Publications by authors named "Andreas Tholey"

124 Publications

A 5,000-year-old hunter-gatherer already plagued by Yersinia pestis.

Cell Rep 2021 Jun;35(13):109278

Institute of Clinical Molecular Biology, Kiel University, Rosalind-Franklin-Str. 12, 24105 Kiel, Germany. Electronic address:

A 5,000-year-old Yersinia pestis genome (RV 2039) is reconstructed from a hunter-fisher-gatherer (5300-5050 cal BP) buried at Riņņukalns, Latvia. RV 2039 is the first in a series of ancient strains that evolved shortly after the split of Y. pestis from its antecessor Y. pseudotuberculosis ∼7,000 years ago. The genomic and phylogenetic characteristics of RV 2039 are consistent with the hypothesis that this very early Y. pestis form was most likely less transmissible and maybe even less virulent than later strains. Our data do not support the scenario of a prehistoric pneumonic plague pandemic, as suggested previously for the Neolithic decline. The geographical and temporal distribution of the few prehistoric Y. pestis cases reported so far is more in agreement with single zoonotic events.
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http://dx.doi.org/10.1016/j.celrep.2021.109278DOI Listing
June 2021

Bottom-up and top-down proteomic approaches for the identification, characterization, and quantification of the low molecular weight proteome with focus on short open reading frame-encoded peptides.

Proteomics 2021 Jun 19:e2100008. Epub 2021 Jun 19.

Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.

The recent discovery of alternative open reading frames creates a need for suitable analytical approaches to verify their translation and to characterize the corresponding gene products at the molecular level. As the analysis of small proteins within a background proteome by means of classical bottom-up proteomics is challenging, method development for the analysis of small open reading frame encoded peptides (SEPs) have become a focal point for research. Here, we highlight bottom-up and top-down proteomics approaches established for the analysis of SEPs in both pro- and eukaryotes. Major steps of analysis, including sample preparation and (small) proteome isolation, separation and mass spectrometry, data interpretation and quality control, quantification, the analysis of post-translational modifications, and exploration of functional aspects of the SEPs by means of proteomics technologies are described. These methods do not exclusively cover the analytics of SEPs but simultaneously include the low molecular weight proteome, and moreover, can also be used for the proteome-wide analysis of proteolytic processing events.
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http://dx.doi.org/10.1002/pmic.202100008DOI Listing
June 2021

Phosphorylation of meprin β controls its cell surface abundance and subsequently diminishes ectodomain shedding.

FASEB J 2021 07;35(7):e21677

Biochemical Institute, Unit for Degradomics of the Protease Web, University of Kiel, Kiel, Germany.

Meprin β is a zinc-dependent metalloprotease exhibiting a unique cleavage specificity with strong preference for acidic amino acids at the cleavage site. Proteomic studies revealed a diverse substrate pool of meprin β including the interleukin-6 receptor (IL-6R) and the amyloid precursor protein (APP). Dysregulation of meprin β is often associated with pathological conditions such as chronic inflammation, fibrosis, or Alzheimer's disease (AD). The extracellular regulation of meprin β including interactors, sheddases, and activators has been intensively investigated while intracellular regulation has been barely addressed in the literature. This study aimed to analyze C-terminal phosphorylation of meprin β with regard to cell surface expression and proteolytic activity. By immunoprecipitation of endogenous meprin β from the colon cancer cell line Colo320 and subsequent LC-MS analysis, we identified several phosphorylation sites in its C-terminal region. Here, T694 in the C-terminus of meprin β was the most preferred residue after phorbol 12-myristate 13-acetate (PMA) stimulation. We further demonstrated the role of protein kinase C (PKC) isoforms for meprin β phosphorylation and identified the involvement of PKC-α and PKC-β. As a result of phosphorylation, the meprin β activity at the cell surface is reduced and, consequently, the extent of substrate cleavage is diminished. Our data indicate that this decrease of the surface activity is caused by the internalization and degradation of meprin β.
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http://dx.doi.org/10.1096/fj.202100271RDOI Listing
July 2021

MALDI mass spectrometry imaging unravels organ and amyloid-type specific peptide signatures in pulmonary and gastrointestinal amyloidosis.

Proteomics Clin Appl 2021 Jun 1:e2000079. Epub 2021 Jun 1.

Department of Pathology, Christian-Albrechts-University, Kiel, Germany.

Purpose: Amyloidosis is a disease group caused by pathological aggregation and deposition of peptides in diverse tissue sites. Recently, matrix-assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI-IMS MSI) was introduced as a novel tool to identify and classify amyloidosis using single sections from formalin-fixed and paraffin-embedded cardiac biopsies. Here, we tested the hypothesis that MALDI-IMS MSI can be applied to lung and gastrointestinal specimens.

Experimental Design: Forty six lung and 65 gastrointestinal biopsy and resection specimens with different types of amyloid were subjected to MALDI-IMS MSI. Ninety three specimens included tissue areas without amyloid as internal negative controls. Nine cases without amyloid served as additional negative controls.

Results: Utilizing a peptide filter method and 21 known amyloid specific tryptic peptides we confirmed the applicability of a universal peptide signature with a sensitivity of 100% and a specificity of 100% for the detection of amyloid deposits in the lung and gastrointestinal tract. Additionally, the frequencies of individual m/z-values of the 21 tryptic marker peptides showed organ- and tissue-type specific differences.

Conclusions And Clinical Relevance: MALDI-IMS MSI adds a valuable analytical approach to diagnose and classify amyloid and the detection frequency of individual tryptic peptides is organ-/tissue-type specific.
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http://dx.doi.org/10.1002/prca.202000079DOI Listing
June 2021

Isobaric Peptide Labeling on Digital Microfluidics for Quantitative Low Cell Number Proteomics.

Anal Chem 2021 04 6;93(15):6278-6286. Epub 2021 Apr 6.

Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel 24105, Germany.

Digital microfluidics (DMF) is a technology suitable for bioanalytical applications requiring miniaturized, automated, and multiplexed liquid handling. Its use in LC-MS-based proteomics, however, has so far been limited to qualitative proteome analyses. This is mainly due to the need for detergents that enable facile, reproducible droplet movement, which are compatible with organic solvents commonly used in targeted chemical modifications of peptides. Aiming to implement isobaric peptide labeling, a widely applied technique allowing multiplexed quantitative proteome studies, on DMF devices, we tested different commercially available detergents. We identified the maltoside-based detergent 3-dodecyloxypropyl-1-β-d-maltopyranoside (DDOPM) to enable facile droplet movement and show micelle formation even in the presence of organic solvent, which is necessary for isobaric tandem mass tag (TMT) labeling. The detergent is fully compatible with reversed phase LC-MS, not interfering with peptide identification. Tryptic digestion in the presence of DDOPM was more efficient than without detergent, resulting in more protein identifications. Using this detergent, we report the first on-DMF chip isobaric labeling strategy, with TMT-labeling efficiency comparable to conventional protocols. The newly developed labeling protocol was evaluated in the multiplexed analyses of a protein standard digest spiked into 25 cells. Finally, using only 75 cells per biological replicate, we were able to identify 39 proteins being differentially abundant after treatment of Jurkat T cells with the anticancer drug doxorubicin. In summary, we demonstrate an important step toward multiplexed quantitative proteomics on DMF, which, in combination with larger chip arrays and optimized hardware, could enable high throughput low cell number proteomics.
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http://dx.doi.org/10.1021/acs.analchem.1c01205DOI Listing
April 2021

ADP-Ribosylation Regulates the Signaling Function of IFN-γ.

Front Immunol 2021 8;12:642545. Epub 2021 Mar 8.

Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Murine T cells express the GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) on the cell surface. In response to T cell activation or extracellular NAD or ATP-mediated gating of the P2X7 ion channel ARTC2.2 is shed from the cell surface as a soluble enzyme. Shedding alters the target specificity of ARTC2.2 from cell surface proteins to secreted proteins. Here we demonstrate that shed ARTC2.2 potently ADP-ribosylates IFN-γ in addition to other cytokines. Using mass spectrometry, we identify arginine 128 as the target site of ADP-ribosylation. This residue has been implicated to play a key role in binding of IFN-γ to the interferon receptor 1 (IFNR1). Indeed, binding of IFN-γ to IFNR1 blocks ADP-ribosylation of IFN-γ. Moreover, ADP-ribosylation of IFN-γ inhibits the capacity of IFN-γ to induce STAT1 phosphorylation in macrophages and upregulation of the proteasomal subunit ß5i and the proteasomal activator PA28-α in podocytes. Our results show that ADP-ribosylation inhibits the signaling functions of IFN-γ and point to a new regulatory mechanism for controlling signaling by IFN-γ.
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http://dx.doi.org/10.3389/fimmu.2021.642545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7983947PMC
March 2021

Multi-protease Approach for the Improved Identification and Molecular Characterization of Small Proteins and Short Open Reading Frame-Encoded Peptides.

J Proteome Res 2021 05 24;20(5):2895-2903. Epub 2021 Mar 24.

Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel 24105, Germany.

The identification of proteins below approximately 70-100 amino acids in bottom-up proteomics is still a challenging task due to the limited number of peptides generated by proteolytic digestion. This includes the short open reading frame-encoded peptides (SEPs), which are a subset of the small proteins that were not previously annotated or that are alternatively encoded. Here, we systematically investigated the use of multiple proteases (trypsin, chymotrypsin, LysC, LysargiNase, and GluC) in GeLC-MS/MS analysis to improve the sequence coverage and the number of identified peptides for small proteins, with a focus on SEPs, in the archaeon . Combining the data of all proteases, we identified 63 small proteins and additional 28 SEPs with at least two unique peptides, while only 55 small proteins and 22 SEP could be identified using trypsin only. For 27 small proteins and 12 SEPs, a complete sequence coverage was achieved. Moreover, for five SEPs, incorrectly predicted translation start points or potential proteolytic processing were identified, confirming the data of a previous top-down proteomics study of this organism. The results show clearly that a multi-protease approach allows to improve the identification and molecular characterization of small proteins and SEPs. LC-MS data: ProteomeXchange PXD023921.
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http://dx.doi.org/10.1021/acs.jproteome.1c00115DOI Listing
May 2021

High complexity of Glutamine synthetase regulation in Methanosarcina mazei: Small protein 26 interacts and enhances glutamine synthetase activity.

FEBS J 2021 Mar 4. Epub 2021 Mar 4.

Institute for General Microbiology, Christian-Albrechts-University, Kiel, Germany.

Small ORF (sORF)-encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding- and noncoding-predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein. spRNA26 level is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. Using heterologously expressed and purified sP26 in independent biochemical approaches [pull-down by affinity chromatography followed by MS analysis, reverse pull-down, microscale thermophoresis, size-exclusion chromatography, and nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA ) with high affinity (app. K  = 0.76 µm ± 0.29 µm). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA . Upon interaction with sP26, GlnA activity is significantly stimulated, independently and in addition to the known activation by the metabolite 2-oxoglutarate (2-OG). Besides, strong interaction of sP26 with the PII-like protein GlnK was demonstrated (app. K  = 2.9 µm ± 0.9 µm). On the basis of these findings, we propose that in addition to 2-OG, sP26 enhances GlnA activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA .
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http://dx.doi.org/10.1111/febs.15799DOI Listing
March 2021

N-Terminomics for the Identification of In Vitro Substrates and Cleavage Site Specificity of the SARS-CoV-2 Main Protease.

Proteomics 2021 01 17;21(2):e2000246. Epub 2020 Nov 17.

Systematic Proteome Research and Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, 24105, Germany.

The genome of coronaviruses, including SARS-CoV-2, encodes for two proteases, a papain like (PL ) protease and the so-called main protease (M ), a chymotrypsin-like cysteine protease, also named 3CL or non-structural protein 5 (nsp5). M is activated by autoproteolysis and is the main protease responsible for cutting the viral polyprotein into functional units. Aside from this, it is described that M proteases are also capable of processing host proteins, including those involved in the host innate immune response. To identify substrates of the three main proteases from SARS-CoV, SARS-CoV-2, and hCoV-NL63 coronviruses, an LC-MS based N-terminomics in vitro analysis is performed using recombinantly expressed proteases and lung epithelial and endothelial cell lysates as substrate pools. For SARS-CoV-2 M , 445 cleavage events from more than 300 proteins are identified, while 151 and 331 M derived cleavage events are identified for SARS-CoV and hCoV-NL63, respectively. These data enable to better understand the cleavage site specificity of the viral proteases and will help to identify novel substrates in vivo. All data are available via ProteomeXchange with identifier PXD021406.
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http://dx.doi.org/10.1002/pmic.202000246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645863PMC
January 2021

The E3 ubiquitin ligase HectD3 attenuates cardiac hypertrophy and inflammation in mice.

Commun Biol 2020 10 9;3(1):562. Epub 2020 Oct 9.

Department of Internal Medicine III (Cardiology, Angiology, Intensive Care), University Medical Center Kiel, Kiel, Germany.

Myocardial inflammation has recently been recognized as a distinct feature of cardiac hypertrophy and heart failure. HectD3, a HECT domain containing E3 ubiquitin ligase has previously been investigated in the host defense against infections as well as neuroinflammation; its cardiac function however is still unknown. Here we show that HectD3 simultaneously attenuates Calcineurin-NFAT driven cardiomyocyte hypertrophy and the pro-inflammatory actions of LPS/interferon-γ via its cardiac substrates SUMO2 and Stat1, respectively. AAV9-mediated overexpression of HectD3 in mice in vivo not only reduced cardiac SUMO2/Stat1 levels and pathological hypertrophy but also largely abolished macrophage infiltration and fibrosis induced by pressure overload. Taken together, we describe a novel cardioprotective mechanism involving the ubiquitin ligase HectD3, which links anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and Stat1. In a broader perspective, these findings support the notion that cardiomyocyte growth and inflammation are more intertwined than previously anticipated.
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http://dx.doi.org/10.1038/s42003-020-01289-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547098PMC
October 2020

Multidimensional separation schemes enhance the identification and molecular characterization of low molecular weight proteomes and short open reading frame-encoded peptides in top-down proteomics.

J Proteomics 2021 01 17;230:103988. Epub 2020 Sep 17.

Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105 Kiel, Germany. Electronic address:

Short open reading frame-encoded peptides (SEP) represent a widely undiscovered part of the proteome. The detailed analysis of SEP has, despite inherent limitations such as incomplete sequence coverage, challenges encountered with protein inference, the identification of posttranslational modifications and the assignment of potential N- and C-terminal truncations, predominantly been assessed using bottom-up proteomic workflows. The use of top-down based proteomic workflows is capable of providing an unparalleled level of characterization information, which is of increased importance in the case of alternatively encoded protein products. However, top-down based analysis is not without its own limitations, for which efficient separation prior to MS analysis is a major issue. We established a sample preparation approach for the combined bottom-up and top-down proteomic analysis of SEP. Key improvements were made by the application of solid phase extraction (SPE), which supported enrichment of proteins below ca. 20 kDa, followed by 2D-LC-MS top-down analysis encompassing both HCD and EThcD ion activation. Bottom-up experiments were used to support and confirm top-down data interpretation. This strategy allowed for the top-down characterization of 36 proteoforms mapping to 12 SEP from the archaeon Methanosarcina mazei strain Gö1, with the concurrent detection and identification of several posttranslational modifications in SEP. BIOLOGICAL SIGNIFICANCE: Small or short open reading frames (sORF) have been widely neglected in genome research in the past. With their increasing discovery, the question about the presence and molecular function of their translation products, the short open reading frame-encoded peptides (SEP), arises. As these small proteins are usually below the 10 kDa range, the number of peptides identifiable by bottom-up proteomics is limited which hampers both the identification and the recognition of potential posttranslational modifications. The presented top-down approach allowed for the detection of full length SEP, as well as of terminally truncated proteoforms, and further enabled the identification of disulfide bonds in these small proteins. This demonstrates, that this yet widely undiscovered part of the proteome undergoes the same modifications as classical proteins which is an essential step for future understanding of the biological functions of these molecules.
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http://dx.doi.org/10.1016/j.jprot.2020.103988DOI Listing
January 2021

Biological functions, genetic and biochemical characterization, and NMR structure determination of the small zinc finger protein HVO_2753 from Haloferax volcanii.

FEBS J 2021 03 24;288(6):2042-2062. Epub 2020 Sep 24.

Institute for Molecular Biosciences, Goethe-University, Frankfurt, Germany.

The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
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http://dx.doi.org/10.1111/febs.15559DOI Listing
March 2021

Complementarity of Different SDS-PAGE Gel Staining Methods for the Identification of Short Open Reading Frame-Encoded Peptides.

Proteomics 2020 10 19;20(19-20):e2000084. Epub 2020 Aug 19.

Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, 24105, Germany.

Short open reading frame-encoded peptides (SEP) have been identified across all domains of life and are predicted to be involved in many biochemical processes, however, for the vast majority of SEP their biological function is still unknown. Optimized methodologies have to be used for the mass spectrometric analysis of SEP, because traditional methods of bottom-up proteomics show a bias against small proteins. Here, different staining methods for SDS-PAGE gels prior in-gel digestion following LC-MS/MS analysis for the identification of SEP in the archaeon Methanosarcina mazei are investigated. In total, 45 SEP with at least one high confidence (FDR <1%) unique peptide and five consecutive b- or y-ions in the MS2 spectrum are identified. The staining methods provide complementary data. The highest number of SEP are identified in the samples stained with Coomassie brilliant blue. However, the highest quality of the identified SEP is achieved in the samples without staining. These comprehensive data sets demonstrate that in-gel digestion is well suited for the identification of SEP.
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http://dx.doi.org/10.1002/pmic.202000084DOI Listing
October 2020

Meprin β cleaves TREM2 and controls its phagocytic activity on macrophages.

FASEB J 2020 05 1;34(5):6675-6687. Epub 2020 Apr 1.

Unit for Degradomics of the Protease Web, Biochemical Institute, University of Kiel, Kiel, Germany.

The triggering receptor expressed on myeloid cells 2 (TREM2) is a multifunctional surface protein that affects survival, migration, and phagocytic capacity of myeloid cells. Soluble TREM2 levels were found to be increased in early stages of sporadic and familial Alzheimer's disease (AD) probably reflecting a defensive microglial response to some initial brain damage. The disintegrin and metalloproteases (ADAM) 10 and 17 were identified as TREM2 sheddases. We demonstrate that meprin β is a direct TREM2 cleaving enzyme using ADAM10/17 deficient HEK293 cells. LC-MS/MS analysis of recombinant TREM2 incubated with meprin β revealed predominant cleavage between Arg136 and Asp137, distant to the site identified for ADAM10/17. We further demonstrate that the metalloprotease meprin β cleaves TREM2 on macrophages concomitant with decreased levels of soluble TREM2 in the serum of Mep1b mice compared to WT controls. Isolated BMDMs from Mep1b mice showed significantly increased full-length TREM2 levels and enhanced phagocytosis efficiency compared to WT cells. The diminished constitutive shedding of TREM2 on meprin β deficient macrophages could be rescued by ADAM stimulation through LPS treatment. Our data provide evidence that meprin β is a TREM2 sheddase on macrophages and suggest that multiple proteases may be involved in the generation of soluble TREM2.
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http://dx.doi.org/10.1096/fj.201902183RDOI Listing
May 2020

Distance dependent shedding of IL-6R.

Biochem Biophys Res Commun 2020 05 26;526(2):355-360. Epub 2020 Mar 26.

Department of Structural Biology, Institute of Zoology, Am Botanischen Garten 1-9, 24118, Kiel, Germany.

Proteolytic processing of membrane proteins by A disintegrin and metalloprotease-17 (ADAM17) is a key regulatory step in many physiological and pathophysiological processes. This so-called shedding is essential for development, regeneration and immune defense. An uncontrolled ADAM17 activity promotes cancer development, chronic inflammation and autoimmune diseases. Consequently, the ADAM17 activity is tightly regulated. As a final trigger for the shedding event a phosphatidylserine (PS) flip to the outer leaflet of the cell membrane was recently described. PS interacts with the extracellular part of ADAM17, which results in the shedding event by shifting the catalytic domain towards the membrane close to the cleavage sites within ADAM17 substrates. Our data indicate that the intrinsic proteolytic activity of the catalytic domain is prerequisite for the shedding activity and constantly present. However, the accessibility for substrate cleavage sites is controlled on several levels. In this report, we demonstrate that the positioning of the catalytic domain towards the cleavage sites is a crucial part of the shedding process. This finding contributes to the understanding of the complex and multilayered regulation of ADAM17 at the cell surface.
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http://dx.doi.org/10.1016/j.bbrc.2020.03.093DOI Listing
May 2020

Exopeptidase Assisted N- and C-Terminal Proteome Sequencing.

Anal Chem 2020 04 25;92(7):5023-5032. Epub 2020 Mar 25.

Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.

Due to mechanisms such as proteolytic processing or alternative translation starts, in vivo proteoforms do not necessarily correspond directly to those encoded in the genome. Therefore, the knowledge of protein termini is an indispensable prerequisite to understand protein functions. So far, sequencing of protein N- and C-termini has been limited to single purified protein species, while the proteome-wide identification of N- and C-termini relies on the generation of single, terminal proteotypic peptides followed by chemical enrichment or depletion strategies to facilitate their detection via mass spectrometry (MS). To overcome the numerous limitations in such approaches, we present an alternative concept that readily enables unbiased ladder sequencing of protein N- and C-termini. The approach combines exopeptidase digestions of the proteome with two-dimensional chromatographic separation and tandem-MS. We demonstrate the potential of the methodology by analyzing the N- and C-terminome of , identifying 2190 N-termini and 1562 C-termini. In conclusion, the presented method largely expands the proteomics toolbox enabling N- and C-terminal sequential characterization of entire proteomes.
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http://dx.doi.org/10.1021/acs.analchem.9b05288DOI Listing
April 2020

Evaluation and improvement of protein extraction methods for analysis of skin proteome by noninvasive tape stripping.

J Proteomics 2020 04 6;217:103678. Epub 2020 Feb 6.

Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, Germany. Electronic address:

Analysis of the human skin proteome is key to understand molecular mechanisms maintaining health or leading to diseases of this important organ. For minimal invasive sampling of skin proteomes, the use of self-adhesive tape strips has been successfully applied. However, the methods previously presented were evaluated on different types of skin samples (e.g. healthy, diseased) and used a variety of cell lysis/protein extraction methods, which renders a systematic comparison and thus the identification of the most efficient protocols difficult. Here, we present a study comparing five different approaches for cell lysis and protein extraction from single tape strip biopsies. Extraction using a detergent mix or 1% SDS proved to be most efficient. Further, we replaced protein precipitation by single-pot, solid-phase-enhanced sample preparation (SP3), which strongly enhanced the number of identified proteins. This fully LC-MS compatible methodology provides a fast and reproducible approach for minimal invasive sampling of human skin proteomes. BIOLOGICAL SIGNIFICANCE: Fast and reproducible minimal invasive sampling of human skin proteomes is a major prerequisite for clinical proteomics studies aiming to decipher molecular mechanisms involved in the homeostasis as well as in the development of diseases. By optimization of tape strip sampling, e.g. the introduction of SP3 sample cleanup prior to LC-MS analysis, the presented protocol leads to yet not reported numbers of protein identifications from healthy human skin. Further, due to its efficiency it allows analysis from minimal sample amounts, e.g. from single tape strips, while established protocols relied on pooling of multiple tape strips. This provides the opportunity to perform spatially (lateral) resolved proteome analyses from different depths of the skin by analysis of consecutive strips.
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http://dx.doi.org/10.1016/j.jprot.2020.103678DOI Listing
April 2020

Prdx4 limits caspase-1 activation and restricts inflammasome-mediated signaling by extracellular vesicles.

EMBO J 2019 10 23;38(20):e101266. Epub 2019 Sep 23.

Institute of Clinical Molecular Biology, Christian-Albrechts-University and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.

Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL-1β by proteolytic cleavage via caspase-1. Although many principles of inflammasome activation have been described, mechanisms that limit inflammasome-dependent immune responses remain poorly defined. Here, we show that the thiol-specific peroxidase peroxiredoxin-4 (Prdx4) directly regulates IL-1β generation by interfering with caspase-1 activity. We demonstrate that caspase-1 and Prdx4 form a redox-sensitive regulatory complex via caspase-1 cysteine 397 that leads to caspase-1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS-induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4-ΔLysMCre). Strikingly, we demonstrate that Prdx4 co-localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome-activated macrophages. Purified EVs are able to transmit a robust IL-1β-dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro-inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell-to-cell communication function of inflammasomes via macrophage-derived EVs.
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http://dx.doi.org/10.15252/embj.2018101266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6792017PMC
October 2019

Miniaturized sample preparation on a digital microfluidics device for sensitive bottom-up microproteomics of mammalian cells using magnetic beads and mass spectrometry-compatible surfactants.

Lab Chip 2019 10;19(20):3490-3498

Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105 Kiel, Germany.

While LC-MS-based proteomics with high nanograms to micrograms of total protein has become routine, the analysis of samples derived from low cell numbers is challenged by factors such as sample losses, or difficulties encountered with the manual manipulation of small liquid volumes. Digital microfluidics (DMF) is an emerging technique for miniaturized and automated droplet manipulation, which has been proposed as a promising tool for proteomic sample preparation. However, proteome analysis of samples prepared on-chip by DMF has previously been unfeasible, due to incompatibility with down-stream LC-MS instrumentation. To overcome these limitations, we here developed protocols for bottom-up LC-MS based proteomics sample preparation of as little as 100 mammalian cells on a commercially available digital microfluidics device. To this end, we developed effective cell lysis conditions optimized for DMF, as well as detergent-buffer systems compatible with downstream proteolytic digestion on DMF chips and subsequent LC-MS analysis. A major step was the introduction of the single-pot, solid-phase-enhanced sample preparation (SP3) approach on-chip, which allowed the removal of salts and anti-fouling polymeric detergents, thus rendering sample preparation by DMF compatible with LC-MS-based proteome analysis. Application of DMF-SP3 to the proteome analysis of Jurkat T cells led to the identification of up to 2500 proteins from approximately 500 cells, and up to 1200 proteins from approximately 100 cells on an Orbitrap mass spectrometer, emphasizing the high compatibility of DMF-SP3 with low protein input and minute volumes handled by DMF. Taken together, we demonstrate the first sample preparation workflow for proteomics on a DMF chip device reported so far, allowing the sensitive analysis of limited biological material.
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http://dx.doi.org/10.1039/c9lc00715fDOI Listing
October 2019

Proteomic investigation of the blue mussel larval shell organic matrix.

J Struct Biol 2019 12 7;208(3):107385. Epub 2019 Sep 7.

GEOMAR Helmholtz Centre for Ocean Research, 24105 Kiel, Germany; Sven Lovén Centre for Marine Infrastructure, Gothenburg University, 45178 Fiskebäckskil, Sweden.

Shell matrix proteins (SMPs) are occluded within molluscan shells and are fundamental to the biological control over mineralization. While many studies have been performed on adult SMPs, those of larval stages remain largely undescribed. Therefore, this study aimed to characterize the larval shell proteome of the blue mussel for the first time and to compare it to adult mussel shell proteomes. Following development of a method for cleaning larval shells of tissue contaminants, 49 SMPs were identified using shotgun proteomics. Twenty-one proteins were independently identified in all samples indicating that they form a subset of the core larval shell proteome. These included: the blue mussel shell protein, a peroxidase domain-containing sequence, a laminin G domain-containing sequence, a ZIP domain-containing sequence and a ferric-chelate reductase 1-like sequence. Additional SMP domains identified were: fibronectin type III, BPTI/Kunitz, chitin-binding type 3, thyroglobulin and EF-hand. While key predictable molluscan shell matrix functions are identified, 67% of sequences remain unknown or uncharacterized, indicating that this shell proteome is unique to mussel larvae. Specifically, comparison with adult mytilids reveals that nine domains are exclusive to the larval shell proteome and only four domains are conserved among species and developmental stages. Thus, strong species-specific and ontogenetic variation exists in shell proteome composition.
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http://dx.doi.org/10.1016/j.jsb.2019.09.002DOI Listing
December 2019

The Inducible Response of the Nematode to Members of Its Natural Microbiota Across Development and Adult Life.

Front Microbiol 2019 7;10:1793. Epub 2019 Aug 7.

Research Group Evolutionary Ecology and Genetics, Zoological Institute, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.

The biology of all organisms is influenced by the associated community of microorganisms. In spite of its importance, it is usually not well understood how exactly this microbiota affects host functions and what are the underlying molecular processes. To rectify this knowledge gap, we took advantage of the nematode as a tractable, experimental model system and assessed the inducible transcriptome response after colonization with members of its native microbiota. For this study, we focused on two isolates of the genus . These bacteria are known to be abundant in the nematode's microbiota and are capable of colonizing and persisting in the nematode gut, even under stressful conditions. The transcriptome response was assessed across development and three time points of adult life, using general and -specific enrichment analyses to identify affected functions. Our assessment revealed an influence of the microbiota members on the nematode's dietary response, development, fertility, immunity, and energy metabolism. This response is mainly regulated by a GATA transcription factor, most likely ELT-2, as indicated by the enrichment of (i) the GATA motif in the promoter regions of inducible genes and (ii) of ELT-2 targets among the differentially expressed genes. We compared our transcriptome results with a corresponding previously characterized proteome data set, highlighting a significant overlap in the differentially expressed genes, the affected functions, and ELT-2 target genes. Our analysis further identified a core set of 86 genes that consistently responded to the microbiota members across development and adult life, including several C-type lectin-like genes and genes known to be involved in energy metabolism or fertility. We additionally assessed the consequences of induced gene expression with the help of metabolic network model analysis, using a previously established metabolic network for . This analysis complemented the enrichment analyses by revealing an influence of the isolates on energy metabolism and furthermore metabolism of specific amino acids, fatty acids, and also folate biosynthesis. Our findings highlight the multifaceted impact of naturally colonizing microbiota isolates on life history and thereby provide a framework for further analysis of microbiota-mediated host functions.
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http://dx.doi.org/10.3389/fmicb.2019.01793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693516PMC
August 2019

Meprin β induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.

FASEB J 2019 11 9;33(11):11925-11940. Epub 2019 Aug 9.

Institute of Biochemistry, University of Kiel, Kiel, Germany.

Meprin β is a membrane-bound metalloprotease involved in extracellular matrix assembly and inflammatory processes in health and disease. A disintegrin and metalloproteinase (ADAM)10 and ADAM17 are physiologic relevant sheddases of inactive promeprin β, which influences its substrate repertoire and subsequent biologic functions. Proteomic analysis also revealed several ADAMs as putative meprin β substrates. Here, we demonstrate specific N-terminal processing of ADAM9, 10, and 17 by meprin β and identify cleavage sites within their prodomains. Because ADAM prodomains can act as specific inhibitors, we postulate a role for meprin β in the regulation of ADAM activities. Indeed, prodomain cleavage by meprin β caused increased ADAM protease activities, as observed by peptide-based cleavage assays and demonstrated by increased ectodomain shedding activity. Direct interaction of meprin β and ADAM proteases could be shown by immunofluorescence microscopy and immunoprecipitation experiments. As demonstrated by a bacterial activator of meprin β and additional measurement of TNF-α shedding on bone marrow-derived macrophages, meprin β/ADAM protease interactions likely influence inflammatory conditions. Thus, we identified a novel proteolytic pathway of meprin β with ADAM proteases to control protease activities at the cell surface as part of the protease web.-Wichert, R., Scharfenberg, F., Colmorgen, C., Koudelka, T., Schwarz, J., Wetzel, S., Potempa, B., Potempa, J., Bartsch, J. W., Sagi, I., Tholey, A., Saftig, P., Rose-John, S., Becker-Pauly, C. Meprin β induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.
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http://dx.doi.org/10.1096/fj.201801371RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902663PMC
November 2019

Degradome of soluble ADAM10 and ADAM17 metalloproteases.

Cell Mol Life Sci 2020 Jan 17;77(2):331-350. Epub 2019 Jun 17.

Unit for Degradomics of the Protease Web, Biochemical Institute, University of Kiel, Kiel, Germany.

Disintegrin and metalloproteinases (ADAMs) 10 and 17 can release the extracellular part of a variety of membrane-bound proteins via ectodomain shedding important for many biological functions. So far, substrate identification focused exclusively on membrane-anchored ADAM10 and ADAM17. However, besides known shedding of ADAM10, we identified ADAM8 as a protease capable of releasing the ADAM17 ectodomain. Therefore, we investigated whether the soluble ectodomains of ADAM10/17 (sADAM10/17) exhibit an altered substrate spectrum compared to their membrane-bound counterparts. A mass spectrometry-based N-terminomics approach identified 134 protein cleavage events in total and 45 common substrates for sADAM10/17 within the secretome of murine cardiomyocytes. Analysis of these cleavage sites confirmed previously identified amino acid preferences. Further in vitro studies verified fibronectin, cystatin C, sN-cadherin, PCPE-1 as well as sAPP as direct substrates of sADAM10 and/or sADAM17. Overall, we present the first degradome study for sADAM10/17, thereby introducing a new mode of proteolytic activity within the protease web.
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http://dx.doi.org/10.1007/s00018-019-03184-4DOI Listing
January 2020

Combination of SCX Fractionation and Charge-Reversal Derivatization Facilitates the Identification of Nontryptic Peptides in C-Terminomics.

J Proteome Res 2019 07 25;18(7):2954-2964. Epub 2019 Jun 25.

Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine , Christian-Albrechts-Universität zu Kiel , 24105 Kiel , Germany.

The proteome wide, mass spectrometry based identification of protein C-termini is hampered by factors such as poor ionization efficiencies, low yielding labeling strategies, or the need for enrichment procedures. We present a bottom-up proteomics workflow to identify protein C-termini utilizing a combination of strong cation exchange chromatography, on-solid phase charge-reversal derivatization and LC-MS/MS analysis. Charge-reversal improved both MS and MS/MS spectra quality of peptides carrying nonbasic C-terminal residues, allowing the identification of a high number of noncanonical C-termini not identified in nonderivatized samples. Further, we could show that C-terminal O labeling introduced during proteolytic processing of the samples is not suitable to distinguish internal from C-terminal peptides. The presented workflow enables the simultaneous identification of proteins by internal peptides and additionally provides data for the C- and N-terminome. Applying the developed workflow for the analysis of a Saccharomyces cerevisiae proteome allowed the identification of 734 protein C-termini in three independent biological replicates, and additional 789 candidate C-termini identified in two or one of three biological replicates, respectively. The developed analytical workflow allowed us to chart the nature of the yeast C-terminome in unprecedented depth and provides an alternative methodology to assess C-terminal proteolytic protein processing.
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http://dx.doi.org/10.1021/acs.jproteome.9b00264DOI Listing
July 2019

Cationic Intrinsically Disordered Antimicrobial Peptides (CIDAMPs) Represent a New Paradigm of Innate Defense with a Potential for Novel Anti-Infectives.

Sci Rep 2019 03 4;9(1):3331. Epub 2019 Mar 4.

Department of Dermatology, University-Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.

In the search for potential mechanisms underlying the remarkable resistance of healthy skin against infection by soil bacteria like Pseudomonas (P.) aeruginosa we identified fragments of the intrinsically disordered protein hornerin as potent microbicidal agents in the stratum corneum. We found that, independent of the amino acid (AA)-sequence, any tested linear cationic peptide containing a high percentage of disorder-promoting AA and a low percentage of order-promoting AA is a potent microbicidal antimicrobial. We further show that the antimicrobial activity of these cationic intrinsically disordered antimicrobial peptides (CIDAMPs) depends on the peptide chain length, its net charge, lipidation and environmental conditions. The ubiquitous presence of latent CIDAMP sources in nature suggests a common and yet overlooked adapted innate disinfection system of body surfaces. The simple structure and virtually any imaginable sequence or composition of disorder-promoting AA allow the generation of a plethora of CIDAMPs. These are potential novel microbicidal anti-infectives for various bacterial pathogens, including P. aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA) and fungal pathogens like Candida albicans and Cryptococcus neoformans.
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http://dx.doi.org/10.1038/s41598-019-39219-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399351PMC
March 2019

Atherogenic LOX-1 signaling is controlled by SPPL2-mediated intramembrane proteolysis.

J Exp Med 2019 04 28;216(4):807-830. Epub 2019 Feb 28.

Institute of Physiological Chemistry, Technische Universität Dresden, Dresden, Germany

The lectin-like oxidized LDL receptor 1 (LOX-1) is a key player in the development of atherosclerosis. LOX-1 promotes endothelial activation and dysfunction by mediating uptake of oxidized LDL and inducing pro-atherogenic signaling. However, little is known about modulators of LOX-1-mediated responses. Here, we show that the function of LOX-1 is controlled proteolytically. Ectodomain shedding by the metalloprotease ADAM10 and lysosomal degradation generate membrane-bound N-terminal fragments (NTFs), which we identified as novel substrates of the intramembrane proteases signal peptide peptidase-like 2a and b (SPPL2a/b). SPPL2a/b control cellular LOX-1 NTF levels which, following self-association via their transmembrane domain, can activate MAP kinases in a ligand-independent manner. This leads to an up-regulation of several pro-atherogenic and pro-fibrotic targets including ICAM-1 and the connective tissue growth factor CTGF. Consequently, SPPL2a/b-deficient mice, which accumulate LOX-1 NTFs, develop larger and more advanced atherosclerotic plaques than controls. This identifies intramembrane proteolysis by SPPL2a/b as a novel atheroprotective mechanism via negative regulation of LOX-1 signaling.
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http://dx.doi.org/10.1084/jem.20171438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446863PMC
April 2019

Depletion of High-Molecular-Mass Proteins for the Identification of Small Proteins and Short Open Reading Frame Encoded Peptides in Cellular Proteomes.

J Proteome Res 2019 04 27;18(4):1725-1734. Epub 2019 Feb 27.

Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine , Christian-Albrechts-Universität zu Kiel , 24105 Kiel , Germany.

The identification of small proteins and peptides (below ca. 100-150 amino acids) in complex biological samples is hampered by the dominance of higher-molecular-weight proteins. On the contrary, the increasing knowledge about alternative or short open reading frames creates a need for methods that allow the existence of the corresponding gene products to be proven in proteomics experiments. We present an acetonitrile-based precipitation methodology that depletes the majority of proteins above ca. 15 kDa. Parameters such as depletion mixture composition, pH, and temperature were optimized using a model protein mixture, and the method was evaluated in comparison with the established differential solubility method. The approach was applied to the analysis of the low-molecular-weight proteome of the archaea Methanosarcina mazei by means of LC-MS. The data clearly show a beneficial effect from a reduction of complexity, especially in terms of the quality of MS/MS-based identification of small proteins. This fast, detergent-free method allowed for, with minimal sample manipulation, the successful identification of several not yet identified short open reading frame encoded peptides in M. mazei.
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http://dx.doi.org/10.1021/acs.jproteome.8b00948DOI Listing
April 2019

Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in .

Metab Eng Commun 2019 Jun 18;8:e00086. Epub 2019 Jan 18.

LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France.

Spatial clustering of enzymes has proven an elegant approach to optimize metabolite transfer between enzymes in synthetic metabolic pathways. Among the multiple methods used to promote colocalisation, enzyme fusion is probably the simplest. Inspired by natural systems, we have explored the metabolic consequences of spatial reorganizations of the catalytic domains of carotenoid enzymes produced in . Synthetic genes encoding bidomain enzymes composed of CrtI and CrtB domains from the natural CrtYB fusion were connected in the two possible orientations, using natural and synthetic linkers. A tridomain enzyme (CrtB, CrtI, CrtY) harboring the full β-carotene producing pathway was also constructed. Our results demonstrate that domain order and linker properties considerably impact both the expression and/or stability of the constructed proteins and the functionality of the catalytic domains, all concurring to either diminish or boost specific enzymatic steps of the metabolic pathway. Remarkably, the yield of β-carotene production doubled with the tridomain fusion while precursor accumulation decreased, leading to an improvement of the pathway efficiency, when compared to the natural system. Our data strengthen the idea that fusion of enzymatic domains is an appropriate technique not only to achieve spatial confinement and enhance the metabolic flux but also to produce molecules not easily attainable with natural enzymatic configurations, even with membrane bound enzymes.
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http://dx.doi.org/10.1016/j.mec.2019.e00086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350077PMC
June 2019

Hornerin contains a Linked Series of Ribosome-Targeting Peptide Antibiotics.

Sci Rep 2018 11 1;8(1):16158. Epub 2018 Nov 1.

Department of Dermatology, University-Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.

Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) belong to a novel class of epithelial peptide antibiotics with microbicidal activity against various pathogens, including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Here we show that treatment of distinct bacteria with different hornerin (HRNR)-derived CIDAMPs cause formation of unique cytoplasmic protein aggregates, suggesting a common intracellular mode of action. We further found that, unlike most amphipathic antimicrobial peptides, HRNR traverses bacterial membranes energy-dependently and accumulates within the cytoplasm. Strikingly, certain structurally different, HRNR-based CIDAMPs were found to bind to an identical panel of distinct bacterial ribosomal proteins, thereby manifesting features of several known classes of antibiotics. This may cause the formation of aberrant proteins and toxic protein aggregates in HRNR-treated pathogens which eventually may induce its death. Our study reveals evidence that structurally distinct CIDAMPs of an abundant body surface protein simultaneously target multiple sites of the bacterial protein synthesis machinery.
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http://dx.doi.org/10.1038/s41598-018-34467-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212518PMC
November 2018

Diazirine-functionalized mannosides for photoaffinity labeling: trouble with FimH.

Beilstein J Org Chem 2018 24;14:1890-1900. Epub 2018 Jul 24.

Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel, Otto-Hahn-Platz 3/4, 24118 Kiel, Germany.

Photoaffinity labeling is frequently employed for the investigation of ligand-receptor interactions in solution. We have employed an interdisciplinary methodology to achieve facile photolabeling of the lectin FimH, which is a bacterial protein, crucial for adhesion, colonization and infection. Following our earlier work, we have here designed and synthesized diazirine-functionalized mannosides as high-affinity FimH ligands and performed an extensive study on photo-crosslinking of the best ligand (mannoside ) with a series of model peptides and FimH. Notably, we have employed high-performance mass spectrometry to be able to detect radiation results with the highest possible accuracy. We are concluding from this study that photolabeling of FimH with sugar diazirines has only very limited success and cannot be regarded a facile approach for covalent modification of FimH.
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http://dx.doi.org/10.3762/bjoc.14.163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071696PMC
July 2018