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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.
The scaffolding protein family Fe65, composed of Fe65, Fe65L1, and Fe65L2, was identified as an interaction partner of the amyloid precursor protein (APP), which plays a key function in Alzheimer’s disease. All three Fe65 family members possess three highly conserved interaction domains, forming complexes with diverse binding partners that can be assigned to different cellular functions, such as transactivation of genes in the nucleus, modulation of calcium homeostasis and lipid metabolism, and regulation of the actin cytoskeleton. In this article, we rule out putative new intracellular signaling mechanisms of the APP-interacting protein Fe65 in the regulation of actin cytoskeleton dynamics in the context of various neuronal functions, such as cell migration, neurite outgrowth, and synaptic plasticity.
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.
The handling of oxygen sensitive samples and growth of obligate anaerobic organisms
requires the stringent exclusion of oxygen, which is omnipresent in our normal atmospheric
environment. Anaerobic workstations (aka. Glove boxes) enable the handling of
oxygen sensitive samples during complex procedures, or the long-term incubation of
anaerobic organisms. Depending on the application requirements, commercial workstations
can cost up to 60.000 €. Here we present the complete build instructions for a highly
adaptive, Arduino based, anaerobic workstation for microbial cultivation and sample handling,
with features normally found only in high cost commercial solutions. This build can
automatically regulate humidity, H2 levels (as oxygen reductant), log the environmental
data and purge the airlock. It is built as compact as possible to allow it to fit into regular
growth chambers for full environmental control. In our experiments, oxygen levels during
the continuous growth of oxygen producing cyanobacteria, stayed under 0.03 % for 21 days
without needing user intervention. The modular Arduino controller allows for the easy
incorporation of additional regulation parameters, such as CO2 concentration or air pressure.
This paper provides researchers with a low cost, entry level workstation for anaerobic
sample handling with the flexibility to match their specific experimental needs.
Botrytis cinerea is a world-wide occurring plant pathogen, causing pre- and post-harvest gray mold rot on a large number of fruit, vegetable, and flower crops. B. cinerea is closely related to Botrytis pseudocinerea, another broad host range species which often occurs in sympatry with B. cinerea, and to several host-specific species including Botrytis fabae and Botrytis calthae. B. cinerea populations have been shown to be genetically heterogeneous, and attempts have been made to correlate genetic markers to virulence and host adaptation. Here, we present the development of a multilocus sequence typing (MLST) scheme, with 10 genes selected for high variability and phylogenetic congruence, to evaluate the genetic diversity of B. cinerea, B. fabae, and B. pseudocinerea. Using PacBio-assisted simultaneous mass sequencing of PCR products, MLST analysis of about 100 strains from diverse geographical origins and years of isolation was performed, which resulted in high-resolution strain differentiation and robust species separation. Several B. cinerea strains formed an as yet unknown population, referred to as group B, which was well separated from all other B. cinerea strains. Furthermore, the gene cluster for biosynthesis of the phytotoxin botcinic acid was missing in B. cinerea B strains. B. cinerea strains from the monocot Iris pseudacorus were found to form a genetically distinct population, and contained an intact gene cluster for production of the red pigment bikaverin, which is usually degenerated in B. cinerea. Remarkably, these strains were much more aggressive on Iris than other B. cinerea strains, which is the first unequivocal example for host specialization in B. cinerea. Our data reveal new insights into the genetic diversity of B. cinerea and provide evidence for intraspecific differentiation and different degrees of host adaptation of this polyphagous necrotrophic pathogen.
Most mitochondrial proteins are synthesized in the cytosol and targeted to the mitochondrial
surface in a post-translational manner. The surface of the endoplasmic reticulum (ER) plays an
active role in this targeting reaction. ER-associated chaperones interact with certain mitochondrial
membrane protein precursors and transfer them onto receptor proteins of the mitochondrial surface
in a process termed ER-SURF. ATP-driven proteins in the membranes of mitochondria (Msp1, ATAD1)
and the ER (Spf1, P5A-ATPase) serve as extractors for the removal of mislocalized proteins. If the
re-routing to mitochondria fails, precursors can be degraded by ER or mitochondria-associated degradation
(ERAD or MAD respectively) in a proteasome-mediated reaction. This review summarizes the
current knowledge about the cooperation of the ER and mitochondria in the targeting and quality
control of mitochondrial precursor proteins.
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.