Scaffolding protein Disheveled (Dvl) is a key component of Wnt signaling cascades. Dvl participates in a number of biological processes, such as cell proliferation, differentiation and migration, determination of cell polarity, and also stem cell self-renewal. It is therefore indispensable for the correct embryo development and tissue homeostasis in adulthood. The protein regulator of cytokinesis (PRC1) is a microtubule-associated protein. PRC1 is involved in spindle midzone formation during cell division. Spindle midzone precedes the contractile ring assembly and is essential for normal cell cleavage. In our laboratory, PRC1 was identified as a putative interaction partner of DVL3. This master thesis is focused on delineation of the interaction interface between DVL3 and PRC1 using TIRF microscopy (Total Internal Reflection Fluorescence microscopy). To this end, full-length DVL and PRC1 proteins together with their truncated variants were designed, expressed and purified. It was discovered that PRC1 interacts with all three DVL isoforms and the N-terminal part of PRC1 is required for the interaction between PRC1 and DVL3. Furthermore, the DEP domain of DVL3 is likely involved in PRC1interactions. Key words: Dishevelled 3, DVL3, Protein regulator of cytokinesis 1, PRC1, interaction interface, TIRF...

>Magister Scientiae - MSc / Retinoblastoma Binding Protein 6 (RBBP6) is a 200 kDa human protein known to play an essential role in mRNA 3’-end processing, as well as functioning as an E3 ligase to catalyze ubiquitination and suppression of p53 and other cancer-associated proteins. A RBBP6 knockout mouse model previously suggested that RBBP6 cooperates with MDM2 in polyubiquitinating p53, but is not able to ubiquitinate p53 without the assistance of MDM2. However, unpublished studies from our laboratory suggest that the N-terminal 335 residues of RBBP6, known as R3, are able to ubiquitinate p53 in full in vitro assays, and that the isolated RING finger of RBBP6 is able to catalyse ubiquitination of itself, a phenomenon known as auto-ubiquitination. It is, however, possible that other domains within RBBP6, in particular the ubiquitin-like DWNN domain situated near to the RING finger, may modulate the autoubiquitination and substrate-ubiquitination potentials of the complete protein. / 2022

Deformed epidermal autoregulatory factor 1 (DEAF1) encodes a transcription factor essential in early embryonic and neuronal development. In humans, mutations in the DNA binding domain of DEAF1 cause intellectual disability together with clinical characteristics collectively termed DEAF1-associated neurodevelopmental disorders (DAND). The objective of this study is to 1) assess the pathogenicity of newly identified variants using established functional assays, and 2) confirm and map the interaction domain of DEAF1 with HDAC1 and evaluate the importance of DEAF1-HDAC1 interaction on DEAF1-mediated transcriptional repression. Exome sequencing analysis identified six de novo DEAF1 mutations (p.D200Y, p.S201R, p.K250E, p.D251N, p.K253E, and p.F297S). Promoter activity experiments indicate DEAF1 transcriptional repression activity was altered by p.K250E, p.K253E, and p.F297S. Transcriptional activation activity was altered by p.K250E, p.K253E, p.F297S, and p.D251N. Combined with clinical phenotype of the patients, this work establishes the pathogenicity of new DEAF1 variants. Previous studies identified a potential protein interaction between DEAF1 and several proteins of the nucleosome remodeling and deacetylating (NuRD) complex including Histone Deacetylase 1 (HDAC1), Retinoblastoma Binding Protein 4 (RBBP4), Methyl CpG Binding Domain Protein 3 (MBD3). GST pull-down and co-immunoprecipitation (CoIP) assays confirmed and mapped the interaction with HDAC1 between amino acids 113 – 176 of DEAF1. To determine whether DEAF1-mediated repression requires HDAC1 activity, HEK293t wild type or CRISPR/Cas9-mediated DEAF1 knockout cells were treated with the HDAC inhibitor Trichostatin A (TSA). Interestingly, this study demonstrates that the requirement of HDAC1 activity on DEAF1-mediated transcriptional repression activity is target gene specific and expands our understanding of DEAF1 mediated transcriptional repression.

The PDZ domain of proteins mediates a protein-protein interaction by recognizing the hydrophobic C-terminal tail of the target protein. One of the challenges put forth by the DREAM (Discussions on Reverse Engineering Assessment and Methods) 2009 Challenge consists of predicting a position weight matrix (PWM) that describes the specificity profile of five PDZ domains to their target peptides. We consider the primary structures of each of the five PDZ domains as a numerical sequence derived from graph-theoretic models of each of the individual amino acids in the protein sequence. Using available PDZ domain databases to obtain known targets, the graph-theoretic based numerical sequences are then used to train a neural network to recognize their targets. Given the challenge sequences, the target probabilities are computed and a corresponding position weight matrix is derived. In this work we present our method. The results of our method placed second in the DREAM 2009 challenge.

Functional immobilization and lateral organization of proteins into micro- and nanopatterns is an important prerequisite for miniaturizing analytical and biotechnological devices. In this thesis I present novel and versatile approaches for high contrast surface micropatterning of proteins, artificial membranes and live cells based on maleimide photochemistry. The patterning strategy is carried-out on glass substrates exploiting a poly(ethylene glycol) PEG polymer layer as a compatible scaffold. The flexible PEG cushion prevents unspecific proteins attachment and cell adhesion to surfaces. The versatility of this method is demonstrated by means of different orthogonal chemistries using covalent- and affinity- based interactions for protein immobilization. Furthermore, using maleimide based alkyl-thiol chemistry, I utilized the patterning approach for capturing liposomes and proteoliposomes onto surfaces. Formation of fluid patterned polymer-supported membranes demonstrating lateral diffusion of lipids and proteins was confirmed by biophysical assays. A similar approach was used for micropatterning of transmembrane proteins in surface adhered live cells.

Indiana University-Purdue University Indianapolis (IUPUI) / Protein-protein interactions are fundamental in cell signaling and cancer progression. An increasing prevalent idea in cancer therapy is the development of small molecules to disrupt protein-protein interactions. Small molecules impart their action by binding to pockets on the protein surface of their physiological target. At protein-protein interactions, these pockets are often too large and tight to be disrupted by conventional design techniques. Residues that contribute a disproportionate amount of energy at these interfaces are known as hot spots. The successful disruption of protein-protein interactions with small molecules is attributed to the ability of small molecules to mimic and engage these hot spots. Here, the role of hot spots is explored in existing inhibitors and compared with the native protein ligand to explore how hot spot residues can be leveraged in protein-protein interactions. Few studies have explored the use of interface residues for the identification of hit compounds from structure-based virtual screening. The tight uPAR•uPA interaction offers a platform to test methods that leverage hot spots on both the protein receptor and ligand. A method is described that enriches for small molecules that both engage hot spots on the protein receptor uPAR and mimic hot spots on its protein ligand uPA. In addition, differences in chemical diversity in mimicking ligand hot spots is explored. In addition to uPAR•uPA, there are additional opportunities at unperturbed protein-protein interactions implicated in cancer. Projects such as TCGA, which systematically catalog the hallmarks of cancer across multiple platforms, provide opportunities to identify novel protein-protein interactions that are paramount to cancer progression. To that end, a census of cancer-specific binding sites in the human proteome are identified to provide opportunities for drug discovery at the system level. Finally, tumor genomic, protein-protein interaction, and protein structural data is integrated to create chemogenomic libraries for phenotypic screening to uncover novel GBM targets and generate starting points for the development of GBM therapeutic agents. / 2020-10-03

Bovine Infected Cell Protein 0 (bICP0) is an immediate-early protein encoded by Bovine Herpesvirus-1 that modulates host immune response, activates transcription for all viral promoters, and causes ubiquitin-dependent degradation of proteins. Presented herein is a bICP0 protein-protein interaction (PPI) profile, consisting of 98 cellular and 15 viral proteins, generated through co-immunoprecipitation of bICP0 and its binding partners. The PPI profile was analyzed computationally to identify potential sites of interaction with bICP0 and any cellular pathways that may be influenced by bICP0. Some interactors fall in conjunction with bICP0’s known roles during infection, and others are consistent with known associations of bICP0 homologs. However, some proteins in the PPI profile are involved in apoptosis signaling and mRNA spicing—processes both significant during viral infection and novel to the known functions of bICP0 and its homologs. The interaction and co-localization of some of these proteins with bICP0 was further examined.

No description available.

No description available.

Die Interaktion des onkogenen Transkriptionsfaktors MYCN mit der Ser/Thr Kinase Aurora-A verhindert dessen Abbau über das Ubiquitin Proteasomsystem indem die Rekrutierung des SCF FbxW7 Komplexes verhindert wird. Die Kinase nimmt mit der Bindung an MYCN eine aktive Konformation ein und erhält somit die Fähigkeit zur Kinaseaktivität ohne die sonst notwendige Phosphorylierung von Thr288 oder die Anwesenheit eines Aktivators wie TPX2. Da hohe MYCN Konzentrationen Tumore wie Neuroblastome antreiben, ist die Störung der Komplexbildung mit Aurora-A eine valide Strategie zur Entwicklung von Chemotherapeutika. Einige Inhibitoren von Aurora-A wie Alisertib (MLN8237) sind in der Lage, eine Konformationsänderung in der Kinase zu verursachen, die mit der Bindung von MYCN inkompatibel ist und auf diese Weise den Abbau des Transkriptionsfaktors induziert. Da Aurora-A wichtige Funktionen in der Mitose übernimmt, könnte eine direkte Adressierung des Komplexes anstelle einer systemischen Inhibition der Kinase vielversprechender sein. Ziel des Projektes war die Identifizierung von Molekülen, die selektiv an das Interface des Aurora-A – MYCN Komplexes binden und weiter optimiert werden können, um einen gezielten Abbau des Transkriptionsfaktors über einen PROTAC Ansatz zu ermöglichen. Virtuelle Screenings und molekulardynamische Simulationen wurden durchgeführt, um kommerziell erhältliche Verbindungen zu identifizieren, welche mit einer Bindetasche des Komplexes interagieren, die nur zustande kommt, wenn beide Proteine miteinander interagieren. Aus einem ersten Set von zehn potentiellen Liganden wurde für vier eine selektive Interaktion mit dem Protein – Protein Komplex gegenüber Aurora-A oder MYCN alleine in STD-NMR Experimenten bestätigt. Zwei der Hits besaßen ein identisches Grundgerüst und wurden als Ausganspunkt für die Optimierung zu potenteren Liganden genutzt. Das Gerüst wurde fragmentweise vergrößert und in Richtung besserer in-silico Ergebnisse und Funktionalisierung zur Anbringung von E3-Ligase-Liganden optimiert. Neun dieser Liganden der zweiten Generation wurden synthetisiert. Um quantitative Bindungsdaten zu erhalten, wurde ein kovalent verknüpftes Aurora-A – MYCN Konstrukt entworfen. Die strukturelle und funktionale Integrität wurde in STD-NMR und BLI Experimenten mit bekannten Aurora-A Inhibitoren bestätigt, sowie in NMR-basierten ATPase Assays. Zusätzlich konnte die Kristallstruktur des Konstrukts gelöst und damit die Validität des Designs bestätigt werden. Quantitative Messungen der synthetisierten Moleküle identifizierten HD19S als Hit mit einer zehnfach höheren Affinität für das Aurora-A – MYCN Konstrukt im Vergleich zu der Kinase allein. Zusätzlich wurden in-silico Untersuchungen zu PROTACs der Aurora-A Kinase durchgeführt. Interaktionen zwischen Aurora-A, der E3-Ligase Cereblon und den Liganden wurden modelliert und für die Erklärung unterschiedlicher Aktivitäten der eingesetzten PROTACs verwendet. Zudem zeigte das aktivste PROTAC eine hohe Selektivität für Aurora-A gegenüber Aurora-B, obwohl die verwendete Erkennungseinheit (Alisertib) an beide Aurora-Proteine bindet. Dieser Umstand konnte durch energetische Analysen von molekulardynamischen Simulationen der ternären Komplexe erklärt werden. Optimierungsmöglichkeiten für eine effizientere Degradation von Aurora-A durch die PROTACs wurden basierend auf modifizierten Erkennungseinheiten und verbesserten Linkern untersucht. / The association of the oncogenic transcription factor MYCN with the Ser/Thr kinase Aurora-A prevents its degradation via the ubiquitin proteasome system by preventing the SCF FbxW7 complex from binding. The kinase adopts an active conformation when bound to MYCN, enabling kinase activity without prior phosphorylation on Thr288 or the presence of an activator like TPX2, and therefore at inappropriate times during the cell cycle. As high levels of MYCN have been shown to drive cancers like neuroblastoma, disrupting the complex formation is thought to be a viable development strategy for chemotherapeutics. Several small-molecule inhibitors of Aurora-A, like Alisertib (MLN8237), are able to induce a conformational change in the kinase, preventing the formation of the protein – protein complex and therefore promoting MYCN degradation. However, since Aurora-A has important roles during mitosis targeting only the complex could be a more promising approach than the systemic inhibition of the kinase. This project aimed to identify small molecules which selectively bind at the Aurora-A – MYCN interface and can be further optimized to induce targeted degradation via a PROTAC approach. Virtual screenings and molecular dynamics simulations were performed to identify commercially available compounds which should bind to a pocket formed only when the two proteins come together. Of a first set of ten potential binders, four showed binding to the Aurora-A – MYCN complex but not the individual proteins in STD-NMR experiments. Two of these hit molecules contained the same scaffold and were used as a starting point for optimization towards more potent ligands. In a fragment-based fashion, the scaffold was grown to achieve better affinity in-silico and provide linkage points for functionalization such as the attachment of E3 ligase ligands to create PROTACs. Nine of these second-generation compounds were then synthesized. In order to obtain quantitative binding data a covalently linked Aurora-A – MYCN construct was designed. Its structural and functional validity was shown in STD-NMR and BLI experiments with known Aurora-A inhibitors and in NMR-based ATPase assays. In addition, a crystal structure of the construct was solved, validating the designed structure. Quantitative measurements with the synthesized compounds revealed a positive hit (HD19S) with a ten-fold higher affinity to the covalently linked AuroraA – MYCN as compared to Aurora-A alone. Additionally, effects of PROTACs designed to degrade Aurora-A were studied in-silico. Interactions between Aurora-A, the E3-ligase Cereblon and small molecules were modelled and successfully used to explain the differences in activities observed with different PROTACs. The most active PROTAC also showed a high selectivity for Aurora-A over Aurora-B, even though the recognition unit (Alisertib) can bind both family members. Through energetic analysis of molecular dynamics simulations of the ternary complexes, these differences could be explained. Optimizations for a more efficient degradation of Aurora-A by the PROTACs were examined by changing the recognition unit and improving linkers.