Publications by authors named "Christoph Weise"

96 Publications

Antimicrobial Peptide Induced-Stress Renders Susceptible to Toxic Nucleoside Analogs.

Front Immunol 2020 29;11:1686. Epub 2020 Sep 29.

Institut für Biologie, Evolutionary Biology, Freie Universität Berlin, Berlin, Germany.

Cationic antimicrobial peptides (AMPs) are active immune effectors of multicellular organisms and are also considered as new antimicrobial drug candidates. One of the problems encountered when developing AMPs as drugs is the difficulty of reaching sufficient killing concentrations under physiological conditions. Here, using pexiganan, a cationic peptide derived from a host defense peptide of the African clawed frog and the first AMP developed into an antibacterial drug, we studied whether sub-lethal effects of AMPs can be harnessed to devise treatment combinations. We studied the pexiganan stress response of at sub-lethal concentrations using quantitative proteomics. Several proteins involved in nucleotide metabolism were elevated, suggesting a metabolic demand. We then show that is highly susceptible to antimetabolite nucleoside analogs when exposed to pexiganan, even at sub-inhibitory concentrations. These findings could be used to enhance pexiganan potency while decreasing the risk of resistance emergence, and our findings can likely be extended to other antimicrobial peptides.
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http://dx.doi.org/10.3389/fimmu.2020.01686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550632PMC
September 2020

Combined Methylome, Transcriptome and Proteome Analyses Document Rapid Acclimatization of a Bacterium to Environmental Changes.

Front Microbiol 2020 15;11:544785. Epub 2020 Sep 15.

Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Stechlin, Germany.

strain QLW-P1DMWA-1 represents a group of highly successful heterotrophic ultramicrobacteria that is frequently very abundant (up to 70% of total bacterioplankton) in freshwater habitats across all seven continents. This strain was originally isolated from a shallow Alpine pond characterized by rapid changes in water temperature and elevated UV radiation due to its location at an altitude of 1300 m. To elucidate the strain's adjustment to fluctuating environmental conditions, we recorded changes occurring in its transcriptomic and proteomic profiles under contrasting experimental conditions by simulating thermal conditions in winter and summer as well as high UV irradiation. To analyze the potential connection between gene expression and regulation via methyl group modification of the genome, we also analyzed its methylome. The methylation pattern differed between the three treatments, pointing to its potential role in differential gene expression. An adaptive process due to evolutionary pressure in the genus was deduced by calculating the ratios of non-synonymous to synonymous substitution rates for 20 spp. genomes obtained from geographically diverse isolates. The results indicate purifying selection.
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http://dx.doi.org/10.3389/fmicb.2020.544785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522526PMC
September 2020

An unusual type I ribosome-inactivating protein from Agrostemma githago L.

Sci Rep 2020 09 21;10(1):15377. Epub 2020 Sep 21.

Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195, Berlin, Germany.

Agrostemma githago L. (corn cockle) is an herbaceous plant mainly growing in Europe. The seeds of the corn cockle are toxic and poisonings were widespread in the past by consuming contaminated flour. The toxic principle of Agrostemma seeds was attributed to triterpenoid secondary metabolites. Indeed, this is in part true. However Agrostemma githago L. is also a producer of ribosome-inactivating proteins (RIPs). RIPs are N-glycosylases that inactivate the ribosomal RNA, a process leading to an irreversible inhibition of protein synthesis and subsequent cell death. A widely known RIP is ricin from Ricinus communis L., which was used as a bioweapon in the past. In this study we isolated agrostin, a 27 kDa RIP from the seeds of Agrostemma githago L., and determined its full sequence. The toxicity of native agrostin was investigated by impedance-based live cell imaging. By RNAseq we identified 7 additional RIPs (agrostins) in the transcriptome of the corn cockle. Agrostin was recombinantly expressed in E. coli and characterized by MALDI-TOF-MS and adenine releasing assay. This study provides for the first time a comprehensive analysis of ribosome-inactivating proteins in the corn cockle and complements the current knowledge about the toxic principles of the plant.
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http://dx.doi.org/10.1038/s41598-020-72282-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7506001PMC
September 2020

Novel Bradykinin-Potentiating Peptides and Three-Finger Toxins from Viper Venom: Combined NGS Venom Gland Transcriptomics and Quantitative Venom Proteomics of the Viper.

Biomedicines 2020 Jul 28;8(8). Epub 2020 Jul 28.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 117997 Moscow, Russia.

Feae's viper belongs to the Azemiopinae subfamily of the Viperidae family. The effects of Viperidae venoms are mostly coagulopathic with limited neurotoxicity manifested by phospholipases A2. From venom, we have earlier isolated azemiopsin, a novel neurotoxin inhibiting the nicotinic acetylcholine receptor. To characterize other toxins, we applied label-free quantitative proteomics, which revealed 120 unique proteins, the most abundant being serine proteinases and phospholipases A2. In total, toxins representing 14 families were identified, among which bradykinin-potentiating peptides with unique amino acid sequences possessed biological activity in vivo. The proteomic analysis revealed also basal (commonly known as non-conventional) three-finger toxins belonging to the group of those possessing neurotoxic activity. This is the first indication of the presence of three-finger neurotoxins in viper venom. In parallel, the transcriptomic analysis of venom gland performed by Illumina next-generation sequencing further revealed 206 putative venom transcripts. Together, the study unveiled the venom proteome and venom gland transciptome of , which in general resemble those of other snakes from the Viperidae family. However, new toxins not found earlier in viper venom and including three-finger toxins and unusual bradykinin-potentiating peptides were discovered.
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http://dx.doi.org/10.3390/biomedicines8080249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460416PMC
July 2020

Pan-Proteomic Analysis and Elucidation of Protein Abundance among the Closely Related Species, and .

Biomolecules 2020 05 30;10(6). Epub 2020 May 30.

Friedrich-Loeffler-Institute, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07743 Jena, Germany.

Brucellosis is a zoonotic infection caused by bacteria of the genus . The species, and , major causative agents of human brucellosis, share remarkably similar genomes, but they differ in their natural hosts, phenotype, antigenic, immunogenic, proteomic and metabolomic properties. In the present study, label-free quantitative proteomic analysis was applied to investigate protein expression level differences. Type strains and field strains were each cultured six times, cells were harvested at a midlogarithmic growth phase and proteins were extracted. Following trypsin digestion, the peptides were desalted, separated by reverse-phase nanoLC, ionized using electrospray ionization and transferred into an linear trap quadrapole (LTQ) Orbitrap Velos mass spectrometer to record full scan MS spectra ( 300-1700) and tandem mass spectrometry (MS/MS) spectra of the 20 most intense ions. Database matching with the reference proteomes resulted in the identification of 826 proteins. The Cluster of Gene Ontologies of the identified proteins revealed differences in bimolecular transport and protein synthesis mechanisms between these two strains. Among several other proteins, antifreeze proteins, Omp10, superoxide dismutase and 30S ribosomal protein S14 were predicted as potential virulence factors among the proteins differentially expressed. All mass spectrometry data are available via ProteomeXchange with identifier PXD006348.
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http://dx.doi.org/10.3390/biom10060836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355635PMC
May 2020

TMEM87a/Elkin1, a component of a novel mechanoelectrical transduction pathway, modulates melanoma adhesion and migration.

Elife 2020 04 1;9. Epub 2020 Apr 1.

EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia.

Mechanoelectrical transduction is a cellular signalling pathway where physical stimuli are converted into electro-chemical signals by mechanically activated ion channels. We describe here the presence of mechanically activated currents in melanoma cells that are dependent on TMEM87a, which we have renamed Elkin1. Heterologous expression of this protein in PIEZO1-deficient cells, that exhibit no baseline mechanosensitivity, is sufficient to reconstitute mechanically activated currents. Melanoma cells lacking functional Elkin1 exhibit defective mechanoelectrical transduction, decreased motility and increased dissociation from organotypic spheroids. By analysing cell adhesion properties, we demonstrate that Elkin1 deletion is associated with increased cell-substrate adhesion and decreased homotypic cell-cell adhesion strength. We therefore conclude that Elkin1 supports a PIEZO1-independent mechanoelectrical transduction pathway and modulates cellular adhesions and regulates melanoma cell migration and cell-cell interactions.
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http://dx.doi.org/10.7554/eLife.53308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173973PMC
April 2020

Non-lethal exposure to H2O2 boosts bacterial survival and evolvability against oxidative stress.

PLoS Genet 2020 03 12;16(3):e1008649. Epub 2020 Mar 12.

Freie Universität Berlin, Institute of Biology, Evolutionary Biology, Berlin, Germany.

Unicellular organisms have the prevalent challenge to survive under oxidative stress of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). ROS are present as by-products of photosynthesis and aerobic respiration. These reactive species are even employed by multicellular organisms as potent weapons against microbes. Although bacterial defences against lethal and sub-lethal oxidative stress have been studied in model bacteria, the role of fluctuating H2O2 concentrations remains unexplored. It is known that sub-lethal exposure of Escherichia coli to H2O2 results in enhanced survival upon subsequent exposure. Here we investigate the priming response to H2O2 at physiological concentrations. The basis and the duration of the response (memory) were also determined by time-lapse quantitative proteomics. We found that a low level of H2O2 induced several scavenging enzymes showing a long half-life, subsequently protecting cells from future exposure. We then asked if the phenotypic resistance against H2O2 alters the evolution of resistance against oxygen stress. Experimental evolution of H2O2 resistance revealed faster evolution and higher levels of resistance in primed cells. Several mutations were found to be associated with resistance in evolved populations affecting different loci but, counterintuitively, none of them was directly associated with scavenging systems. Our results have important implications for host colonisation and infections where microbes often encounter reactive oxygen species in gradients.
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http://dx.doi.org/10.1371/journal.pgen.1008649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093028PMC
March 2020

Protein corona formation and its influence on biomimetic magnetite nanoparticles.

J Mater Chem B 2020 06;8(22):4870-4882

Freie Universität Berlin, Institute of Pharmacy, Königin-Luise-Str. 2-4, 14195 Berlin, Germany and University of British Columbia, Faculty of Pharmaceutical Sciences, 2405 Wesbrook Mall, Vancouver, BC, Canada.

Biomimetic magnetite nanoparticles (BMNPs) synthesized in the presence of MamC, a magnetosome-associated protein from Magnetoccus marinus MC-1, have gained interest for biomedical applications because of their unique magnetic properties. However, their behavior in biological systems, like their interaction with proteins, still has to be evaluated prior to their use in clinics. In this study, doxorubicin (DOXO) as a model drug was adsorbed onto BMNPs to form nanoassemblies. These were incubated with human plasma to trigger protein corona (PC) formation. Proteins from the human plasma stably attached to either BMNPs or DOXO-BMNP nanoassemblies. In particular, fibrinogen was detected as the main component in the PC of DOXO-BMNPs that potentially provides advantages, e.g. protecting the particles from phagocytosis, thus prolonging their circulation time. Adsorption of PC to the BMNPs did not alter their magnetic properties but improved their colloidal stability, thus reducing their toxicity in human macrophages. In addition, PC formation enhanced cellular internalization and did not interfere with DOXO activity. Overall, our data indicate that the adsorption of PC onto DOXO-BMNPs in biological environment even increases their efficiency as drug carrier systems.
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http://dx.doi.org/10.1039/c9tb02480hDOI Listing
June 2020

Treatment of Yersinia similis with the cationic lipid DOTAP enhances adhesion to and invasion into intestinal epithelial cells - A proof-of-principle study.

Biochem Biophys Res Commun 2020 04 22;525(2):378-383. Epub 2020 Feb 22.

Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute for Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07743 Jena, Germany. Electronic address:

The monocationic quaternary surfactant DOTAP has been used for the delivery of nucleic acids and peptides into mammalian cells. This study tested the applicability of DOTAP for the enhancement of adhesion and invasion frequencies of Yersinia (Y.) similis to enable the analysis of the effects of low-pathogenic bacteria on intestinal epithelial cells. Incubation of Y. similis with DOTAP ahead of infection of C2BBe1 intestinal epithelial cells increased invasion and adhesion frequency four- and five-fold, respectively, in plating assays. Proteomic approaches confirmed the increased bacterial load on infected cells: analysis of protein extracts by two-dimensional difference gel electrophoresis (2D-DIGE) revealed higher amounts of bacterial proteins present in the cells infected with DOTAP-treated bacteria. MALDI-TOF mass spectrometry of selected spots from gel-separated protein extracts confirmed the presence of both bacterial and human cell proteins in the samples. Label-free quantitative proteomics analysis identified 1170 human cell proteins and 699 bacterial proteins. Three times more bacterial proteins (279 vs. 93) were detected in C2BBe1 cells infected with DOTAP-treated bacteria compared to infections with untreated bacteria. Infections with DOTAP-treated Y. similis led to a significant upregulation of the stress-inducible ubiquitin-conjugating enzyme UBE2M in C2BBe1 cells. This points towards a stronger impact of the stress and infection responsive transcription factor AP-1 by enhanced bacterial load. DOTAP-treatment of uninfected C2BBe1 cells led to a significant downregulation of the transmembrane trafficking protein TMED10. The application of DOTAP could be helpful for investigating the impact of otherwise low adherent or invasive bacteria on cultivated mammalian cells without utilisation of genetic modifications.
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http://dx.doi.org/10.1016/j.bbrc.2020.02.081DOI Listing
April 2020

The inactive C-terminal cassette of the dual-cassette RNA helicase BRR2 both stimulates and inhibits the activity of the N-terminal helicase unit.

J Biol Chem 2020 02 30;295(7):2097-2112. Epub 2019 Dec 30.

Structural Biochemistry Group, Department of Biochemistry, Freie Universität Berlin, Takustrasse 63, D-14195 Berlin, Germany

The RNA helicase bad response to refrigeration 2 homolog (BRR2) is required for the activation of the spliceosome before the first catalytic step of RNA splicing. BRR2 represents a distinct subgroup of Ski2-like nucleic acid helicases whose members comprise tandem helicase cassettes. Only the N-terminal cassette of BRR2 is an active ATPase and can unwind substrate RNAs. The C-terminal cassette represents a pseudoenzyme that can stimulate RNA-related activities of the N-terminal cassette. However, the molecular mechanisms by which the C-terminal cassette modulates the activities of the N-terminal unit remain elusive. Here, we show that N- and C-terminal cassettes adopt vastly different relative orientations in a crystal structure of BRR2 in complex with an activating domain of the spliceosomal Prp8 protein at 2.4 Å resolution compared with the crystal structure of BRR2 alone. Likewise, inspection of BRR2 structures within spliceosomal complexes revealed that the cassettes occupy different relative positions and engage in different intercassette contacts during different splicing stages. Engineered disulfide bridges that locked the cassettes in two different relative orientations had opposite effects on the RNA-unwinding activity of the N-terminal cassette, with one configuration enhancing and the other configuration inhibiting RNA unwinding compared with the unconstrained protein. Moreover, we found that differences in relative positioning of the cassettes strongly influence RNA-stimulated ATP hydrolysis by the N-terminal cassette. Our results indicate that the inactive C-terminal cassette of BRR2 can both positively and negatively affect the activity of the N-terminal helicase unit from a distance.
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http://dx.doi.org/10.1074/jbc.RA119.010964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029114PMC
February 2020

Mass Spectrometry-Based Method for Detection and Identification of Free Thiol Groups in Proteins.

Methods Mol Biol 2019 ;1934:179-189

Recombinant Research and Development, Octapharma Biopharmaceuticals GmbH, Heidelberg, Germany.

Many proteins contain free sulfhydryl groups which can be involved in a variety of biochemical reactions. Reactive thiol groups can either reside within the active center of oxidoreductases or represent a part of a thiol-based redox switch in proteins. Therefore, the exact position of a free sulfhydryl within a protein is mostly very important.This chapter describes a mass spectrometry-based method to determine the location of protein sulfhydryl groups exemplary shown for a synthetic decapeptide and the plasma glycoprotein von Willebrand factor (VWF). We outline (1) labeling of free sulfhydryl groups, (2) enrichment of labeled peptides, and (3) detection and identification of labeled peptides by mass spectrometry.
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http://dx.doi.org/10.1007/978-1-4939-9055-9_12DOI Listing
January 2020

A homozygous genome-edited Sept2-EGFP fibroblast cell line.

Cytoskeleton (Hoboken) 2019 01 19;76(1):73-82. Epub 2019 Apr 19.

Randall Division of Cell and Molecular Biophysics, King's College London, London, UK.

Septins are a conserved, essential family of GTPases that interact with actin, microtubules, and membranes and form scaffolds and diffusion barriers in cells. Several of the 13 known mammalian septins assemble into nonpolar, multimeric complexes that can further polymerize into filamentous structures. While some GFP-coupled septins have been described, overexpression of GFP-tagged septins often leads to artifacts in localization and function. To overcome this ubiquitous problem, we have here generated a genome-edited rat fibroblast cell line expressing Septin 2 (Sept2) coupled to enhanced green fluorescent protein (EGFP) from both chromosomal loci. We characterize these cells by genomic polymerase chain reaction (PCR) for genomic integration, by western blot and reverse transcriptase-PCR for expression, by immunofluorescence and immunoprecipitation for the colocalization of septins with one another and cellular structures and for complex formation of different septins. By live cell imaging, proliferation and migration assays we investigate proper function of septins in these cells. We find that EGFP is incorporated into both chromosomal loci and only EGFP-coupled Sept2 is expressed in homozygous cells. We find that endogenous Sept2-EGFP exhibits expression levels, localization and incorporation into cellular septin complexes similar to the wt in these cells. The expression level of other septins is not perturbed and cell division and cell migration proceed normally. We expect our cell line to be a useful tool for the cell biology of septins, especially for quantitative biology.
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http://dx.doi.org/10.1002/cm.21518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593442PMC
January 2019

Phosphorylation of the Bruchpilot N-terminus in unlocks axonal transport of active zone building blocks.

J Cell Sci 2019 03 18;132(6). Epub 2019 Mar 18.

Laboratory of Structural Biochemistry, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustraße 6, D-14195 Berlin, Germany

Protein scaffolds at presynaptic active zone membranes control information transfer at synapses. For scaffold biogenesis and maintenance, scaffold components must be safely transported along axons. A spectrum of kinases has been suggested to control transport of scaffold components, but direct kinase-substrate relationships and operational principles steering phosphorylation-dependent active zone protein transport are presently unknown. Here, we show that extensive phosphorylation of a 150-residue unstructured region at the N-terminus of the highly elongated Bruchpilot (BRP) active zone protein is crucial for ordered active zone precursor transport in Point mutations that block SRPK79D kinase-mediated phosphorylation of the BRP N-terminus interfered with axonal transport, leading to BRP-positive axonal aggregates that also contain additional active zone scaffold proteins. Axonal aggregates formed only in the presence of non-phosphorylatable BRP isoforms containing the SRPK79D-targeted N-terminal stretch. We assume that specific active zone proteins are pre-assembled in transport packages and are thus co-transported as functional scaffold building blocks. Our results suggest that transient post-translational modification of a discrete unstructured domain of the master scaffold component BRP blocks oligomerization of these building blocks during their long-range transport.
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http://dx.doi.org/10.1242/jcs.225151DOI Listing
March 2019

RNAi-mediated knockdown of the Rhau helicase preferentially depletes proteins with a Guanine-quadruplex motif in the 5'-UTR of their mRNA.

Biochem Biophys Res Commun 2019 01 6;508(3):756-761. Epub 2018 Dec 6.

Technische Universität Berlin, Institute of Biotechnology, Department of Applied Biochemistry, TUB 4/3-2, Gustav-Meyer-Allee 25, 13355, Berlin, Germany. Electronic address:

Guanine-quadruplex (G-quadruplex) structures in mRNAs have been shown to modulate gene expression. However, the overall biological relevance of this process is under debate, as cellular helicases unwind G-quadruplex structures. The helicase Rhau (encoded by the DHX36 gene) was reported to be the major source of RNA G-quadruplex resolving activity in lysates of human cells. In the current study, we depleted Rhau by RNAi-mediated silencing and analyzed the effect on proteins whose mRNAs harbor a G-quadruplex motif in their 5'-UTRs. A targeted investigation of the proto-oncogenes Bcl-2 and NRAS, which are well-known examples for the translational repression of G-quadruplex structures, did not reveal effects caused by Rhau silencing. We therefore carried out a global analysis of changes in protein levels by label-free quantification using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Following Rhau knockdown, of all the identified proteins, only 1.9% were significantly downregulated to at least 70%. According to a bioinformatic analysis with the QGRS mapper, 33% of the downregulated proteins were predicted to harbor a G-quadruplex motif in the 5'-UTR of their respective mRNAs, compared to only 11% in the complete dataset. This indicates that in an unexpectedly small set of genes, in which G-quadruplex motifs are unusually common in the 5'-UTR of their mRNAs, Rhau helicase is responsible for the regulation of their expression.
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http://dx.doi.org/10.1016/j.bbrc.2018.11.186DOI Listing
January 2019

A new type 1 ribosome-inactivating protein from the seeds of Gypsophila elegans M.Bieb.

Phytochemistry 2019 Jan 3;157:121-127. Epub 2018 Nov 3.

Institut für Pharmazie, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany. Electronic address:

Ribosome-inactivating proteins (RIPs) are enzymes with N-glycosylase activity that remove adenine bases from the ribosomal RNA. In theory, one single RIP molecule internalized into a cell is sufficient to induce cell death. For this reason, RIPs are of high potential as toxic payload for anti-tumor therapy. A considerable number of RIPs are synthesized by plants that belong to the carnation family (Caryophyllaceae). Prominent examples are the RIPs saporin from Saponaria officinalis L. or dianthin from Dianthus caryophyllus L. In this study, we have isolated and characterized a novel RIP (termed gypsophilin-S) from the tiny seeds of Gypsophila elegans M. Bieb. (Caryophyllaceae). It is noteworthy that this is the first study presenting the complete amino acid sequence of a RIP from a Gypsophila species. Gypsophilin-S was isolated from the defatted seed material following ammonium sulphate precipitation and HPLC-based ion exchange chromatography. Gypsophilin-S-containing fractions were analysed by SDS-PAGE and mass spectrometry. The full amino acid sequence of gypsophilin-S was assembled by MALDI-TOF-MS-MS and PCR. Gypsophilin-S exhibited strong adenine releasing activity and its cytotoxicity in human glioblastoma cells was investigated using an impedance-based real-time assay in comparison with recombinant saporin and dianthin.
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http://dx.doi.org/10.1016/j.phytochem.2018.10.024DOI Listing
January 2019

Understanding the elusive protein corona of thermoresponsive nanogels.

Nanomedicine (Lond) 2018 10 18;13(20):2657-2668. Epub 2018 Oct 18.

Freie Universität Berlin, Institute of Chemistry & Biochemistry, Takustr. 3, 14195 Berlin, Germany.

Aim: We analyzed the protein corona of thermoresponsive, poly(N-isopropylacrylamide)- or poly(N-isopropylmethacrylamide)-based nanogels.

Materials & Methods: Traces of protein corona detected after incubation in human serum were characterized by proteomics and dynamic light scattering in undiluted serum.

Results: Apolipoprotein B-100 and albumin were the main components of the protein coronae. For dendritic polyglycerol-poly(N-isopropylacrylamide) nanogels at 37°C, an increase in adsorbed immunoglobulin light chains was detected, followed by partially reversible nanogel aggregation. All nanogels in their hydrophilic state are colloidally stable in serum and bear a dysopsonin-rich protein corona.

Conclusion: We observed strong changes in NG stability upon slight alterations in the composition of the protein coronae according to nanogel solvation state. Nanogels in their hydrophilic state possess safe protein coronae.
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http://dx.doi.org/10.2217/nnm-2018-0217DOI Listing
October 2018

Medical phycology 2017.

Med Mycol 2018 Apr;56(suppl_1):S188-S204

Department of Medical Microbiology, Immunology, and Cell Biology, West Virginia University School of Medicine, Morgantown, West Virginia, USA.

In 2014, ISHAM formed a new working group: "Medical Phycology: Protothecosis and Chlorellosis." The purpose of this working group is to help facilitate collaboration and communication among people interested in the pathogenic algae, to share ideas and work together. Here we present reports on recent work we have done in five areas. 1. The history of medical phycology as a branch of science. 2. Aspects of the genetics of Prototheca. 3. Aspects of the proteins of Prototheca. 4. Human infections caused by Prototheca. 5. Dairy cow mastitis caused by Prototheca.
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http://dx.doi.org/10.1093/mmy/myx162DOI Listing
April 2018

Proteome Analysis of a M. avium Mutant Exposes a Novel Role of the Bifunctional Protein LysX in the Regulation of Metabolic Activity.

J Infect Dis 2018 06;218(2):291-299

Division 16, Mycotic and Parasitic Agents and Mycobacteria, Robert Koch Institute, Berlin, Germany.

Lysyl-phosphatidylglycerol is one of the components of the mycobacterial membrane that contributes to the resistance to cationic antimicrobial peptides, a host-induced frontline defense against invading pathogens. Its production is catalyzed by LysX, a bifunctional protein with lysyl transferase and lysyl transfer RNA synthetase activity. Comparative proteome analysis of a lysX mutant of Mycobacterium avium strain 104 and the wild type indicated that the lysX mutant strain undergoes a transition in phenotype by switching the carbon metabolism to β-oxidation of fatty acids, along with accumulation of lipid inclusions. Surprisingly, proteins associated with intracellular survival were upregulated in the lysX mutant, even during extracellular growth, preparing bacteria for the conditions occurring inside host cells. In line with this, the lysX mutant exhibited enhanced intracellular growth in human-blood-derived monocytes. Thus, our study exposes the significance of lysX in the metabolism and virulence of the environmental pathogen M. avium hominissuis.
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http://dx.doi.org/10.1093/infdis/jiy100DOI Listing
June 2018

Optimizing Circulating Tumor Cells' Capture Efficiency of Magnetic Nanogels by Transferrin Decoration.

Polymers (Basel) 2018 Feb 11;10(2). Epub 2018 Feb 11.

Freie Universität Berlin, Institute of Chemistry and Biochemistry, Takustr. 3, 14195 Berlin, Germany.

Magnetic nanogels (MNGs) are designed to have all the required features for their use as highly efficient trapping materials in the challenging task of selectively capturing circulating tumor cells (CTCs) from the bloodstream. Advantageously, the discrimination of CTCs from hematological cells, which is a key factor in the capturing process, can be optimized by finely tuning the polymers used to link the targeting moiety to the MNG. We describe herein the relationship between the capturing efficiency of CTCs with overexpressed transferrin receptors and the different strategies on the polymer used as linker to decorate these MNGs with transferrin (Tf). Heterobifunctional polyethylene glycol (PEG) linkers with different molecular weights were coupled to Tf in different ratios. Optimal values over 80% CTC capture efficiency were obtained when 3 PEG linkers with a length of 8 ethylene glycol (EG) units were used, which reveals the important role of the linker in the design of a CTC-sorting system.
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http://dx.doi.org/10.3390/polym10020174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414968PMC
February 2018

APLP1 is endoproteolytically cleaved by γ-secretase without previous ectodomain shedding.

Sci Rep 2018 01 30;8(1):1916. Epub 2018 Jan 30.

Institut für Chemie und Biochemie, Freie Universität Berlin, Thielallee 63, 14195, Berlin, Germany.

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) and its homologs, the APP like proteins APLP1 and APLP2, is typically a two-step process, which is initiated by ectodomain-shedding of the substrates by α- or β-secretases. Growing evidence, however, indicates that the cleavage process for APLP1 is different than for APP. Here, we describe that full-length APLP1, but not APP or APLP2, is uniquely cleaved by γ-secretase without previous ectodomain shedding. The new fragment, termed sAPLP1γ, was exclusively associated with APLP1, not APP, APLP2. We provide an exact molecular analysis showing that sAPLP1γ was uniquely generated by γ-secretase from full-length APLP1. Mass spectrometry analysis showed that the sAPLP1γ fragment and the longest Aβ-like peptide share the C-terminus. This novel mechanism of γ-secretase action is consistent with an ϵ-cut based upon the nature of the reaction in APP. We further demonstrate that the APLP1 transmembrane sequence is the critical determinant for γ-shedding and release of full-length APLP1. Moreover, the APLP1 TMS is sufficient to convert larger type-I membrane proteins like APP into direct γ-secretase substrates. Taken together, the direct cleavage of APLP1 is a novel feature of the γ-secretase prompting a re-thinking of γ-secretase activity modulation as a therapeutic strategy for Alzheimer disease.
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http://dx.doi.org/10.1038/s41598-018-19530-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789831PMC
January 2018

Redox-Sensing Under Hypochlorite Stress and Infection Conditions by the Rrf2-Family Repressor HypR in Staphylococcus aureus.

Antioxid Redox Signal 2018 09 30;29(7):615-636. Epub 2018 Jan 30.

1 Institute for Biology-Microbiology, Freie Universität Berlin , Berlin, Germany .

Aims: Staphylococcus aureus is a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections, which requires efficient protection mechanisms to avoid destruction. Here, we have investigated the changes in the RNA-seq transcriptome by the strong oxidant sodium hypochlorite (NaOCl) in S. aureus USA300 to identify novel redox-sensing mechanisms that provide protection under infection conditions.

Results: NaOCl stress caused an oxidative stress response in S. aureus as indicated by the induction of the PerR, QsrR, HrcA, and SigmaB regulons in the RNA-seq transcriptome. The hypR-merA (USA300HOU_0588-87) operon was most strongly upregulated under NaOCl stress, which encodes for the Rrf2-family regulator HypR and the pyridine nucleotide disulfide reductase MerA. We have characterized HypR as a novel redox-sensitive repressor that controls MerA expression and directly senses and responds to NaOCl and diamide stress via a thiol-based mechanism in S. aureus. Mutational analysis identified Cys33 and the conserved Cys99 as essential for NaOCl sensing, while Cys99 is also important for repressor activity of HypR in vivo. The redox-sensing mechanism of HypR involves Cys33-Cys99 intersubunit disulfide formation by NaOCl stress both in vitro and in vivo. Moreover, the HypR-controlled flavin disulfide reductase MerA was shown to protect S. aureus against NaOCl stress and increased survival in J774A.1 macrophage infection assays. Conclusion and Innovation: Here, we identified a new member of the widespread Rrf2 family as redox sensor of NaOCl stress in S. aureus that uses a thiol/disulfide switch to regulate defense mechanisms against the oxidative burst under infections in S. aureus. Antioxid. Redox Signal. 29, 615-636.
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http://dx.doi.org/10.1089/ars.2017.7354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067689PMC
September 2018

The glyceraldehyde-3-phosphate dehydrogenase GapDH of Corynebacterium diphtheriae is redox-controlled by protein S-mycothiolation under oxidative stress.

Sci Rep 2017 07 10;7(1):5020. Epub 2017 Jul 10.

Institute for Biology-Microbiology, Freie Universität Berlin, D-14195, Berlin, Germany.

Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modification by protein S-mycothiolation as emerging thiol-protection and redox-regulatory mechanism. Here, we have used shotgun-proteomics to identify 26 S-mycothiolated proteins in the pathogen Corynebacterium diphtheriae DSM43989 under hypochlorite stress that are involved in energy metabolism, amino acid and nucleotide biosynthesis, antioxidant functions and translation. The glyceraldehyde-3-phosphate dehydrogenase (GapDH) represents the most abundant S-mycothiolated protein that was modified at its active site Cys153 in vivo. Exposure of purified GapDH to HO and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with HO or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro which was faster compared to the overoxidation pathway. Reactivation of S-mycothiolated GapDH could be catalyzed by both, the Trx and the Mrx1 pathways in vitro, but demycothiolation by Mrx1 was faster compared to Trx. In summary, we show here that S-mycothiolation can function in redox-regulation and protection of the GapDH active site against overoxidation in C. diphtheriae which can be reversed by both, the Mrx1 and Trx pathways.
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http://dx.doi.org/10.1038/s41598-017-05206-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5504048PMC
July 2017

Protein Corona Formation on Colloidal Polymeric Nanoparticles and Polymeric Nanogels: Impact on Cellular Uptake, Toxicity, Immunogenicity, and Drug Release Properties.

Biomacromolecules 2017 Jun 26;18(6):1762-1771. Epub 2017 May 26.

Institute for Pharmacy, Freie Universität Berlin , Königin-Luise-Str. 2-4, 14195 Berlin, Germany.

The adsorption of biomolecules to the surface of nanoparticles (NPs) following administration into biological environments is widely recognized. In particular, the "protein corona" is well understood in terms of formation kinetics and impact upon the biological interactions of NPs. Its presence is an essential consideration in the design of therapeutic NPs. In the present study, the protein coronas of six polymeric nanoparticles of prospective therapeutic use were investigated. These included three colloidal NPs-soft core-multishell (CMS) NPs, plus solid cationic Eudragit RS (EGRS), and anionic ethyl cellulose (EC) nanoparticles-and three nanogels (NGs)-thermoresponsive dendritic-polyglycerol (dPG) nanogels (NGs) and two amino-functionalized dPG-NGs. Following incubation with human plasma, protein coronas were characterized and their biological interactions compared with pristine NPs. All NPs demonstrated protein adsorption and increased hydrodynamic diameters, although the solid EGRS and EC NPs bound notably more protein than the other tested particles. Shifts toward moderately negative surface charges were also observed for all corona bearing NPs, despite varied zeta potentials in their pristine states. While the uptake and cellular adhesion of the colloidal NPs in primary human keratinocytes and human umbilical vein endothelial cells were significantly decreased when bearing the protein corona, no obvious impact was seen in the NGs. By contrast, corona bearing NGs induced marked increases in cytokine release from primary human macrophages not seen with corona bearing colloidal NPs. Despite this, no apparent enhancement to in vitro toxicity was noted. Finally, drug release from EGRS and EC NPs was assessed, where a decrease was seen in the EGRS NPs alone. Together these results provide a direct comparison of the physical and biological impact the protein corona has on NPs of widely varied character and in particular highlights a distinction between the corona's effects on NGs and colloidal NPs.
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http://dx.doi.org/10.1021/acs.biomac.7b00158DOI Listing
June 2017

Mass spectrometry data from label-free quantitative proteomic analysis of harmless and pathogenic strains of infectious microalgae, .

Data Brief 2017 Jun 11;12:320-326. Epub 2017 Apr 11.

Institute of Animal Hygiene and Environmental Health, Centre for Infectious Medicine, Freie Universitaet Berlin, Berlin, Germany.

Here, we provide the dataset associated with our research article 'label-free quantitative proteomic analysis of harmless and pathogenic strains of infectious microalgae, spp.' (Murugaiyan et al., 2017) [1]. This dataset describes liquid chromatography-mass spectrometry (LC-MS)-based protein identification and quantification of a non-infectious strain, genotype 1 and two strains associated with severe and mild infections, respectively, genotype 2 and . Protein identification and label-free quantification was carried out by analysing MS raw data using the MaxQuant-Andromeda software suit. The expressional level differences of the identified proteins among the strains were computed using Perseus software and the results were presented in [1]. This DiB provides the MaxQuant output file and raw data deposited in the PRIDE repository with the dataset identifier PXD005305.
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http://dx.doi.org/10.1016/j.dib.2017.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5407497PMC
June 2017

Label-Free Quantitative Proteomic Analysis of Harmless and Pathogenic Strains of Infectious Microalgae, Prototheca spp.

Int J Mol Sci 2016 Dec 29;18(1). Epub 2016 Dec 29.

Institute of Animal Hygiene and Environmental Health, Centre for Infectious Medicine, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany.

Microalgae of the genus () spp are associated with rare algal infections of invertebrates termed protothecosis. Among the seven generally accepted species, genotype 2 (GT2) is associated with a severe form of bovine mastitis while causes the mild and sub-clinical form of mastitis. The reason behind the infectious nature of GT2, while genotype 1 (GT1) remains non-infectious, is not known. Therefore, in the present study we investigated the protein expression level difference between the genotypes of and Cells were cultured to the mid-exponential phase, harvested, and processed for LC-MS analysis. Peptide data was acquired on an LTQ Orbitrap Velos, raw spectra were quantitatively analyzed with MaxQuant software and matching with the reference database of and resulted in the identification of 226 proteins. Comparison of an environmental strain with infectious strains resulted in the identification of 51 differentially expressed proteins related to carbohydrate metabolism, energy production and protein translation. The expression level of Hsp70 proteins and their role in the infectious process is worth further investigation. All mass spectrometry data are available via ProteomeXchange with identifier PXD005305.
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http://dx.doi.org/10.3390/ijms18010059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297694PMC
December 2016

Tuning the Surface of Nanoparticles: Impact of Poly(2-ethyl-2-oxazoline) on Protein Adsorption in Serum and Cellular Uptake.

Macromol Biosci 2016 09 9;16(9):1287-300. Epub 2016 Jun 9.

Fraunhofer ICT-IMM, Carl-Zeiss-Str. 18-20, 55129, Mainz, Germany.

Due to the adsorption of biomolecules, the control of the biodistribution of nanoparticles is still one of the major challenges of nanomedicine. Poly(2-ethyl-2-oxazoline) (PEtOx) for surface modification of nanoparticles is applied and both protein adsorption and cellular uptake of PEtOxylated nanoparticles versus nanoparticles coated with poly(ethylene glycol) (PEG) and non-coated positively and negatively charged nanoparticles are compared. Therefore, fluorescent poly(organosiloxane) nanoparticles of 15 nm radius are synthesized, which are used as a scaffold for surface modification in a grafting onto approach. With multi-angle dynamic light scattering, asymmetrical flow field-flow fractionation, gel electrophoresis, and liquid chromatography-mass spectrometry, it is demonstrated that protein adsorption on PEtOxylated nanoparticles is extremely low, similar as on PEGylated nanoparticles. Moreover, quantitative microscopy reveals that PEtOxylation significantly reduces the non-specific cellular uptake, particularly by macrophage-like cells. Collectively, studies demonstrate that PEtOx is a very effective alternative to PEG for stealth modification of the surface of nanoparticles.
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http://dx.doi.org/10.1002/mabi.201600074DOI Listing
September 2016

Significantly enhanced proteolytic activity of cyclen complexes by monoalkylation.

Dalton Trans 2016 Jul 9;45(26):10500-4. Epub 2016 Jun 9.

Institut für Chemie und Biochemie, Freie Universität Berlin, Fabeckstr. 34/36, 14195 Berlin, Germany.

A simple approach towards efficient artificial proteases based on the cyclen ligand is presented. We thus achieved an increase of the proteolytic activity of two orders of magnitude when compared to the unsubstituted cyclen complex. Amphiphilic Cu(ii) and Co(iii) complexes cut BSA and myoglobin as model substrates at μM concentrations. MALDI-ToF MS is used to identify the cleavage fragments.
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http://dx.doi.org/10.1039/c6dt00681gDOI Listing
July 2016

Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts.

Int J Mol Sci 2016 Apr 30;17(5). Epub 2016 Apr 30.

Institute of Animal Hygiene and Environmental Health, Centre for Infectious Medicine, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, Berlin 14163, Germany.

Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.
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http://dx.doi.org/10.3390/ijms17050659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881485PMC
April 2016

Free thiol groups in von Willebrand factor (VWF) are required for its full function under physiological flow conditions.

Thromb Res 2016 Jan 10;137:202-210. Epub 2015 Nov 10.

Octapharma Biopharmaceuticals GmbH, Molecular Biochemistry, Berlin, Germany. Electronic address:

Introduction: von Willebrand factor (VWF) is rich in cysteine; next to important structural disulfide bonds, free thiol groups are present. Free thiols on the surface of plasmatic VWF have been shown to play a role in VWF self-association and in platelet binding under pathologically high levels of shear stress. The present study explores the role of VWF free thiol groups under physiological levels of shear stress and in interactions with collagen and platelet-GPIbα receptor.

Materials And Methods: Free and accessible thiol groups were blocked with N-ethylmaleimide (NEM) and the derivatized molecule was evaluated in functional assays. Reduced cysteine residues were identified using biotin-linked maleimide (MPB) followed by analysis of multimer and domain incorporation and by analysis of derivatized tryptic peptides by mass spectrometry.

Results: Blockade of free thiol groups significantly reduced VWF-mediated platelet recruitment to collagen under physiological flow conditions. This resulted from inhibition of VWF binding to both collagen and the platelet GPIb receptor. Evaluation of derivatization sites revealed a high level of derivatization in the cysteine-rich N- and C-termini of VWF. 19 MPB-derivatized peptides, 13 of which are described here for the first time, were identified by mass spectrometry.

Conclusions: This study shows a significant contribution of free thiol groups in VWF to the mediation of platelet adhesion under physiological shear stress conditions. The free thiol groups are shown to be involved in VWF binding to both collagen III and platelet GP1b receptor.
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http://dx.doi.org/10.1016/j.thromres.2015.10.037DOI Listing
January 2016