more detail. Docking of Survivin wild type (wt) and a X-linked IAP (XIAP) binding site deletion mutant (ΔXIAP) of Survivin to DNA-PKcs was evaluated in colorectal cancer SW480 and glioblastoma LN-229 cells via immunoprecipitation experiments. These experiments indicated that recombinant Survivin (wt) was able to co-immunoprecipitate with DNA-PKcs in both lines while the ΔXIAP mutant of Survivin did not show complexation to DNA-PKcs. In case of the Aurora-B kinase it has been reported that Survivin stimulates Aurora-B kinase activity by binding to the catalytic domain. In analogy, an interaction of Survivin with the kinase domain of DNA-PKcs (PI3K) was analysed by different methods, including GST pulldown assay, NanoLuc Binary Technology (NanoBiT®) complementation assay and flow cytometry-based Förster resonance energy transfer (FRET). All of these methods confirmed an interaction between Survivin and the PI3K domain of DNA-PKcs, indicating that Survivin is binding directly to the kinase domain but not to other domains like the HEAT1 and FATC domain. Additionally, functional analysis, such as autophosphorylation of serine 2056 of DNA-PKcs, revealed a decreased DNA-PK activity after Survivin knockdown in both SW480 and LN-229 cells. Finally, attenuation of endogenous Survivin in the ΔXIAP mutant of Survivin resulted in a decreased DNA-PK activity measured by SignaTECT kinase assay, while recombinant Survivin (wt) rescued DNA-PK activity following irradiation with 4 Gy. In conclusion these findings for the first time indicate that Survivin not only interacts with DNA-PKcs but directly binds to its kinase domain. Thus, it modulates DNA-PKcs kinase activity and as a consequence repair of radiation induced DNA double-strand breaks. These results add a further facet to the plethora of functions exerted by the nodal protein Survivin in the cellular radiation response in cancer cells.

Place of Publication: Darmstadt
Divisions: 10 Department of Biology
DFG-Graduiertenkollegs
DFG-Graduiertenkollegs > Research Training Group 1657 Molecular and cellular responses to ionizing radiation
Date Deposited: 22 Jul 2018 19:55
Official URL: http://tuprints.ulb.tu-darmstadt.de/7473
URN: urn:nbn:de:tuda-tuprints-74734
Referees: Laube, Prof. Dr. Bodo and Löbrich, Prof. Dr. Markus and Rödel, Prof. Dr. Franz
Refereed / Verteidigung / mdl. Prüfung: 30 May 2018
Alternative Abstract:
Alternative abstract Language
In den vergangenen Jahren wurde die Fähigkeit von Tumorzellen Apoptose zu umgehen bzw. zu vermeiden als zentraler Mechanismus einer Therapieresistenz erkannt. In diesem Zusammenhang gewannen auch Vertreter der inhibitor of apoptosis protein (IAP) Familie immer mehr an Bedeutung. Dabei spielt unter anderem Survivin, der kleinste Vertreter dieser Proteinfamilie, eine bedeutende Rolle aufgrund der universellen Überexpression in Tumorzellen. Survivin ist an einer Vielzahl molekularer Mechanismen und zellulärer Netzwerke wie beispielsweise Zellproliferation, intrazellulärer Signaltransduktion, Apoptoseregulation und Metastasierung beteiligt. Darüber hinaus konnte eine Überexpression von Survivin mit der Tumorprogression und einem schlechteren Überleben der Patienten korreliert werden. Neben der Hemmung von Apoptose und seiner Rolle in der Zellzyklusregulation ist Survivin ebenfalls bekannt dafür nach Bestrahlung im Nukleus zu akkumulieren. Dies steht im Zusammenhang mit der Rolle von Survivin bei der Regulation der DNA-Reparatur als Teil der zellulären Strahlenantwort. In dieser Arbeit sollte der genaue molekulare Mechanismus einer Survivin-vermittelten Regulation der DNA-Reparatur untersucht werden. Dafür wurde die Interaktion zwischen Survivin und der katalytischen Untereinheit der DNA-abhängigen Proteinkinase (DNA-PKcs), einem wichtigen Faktor im non-homologous end-joining (NHEJ) genauer untersucht. Beim NHEJ handelt es sich um einen der beiden Hauptwege der DNA-Doppelstrangbruchreparatur. Um die Interaktion zwischen Survivin und DNA-PKcs genauer zu verstehen, wurde die Bindung einer Deletionsmutante der X-linked IAP-Bindestelle (ΔXIAP) von Survivin an DNA-PKcs mittels Immunpräzipitation untersucht. Hierbei zeigte sich sowohl in kolorektalen SW480 Karzinomzellen als auch in LN-229 Glioblastomzellen, eine Bindung des Survivin-Wildtyps an DNA-PKcs, während die Deletionsmutante der XIAP Bindestelle von Survivin nicht mehr mit DNA-PKcs präzipitieren konnte. Im Falle der Aurora-B Kinase ist bekannt, dass Survivin die Kinaseaktivität durch das Binden an deren katalytische Domäne stimuliert. Aus diesem Grund sollte im weiteren Verlauf dieser Studie eine Interaktion von Survivin mit der Kinasedomäne PI3K von DNA-PKcs mittels verschiedener Methoden wie GST pulldown Assay, NanoLuc Binary Technology (NanoBiT®) Komplementationsassay und Förster-Resonanzenergietransfer (FRET) am Durchflusszytometer untersucht werden. Durch alle verwendeten Analysemethoden konnte eine eindeutige Interaktion zwischen Survivin und der PI3K-Domäne, nicht jedoch mit anderen Domänen wie HEAT1 und FATC gezeigt werden, was auf eine direkte Bindung Survivins an die Kinasedomäne von DNA-PKcs schließen lässt. Zusätzlich zeigten funktionelle Analysen der Interaktion zwischen Survivin und DNA-PKcs, wie zum Beispiel die Detektion der Autophosphorylierung von DNA-PKcs an Serin 2056, eine verminderte Kinaseaktivität von DNA-PK nach Herunterregulation von Survivin in kolorektalen SW480 Karzinomzellen und LN-229 Glioblastomzellen. Eine Herunterregulation des endogenen Survivins in ΔXIAP-exprimierenden Zellen und Bestrahlung mit 4 Gy zeigte eine verminderte DNA-PK-Aktivität im SignaTECT Kinase-Assay, wohingegen der rekombinante Survivin Wildtyp die Kinaseaktivität wiederherstellen konnte. In dieser Studie konnte zum ersten Mal gezeigt werden, dass Survivin nicht nur mit DNA-PKcs interagiert, sondern direkt an dessen Kinasedomäne bindet. Durch diese Interaktion moduliert Survivin die Kinaseaktivität von DNA-PKcs und damit die Reparatur von strahleninduzierten DNA-Doppelstrangbrüchen. Alles in allem wird Survivin einmal mehr seiner Rolle als Knotenpunkt-Protein in der zellulären Strahlenantwort gerecht.German
Export:

Optionen (nur für Redakteure)

View Item View Item