New cysteine protease inhibitors : electrophilic (het)arenes and unexpected prodrug identification for the trypanosoma protease rhodesain

Electrophilic (het)arenes can undergo reactions with nucleophiles yielding π- or Meisenheimer (σ-) complexes or the products of the SNAr addition/elimination reactions. Such building blocks have only rarely been employed for the design of enzyme inhibitors. Herein, we demonstrate the combination of a peptidic recognition sequence with such electrophilic (het)arenes to generate highly active inhibitors of disease-relevant proteases. We further elucidate an unexpected mode of action for the trypanosomal protease rhodesain using NMR spectroscopy and mass spectrometry, enzyme kinetics and various types of simulations. After hydrolysis of an ester function in the recognition sequence of a weakly active prodrug inhibitor, the liberated carboxylic acid represents a highly potent inhibitor of rhodesain (Ki = 4.0 nM). The simulations indicate that, after the cleavage of the ester, the carboxylic acid leaves the active site and re-binds to the enzyme in an orientation that allows the formation of a very stable π-complex between the catalytic dyad (Cys-25/His-162) of rhodesain and the electrophilic aromatic moiety. The reversible inhibition mode results because the SNAr reaction, which is found in an alkaline solvent containing a low molecular weight thiol, is hindered within the enzyme due to the presence of the positively charged imidazolium ring of His-162. Comparisons between measured and calculated NMR shifts support this interpretation

Naphthoquinones as covalent reversible inhibitors of cysteine proteases : studies on inhibition mechanism and kinetics

The facile synthesis and detailed investigation of a class of highly potent protease inhibitors based on 1,4-naphthoquinones with a dipeptidic recognition motif (HN-l-Phe-l-Leu-OR) in the 2-position and an electron-withdrawing group (EWG) in the 3-position is presented. One of the compound representatives, namely the acid with EWG = CN and with R = H proved to be a highly potent rhodesain inhibitor with nanomolar affinity. The respective benzyl ester (R = Bn) was found to be hydrolyzed by the target enzyme itself yielding the free acid. Detailed kinetic and mass spectrometry studies revealed a reversible covalent binding mode. Theoretical calculations with different density functionals (DFT) as well as wavefunction-based approaches were performed to elucidate the mode of action

REGGAE: a novel approach for the identification of key transcriptional regulators

Motivation: Transcriptional regulators play a major role in most biological processes. Alterations in their activities are associated with a variety of diseases and in particular with tumor development and progres sion. Hence, it is important to assess the effects of deregulated regulators on pathological processes. Results: Here, we present REGulator-Gene Association Enrichment (REGGAE), a novel method for the identification of key transcriptional regulators that have a significant effect on the expression of a given set of genes, e.g. genes that are differentially expressed between two sample groups. REGGAE uses a Kolmogorov–Smirnov-like test statistic that implicitly combines associations be tween regulators and their target genes with an enrichment approach to prioritize the influence of transcriptional regulators. We evaluated our method in two different application scenarios, which demonstrate that REGGAE is well suited for uncovering the influence of transcriptional regulators and is a valuable tool for the elucidation of complex regulatory mechanisms

MaxDIA enables library-based and library-free data-independent acquisition proteomics

MaxDIA is a software platform for analyzing data-independent acquisition (DIA) proteomics data within the MaxQuant software environment. Using spectral libraries, MaxDIA achieves deep proteome coverage with substantially better coefficients of variation in protein quantification than other software. MaxDIA is equipped with accurate false discovery rate (FDR) estimates on both library-to-DIA match and protein levels, including when using whole-proteome predicted spectral libraries. This is the foundation of discovery DIA—hypothesis-free analysis of DIA samples without library and with reliable FDR control. MaxDIA performs three- or four-dimensional feature detection of fragment data, and scoring of matches is augmented by machine learning on the features of an identification. MaxDIA’s bootstrap DIA workflow performs multiple rounds of matching with increasing quality of recalibration and stringency of matching to the library. Combining MaxDIA with two new technologies—BoxCar acquisition and trapped ion mobility spectrometry—both lead to deep and accurate proteome quantification.publishedVersio

Synaptic Phospholipids as a New Target for Cortical Hyperexcitability and E/I Balance in Psychiatric Disorders

Lysophosphatidic acid (LPA) is a synaptic phospholipid, which regulates cortical excitation/inhibition (E/I) balance and controls sensory information processing in mice and man. Altered synaptic LPA signaling was shown to be associated with psychiatric disorders. Here, we show that the LPA-synthesizing enzyme autotaxin (ATX) is expressed in the astrocytic compartment of excitatory synapses and modulates glutamatergic transmission. In astrocytes, ATX is sorted toward fine astrocytic processes and transported to excitatory but not inhibitory synapses. This ATX sorting, as well as the enzymatic activity of astrocyte-derived ATX are dynamically regulated by neuronal activity via astrocytic glutamate receptors. Pharmacological and genetic ATX inhibition both rescued schizophrenia-related hyperexcitability syndromes caused by altered bioactive lipid signaling in two genetic mouse models for psychiatric disorders. Interestingly, ATX inhibition did not affect naive animals. However, as our data suggested that pharmacological ATX inhibition is a general method to reverse cortical excitability, we applied ATX inhibition in a ketamine model of schizophrenia and rescued thereby the electrophysiological and behavioral schizophrenia-like phenotype. Our data show that astrocytic ATX is a novel modulator of glutamatergic transmission and that targeting ATX might be a versatile strategy for a novel drug therapy to treat cortical hyperexcitability in psychiatric disorders

Shiga toxin receptor Gb3Cer/CD77:tumor-association and promising therapeutic target in pancreas and colon cancer

BACKGROUND: Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas. METHODOLOGY/PRINCIPAL FINDINGS: Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors. CONCLUSIONS/SIGNIFICANCE: Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies

Molecular Cause and Functional Impact of Altered Synaptic Lipid Signaling Due to a \u3cem\u3eprg-1\u3c/em\u3e Gene SNP

Loss of plasticity‐related gene 1 (PRG‐1), which regulates synaptic phospholipid signaling, leads to hyperexcitability via increased glutamate release altering excitation/inhibition (E/I) balance in cortical networks. A recently reported SNP in prg‐1 (R345T/mutPRG‐1) affects ~5 million European and US citizens in a monoallelic variant. Our studies show that this mutation leads to a loss‐of‐PRG‐1 function at the synapse due to its inability to control lysophosphatidic acid (LPA) levels via a cellular uptake mechanism which appears to depend on proper glycosylation altered by this SNP. PRG‐1+/− mice, which are animal correlates of human PRG‐1+/mut carriers, showed an altered cortical network function and stress‐related behavioral changes indicating altered resilience against psychiatric disorders. These could be reversed by modulation of phospholipid signaling via pharmacological inhibition of the LPA‐synthesizing molecule autotaxin. In line, EEG recordings in a human population‐based cohort revealed an E/I balance shift in monoallelic mutPRG‐1 carriers and an impaired sensory gating, which is regarded as an endophenotype of stress‐related mental disorders. Intervention into bioactive lipid signaling is thus a promising strategy to interfere with glutamate‐dependent symptoms in psychiatric diseases

Tumorassoziierte Glykosphingolipide in Pankreas- und Kolonkarzinomen : potentielle Targetstrukturen für adjuvante Therapien

Distler U. Tumorassoziierte Glykosphingolipide in Pankreas- und Kolonkarzinomen : potentielle Targetstrukturen für adjuvante Therapien. Bielefeld (Germany): Bielefeld University; 2009.Glykosphingolipide (GSL) spielen eine Schlüsselrolle in vielen biologischen Prozessen, einschließlich der Tumorgenese. Die GSL-Analytik erfährt dank moderner, massenspektrometrischer Methoden einen rasanten Fortschritt. In der vorliegenden Arbeit wurde eine neue Technik zur strukturellen Charakterisierung von GSL entwickelt, die auf der direkten Kopplung der IR-MALDI-o-TOF-MS und des DC-Overlay-Assays beruht. Hierbei werden drei komplementäre Methoden miteinander kombiniert: 1) die Trennung der GSL mittels DC, 2) ihre Detektion mit Oligosaccharid-spezifischen Proteinen und 3) die in situ MS von Protein-detektierten GSL. Die hohe Spezifität und Sensitivität der DC-IR-MALDI-o-TOF-MS konnten anhand verschiedener Detektionssysteme demonstriert werden. Es ließen sich sowohl GSL, die mit poly- und monoklonalen Antikörpern auf der DC-Platte detektiert wurden, als auch GSL, die mit verschiedenen Toxinen nachgewiesen wurden, massenspektrometrisch charakterisieren. Die Methode ermöglicht die Messung von GSL direkt auf der DC-Platte bis in den unteren Nanogrammbereich. Im besten Fall lag das Detektionslimit bei 0,6 ng. Zudem konnte gezeigt werden, dass für die DC-IR-MALDI-o-TOF-MS lediglich geringe Mengen an GSL aus Geweberohextrakten erforderlich sind und somit arbeitsintensive Aufreinigungsverfahren, die mit einem Materialverlust einhergehen, entfallen. Die Technik lässt sich erfolgreich zur Identifikation von tumorassoziierten GSL aus allen bisher untersuchten humanen Karzinomen einsetzen. Somit eröffnet die in situ DC-IR-MALDI-o-TOF-MS immundetektierter GSL neue Möglichkeiten zur spezifischen Charakterisierung von Nanogrammmengen an GSL aus sehr kleinen Gewebeproben im Milligrammbereich ohne aufwendige GSL-Isolierungsverfahren. Nach Etablierung dieser Technik wurden GSL-Expressionsanalysen in humanen Pankreas- und Kolonkarzinomgeweben durchgeführt. Mittels DC-Immundetektion konnte gezeigt werden, dass es sich im Falle der [alpha]2-6-sialylierten CD75s-1-Ganglioside sowie der isomeren [alpha]2-3-sialylierten iso-CD75s-1-Ganglioside in Pankreaskarzinomgeweben um tumorassoziierte Strukturen handelt. Im Vergleich zu gesundem Pankreasgewebe zeigten 42,1 Prozent der Pankreaskarzinome eine Überexpression an CD75s-1-Gangliosiden und 65,8 Prozent einen erhöhten Gehalt an iso-CD75s-1-Gangliosiden. Neben diesen beiden Gangliosiden konnte auch das neutrale GSL Gb3Cer/CD77 als tumorassoziiertes Molekül identifiziert werden. Insgesamt wurde in 61,9 Prozent der Pankreastumoren und in 81,3 Prozent der Kolonkarzinome eine erhöhte Gb3Cer/CD77-Expression im Vergleich zu gesundem Kontrollgewebe nachgewiesen. In allen Fällen konnten die Daten der DC-Overlay-Assays mittels fluoreszenzmikroskopischer und massenspektrometrischer Analysen bestätigt werden. Sowohl bei den CD75s-1-Gangliosiden als auch bei Gb3Cer/CD77 könnte es sich um potentielle Marker für eine ungünstige Prognose bei einer Erkrankung an Pankreaskrebs handeln, da ihre Expression mit einem schlecht- beziehungsweise undifferenzierten histomorphologischen Phänotyp korreliert. CD75s-1-Ganglioside und das neutrale GSL Gb3Cer/CD77 sind von besonderem Interesse, da es sich bei ihnen um die Rezeptoren der Ribosomen-inaktivierenden Proteine ML-I/rViscumin beziehungsweise Stx1 handelt, deren zyotoxische Effekte auf Tumorzellen in vitro und in vivo bereits von verschiedenen Arbeitsgruppen nachgewiesen werden konnten. Die Verträglichkeit von rViscumin wurde unlängst in klinischen Phase-I-Studien an Patienten erfolgreich getestet. Zusammenfassend lässt sich festhalten, dass die tumorassoziierten CD75s-1- und iso-CD75s-1-Ganglioside sowie Gb3Cer/CD77 vielversprechende, hocheffiziente Targetstrukturen für potentielle Adjuvanztherapien zur Behandlung von Pankreas- und, im Falle von Gb3Cer/CD77, auch für Kolonkarzinome darstellen. Die Ergebnisse der Expressionsanalysen verdeutlichen aber auch, dass eine Behandlung mit ML-I/rViscumin oder Stx1 nicht für alle Patienten gleichermaßen in Frage kommt, da nicht alle Tumoren eine erhöhte CD75s-1-Gangliosid- beziehungsweise Gb3Cer/CD77-Expression gegenüber gesundem Gewebe aufwiesen. Dies ist ein weiteres Beispiel für die Notwendigkeit, Therapiekonzepte in Zukunft individuell auf den Patienten zuzuschneiden, um somit möglichst effektiv und nachhaltig Pankreas- und Kolonkarzinome mit GSL-basierten Konzepten therapieren zu können

Purification and Properties of Yeast Proteases Secreted by Wickerhamomyces anomalus 227 and Metschnikovia pulcherrima 446 during Growth in a White Grape Juice

Aspartic proteases are of significant importance for medicine and biotechnology. In spite of sufficient evidence that many non-Saccharomyces yeasts produce extracellular proteases, previous research has focused on the enzymes of Candida species because of their role as virulence factors. Nowadays, there is also increasing interest for their applications in industrial processes, mainly because of their activities at low pH values. Here, we report the features of new acid proteases isolated from wine-relevant yeasts Metschnikovia pulcherrima and Wickerhamomyces anomalus. To our knowledge, this is the first detailed description of such an enzyme derived from strains of W. anomalus. Deviating to most former studies, we could demonstrate that the yeasts produce these enzymes in a natural substrate (grape juice) during the active growth phase. The enzymes were purified from concentrated grape juice by preparative isoelectric focusing. Biochemical data (maximum activity at ≈ pH 3.0, inhibition by pepstatin A) classify them as aspartic proteases. For W. anomalus 227, this assumption was confirmed by the protein sequence of WaAPR1 determined by LC-MS/MS. The sequence revealed a signal peptide for secretion, as well as a peptidase A1 domain with two aspartate residues in the active site. The enzyme has a calculated molecular mass of 47 kDa and an isolelectric point of 4.11

Apically-located P4-ATPase1-Lem1 complex internalizes phosphatidylserine and regulates motility-dependent invasion and egress in Toxoplasma gondii

The membrane asymmetry regulated by P4-ATPases is crucial for the functioning of eukaryotic cells. The underlying spatial translocation or flipping of specific lipids is usually assured by respective P4-ATPases coupled to conforming non-catalytic subunits. Our previous work has identified five P4-ATPases (TgP4-ATPase1–5) and three non-catalytic partner proteins (TgLem1–3) in the intracellular protozoan pathogen Toxoplasma gondii. However, their flipping activity, physiological relevance and functional coupling remain unknown. Herein, we demonstrate that TgP4-ATPase1 and TgLem1 work together to translocate phosphatidylserine (PtdSer) during the lytic cycle of T. gondii. Both proteins localize in the plasma membrane at the invasive (apical) end of its acutely-infectious tachyzoite stage. The genetic knockout of P4-ATPase1 and conditional depletion of Lem1 in tachyzoites severely disrupt the asexual reproduction and translocation of PtdSer across the plasma membrane. Moreover, the phenotypic analysis of individual mutants revealed a requirement of lipid flipping for the motility, egress and invasion of tachyzoites. Not least, the proximity-dependent biotinylation and reciprocal immunoprecipitation assays demonstrated the physical interaction of P4-ATPase1 and Lem1. Our findings disclose the mechanism and significance of PtdSer flipping during the lytic cycle and identify the P4-ATPase1-Lem1 heterocomplex as a potential drug target in T. gondii