Neue Inhibitoren für die Protease Taspase1

Abstract The threonine protease Taspase1 is of high interest as new target for cancer therapeutics, due to its implication in leukemias and solid tumors. Although Taspase is not an oncogene itself, it is crucially required for the proteolytic activation of the MLL oncogene as well as MLL fusion proteins generated by chromosomal translocations. Hence, MLL‐dependent cells can successfully be targeted by inhibition of Taspase activity. However, Taspase is insensitive to inhibition by general serine, cysteine or metalloprotease inhibitors. Furthermore, the limited number of available Taspase inhibitors display either low inhibitory potential or broad bioactivity. Thus, novel Taspase inhibitors are still urgently required. In a first step, detailed characterization revealed for the first time that recombinant Taspase and protein overexpressed in eukaryotic cells are comparable with respect to their catalytic parameters. Moreover, the specific catalytic activity (0.03 U/mg) was determined, as well as a 20‐fold higher efficiency regarding cleavage of the CS2 cleavage site of MLL over CS1. Regarding stability, Taspase rapidly loses its catalytic activity (half‐life < 3 h), unless it is stabilized by high concentrations of NaCl (≥ 450 mM). Additionally, the autocatalytic processing event required for catalytic competence was found to occur on a time scale of 2‐3 days. The flexible loop region between glycine 178 and aspartate 233 generated by this activation event is not resolved in the crystal structure. Hence, NMR and CD spectroscopy were applied and the results suggest a helixturn‐helix structure. Furthermore, analytical gel filtration pinpointed that Taspase is active as a hetero‐tetramer in solution. With these data, a fluorogenic in vitro assay was optimized for initial inhibitor tests. While the controversially discussed bisarsonic inhibitor NSC48300 and compounds derived from virtual docking exhibited no inhibitory effect, both silica nanoparticles and succinimide peptides were discovered as novel Taspase inhibitors. In fact, silica nanoparticles are known to bind proteins, albeit nanoparticle‐mediated inhibition of enzyme activity is scarcely described. Surprisingly, IC50 values in the nanomolar and picomolar range indicate high affinity binding, which was observed in vitro and in cells. In addition, comparison of nanoparticles with different diameters (8‐125 nm) suggests a positive correlation of size and affinity. Subsequent analyses characterized the inhibition type as noncompetitive and pinpointed that Taspase does not change its structure upon binding. Importantly, inhibition by nanoparticles is no general feature, as three other enzymes (lactate dehydrogenase, chymotrypsin, and proteinase K) showed no or only weak inhibition by silica nanoparticles. Another approach for Taspase inhibition exploited the cleavage sequence and the proposed mechanism to design substrate analog peptides with a succinimide moiety at the cleavage site. The most promising compound exhibited a Ki value of 400 ± 88 nM and displays thus a tenfold better inhibitory potential than the best reported Taspase inhibitor. Moreover, the competitive inhibition type and the resistance against Taspase cleavage were verified. However, degradation by other proteases and/or limited cellular uptake as well as competition with chloride ions prevents the in vivo inhibition of Taspase. Altogether, these findings demonstrate the potential of silica nanoparticles as well as succinimide peptides as Taspase inhibitors and tools for subsequent studies.Abstract Die Threonin‐Protease Taspase1 stellt aufgrund ihrer Beteiligung an Leukämien und soliden Tumoren einen wichtigen neuen Ansatzpunkt für Krebsmedikamente dar. Obwohl Taspase selbst Onkogen ist, ist sie unabdingbar für die proteolytische Aktivierung des Onkogens MLL und der durch Translokationen entstandenen MLL‐Fusionsproteine. Deshalb reagieren MLLabhängige Zellen besonders sensitiv auf Taspase‐Inhibition. Allerdings sind sämtliche allgemeinen Serin‐, Cystein‐ und Metalloprotease‐Inhibitoren unwirksam gegen Taspase, und auch die wenigen verfügbaren Taspase‐Inhibitoren besitzen entweder nur geringes Inhibitorpotential oder ein breites Wirkspektrum. Daher sind neue Taspase‐Inhibitoren nach wie vor von hoherRelevanz. Ein Vergleich rekombinanter Taspase mit in eukaryotischen Zellen überexprimiertem Protein zeigte eine vergleichbare spezifische katalytische Aktivität (0.03 U/mg). Weiterhin konnte eine 20‐fach höhere Effizienz hinsichtlich der Spaltung der CS2‐Schnittstelle von MLL gegenüber der CS1‐Schnittstelle festgestellt werden. Bei Betrachtung der Proteinstabilität fiel auf, dass Taspase sehr schnell an Aktivität verliert (Halbwertszeit < 3 h), sofern das Protein nicht durch hohe Salzkonzentrationen (≥ 450 mM NaCl) stabilisiert wird. Zudem wurde für die autokatalytische Prozessierung des Proenzyms, welche wichtig zur Erlangung katalytischer Kompetenz ist, eine Zeitspanne von 2‐3 Tagen ermittelt. Der dabei entstehende flexible Bereich zwischen Glycin 178 und Aspartat 233 ist in der Kristallstruktur ungeordnet, konnte aber dank NMR‐ und CDSpektroskopie als potentielles Helix‐Turn‐Helix‐Motiv identifiziert werden. Des Weiteren belegten analytische Gelfiltrationen die Aktivität von Taspase als Hetero‐Tetramer. Mithilfe dieser Informationen wurde ein in vitro Assay optimiert und für Inhibitortests verwendet. Während der kontrovers diskutierte Inhibitor NSC48300 und die mittels virtuellen Dockings ermittelten Substanzen keine Inhibition zeigten, konnten Silikatnanopartikel und Succinimid‐Peptide als neue Taspase‐Inhibitoren identifiziert werden. Obwohl Proteinbindung an Nanopartikel bekannt ist, ist eine durch diese vermittelte Enzyminhibition kaum beschrieben. Umso überraschender sind die IC50‐Werte im nano‐ und picomolaren Bereich, die sowohl in vitro als auch in Zellen beobachtet wurden. Weiterhin konnte mit verschiedenen Nanopartikelgrößen eine positive Korrelation von Durchmesser und Affinität belegt werden. Weitere Analysen ergaben eine nichtkompetitive Inhibition und keine Strukturänderung bei Bindung. Bemerkenswerterweise stellt Enzyminhibition keine generelle Eigenschaft von Nanopartikeln dar, da drei Kontrollenzyme (Lactatdehydrogenase, Chymotrypsin und Proteinase K) kaum durch Nanopartikel inhibiert wurden. In einem anderen Ansatz wurde die Taspase‐Schnittsequenz und der vermutete Mechanismus für das Design peptidischer Substratanaloga mit Succinimid‐Motiv an der Schnittstelle genutzt. Die beste Verbindung zeigte einen Ki‐Wert von 400 ± 88 nM und ist damit ein zehnfach besserer Hemmstoff als der beste zuvor beschriebene Taspase‐Inhibitor. Des Weiteren konnte die kompetitive Hemmung und die Stabilität gegenüber Abbau durch Taspase gezeigt werden. Allerdings verhindern schlechte Zellaufnahme und/oder Abbau durch andere Proteasen sowie Kompetition mit Chlorid‐Ionen eine Inhibition in vivo. Insgesamt belegen diese Ergebnisse das Potential von Silikatnanopartikel sowie Succinimid‐Peptiden als Taspase‐Inhibitoren und Werkzeuge für nachfolgende Studien

Structural Characterization of the Loop at the Alpha-Subunit C-Terminus of the Mixed Lineage Leukemia Protein Activating Protease Taspase1.

Acknowledgments We acknowledge the Fonds of Chemical Industry for funding JvdB by their Chemiefonds grant and the DFG for funding PB and CB (CRC 1093). Additional gratitude goes to Shirley Knauer for providing Taspase1 expression clones. Funding: The authors acknowledge the Fonds of Chemical Industry for funding JvdB by their Chemiefonds grant and the DFG for funding PB and CB (CRC 1093).Peer reviewedPublisher PD

Single-Domain Parvulins Constitute a Specific Marker for Recently Proposed Deep-Branching Archaeal Subgroups

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes assisting protein folding and protein quality control in organisms of all kingdoms of life. In contrast to the other sub-classes of PPIases, the cyclophilins and the FK-506 binding proteins, little was formerly known about the parvulin type of PPIase in Archaea. Recently, the first solution structure of an archaeal parvulin, the PinA protein from Cenarchaeum symbiosum, was reported. Investigation of occurrence and frequency of PPIase sequences in numerous archaeal genomes now revealed a strong tendency for thermophilic microorganisms to reduce the number of PPIases. Single-domain parvulins were mostly found in the genomes of recently proposed deep-branching archaeal subgroups, the Thaumarchaeota and the ARMANs (archaeal Richmond Mine acidophilic nanoorganisms). Hence, we used the parvulin sequence to reclassify available archaeal metagenomic contigs, thereby, adding new members to these subgroups. A combination of genomic background analysis and phylogenetic approaches of parvulin sequences suggested that the assigned sequences belong to at least two distinct groups of Thaumarchaeota. Finally, machine learning approaches were applied to identify amino acid residues that separate archaeal and bacterial parvulin proteins from each other. When mapped onto the recent PinA solution structure, most of these positions form a cluster at one site of the protein possibly indicating a different functionality of the two groups of parvulin proteins

Transanal endoscopic microsurgery versus endoscopic mucosal resection for large rectal adenomas (TREND-study)

Background: Recent non-randomized studies suggest that extended endoscopic mucosal resection (EMR) is equally effective in removing large rectal adenomas as transanal endoscopic microsurgery (TEM). If equally effective, EMR might be a more cost-effective approach as this strategy does not require expensive equipment, general anesthesia and hospital admission. Furthermore, EMR appears to be associated with fewer complications. The aim of this study is to compare the cost-effectiveness and cost-utility of TEM and EMR for the resection of large rectal adenomas. Methods/design. Multicenter randomized trial among 15 hospitals in the Netherlands. Patients with a rectal adenoma 3 cm, located between 115 cm ab ano, will be randomized to a TEM- or EMR-treatment strategy. For TEM, patients will be treated under general anesthesia, adenomas will be dissected en-bloc by a full-thickness excision, and patients will be admitted to the hospital. For EMR, no or conscious sedation is used, lesions will be resected through the submucosal plane i

Potential of Core-Collapse Supernova Neutrino Detection at JUNO

JUNO is an underground neutrino observatory under construction in Jiangmen, China. It uses 20kton liquid scintillator as target, which enables it to detect supernova burst neutrinos of a large statistics for the next galactic core-collapse supernova (CCSN) and also pre-supernova neutrinos from the nearby CCSN progenitors. All flavors of supernova burst neutrinos can be detected by JUNO via several interaction channels, including inverse beta decay, elastic scattering on electron and proton, interactions on C12 nuclei, etc. This retains the possibility for JUNO to reconstruct the energy spectra of supernova burst neutrinos of all flavors. The real time monitoring systems based on FPGA and DAQ are under development in JUNO, which allow prompt alert and trigger-less data acquisition of CCSN events. The alert performances of both monitoring systems have been thoroughly studied using simulations. Moreover, once a CCSN is tagged, the system can give fast characterizations, such as directionality and light curve

Detection of the Diffuse Supernova Neutrino Background with JUNO

As an underground multi-purpose neutrino detector with 20 kton liquid scintillator, Jiangmen Underground Neutrino Observatory (JUNO) is competitive with and complementary to the water-Cherenkov detectors on the search for the diffuse supernova neutrino background (DSNB). Typical supernova models predict 2-4 events per year within the optimal observation window in the JUNO detector. The dominant background is from the neutral-current (NC) interaction of atmospheric neutrinos with 12C nuclei, which surpasses the DSNB by more than one order of magnitude. We evaluated the systematic uncertainty of NC background from the spread of a variety of data-driven models and further developed a method to determine NC background within 15\% with {\it{in}} {\it{situ}} measurements after ten years of running. Besides, the NC-like backgrounds can be effectively suppressed by the intrinsic pulse-shape discrimination (PSD) capabilities of liquid scintillators. In this talk, I will present in detail the improvements on NC background uncertainty evaluation, PSD discriminator development, and finally, the potential of DSNB sensitivity in JUNO

Structure of the PUB Domain from Ubiquitin Regulatory X Domain Protein 1 (UBXD1) and Its Interaction with the p97 AAA+ ATPase

AAA+ ATPase p97/valosin-containing protein (VCP)/Cdc48 is a key player in various cellular stress responses in which it unfolds ubiquitinated proteins to facilitate their degradation by the proteasome. P97 works in different cellular processes using alternative sets of cofactors and is implicated in multiple degenerative diseases. Ubiquitin regulatory X domain protein 1 (UBXD1) has been linked to pathogenesis and is unique amongst p97 cofactors because it interacts with both termini of p97. Its N-domain binds to the N-domain and N/D1 interface of p97 and regulates its ATPase activity. The PUB (peptide:N-glycanase and UBA or UBX-containing proteins) domain binds the p97 C-terminus, but how it controls p97 function is still unknown. Here we present the NMR structure of UBXD1-PUB together with binding studies, mutational analysis, and a model of UBXD1-PUB in complex with the p97 C-terminus. While the binding pocket is conserved among PUB domains, UBXD1-PUB features a unique loop and turn regions suggesting a role in coordinating interaction with downstream regulators and substrate processin