Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion

Hung, Chien‐Wen; Klein, Tobias; Cassidy, Liam; Linke, Dennis; Lange, Sabrina; Anders, Uwe; Bureik, Matthias; Heinzle, Elmar; Schneider, Konstantin; Tholey, Andreas

doi:10.1074/mcp.m115.051474

Cited by 9 publications

(7 citation statements)

References 58 publications

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“…A FC cutoff of ≥1.25 for overexpression and ≤0.8 for the downregulation of proteins was selected to be biologically significant, and an adjusted p -value, that is, q ≤ 0.05, was selected for statistical significance. A similar range of FCs has been previously reported by studies using isobaric labeling. − Rotenone treatment highlighted 421 differentially regulated proteins (FC ≥1.25 or ≤0.8), of which 144 were significantly altered ( q ≤ 0.05, with respect to untreated cell control, Figure B). Yashtimadhu choorna extract cotreatment was found to significantly regulate 88 proteins ( q ≤ 0.05, with respect to rotenone), which included restoration of 84 proteins that were dysregulated by rotenone and an additional regulation of 4 proteins (Figure C).…”

Section: Results and Discussionmentioning

confidence: 99%

Identification of Molecular Network Associated with Neuroprotective Effects of Yashtimadhu (Glycyrrhiza glabra L.) by Quantitative Proteomics of Rotenone-Induced Parkinson’s Disease Model

et al. 2020

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Parkinson’s disease (PD) is a progressive neurodegenerative disorder, whose treatment with modern therapeutics leads to a plethora of side effects with prolonged usage. Therefore, the management of PD with complementary and alternative medicine is often pursued. In the Ayurveda system of alternative medicine, Yashtimadhu choorna, a Medhya Rasayana (nootropic), prepared from the dried roots of Glycyrrhiza glabra L. (licorice), is prescribed for the management of PD with a favorable outcome. We pursued to understand the neuroprotective effects of Yashtimadhu choorna against a rotenone-induced cellular model of PD using differentiated IMR-32 cells. Cotreatment with Yashtimadhu choorna extract rescued rotenone-induced apoptosis and hyperphosphorylation of ERK-1/2. Quantitative proteomic analysis of six peptide fractions from independent biological replicates acquired 1,561,169 mass spectra, which when searched resulted in 565,008 peptide-spectrum matches mapping to 30,554 unique peptides that belonged to 4864 human proteins. Proteins commonly identified in biological replicates and >4 PSMs were considered for further analysis, leading to a refined set of 3720 proteins. Rotenone treatment differentially altered 144 proteins (fold ≥1.25 or ≤0.8), involved in mitochondrial, endoplasmic reticulum, and autophagy functions. Cotreatment with Yashtimadhu choorna extract rescued 84 proteins from the effect of rotenone and an additional regulation of 4 proteins. Network analysis highlighted the interaction of proteins and pathways regulated by them, which can be targeted for neuroprotection. Validation of proteomics data highlighted that Yashtimadhu confers neuroprotection by preventing mitochondrial oxidative stress and apoptosis. This discovery will pave the way for understanding the molecular action of Ayurveda drugs and developing novel therapeutics for PD.

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Section: Results and Discussionmentioning

confidence: 99%

Identification of Molecular Network Associated with Neuroprotective Effects of Yashtimadhu (Glycyrrhiza glabra L.) by Quantitative Proteomics of Rotenone-Induced Parkinson’s Disease Model

et al. 2020

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“…To identify proteins released by OEC in response to stimulation with LPS into the conditioned media, nano‐liquid chromatography–mass spectrometry (LC‐MS) was performed using a set of cell culture supernatants from four OEC donors. To remove low molecular weight contaminants and salts from the samples and to concentrate proteins, supernatants were supplied to a SDS‐PAGE but only migrated into the stacking gel without further protein separation . After tryptic digestion, the resulting peptides were separated via ion‐paring reversed phase chromatography and analysed online by electrospray Orbitrap‐tandem MS, followed by database search for protein identification.…”

Section: Resultsmentioning

confidence: 99%

“…LC‐MS analysis was performed using a Dionex UltiMate 3000 RSLCnano UPLC system (Thermo, Germany) equipped with an Acclaim PepMap RSLC nano column (C18, 100 Å, 2 μm, 75 μm × 500 mm) (Thermo, Germany) coupled online to a QExative Plus mass spectrometer (Thermo, Germany) as described previously . The solvents used were buffer A: 0.05% formic acid (FA), buffer B: 80% ACN + 0.04% FA; the flow rate was 300 nl/min.…”

Section: Methodsmentioning

confidence: 99%

Site‐specific and endothelial‐mediated dysfunction of the alveolar‐capillary barrier in response to lipopolysaccharides

Janga

Cassidy

Wang

et al. 2017

J Cellular Molecular Medi

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Infectious agents such as lipopolysaccharides (LPS) challenge the functional properties of the alveolar‐capillary barrier (ACB) in the lung. In this study, we analyse the site‐specific effects of LPS on the ACB and reveal the effects on the individual cell types and the ACB as a functional unit. Monocultures of H441 epithelial cells and co‐cultures of H441 with endothelial cells cultured on Transwells® were treated with LPS from the apical or basolateral compartment. Barrier properties were analysed by the transepithelial electrical resistance (TEER), by transport assays, and immunostaining and assessment of tight junctional molecules at protein level. Furthermore, pro‐inflammatory cytokines and immune‐modulatory molecules were evaluated by ELISA and semiquantitative real‐time PCR. Liquid chromatography–mass spectrometry‐based proteomics (LS‐MS) was used to identify proteins and effector molecules secreted by endothelial cells in response to LPS. In co‐cultures treated with LPS from the basolateral compartment, we noticed a significant reduction of TEER, increased permeability and induction of pro‐inflammatory cytokines. Conversely, apical treatment did not affect the barrier. No changes were noticed in H441 monoculture upon LPS treatment. However, LPS resulted in an increased expression of pro‐inflammatory cytokines such as IL‐6 in OEC and in turn induced the reduction of TEER and an increase in SP‐A expression in H441 monoculture, and H441/OEC co‐cultures after LPS treatment from basolateral compartment. LS‐MS‐based proteomics revealed factors associated with LPS‐mediated lung injury such as ICAM‐1, VCAM‐1, Angiopoietin 2, complement factors and cathepsin S, emphasizing the role of epithelial–endothelial crosstalk in the ACB in ALI/ARDS.

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“…Additionally, stacking gel-based SDS-PAGE followed by LC-MS analysis (28) was employed for the analysis of the proteome of two MetSV samples. Briefly, 10-g aliquots of the protein extract, either without or with washing of the filtered extract (samples 1 and 2, respectively) were suspended in SDS-PAGE loading buffer, heated at 95°C for 5 min, and loaded onto 12% SDS-PAGE gels.…”

Section: Methodsmentioning

confidence: 99%

Methanosarcina Spherical Virus, a Novel Archaeal Lytic Virus Targeting Methanosarcina Strains

Weidenbach

Nickel

Neve

et al. 2017

J Virol

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A novel archaeal lytic virus targeting species of the genus was isolated using strain Gö1 as the host. Due to its spherical morphology, the virus was designated hanosarcinapherical irus (MetSV). Molecular analysis demonstrated that MetSV contains double-stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs), all oriented in the same direction. Functions were predicted for some of these ORFs, i.e., such as DNA polymerase, ATPase, and DNA-binding protein as well as envelope (structural) protein. MetSV-derived spacers in CRISPR loci were detected in several published draft genomes using bioinformatic tools, revealing a potential protospacer-adjacent motif (PAM) motif (TTA/T). Transcription and expression of several predicted viral ORFs were validated by reverse transcription-PCR (RT-PCR), PAGE analysis, and liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Analysis of core lipids by atmospheric pressure chemical ionization (APCI) mass spectrometry showed that MetSV and both contain archaeol and glycerol dialkyl glycerol tetraether without a cyclopentane moiety (GDGT-0). The MetSV host range is limited to strains growing as single cells (, and). In contrast, strains growing as sarcina-like aggregates were apparently protected from infection. Heterogeneity related to morphology phases in cultures allowed acquisition of resistance to MetSV after challenge by growing cultures as sarcina-like aggregates. CRISPR/Cas-mediated resistance was excluded since neither of the two CRISPR arrays showed MetSV-derived spacer acquisition. Based on these findings, we propose that changing the morphology from single cells to sarcina-like aggregates upon rearrangement of the envelope structure prevents infection and subsequent lysis by MetSV. Methanoarchaea are among the most abundant organisms on the planet since they are present in high numbers in major anaerobic environments. They convert various carbon sources, e.g., acetate, methylamines, or methanol, to methane and carbon dioxide; thus, they have a significant impact on the emission of major greenhouse gases. Today, very little is known about viruses specifically infecting methanoarchaea that most probably impact the abundance of methanoarchaea in microbial consortia. Here, we characterize the first identified -infecting virus (MetSV) and show a mechanism for acquiring resistance against MetSV. Based on our results, we propose that growth as sarcina-like aggregates prevents infection and subsequent lysis. These findings allow new insights into the virus-host relationship in methanogenic community structures, their dynamics, and their phase heterogeneity. Moreover, the availability of a specific virus provides new possibilities to deepen our knowledge of the defense mechanisms of potential hosts and offers tools for genetic manipulation.

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Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion

Cited by 9 publications

References 58 publications

Identification of Molecular Network Associated with Neuroprotective Effects of Yashtimadhu (Glycyrrhiza glabra L.) by Quantitative Proteomics of Rotenone-Induced Parkinson’s Disease Model

Identification of Molecular Network Associated with Neuroprotective Effects of Yashtimadhu (Glycyrrhiza glabra L.) by Quantitative Proteomics of Rotenone-Induced Parkinson’s Disease Model

Site‐specific and endothelial‐mediated dysfunction of the alveolar‐capillary barrier in response to lipopolysaccharides

Methanosarcina Spherical Virus, a Novel Archaeal Lytic Virus Targeting Methanosarcina Strains

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