Dynamics of Protein Ubiquitination upon Proteasome Modulation : A Quantitative Mass Spectrometry Approach

__Scope of the Thesis__ The proteasome is a protein complex mostly known for its role in the degradation of unneeded, damaged or misfolded proteins. The proteasome plays a central role in all cells and hence a widely studied protein assembly. Malfunctioning of this protein complex has major effects on cellular processes and is known to lead to the development of a variety of diseases such as cancer and neurodegenerative disorders. The proteasome is also an important target for drug discovery; for instance, proteasome inhibitors are used for the treatment of multiple myeloma. However, not much is known about the biological mechanisms behind these treatments. In this project we monitored the cellular responses in terms of protein abundance and protein ubiquitination dynamics upon proteasome malfunctioning (Chapter 3). In order to gain more insight into the specificity and function of individual proteasome complex components, we also manipulated single proteasome subunits, i.e., the proteasome-bound deubiquitinating enzymes (DUBs) and monitored the effects on the cellular (modified) proteome (Chapter 4). The proteasome is a key player in maintaining a balance in proteostasis under both normal and abnormal cellular conditions. In order to gain further knowledge about the functioning of the proteasome under such conditions we characterized the proteasome interactome under different stress conditions, such as oxidative stress, endoplasmatic reticulum stress and proteasome inhibition (Chapter 5). Large scale quantitative mass spectrometry is the central methodology applied in all studies described in this thesis. These types of global and unbiased approaches make it possible to study the relation of a protein complex with its direct cellular protein environment. In Chapter 6 we have monitored changes in the cellular environment upon activation of ecdysone-responsive genes, in terms of global transcriptome and global proteome dynamics, as well as in terms of ecdysone-receptor interactome dynamics. As such, this work provides several clues to address the relationship between mRNA and protein abundances in Drosophila

Improvement of ubiquitylation site detection by Orbitrap mass spectrometry

Ubiquitylation is an important posttranslational protein modification that is involved in many cellular events. Immunopurification of peptides containing a K-ε-diglycine (diGly) remnant as a mark of ubiquitylation combined with mass spectrometric detection has resulted in an explosion of the number of identified ubiquitylation sites. Here, we present several significant improvements to this workflow, including fast, offline and crude high pH reverse-phase fractionation of tryptic peptides into only three fractions with simultaneous desalting prior to immunopurification and better control of the peptide fragmentation settings in the Orbitrap HCD cell. In addition, more efficient sample cleanup using a filter plug to retain the antibody beads results in a higher specificity for diGly peptides and less non-specific binding. These relatively simple modifications of the protocol result in the routine detection of over 23,000 diGly peptides from HeLa cells upon proteasome inhibition. The efficacy of this strategy is shown for lysates of both non-labeled and SILAC labeled cell lines. Furthermore, we demonstrate that this strategy is useful for the in-depth analysis of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. This study presents a valuable addition to the toolbox for ubiquitylation site analysis to uncover the deep ubiquitinome. Significance: A K-ε-diglycine (diGly) mark on peptides after tryptic digestion of proteins indicates a site of ubiquitylation, a posttranslational modification involved in a wide range of cellular processes. Here, we report several improvements to methods for the isolation and detection of diGly peptides from complex biological mixtures such as cell lysates and brain tissue. This adapted method is robust, reproducible and outperforms previously published methods in terms of number of modified peptide identifications from a single sample. In-depth analysis of the ubiquitinome using mass spectrometry will lead to a better understanding of the roles of protein ubiquitylation in cellular events

Histone Chaperone NAP1 Mediates Sister Chromatid Resolution by Counteracting Protein Phosphatase 2A

Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. We found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. We conclude that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis