Sex-specific pharmacokinetic response to phytoestrogens in Drosophila melanogaster.

Drosophila melanogaster, or the fruit fly, is widely used for modeling numerous human diseases, such as neurodegeneration, tumor development, cachexia, and intestinal dysfunction. It is a suitable model organism for research targeting the physiology and pathophysiology of the intestinal epithelial barrier and has also been used as a model organism for preliminary drug and bioactive nutrient screening. However, the application of D. melanogaster in research on drug bioavailability and pharmacokinetic properties has not yet been well explored. In this study, we applied D. melanogaster to investigate the absorption and excretion of the orally administered phytoestrogens daidzein, glycitein, genistein, and their glycosides. Therefore, we established a quick, noninvasive method to quantify compound retention in D. melanogaster, suitable for the investigation of a broad variety of potentially bioactive substances. We showed that fruit fly sex plays a key role in the metabolization, transportation, and excretion of phytoestrogenic isoflavones. In particular, female fruit flies retained significantly more isoflavones than male fruit flies, which was reflected in the greater metabolic impact of isoflavones on females. Male fruit flies excreted more isoflavones than females did, which was linked to the upregulation of the xenobiotic transporter gene Mdr50. We also demonstrated that micellized isoflavones were more bioavailable than powdered isoflavones, independent of sex, age or the addition of dietary fibers.

Targeted and untargeted screening of a plant extract library established from raw materials originating from Upper Austria.

Plant extracts rich in phytochemicals are known for their health benefits. Plant extract library from edible plants obtained from the region of Upper Austria was prepared. Food grade extraction procedures were applied, and relevant physico-chemical parameters measured. A focus on polyphenolic compounds revealed a significant correlation between the total phenolic content (measured by a colorimetric assay) and the cumulated concentration of main individual polyphenols (measured by HPLC-DAD), demonstrating the comparability of these parameters. Targeted screening was performed by HPLC-FLD and -MS for the presence of phytomelatonin. 20 extracts were identified with concentrations of up to 1.4 µg/mL of this phytochemical, which attracts much attention from the food industry. Finally, chemometric methods were employed to cluster extracts based on their phenolic compound profile. This approach allows for an informed preselection of extracts without the need for comprehensive chemical analysis.

Identification of Oxidative-Stress-Reducing Plant Extracts from a Novel Extract Library-Comparative Analysis of Cell-Free and Cell-Based In Vitro Assays to Quantitate Antioxidant Activity.

Numerous underexplored plant species are believed to possess considerable potential in combating oxidative stress and its associated health impacts, emphasizing the need for a comprehensive methodological screening approach to assess their antioxidant capacity. This study investigated 375 plant extracts, utilizing both cell-free and cellular methods to evaluate their antioxidant properties. Target-based antioxidant capacity was evaluated by the total phenolic content (TPC) and ferric reducing antioxidant power (FRAP) assays. Cell-based assays employed the H2DCF-DA probe to measure reactive oxygen species (ROS) levels and the Griess assay to quantify nitric oxide (NO) levels in stressed Caco-2 and RAW264.7 cells, respectively. The highest TPC and FRAP values were found in extracts of Origanum vulgare and Fragaria × ananassa leaves. Several plant extracts significantly reduced stress-induced ROS or NO levels by at least 30%. Distinctive selectivity was noted in certain extracts, favoring the significant reduction of NO (e.g., Helianthus tuberosus extract), of ROS (e.g., Prunus domestica subsp. Syriaca extract), or of both (e.g., Fragaria × ananassa leaf extract). A strong correlation between TPC and FRAP values and moderate correlations between the results of the cell-free and cell-based assays were evident. These findings highlight the great antioxidant potential of underexplored plant extracts and the diversity of the underlying mechanisms, emphasizing the importance of a multifaceted approach for a comprehensive assessment.

Improved Bioavailability and Bioaccessibility of Lutein and Isoflavones in Cultured Cells In Vitro through Interaction with Ginger, Curcuma and Black Pepper Extracts.

Intestinal absorption is intrinsically low for lipophilic micronutrients and phytochemicals. Plant extracts acting as bioavailability enhancers can complement for this deficiency by modulation of both, physicochemical and biochemical parameters, in the absorption process. However, these interactions often are limited to specific conditions and the mechanisms and potential synergisms are poorly understood. In this work, we used a human intestinal cell line to characterize the impact of extracts from C. longa (curcuma), Z. officinale (ginger) and P.nigrum (black pepper) on uptake and transport rates of the xanthophylls lutein and zeaxanthin as well as soy isoflavones measured by HPLC-DAD. We found a significant increase in the uptake of lutein in the presence of curcuma extract and enhanced isoflavone transport rates mediated by curcuma and ginger extracts. Combinations of the plant extracts did not lead to any additional increase in uptake or transport rates. By investigation of mixed micelle incorporation efficiency, we could dismiss changes in bioaccessibility as a potential enhancing mechanism in our experimental setup. We further conducted a rhodamine 123 efflux assay and discovered inhibition of P-glycoproteins by the ginger and black pepper extracts, highlighting a plausible route of action leading to increased isoflavone bioavailability.

Anti-Hyperglycemic Effects of Oils and Extracts Derived from Sea Buckthorn - A Comprehensive Analysis Utilizing In Vitro and In Vivo Models.

SCOPE: Sea buckthorn (Hippophaes rhamnoides) is capable of ameliorating disturbed glucose metabolism in animal models and human subjects. Here, the effect of sea buckthorn oil as well as of extracts of fruits, leaves, and press cake on postprandial glucose metabolism is systematically investigated. METHODS AND RESULTS: Sea buckthorn did neither exert decisive effects in an in vitro model of intestinal glucose absorption nor did it alter insulin secretion. However, sea buckthorn stimulates GLUT4 translocation to the plasma membrane comparable to insulin, indicative of increased glucose clearance from the circulation. Isorhamnetin is identified in all sea buckthorn samples investigated and is biologically active in triggering GLUT4 cell surface localization. Consistently, sea buckthorn products lower circulating glucose by ≈10% in a chick embryo model. Moreover, sea buckthorn products fully revert hyperglycemia in the nematode Caenorhabditis elegans while they are ineffective in Drosophila melanogaster under euglycemic conditions. CONCLUSION: These data indicate that edible sea buckthorn products as well as by-products are promising resources for hypoglycemic nutrient supplements that increase cellular glucose clearance into target tissues.

Mono-ADP-ribosylation sites of human CD73 inhibit its adenosine-generating enzymatic activity.

CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD+ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD+ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD+ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD+ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity.

Targeted Quantification of Phosphorylation Sites Identifies STRIPAK-Dependent Phosphorylation of the Hippo Pathway-Related Kinase SmKIN3.

We showed recently that the germinal center kinase III (GCKIII) SmKIN3 from the fungus Sordaria macrospora is involved in sexual development and hyphal septation. Our recent extensive global proteome and phosphoproteome analysis revealed that SmKIN3 is a target of the striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex. Here, using protein samples from the wild type and three STRIPAK mutants, we applied absolute quantification by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in SmKIN3 and other septation initiation network (SIN) components, such as CDC7 and DBF2, as well as BUD4, acting downstream of SIN. For SmKIN3, we show that phosphorylation of S668 and S686 is decreased in mutants lacking distinct subunits of STRIPAK, while a third phosphorylation site, S589, was not affected. We constructed SmKIN3 mutants carrying phospho-mimetic and phospho-deficient codons for phosphorylation sites S589, S668, and S686. Investigation of hyphae in a ΔSmkin3 strain complemented by the S668 and S686 mutants showed a hyper-septation phenotype, which was absent in the wild type, the ΔSmkin3 strain complemented with the wild-type gene, and the S589 mutant. Furthermore, localization studies with SmKIN3 phosphorylation variants and STRIPAK mutants showed that SmKIN3 preferentially localizes at the terminal septa, which is distinctly different from the localization of the wild-type strains. We conclude that STRIPAK-dependent phosphorylation of SmKIN3 has an impact on controlled septum formation and on the time-dependent localization of SmKIN3 on septa at the hyphal tip. Thus, STRIPAK seems to regulate SmKIN3, as well as DBF2 and BUD4 phosphorylation, affecting septum formation.IMPORTANCE Phosphorylation and dephosphorylation of proteins are fundamental posttranslational modifications that determine the fine-tuning of their biological activity. Involved in this modification process is the recently identified striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex, which is evolutionarily conserved from fungi to humans. STRIPAK functions as a macromolecular assembly communicating through physical interactions with other conserved signaling protein complexes to constitute larger dynamic protein networks. Its function is implied in many cellular processes, such as signal transduction pathways, growth, and cellular differentiation. We applied absolute quantification of protein phosphorylation by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in signaling components that are linked to the STRIPAK complex. Using the filamentous fungus Sordaria macrospora, we provide evidence for the phosphorylation-dependent role of the Hippo-like germinal center kinase SmKIN3, which controls septum formation, and localize it in a time-dependent manner on septa at the hyphal tip.

The STRIPAK signaling complex regulates dephosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes.

The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22, Δpp2Ac1Δpro22) from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343 served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microscopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth.

Cutting the Gordian knot: early and complete amino acid sequence confirmation of class II lasso peptides by HCD fragmentation.

Lasso peptides are a diverse class of ribosomally synthesized and post-translationally modified peptides (RiPPs). Their proteolytic and thermal stability alongside their growing potential as therapeutics has increased attention to these antimicrobial peptides. With the advent of genome mining, the discovery of RiPPs allows for the accurate prediction of putatively encoded structures, however, MSn experiments only provide partial sequence confirmation, therefore 2D NMR experiments are necessary for characterisation. Multiple MS/MS techniques were applied to two structurally characterized lasso peptides, huascopeptin and leepeptin, and one uncharacterized lasso peptide, citrulassin C, which was not isolable in sufficient quantity for NMR analysis. We have shown that MS2 can be used to elucidate the full amino acid sequences previously predicted with genome mining for this compound class. HCD was able to open the macrocycles and fragment the newly opened linear peptides, confirming the complete amino acid sequences of the characterised lasso peptides. In addition, to determine if this technique could be applied at the earliest stages of the isolation process, we targeted a lasso peptide found by genome mining, citrulassin C, and were able to fully elucidate the amino acid sequence using only MS2 from a semi-crude extract of Streptomyces huasconensis HST28T.

Phosphoproteomic analysis of STRIPAK mutants identifies a conserved serine phosphorylation site in PAK kinase CLA4 to be important in fungal sexual development and polarized growth.

The highly conserved striatin-interacting phosphatases and kinases (STRIPAK) complex regulates phosphorylation/dephosphorylation of developmental proteins in eukaryotic microorganisms, animals and humans. To first identify potential targets of STRIPAK, we performed extensive isobaric tags for relative and absolute quantification-based proteomic and phosphoproteomic analyses in the filamentous fungus Sordaria macrospora. In total, we identified 4,193 proteins and 2,489 phosphoproteins, which are represented by 10,635 phosphopeptides. By comparing phosphorylation data from wild type and mutants, we identified 228 phosphoproteins to be regulated in all three STRIPAK mutants, thus representing potential targets of STRIPAK. To provide an exemplarily functional analysis of a STRIPAK-dependent phosphorylated protein, we selected CLA4, a member of the conserved p21-activated kinase family. Functional characterization of the ∆cla4 deletion strain showed that CLA4 controls sexual development and polarized growth. To determine the functional relevance of CLA4 phosphorylation and the impact of specific phosphorylation sites on development, we next generated phosphomimetic and -deficient variants of CLA4. This analysis identified (de)phosphorylation of a highly conserved serine (S685) residue in the catalytic domain of CLA4 as being important for fungal cellular development. Collectively, these analyses significantly contribute to the understanding of the mechanistic function of STRIPAK as a phosphatase and kinase signaling complex.

Inhibition of osimertinib-resistant epidermal growth factor receptor EGFR-T790M/C797S.

Precision medicine has revolutionized the treatment of patients in EGFR driven non-small cell lung cancer (NSCLC). Targeted drugs show high response rates in genetically defined subsets of cancer patients and markedly increase their progression-free survival as compared to conventional chemotherapy. However, recurrent acquired drug resistance limits the success of targeted drugs in long-term treatment and requires the constant development of novel efficient inhibitors of drug resistant cancer subtypes. Herein, we present covalent inhibitors of the drug resistant gatekeeper mutant EGFR-L858R/T790M based on the pyrrolopyrimidine scaffold. Biochemical and cellular characterization, as well as kinase selectivity profiling and western blot analysis, substantiate our approach. Moreover, the developed compounds possess high activity against multi drug resistant EGFR-L858R/T790M/C797S in biochemical assays due to their highly reversible binding character, that was revealed by characterization of the binding kinetics. In addition, we present the first X-ray crystal structures of covalent inhibitors in complex with C797S-mutated EGFR which provide detailed insight into their binding mode.

Proteogenomics of Colorectal Cancer Liver Metastases: Complementing Precision Oncology with Phenotypic Data.

: Hotspot testing for activating KRAS mutations is used in precision oncology to select colorectal cancer (CRC) patients who are eligible for anti-EGFR treatment. However, even for KRASwildtype tumors anti-EGFR response rates are <30%, while mutated-KRAS does not entirely rule out response, indicating the need for improved patient stratification. We performed proteogenomic phenotyping of KRASwildtype and KRASG12V CRC liver metastases (mCRC). Among >9000 proteins we detected considerable expression changes including numerous proteins involved in progression and resistance in CRC. We identified peptides representing a number of predicted somatic mutations, including KRASG12V. For eight of these, we developed a multiplexed parallel reaction monitoring (PRM) mass spectrometry assay to precisely quantify the mutated and canonical protein variants. This allowed phenotyping of eight mCRC tumors and six paired healthy tissues, by determining mutation rates on the protein level. Total KRAS expression varied between tumors (0.47-1.01 fmol/µg total protein) and healthy tissues (0.13-0.64 fmol/µg). In KRASG12V-mCRC, G12V-mutation levels were 42-100%, while one patient had only 10% KRASG12V but 90% KRASwildtype. This might represent a missed therapeutic opportunity: based on hotspot sequencing, the patient was excluded from anti-EGFR treatment and instead received chemotherapy, while PRM-based tumor-phenotyping indicates the patient might have benefitted from anti-EGFR therapy.

Short Peptides with Uncleavable Peptide Bond Mimetics as Photoactivatable Caspase-3 Inhibitors.

Chemical probes that covalently interact with proteases have found increasing use for the study of protease function and localization. The design and synthesis of such probes is still a bottleneck, as the strategies to target different families are highly diverse. We set out to design and synthesize chemical probes based on protease substrate specificity with inclusion of an uncleavable peptide bond mimic and a photocrosslinker for covalent modification of the protease target. With caspase-3 as a model target protease, we designed reduced amide and triazolo peptides as substrate mimetics, whose sequences can be conveniently constructed by modified solid phase peptide synthesis. We found that these probes inhibited the caspase-3 activity, but did not form a covalent bond. It turned out that the reduced amide mimics, upon irradiation with a benzophenone as photosensitizer, are oxidized and form low concentrations of peptide aldehydes, which then act as inhibitors of caspase-3. This type of photoactivation may be utilized in future photopharmacology experiments to form protease inhibitors at a precise time and location.

Disentangling thermal stress responses in a reef-calcifier and its photosymbionts by shotgun proteomics.

The proliferation of key marine ecological engineers and carbonate producers often relies on their association with photosymbiotic algae. Evaluating stress responses of these organisms is important to predict their fate under future climate projections. Physiological approaches are limited in their ability to resolve the involved molecular mechanisms and attribute stress effects to the host or symbiont, while probing and partitioning of proteins cannot be applied in organisms where the host and symbiont are small and cannot be physically separated. Here we apply a label-free quantitative proteomics approach to detect changes of proteome composition in the diatom-bearing benthic foraminifera Amphistegina gibbosa experimentally exposed to three thermal-stress scenarios. We developed a workflow for protein extraction from less than ten specimens and simultaneously analysed host and symbiont proteomes. Despite little genomic data for the host, 1,618 proteins could be partially assembled and assigned. The proteomes revealed identical pattern of stress response among stress scenarios as that indicated by physiological measurements, but allowed identification of compartment-specific stress reactions. In the symbiont, stress-response and proteolysis-related proteins were up regulated while photosynthesis-related proteins declined. In contrast, host homeostasis was maintained through chaperone up-regulation associated with elevated proteosynthesis and proteolysis, and the host metabolism shifted to heterotrophy.

Comparison and Evaluation of Clustering Algorithms for Tandem Mass Spectra.

In proteomics, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is established for identifying peptides and proteins. Duplicated spectra, that is, multiple spectra of the same peptide, occur both in single MS/MS runs and in large spectral libraries. Clustering tandem mass spectra is used to find consensus spectra, with manifold applications. First, it speeds up database searches, as performed for instance by Mascot. Second, it helps to identify novel peptides across species. Third, it is used for quality control to detect wrongly annotated spectra. We compare different clustering algorithms based on the cosine distance between spectra. CAST, MS-Cluster, and PRIDE Cluster are popular algorithms to cluster tandem mass spectra. We add well-known algorithms for large data sets, hierarchical clustering, DBSCAN, and connected components of a graph, as well as the new method N-Cluster. All algorithms are evaluated on real data with varied parameter settings. Cluster results are compared with each other and with peptide annotations based on validation measures such as purity. Quality control, regarding the detection of wrongly (un)annotated spectra, is discussed for exemplary resulting clusters. N-Cluster proves to be highly competitive. All clustering results benefit from the so-called DISMS2 filter that integrates additional information, for example, on precursor mass.

Combining De Novo Peptide Sequencing Algorithms, A Synergistic Approach to Boost Both Identifications and Confidence in Bottom-up Proteomics.

Complex mass spectrometry based proteomics data sets are mostly analyzed by protein database searches. While this approach performs considerably well for sequenced organisms, direct inference of peptide sequences from tandem mass spectra, i.e., de novo peptide sequencing, oftentimes is the only way to obtain information when protein databases are absent. However, available algorithms suffer from drawbacks such as lack of validation and often high rates of false positive hits (FP). Here we present a simple method of combining results from commonly available de novo peptide sequencing algorithms, which in conjunction with minor tweaks in data acquisition ensues lower empirical FDR compared to the analysis using single algorithms. Results were validated using state-of-the art database search algorithms as well specifically synthesized reference peptides. Thus, we could increase the number of PSMs meeting a stringent FDR of 5% more than 3-fold compared to the single best de novo sequencing algorithm alone, accounting for an average of 11 120 PSMs (combined) instead of 3476 PSMs (alone) in triplicate 2 h LC-MS runs of tryptic HeLa digestion.

DISMS2: A flexible algorithm for direct proteome- wide distance calculation of LC-MS/MS runs.

BACKGROUND: The classification of samples on a molecular level has manifold applications, from patient classification regarding cancer treatment to phylogenetics for identifying evolutionary relationships between species. Modern methods employ the alignment of DNA or amino acid sequences, mostly not genome-wide but only on selected parts of the genome. Recently proteomics-based approaches have become popular. An established method for the identification of peptides and proteins is liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, protein sequences from MS/MS spectra are identified by means of database searches, given samples with known genome-wide sequence information, then sequence based methods are applied. Alternatively, de novo peptide sequencing algorithms annotate MS/MS spectra and deduce peptide/protein information without a database. A newer approach independent of additional information is to directly compare unidentified tandem mass spectra. The challenge then is to compute the distance between pairwise MS/MS runs consisting of thousands of spectra. METHODS: We present DISMS2, a new algorithm to calculate proteome-wide distances directly from MS/MS data, extending the algorithm compareMS2, an approach that also uses a spectral comparison pipeline. RESULTS: Our new more flexible algorithm, DISMS2, allows for the choice of the spectrum distance measure and includes different spectra preprocessing and filtering steps that can be tailored to specific situations by parameter optimization. CONCLUSIONS: DISMS2 performs well for samples from species with and without database annotation and thus has clear advantages over methods that are purely based on database search.