Publications by authors named "Markus Kaiser"

147 Publications

Targeted substrate loop insertion by VCP/p97 during PP1 complex disassembly.

Nat Struct Mol Biol 2021 Nov 25. Epub 2021 Nov 25.

Centre for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.

The AAA-ATPase VCP/p97/Cdc48 unfolds proteins by threading them through its central pore, but how substrates are recognized and inserted into the pore in diverse pathways has remained controversial. Here, we show that p97, with its adapter p37, binds an internal recognition site (IRS) within inhibitor-3 (I3) and then threads a peptide loop into its channel to strip I3 off protein phosphatase-1 (PP1). Of note, the IRS is adjacent to the prime interaction site of I3 to PP1, and IRS mutations block I3 processing both in vitro and in cells. In contrast, amino- and carboxy-terminal regions of I3 are not required, and even circularization of I3 does not prevent I3 processing. This was confirmed by an in vitro Förster resonance energy transfer assay that allowed kinetic analysis of the reaction. Thus, our data uncover how PP1 is released from its inhibitory partner for activation and demonstrate a remarkable plasticity in substrate threading by p97.
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http://dx.doi.org/10.1038/s41594-021-00684-5DOI Listing
November 2021

Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis.

Cell Rep 2021 Nov;37(8):110056

Department of Medical Oncology, West German Cancer Center, University Hospital Essen at the University Duisburg-Essen, Duisburg, Germany; German Cancer Consortium (DKTK) partner site Essen, Essen, Germany. Electronic address:

Statins are among the most commonly prescribed drugs, and around every fourth person above the age of 40 is on statin medication. Therefore, it is of utmost clinical importance to understand the effect of statins on cancer cell plasticity and its consequences to not only patients with cancer but also patients who are on statins. Here, we find that statins induce a partial epithelial-to-mesenchymal transition (EMT) phenotype in cancer cells of solid tumors. Using a comprehensive STRING network analysis of transcriptome, proteome, and phosphoproteome data combined with multiple mechanistic in vitro and functional in vivo analyses, we demonstrate that statins reduce cellular plasticity by enforcing a mesenchymal-like cell state that increases metastatic seeding ability on one side but reduces the formation of (secondary) tumors on the other due to heterogeneous treatment responses. Taken together, we provide a thorough mechanistic overview of the consequences of statin use for each step of cancer development, progression, and metastasis.
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http://dx.doi.org/10.1016/j.celrep.2021.110056DOI Listing
November 2021

Leaf Apoplast of Field-Grown Potato Analyzed by Quantitative Proteomics and Activity-Based Protein Profiling.

Int J Mol Sci 2021 Nov 6;22(21). Epub 2021 Nov 6.

Department of Plant Protection Biology, Swedish University of Agricultural Sciences, SE-234 22 Lomma, Sweden.

Multiple biotic and abiotic stresses challenge plants growing in agricultural fields. Most molecular studies have aimed to understand plant responses to challenges under controlled conditions. However, studies on field-grown plants are scarce, limiting application of the findings in agricultural conditions. In this study, we investigated the composition of apoplastic proteomes of potato cultivar Bintje grown under field conditions, i.e., two field sites in June-August across two years and fungicide treated and untreated, using quantitative proteomics, as well as its activity using activity-based protein profiling (ABPP). Samples were clustered and some proteins showed significant intensity and activity differences, based on their field site and sampling time (June-August), indicating differential regulation of certain proteins in response to environmental or developmental factors. Peroxidases, class II chitinases, pectinesterases, and osmotins were among the proteins more abundant later in the growing season (July-August) as compared to early in the season (June). We did not detect significant differences between fungicide Shirlan treated and untreated field samples in two growing seasons. Using ABPP, we showed differential activity of serine hydrolases and β-glycosidases under greenhouse and field conditions and across a growing season. Furthermore, the activity of serine hydrolases and β-glycosidases, including proteins related to biotic stress tolerance, decreased as the season progressed. The generated proteomics data would facilitate further studies aiming at understanding mechanisms of molecular plant physiology in agricultural fields and help applying effective strategies to mitigate biotic and abiotic stresses.
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http://dx.doi.org/10.3390/ijms222112033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8584485PMC
November 2021

The chemical compound 'Heatin' stimulates hypocotyl elongation and interferes with the Arabidopsis NIT1-subfamily of nitrilases.

Plant J 2021 06 6;106(6):1523-1540. Epub 2021 May 6.

Molecular Plant Physiology, Institute of Environmental Biology, Utrecht University, Padualaan 8, Utrecht, 3584 CH, the Netherlands.

Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.
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http://dx.doi.org/10.1111/tpj.15250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8360157PMC
June 2021

Protein Phosphatase-1 Complex Disassembly by p97 is Initiated through Multivalent Recognition of Catalytic and Regulatory Subunits by the p97 SEP-domain Adapters.

J Mol Biol 2020 11 12;432(23):6061-6074. Epub 2020 Oct 12.

Centre for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany. Electronic address:

The AAA-ATPase VCP/p97 cooperates with the SEP-domain adapters p37, UBXN2A and p47 in stripping inhibitor-3 (I3) from protein phosphatase-1 (PP1) for activation. In contrast to p97-mediated degradative processes, PP1 complex disassembly is ubiquitin-independent. It is therefore unclear how selective targeting is achieved. Using biochemical reconstitution and crosslink mass spectrometry, we show here that SEP-domain adapters use a multivalent substrate recognition strategy. An N-terminal sequence element predicted to form a helix, together with the SEP-domain, binds and engages the direct target I3 in the central pore of p97 for unfolding, while its partner PP1 is held by a linker between SHP box and UBX domain locked onto the peripheral N-domain of p97. Although the I3-binding element is functional in p47, p47 in vitro requires a transplant of the PP1-binding linker from p37 for activity stressing that both sites are essential to control specificity. Of note, unfolding is then governed by an inhibitory segment in the N-terminal region of p47, suggesting a regulatory function. Together, this study reveals how p97 adapters engage a protein complex for ubiquitin-independent disassembly while ensuring selectivity for one subunit.
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http://dx.doi.org/10.1016/j.jmb.2020.10.001DOI Listing
November 2020

Structure-based evolution of a promiscuous inhibitor to a selective stabilizer of protein-protein interactions.

Nat Commun 2020 08 7;11(1):3954. Epub 2020 Aug 7.

Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands.

The systematic stabilization of protein-protein interactions (PPI) has great potential as innovative drug discovery strategy to target novel and hard-to-drug protein classes. The current lack of chemical starting points and focused screening opportunities limits the identification of small molecule stabilizers that engage two proteins simultaneously. Starting from our previously described virtual screening strategy to identify inhibitors of 14-3-3 proteins, we report a conceptual molecular docking approach providing concrete entries for discovery and rational optimization of stabilizers for the interaction of 14-3-3 with the carbohydrate-response element-binding protein (ChREBP). X-ray crystallography reveals a distinct difference in the binding modes between weak and general inhibitors of 14-3-3 complexes and a specific, potent stabilizer of the 14-3-3/ChREBP complex. Structure-guided stabilizer optimization results in selective, up to 26-fold enhancement of the 14-3-3/ChREBP interaction. This study demonstrates the potential of rational design approaches for the development of selective PPI stabilizers starting from weak, promiscuous PPI inhibitors.
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http://dx.doi.org/10.1038/s41467-020-17741-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414219PMC
August 2020

Natural brominated phenoxyphenols kill persistent and biofilm-incorporated cells of MRSA and other pathogenic bacteria.

Appl Microbiol Biotechnol 2020 Jul 16;104(13):5985-5998. Epub 2020 May 16.

Institute of Pharmaceutical Biology and Biotechnology, Heinrich Heine University Düsseldorf, Dusseldorf, Germany.

Due to a high unresponsiveness to chemotherapy, biofilm formation is an important medical problem that frequently occurs during infection with many bacterial pathogens. In this study, the marine sponge-derived natural compounds 4,6-dibromo-2-(2',4'-dibromophenoxy)phenol and 3,4,6-tribromo-2-(2',4'-dibromophenoxy)phenol were found to exhibit broad antibacterial activity against medically relevant gram-positive and gram-negative pathogens. The compounds were not only bactericidal against both replicating and stationary phase-persistent planktonic cells of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa; they also killed biofilm-incorporated cells of both species while not affecting biofilm structural integrity. Moreover, these compounds were active against carbapenemase-producing Enterobacter sp. This simultaneous activity of compounds against different growth forms of both gram-positive and gram-negative bacteria is rare. Genome sequencing of spontaneous resistant mutants and proteome analysis suggest that resistance is mediated by downregulation of the bacterial EIIBC phosphotransferase components scrA and mtlA in MRSA likely leading to a lower uptake of the molecules. Due to their only moderate cytotoxicity against human cell lines, phenoxyphenols provide an interesting new scaffold for development of antimicrobial agents with activity against planktonic cells, persisters and biofilm-incoporated cells of ESKAPE pathogens. KEY POINTS: • Brominated phenoxyphenols kill actively replicating and biofilm-incorporated bacteria. • Phosphotransferase systems mediate uptake of brominated phenoxyphenols. • Downregulation of phosphotransferase systems mediate resistance.
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http://dx.doi.org/10.1007/s00253-020-10654-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217011PMC
July 2020

Discovering the RNA-Binding Proteome of Plant Leaves with an Improved RNA Interactome Capture Method.

Biomolecules 2020 04 24;10(4). Epub 2020 Apr 24.

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK.

RNA-binding proteins (RBPs) play a crucial role in regulating RNA function and fate. However, the full complement of RBPs has only recently begun to be uncovered through proteome-wide approaches such as RNA interactome capture (RIC). RIC has been applied to various cell lines and organisms, including plants, greatly expanding the repertoire of RBPs. However, several technical challenges have limited the efficacy of RIC when applied to plant tissues. Here, we report an improved version of RIC that overcomes the difficulties imposed by leaf tissue. Using this improved RIC method in Arabidopsis leaves, we identified 717 RBPs, generating a deep RNA-binding proteome for leaf tissues. While 75% of these RBPs can be linked to RNA biology, the remaining 25% were previously not known to interact with RNA. Interestingly, we observed that a large number of proteins related to photosynthesis associate with RNA in vivo, including proteins from the four major photosynthetic supercomplexes. As has previously been reported for mammals, a large proportion of leaf RBPs lack known RNA-binding domains, suggesting unconventional modes of RNA binding. We anticipate that this improved RIC method will provide critical insights into RNA metabolism in plants, including how cellular RBPs respond to environmental, physiological and pathological cues.
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http://dx.doi.org/10.3390/biom10040661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226388PMC
April 2020

Zelkovamycin is an OXPHOS Inhibitory Member of the Argyrin Natural Product Family.

Chemistry 2020 Jul 17;26(39):8524-8531. Epub 2020 Jun 17.

Chemische Biologie, Universität Duisburg-Essen, ZMB, Fakultät für Biologie, Universitätsstr. 2, 45117, Essen, Germany.

Natural products (NPs) are an important inspirational source for developing drugs and chemical probes. In 1999, the group of Ōmura reported the constitutional elucidation of zelkovamycin. Although largely unrecognized so far, this NP displays structural similarities as well as differences to the argyrin NP family, a class of peptidic NPs with promising anticancer activities and diverse mode-of-action at the molecular level. By a combination of structure elucidation experiments, the first total synthesis of zelkovamycin and bioassays, the zelkovamycin configuration was determined and its previously proposed molecular structure was revised. The full structure assignment proves zelkovamycin as an additional member of the argyrins with however unique OXPHOS inhibitory properties. Zelkovamycin may therefore not only serve as a new starting point for chemical inhibitors of the OXPHOS system, but also guide customized argyrin NP isolation and biosynthesis studies.
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http://dx.doi.org/10.1002/chem.202001577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383741PMC
July 2020

Targeting of parvulin interactors by diazirine mediated cross-linking discloses a cellular role of human Par14/17 in actin polymerization.

Biol Chem 2020 07;401(8):955-968

Structural and Medicinal Biochemistry, Center for Medical Biotechnology (ZMB), University of Duisburg-Essen, Universitätsstrasse 2-5, D-45117 Essen, Germany.

The peptidyl-prolyl cis/trans isomerases (PPIases) Parvulin 14 (Par14) and Parvulin 17 (Par17) result from alternative transcription initiation of the PIN4 gene. Whereas Par14 is present in all metazoan, Par17 is only expressed in Hominidae. Par14 resides mainly within the cellular nucleus, while Par17 is translocated into mitochondria. Using photo-affinity labeling, cross-linking and mass spectrometry (MS) we identified binding partners for both enzymes from HeLa lysates and disentangled their cellular roles. Par14 is involved in biogenesis of ribonucleoprotein (RNP)-complexes, RNA processing and DNA repair. Its elongated isoform Par17 participates in protein transport/translocation and in cytoskeleton organization. Nuclear magnetic resonance (NMR) spectroscopy reveals that Par17 binds to β-actin with its N-terminal region, while both parvulins initiate actin polymerization depending on their PPIase activity as monitored by fluorescence spectroscopy. The knockdown (KD) of Par17 in HCT116 cells results in a defect in cell motility and migration.
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http://dx.doi.org/10.1515/hsz-2019-0423DOI Listing
July 2020

Activation by substoichiometric inhibition.

Proc Natl Acad Sci U S A 2020 01 6;117(3):1414-1418. Epub 2020 Jan 6.

Center of Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, 45117 Essen, Germany;

Startling reports described the paradoxical triggering of the human mitogen-activated protein kinase pathway when a small-molecule inhibitor specifically inactivates the BRAF V600E protein kinase but not wt-BRAF. We performed a conceptual analysis of the general phenomenon "activation by inhibition" using bacterial and human HtrA proteases as models. Our data suggest a clear explanation that is based on the classic biochemical principles of allostery and cooperativity. Although substoichiometric occupancy of inhibitor binding sites results in partial inhibition, this effect is overrun by a concomitant activation of unliganded binding sites. Therefore, when an inhibitor of a cooperative enzyme does not reach saturating levels, a common scenario during drug administration, it may cause the contrary of the desired effect. The implications for drug development are discussed.
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http://dx.doi.org/10.1073/pnas.1918721117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983408PMC
January 2020

A homology-guided, genome-based proteome for improved proteomics in the alloploid Nicotiana benthamiana.

BMC Genomics 2019 Oct 4;20(1):722. Epub 2019 Oct 4.

Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK.

Background: Nicotiana benthamiana is an important model organism of the Solanaceae (Nightshade) family. Several draft assemblies of the N. benthamiana genome have been generated, but many of the gene-models in these draft assemblies appear incorrect.

Results: Here we present an improved proteome based on the Niben1.0.1 draft genome assembly guided by gene models from other Nicotiana species. Due to the fragmented nature of the Niben1.0.1 draft genome, many protein-encoding genes are missing or partial. We complement these missing proteins by similarly annotating other draft genome assemblies. This approach overcomes problems caused by mis-annotated exon-intron boundaries and mis-assigned short read transcripts to homeologs in polyploid genomes. With an estimated 98.1% completeness; only 53,411 protein-encoding genes; and improved protein lengths and functional annotations, this new predicted proteome is better in assigning spectra than the preceding proteome annotations. This dataset is more sensitive and accurate in proteomics applications, clarifying the detection by activity-based proteomics of proteins that were previously predicted to be inactive. Phylogenetic analysis of the subtilase family of hydrolases reveal inactivation of likely homeologs, associated with a contraction of the functional genome in this alloploid plant species. Finally, we use this new proteome annotation to characterize the extracellular proteome as compared to a total leaf proteome, which highlights the enrichment of hydrolases in the apoplast.

Conclusions: This proteome annotation provides the community working with Nicotiana benthamiana with an important new resource for functional proteomics.
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http://dx.doi.org/10.1186/s12864-019-6058-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778390PMC
October 2019

Cathepsins Drive Anti-Inflammatory Activity by Regulating Autophagy and Mitochondrial Dynamics in Macrophage Foam Cells.

Cell Physiol Biochem 2019 ;53(3):550-572

Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University, Jerusalem, Israel,

Background/aims: Atherosclerosis underlies the majority of cardiovascular events, consequent to non-resolving inflammation. Considerable evidence implicates autophagy dysfunction at the core of this inflammatory condition, but the basis of this dysfunction is not fully understood.

Methods: Using an in vitro model of lipid-laden macrophages, activity-based probes and high-throughput techniques, we studied the role of the cysteine proteases cathepsins in autophagy.

Results: We showed that cathepsin activity is suppressed by oxidized lipids and that cathepsin has an indispensable role in the autophagy-lysosomal degradation pathway. Accordingly, loss of cathepsin function resulted in autophagy derangement. Shotgun proteomics confirmed autophagy dysfunction and unveiled a pivotal role of cathepsin L in a putative cathepsin degradation network. At the physiological level, cathepsin inhibition resulted in mitochondrial stress, which translated into impaired oxidative metabolism, excessive production of reactive oxygen species and activation of the cellular stress response, driven by ATF4-CHOP transcription factors. In addition, transcriptomic analysis of these cells uncovered some genetic similarities with the inflammatory macrophage phenotype (a.k.a M1 macrophages) and increased expression of inflammatory cytokines.

Conclusion: Our data highlight the importance of cathepsins for mitochondrial quality control mechanisms and amelioration of vascular inflammation.
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http://dx.doi.org/10.33594/000000157DOI Listing
December 2019

From dolastatin 13 to cyanopeptolins, micropeptins, and lyngbyastatins: the chemical biology of Ahp-cyclodepsipeptides.

Nat Prod Rep 2020 02;37(2):163-174

Chemical Biology, Zentrum für Medizinische Biotechnologie (ZMB), Faculty of Biology, Universität Duisburg-Essen, Universitätsstr. 2, 45117 Essen, Germany.

Covering: 1989 up to 2019 Ahp-cyclodepsipeptides (also known as Ahp-containing cyclodepsipeptides, cyanopeptolins, micropeptins, microginines, and lyngbyastatins, and by many other names) are a family of non-ribosomal peptide synthesis (NRPS)-derived natural products with potent serine protease inhibitory properties. Here, we review their isolation and structural elucidation from natural sources as well as studies of their biosynthesis, molecular mode of action, and use in drug discovery efforts. Accordingly, this summary aims to provide a comprehensive overview of the current state-of-the-art Ahp-cyclodepsipeptide research.
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http://dx.doi.org/10.1039/c9np00033jDOI Listing
February 2020

Plant chemical genetics reveals colistin sulphate as a SA and NPR1-independent PR1 inducer functioning via a p38-like kinase pathway.

Sci Rep 2019 08 1;9(1):11196. Epub 2019 Aug 1.

Chemical Biology, Centre of Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, Universitätsstr. 2, 45141, Essen, Germany.

In plants, low-dose of exogenous bacterial cyclic lipopeptides (CLPs) trigger transient membrane changes leading to activation of early and late defence responses. Here, a forward chemical genetics approach identifies colistin sulphate (CS) CLP as a novel plant defence inducer. CS uniquely triggers activation of the PATHOGENESIS-RELATED 1 (PR1) gene and resistance against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis thaliana (Arabidopsis) independently of the PR1 classical inducer, salicylic acid (SA) and the key SA-signalling protein, NON-EXPRESSOR OF PR1 (NPR1). Low bioactive concentration of CS does not trigger activation of early defence markers such as reactive oxygen species (ROS) and mitogen activated protein kinase (MAPK). However, it strongly suppresses primary root length elongation. Structure activity relationship (SAR) assays and mode-of-action (MoA) studies show the acyl chain and activation of a ∼46 kDa p38-like kinase pathway to be crucial for CS' bioactivity. Selective pharmacological inhibition of the active p38-like kinase pathway by SB203580 reverses CS' effects on PR1 activation and root length suppression. Our results with CS as a chemical probe highlight the existence of a novel SA- and NPR1-independent branch of PR1 activation functioning via a membrane-sensitive p38-like kinase pathway.
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http://dx.doi.org/10.1038/s41598-019-47526-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6671972PMC
August 2019

Triazine Probes Target Ascorbate Peroxidases in Plants.

Plant Physiol 2019 08 28;180(4):1848-1859. Epub 2019 May 28.

Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom

Though they are rare in nature, anthropogenic 1,3,5-triazines have been used in herbicides as chemically stable scaffolds. Here, we show that small 1,3,5-triazines selectively target ascorbate peroxidases (APXs) in Arabidopsis (), tomato (), rice (), maize (), liverwort (), and other plant species. The alkyne-tagged 2-chloro-4-methyl-1,3,5-triazine probe KSC-3 selectively binds APX enzymes, both in crude extracts and in living cells. KSC-3 blocks APX activity, thereby reducing photosynthetic activity under moderate light stress, even in mutant plants. This suggests that APX enzymes in addition to APX1 protect the photosystem against reactive oxygen species. Profiling APX1 with KCS-3 revealed that the catabolic products of atrazine (a 1,3,5-triazine herbicide), which are common soil pollutants, also target APX1. Thus, KSC-3 is a powerful chemical probe to study APX enzymes in the plant kingdom.
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http://dx.doi.org/10.1104/pp.19.00481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6670103PMC
August 2019

Proteases Underground: Analysis of the Maize Root Apoplast Identifies Organ Specific Papain-Like Cysteine Protease Activity.

Front Plant Sci 2019 30;10:473. Epub 2019 Apr 30.

Center of Excellence on Plant Sciences (CEPLAS), Botanical Institute, University of Cologne, Cologne, Germany.

Plant proteases are key regulators of plant cell processes such as seed development, immune responses, senescence and programmed cell death (PCD). Apoplastic papain-like cysteine proteases (PL) are hubs in plant-microbe interactions and play an important role during abiotic stresses. The apoplast is a crucial interface for the interaction between plant and microbes. So far, apoplastic maize PL and their function have been mostly described for aerial parts. In this study, we focused on apoplastic PLCPs in the roots of maize plants. We have analyzed the phylogeny of maize PLCPs and investigated their protein abundance after salicylic acid (SA) treatment. Using activity-based protein profiling (ABPP) we have identified a novel root-specific PLCP belonging to the RD21-like subfamily, as well as three SA activated PLCPs. The root specific PLCP CP1C shares sequence and structural similarities to known CP1-like proteases. Biochemical analysis of recombinant CP1C revealed different substrate specificities and inhibitor affinities compared to the related proteases. This study characterized a root-specific PLCP and identifies differences between the SA-dependent activation of PLCPs in roots and leaves.
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http://dx.doi.org/10.3389/fpls.2019.00473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503450PMC
April 2019

Cathepsin L Regulates Metabolic Networks Controlling Rapid Cell Growth and Proliferation.

Mol Cell Proteomics 2019 07 22;18(7):1330-1344. Epub 2019 Apr 22.

From the ‡Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University, Jerusalem, Israel, 9112001;. Electronic address:

Rapidly proliferating cells reshape their metabolism to satisfy their ever-lasting need for cellular building blocks. This phenomenon is exemplified in certain malignant conditions such as cancer but also during embryonic development when cells rely heavily on glycolytic metabolism to exploit its metabolic intermediates for biosynthetic processes. How cells reshape their metabolism is not fully understood. Here we report that loss of cathepsin L (Cts L) is associated with a fast proliferation rate and enhanced glycolytic metabolism that depend on lactate dehydrogenase A (LDHA) activity. Using mass spectrometry analysis of cells treated with a pan cathepsin inhibitor, we observed an increased abundance of proteins involved in central carbon metabolism. Further inspection of putative Cts L targets revealed an enrichment for glycolytic metabolism that was independently confirmed by metabolomic and biochemical analyses. Moreover, proteomic analysis of Cts L-knockout cells identified LDHA overexpression that was demonstrated to be a key metabolic junction in these cells. Lastly, we show that Cts L inhibition led to increased LDHA protein expression, suggesting a causal relationship between LDHA expression and function. In conclusion, we propose that Cts L regulates this metabolic circuit to keep cell division under control, suggesting the therapeutic potential of targeting this protein and its networks in cancer.
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http://dx.doi.org/10.1074/mcp.RA119.001392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601214PMC
July 2019

Glycosidase and glycan polymorphism control hydrolytic release of immunogenic flagellin peptides.

Science 2019 Apr;364(6436)

Department of Plant Sciences, University of Oxford, Oxford, UK.

Plants and animals recognize conserved flagellin fragments as a signature of bacterial invasion. These immunogenic elicitor peptides are embedded in the flagellin polymer and require hydrolytic release before they can activate cell surface receptors. Although much of flagellin signaling is understood, little is known about the release of immunogenic fragments. We discovered that plant-secreted β-galactosidase 1 (BGAL1) of promotes hydrolytic elicitor release and acts in immunity against pathogenic strains only when they carry a terminal modified viosamine (mVio) in the flagellin -glycan. In counter defense, pathovars evade host immunity by using BGAL1-resistant -glycans or by producing a BGAL1 inhibitor. Polymorphic glycans on flagella are common to plant and animal pathogenic bacteria and represent an important determinant of host immunity to bacterial pathogens.
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http://dx.doi.org/10.1126/science.aav0748DOI Listing
April 2019

Chemical Validation of DegS As a Target for the Development of Antibiotics with a Novel Mode of Action.

ChemMedChem 2019 06 24;14(11):1074-1078. Epub 2019 Apr 24.

Chemical Biology, Faculty of Biology, Center of Medical Biotechnology, University Duisburg-Essen, Universitätsstr. 2, 45117, Essen, Germany.

Despite the availability of hundreds of antibiotic drugs, infectious diseases continue to remain one of the most notorious health issues. In addition, the disparity between the spread of multidrug-resistant pathogens and the development of novel classes of antibiotics exemplify an important unmet medical need that can only be addressed by identifying novel targets. Herein we demonstrate, by the development of the first in vivo active DegS inhibitors based on a pyrazolo[1,5-a]-1,3,5-triazine scaffold, that the serine protease DegS and the cell envelope stress-response pathway σE represent a target for generating antibiotics with a novel mode of action. Moreover, DegS inhibition is synergistic with well-established membrane-perturbing antibiotics, thereby opening promising avenues for rational antibiotic drug design.
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http://dx.doi.org/10.1002/cmdc.201900193DOI Listing
June 2019

Dynamic hydrolase labelling as a marker for seed quality in Arabidopsis seeds.

Biochem J 2019 03 12;476(5):843-857. Epub 2019 Mar 12.

Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany

Seed quality is affected by different constituents of the seed. In general, seed lots are considered to be of high quality when they exhibit fast and homogeneous germination. When seeds are stored, they undergo different degrees of damage that have detrimental effects on their quality. Therefore, accurate prediction of the seed quality and viability levels of a seed lot is of high importance in the seed-producing industry. Here, we describe the use of activity-based protein profiling of proteases to evaluate the quality of artificially and naturally aged seeds of Using this approach, we have identified two protease activities with opposite behaviours in aged seeds of Arabidopsis that correlate with the quality status of the seeds. We show that vacuolar processing enzymes (VPEs) become more active during the ageing process, in both artificial and natural ageing treatments. Secondly, we demonstrate that serine hydrolases are active at the beginning of our artificial ageing treatment, but their labelling decreases along with seed viability. We present a list of candidate hydrolases active during seed germination and propose that these protease activities can be used in combination with VPEs to develop novel markers of seed quality.
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http://dx.doi.org/10.1042/BCJ20180911DOI Listing
March 2019

Activity-based proteomics reveals nine target proteases for the recombinant protein-stabilizing inhibitor SlCYS8 in Nicotiana benthamiana.

Plant Biotechnol J 2019 08 14;17(8):1670-1678. Epub 2019 Mar 14.

Department of Plant Sciences, Plant Chemetics Laboratory, University of Oxford, Oxford, UK.

Co-expression of protease inhibitors like the tomato cystatin SlCYS8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity-based protein profiling to identify proteases that are inhibited by SlCYS8 in agroinfiltrated Nicotiana benthamiana. We discovered that SlCYS8 selectively suppresses papain-like cysteine protease (PLCP) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar-processing enzyme and serine hydrolase activity. A robust concentration-dependent inhibition of PLCPs occurred in vitro when purified SlCYS8 was added to leaf extracts, indicating direct cystatin-PLCP interactions. Activity-based proteomics revealed that nine different Cathepsin-L/-F-like PLCPs are strongly inhibited by SlCYS8 in leaves. By contrast, the activity of five other Cathepsin-B/-H-like PLCPs, as well as 87 Ser hydrolases, was unaffected by SlCYS8. SlCYS8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin-containing proteases from the Resistant-to-Desiccation-21 (RD21) PLCP subfamily. Our data underline the key role of endogenous PLCPs on recombinant protein degradation and reveal candidate proteases for depletion strategies.
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http://dx.doi.org/10.1111/pbi.13092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662110PMC
August 2019

Metabolic reconstruction of the genome of candidate Desulfatiglans TRIP_1 and identification of key candidate enzymes for anaerobic phenanthrene degradation.

Environ Microbiol 2019 04 7;21(4):1267-1286. Epub 2019 Feb 7.

Biofilm Centre, Aquatic Microbiology Department, Faculty of Chemistry, University Duisburg-Essen, Essen, Germany.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed pollutants. As oxygen is rapidly depleted in water-saturated PAH-contaminated sites, anaerobic microorganisms are crucial for their consumption. Here, we report the metabolic pathway for anaerobic degradation of phenanthrene by a sulfate-reducing enrichment culture (TRIP) obtained from a natural asphalt lake. The dominant organism of this culture belongs to the Desulfobacteraceae family of Deltaproteobacteria and genome-resolved metagenomics led to the reconstruction of its genome along with a handful of genomes from lower abundance bacteria. Proteogenomic analyses confirmed metabolic capabilities for dissimilatory sulfate reduction and indicated the presence of the Embden-Meyerhof-Parnas pathway, a complete tricarboxylic acid cycle as well as a complete Wood-Ljungdahl pathway. Genes encoding enzymes putatively involved in the degradation of phenanthrene were identified. This includes two gene clusters encoding a multisubunit carboxylase complex likely involved in the activation of phenanthrene, as well as genes encoding reductases potentially involved in subsequent ring dearomatization and reduction steps. The predicted metabolic pathways were corroborated by transcriptome and proteome analyses, and provide the first insights into the metabolic pathway responsible for the anaerobic degradation of three-ringed PAHs.
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http://dx.doi.org/10.1111/1462-2920.14527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849830PMC
April 2019

Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

Mol Cell 2018 11 18;72(4):766-777.e6. Epub 2018 Oct 18.

Centre for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany. Electronic address:

The functional diversity of protein phosphatase-1 (PP1), with its countless substrates, relies on the ordered assembly of alternative PP1 holoenzymes. Here, we show that newly synthesized PP1 is first held by its partners SDS22 and inhibitor-3 (I3) in an inactive complex, which needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Unlike p97-mediated degradative processes that require the Ufd1-Npl4 ubiquitin adapters, p97 is targeted to PP1 by p37 and related adapter proteins. Reconstitution with purified components revealed direct interaction of the p37 SEP domain with I3 without the need for ubiquitination, and ATP-driven pulling of I3 into the central channel of the p97 hexamer, which triggers dissociation of I3 and SDS22. Thus, we establish regulatory ubiquitin-independent protein complex disassembly as part of the functional arsenal of p97 and define an unanticipated essential step in PP1 biogenesis that illustrates the molecular challenges of ordered subunit exchange.
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http://dx.doi.org/10.1016/j.molcel.2018.09.020DOI Listing
November 2018

Utilities for Mass Spectrometry Analysis of Proteins (UMSAP): Fast post-processing of mass spectrometry data.

Rapid Commun Mass Spectrom 2018 Oct;32(19):1659-1667

Centre of Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstraße, 45117, Essen, Germany.

Rationale: Mass spectrometry (MS) is an invaluable tool for the analysis of proteins. However, the sheer amount of data generated in MS studies demands dedicated data-processing tools that are efficient and require minimal user intervention.

Methods: Utilities for Mass Spectrometry Analysis of Proteins (UMSAP) is a graphical user interface designed for efficient post-processing of MS result files. The software is written in Tcl/Tk and can be used in Windows, OS X or Linux. No third party programs or libraries are required. Currently, UMSAP can process data obtained from proteolytic degradation experiments and generates graphical outputs allowing a straightforward interpretation of statistically relevant results.

Results: UMSAP is used here to analyze the proteolytic degradation of glycerophosphoryl diester phosphodiesterase GlpQ by the protein quality control protease DegP. Mass spectrometry was used to monitor proteolysis over time in the absence and presence of a peptidic allosteric activator of DegP. The software's output clearly shows the increased proteolytic activity of DegP in the presence of the activating peptide, identifies statistically significant products of the proteolysis and offers valuable insights into substrate specificity.

Conclusions: Utilities for Mass Spectrometry Analysis of Proteins is an open-source software designed for efficient post-processing of large datasets obtained by MS analyses of proteins. In addition, the modular architecture of the software allows easy incorporation of new modules to analyze various experimental mass spectrometry setups.
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http://dx.doi.org/10.1002/rcm.8243DOI Listing
October 2018

Identification of the Natural Product Rotihibin A as a TOR Kinase Signaling Inhibitor by Unbiased Transcriptional Profiling.

Chemistry 2018 Aug 27;24(48):12500-12504. Epub 2018 Jul 27.

Chemical Biology, Zentrum für Medizinische Biotechnologie, Universität Duisburg-Essen, Universitätsstrasse 2, 45117, Essen, Germany.

Bioactive natural products are important starting points for developing chemical tools for biological research. For elucidating their bioactivity profile, biological systems with concise complexity such as cell culture systems are frequently used, whereas unbiased investigations in more complex multicellular systems are only rarely explored. Here, we demonstrate with the natural product Rotihibin A and the plant research model system Arabidopsis thaliana that unbiased transcriptional profiling enables a rapid, label-free, and compound economic evaluation of a natural product's bioactivity profile in a complex multicellular organism. To this end, we established a chemical synthesis of Rotihibin A as well as that of structural analogues, followed by transcriptional profiling-guided identification and validation of Rotihibin A as a TOR signaling inhibitor (TOR=target of rapamycin). These findings illustrate that a combined approach of transcriptional profiling and natural product research may represent a technically simple approach to streamline the development of chemical tools from natural products even for biologically complex multicellular biological systems.
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http://dx.doi.org/10.1002/chem.201802647DOI Listing
August 2018

Mono- and Bivalent 14-3-3 Inhibitors for Characterizing Supramolecular "Lysine Wrapping" of Oligoethylene Glycol (OEG) Moieties in Proteins.

Chemistry 2018 Sep 22;24(52):13807-13814. Epub 2018 Aug 22.

Chemical Biology, Zentrum für Medizinische Biotechnologie, Fakultät für Biologie, Universität Duisburg-Essen, Universitätsstr. 2, 45117, Essen, Germany.

Previous studies have indicated the presence of defined interactions between oligo or poly(ethylene glycol) (OEG or PEG) and lysine residues. In these interactions, the OEG or PEG residues "wrap around" the lysine amino group, thereby enabling complexation of the amino group by the ether oxygen residues. The resulting biochemical binding affinity and thus biological relevance of this supramolecular interaction however remains unclear so far. Here, we report that OEG-containing phosphophenol ether inhibitors of 14-3-3 proteins also display such a "lysine-wrapping" binding mode. For better investigating the biochemical relevance of this binding mode, we made use of the dimeric nature of 14-3-3 proteins and designed as well as synthesized a set of bivalent 14-3-3 inhibitors for biochemical and X-ray crystallography-based structural studies. We found that all synthesized derivatives adapted the "lysine-wrapping" binding mode in the crystal structures; in solution, a different binding mode is however observed, most probably as the "lysine-wrapping" binding mode turned out to be a rather weak interaction. Accordingly, our studies demonstrate that structural studies of OEG-lysine interactions are difficult to interpret and their presence in structural studies may not automatically be correlated with a relevant interaction also in solution but requires further biochemical studies.
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http://dx.doi.org/10.1002/chem.201801074DOI Listing
September 2018

HTRA1-Dependent Cell Cycle Proteomics.

J Proteome Res 2018 08 13;17(8):2679-2694. Epub 2018 Jul 13.

Centre of Medical Biotechnology, Faculty of Biology , University Duisburg-Essen, Universitaetsstrasse , 45141 Essen , Germany.

The HTRA1 gene encoding an evolutionary conserved protein quality-control factor can be epigenetically silenced or inactivated by mutation under pathologic conditions such as cancer. Recent evidence suggests that the loss of HTRA1 function causes multiple phenotypes, including the acceleration of cell growth, delayed onset of senescence, centrosome amplification, and polyploidy, suggesting an implication in the regulation of the cell cycle. To address this model, we performed a large-scale proteomics study to correlate the abundance of proteins and HTRA1 levels in various cell cycle phases using label-free-quantification mass spectrometry. These data indicate that the levels of 4723 proteins fluctuated in a cell-cycle-dependent manner, 2872 in a HTRA1-dependent manner, and 1530 in a cell-cycle- and HTRA1-dependent manner. The large number of proteins affected by the modulation of HTRA1 levels supports its general role in protein homeostasis. Moreover, the detected changes in protein abundance, in combination with pull-down data, implicate HTRA1 in various cell cycle events such as DNA replication, chromosome segregation, and cell-cycle-dependent apoptosis. These results highlight the wide implications of HTRA1 in cellular physiology.
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http://dx.doi.org/10.1021/acs.jproteome.8b00129DOI Listing
August 2018

Fermentative Spirochaetes mediate necromass recycling in anoxic hydrocarbon-contaminated habitats.

ISME J 2018 08 30;12(8):2039-2050. Epub 2018 May 30.

University of Duisburg-Essen, Biofilm Centre, Universitätsstrasse 5, 45141, Essen, Germany.

Spirochaetes are frequently detected in anoxic hydrocarbon- and organohalide-polluted groundwater, but their role in such ecosystems has remained unclear. To address this, we studied a sulfate-reducing, naphthalene-degrading enrichment culture, mainly comprising the sulfate reducer Desulfobacterium N47 and the rod-shaped Spirochete Rectinema cohabitans HM. Genome sequencing and proteome analysis suggested that the Spirochete is an obligate fermenter that catabolizes proteins and carbohydrates, resulting in acetate, ethanol, and molecular hydrogen (H) production. Physiological experiments inferred that hydrogen is an important link between the two bacteria in the enrichment culture, with H derived from fermentation by R. cohabitans used as reductant for sulfate reduction by Desulfobacterium N47. Differential proteomics and physiological experiments showed that R. cohabitans utilizes biomass (proteins and carbohydrates) released from dead cells of Desulfobacterium N47. Further comparative and community genome analyses indicated that other Rectinema phylotypes are widespread in contaminated environments and may perform a hydrogenogenic fermentative lifestyle similar to R. cohabitans. Together, these findings indicate that environmental Spirochaetes scavenge detrital biomass and in turn drive necromass recycling at anoxic hydrocarbon-contaminated sites and potentially other habitats.
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http://dx.doi.org/10.1038/s41396-018-0148-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052044PMC
August 2018
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