Strukturelle und funktionelle Untersuchung der p53-Familie, im Besonderen p63

The transcription factor p63 is part of the p53 protein family, which consists of three members, p53, p63 and p73. P63 shares structural similarity with all family members, but is associated to different biological functions than p53 or p73. While p53 is mainly linked to tumor suppression and p73 is connected with neuronal development, p63 has been connected to critical biological roles within ectodermal development and skin stem cell biology as well as supervision of the genetic stability of oocytes. Due to its gene structure p63 is expressed as at least six different isoforms, three of them containing a N-terminal transactivation domain. The isoforms that are of biological relevance both have a C-terminal inhibitory domain that negatively regulates the transcriptional activity. This inhibitory domain is supposed to contain two individual components of which one is internally binding and masking the transactivation domain while the other one can be sumoylated. To further investigate this domain a mutational analysis with the help of transactivation assays in SAOS2 cells was carried out to identify the critical amino acids within the inhibitory domain and the impact on transcriptional activity of TAp63alpha, the p63-isoform which is essential for the integrity of the female germline. The results of these experiments show that a stretch of approximately 13 amino acids seems to be important for the regulation of transcriptional activity in TAp63alpha, due to the increased transcriptional activity occurring in this region after mutation. Additional experiments showed that this mechanism is distinct from sumoylation, which seems to have only implications for the intracellular level of TAp63alpha. As a conclusion, the C-terminus of the Tap63alpha is essential for two different mechanisms, which control the transcriptional activity of the protein. Both regulatory elements are independent from each other and can now be restricted to certain amino acids. Activation of the wild type protein might take place in the identified region via post-translational modification. Furthermore an inhibition assay was carried out to test if the same region might have implications on the second biological relevant isoform deltaNp63alpha. The results show that the same amino acids which show an impact on transcriptional activity in Tap63alpha lead to a significant change in functional behaviour of deltaNp63alpha. There is a possibility that both proteins are regulated with opposite effects via the same mechanisms, based at the C-terminus of the p63alpha-isoforms. In both cases a modification of these residues could lead to a more opened conformation of the protein with consequences on promoter binding, which can be even important for deltaNp63alpha with respect to promoter squelching. Both alpha-isoforms seem to be regulated via the C-terminus and to elucidate if that is also the case for TAp63gamma a deletion analysis was carried out. The results show that there are also amino acids within the C-terminus of TAp63gamma, which have implications on the transcriptional activity of the protein. Therefore the C-terminus seems to play a major role for regulation of diverse p63 isoforms.Der Transkriptionsfaktor p63 ist Mitglied der p53-Familie. Zu dieser zählen auch die Transkriptionsfaktoren p53 und p73, wobei diese Proteine trotz struktureller Homologien in unterschiedlichen biologischen Vorgängen innerhalb der Zelle involviert sind. Während p53 als Tumorsuppressor agiert, wird p73 hauptsächlich mit der neuronalen Entwicklung in Verbindung gebracht. P63 spielt wiederum eine kritische Rolle in der Entwicklung des Epithelgewebes als auch der Aufrechterhaltung der genetischen Integrität in Oocyten. P63 kann aufgrund der Genstruktur in sechs verschiedenen Isoformen in der Zelle vorliegen. Von den möglichen Isoformen sind zwei in den genannten biologischen Vorgängen involviert. Drei der sechs möglichen Isoformen verfügen über eine N-terminale Transaktivierungsdomäne (TA-Isoformen), die deltaN-Isoformen dagegen nicht. Die längste Isoform TAp63alpha besteht aus insgesamt sechs Domänen, Transaktivierungsdomäne (TA), DNA-Binde-Domäne (DBD), Oligomerisierungs-domäne (OD), SAM- (Sterile alpha motif) Domäne, QP- (Glutamine and Proline rich region) Domäne und die Transaktivierungs-inhibierende Domäne (TID). Des Weiteren gibt es am C-terminus von TAp63alpha noch eine Sumoylierungssequenz. Die beta- und gamma- Isoformen von p63 unterscheiden sich in ihrem C-terminus und von ihrem Molekulargewicht von den alpha-Isoformen. Daher verfügen nur die beiden biologisch relevanten alpha-Isoformen C-terminal über die TI-Domäne, welche Einfluss auf die transkriptionelle Aktivität über die Bindung der TA-Domäne hat. Ebenso beeinflusst die Sumoylierung am C-terminus die transkriptionelle Aktivität. Um den Zusammenhang zwischen beiden Mechanismen zu untersuchen, sowie die für die Inhibition wichtigen Aminosäuren zu identifizieren wurde in dieser Doktorarbeit eine Mutationsstudie von TAp63alpha mit Hilfe von Transaktivierungsassays in SAOS2 Zellen durchgeführt. Das Ergebnis dieser Experimente führte zu der Identifizierung einer Folge von etwa 13 Aminosäuren, welche Auswirkungen auf die transkriptionelle Aktivität des Proteins haben. Diese Auswirkungen auf die intrinsische Aktivität von TAp63alpha unterscheiden sich auch von den Effekten der Sumoylierung, da gezeigt werden konnte dass sich die transkriptionelle Aktivität in diesem Fall nur indirekt über die Steuerung der intrazellulären Proteinkonzentration ändert. Es lässt sich daher schlussfolgern, dass der C-terminus von TAp63alpha essentiell für zwei unterschiedliche und unabhängige Vorgänge ist, welche Auswirkungen auf die transkriptionelle Aktivität von TAp63alpha haben. Beide Vorgänge lassen sich nun auf bestimmte Aminosäuren beschränken. Um festzustellen, ob dieselben Aminosäuren, welche Auswirkungen auf die transkriptionelle Aktivität von TAp63alpha haben ebenso die zweite biologisch relevante Isoform deltaNp63alpha beeinflussen, wurden Inhibierungsassays durchgeführt. Die analogen Aminosäuren haben in diesem Fall massive Auswirkungen auf die direkten inhibitorischen Fähigkeiten von deltaNp63alpha. Es ist denkbar, dass beide Isoformen über denselben Mechanismus reguliert werden, mit jeweils unterschiedlichen Auswirkungen. In beiden Fällen könnte eine Modifikation dieser Aminosäuren zu einer offeneren Konformation führen, was zu erhöhter Promoter-Affinität führen könnte. Dies hätte auch Auswirkungen auf deltaNp63alpha im Sinne von Promoter Squelching. Zusammenfassend werden beide alpha-Isoformen über den C-terminus reguliert. Ob dies analog auch für TAp63gamma mit einem zu den alpha-Isoformen unterschiedlichen C-terminus gilt, wurde anhand einer Deletionsstudie überprüft. Hierbei führte die Deletion bestimmter Aminosäuren auch zu einer signifikanten Änderung der transkriptionellen Aktivität. Anhand der durchgeführten Experimente lässt sich daher schlussfolgern, dass bestimmte Elemente im C-terminus der untersuchten p63-Isoformen sehr wichtig für die Regulation dieser p63-Isoformen Ist

Identification of Exhaled Metabolites in Children with Cystic Fibrosis

The early detection of inflammation and infection is important to prevent irreversible lung damage in cystic fibrosis. Novel and non-invasive monitoring tools would be of high benefit for the quality of life of patients. Our group previously detected over 100 exhaled mass-to-charge (m/z) features, using on-line secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS), which distinguish children with cystic fibrosis from healthy controls. The aim of this study was to annotate as many m/z features as possible with putative chemical structures. Compound identification was performed by applying a rigorous workflow, which included the analysis of on-line MS2 spectra and a literature comparison. A total of 49 discriminatory exhaled compounds were putatively identified. A group of compounds including glycolic acid, glyceric acid and xanthine were elevated in the cystic fibrosis group. A large group of acylcarnitines and aldehydes were found to be decreased in cystic fibrosis. The proposed compound identification workflow was used to identify signatures of volatile organic compounds that discriminate children with cystic fibrosis from healthy controls, which is the first step for future non-invasive and personalized applications. Keywords: SESI-HRMS; breath analysis; children; cystic fibrosis; infection; inflammation; putative compound identificatio

The C-terminus of p63 contains multiple regulatory elements with different functions

The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation. We have carried out an extensive alanine scanning study to identify critical regions within the inhibitory domain. These experiments show that a stretch of ~13 amino acids is crucial for the binding function. Further, investigation of transcriptional activity and the intracellular level of mutants that cannot be sumoylated suggests that sumoylation reduces the concentration of p63. We therefore propose that the inhibitory function of the C-terminal domain is in part due to direct inhibition of the transcriptional activity of the protein and in part due to indirect inhibition by controlling the concentration of p63. Keywords: p63, transcriptional regulation, auto-inhibition, sumoylatio

Synthesis and characterization of metastable transition metal oxides and oxide nitrides

New routes to vanadium sesquioxide and tantalum oxide nitride (γ- and δ-phase) are presented. Phase pure V2O3 with bixbyite-type structure, a metastable polymorph, was obtained from vanadium fluoride hydrates at ~750 K. It crystallizes in the cubic crystal system in space group Ia3¯ with lattice parameter a=939.30(5) pm. The catalytical properties of the corresponding oxide nitride phases and their oxidation and reduction solid-state kinetics were investigated. The preparation of γ-TaON as a phase pure sample can be realized by ammonolysis of X-ray amorphous tantalum oxide precursors at 1073 K. This metastable tantalum oxide nitride crystallizes in the monoclinic VO2(B)-type structure in space group C2/m. The same precursors can be used to synthesize the δ-modification with an anatase-type structure at 1023 K. It crystallizes in the tetragonal crystal system in space group I41/amd. A maximum yield of 82 m % could be obtained. The fundamental band gaps of the synthesized and of other metastable TaON polymorphs were calculated from first principles using the GW method. The present results are compared to experimental data and to previous calculations at hybrid DFT level

Vertical Scanning Interferometry for Label-Free Detection of Peptide-Antibody Interactions

Peptide microarrays are a fast-developing field enabling the mapping of linear epitopes in the immune response to vaccinations or diseases and high throughput studying of protein-protein interactions. In this respect, a rapid label-free measurement of protein layer topographies in the array format is of great interest but is also a great challenge due to the extremely low aspect ratios of the peptide spots. We have demonstrated the potential of vertical scanning interferometry (VSI) for a detailed morphological analysis of peptide arrays and binding antibodies. The VSI technique is shown to scan an array area of 5.1 square millimeters within 3–4 min at a resolution of 1.4 μm lateral and 0.1 nm vertical in the full automation mode. Topographies obtained by VSI do match the one obtained by AFM measurements, demonstrating the accuracy of the technique. A detailed topology of peptide-antibody layers on single spots was measured. Two different measurement regions are distinguished according to the antibody concentration. In the case of weakly diluted serum, the thickness of the antibody layer is independent of the serum dilution and corresponds to the physical thickness of the accumulated antibody layer. In strongly diluted serum, the thickness measured via VSI is linearly proportional to the serum dilution

A Circulating MicroRNA Profile in a Laser-Induced Mouse Model of Choroidal Neovascularization

Funding: This research was funded by the Deutsche Forschungsgemeinschaft (GR5065/1-1). Author Contributions: Conceptualization, F.G. and B.H.F.W.; Data curation, T.S.; Formal analysis, P.B., M.K., A.A., and T.S.; Funding acquisition, C.K. and F.G.; Investigation, M.K. and B.H.F.W.; Methodology, C.K. and A.A.;Project administration, B.H.F.W.; Resources, M.K., A.A., T.L., and F.G.; Software, C.K. and T.S.; Supervision, T.L., F.G., and B.H.F.W.; Validation, P.B.; Visualization, C.K.; Writing—original draft, C.K. and P.B.; Writing—review & editing, B.H.F.W. All authors have read and agreed to the published version of the manuscript.Peer reviewedPublisher PD

Low Serum Levels of Soluble Receptor Activator of Nuclear Factor κ B Ligand (sRANKL) Are Associated with Metabolic Dysregulation and Predict Long-Term Mortality in Critically Ill Patients

Soluble receptor activator of nuclear factor κ B ligand (sRANKL) is a member of the tumor necrosis factor receptor superfamily, and therefore, involved in various inflammatory processes. The role of sRANKL in the course of bone remodeling via activation of osteoclasts as well as chronic disease progression has been described extensively. However, the potential functional importance of sRANKL in critically ill or septic patients remained unknown. Therefore, we measured sRANKL serum concentrations in 303 critically ill patients, including 203 patients with sepsis and 100 with non-sepsis critical illness. Results were compared to 99 healthy controls. Strikingly, in critically ill patients sRANKL serum levels were significantly decreased at intensive care unit (ICU) admission (p = 0.011) without differences between sepsis and non-sepsis patients. Inline, sRANKL was correlated with markers of metabolic dysregulation, such as pre-existing diabetes and various adipokines (e.g., adiponectin, leptin receptor). Importantly, overall mortality of critically ill patients in a three-year follow-up was significantly associated with decreased sRANKL serum concentrations at ICU admission (p = 0.038). Therefore, our study suggests sRANKL as a biomarker in critically ill patients which is associated with poor prognosis and overall survival beyond ICU stay

Systematic Studies of the Micro-Bunching Instability at Very Low Bunch Charges

At KARA, the KArlsruhe Research Accelerator of the KIT synchrotron, the so called short bunch operation mode allows the reduction of the bunch length down to a few picoseconds. The micro- bunching instability resulting from the high degree of longitudinal compression leads to fluctuations in the emitted THz radiation, referred to as bursting. For extremely compressed bunches at KARA, bursting occurs not only in one but in two different bunch-current ranges that are separated by a stable region. This work presents measurements of the bursting behavior in both regimes. Good agreement is found between data and numerical solutions of the Vlasov-Fokker-Planck equation.Comment: 6 pages, 5 figures, to be submitte