Isolierung und Charakterisierung hochaffiner Cystinknoten- basierter Matriptase-1 Inhibitoren

Die vorliegende Arbeit beschreibt die Isolierung und Charakterisierung neuartiger hochaffiner Inhibitoren der humanen Typ II transmembranen Serinprotease (TTSP) Matriptase-1. In Publikationen der letzten Jahre wurde dieses proteolytische Enzym als neuartiger Biomarker verschiedener humaner Karzinome beschrieben und stellt somit einen möglichen diagnostischen sowie therapeutischen Ansatzpunkt dar. Ausgehend von den Cystinknoten-Peptiden SOTI-III sowie MCoTI-II sollten mittels Gerichteter Evolution neuartige Inhibitoren der Protease isoliert und anschließend charakterisiert werden. Hierfür wurde zunächst die Struktur des Cystinknotenpeptids SOTI-III aufgeklärt. Der Trypsininhibitor wurde zu diesem Zweck mittels Festphasenpeptidsynthese (SPPS) chemisch synthetisiert, oxidativ gefaltet, gereinigt und im Komplex mit Rinderpankreastrypsin kristallisiert. In Kenntnis detaillierter Strukturinformationen wurden für beide Ausgangsmoleküle strukturbasierte Bibliotheken mit bis zu 2 • 10^8 Varianten erstellt und diese auf der Oberfläche von Zellen der Bäckerhefe (S. cerivisiae) zur Durchmusterung bereit gestellt. Das Präsentationsverfahren wurde durch eine erfolgreiche Durchmusterung der SOTI-III Bibliothek nach Bindemolekülen der Protease Trypsin mittels fluoreszenzassoziierter Zellsortierung (FACS) validiert. Aus der Durchmusterung und Charakterisierung von Einzelklonen gingen neuartige Bindemoleküle hervor, wobei sich eine Inhibitionskonstante von 100 nM für eine synthetisierte Variante ergab. Für die Durchmusterung der Bibliotheken auf Bindemoleküle der humanen Matriptase-1 wurde die katalytische Domäne der Protease in E. coli als Einschlußkörper produziert und via Dialyse gefaltet. Im Anschluss wurden beide Variantenbibliotheken in jeweils 4 Selektionsrunden auf Bindemoleküle der katalytischen Matriptase-1 Domäne mittels FACS durchmustert. Gehäuft auftretende Einzelklone wurden im Folgenden für eine weitere Charakterisierung via SPPS synthetisiert, oxidativ gefaltet und gereinigt. Die potenteste Variante zeigte eine Ki von 0,8 nM sowie eine hohe Selektivität gegenüber Matriptase-1. Für die dominanteste Variante der SOTI-III Bibliothek lag eine Inhibitionskonstante von 29 nM vor. Um nun eine Aktivität der isolierten Inhibitoren gegenüber nativer Protease in vivo nachzuweisen, erfolgte eine Dosiswirkungsstudie mit einer Prostatakrebszellen (PC-3) unter Zellkulturbedingungen. Hierbei ergab sich eine halbmaximale Inhibitorkonzentration von 200 nM für den potentesten Inhibitor auf McoTI Basis, wohingegen nur eine geringe inhibitorische Aktivität der SOTI-Variante festgestellt wurde. Somit konnten aus den erstellten Variantenbibliotheken neuartige, potente sowie selektive Inhibitoren der humanen Matriptase-1 isoliert werden, die zusätzlich Aktivität gegenüber der nativen Protease aufweisen

Home parenteral nutrition with an omega-3-fatty-acid-enriched MCT/LCT lipid emulsion in patients with chronic intestinal failure (the HOME study):study protocol for a randomized, controlled, multicenter, international clinical trial

BACKGROUND: Home parenteral nutrition (HPN) is a life-preserving therapy for patients with chronic intestinal failure (CIF) indicated for patients who cannot achieve their nutritional requirements by enteral intake. Intravenously administered lipid emulsions (ILEs) are an essential component of HPN, providing energy and essential fatty acids, but can become a risk factor for intestinal-failure-associated liver disease (IFALD). In HPN patients, major effort is taken in the prevention of IFALD. Novel ILEs containing a proportion of omega-3 polyunsaturated fatty acids (n-3 PUFA) could be of benefit, but the data on the use of n-3 PUFA in HPN patients are still limited. METHODS/DESIGN: The HOME study is a prospective, randomized, controlled, double-blind, multicenter, international clinical trial conducted in European hospitals that treat HPN patients. A total of 160 patients (80 per group) will be randomly assigned to receive the n-3 PUFA-enriched medium/long-chain triglyceride (MCT/LCT) ILE (Lipidem/Lipoplus® 200 mg/ml, B. Braun Melsungen AG) or the MCT/LCT ILE (Lipofundin® MCT/LCT/Medialipide® 20%, B. Braun Melsungen AG) for a projected period of 8 weeks. The primary endpoint is the combined change of liver function parameters (total bilirubin, aspartate transaminase and alanine transaminase) from baseline to final visit. Secondary objectives are the further evaluation of the safety and tolerability as well as the efficacy of the ILEs. DISCUSSION: Currently, there are only very few randomized controlled trials (RCTs) investigating the use of ILEs in HPN, and there are very few data at all on the use of n-3 PUFAs. The working hypothesis is that n-3 PUFA-enriched ILE is safe and well-tolerated especially with regard to liver function in patients requiring HPN. The expected outcome is to provide reliable data to support this thesis thanks to a considerable number of CIF patients, consequently to broaden the present evidence on the use of ILEs in HPN. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT03282955. Registered on 14 September 2017

Protein Production in Yarrowia lipolytica Via Fusion to the Secreted Lipase Lip2p.

We established a strategy for protein production and purification via expression in Yarrowia lipolytica as Lip2p fusion protein. To evaluate the expression system a cysteine-rich miniprotein, an antibody fragment and an enzyme showing galactose oxidase activity were chosen. These proteins have varying disulfide bond content, size, and structural complexity. Endogenous lipase Lip2p was used as a fusion partner to direct the fused proteins to the extracellular medium. A linker sequence was introduced at the junction of Lip2p and the respective fused protein that contains a hexahistidine tag followed by a TEV protease cleavage site. This allows for a specific and simple purification via IMAC for capturing the secreted proteins from the supernatant followed by a second IMAC for removing all contaminants after proteolytic release of the protein of interest. Up to 174 mg/L fusion protein was obtained using shake flask cultivation. Functionality of each of the purified proteins was confirmed by individual assays. Expression of proteins of interest via Lip2p fusion not only provides a convenient expression and purification scheme but also enables for an online monitoring of accumulation of secreted fusion proteins in the medium by exploiting the intrinsic lipase activity of the fusion

Combinatorial Optimization of Cystine-Knot Peptides towards High-Affinity Inhibitors of Human Matriptase-1

<div><p>Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain cystine-knot trypsin inhibitors of the MCoTI (<i>Momordica cochinchinensis</i> trypsin inhibitor) and SOTI (<i>Spinacia oleracea</i> trypsin inhibitor) families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of cystine-knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (K_i = 0.83 nM) with an inverted selectivity towards trypsin and matriptase-1 was identified.</p></div

Late-Pleistocene catchment-wide denudation patterns across the European Alps

We compile detrital 10Be concentrations of Alpine rivers, representing the denudation rates pattern for 375 catchments across the entire European Alps. Using a homogeneized framework, we employ state-of-the-art techniques for inverting in-situ 10Be concentrations into denudation rates. From our compilation, we find that (i) while lithologic properties and precipitation/runoff do influence erosion mechanisms and rates at the scale of individual catchments and in some specific Alpine regions, such controls do not directly stand for the entire Alps, (ii) as also previously suggested, catchment-wide denudation rates across the entire European Alps closely follow first-order Alpine topographic metrics at the scale of individual catchments or selected Alpine sub-regions. However, in addition to previous local-scale studies conducted in the European Alps, our large-scale compilation highlights a functional relationship between catchment-wide denudation and mean catchment slope angle. Catchment-wide denudation positively correlates with mean catchment slope up to a threshold angle (25–30°). Above this threshold, any correlation between catchment-wide denudation and slope as well as other catchment metrics breaks apart. We can reconcile these systematic patterns by proposing a regional erosion model based on diffusive-transport laws for catchments located below the slope threshold angle. In oversteepened catchments situated above-threshold slopes, erosion is stochastic in nature, as glacial carving likely caused a partial decoupling between hillslope and fluvial domains with complex topographic relationships and sediment connectivity patterns. Finally, we identify a first-order positive relationship between modern geodetic rock uplift and catchment-wide denudation for the European Alps. The observed spatial pattern is highly variable and possibly reflects the surface response to deep geodynamic mechanisms prevailing in the different Alpine regions. We conclude that today's topography and geomorphic features of the entire Alps are the result of a millenial-scale geomorphic response to past glacial processes and active rock uplift, highlighting a link between external and internal drivers for mountain erosion.ISSN:0012-8252ISSN:1872-682

Sequences and structures of cystine-knot trypsin inhibitors.

<p>(<b>A</b>) Knottin oMCoTI-II (pdb: 1ha9). (<b>B</b>) SOTI-III (pdb: 4aor). Secondary structure is shown as cartoon with surface, and cysteine residues are depicted as yellow sticks; protease-binding regions (or inhibitor loops) are depicted in red, β-sheets - in blue, and α-helices - in green. Cystine-forming residues are marked bold, and the numbering of respective cysteines is according to their appearance in the sequence.</p

Selectivity profile of MCoTI-based inhibitor Var. 4.

a<p>No inhibition was observed at 10 µM inhibitor concentration.</p

Yeast surface display of SOTI-III <i>wild type</i> and screening against matriptase-1.

<p>(<b>A</b>) Schematic illustration of Aga1p/Aga2p surface-displayed inhibitor (red) flanked by the amino terminal HA (Human influenza hemagglutinin) epitope (green) and the carboxy terminal cMyc epitope (purple). Functional display of the inhibitor is monitored by incubation with biotinylated trypsin followed by fluorescence labeling with streptavidin, R-phycoreythrin conjugate (SPE). (<b>B</b>) FACS histogram overlay of yeast surface presented SOTI-III <i>wild type</i> labeled with anti-cMyc antibody (yellow), trypsin (blue), matriptase-1 (green) and chymotrypsin (brown). (<b>C</b>) FACS overlays of matriptase-1 binder enrichment. The sorting round (R) and the matriptase-1 concentration used in each round (µM) is given in the figures. Dark grey: FACS histogram during sorting. Light grey: FACS histogram during resort (only rounds 2 and 4). (<b>D</b>) Sequence alignment of SOTI-III <i>wild type</i> and matriptase-1-binding SOTI variant 1. Randomized residues are colored in red. Cysteines are depicted in bold letters, while cystine connections are omitted for clarity.</p

Inhibition constants of inhibitors studied in this work.

a<p>Structural information for reference compounds S1 and S2 are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076956#pone.0076956.s003" target="_blank">Figure S3</a>.</p