41 results on '"Barkovits K"'
Search Results
2. Changes in the Proteome of Platelets from Patients with Critical Progression of COVID-19.
- Author
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Zimmermann, M., Wolny, M., Rozanova, S., Knabbe, C., Flieder, T., Hohbein, A., Pfeiffer, K., Barkovits, K., Marcus, K., and Birschmann, I.
- Published
- 2024
- Full Text
- View/download PDF
3. New insights into the protein aggregation pathology in myotilinopathy by combined proteomic and immunolocalization analyses
- Author
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Maerkens, A., primary, Olivé, M., additional, Schreiner, A., additional, Feldkirchner, S., additional, Schessl, J., additional, Uszkoreit, J., additional, Barkovits, K., additional, Güttsches, A. K., additional, Theis, V., additional, Eisenacher, M., additional, Tegenthoff, M., additional, Goldfarb, L. G., additional, Schröder, R., additional, Schoser, B., additional, van der Ven, P. F. M., additional, Fürst, D. O., additional, Vorgerd, M., additional, Marcus, K., additional, and Kley, R. A., additional
- Published
- 2016
- Full Text
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4. Changes in the Proteome of Platelets from Patients with Critical Progression of COVID-19.
- Author
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Wolny M, Rozanova S, Knabbe C, Pfeiffer K, Barkovits K, Marcus K, and Birschmann I
- Subjects
- Humans, Proteome, B-Lymphocytes, Cytokine Release Syndrome, Blood Platelets, COVID-19
- Abstract
Platelets, the smallest cells in human blood, known for their role in primary hemostasis, are also able to interact with pathogens and play a crucial role in the immune response. In severe coronavirus disease 2019 (COVID-19) cases, platelets become overactivated, resulting in the release of granules, exacerbating inflammation and contributing to the cytokine storm. This study aims to further elucidate the role of platelets in COVID-19 progression and to identify predictive biomarkers for disease outcomes. A comparative proteome analysis of highly purified platelets from critically diseased COVID-19 patients with different outcomes (survivors and non-survivors) and age- and sex-matched controls was performed. Platelets from critically diseased COVID-19 patients exhibited significant changes in the levels of proteins associated with protein folding. In addition, a number of proteins with isomerase activity were found to be more highly abundant in patient samples, apparently exerting an influence on platelet activity via the non-genomic properties of the glucocorticoid receptor (GR) and the nuclear factor κ-light-chain-enhancer of activated B cells (NFκB). Moreover, carbonic anhydrase 1 (CA-1) was found to be a candidate biomarker in platelets, showing a significant increase in COVID-19 patients.
- Published
- 2023
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5. LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.
- Author
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Wu Z, Berlemann LA, Bader V, Sehr DA, Dawin E, Covallero A, Meschede J, Angersbach L, Showkat C, Michaelis JB, Münch C, Rieger B, Namgaladze D, Herrera MG, Fiesel FC, Springer W, Mendes M, Stepien J, Barkovits K, Marcus K, Sickmann A, Dittmar G, Busch KB, Riedel D, Brini M, Tatzelt J, Cali T, and Winklhofer KF
- Subjects
- Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Signal Transduction physiology, Mitochondria metabolism, Ubiquitination, NF-kappa B genetics, NF-kappa B metabolism, Ubiquitin metabolism
- Abstract
Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)
- Published
- 2022
- Full Text
- View/download PDF
6. The Proteome of Neuromelanin Granules in Dementia with Lewy Bodies.
- Author
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Wulf M, Barkovits K, Schork K, Eisenacher M, Riederer P, Gerlach M, Eggers B, and Marcus K
- Subjects
- Humans, alpha-Synuclein, Proteomics, Proteome, Lewy Body Disease
- Abstract
Neuromelanin granules (NMGs) are organelle-like structures present in the human substantia nigra pars compacta . In addition to neuromelanin, NMGs contain proteins, lipids and metals. As NMG-containing dopaminergic neurons are preferentially lost in Parkinson's disease and dementia with Lewy bodies (DLB), it is assumed that NMGs may play a role in neurodegenerative processes. Until now, this role is not completely understood and needs further investigation. We therefore set up an exploratory proteomic study to identify differences in the proteomic profile of NMGs from DLB patients (n = 5) compared to healthy controls (CTRL, n = 5). We applied a laser microdissection and mass-spectrometry-based approach, in which we used targeted mass spectrometric experiments for validation. In NMG-surrounding (SN
Surr. ) tissue of DLB patients, we found evidence for ongoing oxidative damage and an impairment of protein degradation. As a potentially disease-related mechanism, we found α-synuclein and protein S100A9 to be enriched in NMGs of DLB cases, while the abundance of several ribosomal proteins was significantly decreased. As S100A9 is known to be able to enhance the formation of toxic α-synuclein fibrils, this finding points towards an involvement of NMGs in pathogenesis, however the exact role of NMGs as either neuroprotective or neurotoxic needs to be further investigated. Nevertheless, our study provides evidence for an impairment of protein degradation, ongoing oxidative damage and accumulation of potentially neurotoxic protein aggregates to be central mechanisms of neurodegeneration in DLB.- Published
- 2022
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7. Neuromelanin granules of the substantia nigra: proteomic profile provides links to tyrosine hydroxylase, stress granules and lysosomes.
- Author
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Wulf M, Barkovits K, Schork K, Eisenacher M, Riederer P, Gerlach M, Eggers B, and Marcus K
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- Humans, Lysosomes metabolism, Melanins metabolism, Stress Granules, Substantia Nigra metabolism, Proteomics methods, Tyrosine 3-Monooxygenase metabolism
- Abstract
Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in the cell bodies of dopaminergic neurons in the substantia nigra (SN) pars compacta. These neurons are lost in neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies. Although it is known that lipids, proteins, and environmental toxins accumulate in NMGs, the function of NMGs has not yet been finally clarified as well as their origin and the synthesis of neuromelanin. We, therefore, isolated NMGs and surrounding SN tissue from control patients by laser microdissection and analyzed the proteomic profile by tandem mass spectrometry. With our improved workflow, we were able to (1) strengthen the regularly reported link between NMGs and lysosomes, (2) detect tyrosine hydroxylase to be highly abundant in NMGs, which may be related to neuromelanin synthesis and (3) indicate a yet undescribed link between stress granules (SGs) and NMGs. Based on our findings, we cautiously hypothesize, that SGs may be the origin of NMGs or form in close proximity to them, potentially due to the oxidative stress caused by neuromelanin-bound metals., (© 2022. The Author(s).)
- Published
- 2022
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8. Dataset containing physiological amounts of spike-in proteins into murine C2C12 background as a ground truth quantitative LC-MS/MS reference.
- Author
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Uszkoreit J, Barkovits K, Pacharra S, Pfeiffer K, Steinbach S, Marcus K, and Eisenacher M
- Abstract
In this article, we present a data dependent acquisition (DDA) dataset which was generated as a reference and ground truth quantitative dataset. While initially used to compare samples measured with DDA and data independent acquisition (DIA) (Barkovits et al., 2020), the presented dataset holds potential value as a benchmark reference for any workflows working on DDA data. The entire dataset consists of 15 LC-MS/MS measurements composed of five distinct spike-in-states, each with three replicates. To generate the data set, a C2C12 (immortalized mouse myoblast) cell lysate was used as a complex background for five different states which were simulated by spiking 13 defined proteins at different concentrations. For this purpose, the cell lysate was used in a constant amount of 20 µg for all samples and different amounts of the 13 selected proteins ranging from 0.1 to 10 pmol were added, reflecting physiological amounts of proteins. Afterwards, all samples were tryptically digested using the same method. From each sample 200 ng tryptic peptides were measured in triplicates on a Q Exactive HF (Thermo Fisher Scientific). The mass range for MS1 was set to 350-1400 m/z with a resolution of 60,000 at 200 m/z. HCD fragmentation of the Top10 abundant precursor ions was performed at 27% NCE. The fragment analysis (MS2) was performed with a resolution of 30,000 at 200 m/z. Additionally to the raw files, the dataset contains centroided mzML files and spectrum identification results for peptide identifications performed by Mascot (Perkins et al., 1999), MS-GF+ (Kim et al., 2010) and X!Tandem (Craig and Beavis, 2004) for each separate MS analysis. The corresponding FASTA containing protein sequences as well as a combination of all identification runs performed by PIA (Uszkoreit et al., 2019, 2015) and a peptide and protein quantification performed by OpenMS (Pfeuffer et al., 2017) is included. All data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2018) with the dataset identifier PXD012986., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)
- Published
- 2022
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9. Nanoenviroments of the β-Subunit of L-Type Voltage-Gated Calcium Channels in Adult Cardiomyocytes.
- Author
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Cruz-Garcia Y, Barkovits K, Kohlhaas M, Pickel S, Gulentz M, Heindl C, Pfeiffer K, Eder-Negrin P, Maack C, Marcus K, Kuhn M, and Miranda-Laferte E
- Abstract
In cardiomyocytes, Ca
2+ influx through L-type voltage-gated calcium channels (LTCCs) following membrane depolarization regulates crucial Ca2+ -dependent processes including duration and amplitude of the action potentials and excitation-contraction coupling. LTCCs are heteromultimeric proteins composed of the Cav α1 , Cav β, Cav α2 δ and Cav γ subunits. Here, using ascorbate peroxidase (APEX2)-mediated proximity labeling and quantitative proteomics, we identified 61 proteins in the nanoenvironments of Cav β2 in cardiomyocytes. These proteins are involved in diverse cellular functions such as cellular trafficking, cardiac contraction, sarcomere organization and excitation-contraction coupling. Moreover, pull-down assays and co-immunoprecipitation analyses revealed that Cav β2 interacts with the ryanodine receptor 2 (RyR2) in adult cardiomyocytes, probably coupling LTCCs and the RyR2 into a supramolecular complex at the dyads. This interaction is mediated by the Src-homology 3 domain of Cav β2 and is necessary for an effective pacing frequency-dependent increase of the Ca2+ -induced Ca2+ release mechanism in cardiomyocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cruz-Garcia, Barkovits, Kohlhaas, Pickel, Gulentz, Heindl, Pfeiffer, Eder-Negrin, Maack, Marcus, Kuhn and Miranda-Laferte.)- Published
- 2022
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10. Nematode CDC-37 and DNJ-13 form complexes and can interact with HSP-90.
- Author
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Schmauder L, Absmeier E, Bepperling A, Barkovits K, Marcus K, and Richter K
- Subjects
- Animals, Caenorhabditis elegans chemistry, Caenorhabditis elegans Proteins chemistry, Cell Cycle Proteins chemistry, HSP40 Heat-Shock Proteins chemistry, HSP90 Heat-Shock Proteins chemistry, Models, Molecular, Protein Binding, Protein Folding, Protein Interaction Maps, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, Cell Cycle Proteins metabolism, HSP40 Heat-Shock Proteins metabolism, HSP90 Heat-Shock Proteins metabolism
- Abstract
The molecular chaperones Hsc70 and Hsp90 are required for proteostasis control and specific folding of client proteins in eukaryotic and prokaryotic organisms. Especially in eukaryotes these ATP-driven molecular chaperones are interacting with cofactors that specify the client spectrum and coordinate the ATPase cycles. Here we find that a Hsc70-cofactor of the Hsp40 family from nematodes, DNJ-13, directly interacts with the kinase-specific Hsp90-cofactor CDC-37. The interaction is specific for DNJ-13, while DNJ-12 another DnaJ-like protein of C. elegans, does not bind to CDC-37 in a similar manner. Analytical ultracentrifugation is employed to show that one CDC-37 molecule binds to a dimeric DNJ-13 protein with low micromolar affinity. We perform cross-linking studies with mass spectrometry to identify the interaction site and obtain specific cross-links connecting the N-terminal J-domain of DNJ-13 with the N-terminal domain of CDC-37. Further AUC experiments reveal that both, the N-terminal part of CDC-37 and the C-terminal domain of CDC-37, are required for efficient interaction. Furthermore, the presence of DNJ-13 strengthens the complex formation between CDC-37 and HSP-90 and modulates the nucleotide-dependent effects. These findings on the interaction between Hsp40 proteins and Hsp90-cofactors provide evidence for a more intricate interaction between the two chaperone systems during client processing., (© 2021. The Author(s).)
- Published
- 2021
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11. CSF Diagnostics: A Potentially Valuable Tool in Neurodegenerative and Inflammatory Disorders Involving Motor Neurons: A Review.
- Author
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Krause K, Wulf M, Sommer P, Barkovits K, Vorgerd M, Marcus K, and Eggers B
- Abstract
Cerebrospinal fluid (CSF) diagnostics has emerged as a valid tool for a variety of neurological diseases. However, CSF diagnostics has been playing a subordinate role in the diagnosis of many neurological conditions. Thus, in the multitude of neuromuscular diseases in which motor neurons are affected, a CSF sample is rarely taken routinely. However, CSF diagnostics has the potential to specify the diagnosis and monitor the treatment of neuromuscular disorders. In this review, we therefore focused on a variety of neuromuscular diseases, among them amyotrophic lateral sclerosis (ALS), peripheral neuropathies, and spinal muscular atrophy (SMA), for which CSF diagnostics has emerged as a promising option for determining the disease itself and its progression. We focus on potentially valuable biomarkers among different disorders, such as neurofilaments, cytokines, other proteins, and lipids to determine their suitability, differentiating between different neurological disorders and their potential to determine early disease onset, disease progression, and treatment outcome. We further recommend novel approaches, e.g., the use of mass spectrometry as a promising alternative techniques to standard ELISA assays, potentially enhancing biomarker significance in clinical applications.
- Published
- 2021
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12. The β 2 -Subunit of Voltage-Gated Calcium Channels Regulates Cardiomyocyte Hypertrophy.
- Author
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Pickel S, Cruz-Garcia Y, Bandleon S, Barkovits K, Heindl C, Völker K, Abeßer M, Pfeiffer K, Schaaf A, Marcus K, Eder-Negrin P, Kuhn M, and Miranda-Laferte E
- Abstract
L-type voltage-gated calcium channels (LTCCs) regulate crucial physiological processes in the heart. They are composed of the Ca
v α1 pore-forming subunit and the accessory subunits Cav β, Cav α2 δ, and Cav γ. Cav β is a cytosolic protein that regulates channel trafficking and activity, but it also exerts other LTCC-independent functions. Cardiac hypertrophy, a relevant risk factor for the development of congestive heart failure, depends on the activation of calcium-dependent pro-hypertrophic signaling cascades. Here, by using shRNA-mediated Cav β silencing, we demonstrate that Cav β2 downregulation enhances α1-adrenergic receptor agonist-induced cardiomyocyte hypertrophy. We report that a pool of Cav β2 is targeted to the nucleus in cardiomyocytes and that the expression of this nuclear fraction decreases during in vitro and in vivo induction of cardiac hypertrophy. Moreover, the overexpression of nucleus-targeted Cav β2 in cardiomyocytes inhibits in vitro -induced hypertrophy. Quantitative proteomic analyses showed that Cav β2 knockdown leads to changes in the expression of diverse myocyte proteins, including reduction of calpastatin, an endogenous inhibitor of the calcium-dependent protease calpain. Accordingly, Cav β2 -downregulated cardiomyocytes had a 2-fold increase in calpain activity as compared to control cells. Furthermore, inhibition of calpain activity in Cav β2 -downregulated cells abolished the enhanced α1-adrenergic receptor agonist-induced hypertrophy observed in these cells. Our findings indicate that in cardiomyocytes, a nuclear pool of Cav β2 participates in cellular functions that are independent of LTCC activity. They also indicate that a downregulation of nuclear Cav β2 during cardiomyocyte hypertrophy promotes the activation of calpain-dependent hypertrophic pathways., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pickel, Cruz-Garcia, Bandleon, Barkovits, Heindl, Völker, Abeßer, Pfeiffer, Schaaf, Marcus, Eder-Negrin, Kuhn and Miranda-Laferte.)- Published
- 2021
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13. HSP-90/kinase complexes are stabilized by the large PPIase FKB-6.
- Author
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Sima S, Barkovits K, Marcus K, Schmauder L, Hacker SM, Hellwig N, Morgner N, and Richter K
- Subjects
- Animals, Binding Sites, Cell Cycle Proteins metabolism, Chaperonins metabolism, HSP90 Heat-Shock Proteins metabolism, Humans, Protein Binding, Protein Stability, Tacrolimus Binding Proteins metabolism, Cell Cycle Proteins chemistry, Chaperonins chemistry, HSP90 Heat-Shock Proteins chemistry, Tacrolimus Binding Proteins chemistry
- Abstract
Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase•Hsp90•Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex. The synergistic interaction between the participating proteins and the conserved nature of the interaction suggests functions of the large PPIases Fkbp51/Fkbp52 and their nematode homolog FKB-6 as contributing factors to the kinase cycle of the Hsp90 machinery.
- Published
- 2021
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14. Advanced Fiber Type-Specific Protein Profiles Derived from Adult Murine Skeletal Muscle.
- Author
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Eggers B, Schork K, Turewicz M, Barkovits K, Eisenacher M, Schröder R, Clemen CS, and Marcus K
- Abstract
Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. All data are available via ProteomeXchange with the identifier PXD025359. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach, identified a discriminative peptide panel, and confirmed our panel using a public data set.
- Published
- 2021
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15. MaCPepDB: A Database to Quickly Access All Tryptic Peptides of the UniProtKB.
- Author
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Uszkoreit J, Winkelhardt D, Barkovits K, Wulf M, Roocke S, Marcus K, and Eisenacher M
- Subjects
- Databases, Protein, Proteins, Proteomics, Peptides, Tandem Mass Spectrometry
- Abstract
Protein sequence databases play a crucial role in the majority of the currently applied mass-spectrometry-based proteomics workflows. Here UniProtKB serves as one of the major sources, as it combines the information of several smaller databases and enriches the entries with additional biological information. For the identification of peptides in a sample by tandem mass spectra, as generated by data-dependent acquisition, protein sequence databases provide the basis for most spectrum identification search engines. In addition, for targeted proteomics approaches like selected reaction monitoring (SRM) and parallel reaction monitoring (PRM), knowledge of the peptide sequences, their masses, and whether they are unique for a protein is essential. Because most bottom-up proteomics approaches use trypsin to cleave the proteins in a sample, the tryptic peptides contained in a protein database are of great interest. We present a database, called MaCPepDB (mass-centric peptide database), that consists of the complete tryptic digest of the Swiss-Prot and TrEMBL parts of UniProtKB. This database is especially designed to not only allow queries of peptide sequences and return the respective information about connected proteins and thus whether a peptide is unique but also allow queries of specific masses of peptides or precursors of MS/MS spectra. Furthermore, posttranslational modifications can be considered in a query as well as different mass deviations for posttranslational modifications. Hence the database can be used by a sequence query not only to, for example, check in which proteins of the UniProt database a tryptic peptide can be found but also to find possibly interfering peptides in PRM/SRM experiments using the mass query. The complete database contains currently 5 939 244 990 peptides from 185 561 610 proteins (UniProt version 2020_03), for which a single query usually takes less than 1 s. For easy exploration of the data, a web interface was developed. A REST application programming interface (API) for programmatic and workflow access is also available at https://macpepdb.mpc.rub.de.
- Published
- 2021
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16. Targeted Protein Quantification Using Parallel Reaction Monitoring (PRM).
- Author
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Barkovits K, Chen W, Kohl M, and Bracht T
- Subjects
- Animals, Calibration, Humans, Limit of Detection, Reference Standards, Research Design, Isotope Labeling standards, Proteins analysis, Proteome, Proteomics standards, Tandem Mass Spectrometry standards
- Abstract
Targeted proteomics represents an efficient method to quantify proteins of interest with high sensitivity and accuracy. Targeted approaches were first established for triple quadrupole instruments, but the emergence of hybrid instruments allowing for high-resolution and accurate-mass measurements of MS/MS fragment ions enabled the development of parallel reaction monitoring (PRM). In PRM analysis, specific peptides are measured as representatives of proteins in complex samples, with the full product ion spectra being acquired, allowing for identification and quantification of the peptides. Ideally, corresponding stable isotope-labeled peptides are spiked into the analyzed samples to account for technical variation and enhance the precision. Here, we describe the development of a PRM assay including the selection of appropriate peptides that fulfill the criteria to serve as unique surrogates of the targeted proteins. We depict the sequential steps of method development and the generation of calibration curves. Furthermore, we present the open-access tool CalibraCurve for the determination of the linear concentration ranges and limits of quantification (LOQ).
- Published
- 2021
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17. Quantitative Mass Spectrometry-Based Proteomics: An Overview.
- Author
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Rozanova S, Barkovits K, Nikolov M, Schmidt C, Urlaub H, and Marcus K
- Subjects
- Animals, Humans, Mass Spectrometry, Proteins analysis, Proteome, Proteomics
- Abstract
In recent decades, mass spectrometry has moved more than ever before into the front line of protein-centered research. After being established at the qualitative level, the more challenging question of quantification of proteins and peptides using mass spectrometry has become a focus for further development. In this chapter, we discuss and review actual strategies and problems of the methods for the quantitative analysis of peptides, proteins, and finally proteomes by mass spectrometry. The common themes, the differences, and the potential pitfalls of the main approaches are presented in order to provide a survey of the emerging field of quantitative, mass spectrometry-based proteomics.
- Published
- 2021
- Full Text
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18. Protein Quantification Using the "Rapid Western Blot" Approach.
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Barkovits K, Pfeiffer K, Eggers B, and Marcus K
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- Animals, Calibration, Humans, Reference Standards, Research Design, Time Factors, Workflow, Blotting, Western standards, Proteins analysis, Proteome, Proteomics standards
- Abstract
For the quantification of certain proteins of interest within a complex sample, Western blot analysis is the most widely used method. It enables detection of a target protein based on the use of specific antibodies. However, the whole procedure is often very time-consuming. Nevertheless, with the development of fast blotting systems and further development of immunostaining methods, a reduction of the processing time can be achieved. Major challenges for the reliable protein quantification by Western blotting are adequate data normalization and stable protein detection. Usually, normalization of the target protein signal is performed based on housekeeping proteins (e.g., glyceraldehyde 3-phosphate dehydrogenase, ß-actin) with the assumption that those proteins are expressed constitutively at the same level across experiments. However, several studies have already shown that this is not always the case making this approach suboptimal. Another strategy uses total protein normalization where the abundance of the target protein is related to the total protein amount in each lane. This approach is independent of a single loading control, and precision of quantification and reliability is increased. For Western blotting several detection methods are available, e.g., colorimetric, chemiluminescent, radioactive, fluorescent detection. Conventional colorimetric staining tends to suffer from low sensitivity, limited dynamic range, and low reproducibility. Chemiluminescence-based methods are straightforward, but the detected signal does not linearly correlate to protein abundance (from protein amounts >5μg) and have a relatively narrow dynamic range. Radioactivity is harmful to health. To overcome these limitations, stain-free methods were developed allowing the combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer to the membrane. Here, we present a rapid Western blot protocol, which combines fast blotting using the iBlot system and fast immunostaining utilizing ReadyTector
® all-in-one solution with the Smart Protein Layers (SPL) approach.- Published
- 2021
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19. Glucocorticoid receptor complexes form cooperatively with the Hsp90 co-chaperones Pp5 and FKBPs.
- Author
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Kaziales A, Barkovits K, Marcus K, and Richter K
- Subjects
- Animals, Caenorhabditis elegans, Humans, Protein Binding, Protein Interaction Domains and Motifs, Caenorhabditis elegans Proteins metabolism, Glycoproteins metabolism, HSP90 Heat-Shock Proteins metabolism, Receptors, Glucocorticoid metabolism, Tacrolimus Binding Proteins metabolism
- Abstract
The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies. We find that the nematode phosphatase PPH-5 and the prolyl isomerase FKB-6 facilitate the formation of glucocorticoid receptor (GR) complexes with Hsp90. Within these complexes, Hsp90 can perform its closing reaction more efficiently. By combining chemical crosslinking and mass spectrometry, we define contact sites within these assemblies. Compared to the nematode Hsp90 system, the human system shows less cooperative client interaction and a stricter requirement for the co-chaperone p23 to complete the closing reaction of GR·Hsp90·Pp5/Fkbp51/Fkbp52 complexes. In both systems, hormone binding to GR is accelerated by Hsp90 alone and in the presence of its cofactors. Our results show that cooperative complex formation and hormone binding patterns are, in many aspects, conserved between the nematode and human systems.
- Published
- 2020
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20. Blood Contamination in CSF and Its Impact on Quantitative Analysis of Alpha-Synuclein.
- Author
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Barkovits K, Kruse N, Linden A, Tönges L, Pfeiffer K, Mollenhauer B, and Marcus K
- Subjects
- Biomarkers blood, Enzyme-Linked Immunosorbent Assay, Hemoglobins metabolism, Humans, Mass Spectrometry, Proteome metabolism, Blood metabolism, Cerebrospinal Fluid metabolism, alpha-Synuclein blood
- Abstract
Analysis of cerebrospinal fluid (CSF) is important for diagnosis of neurological diseases. Especially for neurodegenerative diseases, abnormal protein abundance in CSF is an important biomarker. However, the quality of CSF is a key factor for the analytic outcome. Any external contamination has tremendous impact on the analysis and the reliability of the results. In this study, we evaluated the effect of blood contamination in CSF with respect to protein biomarker identification. We compared three distinct measures: Combur10-Test
® strips, a specific hemoglobin ELISA, and bottom-up mass spectrometry (MS)-based proteomics for the determination of the general blood contamination level. In parallel, we studied the impact of blood contamination on the detectability of alpha-synuclein (aSyn), a highly abundant protein in blood/erythrocytes and a potential biomarker for Parkinson's disease. Comparable results were achieved, with all three approaches enabling detection of blood levels in CSF down to 0.001%. We found higher aSyn levels with increasing blood contamination, highlighting the difficulty of authentic quantification of this protein in CSF. Based on our results, we identified other markers for blood contamination beyond hemoglobin and defined a grading system for blood levels in CSF samples, including a lower limit of tolerable blood contamination for MS-based biomarker studies., Competing Interests: The authors declare no conflict of interest.- Published
- 2020
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21. The parkin-coregulated gene product PACRG promotes TNF signaling by stabilizing LUBAC.
- Author
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Meschede J, Šadić M, Furthmann N, Miedema T, Sehr DA, Müller-Rischart AK, Bader V, Berlemann LA, Pilsl A, Schlierf A, Barkovits K, Kachholz B, Rittinger K, Ikeda F, Marcus K, Schaefer L, Tatzelt J, and Winklhofer KF
- Subjects
- Animals, Cell Line, Tumor, Cells, Cultured, HEK293 Cells, HeLa Cells, Humans, Mice, Knockout, Microfilament Proteins genetics, Mitophagy genetics, Molecular Chaperones genetics, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Ubiquitin-Protein Ligases genetics, Microfilament Proteins metabolism, Molecular Chaperones metabolism, NF-kappa B metabolism, Signal Transduction, Tumor Necrosis Factor-alpha metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
The Parkin-coregulated gene ( PACRG ), which encodes a protein of unknown function, shares a bidirectional promoter with Parkin ( PRKN ), which encodes an E3 ubiquitin ligase. Because PRKN is important in mitochondrial quality control and protection against stress, we tested whether PACRG also affected these pathways in various cultured human cell lines and in mouse embryonic fibroblasts. PACRG did not play a role in mitophagy but did play a role in tumor necrosis factor (TNF) signaling. Similarly to Parkin, PACRG promoted nuclear factor κB (NF-κB) activation in response to TNF. TNF-induced nuclear translocation of the NF-κB subunit p65 and NF-κB-dependent transcription were decreased in PACRG-deficient cells. Defective canonical NF-κB activation in the absence of PACRG was accompanied by a decrease in linear ubiquitylation mediated by the linear ubiquitin chain assembly complex (LUBAC), which is composed of the two E3 ubiquitin ligases HOIP and HOIL-1L and the adaptor protein SHARPIN. Upon TNF stimulation, PACRG was recruited to the activated TNF receptor complex and interacted with LUBAC components. PACRG functionally replaced SHARPIN in this context. In SHARPIN-deficient cells, PACRG prevented LUBAC destabilization, restored HOIP-dependent linear ubiquitylation, and protected cells from TNF-induced apoptosis. This function of PACRG in positively regulating TNF signaling may help to explain the association of PACRG and PRKN polymorphisms with an increased susceptibility to intracellular pathogens., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2020
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22. Reproducibility, Specificity and Accuracy of Relative Quantification Using Spectral Library-based Data-independent Acquisition.
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Barkovits K, Pacharra S, Pfeiffer K, Steinbach S, Eisenacher M, Marcus K, and Uszkoreit J
- Subjects
- Animals, Cell Line, Chromatography, Liquid methods, Databases, Protein, Mice, Myoblasts metabolism, Peptides analysis, Proteins analysis, Reproducibility of Results, Sensitivity and Specificity, Sequence Analysis, Protein, Software, Tandem Mass Spectrometry methods, Data Accuracy, Peptide Library, Proteome analysis, Proteomics methods
- Abstract
Currently data-dependent acquisition (DDA) is the method of choice for mass spectrometry-based proteomics discovery experiments, but data-independent acquisition (DIA) is steadily becoming more important. One of the most important requirements to perform a DIA analysis is the availability of suitable spectral libraries for peptide identification and quantification. Several studies were performed addressing the evaluation of spectral library performance for protein identification in DIA measurements. But so far only few experiments estimate the effect of these libraries on the quantitative level.In this work we created a gold standard spike-in sample set with known contents and ratios of proteins in a complex protein matrix that allowed a detailed comparison of DIA quantification data obtained with different spectral library approaches. We used in-house generated sample-specific spectral libraries created using varying sample preparation approaches and repeated DDA measurement. In addition, two different search engines were tested for protein identification from DDA data and subsequent library generation. In total, eight different spectral libraries were generated, and the quantification results compared with a library free method, as well as a default DDA analysis. Not only the number of identifications on peptide and protein level in the spectral libraries and the corresponding DIA analysis results was inspected, but also the number of expected and identified differentially abundant protein groups and their ratios.We found, that while libraries of prefractionated samples were generally larger, there was no significant increase in DIA identifications compared with repetitive non-fractionated measurements. Furthermore, we show that the accuracy of the quantification is strongly dependent on the applied spectral library and whether the quantification is based on peptide or protein level. Overall, the reproducibility and accuracy of DIA quantification is superior to DDA in all applied approaches.Data has been deposited to the ProteomeXchange repository with identifiers PXD012986, PXD012987, PXD012988 and PXD014956., (© 2020 Barkovits et al.)
- Published
- 2020
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23. CRN2 binds to TIMP4 and MMP14 and promotes perivascular invasion of glioblastoma cells.
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Solga R, Behrens J, Ziemann A, Riou A, Berwanger C, Becker L, Garrett L, de Angelis MH, Fischer L, Coras R, Barkovits K, Marcus K, Mahabir E, Eichinger L, Schröder R, Noegel AA, and Clemen CS
- Subjects
- Animals, Glioblastoma diagnostic imaging, Mice, Mice, Knockout, Microfilament Proteins deficiency, Tumor Cells, Cultured, Tumor Microenvironment, Tissue Inhibitor of Metalloproteinase-4, Glioblastoma metabolism, Matrix Metalloproteinase 14 metabolism, Microfilament Proteins metabolism, Tissue Inhibitor of Metalloproteinases metabolism
- Abstract
CRN2 is an actin filament binding protein involved in the regulation of various cellular processes including cell migration and invasion. CRN2 has been implicated in the malignant progression of different types of human cancer. We used CRN2 knock-out mice for analyses as well as for crossbreeding with a Tp53/Pten knock-out glioblastoma mouse model. CRN2 knock-out mice were subjected to a phenotyping screen at the German Mouse Clinic. Murine glioblastoma tissue specimens as well as cultured murine brain slices and glioblastoma cell lines were investigated by immunohistochemistry, immunofluorescence, and cell biological experiments. Protein interactions were studied by immunoprecipitation, pull-down, and enzyme activity assays. CRN2 knock-out mice displayed neurological and behavioural alterations, e.g. reduced hearing sensitivity, reduced acoustic startle response, hypoactivity, and less frequent urination. While glioblastoma mice with or without the additional CRN2 knock-out allele exhibited no significant difference in their survival rates, the increased levels of CRN2 in transplanted glioblastoma cells caused a higher tumour cell encasement of murine brain slice capillaries. We identified two important factors of the tumour microenvironment, the tissue inhibitor of matrix metalloproteinase 4 (TIMP4) and the matrix metalloproteinase 14 (MMP14, synonym: MT1-MMP), as novel binding partners of CRN2. All three proteins mutually interacted and co-localised at the front of lamellipodia, and CRN2 was newly detected in exosomes. On the functional level, we demonstrate that CRN2 increased the secretion of TIMP4 as well as the catalytic activity of MMP14. Our results imply that CRN2 represents a pro-invasive effector within the tumour cell microenvironment of glioblastoma multiforme., (Copyright © 2019 The Author(s). Published by Elsevier GmbH.. All rights reserved.)
- Published
- 2019
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24. Intricate Crosstalk Between Lipopolysaccharide, Phospholipid and Fatty Acid Metabolism in Escherichia coli Modulates Proteolysis of LpxC.
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Thomanek N, Arends J, Lindemann C, Barkovits K, Meyer HE, Marcus K, and Narberhaus F
- Abstract
Lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria provide the first line of defense against antibiotics and other harmful compounds. LPS biosynthesis critically depends on LpxC catalyzing the first committed enzyme in this process. In Escherichia coli , the cellular concentration of LpxC is adjusted in a growth rate-dependent manner by the FtsH protease making sure that LPS biosynthesis is coordinated with the cellular demand. As a result, LpxC is stable in fast-growing cells and prone to degradation in slow-growing cells. One of the factors involved in this process is the alarmone guanosine tetraphosphate (ppGpp) but previous studies suggested the involvement of yet unknown factors in LpxC degradation. We established a quantitative proteomics approach aiming at the identification of proteins that are associated with LpxC and/or FtsH at high or low growth rates. The identification of known LpxC and FtsH interactors validated our approach. A number of proteins involved in fatty acid biosynthesis and degradation, including the central regulator FadR, were found in the LpxC and/or FtsH interactomes. Another protein associated with LpxC and FtsH was WaaH, a LPS-modifying enzyme. When overproduced, several members of the LpxC/FtsH interactomes were able to modulate LpxC proteolysis. Our results go beyond the previously established link between LPS and phospholipid biosynthesis and uncover a far-reaching network that controls LPS production by involving multiple enzymes in fatty acid metabolism, phospholipid biosynthesis and LPS modification.
- Published
- 2019
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25. Co-extraction for Metabolomics and Proteomics from a Single CSF Sample.
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Hörmann P, Barkovits K, Marcus K, and Hiller K
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- Brain metabolism, Cerebrospinal Fluid metabolism, Cerebrospinal Fluid Proteins metabolism, Chromatography, Gas methods, Chromatography, High Pressure Liquid methods, Humans, Mass Spectrometry methods, Methanol chemistry, Proteolysis, Trypsin, Workflow, Cerebrospinal Fluid Proteins isolation & purification, Metabolomics methods, Proteomics methods
- Abstract
In the field of neurodegeneration, it is important to identify biomarkers that enable early disease prediction, since these disorders start decades before clinical symptoms manifest. Cerebrospinal fluid (CSF) is considered an excellent source for biomarker discovery since it is in direct contact with the extracellular space of the brain and directly reflects disease-specific changes.While the liquor drainage is no major risk factor for patients, it is still not as easy and popular as simple blood sampling and less liquid can be collected. Especially when a variety of experiments for one cohort is planned, the volume of CSF can be a limiting factor. Therefore, it is essential that extraction and analytical methods are adapted to low amounts of liquor. If in follow-up studies, additional replicates to increase statistical significance or different extraction approaches are planned, the required amounts have to be minimized.With this extraction method, a combined proteomics and metabolomics approach is possible. This opportunity implies a variety of advantages. First, a classification matrix based on the comprehensive data set has a potentially higher accuracy even without a deeper understanding of the biological meaning of the different omics changes. If the proteome and metabolome differences can be linked to each other, this approach can conceivably open so far unknown doors regarding the cause or progression of different diseases like Alzheimer's or Parkinson's disease.
- Published
- 2019
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26. CSF Sample Preparation for Data-Independent Acquisition.
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Barkovits K, Tönges L, and Marcus K
- Subjects
- Biomarkers blood, Biomarkers metabolism, Cerebrospinal Fluid Proteins chemistry, Erythrocyte Count, Erythrocytes chemistry, Erythrocytes metabolism, Humans, Reproducibility of Results, Software, Tandem Mass Spectrometry methods, Biomarkers cerebrospinal fluid, Cerebrospinal Fluid Proteins metabolism, Proteome metabolism, Proteomics methods
- Abstract
To study changes in neurological diseases and to identify disease-related mechanisms or biomarkers for diagnosis, cerebrospinal fluid (CSF) is frequently used for proteomic-based discovery. In the last years, development and application of mass spectrometry (MS) techniques have made essential contributions to proteomic studies including protein identification as well as quantification. Until recently, biomarker discovery studies were performed through bottom-up proteomics utilizing data-dependent acquisition. However, drawbacks like stochastic selection of precursor ions cause the exclusion of low-abundant ions from fragmentation as well as from data analysis leading to technical variances among different samples and result in inconsistent data sets. In contrast, data-independent acquisition (DIA) enables almost complete and reproducible quantitative analysis gaining more and more interest as a method for reliable MS-based protein quantification. Besides the utilization of a proper analysis platform, a prerequisite for biomarker studies is the selection of suitable samples and sample processing strategies. Especially for CSF, blood contamination has tremendous impact on the quantitative analysis. In addition, complex processing methods such as protein or peptide fractionation prior to MS analysis can lead to variabilities that affect the reliability of the quantitative results. Here we present methods to evaluate in a first step the CSF quality in regard to blood contamination for the subsequent MS-based sample preparation and finally a DIA method for the analysis of CSF.
- Published
- 2019
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27. Protein variability in cerebrospinal fluid and its possible implications for neurological protein biomarker research.
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Schilde LM, Kösters S, Steinbach S, Schork K, Eisenacher M, Galozzi S, Turewicz M, Barkovits K, Mollenhauer B, Marcus K, and May C
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- Adult, Aged, Aged, 80 and over, Biomarkers cerebrospinal fluid, Female, Healthy Volunteers, Humans, Longitudinal Studies, Male, Middle Aged, Proteomics, Cerebrospinal Fluid Proteins metabolism, Nervous System Diseases cerebrospinal fluid
- Abstract
Cerebrospinal fluid is investigated in biomarker studies for various neurological disorders of the central nervous system due to its proximity to the brain. Currently, only a limited number of biomarkers have been validated in independent studies. The high variability in the protein composition and protein abundance of cerebrospinal fluid between as well as within individuals might be an important reason for this phenomenon. To evaluate this possibility, we investigated the inter- and intraindividual variability in the cerebrospinal fluid proteome globally, with a specific focus on disease biomarkers described in the literature. Cerebrospinal fluid from a longitudinal study group including 12 healthy control subjects was analyzed by label-free quantification (LFQ) via LC-MS/MS. Data were quantified via MaxQuant. Then, the intra- and interindividual variability and the reference change value were calculated for every protein. We identified and quantified 791 proteins, and 216 of these proteins were abundant in all samples and were selected for further analysis. For these proteins, we found an interindividual coefficient of variation of up to 101.5% and an intraindividual coefficient of variation of up to 29.3%. Remarkably, these values were comparably high for both proteins that were published as disease biomarkers and other proteins. Our results support the hypothesis that natural variability greatly impacts cerebrospinal fluid protein biomarkers because high variability can lead to unreliable results. Thus, we suggest controlling the variability of each protein to distinguish between good and bad biomarker candidates, e.g., by utilizing reference change values to improve the process of evaluating potential biomarkers in future studies., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2018
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28. Characterization of Cerebrospinal Fluid via Data-Independent Acquisition Mass Spectrometry.
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Barkovits K, Linden A, Galozzi S, Schilde L, Pacharra S, Mollenhauer B, Stoepel N, Steinbach S, May C, Uszkoreit J, Eisenacher M, and Marcus K
- Subjects
- Biomarkers cerebrospinal fluid, Biomarkers metabolism, Humans, Reproducibility of Results, Substantia Nigra metabolism, Hydrocephalus cerebrospinal fluid, Mass Spectrometry methods, Proteome metabolism, Proteomics methods
- Abstract
Cerebrospinal fluid (CSF) is in direct contact with the brain and serves as a valuable specimen to examine diseases of the central nervous system through analyzing its components. These include the analysis of metabolites, cells as well as proteins. For identifying new suitable diagnostic protein biomarkers bottom-up data-dependent acquisition (DDA) mass spectrometry-based approaches are most popular. Drawbacks of this method are stochastic and irreproducible precursor ion selection. Recently, data-independent acquisition (DIA) emerged as an alternative method. It overcomes several limitations of DDA, since it combines the benefits of DDA and targeted methods like selected reaction monitoring (SRM). We established a DIA method for in-depth proteome analysis of CSF. For this, four spectral libraries were generated with samples from native CSF ( n = 5), CSF fractionation (15 in total) and substantia nigra fractionation (54 in total) and applied to three CSF DIA replicates. The DDA and DIA methods for CSF were conducted with the same nanoLC parameters using a 180 min gradient. Compared to a conventional DDA method, our DIA approach increased the number of identified protein groups from 648 identifications in DDA to 1574 in DIA using a comprehensive spectral library generated with DDA measurements from five native CSF and 54 substantia nigra fractions. We also could show that a sample specific spectral library generated from native CSF only increased the identification reproducibility from three DIA replicates to 90% (77% with a DDA method). Moreover, by utilizing a substantia nigra specific spectral library for CSF DIA, over 60 brain-originated proteins could be identified compared to only 11 with DDA. In conclusion, the here presented optimized DIA method substantially outperforms DDA and could develop into a powerful tool for biomarker discovery in CSF. Data are available via ProteomeXchange with the identifiers PXD010698, PXD010708, PXD010690, PXD010705, and PXD009624.
- Published
- 2018
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29. Label-free identification of myopathological features with coherent anti-Stokes Raman scattering.
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Niedieker D, GrosserÜschkamp F, Schreiner A, Barkovits K, Kötting C, Marcus K, Gerwert K, and Vorgerd M
- Subjects
- Child, Preschool, Female, Humans, Infant, Male, Middle Aged, Spectrum Analysis, Raman methods, Glycogen Storage Disease Type V diagnostic imaging, Glycogen Storage Disease Type V metabolism, Nonlinear Optical Microscopy methods
- Abstract
Introduction: The aim of this study was the label-free identification of distinct myopathological features with coherent anti-Stokes Raman scattering (CARS) imaging, which leaves the sample intact for further analysis., Methods: The protein distribution was determined without labels by CARS at 2,930 cm
-1 and was compared with the results of standard histological staining., Results: CARS imaging allowed the visualization of glycogen accumulation in glycogen storage disease type 5 (McArdle disease) and of internal nuclei in centronuclear myopathy. CARS identified an inhomogeneous protein distribution within muscle fibers in sporadic inclusion body myositis that was not shown with standard staining. In Duchenne muscular dystrophy, evidence for a higher protein content at the border of hypercontracted fibers was detected., Discussion: CARS enables the label-free identification of distinct myopathological features, possibly paving the way for subsequent proteomic, metabolic, and genomic analyses. Muscle Nerve 58: 457-460, 2018., (© 2018 Wiley Periodicals, Inc.)- Published
- 2018
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30. Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype.
- Author
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Minakaki G, Menges S, Kittel A, Emmanouilidou E, Schaeffner I, Barkovits K, Bergmann A, Rockenstein E, Adame A, Marxreiter F, Mollenhauer B, Galasko D, Buzás EI, Schlötzer-Schrehardt U, Marcus K, Xiang W, Lie DC, Vekrellis K, Masliah E, Winkler J, and Klucken J
- Subjects
- Animals, Autophagosomes drug effects, Autophagy drug effects, Biomarkers metabolism, Cells, Cultured, Chloroquine pharmacology, Exosomes drug effects, Exosomes ultrastructure, Humans, Lewy Body Disease metabolism, Lysosomes drug effects, Macrolides pharmacology, Mice, Neurons drug effects, Neurons pathology, Parkinson Disease metabolism, Protein Transport, Rats, Sprague-Dawley, Autophagosomes metabolism, Autophagy physiology, Exosomes metabolism, Lysosomes metabolism, Neurons metabolism, alpha-Synuclein metabolism
- Abstract
The autophagy-lysosome pathway (ALP) regulates intracellular homeostasis of the cytosolic protein SNCA/alpha-synuclein and is impaired in synucleinopathies, including Parkinson disease and dementia with Lewy bodies (DLB). Emerging evidence suggests that ALP influences SNCA release, but the underlying cellular mechanisms are not well understood. Several studies identified SNCA in exosome/extracellular vesicle (EV) fractions. EVs are generated in the multivesicular body compartment and either released upon its fusion with the plasma membrane, or cleared via the ALP. We therefore hypothesized that inhibiting ALP clearance 1) enhances SNCA release via EVs by increasing extracellular shuttling of multivesicular body contents, 2) alters EV biochemical profile, and 3) promotes SNCA cell-to-cell transfer. Indeed, ALP inhibition increased the ratio of extra- to intracellular SNCA and upregulated SNCA association with EVs in neuronal cells. Ultrastructural analysis revealed a widespread, fused multivesicular body-autophagosome compartment. Biochemical characterization revealed the presence of autophagosome-related proteins, such as LC3-II and SQSTM1. This distinct "autophagosome-exosome-like" profile was also identified in human cerebrospinal fluid (CSF) EVs. After a single intracortical injection of SNCA-containing EVs derived from CSF into mice, human SNCA colocalized with endosome and neuronal markers. Prominent SNCA immunoreactivity and a higher number of neuronal SNCA inclusions were observed after DLB patient CSF EV injections. In summary, this study provides compelling evidence that a) ALP inhibition increases SNCA in neuronal EVs, b) distinct ALP components are present in EVs, and c) CSF EVs transfer SNCA from cell to cell in vivo. Thus, macroautophagy/autophagy may regulate EV protein composition and consequently progression in synucleinopathies.
- Published
- 2018
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31. Distinct metabolomic signature in cerebrospinal fluid in early parkinson's disease.
- Author
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Trezzi JP, Galozzi S, Jaeger C, Barkovits K, Brockmann K, Maetzler W, Berg D, Marcus K, Betsou F, Hiller K, and Mollenhauer B
- Subjects
- Adult, Aged, Butyrates cerebrospinal fluid, Case-Control Studies, Cohort Studies, Dehydroascorbic Acid cerebrospinal fluid, Female, Fructose cerebrospinal fluid, Gas Chromatography-Mass Spectrometry, Humans, Logistic Models, Male, Mannose cerebrospinal fluid, Middle Aged, Biomarkers cerebrospinal fluid, Metabolomics methods, Parkinson Disease cerebrospinal fluid, Parkinson Disease diagnosis
- Abstract
Objective: The purpose of this study was to profile cerebrospinal fluid (CSF) from early-stage PD patients for disease-related metabolic changes and to determine a robust biomarker signature for early-stage PD diagnosis., Methods: By applying a non-targeted and mass spectrometry-driven approach, we investigated the CSF metabolome of 44 early-stage sporadic PD patients yet without treatment (DeNoPa cohort). We compared all detected metabolite levels with those measured in CSF of 43 age- and gender-matched healthy controls. After this analysis, we validated the results in an independent PD study cohort (Tübingen cohort)., Results: We identified that dehydroascorbic acid levels were significantly lower and fructose, mannose, and threonic acid levels were significantly higher (P < .05) in PD patients when compared with healthy controls. These changes reflect pathological oxidative stress responses, as well as protein glycation/glycosylation reactions in PD. Using a machine learning approach based on logistic regression, we successfully predicted the origin (PD patients vs healthy controls) in a second (n = 18) as well as in a third and completely independent validation set (n = 36). The biomarker signature is composed of the three markers-mannose, threonic acid, and fructose-and allows for sample classification with a sensitivity of 0.790 and a specificity of 0.800., Conclusion: We identified PD-specific metabolic changes in CSF that were associated with antioxidative stress response, glycation, and inflammation. Our results disentangle the complexity of the CSF metabolome to unravel metabolome changes related to early-stage PD. The detected biomarkers help understanding PD pathogenesis and can be applied as biomarkers to increase clinical diagnosis accuracy and patient care in early-stage PD. © 2017 International Parkinson and Movement Disorder Society., (© 2017 International Parkinson and Movement Disorder Society.)
- Published
- 2017
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32. Metabolic profiling of body fluids and multivariate data analysis.
- Author
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Trezzi JP, Jäger C, Galozzi S, Barkovits K, Marcus K, Mollenhauer B, and Hiller K
- Abstract
Metabolome analyses of body fluids are challenging due pre-analytical variations, such as pre-processing delay and temperature, and constant dynamical changes of biochemical processes within the samples. Therefore, proper sample handling starting from the time of collection up to the analysis is crucial to obtain high quality samples and reproducible results. A metabolomics analysis is divided into 4 main steps: 1) Sample collection, 2) Metabolite extraction, 3) Data acquisition and 4) Data analysis. Here, we describe a protocol for gas chromatography coupled to mass spectrometry (GC-MS) based metabolic analysis for biological matrices, especially body fluids. This protocol can be applied on blood serum/plasma, saliva and cerebrospinal fluid (CSF) samples of humans and other vertebrates. It covers sample collection, sample pre-processing, metabolite extraction, GC-MS measurement and guidelines for the subsequent data analysis. Advantages of this protocol include: •Robust and reproducible metabolomics results, taking into account pre-analytical variations that may occur during the sampling process•Small sample volume required•Rapid and cost-effective processing of biological samples•Logistic regression based determination of biomarker signatures for in-depth data analysis.
- Published
- 2017
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33. Mutant desmin substantially perturbs mitochondrial morphology, function and maintenance in skeletal muscle tissue.
- Author
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Winter L, Wittig I, Peeva V, Eggers B, Heidler J, Chevessier F, Kley RA, Barkovits K, Strecker V, Berwanger C, Herrmann H, Marcus K, Kornblum C, Kunz WS, Schröder R, and Clemen CS
- Subjects
- Animals, Cytoskeleton metabolism, Cytoskeleton pathology, Desmin metabolism, Humans, Intermediate Filaments genetics, Mice, Transgenic, Mitochondria pathology, Muscular Diseases pathology, Mutation genetics, Desmin genetics, Intermediate Filaments pathology, Mitochondria metabolism, Muscle, Skeletal pathology, Muscular Diseases genetics
- Abstract
Secondary mitochondrial dysfunction is a feature in a wide variety of human protein aggregate diseases caused by mutations in different proteins, both in the central nervous system and in striated muscle. The functional relationship between the expression of a mutated protein and mitochondrial dysfunction is largely unknown. In particular, the mechanism how this dysfunction drives the disease process is still elusive. To address this issue for protein aggregate myopathies, we performed a comprehensive, multi-level analysis of mitochondrial pathology in skeletal muscles of human patients with mutations in the intermediate filament protein desmin and in muscles of hetero- and homozygous knock-in mice carrying the R349P desmin mutation. We demonstrate that the expression of mutant desmin causes disruption of the extrasarcomeric desmin cytoskeleton and extensive mitochondrial abnormalities regarding subcellular distribution, number and shape. At the molecular level, we uncovered changes in the abundancy and assembly of the respiratory chain complexes and supercomplexes. In addition, we revealed a marked reduction of mtDNA- and nuclear DNA-encoded mitochondrial proteins in parallel with large-scale deletions in mtDNA and reduced mtDNA copy numbers. Hence, our data demonstrate that the expression of mutant desmin causes multi-level damage of mitochondria already in early stages of desminopathies.
- Published
- 2016
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34. Statically Adsorbed Coatings for High Separation Efficiency and Resolution in CE-MS Peptide Analysis: Strategies and Implementation.
- Author
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Pattky M, Barkovits K, Marcus K, Weiergräber OH, and Huhn C
- Subjects
- Adsorption, Cations chemistry, Hydrogen-Ion Concentration, Peptides chemistry, Proteins chemistry, Electrophoresis, Capillary methods, Peptides isolation & purification, Proteins isolation & purification, Spectrometry, Mass, Electrospray Ionization methods
- Abstract
Coatings are necessary to prevent protein and peptide adsorption to the capillary surface and obtain high intermediate precision. In this protocol, we first present our basic strategy to address peptide separation using three different coatings: one neutral and two cationic coatings, the latter largely differing in their induced electroosmotic mobility. In detail, we will describe how we apply the statically adsorbed coatings to obtain very high plate numbers and high repeatability.With some model examples, we clearly describe the scope of the method for the analysis of peptide samples: tryptic digests are addressed as well as small glycoproteins and glycopeptides largely differing in their effective electrophoretic mobility. We also show that the method is suitable for a fast screening of peptide samples despite a high matrix load comprising of up to 500 mmol/L sodium chloride. We demonstrate that this basic CE-MS method is rather independent of the polarity of the analytes with a very fast near-baseline separation of very hydrophobic Aβ peptides related to the onset of Alzheimer's disease. These examples will give an impression, which coating is most suitable for a specific analytical application.Special attention is paid to difficult aspects of the coating procedure and the CE-MS method, e.g., the potential of cross-contamination when changing the coatings.
- Published
- 2016
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35. Amyloid-β as a biomarker for Alzheimer's disease: quantification methods in body fluids.
- Author
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Galozzi S, Marcus K, and Barkovits K
- Subjects
- Alzheimer Disease blood, Alzheimer Disease cerebrospinal fluid, Amyloid beta-Peptides blood, Amyloid beta-Peptides cerebrospinal fluid, Body Fluids metabolism, Humans, Alzheimer Disease metabolism, Amyloid beta-Peptides metabolism, Biomarkers metabolism
- Abstract
Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by neuronal impairment leading to dramatic changes in brain. Amyloid-β peptides and tau protein are the most promising biomarkers for AD. Cerebrospinal fluid and plasma are used to determine the concentration of these species. Since the pathological processes of AD start decades before the first symptoms, biomarkers may provide the possibility of early disease detection. The application of rapidly emerging technology, such as mass spectrometry, has opened new avenues to accelerate biomarker discovery, both for diagnostic as well as for prognostic purposes. This review summarizes AD biomarker studies with focus on amyloid-β peptides in biological fluids and their quantification with immunoassays as well as the latest mass spectrometry-based methods.
- Published
- 2015
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36. Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies.
- Author
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Chen J, Shinde S, Koch MH, Eisenacher M, Galozzi S, Lerari T, Barkovits K, Subedi P, Krüger R, Kuhlmann K, Sellergren B, Helling S, and Marcus K
- Subjects
- Amino Acid Sequence, Animals, Cerebrospinal Fluid metabolism, Chromatography, Ion Exchange, Chromatography, Liquid, HEK293 Cells, Humans, Mice, Molecular Sequence Data, Phosphopeptides chemistry, Phosphorylation, Phosphoserine metabolism, Proteomics, Solid Phase Extraction, Spectrometry, Mass, Electrospray Ionization, Antibodies metabolism, Molecular Imprinting methods, Phosphopeptides metabolism, Plastics chemistry
- Abstract
Phosphospecific enrichment techniques and mass spectrometry (MS) are essential tools for comprehending the cellular phosphoproteome. Here, we report a fast and simple approach for low sequence-bias phosphoserine (pS) peptide capture and enrichment that is compatible with low biological or clinical sample input. The approach exploits molecularly imprinted polymers (MIPs, "plastic antibodies") featuring tight neutral binding sites for pS or pY that are capable of cross-reacting with phosphopeptides of protein proteolytic digests. The versatility of the resulting method was demonstrated with small samples of whole-cell lysate from human embryonic kidney (HEK) 293T cells, human neuroblastoma SH-SY5Y cells, mouse brain or human cerebrospinal fluid (CSF). Following pre-fractionation of trypsinized proteins by strong cation exchange (SCX) chromatography, pS-MIP enrichment led to the identification of 924 phosphopeptides in the HEK 293T whole-cell lysate, exceeding the number identified by TiO2-based enrichment (230). Moreover, the phosphopeptides were extracted with low sequence bias and showed no evidence for the characteristic preference of TiO2 for acidic amino acids (aspartic and glutamic acid). Applying the method to human CSF led to the discovery of 47 phosphopeptides belonging to 24 proteins and revealed three previously unknown phosphorylation sites.
- Published
- 2015
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37. Analysis of disease-associated protein expression using quantitative proteomics—fibulin-5 is expressed in association with hepatic fibrosis.
- Author
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Bracht T, Schweinsberg V, Trippler M, Kohl M, Ahrens M, Padden J, Naboulsi W, Barkovits K, Megger DA, Eisenacher M, Borchers CH, Schlaak JF, Hoffmann AC, Weber F, Baba HA, Meyer HE, and Sitek B
- Subjects
- Biomarkers metabolism, Biopsy, Carrier Proteins genetics, Carrier Proteins metabolism, Chondroitin Sulfate Proteoglycans genetics, Chondroitin Sulfate Proteoglycans metabolism, Cohort Studies, Collagen genetics, Collagen metabolism, Extracellular Matrix Proteins metabolism, Female, Glycoproteins genetics, Glycoproteins metabolism, Hepatitis B complications, Hepatitis B genetics, Hepatitis B virology, Hepatitis C complications, Hepatitis C genetics, Hepatitis C virology, Humans, Keratan Sulfate genetics, Keratan Sulfate metabolism, Liver pathology, Liver virology, Liver Cirrhosis complications, Liver Cirrhosis genetics, Liver Cirrhosis virology, Lumican, Male, Middle Aged, Proteomics methods, Extracellular Matrix Proteins genetics, Hepatitis B diagnosis, Hepatitis C diagnosis, Liver metabolism, Liver Cirrhosis diagnosis, Transcriptome
- Abstract
Hepatic fibrosis and cirrhosis are major health problems worldwide. Until now, highly invasive biopsy remains the diagnostic gold standard despite many disadvantages. To develop noninvasive diagnostic assays for the assessment of liver fibrosis, it is urgently necessary to identify molecules that are robustly expressed in association with the disease. We analyzed biopsied tissue samples from 95 patients with HBV/HCV-associated hepatic fibrosis using three different quantification methods. We performed a label-free proteomics discovery study to identify novel disease-associated proteins using a subset of the cohort (n = 27). Subsequently, gene expression data from all available clinical samples were analyzed (n = 77). Finally, we performed a targeted proteomics approach, multiple reaction monitoring (MRM), to verify the disease-associated expression in samples independent from the discovery approach (n = 68). We identified fibulin-5 (FBLN5) as a novel protein expressed in relation to hepatic fibrosis. Furthermore, we confirmed the altered expression of microfibril-associated glycoprotein 4 (MFAP4), lumican (LUM), and collagen alpha-1(XIV) chain (COL14A1) in association to hepatic fibrosis. To our knowledge, no tissue-based quantitative proteomics study for hepatic fibrosis has been performed using a cohort of comparable size. By this means, we add substantial evidence for the disease-related expression of the proteins examined in this study.
- Published
- 2015
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38. MS-based methods for biomarkers of Parkinson's disease: what is the future?
- Author
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Barkovits K, Helling S, and Marcus K
- Subjects
- Biomarkers cerebrospinal fluid, Electrophoresis, Gel, Two-Dimensional, Enzyme-Linked Immunosorbent Assay, Humans, Parkinson Disease metabolism, Proteomics, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, alpha-Synuclein metabolism, Biomarkers analysis, Parkinson Disease diagnosis, Parkinson Disease pathology
- Published
- 2015
- Full Text
- View/download PDF
39. Metabolic flux of extracellular heme uptake in Pseudomonas aeruginosa is driven by the iron-regulated heme oxygenase (HemO).
- Author
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Barker KD, Barkovits K, and Wilks A
- Subjects
- Blotting, Western, Chromatography, High Pressure Liquid, Electrophoresis, Polyacrylamide Gel, Genetic Complementation Test, Heme Oxygenase (Decyclizing) genetics, Spectrometry, Mass, Electrospray Ionization, Heme metabolism, Heme Oxygenase (Decyclizing) metabolism, Iron metabolism, Pseudomonas aeruginosa metabolism
- Abstract
Heme utilization by Pseudomonas aeruginosa involves several proteins required for internalization and degradation of heme. In the following report we provide the first direct in vivo evidence for the specific degradation of extracellular heme to biliverdin (BV) by the iron-regulated HemO. Moreover, through isotopic labeling ((13)C-heme) and electrospray ionization-MS analysis we have confirmed the regioselectivity and ratio of (13)C-δ and β-BV IX (70:30) is identical in vivo to that previously observed for the purified protein. Furthermore, the (13)C-BV IXδ and BV IXβ products are effluxed from the cell by an as yet unidentified transporter. Conversion of extracellular heme to BV is dependent solely on the iron-regulated HemO as evidenced by the lack of BV production in the P. aeruginosa hemO deletion strain. Complementation of P. aeruginosa ΔhemO with a plasmid expressing either the wild type HemO or α-regioselective HemO mutant restored extracellular heme uptake and degradation. In contrast deletion of the gene encoding the cytoplasmic heme-binding protein, PhuS, homologs of which have been proposed to be heme oxygenases, did not eliminate (13)C-BV IXδ and IXβ production. In conclusion the metabolic flux of extracellular heme as a source of iron is driven by the catalytic action of HemO.
- Published
- 2012
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40. Function of the bacteriophytochrome BphP in the RpoS/Las quorum-sensing network of Pseudomonas aeruginosa.
- Author
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Barkovits K, Schubert B, Heine S, Scheer M, and Frankenberg-Dinkel N
- Subjects
- Bacterial Proteins genetics, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Phytochrome genetics, Proteome, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sigma Factor genetics, Trans-Activators genetics, Bacterial Proteins metabolism, Phytochrome metabolism, Pseudomonas aeruginosa growth & development, Quorum Sensing, Sigma Factor metabolism, Trans-Activators metabolism
- Abstract
The bacterial phytochrome of Pseudomonas aeruginosa (PaBphP) is an in vitro-active red/far-red light sensor histidine kinase of a two-component regulatory system. Despite solid biochemical data, its function in this heterotrophic, opportunistic pathogen is still unknown. Previous studies established that the genes encoding the two necessary phytochrome components BphO, a chromophore-producing haem oxygenase, and BphP, the apo-phytochrome, are co-transcribed in a bicistronic operon. Transcription has been shown to be induced in the stationary phase and to be dependent on the alternative sigma factor RpoS. Here we show an additional regulation of bphP expression through the quorum-sensing (QS) regulator LasR. This regulation is also reflected in a combination of expression profile experiments and proteome analyses of wild-type and phytochrome-deficient strains. While PaBphP has a pleiotropic effect on global gene expression, 66 % of the downregulated genes in the phytochrome mutant display a link to the Las QS system. Most of these genes seem to be indirectly regulated by LasR through BphP and the unknown response regulator BphR. A model of phytochrome function within the Las QS network is presented.
- Published
- 2011
- Full Text
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41. Expression of the phytochrome operon in Pseudomonas aeruginosa is dependent on the alternative sigma factor RpoS.
- Author
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Barkovits K, Harms A, Benkartek C, Smart JL, and Frankenberg-Dinkel N
- Subjects
- Biliverdine metabolism, Gene Expression Regulation, Bacterial, Heme metabolism, Heme Oxygenase (Decyclizing) physiology, Operon, Phytochrome chemistry, Phytochrome genetics, Pseudomonas aeruginosa genetics, Quorum Sensing genetics, Phytochrome metabolism, Pseudomonas aeruginosa metabolism, Regulon physiology, Sigma Factor physiology
- Abstract
Phytochromes are red/far-red light photoreceptors found in plants, cyanobacteria and heterotrophic bacteria. Biochemical analyses have established that the genes bphO and bphP (PA4116 and PA4117) of Pseudomonas aeruginosa encode both phytochrome components: BphO, a heme oxygenase that produces the linear tetrapyrrole chromophore biliverdin IXalpha, and BphP, the apo-phytochrome. Reverse transcription-PCR established that both genes form a bicistronic operon. Expression of the bphOP operon was induced in the stationary phase, indicating an involvement of the P. aeruginosa quorum-sensing system and/or the stationary-phase alternative sigma factor RpoS. Bioinformatic analyses of the promoter region revealed a potential binding site for the quorum sensing regulators LasR and/or RhlR. While a direct involvement of the quorum-sensing system could be ruled out, the dependence of bphOP expression on RpoS was clearly demonstrated. Chromosomal knock-out mutants showed identical growth behavior as a wild type under various conditions but increased levels of pyocyanin were detected in the DeltabphO strain. Additionally, this strain showed decreased heat tolerance in the stationary phase, indicating a potential protective role of the BphO reaction product biliverdin. Therefore, BphO might have an additional function besides providing the chromophore for BphP and both proteins are likely to fulfill a task in the stationary phase.
- Published
- 2008
- Full Text
- View/download PDF
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