TU Darmstadt / ULB / TUbiblio

Spatial and functional interrelationship of a heterotetramer Survivin-DNA-PKcs complex in the repair of DNA double-strand breaks

Güllülü, Ömer (2020):
Spatial and functional interrelationship of a heterotetramer Survivin-DNA-PKcs complex in the repair of DNA double-strand breaks. (Publisher's Version)
Darmstadt, Technische Universität,
DOI: 10.25534/tuprints-00015406,
[Ph.D. Thesis]

Abstract

Survivin was discovered as a member of the Inhibitor of Apoptosis Protein (IAP) family showing high expression in almost all human cancers. Although primarily considered as a protein implicated apoptosis and cell cycle/mitotic spindle checkpoint regulation, Survivin is now recognized as a nodal factor involved in a multitude of cellular circuits. By this, Survivin covers a radiation resistance factor in a variety of cancer entities and enhances tumor cell survival upon radiation exposure by impacting DNA double-strand break (DNA DSB) repair. Following irradiation, nuclear accumulation of Survivin was mechanistically been linked to the activity of the DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), a key component of DNA DSB repair pathway non-homologous end joining (NHEJ).

In this study, we aimed to unravel the determinants of the Survivin-DNA-PKcs interrelationship on a molecular level by computational investigations of the regions of interaction and biochemical approaches. 3D crystallographic structures of Survivin and catalytic PI3K domain of DNA-PKcs were virtually docked using advanced global docking algorithms, simulated by molecular dynamics, and were evaluated according to binding free energies (ΔG) and the spatial accessibility/physical proximity. Next, multiple residues derived from these analyses were mutated, and the functional consequences of the mutagenesis were assayed by flow cytometry-based Förster resonance energy transfer (FACS-FRET) and co-immunoprecipitation (co-IP) experiments. Radiation survival and DNA damage repair capacity were assayed by 3D colony formation assays and DNA foci analysis (γH2AX/53BP1), respectively. The effects of the Survivin-DNA-PKcs interrelationship were further analyzed by in vitro DNA-PKcs kinase activity assays and Liquid Chromatography-Mass Spectrometry (LC-MS2/3)-based multi-proteomic techniques. Finally, a virtual drug screening approach was employed in search for novel small-molecule radiosensitizers targeting the Survivin-DNA-PKcs interaction.

Molecular docking and advanced in silico analyses uncovered residues serine(S)20 and tryptophan(W)67 located in the baculovirus inhibitor of apoptosis protein repeat (BIR) domain of dimerized Survivin to interact with the PI3K domain of DNA-PKcs. Mutagenesis of these residues significantly decreased the interaction compared to wild-type (wt) Survivin, was correlated with an increased radiosensitivity of colorectal cancer cells and a hampered DNA repair capacity, measured by γH2AX/53BP1 foci analysis, after knockdown of endogenous Survivin. By contrast, overexpression of wt Survivin rescued radiation survival and DNA repair. In addition, advanced molecular docking and dynamics simulation analyses revealed a heterotetramer model, where Survivin binds to the surface of pre-existing DNA-PKcs dimer. Moreover, by investigating the effects of Survivin on DNA-PKcs’ downstream regulatory functions, differentially abundant phosphopeptides and proteins were identified for multiple pathways, predominantly for DNA damage/repair. Binding of Survivin to a pre-existing DNA-PKcs dimer was lead to a conformational change on the PI3K domain and resulted in a differential change in substrate specificity. Particularly, the previously little-known DNA-PKcs’ S/T-Hydr (hydrophobic residues: G, A, V, L, I, P, F, M, W) motif substrates including the FOXO3 S253 phosphosite displayed high conservation within the detected phosphosites. Further, proteomics analyses indicated that the Survivin-DNA-PKcs interrelationship not only displays post-translational but also protein expression-level regulatory properties. Ultimately, the virtual drug screening approach uncovered small-molecule compounds having strong binding affinity to S20, and W67 residues and consequently might show promise for the development of future radiation sensitizing therapeutic approaches.

In summary, in this study, we identified specific residues of Survivin involved in the interaction with the PI3K domain of DNA-PKcs by implementing in vivo live cell protein interaction quantification and in silico structure-based molecular docking technologies. Besides that, findings on radiosensitivity, DNA foci formation, kinase activity, and phosphoproteomics and proteomics analyses further strengthen the notion that Survivin is a fine-tuning regulator of DNA DSB repair and impacts on substrate specificity by fostering the S/T-Hydr motif phosphorylation. Large-scale proteomics and phosphoproteomics studies further discovered novel candidate proteins and phosphosites, enlightening the underlying mechanistic relation between Survivin and DNA-PKcs in response to irradiation and may pave the way to novel Survivin-related cancer and DNA damage response marker discoveries.

Item Type: Ph.D. Thesis
Erschienen: 2020
Creators: Güllülü, Ömer
Status: Publisher's Version
Title: Spatial and functional interrelationship of a heterotetramer Survivin-DNA-PKcs complex in the repair of DNA double-strand breaks
Language: English
Abstract:

Survivin was discovered as a member of the Inhibitor of Apoptosis Protein (IAP) family showing high expression in almost all human cancers. Although primarily considered as a protein implicated apoptosis and cell cycle/mitotic spindle checkpoint regulation, Survivin is now recognized as a nodal factor involved in a multitude of cellular circuits. By this, Survivin covers a radiation resistance factor in a variety of cancer entities and enhances tumor cell survival upon radiation exposure by impacting DNA double-strand break (DNA DSB) repair. Following irradiation, nuclear accumulation of Survivin was mechanistically been linked to the activity of the DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), a key component of DNA DSB repair pathway non-homologous end joining (NHEJ).

In this study, we aimed to unravel the determinants of the Survivin-DNA-PKcs interrelationship on a molecular level by computational investigations of the regions of interaction and biochemical approaches. 3D crystallographic structures of Survivin and catalytic PI3K domain of DNA-PKcs were virtually docked using advanced global docking algorithms, simulated by molecular dynamics, and were evaluated according to binding free energies (ΔG) and the spatial accessibility/physical proximity. Next, multiple residues derived from these analyses were mutated, and the functional consequences of the mutagenesis were assayed by flow cytometry-based Förster resonance energy transfer (FACS-FRET) and co-immunoprecipitation (co-IP) experiments. Radiation survival and DNA damage repair capacity were assayed by 3D colony formation assays and DNA foci analysis (γH2AX/53BP1), respectively. The effects of the Survivin-DNA-PKcs interrelationship were further analyzed by in vitro DNA-PKcs kinase activity assays and Liquid Chromatography-Mass Spectrometry (LC-MS2/3)-based multi-proteomic techniques. Finally, a virtual drug screening approach was employed in search for novel small-molecule radiosensitizers targeting the Survivin-DNA-PKcs interaction.

Molecular docking and advanced in silico analyses uncovered residues serine(S)20 and tryptophan(W)67 located in the baculovirus inhibitor of apoptosis protein repeat (BIR) domain of dimerized Survivin to interact with the PI3K domain of DNA-PKcs. Mutagenesis of these residues significantly decreased the interaction compared to wild-type (wt) Survivin, was correlated with an increased radiosensitivity of colorectal cancer cells and a hampered DNA repair capacity, measured by γH2AX/53BP1 foci analysis, after knockdown of endogenous Survivin. By contrast, overexpression of wt Survivin rescued radiation survival and DNA repair. In addition, advanced molecular docking and dynamics simulation analyses revealed a heterotetramer model, where Survivin binds to the surface of pre-existing DNA-PKcs dimer. Moreover, by investigating the effects of Survivin on DNA-PKcs’ downstream regulatory functions, differentially abundant phosphopeptides and proteins were identified for multiple pathways, predominantly for DNA damage/repair. Binding of Survivin to a pre-existing DNA-PKcs dimer was lead to a conformational change on the PI3K domain and resulted in a differential change in substrate specificity. Particularly, the previously little-known DNA-PKcs’ S/T-Hydr (hydrophobic residues: G, A, V, L, I, P, F, M, W) motif substrates including the FOXO3 S253 phosphosite displayed high conservation within the detected phosphosites. Further, proteomics analyses indicated that the Survivin-DNA-PKcs interrelationship not only displays post-translational but also protein expression-level regulatory properties. Ultimately, the virtual drug screening approach uncovered small-molecule compounds having strong binding affinity to S20, and W67 residues and consequently might show promise for the development of future radiation sensitizing therapeutic approaches.

In summary, in this study, we identified specific residues of Survivin involved in the interaction with the PI3K domain of DNA-PKcs by implementing in vivo live cell protein interaction quantification and in silico structure-based molecular docking technologies. Besides that, findings on radiosensitivity, DNA foci formation, kinase activity, and phosphoproteomics and proteomics analyses further strengthen the notion that Survivin is a fine-tuning regulator of DNA DSB repair and impacts on substrate specificity by fostering the S/T-Hydr motif phosphorylation. Large-scale proteomics and phosphoproteomics studies further discovered novel candidate proteins and phosphosites, enlightening the underlying mechanistic relation between Survivin and DNA-PKcs in response to irradiation and may pave the way to novel Survivin-related cancer and DNA damage response marker discoveries.

Place of Publication: Darmstadt
Divisions: 10 Department of Biology
10 Department of Biology > Radiation Biology and DNA Repair
DFG-Graduiertenkollegs
DFG-Graduiertenkollegs > Research Training Group 1657 Molecular and cellular responses to ionizing radiation
TU-Projects: DFG|GRK1657|GRK 1657
Date Deposited: 23 Dec 2020 09:09
DOI: 10.25534/tuprints-00015406
Official URL: https://tuprints.ulb.tu-darmstadt.de/15406
URN: urn:nbn:de:tuda-tuprints-154062
Referees: Rödel, Prof. Dr. Franz and Löbrich, Prof. Dr. Markus
Refereed / Verteidigung / mdl. Prüfung: 2 December 2020
Alternative Abstract:
Alternative abstract Language

Survivin, das kleinste Mitglied der Inhibitor of Apoptosis Protein (IAP)-Familie ist durch eine hohe Expression in fast allen untersuchten humanen Tumoren gekennzeichnet. Obwohl Survivin primär als ein Protein mit Funktionalität in der Regulation von Apoptose und Zellzyklus/mitotischen Spindelkontroll- punkt beschrieben ist, wird das Protein aktuell als ein wesentlicher Knotenfaktor in einer Vielzahl zellulärer Reaktionskaskaden angesehen. In diesem Zusammenhang stellt Survivin in einer Vielzahl von Tumorentitäten einen Resistenzfaktor dar, der das Überleben der malignen Zellen nach Strahlenexposition verbessert indem er die Reparatur von DNA-Doppelstrangbrüchen (DNA-DSB) zu modulieren vermag. Dabei konnte eine nukleäre Akkumulation von Survivin nach Bestrahlung mechanistisch mit der Aktivität der DNA-abhängigen Proteinkinase (DNA-PKcs), einer Schlüsselkomponente des DNA-DSB Reparatur- Mechanismus der Nicht-Homologen Endverknüpfung (NHEJ), assoziiert werden.

Ziel der Studie war die Aufklärung der molekularen Determinanten der Survivin-DNA-PKcs Wechsel- Beziehung durch computergestützte Analysen der Interaktionsregionen und biochemische Ansätze. Dazu wurden die Bindung kristallographischer 3D-Strukturen von Survivin und die katalytische PI3Kinase-Domäne der DNA-PKcs virtuell mit Hilfe fortgeschrittener Algorithmen und molekular-dynamischer Berechnungen simuliert und in Abhängigkeit von freien Bindungsenergien (ΔG) und der räumlichen Zugänglichkeit/physischen Nähe bewertet. Anschließend wurden als Ergebnis dieser Analysen unterschiedliche Aminosäuren von Survivin mutiert und die funktionellen Konsequenzen dieser Mutagenese mittels durchflusszytometrischer Förster-Resonanzenergietransfer (FACS-FRET) und Ko-Immunpräzipitations (co-IP) Experimenten untersucht. Das Überleben nach Bestrahlung und die Fähigkeit zur Reparatur von DNA-Schäden wurden mit 3-dimensionalen-Koloniebildungstests bzw. der Quantifizierung von DNA-Schadensmarker γH2AX/53BP1 analysiert, während die Effekte der Interaktion auf die Kinaseaktivität der DNA-PKcs durch in-vitro Kinase-Aktivitätsmessungen und Massenspektrometrie (LC-MS2/3) basierten multi-Proteomik-Ansätzen evaluiert wurden. Schließlich erfolgte mit Hilfe eines virtuellen Wirkstoff-Screening-Ansatzes auf Grundlage der Survivin-DNA-PKcs-Interaktion eine Suche nach neuartigen niedermolekularen Hemmstoffen der Interaktion mit möglicher strahlensensibilisierender Wirkung.

Durch molekulare Bindungs- und in-silico Analysen konnte eine Bindung von Survivin an die PI3Kinase-Domäne der DNA-PKcs nachgewiesen werden, die überwiegend durch eine Interaktion der Aminosäuren Serin (S)20 und Tryptophan (W)67 der Baculovirus-IAP repeat (BIR) Domäne von dimerisierten Survivin vermittelt wird. Im Vergleich zum Wildtyp (wt) Survivin verringerte eine spezifische Mutagenese dieser Aminosäuren signifikant die Interaktion mit der DNA-PKcs, korrelierte mit einer erhöhten Strahlen- sensibilität von kolorektalen Tumorzellen und einer verminderten DNA-Reparaturkapazität nach Hemmung von endogenem Survivin. Im Vergleich dazu konnte durch eine Überexpression von wt Survivin das klonogene Zellüberleben und die DNA-Reparaturkapazität wiederhergestellt werden. Darüber hinaus führten weiterführende molekulare Bindungsanalysen und dynamische Simulationen zur Entwicklung eines Heterotetramer-Modells, bei dem Survivin an die Oberfläche eines bereits existierenden DNA-PKcs-Dimers zu binden vermag. In Untersuchungen des Effekts dieser Bindung auf nachgeschaltete regulatorische Funktionen der DNA-PKcs konnte eine große Anzahl differentiell regulierter Phospho- peptide identifiziert werden, die vorwiegend Reaktionswegen der DNA-Schadensantwort/Reparatur betreffen. Dabei führt die Bindung von Survivin an ein präformiertes DNA-PKcs-Dimer zu einer Konformationsänderung der PI3K-Domäne, resultierte in einer signifikanten Steigerung der Kinaseaktivität und einer differentiellen Änderung der Substratspezifität. Insbesondere bisher wenig beschriebene DNA-PKcs S/T-Hydr (hydrophobe Aminosäuren: G, A, V, L, I, P, F, M, W) Motive einschließlich der FOXO3 Aminosäure S253 zeigten eine hohe Konservierung innerhalb der detektierten Phosphorylierungsstellen. Darüber hinaus zeigten Proteomanalysen, dass die Survivin-DNA-PKcs-Interaktion nicht nur post- translationale, sondern auch regulatorische Funktionen auf Proteinexpressionsebene ausüben kann. Schließlich konnten in ersten virtuellen Wirkstoff-Screening-Ansatz niedermolekulare Verbindungen identifiziert werden, die eine hohe Bindungsaffinität zu den Amiosäuren S20 und W67 aufweisen und für zukünftige strahlensensibilisierende Therapieansätze vielversprechend sein könnten.

Zusammenfassend konnte erstmals unter Verwendung einer Methodik zur Quantifizierung einer Proteineninteraktion in lebenden Zellen und durch in-silico strukturbasierte Bindungsanalysen spezifische Aminosäuren von Survivin identifiziert werden, die an der Wechselwirkung mit der PI3K-Domäne der DNA-PKcs beteiligt sind. Darüber hinaus bestätigen die Ergebnisse der Strahlensensibilitätsuntersuchungen, der Nachweis von DNA Reparatur-Foci, DNA-PKcs Aktivitätsmessungen und (Phospho)proteomik-Analysen die Vorstellung, dass Survivin einen (Fein)regulator der DNA-DSB Reparatur und insbesondere der Substratspezifität der DNA-PKcs hin zu S/T-Hydr-Motiven darstellt. Umfangreiche (Phospho)proteomik-Analysen führten zudem zur Aufdeckung neuer Kandidatenproteine und Phosphorylierungsstellen. Dies könnte dazu beitragen die zugrundeliegenden mechanistischen Beziehungen zwischen Survivin und DNA-PKcs weiter aufzuklären und den Weg zur Entdeckung neuer, Survivin-assoziierter Tumor und DNA-Schadenreaktionsmarkern und Therapieansätze zu ebnen.

German
Export:
Suche nach Titel in: TUfind oder in Google
Send an inquiry Send an inquiry

Options (only for editors)
Show editorial Details Show editorial Details