Dataset Information

Secretome of Bjerkandera adusta

ABSTRACT: The secretome of the white-rot fungus Bjerkandera adusta produced in synthetic Kirk medium was compared to that supplemented with an aqueous phenol-rich extract of olive mill residues (ADOR). Distinct changes in the protein composition of oxidoreductases, namely diverse class- II peroxidases and aryl alcohol oxidases were found. In the secretome produced in ADOR- supplemented medium (ASC) 157 different proteins were identified, whereas only 59 were secreted in cultures without ADOR supplementation (Kirk medium culture; KM). Peptide sequence analysis done by LC-MS turned out that the number of peroxidases produced in ASC was more than doubled (from 4 to 11) compared to KM. Two short manganese peroxidases (MnP1 & MnP6) and one versatile peroxidase (VP1) represented 29% of the relative abundance (NSAF) in ASC. Two of them (MnP1 & VP1) also detected in KM displayed a relative abundance (NSAF) of only 3%. Further peroxidases present in ASC were one lignin peroxidase (LiP2), one generic peroxidase (GP) and three dye-decolorizing peroxidases (DyPs). The relative abundance of DyPs and aryl alcohol oxidases (AAO) were lower in ASC in comparison to KM. In addition to peptide sequence analysis, the secretion of Mn2+-oxidizing peroxidases as well as AAOs were followed by enzyme measurement. The Mn2+-oxidizing activity increased nearly 30-fold (from 10 to 281 U l-1) after ADOR addition. Two enzymes responsible for that activity were successfully purified (BadVPI and BadVPII). To prove a potential involvement of these enzymes in the degradation of aromatic compounds, BadVPI was tested for its ability to degrade the recalcitrant dehydrogenated polymer (DHP, synthetic lignin). These results show that natural phenol-rich materials act as secretome-stimulating additives. Applying these substances enables us to investigate fungal degradation and detoxification processes and gives more insight into the complexity of fungal secretomes, e.g. of. white-rot fungi.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Bjerkandera Adusta

SUBMITTER: Nico Jehmlich

LAB HEAD: Christiane Liers

PROVIDER: PXD000962 | Pride | 2018-02-28

REPOSITORIES: Pride

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Publications

Differences in the secretion pattern of oxidoreductases from Bjerkandera adusta induced by a phenolic olive mill extract.

Reina Rocío R Kellner Harald H Jehmlich Nico N Ullrich René R García-Romera Inmaculada I Aranda Elisabet E Liers Christiane C

Fungal genetics and biology : FG & B 20140725

The secretome of the white-rot fungus Bjerkandera adusta produced in synthetic Kirk medium was compared to that supplemented with an aqueous phenol-rich extract of dry olive mill residues (ADOR). Distinct changes in the protein composition of oxidoreductases, namely diverse class-II peroxidases and aryl alcohol oxidases were found. In the ADOR-supplemented medium (ASC), 157 distinct proteins were identified by the secretome analysis, whereas only 59 of them were identified without ADOR supplemen ...[more]

PMID: 25069088

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Project description:Treatment strategies of critical nerve injuries involving nerve gaps more than 3 cm are still not optimal. The need for an alternative to the standard autologous nerve grafts has led to the exploration of adipose derived stem cells (ASC) seeded in bioartificial nerve conduits for enhancement of peripheral nerve regeneration. ASC can be differentiated into Schwann cell like cells, which is believed to improve their neurotrophic potency. Nevertheless, the differentiation process requires several weeks and there is no evidence that the cells maintain their differentiated phenotype in vivo. Thus, the present study evaluated the synergistic effect of undifferentiated ASC with exogenously added growth factors in vitro with the aim to improve the neurotrophic potency of undifferentiated ASC through short ex-vivo stimulation with growth factors. The study furthermore provides a comparative in vitro assay of the six well-known neurotrophic factors NGF, GDNF, BDNF, CNTF, NT3 and NT4. ASC were cultured and thus stimulated in the presence of the respective exogenous growth factor for three days and resulting conditioned medium was evaluated in an in vitro axonal outgrowth assay. In addition, also unstimulated ASC’s conditioned medium was tested in combination with the respective exogenous growth factor for the same assay, which was perfomed on dorsal root ganglion (DRG) explants from 10 days old embryonic chicken. DRG explants from most promising conditions were further analyzed for upregulated STAT-3 and GAP-43 by qRT-PCR. Furthermore, also proteomics and phosphoproteomics were performed for ASC and DRG explants using mass spectrometry. The quantity of selected proteins contained in the ASC’s conditioned medium was investigated by ELISA. Combination of ASC’s conditioned medium with growth factors generally resulted in significantly enhanced axonal outgrowth when compared with their control of DRG explants cultured with the respective growth factor only. Specifically, conditioned medium derived from NT3-stimulated as well as ASC’s conditioned medium supplemented with NT3 or BDNF elicited significant axonal outgrowth from DRG explants in contrast to all other experimental conditions. Significant upregulation of STAT-3 and GAP-43 was evidenced for DRG explants grown in NT3-stimulated ASC’s conditioned medium and to a certain extent also for DRG cultured in ASC’s conditioned medium supplemented with NT3.

Project description:We have previously reported that the deficiency of p53 alone or in combination with Rb (Rb-/- p53-/-) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53-/- and Rb-/-p53-/- MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53-/- and Rb-/-p53-/- ASCs. In addition, gene expression profiling revealed a link between p53- or Rb-p53-deficient BM-MSCs and ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem a crucial factor in the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless of the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development. BM-MSC and ASC cultures were established from 3 mouse strains: (a) WT, (b) p53loxP/loxP and (c) p53loxP/loxP RbloxP/loxP. The corresponding mutant cells were generated by excision of the LoxP-flanked sequences by infection of all MSC cultures with adenoviral vectors expressing the Cre-recombinase gene (Ad-CMV-Cre). NSG mice were inoculated subcutaneously with 5×10^6 mutant cells. Animals were killed when tumors reached 1 cm3 or 150 days after infusion. Some of the obtained tumors were mechanically disaggregated to establish ex vivo MSC-transformed cell lines. Gene expression analysis was performed using BM-MSCs and ASCs from all genotypes, as well as tumor cell lines derived from p53-/- and p53-/-Rb-/- BM-MSC and ASC cultures.

Dataset Information

Secretome of Bjerkandera adusta

Publications

Differences in the secretion pattern of oxidoreductases from Bjerkandera adusta induced by a phenolic olive mill extract.

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