Novel catalyst for the removal of aromatic sulfur species from refinery streams.

A novel catalyst with hydro-desulfurization and hydrogenation capabilities was tested with the aim of producing ultra-low sulfur and aromatics diesel oil. The catalytically active phase is nickel in a reduced valence state on a carrier made of zinc oxide nanowires and alumina. Based on the reactive adsorption principle, it was speculated that enhanced metal-support interactions and short diffusion paths between nickel and zinc oxide could lead to improved activity and sulfur uptake capacity. Zinc oxide nanowires, proposed here as a novel catalyst support, were produced in appreciable quantities in a microwave-induced plasma jet reactor. After purification and decoration with an active nickel phase, the nanowires underwent extensive characterization, which revealed promising properties. On-stream hydrogenation activity and sulfur uptake was tested on a model diesel oil spiked with difficult-to-remove organic sulfur species. It was observed that the proposed catalyst system, as it was assembled in this project, is inferior to existing hydro-desulfurization products. Nevertheless, this undertaking was a first crude attempt and the concept of reactive adsorption was sufficiently demonstrated, which can hopefully be improved upon with succeeding trials

Resveratrol differentially regulates NAMPT and SIRT1 in hepatocarcinoma cells and primary human hepatocytes

Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells

PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development

Structural basis for the molecular evolution of SRP-GTPase activation by protein

Small G proteins have key roles in signal transduction pathways. They are switched from the signaling 'on' to the non-signaling 'off' state when GTPase-activating proteins (GAPs) provide a catalytic residue. The ancient signal recognition particle (SRP)-type GTPases form GTP-dependent homo- and heterodimers and deviate from the canonical switch paradigm in that no GAPs have been identified. Here we show that the YlxH protein activates the SRP-GTPase FlhF. The crystal structure of the Bacillus subtilis FlhF–effector complex revealed that the effector does not contribute a catalytic residue but positions the catalytic machinery already present in SRP-GTPases. We provide a general concept that might also apply to the RNA-driven activation of the universally conserved, co-translational protein-targeting machinery comprising the SRP-GTPases Ffh and FtsY. Our study exemplifies the evolutionary transition from RNA- to protein-driven activation in SRP-GTPases and suggests that the current view on SRP-mediated protein targeting is incomplete

Transferrin receptor 2 controls bone mass and pathological bone formation via BMP and Wnt signalling

Transferrin receptor 2 (Tfr2) is mainly expressed in the liver and controls iron homeostasis. Here, we identify Tfr2 as a regulator of bone homeostasis that inhibits bone formation. Mice lacking Tfr2 display increased bone mass and mineralization independent of iron homeostasis and hepatic Tfr2. Bone marrow transplantation experiments and studies of cell-specific Tfr2 knockout mice demonstrate that Tfr2 impairs BMP-p38MAPK signaling and decreases expression of the Wnt inhibitor sclerostin specifically in osteoblasts. Reactivation of MAPK or overexpression of sclerostin rescues skeletal abnormalities in Tfr2 knockout mice. We further show that the extracellular domain of Tfr2 binds BMPs and inhibits BMP-2-induced heterotopic ossification by acting as a decoy receptor. These data indicate that Tfr2 limits bone formation by modulating BMP signaling, possibly through direct interaction with BMP either as a receptor or as a co-receptor in a complex with other BMP receptors. Finally, the Tfr2 extracellular domain may be effective in the treatment of conditions associated with pathological bone formation

Recent progress in translational research on neurovascular and neurodegenerative disorders

The already established and widely used intravenous application of recombinant tissue plasminogen activator as a re-opening strategy for acute vessel occlusion in ischemic stroke was recently added by mechanical thrombectomy, representing a fundamental progress in evidence-based medicine to improve the patient’s outcome. This has been paralleled by a swift increase in our understanding of pathomechanisms underlying many neurovascular diseases and most prevalent forms of dementia. Taken together, these current advances offer the potential to overcome almost two decades of marginally successful translational research on stroke and dementia, thereby spurring the entire field of translational neuroscience. Moreover, they may also pave the way for the renaissance of classical neuroprotective paradigms. This review reports and summarizes some of the most interesting and promising recent achievements in neurovascular and dementia research. It highlights sessions from the 9th International Symposium on Neuroprotection and Neurorepair that have been discussed from April 19th to 22nd in Leipzig, Germany. To acknowledge the emerging culture of interdisciplinary collaboration and research, special emphasis is given on translational stories ranging from fundamental research on neurode- and -regeneration to late stage translational or early stage clinical investigations

Substrate binding on the anaphase promoting complex/ cyclosome

Der „Anaphase einleitende Komplex“ oder „Zyklosom“ (APC/C) ist eine molekulare Nanomaschine, die die Anheftung von Ubiquitinketten an Substratproteine katalysiert (Ubiquitin-Ligase). Wenn alle Chromosomen in korrekter Weise mit dem mitotischen Spindelapperat verbunden sind wird der APC/C aktiviert und kann somit die inhibitorischen Proteine Securin und Zyklin-B1 ubiquitinieren. Die Degradation dieser zwei Proteine ermöglicht, dass die genetisch identischen Schwesterchromatiden von den Spindelfasern an die entgegengesetzten Zellpole gezogen werden können, welches eine Voraussetzung für die anschliessende Zellteilung darstellt. Der APC/C kann nur aktiviert werden, wenn er mit einem der zwei Koaktivatorproteinen, Cdh1 oder Cdc20, interagiert. Die Koaktivatorproteine helfen Substratproteine an den APC/C zu binden und haben vermutlich auch eine katalytische Funktion. Der genaue Mechanismus, wie die Substrate durch Koaktivatorproteine zum APC/C rekrutiert werden und was genau dessen katalytische Aktivatät stimuliert, ist nicht vollständig verstanden. Elektronenmikroskopische Methoden wurden verwendet, um zu untersuchen wie der APC/C mit Koaktivatorproteinen und Substraten interagiert. Dafür wurde ein Protokoll etabliert, welches die Aufreinigung von stöchiometrischen APC/C-CDH1-Substrat Komplexen ermöglicht. Die 3D Rekonstruktion einzelner APC/C-CDH1-Substrat Partikel hat gezeigt, dass ein APC/C Molekül mit zwei Koaktivatorproteinen interagiert, wodurch die Interaktion mit zwei Substratmolekülen ermöglicht wird. Eines der beiden Substratmoleküle bindet im katalytischen Zentrum zwischen CDH1 und der APC/C Untereinheit DOC1. Die zweite CDH1/Substrat Bindestelle befindet sich in der Plattformregion des APC/C, vermutlich zwischen den Untereinheiten CDC23 und APC1. Ausserdem scheint die Bindung von Substratmolekülen konformationelle Änderungen zu begünstigen, die in den katalytischen Untereinheiten APC2 und/oder APC11 stattfinden, welche wichtig sein könnten um die katalyische Aktivität des APC/C zu stimulieren.The anaphase promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin-protein ligase which is essential for cell division. In metaphase, when all chromosomes are bioriented on the mitotic spindle, APC/C becomes active and polyubiquitinates proteins that inhibit cell cycle progression, which targets them for proteasomal degradation. Removal of Securin and Cyclin B1 initiates sister chromatid separation in anaphase and mitotic exit. Ubiquitination of substrate proteins by the APC/C depends on either of two co-activator proteins, called Cdc20 and Cdh1. These proteins have been implicated in substrate recruitment and allosteric activation of the APC/C. However, the exact mechanism of how APC/C and the co-activator proteins recruit substrates is incompletely understood. To obtain insight into this process electron microscopy is used to determine how Cdh1 and substrate proteins interact with APC/C. A protocol is established to isolate human APC/C which is bound to stoichiometric amounts of CDH1 and substrate proteins. Single particle electron microscopy and angular reconstruction suggest the existence of two CDH1 binding sites that mediate binding of two substrate molecules, which is confirmed by biochemical analysis. One substrate molecule is intercalated between CDH1 and DOC1 in the central cavity of APC/C. The second CDH1/substrate binding site is located in the platform region, presumably between CDC23 and APC1. Furthermore, conformational changes in the catalytic subunits APC2 and/or APC11 could be observed which upon substrate binding contact the proximal co-activator protein. This conformational rearrangement could reflect a co-activator induced stimulation of APC/C’s E3 activity