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Characterisation of Nuclear Events in Apoptosis by a Comprehensive Proteome Approach

Characterisation of Nuclear Events in Apoptosis by a Comprehensive Proteome Approach

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TABBERT, Anja, 2004. Characterisation of Nuclear Events in Apoptosis by a Comprehensive Proteome Approach [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Tabbert2004Chara-7771, title={Characterisation of Nuclear Events in Apoptosis by a Comprehensive Proteome Approach}, year={2004}, author={Tabbert, Anja}, address={Konstanz}, school={Universität Konstanz} }

2004 Tabbert, Anja Tabbert, Anja terms-of-use 2011-03-24T17:37:24Z application/pdf 2011-03-24T17:37:24Z Characterisation of Nuclear Events in Apoptosis by a Comprehensive Proteome Approach eng Apoptosis is a controlled process of cell demise which plays an essential role in development, organ homeostasis and disease.<br />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.<br /><br />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.<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. Charakterisierung von apoptose-spezifischen Kernveränderungen mittels einer umfassenden Proteomanalyse

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