Publikation: Characterisation of Nuclear Events in Apoptosis by a Comprehensive Proteome Approach
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Zusammenfassung
Apoptosis is a controlled process of cell demise which plays an essential role in development, organ homeostasis and disease.
Shrinkage and fragmentation of the nucleus are among the most striking morphological features of cell death by apoptosis, but little is known about the underlying mechanisms.
The objective of the present study was to perform a comprehensive analysis of the nuclear proteome during apoptosis using a mass spectrometry based analysis platform. Subsequently, the candidate proteins should be characterised biochemically.
The work was divided into three major parts. In the first part, a cell-free apoptosis reaction consisting of isolated nuclei and cytosolic extracts had to be established. First, CD95-L induced apoptosis in Jurkat T-cells was characterised. Then, cytosolic extracts from control cells as well as from cells undergoing CD95-L induced apoptosis were isolated using the detergent digitonin. These extracts were free from nuclear, mitochondrial and ER-proteins.
Nuclei in high-purity were prepared from mouse liver. The successful induction of cell-free apoptosis was monitored by the caspase-dependent cleavage of the nuclear proteins PARP-1 and lamin B as well as by oligonucleosomal DNA fragmentation.
In the second part, a method had to be established which was suitable for the relative quantification of the highly positive charged nuclear proteins. The method applied here is named Isotope Coded Protein Label (ICPL) and is based upon the differential isotopic labelling of all free amino groups, at lysines and at the N-terminus, followed by the identification and quantification of peptides using mass spectrometry. The proteins were labelled with two different isoforms of the reagent nicotinoyloxy-succinimide, a light (4 hydrogen, 4H) and a deuterated, heavy form (4 deuteriums, 4D). The chemical reaction of free amino groups with nicotinoyloxy-succinimide leads to their modification with nicotinic acid resulting in a neutralisation of the positively charged lysines and thus shifting the isoelectric point towards the acidic area. This shift improved the separation of nuclear alkaline proteins (> pI 10) in the following 2D-gel-electrophoresis that followed.
In a high-throughput approach, nuclei from cell-free control reactions were labelled with the light ICPL-reagent (H4) and nuclei from apoptotic reactions were reacted with the heavy reagent (D4) before both samples were combined. Then, the complexity of this protein mixture was reduced by 2D-gel-electrophoresis. The most prominent 384 spots were excised from the stained gels, digested with trypsin and analysed by mass spectrometry. Three independent sets of experiments, consisting of freshly isolated components were performed. This led to the identification of 13 nuclear proteins the level of which were reproducibly altered between the control and apoptosis reaction.
These identified proteins can be classified mainly into two groups of protein: proteins involved in chromatin organisation and architecture (HMG B1/B2, DEK, HCC-1, Histone H1.0/H1.2/H4) and proteins involved in RNA-transport and -metabolism (hnRNP A2/B1, hnRNP C1/C2, U2 snRNPA ). Three further proteins were identified which did not belong to either group: hsp 70, lamin B2 and PP1.
In the third part of this work, some of the identified proteins were characterised using biochemical and immunochemical methods, leading to the following conclusions:
1) Neither HMGB1 nor HMGB2 are proteolytically processed in apoptosis. In addition, no translocation between the nucleus and the cytoplasm takes place. Most probably, both proteins are post-translationally modified in the course of apoptosis.
2) Proteolysis of hnRNP A2/B1 was not observed in apoptosis. A translocation of hnRNP A2/B1 out of the nucleus explains most likely constant reduction of its protein level observed by the proteome approach.
3) No proteolytic cleavage of hnRNP C1/C2 was detected in apoptosis, whereas a translocation from the nucleus to the cytoplasm was observed. Additionally, post-translational modification may influence this translocation process.
4) Neither translocation from the nucleus nor proteolytic cleavage was observed for the proto-oncogene DEK. Nevertheless, the binding affinity of DEK toward DNA changed during apoptosis. This effect is dependent on caspases and on the protein kinase CK2 and is most likely due to changes of the phosphorylation status of DEK.
Zusammenfassung in einer weiteren Sprache
Apoptose, die kontrollierte Form des Zelltodes, spielt eine zentrale Rolle in der Embryonalentwicklung, der Organ-Homöostase und zahlreichen Erkrankungen.
Obwohl das Schrumpfen und die Fragmentierung des Zellkerns zu den auffälligsten morphologischen Veränderungen in der apoptotischen Zelle gehören, ist wenig über die Mechanismen, die hierzu führen, bekannt.
Ziel dieser Arbeit war es, mit Hilfe eines neuartigen, auf Massenspektrometrie beruhenden Proteomansatzes eine umfassende Analyse der apoptose-bedingten Veränderungen der Proteine des Zellkerns durchzuführen und diese anschließend biochemisch zu charakterisieren.
Die Untersuchungen ließen sich in drei experimentelle Teile gliedern. Im ersten Teil der Arbeit konnte eine zellfreie Apoptose-Reaktion, bestehend aus isolierten Zellkernen und zytosolischen Extrakten, etabliert werden. Es wurde zunächst die CD95-L stimulierte Apoptose in Jurkat T-Zellen zeitlich charakterisiert. Anschließend wurden zytosolische Extrakte sowohl aus Kontrollzellen als auch aus CD95-L-behandelten Zellen mittels des Detergens Digitonin gewonnen. Diese konnten frei von Kern-, Mitochondrien- und ER-Proteinen hergestellt werden. Hochreine Zellkerne wurden aus Mausleber isoliert. Die erfolgreiche Induktion der Apoptose unter zellfreien Bedingungen wurde anhand der Spaltung der nukleären Caspasesubstrate PARP-1 und Lamin B sowie der oligonukleosomalen DNA Fragmentierung verifiziert.
Im zweiten Teil der Arbeit wurde eine Methode zum Vergleich zweier Proteome etabliert, mit der bevorzugt die stark positiv geladenen Kernproteine untersucht werden konnten. Die gewählte Methode, das Isotope Coded Protein Label (ICPL), beruht auf einer differentiellen isotopischen Markierung aller freien Aminogruppen, an Lysinen und dem N-Terminus, und der anschließenden Identifizierung und Quantifizierung der Proteine mittels Massen-spekrometrie. Hierzu werden die Proteine mit zwei unterschiedlichen Formen des Reagens Nicotinoyloxy-succinimid, einer leichten (4 Wasserstoffe - H4) bzw. einer schweren Form (4 Deuteriums - D4), derivatisiert. Die Reaktion von freien Aminogruppen mit Nicotinoyloxy-succinimid führt zu einer kovalenten Modifizierung mit Nikotinsäure, diese neutralisiert zusätzlich die positive Ladung der Lysine und führt so zu einer Verschiebung des isoelektrischen Punktes in den sauren Bereich. Dies ermöglichte eine verbesserte Trennung basischer Proteine (< pI 10) während der anschließenden 2D-Gel-Elektrophorese.
In einem experimentellen Großansatz wurden zunächst Kernproteine aus zellfreien Kontrollreaktionen mit dem leichten ICPL-Reagenz (H4) und Proteine aus apoptotischen Reaktionen (30 und 60 min CD95-L) mit dem schweren ICPL-Reagenz (D4) markiert und anschließend vereinigt. Um die Proteinkomplexität zu reduzieren, wurde dieses Gemisch mittels 2D-Gel-Elektrophorese aufgetrennt. Die 384 deutlichsten Proteinspots aus dem gefärbten 2D-Gel wurden ausgestochen, tryptisch verdaut und massenspektrometrisch analysiert. Nach insgesamt dreimaliger Durchführung dieses Ansatzes mit jeweils neu und unabhängig voneinander isolierten Komponenten konnten insgesamt 13 Kernproteine identifiziert werden, deren Niveau zwischen kontroll- und apoptotischem Ansatz reproduzierbar verändert war.
Die identifizierten Kandidaten ließen sich vorrangig zwei Proteingruppen zuordnen: Proteine, die an der Organisation und Architektur von Chromatin beteiligt sind (HMG B1/B2, DEK, HCC-1, Histone H1.0/H1.2/H4) und Proteine, die bei RNA-Transport und -Metabolismus eine Rolle spielen (hnRNP A2/B1, hnRNP C1/C2, U2 snRNPA ). Zusätzlich konnten noch drei weitere Proteine identifiziert werden: Hsp 70, Lamin B2, PP1.
Im dritten Teil der Arbeit wurden einige Kandidaten biochemisch charakterisiert, woraus folgende Teilergebnisse abgeleitet werden konnten:
1. Weder HMGB1 noch HMGB2 werden während der Apoptose proteolytisch gespalten, noch kommt es zu einer Translokation zwischen Kern und Zytoplasma. Wahrscheinlich ist, daß beide Proteine im Laufe der Apoptose posttranslational modifiziert werden.
2. Proteolyse von hnRNP A2/B1 in der Apoptose konnte nicht beobachtet weren. Eine Translokation von hnRNP A2/B1 vom Zellkern ins Zytoplasma ist vermutlich der Grund für die stetige Abnahme des Proteinlevels im Proteomansatz.
3. Eine proteolytische Spaltung von hnRNP C1/C2 in der Apoptose wurde nicht detektiert. Hingegen konnte eine Translokation von hnRNP C1/C2 während der Apoptose in das Zytoplasma beobachtet werden. Ein Einfluß von posttranslationalen Modifikationen auf diese Translokation ist nicht auszuschließen.
4. Weder eine Translokation aus dem Kern noch eine proteolytische Spaltung konnte für das Proto-Onkogen DEK in der Apoptose beobachtet werden. Vielmehr nimmt im Laufe der Apoptose die Bindungsaffinität von DEK für DNA ab, was wahrscheinlich auf eine Veränderung des Phosphorylierungsstatus des Proteins zurückzuführen ist. Außerdem ist diese apoptose-spezifische Veränderung der Bindungsaffinität abhängig von Caspasen und der Protein Kinase CK2.
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TABBERT, Anja, 2004. Characterisation of Nuclear Events in Apoptosis by a Comprehensive Proteome Approach [Dissertation]. Konstanz: University of KonstanzBibTex
@phdthesis{Tabbert2004Chara-7771, year={2004}, title={Characterisation of Nuclear Events in Apoptosis by a Comprehensive Proteome Approach}, author={Tabbert, Anja}, address={Konstanz}, school={Universität Konstanz} }
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Subsequently, the candidate proteins should be characterised biochemically.<br /><br />The work was divided into three major parts. In the first part, a cell-free apoptosis reaction consisting of isolated nuclei and cytosolic extracts had to be established. First, CD95-L induced apoptosis in Jurkat T-cells was characterised. Then, cytosolic extracts from control cells as well as from cells undergoing CD95-L induced apoptosis were isolated using the detergent digitonin. These extracts were free from nuclear, mitochondrial and ER-proteins.<br />Nuclei in high-purity were prepared from mouse liver. The successful induction of cell-free apoptosis was monitored by the caspase-dependent cleavage of the nuclear proteins PARP-1 and lamin B as well as by oligonucleosomal DNA fragmentation.<br /><br />In the second part, a method had to be established which was suitable for the relative quantification of the highly positive charged nuclear proteins. The method applied here is named Isotope Coded Protein Label (ICPL) and is based upon the differential isotopic labelling of all free amino groups, at lysines and at the N-terminus, followed by the identification and quantification of peptides using mass spectrometry. The proteins were labelled with two different isoforms of the reagent nicotinoyloxy-succinimide, a light (4 hydrogen, 4H) and a deuterated, heavy form (4 deuteriums, 4D). The chemical reaction of free amino groups with nicotinoyloxy-succinimide leads to their modification with nicotinic acid resulting in a neutralisation of the positively charged lysines and thus shifting the isoelectric point towards the acidic area. This shift improved the separation of nuclear alkaline proteins (> pI 10) in the following 2D-gel-electrophoresis that followed.<br />In a high-throughput approach, nuclei from cell-free control reactions were labelled with the light ICPL-reagent (H4) and nuclei from apoptotic reactions were reacted with the heavy reagent (D4) before both samples were combined. Then, the complexity of this protein mixture was reduced by 2D-gel-electrophoresis. The most prominent 384 spots were excised from the stained gels, digested with trypsin and analysed by mass spectrometry. Three independent sets of experiments, consisting of freshly isolated components were performed. This led to the identification of 13 nuclear proteins the level of which were reproducibly altered between the control and apoptosis reaction.<br />These identified proteins can be classified mainly into two groups of protein: proteins involved in chromatin organisation and architecture (HMG B1/B2, DEK, HCC-1, Histone H1.0/H1.2/H4) and proteins involved in RNA-transport and -metabolism (hnRNP A2/B1, hnRNP C1/C2, U2 snRNPA ). Three further proteins were identified which did not belong to either group: hsp 70, lamin B2 and PP1.<br /><br />In the third part of this work, some of the identified proteins were characterised using biochemical and immunochemical methods, leading to the following conclusions:<br /><br />1) Neither HMGB1 nor HMGB2 are proteolytically processed in apoptosis. In addition, no translocation between the nucleus and the cytoplasm takes place. Most probably, both proteins are post-translationally modified in the course of apoptosis.<br />2) Proteolysis of hnRNP A2/B1 was not observed in apoptosis. A translocation of hnRNP A2/B1 out of the nucleus explains most likely constant reduction of its protein level observed by the proteome approach.<br />3) No proteolytic cleavage of hnRNP C1/C2 was detected in apoptosis, whereas a translocation from the nucleus to the cytoplasm was observed. Additionally, post-translational modification may influence this translocation process.<br />4) Neither translocation from the nucleus nor proteolytic cleavage was observed for the proto-oncogene DEK. Nevertheless, the binding affinity of DEK toward DNA changed during apoptosis. This effect is dependent on caspases and on the protein kinase CK2 and is most likely due to changes of the phosphorylation status of DEK.</dcterms:abstract> <dc:contributor>Tabbert, Anja</dc:contributor> <dcterms:rights rdf:resource="https://rightsstatements.org/page/InC/1.0/"/> <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/> <dcterms:title>Characterisation of Nuclear Events in Apoptosis by a Comprehensive Proteome Approach</dcterms:title> <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/> <dcterms:issued>2004</dcterms:issued> <dc:format>application/pdf</dc:format> <dc:rights>terms-of-use</dc:rights> </rdf:Description> </rdf:RDF>