Kaiserslautern - Fachbereich Biologie
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Chromosomal aberrations are manifold changes in the configuration of the DNA. Each cell in a tumor
may accumulate different karyotype changes, making it challenging to determine the causes and
consequences of this instability. Therefore, model systems have been developed in the past to
generate and study specific genome alterations. In this thesis, I present the results of my studies on
three types of chromosomal aberrations, all of which may contribute to tumor development or
progression.
Chromothripsis is a phenomenon that describes a one-off massive chromosomal disruption and
reassembly, perhaps arising via DNA damage micronuclei (MN). MN are small DNA-packed nuclear
envelopes. I tested potential causes of DNA damage in MN and found that the rupture of the MN
envelope and the entry of cytosolic fractions increase DNA damage in MN. Furthermore, I addressed
the question of what physiological consequences cell lines with an additional rearranged chromosome
have compared to those with an intact extra chromosome. Strikingly, the cells with more
rearrangements showed a functional advantage resulting in an improved fitness potential.
However, the engineering of polysomic cell lines with fully intact additional chromosomes increases
various cellular stress responses and reduces the proliferation capacity. To investigate how cancer cells
overcome the detrimental consequences of aneuploidy, I explored physiological adaptations of model
cells with a defined additional chromosome that underwent in vivo and in vitro evolution. Interestingly,
unfavorable phenotypes of aneuploid cells, such as the replication stress, were mitigated upon
evolution. Furthermore, I examined the replication on single molecule resolution, showing alteration
after evolution that might underlie the replication stress bypass or tolerance.
In contrast to these unbalanced forms of genomic aberrations, whole genome doubling (WGD) leads
to a full doubled chromosome set, which was shown to evolve into aneuploid karyotypes by
chromosomal instability (CIN), frequently by losing chromosomes. Cells that underwent WGD
accumulate DNA damage in the S phase. I performed a single molecule analysis on the DNA during the
first cell cycle after WGD to elucidate how the DNA damage arises and found that the number of active
origins is not sufficient to replicate the doubled amount of DNA in the first S phase after WGD faithfully.
This starts a genome-destabilizing cascade that eventually promotes tumorigenesis, metastasis, and
poor patient outcome.
Taken together, these studies provide insights into the causes and consequences of three types of
genomic aberrations: chromothripsis, polysomy, and WGD. However different these phenomena may
be, they share one common feature – they contribute to tumor development and progression.
Therefore, elucidating the aberrant cell functions caused by genomic aberrations contributes to a
better understanding of a cancer cell's nature and will perhaps help to find new cancer therapy targets.
Die Sensorkinasen MsmS und RdmS aus dem methanogenen Archaeon M. acetivorans liegen
mit Genen, die für die Regulatoren der Msr-Familie, MsrG, MsrF und MsrC, kodieren, assoziiert
im Genom vor und sind an der Regulation der methylsulfidspezifischen Methyltransferasen
MtsH, MtsD und MtsF beteiligt, die für die Verstoffwechselung von Methylsulfiden innerhalb
der Methanogenese von Bedeutung sind. Diese Systeme eignen sich bestens, um die
Signaltransduktion in Archaea im Allgemeinen besser zu verstehen und wurden daher im
Rahmen dieser Arbeit näher charakterisiert.
Im Vorfeld wurden die Sensorkinasen MsmS und RdmS als Häm-basierte Redoxsensoren
beschrieben, die an Serin- oder Tyrosinresten phosphoryliert werden. Entgegen dieser
Annahme konnte in dieser Arbeit gezeigt werden, dass RdmS keine Autokinaseaktivität
aufweist und dass die in vorangegangenen Studien identifizierten Phosphorylierungen auf
unspezifische Interaktionen der Proteine mit ATP unter den getesteten Bedingungen
zurückzuführen sind. ATP-Binde- und Hydrolyseassays konnten allerdings zeigen, dass RdmS
in der Lage ist, ATP zu binden und dieses auch zu hydrolysieren. Aufgrund der genomischen
Organisation wurde daher ein Phosphatgruppentransfer von RdmS auf den Regulator MsrF
untersucht. Dies konnte jedoch nicht bestätigt werden und auch andere Phosphoakzeptor
konnten im Zuge dieser Arbeit nicht identifiziert werden.
Mit Hilfe von Oberflächenplasmonenresonanz-Spektroskopie und in vivo Crosslinking-Experimenten konnte die Interaktion der Sensorkinase RdmS mit den Regulatoren MsrF und
MsrC und die Interaktion der Sensorkinase MsmS mit dem Regulator MsrG bestätigt werden.
Auf Grundlage dieser und vorangegangener Analysen sind die Kinasen jeweils in der Lage,
mit allen drei Msr-Regulatoren zu interagieren, weshalb angenommen werden kann, dass das
untersuchte Signaltransduktionssystem ein Multi-Komponenten-System darstellt und die
Kinasen und Regulatoren miteinander kreuzregulieren.
Durchgeführte Electrophoretic mobility shift assays zeigen, dass MsrG, MsrF und MsrC
spezifisch an die Promotorbereiche der mts-Gene binden. Dabei konnte eine Bindung jeweils
nur an den mts-Promotorbereich identifiziert werden, der mit dem jeweiligen msr-Gene
assoziiert auf dem Genom vorliegt. Die Bindestellen von MsrF im mtsD-Promotor und die
Bindestelle von MsrG im mtsH-Promotor konnten zudem auf einen ca. 140 bp bzw. 60 bp
großen Bereich stromaufwärts des Transkriptionsstarts begrenzt werden und legen beiden
Regulatoren somit eine Rolle als Transkriptionsaktivator nahe. Durch Sequenzvergleiche
konnte in diesen Bereichen zudem das putative Bindemotiv ATCAA-xxxxxx-TTGAT
ausgemacht werden, welches jedoch in weiterführenden Analysen bestätigt werden muss.
Towards standardized operating procedures for eDNA-based monitoring of marine coastal ecosystems
(2022)
Marine coastal ecosystems are exposed to a variety of anthropogenic impacts, which
often manifest themselves in the pollution of the surrounding ecosystem. Especially on
densely populated coasts or in regions heavily used for aquaculture, changes in the natural
marine habitat can be observed. In order to protect nature and thus its ecosystem services
for humans, more and more environmental protection laws are coming into force.
Exemplary, operators of facilities known to contribute to pollution are obliged to regularly
monitor the condition of the surrounding environment. The purpose of such so-called
compliance monitoring is to determine whether the prescribed regulations are being
followed. The traditional routine involves sampling by ship, during which sediment
samples are taken from the seabed below the aquaculture cages and all macrofauna
organisms found, such as mussels or worms, are taxonomically determined and quantified
by experts. Based on the community of organisms the ecological status of the sample can
then be inferred. Since this method is very labor- and time-consuming, a reorientation of
the scientific community towards alternative monitoring methods is currently taking place.
A bacteria-based eDNA (environmental DNA) metabarcoding system in particular has
proven to be a suitable monitoring tool. With this molecular method, the composition of
the benthic bacterial community is determined using high-throughput sequencing. The
great advantage of this method is that bacteria, due to their short generation times, react
rapidly to various environmental influences. The composition of this community can then
be used to infer the ecological status of the sample under investigation via sequencing
without the need for laborious enumeration and identification of organisms. Additionally,
sequencing costs are more and more decreasing, proposing eDNA metabarcoding-based
monitoring as a faster and cheaper alternative to traditional monitoring. In order to
implement the method in legislation in the long term, standard protocols need to be
developed. Once these are sufficiently validated, the novel methodology can be
incorporated into regulations to support or even replace traditional monitoring. However,
some steps of the eDNA metabarcoding method, from sampling to ecosystem assessment,
are not yet sufficiently standardized, which is why the development of this work was
necessary. Since there is no consensus in the scientific community on (i) the preservation of
environmental samples during transport, (ii) the reproducibility of ecosystem assessment
among different laboratories, (iii) the most appropriate bioinformatic method for ecosystem
assessment, and (iv) the minimum sequencing depth required to determine ecosystem
status, these sub-steps were investigated. It was found that the most common methods
currently used to preserve samples during transport had no discernible effect on the final
ecosystem assessment. Furthermore, sample processing in independent laboratories
allowed the same ecological interpretations based on the bacterial community, which
resulted in concordant ecosystem assessments among laboratories. This indicates the
overall reproducibility of the eDNA metabarcoding-based method, thus enabling its
implementation in standard protocols. Furthermore, it was shown that corresponding
ecosystem assessments can be obtained with the currently used methods for determining
ecological status based on eDNA data. Critical to predictive accuracy is not the method
itself, but a sufficient number of samples that accounts for the natural spatial and temporal
variability of bacterial communities. It was demonstrated that a very shallow sequencing
depth per sample can be sufficient to use machine learning to prediction the ecological
status of the environmental sample. The quality of this classifications did not depend on
the sequencing depth as assumed but was determined by the separability of individual
categories. The results and recommendations of this work contribute directly to the
standardization of ecological assessment of nearshore marine ecosystems. By establishing
these standard protocols, it will be possible to integrate the eDNA metabarcoding-based
method for monitoring compliance of coastal marine ecosystems into legislative
regulations in the future.
Every organism contains a characteristic number of chromosomes that have to be segregated equally into
two daughter cells during mitosis. Any error during chromosome segregation results in daughter cells that
lost or gained a chromosome, a condition known as aneuploidy. Several studies from our laboratory and
across the world have previously shown that aneuploidy per se strongly affects cellular physiology.
However, these studies were limited mainly to the chromosomal gains due to the availability of several
model systems. Strikingly, no systemic study to evaluate the impact of chromosome loss in the human
cells has been performed so far. This is mainly due to the lack of model systems, as chromosome loss is
incompatible with survival and drastically reduces cellular fitness. During my PhD thesis, for the first time,
I used diverse omics and biochemical approaches to investigate the consequences of chromosome losses
in human somatic cells.
Using isogenic monosomic cells derived from the human cell line RPE1 lacking functional p53, we showed
that, similar to the cells with chromosome gains, monosomic cells proliferate slower than the parental
cells and exhibit genomic instability. Transcriptome and proteome analysis revealed that the expression
of genes encoded on the monosomic chromosomes was reduced, as expected, but the abundance was
partially compensated towards diploid levels by both transcriptional and post transcriptional mechanisms.
Furthermore, we showed that monosomy induces global gene expression changes that are distinct to
changes in response to chromosome gains. The most consistently deregulated pathways among the
monosomies were ribosomes and translation, which we validated using polysome profiling and analysis
of translation with puromycin incorporation experiments. We showed that these defects could be
attributed to the haploinsufficiency of ribosomal protein genes (RPGs) encoded on monosomic
chromosomes. Reintroduction of p53 into the monosomic cells uncovered that monosomy is incompatible
with p53 expression and that the monosomic cells expressing p53 are either eliminated or outgrown by
the p53 negative population. Given the RPG haploinsufficiency and ribosome biogenesis defects caused
by monosomy, we show an evidence that the p53 activation in monosomies could be caused by the
defects in ribosomes. These findings were further supported by computational analysis of cancer genomes
revealing those cancers with monosomic karyotype accumulated frequently p53 pathway mutations and
show reduced ribosomal functions.
Together, our findings provide a rationale as to why monosomy is embryonically lethal, but frequently
occurs in p53 deficient cancers.
Membrane proteins are of high pharmacological interest as they are involved in a variety of vital functions. However, to make them accessible to in vitro studies, they often need to be extracted from their natural lipid environment and stabilized with the aid of membrane-mimetic systems. Such membrane mimics can consist of diverse amphiphilic molecules. Small-molecule amphiphiles that can solubilize lipid bilayers, so-called detergents, have been invaluable tools for membrane-protein research in recent decades. Herein, novel small-molecule glyco-amphiphiles embodying three distinct design principles are introduced, and their biophysical and physicochemical properties are investigated. In doing so, the major aims consist in establishing new promising amphiphiles and in determining structure–efficacy relationships for their synthesis and application.
First, the software package D/STAIN was introduced to facilitate the analysis of demicellization curves obtained by isothermal titration calorimetry. The robustness of the underlying algorithm was demonstrated by analyzing demicellization curves representing large variations in amphiphile concentrations and thermodynamic parameters.
Second, the interactions of diastereomeric cyclopentane maltoside amphiphiles (CPMs) with lipid bilayers and membrane proteins were investigated. To this end, lipid model membranes, cellular membranes, and model membrane proteins were treated with different stereoisomer CPMs. These investigations pointed out the importance of stereochemical configuration in the solubilization of lipid bilayers, in the extraction of membrane proteins, and, ultimately, in the stabilization of the latter. Ultimately, CPM C12 could be identified as a particularly stabilizing agent.
Third, the influence of a polymerizable group attached to detergent-like amphiphiles was characterized regarding their micellization, micellar properties, and ability to solubilize lipid membranes. This revealed that such chemical modifications can have different degrees of impact regarding the investigated properties. In particular, micellization was influenced substantially, whereas the sizes of the resulting micelles varied slightly. The polymerizable amphiphiles were shown to solubilize artificial and natural lipid membranes and, consequently, to extract membrane proteins.
Last, the self-assembly of diglucoside amphiphiles bearing either a hydrocarbon or a lipophobic fluorocarbon chain to form native nanodiscs was investigated. It was shown that the presence of a fluorocarbon hydrophobic chain conveys superior stabilization properties onto the amphiphile and the resulting nanodiscs. Moreover, the kinetics of lipid exchange were fundamentally altered by the presence of the fluorocarbon amphiphiles in the nanodisc rim.
On a route from whole genome duplication to aneuploidy and cancer: consequences and adaptations
(2022)
Whole genome duplication (WGD) is commonly accepted as an intermediate state between healthy cells and aneuploid cancer cells. Usually, cells after WGD get removed from the replicating pool by p53-dependent cell cycle arrest or apoptosis. Cells, which are able to bypass these mechanisms exhibit chromosomal instability (CIN) and DNA damage, promoting the formation of highly aneuploid karyotypes. In general, WGD favors several detrimental consequences such as increased drug resistance, transformation and metastasis formation. Therefore, it is of special interest to investigate the limiting factors and consequences of tetraploid proliferation as well as the adaptations to WGD. In the past it has been difficult to study the consequences of such large-scale genomic changes and how cells adapt to tetraploidy in order to survive. Our lab established protocols to generate tetraploids as well as isolated post-tetraploid/aneuploid single cells clones derived from euploid parental cell lines after induction of cytokinesis failure. This system enables to study the consequences and adaptations of WGD in newly generated tetraploid cells and evolved post-tetraploid clones in comparison to their isogenic parental cell line.
Using newly generated tetraploids from HCT116 cells, we identified USP28 and SPINT2 as novel factors limiting the proliferation after WGD. Using mass spectrometry and immunoprecipitation, we revealed an interaction between USP28 and NuMA1 upon WGD, which affects centrosome coalescence of supernumerary centrosomes, an important process that enhances survival of tetraploids. Furthermore, we validated the occurrence of DNA damage in tetraploid cells and found that USP28 depletion diminished the DNA damage dependent checkpoint activation. SPINT2 influences the proliferation after WGD by regulating the transcription of CDKN1A via histone acetylation. Following proliferating tetraploid cells, we confirmed the activation of the DNA damage response (DDR) by immunoblotting and microscopic approaches. Furthermore, we show that the DDR in the arising post-tetraploid clones is reduced. Further experiments verified the appearance of severe mitotic aberrations, replication stress and accumulation of reactive oxygen species in newly generated tetraploids as well as in the aneuploid cancer cells contributing to the occurrence of DNA damage. Using various drug treatments, we observed an increased dependency on the spindle assembly checkpoint in aneuploid cancer cells compared to their diploid parental cell line. Additionally, siRNA knock down experiments revealed the kinesin motor protein KIF18A as an essential protein in aneuploid cells.
Taken together, the results point out cellular consequences of proliferation after tetraploidization as well as the cellular adaptations needed to cope with the increased amount of DNA.
Multi-omics analysis as a tool to investigate causes and consequences of impaired genome integrity
(2022)
Impaired genome integrity has severe consequences for the viability of any cell. Unrepaired DNA lesions can lead to genomically unstable cells, which will often become predisposed for malignant growth and tumorigenesis, where genomic instability turns into a driving factor through the selection of more aggressive clones. Aneuploidy and polyploidy are both poorly tolerated in somatic cells, but frequently observed hallmarks of cancer. Keeping the genome intact requires the concentrated action of cellular metabolism, cell cycle and DNA damage response.
This study presents multi-omics analysis as a versatile tool to understand the various causes and consequences of impaired genome integrity. The possible computational approaches are demonstrated on three different datasets. First, an analysis of a collection of DNA repair experiments is shown, which features the creation of a high-fidelity dataset for the identification and characterization of DNA damage factors. Additionally, a web-application is presented that allows scientists without a computational background to interrogate this dataset. Further, the consequences of chromosome loss in human cells are analyzed by an integrated analysis of TMT labeled mass spectrometry and sequencing data. This analysis revealed heterogeneous cellular responses to chromosome losses that differ from chromosome gains. My analysis further revealed that cells possess both transcriptional and post-transcriptional mechanisms that compensate for the loss of genes encoded on a monosomic chromosome to alleviate the detrimental consequences of reduced gene expression. In my final project, I present a multi-omics analysis of data obtained from SILAC labeled mass spectrometry and dynamic transcriptome analysis of yeast cells of different ploidy, from haploidy to tetraploid. This analysis revealed that unlike cell volume, the proteome of a cell does not scale linearly with increasing ploidy. While the expression of most proteins followed this scaling, several proteins showed ploidy-dependent regulation that could not be explained by transcriptome expression. Hence, this ploidy-dependent regulation occurs mostly on a post-transcriptional level. The analysis uncovered that ribosomal and translation related proteins are downregulated with increasing ploidy, emphasizing a remodeling of the cellular proteome in response to increasing ploidy to ensure survival of cells after whole genome doubling. Altogether this study intends to show how state-of-the-art multi-omics analysis can uncover cellular responses to impaired genome integrity in a highly diverse field of research.
Like many other bacteria, the opportunistic pathogen P. aeruginosa encodes a broad network of enzymes that regulate the intracellular concentration of the second messenger c-di-GMP. One of these enzymes is the phosphodiesterase NbdA that consists of three domains: a membrane anchored, putative sensory MHYT domain, a non-functional diguanylate cyclase domain with degenerated GGDEF motif and an active PDE domain with EAL motif. Analysis of the nbdA open reading frame by 5’-RACE PCR revealed an erroneous annotation of nbdA in the Pseudomonas database with the ORF 170 bp shorter than previously predicted. The newly defined promoter region of nbdA contains recognition sites for the alternative sigma-factor RpoS as well as the transcription factor AmrZ. Promoter analysis within PAO1 wt as well as rpoS and amrZ mutant strains utilizing transcriptional fusions of the nbdA promoter to the reporter gene lacZ revealed transcriptional activation of nbdA by RpoS in stationary growth phase and transcriptional repression by AmrZ. Additionally, no influence of nitrite and neither exogenous nor endogenous NO on nbdA transcription could be shown in this study. However, deletion of the nitrite reductase gene nirS led to a strong increase of nbdA promoter activity which needs to be characterized further. Predicted secondary structures of the 5’-UTR of the nbdA mRNA indicated either an RNA thermometer function of the mRNA or post-transcriptional regulation of nbdA by the RNA binding proteins RsmA and RsmF. Nevertheless, translational studies using fusions of the 5’ UTR of nbdA to the reporter gene bgaB did not verify either of these hypotheses. In general, nbdA translational levels were very low and neither the production of the reporter BgaB nor genomically encoded NbdA could be detected on a western blot. Overproduction of NbdA variants induced many phenotypic changes in motility and biofilm formation. But strains overproducing variants containing the MHYT domain revealed greatly elongated cells and were impaired in surface growth, indicating a misbalance in the membrane protein homeostasis. Therefore, these phenotypes have to be interpreted very critically. Microscopic studies with fluorescently tagged NbdA revealed either a diffuse fluorescent signal of NbdA or the formation of fluorescent foci which were located mainly at the cell poles. Co-localization studies with the polar flagellum and the chemotaxis protein CheA showed that NbdA is not generally localizing to the flagellated cell pole. NbdA localization indicates the control of a specific local c-di-GMP pool in the cell which is most likely involved in MapZ mediated chemotactic flagellar motor switching.
To render membrane proteins amenable to in vitro functional and structural studies, they need to be extracted from cellular membranes and stabilised using membrane-mimetic systems. Amphiphilic copolymers gain considerable interest, because they are able to coextract
membrane proteins and their surrounding lipids from complex cellular membranes to form polymer-bounded nanodiscs. The latter harbour a native-like lipid-bilayer core stabilised by a copolymer rim. Accordingly, these membrane mimics are supposed to provide superior
stability to embedded membrane proteins as compared with conventional detergent micelles.
Herein, the formation of nanodiscs by the most commonly used styrene/maleic acid (SMA)copolymer, termed SMA(2:1), was elucidated in detail. To this end, the equilibrium solubilisation efficiencies towards model and cellular membranes were quantified and
compared with those of the more hydrophobic SMA(3:1) and the more hydrophilic diisobutylene/maleic acid (DIBMA) copolymers. It was shown that, from a thermodynamic viewpoint, SMA(2:1) is the most efficient membrane solubiliser in terms of lipid- and proteinextraction
yields. Solvent properties (pH, ionic strength) or membrane characteristics (lateral pressure, charge, or thickness) can affect the polymers’ solubilisation efficiency to a certain extent. In addition, the lipid transfer behaviour of SMA(2:1) nanodiscs was studied.
Notwithstanding their high effective negative charge, SMA(2:1) nanodiscs exchange phospholipids more rapidly among each other than vesicles or protein-bounded nanodiscs, thus rendering them highly dynamic nano-assemblies. Two alternative electroneutral polymers, namely SMA(2:1)-SB and DIBMA-SB, were introduced in this thesis. They were generated by polymer backbone modifications of SMA(2:1) and DIBMA, respectively. The derivatised polymers were shown to quantitatively solubilise model and biological membranes and, like DIBMA, only had a mild effect on lipidbilayer integrity. Along these lines, DIBMA-SB preserved membrane-protein complexes of distinct structural classes and extracted them from various cellular membranes. Importantly, the electroneutral polymers were amenable to protein/lipid interaction studies otherwise masked by unspecific interactions of their anionic counterparts with target lipids or proteins. Taken together, the in-depth characterisation of nanodiscs formed by anionic and electroneutral polymers allows for adjusting the nanodisc properties to specifically suit experimental requirements or address membrane-protein research questions.
Amino acids, apart from being building blocks of proteins, serve various cellular and metabolic functions1,2. Changes in amino acid handling have been observed in a wide range of human pathologies, including diabetes and various metabolic disorders (aminoacidopathies)3–5. Saccharomyces cerevisiae is used as a model to investigate how increase in amino acid content (in the form of amino acid dropout mix: AAM) in growth medium influences cell growth. Intriguingly, it was observed that increasing the concentration of AAM in the media (double or triple times; 2 X AAM and 3 X AAM respectively), severely affects the growth of auxotrophic but not of prototrophic yeast strains in presence of glucose as carbon substrate. Increased concentration of Ehrlich amino acids, which are degraded to fusel acidic/alcoholic compounds, induced the observed slow growth phenotype of BY4742. These phenotypes can be rescued by either re-establishing the functional leucine biosynthetic pathway in BY4742 (leucine auxotroph) or increasing leucine in proportion to the increased AAM. Interestingly, the amino acid dependent growth phenotypes are absent when cells grow in media containing non-fermentable carbon sources. Furthermore, the deletion of ILV2 or ILV3 (genes encoding enzymes involved in the leucine biosynthetic pathway) also rescues the growth phenotype of BY4742 on 2 X AAM and 3 X AAM growth media. It was found that Ilv3 is the potential switching point and links cellular growth to redox homeostasis. The possibility of leucine limitation per se or transport competition between different Ehrlich amino acids and leucine, as a cause for the observed phenotypes, is ruled out. Upregulation of the branched-chain amino acid pathway inhibits cell growth of BY4742 on 2 X AAM. Although we could not detect KIV, the α-keto acid intermediate formed by the Ilv3. It is proposed that KIV itself (or its unknown downstream product) leads to the onset of the observed phenotypes. Different studies suggest that oxidative stress (due to accumulation of branched-chain amino acids (BCaa) and their α-keto acids) contributes to the neurological damage of MSUD patients6–9. It was also observed that the trigger of the BCaa bio-synthesis pathway on 2 X AAM growth conditions also contributes to the significant oxidative stress in the cell. In conclusion, we propose that yeast can be used as a suitable model system to study how accumulation of BCaa and their α-keto acids are lead to oxidative stress that is potentially toxic to cells. Further, this knowledge and the underlying molecular mechanisms will enhance our understanding of MSUD in humans.