DNA Nanostructures on Membranes as Tools for Synthetic Biology

Over the last decade, functionally designed DNA nanostructures applied to lipid membranes prompted important achievements in the fields of biophysics and synthetic biology. Taking advantage of the universal rules for self-assembly of complementary oligonucleotides, DNA has proven to be an extremely versatile biocompatible building material on the nanoscale. The possibility to chemically integrate functional groups into oligonucleotides, most notably with lipophilic anchors, enabled a widespread usage of DNA as a viable alternative to proteins with respect to functional activity on membranes. As described throughout this review, hybrid DNA-lipid nanostructures can mediate events such as vesicle docking and fusion, or selective partitioning of molecules into phase-separated membranes. Moreover, the major benefit of DNA structural constructs, such as DNA tiles and DNA origami, is the reproducibility and simplicity of their design. DNA nanotechnology can produce functional structures with subnanometer precision and allow for a tight control over their biochemical functionality, e.g., interaction partners. DNA-based membrane nanopores and origami structures able to assemble into two-dimensional networks on top of lipid bilayers are recent examples of the manifold of complex devices that can be achieved. In this review, we will shortly present some of the potentially most relevant avenues and accomplishments of membrane-anchored DNA nanostructures for investigating, engineering, and mimicking lipid membrane-related biophysical processes

Optimization of flotation assay conditions for syndapin binding to phosphatidic acid containing liposomes

Flotation is one of the best method for preliminary identification of protein-lipid interactions. In most widely used approach it utilizes large unilamellar vesicles, that are excellent models of freestanding membranes and do not require any additional components, like solid supports or beads that are needed in other methods commonly used for protein-lipid binding studies. Here we present results obtained during our studies on phosphatidic acid - syndapin interactions and discuss some technical aspects of this method underlying how relatively small changes in the conditions can influence the results

Funktionelle Untersuchung der Vitamin K 2,3-Epoxid-Reduktase

Seit 1941 werden Cumarine als orale Antikoagulantien in der Therapie venöser und arterieller Thrombosen sowie zur Prävention thromboembolischer Ereignisse bei z.B. Herz-Kreislauf-Erkrankungen eingesetzt. Trotz der jahrelangen Erfahrung mit Cumarinen wurde ihr Targetmolekül, die „Vitamin K 2,3-Epoxid-Reduktase-Komplex, Untereinheit 1“ (vitamin K 2,3-epoxide reductase complex, subunit 1; VKORC1), erst im Jahr 2004 entdeckt. VKORC1 reduziert Vitamin K 2,3-Epoxid und Vitamin K-Chinon zur Hydrochinon-Form des Vitamin K. Das Vitamin K-Hydrochinon dient dem Enzym γ-glutamyl-Carboxylase (GGCX) als Substrat um Vitamin K-abhängige Proteine zu γ-carboxylieren, die dadurch ihre biologische Aktivität erhalten. Bei diesem Prozess wird das Vitamin K-Hydrochinon zu Vitamin K 2,3-Epoxid oxidiert, welches wiederum von der VKORC1 zu Vitamin K-Hydrochinon reduziert werden kann. Die Inhibition der VKORC1 durch Cumarine (z.B. Warfarin) führt dazu, dass der GGCX kein Vitamin K-Hydrochinon bereitgestellt werden kann. Dadurch werden unter anderem weniger Vitamin K-abhängige Gerinnungsfaktoren (Faktor II, Faktor VII, Faktor IX, Faktor X) γ-carboxyliert und somit die Gerinnung gehemmt. Der molekulare Mechanismus der oralen Antikoagulation und des Vitamin K-Zyklus ist daher seit der Entdeckung der VKORC1 weitgehend geklärt. Allerdings wurden seither 24 Mutationen in der humanen VKORC1 (hVKORC1) beschrieben die zu einer Cumarinresistenz führen. Warum diese Mutationen zu einer Resistenz führen und welche Strukturen und Regionen in die Cumarinbindung involviert sind, ist weiterhin unklar. Um Fragestellungen bezüglich der Funktion der VKORC1 zu untersuchen wurde bisher der „klassische“ VKOR-DTT Assay genutzt. Jedoch sind die mit diesem Assay generierten Daten in weiten Teilen nicht mit dem klinischen Phänotyp der jeweiligen Patienten vereinbar. Mit dieser Dissertation wurde eine neue Methode zur Untersuchung cumarinresistenter Mutationen in der VKORC1 etabliert, deren Ergebnisse die VKOR-Funktion korrekt widerspiegeln. Alle 24 bekannten humanen cumarinresistenen VKORC1-Varianten zeigen mit dieser Methode eine Resistenz gegenüber Warfarin, die zudem mit dem Patientenphänotyp übereinstimmt. Damit stellt der neue Assay eine erhebliche Verbesserung im Vergleich zum „klassischen“ VKOR-DTT Assay dar, indem dieselben VKORC1-Mutationen größtenteils keine Resistenz zeigen und daher nicht mit dem Patientenphänotyp korrelieren. Die in der neuen Methode generierten Daten, berechnet als IC50-Werte (mittlere inhibitorische Konzentration), erlauben es die jeweiligen Mutationen in milde, mittlere und komplette Resistenzgrade einzuteilen. Zudem kann der Wert des Quotienten aus der jeweiligen IC50 Variante / IC50 wt als erster Anhaltspunkt für den erhöhten Cumarin-Bedarf für die Therapie von Patienten, die die Mutation tragen genutzt werden. Um den Mechansimus der Warfarininhibition und die Auswirkungen der Mutationen besser charakterisieren zu können, wurde anhand der bekannten Kristallstruktur eines bakteriellen VKOR-Homolog ein Proteinmodell für die hVKORC1 erstellt. Zusätzlich wurde an diesem Modell die Bindung von Warfarin simuliert. Die Simulation ergab drei Warfarin-Kontaktflächen in der hVKORC1, die die 310-Helix des ER-lumenalen Loops sowie periplasmatische Anteile der ersten und vierten Transmembrandomäne der hVKORC1 betreffen. Nahezu alle Mutationen, die zu einer Cumarinresistenz führen, sind in oder um eine dieser ermittelten Kontaktflächen lokalisiert. Durch die Interaktion von Warfarin mit den Kontaktflächen der hVKORC1 wird der inter- und/oder intramolekulare Elektronentransfer blockiert und/oder der Eintritt des Vitamin K verhindert. Zudem zeigten weitere Experimente mit verschiedenen Konzentrationen an Vitamin K und Warfarin, dass entgegengesetzt der bisherigen Meinung, Cumarine binden irreversibel an die VKORC1, Warfarin wahrscheinlich ein kompetetiver Inhibitior ist

Evaluation of the In vitro cytotoxic activity of caffeic acid derivatives and liposomal formulation against pancreatic cancer cell lines

Pancreatic cancer belongs to the most aggressive group of cancers, with very poor prognosis. Therefore, there is an important need to find more potent drugs that could deliver an improved therapeutic approach. In the current study we searched for selective and effective caffeic acid derivatives. For this purpose, we analyzed twelve compounds and evaluated their in vitro cytotoxic activity against two human pancreatic cancer cell lines, along with a control, normal fibroblast cell line, by the classic MTT assay. Six out of twelve tested caffeic acid derivatives showed a desirable effect. To improve the therapeutic efficacy of such active compounds, we developed a formulation where caffeic acid derivative (7) was encapsulated into liposomes composed of soybean phosphatidylcholine and DSPE-PEG2000. Subsequently, we analyzed the properties of this formulation in terms of basic physical parameters (such as size, zeta potential, stability at 4 °C and morphology), hemolytic and cytotoxic activity and cellular uptake. Overall, the liposomal formulation was found to be stable, non-hemolytic and had activity against pancreatic cancer cells (IC50 19.44 µM and 24.3 µM, towards AsPC1 and BxPC3 cells, respectively) with less toxicity against normal fibroblasts. This could represent a promising alternative to currently available treatment options

The role of hydrophobic interactions in ankyrin–spectrin complex formation

AbstractSpectrin and ankyrin are the key components of the erythrocyte cytoskeleton. The recently published crystal structure of the spectrin–ankyrin complex has indicated that their binding involves complementary charge interactions as well as hydrophobic interactions. However, only the former is supported by biochemical evidence. We now show that nonpolar interactions are important for high affinity complex formation, excluding the possibility that the binding is exclusively mediated by association of distinctly charged surfaces. Along these lines we report that substitution of a single hydrophobic residue, F917S in ankyrin, disrupts the structure of the binding site and leads to complete loss of spectrin affinity. Finally, we present data showing that minimal ankyrin binding site in spectrin is formed by helix 14C together with the loop between helices 15 B/C

Two enzymes catalyze vitamin K 2,3-epoxide reductase activity in mouse: VKORC1 is highly expressed in exocrine tissues while VKORC1L1 is highly expressed in brain

AbstractVKORC1 and VKORC1L1 are enzymes that both catalyze the reduction of vitamin K2,3-epoxide via vitamin K quinone to vitamin K hydroquinone. VKORC1 is the key enzyme of the classical vitamin K cycle by which vitamin K-dependent (VKD) proteins are γ-carboxylated by the hepatic γ-glutamyl carboxylase (GGCX). In contrast, the VKORC1 paralog enzyme, VKORC1L1, is chiefly responsible for antioxidative function by reduction of vitamin K to prevent damage by intracellular reactive oxygen species. To investigate tissue-specific vitamin K 2,3-epoxide reductase (VKOR) function of both enzymes, we quantified mRNA levels for VKORC1, VKORC1L1, GGCX, and NQO1 and measured VKOR enzymatic activities in 29 different mouse tissues. VKORC1 and GGCX are highly expressed in liver, lung and exocrine tissues including mammary gland, salivary gland and prostate suggesting important extrahepatic roles for the vitamin K cycle. Interestingly, VKORC1L1 showed highest transcription levels in brain. Due to the absence of detectable NQO1 transcription in liver, we assume this enzyme has no bypass function with respect to activation of VKD coagulation proteins. Our data strongly suggest diverse functions for the vitamin K cycle in extrahepatic biological pathways

H3K4me3 Is a Potential Mediator for Antiproliferative Effects of Calcitriol (1α,25(OH)2D3) in Ovarian Cancer Biology

Abstract Posttranslational histone modification plays an important role in tumorigenesis. Histone modification is a dynamic response of chromatin to various signals, such as the exposure to calcitriol (1α,25(OH)2D3). Recent studies suggested that histone modification levels could be used to predict patient outcomes in various cancers. Our study evaluated the expression level of histone 3 lysine 4 trimethylation (H3K4me3) in a cohort of 156 epithelial ovarian cancer (EOC) cases by immunohistochemical staining and analyzed its correlation to patient prognosis. The influence of 1α,25(OH)2D3 on the proliferation of ovarian cancer cells was measured by BrdU proliferation assay in vitro. We could show that higher levels of H3K4me3 were correlated with improved overall survival (median overall survival (OS) not reached vs. 37.0 months, p = 0.047) and identified H3K4me3 as a potential prognostic factor for the present cohort. Ovarian cancer cell 1α,25(OH)2D3 treatment induced H3K4me3 protein expression and exhibited antiproliferative effects. By this, the study suggests a possible impact of H3K4me3 expression on EOC progression as well as its relation to calcitriol (1α,25(OH)2D3) treatment. These results may serve as an explanation on how 1α,25(OH)2D3 mediates its known antiproliferative effects. In addition, they further underline the potential benefit of 1α,25(OH)2D3 supplementation in context of ovarian cancer care

Vitamin D and VDR in Gynecological CancersA Systematic Review

In recent years, a vast amount of studies have centered on the role of vitamin D in the pathogenesis of certain types of cancers such as breast, colorectal and lung cancer. Increasing evidence suggests that vitamin D and its receptor play a crucial role in the development of gynecological cancers. In this review, we systematically analyzed the effect of vitamin D and the vitamin D receptor on endometrial, ovarian, cervical, vulvar and vaginal cancer. Our literature research shows that vitamin D levels and vitamin-D-related pathways affect the risk of gynecological cancers. Numerous ecological studies give evidence on the inverse relationship between UVB exposure and gynecological cancer risk. However, epidemiologic research is still inconclusive for endometrial and ovarian cancer and insufficient for rarer types of gynecological cancers. The vitamin D receptor (VDR) is upregulated in all gynecological cancers, indicating its influence on cancer etiology. The VDR polymorphism FokI (rs2228570) seems to increase the risk of ovarian cancer. Other nuclear receptors, such as the RXR, also influence gynecological cancers. Although there is limited knowledge on the role of the VDR/RXR on the survival of endometrial, cervical, vulvar or vaginal cancer patients, some studies showed that both receptors influence survival. Therefore, we suggest that further studies should focus on the vitamin D- and its hetero dimer receptor RXR in gynecological cancers

Regulatory T cells with additional COX-2 expression are independent negative prognosticators for vulvar cancer patients

Vulvar cancer incidence numbers have been steadily rising over the past decades. In particular, the number of young patients with vulvar cancer has recently increased. Therefore, the need to identify new prognostic factors and, in addition, therapeutic options for vulvar carcinoma is more apparent. The aim of this study was to analyze the influx of COX-2 positive tumor-infiltrating lymphocytes and monocytes and their influence on prognosis. Using subtyping by immunofluorescence, the majority of COX-2 expressing immune cells were identified as FOXP3-positive regulatory T cells. In addition, peri- and intra-tumoral macrophages in the same tumor tissue were detected simultaneously as M2-polarized macrophages. COX-2 positive immune cells were independent negative prognostic markers in long-term overall survival of patients with vulvar cancer. These results show an influence of immune cell infiltration for vulvar carcinoma patients. Immune cell infiltration and immune checkpoint expression may, therefore, become interesting targets for further research on new vulvar cancer treatment strategies