Eef Dirksen's Quick Files

The Quick Files feature was discontinued and it’s files were migrated into this Project on March 11, 2022. The file URL’s will still resolve properly, and the Quick Files logs are available in the Project’s Recent Activity

Dynamics of the human nuclear proteome in response to DNA damage

The genome is constantly challenged by factors that can induce DNA damage and thereby threaten the viability of the cell. If DNA damage remains unrepaired it can lead to the development of cancer. Although much is known about the role of proteins and protein complexes in the cellular response to DNA damage, an overview of events occurring at the protein level upon DSB induction has never been reported. Here proteomics was used to bring this about. Mapping of cellular events at the proteome level requires methods that allow accurate and reproducible quantitation of protein expression levels in complex biological systems. We compared a 2D gel-based quantitation method, two-dimensional difference in-gel electrophoresis (2D-DiGE), to metabolic stable isotope labelling, one of the most accurate methods. This showed that both methods provide comparable results with excellent correlation. Subsequently, 2D-DiGE was applied to the investigation of effects of bleomycin-induced DNA damage on the nuclear proteome of human lymphoblastoid cell lines. Interestingly, the nuclear levels of three proteins, known to form the inhibitor of acetyltransferase (INHAT) complex involved in transcriptional regulation, were found to decrease rapidly upon DNA DSB induction, suggesting a role for the INHAT complex in chromatin remodelling events that precede DNA repair. Some of the cell lines used in our study show a hypersensitive phenotype, which means they are hypersensitive towards DNA damage induction and the individuals from which they were retrieved have an increased risk to develop cancer. Since the underlying mechanisms for this decreased genome stability are unknown, we attempted to gain insight into the way these cells cope with DNA damage. This revealed differences in nuclear protein levels between the normal and the hypersensitive cell lines and suggested that differences in regulation of chromatin structure can also influence cellular sensitivity towards DNA damage induction. In the tight regulation of the response to DNA damage, protein phosphorylation plays a crucial role. To study this, a method was developed for the specific enrichment of serine- and threonine-phosphorylated peptides, which is based on the base-catalysed b-elimination of phosphate from a serine- or threonine-phosphorylated peptide. The method allowed selective enrichment of a phosphorylated peptide from a peptide mixture. Additionally, DNA damage-induced changes in the sub-proteome of histone-binding proteins were studied. Despite the growing interest in the role of chromatin remodelling in the DNA damage response, not much is known about the exact mechanisms through which these processes are regulated. Our study reveals that phosphorylation plays an important role in activation of histone chaperones that take part in multiple remodelling complexes. Our results provide insight into some complex features of protein networks at the cross roads of nucleosome assembly, DNA transcription and repair. Taken together, the work described in this thesis highlights the strength of proteomics when analysing complex cellular mechanisms, like the DNA damage response and it emphasizes the importance of protein post-translational modifications and relocalization in the regulation of cellular events

Human lymphoblastoid proteome analysis reveals a role for the inhibitor of acetyltransferases complex in DNA double-strand break response

A DNA double-strand break (DSB) is highly cytotoxic; it emerges as the type of DNA damage that most severely affects the genomic integrity of the cell. It is essential that DNA DSBs are recognized and repaired efficiently, in particular, prior to mitosis, to prevent genomic instability and eventually, the development of cancer. To assess the pathways that are induced on DNA DSBs, 14 human lymphoblastoid cell lines were challenged with bleomycin for 30 and 240 minutes to establish the fast and more prolonged response, respectively. The proteomes of 14 lymphoblastoid cell lines were investigated to account for the variation among individuals. The primary DNA DSB response was expected to occur within the nucleus; therefore, the nuclear extracts were considered. Differential analysis was done using two-dimensional difference in gel electrophoresis; paired ANOVA statistics were used to recognize significant changes in time. Many proteins whose nuclear levels changed statistically significantly showed a fast response, i.e., within 30 minutes after bleomycin challenge. A significant number of these proteins could be assigned to known DNA DSB response processes, such as sensing DSBs (Ku70), DNA repair through effectors (high-mobility group protein 1), or cell cycle arrest at the G 2-M phase checkpoint (14-3-3 ζ). Interestingly, the nuclear levels of all three proteins in the INHAT complex were reduced after 30 minutes of bleomycin challenge, suggesting that this complex may have a role in changing the chromatin structure, allowing the DNA repair enzymes to gain access to the DNA lesions

Use of Multi Attribute Method by mass spectrometry as a QC release and stability tool for biopharmaceuticals – Regulatory Considerations

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