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Mass spectrometric approaches to study the composition and assembly of centromere associated complexes
Mass spectrometric approaches to study the composition and assembly of centromere associated complexes
Mitosis is the process of dividing a eukaryotic cell into two identical daughter cells. This part of the cell cycle executes the faithful propagation of the genome. A prerequisite for maintaining genome stability is the assembly of the conserved kinetochore structure at chromosomal loci called centromeres. The kinetochore is a macromolecular protein complex that physically links chromosomes to spindle microtubules. Aberrations in chromosome segregation cause aneuploidy, which has been associated with tumorigenesis, trisomy, and age-related pathologies. To ensure the accurate segregation of sister chromatids, their kinetochores have to be attached to microtubules emanating from opposite spindle poles, a configuration which is known as biorientation of chromosomes. The kinetochore is composed of more than 80 proteins, which are organized in stable subcomplexes and follow a conserved hierarchy of assembly from centromeric chromatin to microtubules: the centromere proximal inner kinetochore or Constitutive Centromere Associated Network (CCAN), the microtubule binding KMN (KNL1/MIS12/NDC80) network at the outer kinetochore and the fibrous corona. The proteins of the CCAN complex build the interface between centromeric chromatin and the microtubule-binding unit. Several kinetochore proteins are conserved among eukaryotes. In contrast, the underlying centromeric chromatin is highly divergent and epigenetically specified. The major epigenetic mark of the centromere are nucleosomes that have H3 replaced by centromere specific histone variant CENP A. Interestingly, the levels of CENP-A are halved during DNA replication by equally distributing CENP-A between sister chromatids. Cells pass through mitosis with half-maximal CENP-A levels until they are replenished during mitotic exit. The underlying molecular pathways of histone redistribution during DNA replication and CENP-A replenishment in the early G1-phase remain largely unknown. In this thesis, I analyzed the protein composition of the human centromere in a time-resolved manner to study the quantitative changes in protein interactions of CENP-A containing oligo-nucleosomes. This proteomic screen detected several proteins that are associated with the centromere in a cell cycle-dependent manner and identified candidates that may regulate CENP-A distribution to the leading and lagging DNA strands subsequent to replication. Besides chromatin-associated proteins, histone remodelers, and readers and writers of histone post-translational modifications (PTMs), I identified an uncharacterized protein. This transcription factor-like protein was selectively associated with CENP-A at levels comparable to CCAN proteins throughout the entire cell cycle, indicating that this protein may have a structural role at the centromere or inner kinetochore. Spatial restraints derived from the mass spectrometric analysis of crosslinked proteins (XLMS) are widely applied in integrative structural biology approaches to determine protein connectivity. I used label-free quantification of crosslink spectral data to show the dependence of crosslink distances and intensities, which facilitated the estimation of protein dissociation constants and aided the prediction of interfaces of budding yeast subunit contacts. The load-bearing link of chromosomes to microtubules through the kinetochore is stabilized through phosphorylation of CCAN and KMN proteins by mitotic kinases. Titration of the assembly of up to 11 budding yeast kinetochore proteins in vitro indicated that phosphorylation of CCAN and KMN proteins induces cooperative stabilization of the kinetochore at the centromeric nucleosome, which is required to withstand the pulling forces of depolymerizing microtubules. Phosphorylation of distinct sites at the outer kinetochore subunit Dsn1 by AuroraBIpl1, and at the inner kinetochore protein Mif2, mediated cooperativity of the kinetochore assembly. These phosphorylation events decreased the KD values of the kinetochore protein-interactions to the centromeric nucleosome by ~200-fold, which was essential for cell viability. This work demonstrates the potential of quantitative XLMS for characterizing mechanistic effects on protein assemblies upon post-translational modifications or cofactor interaction and for biological modeling.
Crosslinking, Mass Spectrometry, Kinetochore, Centromere, CENP-A
Hagemann, Götz-Norman
2020
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Hagemann, Götz-Norman (2020): Mass spectrometric approaches to study the composition and assembly of centromere associated complexes. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Mitosis is the process of dividing a eukaryotic cell into two identical daughter cells. This part of the cell cycle executes the faithful propagation of the genome. A prerequisite for maintaining genome stability is the assembly of the conserved kinetochore structure at chromosomal loci called centromeres. The kinetochore is a macromolecular protein complex that physically links chromosomes to spindle microtubules. Aberrations in chromosome segregation cause aneuploidy, which has been associated with tumorigenesis, trisomy, and age-related pathologies. To ensure the accurate segregation of sister chromatids, their kinetochores have to be attached to microtubules emanating from opposite spindle poles, a configuration which is known as biorientation of chromosomes. The kinetochore is composed of more than 80 proteins, which are organized in stable subcomplexes and follow a conserved hierarchy of assembly from centromeric chromatin to microtubules: the centromere proximal inner kinetochore or Constitutive Centromere Associated Network (CCAN), the microtubule binding KMN (KNL1/MIS12/NDC80) network at the outer kinetochore and the fibrous corona. The proteins of the CCAN complex build the interface between centromeric chromatin and the microtubule-binding unit. Several kinetochore proteins are conserved among eukaryotes. In contrast, the underlying centromeric chromatin is highly divergent and epigenetically specified. The major epigenetic mark of the centromere are nucleosomes that have H3 replaced by centromere specific histone variant CENP A. Interestingly, the levels of CENP-A are halved during DNA replication by equally distributing CENP-A between sister chromatids. Cells pass through mitosis with half-maximal CENP-A levels until they are replenished during mitotic exit. The underlying molecular pathways of histone redistribution during DNA replication and CENP-A replenishment in the early G1-phase remain largely unknown. In this thesis, I analyzed the protein composition of the human centromere in a time-resolved manner to study the quantitative changes in protein interactions of CENP-A containing oligo-nucleosomes. This proteomic screen detected several proteins that are associated with the centromere in a cell cycle-dependent manner and identified candidates that may regulate CENP-A distribution to the leading and lagging DNA strands subsequent to replication. Besides chromatin-associated proteins, histone remodelers, and readers and writers of histone post-translational modifications (PTMs), I identified an uncharacterized protein. This transcription factor-like protein was selectively associated with CENP-A at levels comparable to CCAN proteins throughout the entire cell cycle, indicating that this protein may have a structural role at the centromere or inner kinetochore. Spatial restraints derived from the mass spectrometric analysis of crosslinked proteins (XLMS) are widely applied in integrative structural biology approaches to determine protein connectivity. I used label-free quantification of crosslink spectral data to show the dependence of crosslink distances and intensities, which facilitated the estimation of protein dissociation constants and aided the prediction of interfaces of budding yeast subunit contacts. The load-bearing link of chromosomes to microtubules through the kinetochore is stabilized through phosphorylation of CCAN and KMN proteins by mitotic kinases. Titration of the assembly of up to 11 budding yeast kinetochore proteins in vitro indicated that phosphorylation of CCAN and KMN proteins induces cooperative stabilization of the kinetochore at the centromeric nucleosome, which is required to withstand the pulling forces of depolymerizing microtubules. Phosphorylation of distinct sites at the outer kinetochore subunit Dsn1 by AuroraBIpl1, and at the inner kinetochore protein Mif2, mediated cooperativity of the kinetochore assembly. These phosphorylation events decreased the KD values of the kinetochore protein-interactions to the centromeric nucleosome by ~200-fold, which was essential for cell viability. This work demonstrates the potential of quantitative XLMS for characterizing mechanistic effects on protein assemblies upon post-translational modifications or cofactor interaction and for biological modeling.