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

Die Interaktion von Cytokeratin 18 und ZAP90 in der Retinolsäure-induzierten Apoptose bei hepatozellulären Karzinomzelllinien

Die hohe Sterblichkeit und die geringe Überlebenszeit des hepatozellulären Karzinoms sind bedingt durch unzureichende diagnostische Verfahren zur Früherkennung und die eingeschränkten Therapiemöglichkeiten. Erkenntnisse über molekulare Mechanismen erscheinen daher von besonderem Interesse. Retinoide werden als mögliche Therapieoption für das hepatozelluläre Karzinom vermutet. Um die zu Grunde liegenden biochemischen Mechanismen besser zu verstehen, sollte die Wirkung von all-trans und 9-cis Retinolsäure auf hepatozelluläre Karzinomzelllinien geprüft werden. Ziel der Arbeit war unter anderem die Untersuchung der Retinolsäure-induzierten Apoptose in den humanen Zelllinien HepG2 und Hep3B. Durch die Western-Blot Analyse wurden beteiligte Signaltransduktionswege der Retinoide veranschaulicht. Die gewebsspezifische Expression bestimmter Cytokeratinpaare ermöglicht den Einsatz in der Diagnostik undifferenzierter Karzinome. Weiterhin wird eine Beteiligung von Cytokeratinen an zellulären Regulationsmechanismen vermutet. Vor diesem Hintergrund wurde die Cytokeratin18-Expression in hepatozellulären Karzinomzelllinien sowie die Morphologie während der Apoptose durch die Immunfluoreszenzhistologie und im Western-Blot analysiert. ZAP90 wird in humanen hepatozellulären Karzinomen vermehrt exprimiert. Die funktionelle Bedeutung ist noch nicht geklärt. Eine Verifizierung der Co-Lokalisation von Cytokeratin18 und ZAP90 war Bestandteil dieser Arbeit. Die beschriebenen Proteininteraktionen lassen eine Funktion für ZAP90 innerhalb von Signaltransduktionskaskaden vermuten. Daher sollte die Co-Lokalisation mit CK18 und das Verhalten von ZAP90 in der Apoptose hepatozellulärer Karzinomzelllinien immunhistochemisch verfolgt werden, um Rückschlüsse auf seine Funktion in der Karzinogenese des hepatozellulären Karzinoms zu erzielen

A conserved filamentous assembly underlies the structure of the meiotic chromosome axis.

The meiotic chromosome axis plays key roles in meiotic chromosome organization and recombination, yet the underlying protein components of this structure are highly diverged. Here, we show that 'axis core proteins' from budding yeast (Red1), mammals (SYCP2/SYCP3), and plants (ASY3/ASY4) are evolutionarily related and play equivalent roles in chromosome axis assembly. We first identify 'closure motifs' in each complex that recruit meiotic HORMADs, the master regulators of meiotic recombination. We next find that axis core proteins form homotetrameric (Red1) or heterotetrameric (SYCP2:SYCP3 and ASY3:ASY4) coiled-coil assemblies that further oligomerize into micron-length filaments. Thus, the meiotic chromosome axis core in fungi, mammals, and plants shares a common molecular architecture, and likely also plays conserved roles in meiotic chromosome axis assembly and recombination control

A conserved filamentous assembly underlies the structure of the meiotic chromosome axis

The meiotic chromosome axis plays key roles in meiotic chromosome organization and recombination, yet the underlying protein components of this structure are highly diverged. Here, we show that 'axis core proteins' from budding yeast (Red1), mammals (SYCP2/SYCP3), and plants (ASY3/ASY4) are evolutionarily related and play equivalent roles in chromosome axis assembly. We first identify 'closure motifs' in each complex that recruit meiotic HORMADs, the master regulators of meiotic recombination. We next find that axis core proteins form homotetrameric (Red1) or heterotetrameric (SYCP2:SYCP3 and ASY3:ASY4) coiled-coil assemblies that further oligomerize into micron-length filaments. Thus, the meiotic chromosome axis core in fungi, mammals, and plants shares a common molecular architecture, and likely also plays conserved roles in meiotic chromosome axis assembly and recombination control

C-Terminal Motifs of the MTW1 Complex Cooperatively Stabilize Outer Kinetochore Assembly in Budding Yeast

Kinetochores are macromolecular protein assemblies at centromeres that mediate accurate chromosome segregation during cell division. The outer kinetochore KNL1SPC105, MIS12MTW1, and NDC80NDC80 complexes assemble the KMN network, which harbors the sites of microtubule binding and spindle assembly checkpoint signaling. The buildup of the KMN network that transmits microtubule pulling forces to budding yeast point centromeres is poorly understood. Here, we identify 225 inter-protein crosslinks by mass spectrometry on KMN complexes isolated from Saccharomyces cerevisiae that delineate the KMN subunit connectivity for outer kinetochore assembly. C-Terminal motifs of Nsl1 and Mtw1 recruit the SPC105 complex through Kre28, and both motifs aid tethering of the NDC80 complex by the previously reported Dsn1 C terminus. We show that a hub of three C-terminal MTW1 subunit motifs mediates the cooperative stabilization of the KMN network, which is augmented by a direct NDC80-SPC105 association

A conserved filamentous assembly underlies the structure of the meiotic chromosome axis.

The meiotic chromosome axis plays key roles in meiotic chromosome organization and recombination, yet the underlying protein components of this structure are highly diverged. Here, we show that 'axis core proteins' from budding yeast (Red1), mammals (SYCP2/SYCP3), and plants (ASY3/ASY4) are evolutionarily related and play equivalent roles in chromosome axis assembly. We first identify 'closure motifs' in each complex that recruit meiotic HORMADs, the master regulators of meiotic recombination. We next find that axis core proteins form homotetrameric (Red1) or heterotetrameric (SYCP2:SYCP3 and ASY3:ASY4) coiled-coil assemblies that further oligomerize into micron-length filaments. Thus, the meiotic chromosome axis core in fungi, mammals, and plants shares a common molecular architecture, and likely also plays conserved roles in meiotic chromosome axis assembly and recombination control

A conserved filamentous assembly underlies the structure of the meiotic chromosome axis

The meiotic chromosome axis plays key roles in meiotic chromosome organization and recombination, yet the underlying protein components of this structure are highly diverged. Here, we show that 'axis core proteins' from budding yeast (Red1), mammals (SYCP2/SYCP3), and plants (ASY3/ASY4) are evolutionarily related and play equivalent roles in chromosome axis assembly. We first identify 'closure motifs' in each complex that recruit meiotic HORMADs, the master regulators of meiotic recombination. We next find that axis core proteins form homotetrameric (Red1) or heterotetrameric (SYCP2:SYCP3 and ASY3:ASY4) coiled-coil assemblies that further oligomerize into micron-length filaments. Thus, the meiotic chromosome axis core in fungi, mammals, and plants shares a common molecular architecture, and likely also plays conserved roles in meiotic chromosome axis assembly and recombination control.SU acknowledges past support from the UC San Diego Molecular Biophysics Training Grant (National Institutes of Health T32 GM008326), and current support from the National Science Foundation (Graduate Research Fellowship). IU and IC are supported by grants BIO2015-64216-P and MDM2014-0435 (the Spanish Ministry of Science, Innovation and Universities). AJM acknowledges support from the National Institutes of Health (R15 GM116109). KDC acknowledges past support from the Ludwig Institute for Cancer Research and the National Institutes of Health (R01 GM104141). KDC and FH acknowledge joint support from the Human Frontiers Science Program (RGP0008/2015)