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Penicillium chrysogenum as a model system for studying cellular effects of methylglyoxal.

Scheckhuber CQ - BMC Microbiol. (2015)

Bottom Line: The biologically most important compound of this class, methylglyoxal, results from spontaneous phosphate elimination from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate which are intermediate glycolysis products.Methylglyoxal leads to growth rate reduction of this fungus so that the entry into the stationary phase is delayed.Furthermore, three proteins are identified that are present in lower abundance when methylglyoxal is added to the growth medium (aldo-keto reductase [Pc22g04850], 5-methyl-tetrahydropteroyl-triglutamate-homocysteine S-methyltransferase [Pc22g18630] and NAD-dependent formate dehydrogenase [Pc12g04310]).

View Article: PubMed Central - PubMed

Affiliation: Senckenberg Research Institute, LOEWE Excellence Cluster for Integrative Fungal Research (IPF), Georg-Voigt-Str. 14-16, D-60325, Frankfurt am Main, Germany. Christian.Scheckhuber@senckenberg.de.

ABSTRACT

Background: α-oxoaldehydes are formed as toxic by-products during metabolic activity. The biologically most important compound of this class, methylglyoxal, results from spontaneous phosphate elimination from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate which are intermediate glycolysis products. Methylglyoxal-mediated modification of lipids, nucleic acids and proteins is known to lead to the formation of advanced glycation end products. These modifications contribute to the aetiology of severe diseases like diabetes and neurodegenerative disorders. By using simple model organisms it is possible to conveniently study the effects of methylglyoxal on cellular processes. Here, results are presented on the effects of methylglyoxal on mycelium growth, stationary phase entry (monitored by autophagy induction), mitochondrial morphology and protein composition in the filamentous fungus Penicillium chrysogenum.

Results: Methylglyoxal leads to growth rate reduction of this fungus so that the entry into the stationary phase is delayed. Mitochondrial morphology is not changed by methylglyoxal. However, rapamycin-mediated fragmentation of mitochondria is prevented by methylglyoxal. Furthermore, three proteins are identified that are present in lower abundance when methylglyoxal is added to the growth medium (aldo-keto reductase [Pc22g04850], 5-methyl-tetrahydropteroyl-triglutamate-homocysteine S-methyltransferase [Pc22g18630] and NAD-dependent formate dehydrogenase [Pc12g04310]).

Conclusions: The presented results contribute to the understanding of cellular pathways and mechanisms that are affected by the ubiquitous α-oxoaldehyde methylglyoxal.

No MeSH data available.


Related in: MedlinePlus

Localisation of GFP-SKL in Ws54-1255 (GFP-SKL) treated with methylglyoxal and/or rapamycin. At the indicated times cultures grown on starvation pads supplemented with or without methylglyoxal and/or rapamycin were analysed using fluorescence microscopy. Representative images are shown for each time point. a control; b + 0.05 % (v/v) methylglyoxal; c + 1 μM rapamycin; d + 0.05 % (v/v) methylglyoxal/+ 1 μM rapamycin. Corresponding bright field areas are shown below each fluorescence channel image. White arrows: GFP-SKL localised to vacuoles. Scale bars: 10 μm
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Fig2: Localisation of GFP-SKL in Ws54-1255 (GFP-SKL) treated with methylglyoxal and/or rapamycin. At the indicated times cultures grown on starvation pads supplemented with or without methylglyoxal and/or rapamycin were analysed using fluorescence microscopy. Representative images are shown for each time point. a control; b + 0.05 % (v/v) methylglyoxal; c + 1 μM rapamycin; d + 0.05 % (v/v) methylglyoxal/+ 1 μM rapamycin. Corresponding bright field areas are shown below each fluorescence channel image. White arrows: GFP-SKL localised to vacuoles. Scale bars: 10 μm

Mentions: The observed differences in mycelium growth in the presence or absence of methylglyoxal could be accompanied by a delayed entry into stationary phase. In filamentous fungi, autophagy induction is a marker for stationary phase entry. Therefore degradation of GFP-SKL labelled peroxisomes as an autophagy marker was analysed by fluorescence microscopy (Fig. 2, Table 1). The utilised assay is based on the principle that GFP is not efficiently degraded by proteases localized in vacuoles [28]. Therefore the presence of GFP in vacuoles is a measure for peroxisomal degradation in the Ws54-1255 (GFP-SKL) reporter strain. In untreated samples vacuoles filled with GFP become visible after 40 h of incubation and these are also present after 60 h (Fig. 2a). At the later time point GFP localized to the vacuole lumen is observed in ca. 50 % of hyphae (Table 1). By contrast, in samples subjected to 0.05 % (v/v) methylglyoxal no GFP-labelled vacuoles are observed up to 40 h (Fig. 2b, Table 1) indicating that these mycelia have not entered stationary phase by this time point. At a later time point (60 h) GFP-labelled vacuoles become visible (Fig. 2b) but only in approximately 25 % of hyphae (Table 1). After 40 h pronounced vacuolation of hyphae is observed when 1 μM rapamycin was added to the samples (Fig. 2c, Table 1). These vacuoles contain GFP suggesting peroxisomal degradation by autophagy (pexophagy). At 60 h almost all hyphae contain GFP-labelled vacuoles (Table 1) which are differing in morphology from vacuoles observed at 40 h (their structure seems less regular and they are filled with granulate matter which might be due to the high level of degradation occurring in strains that are subjected to both starvation and rapamycin). When rapamycin and methylglyoxal are synergistically applied the following observations are made (Fig. 2d): (i) at 20 h, there is no apparent difference to the untreated control and exclusive methylglyoxal treatment, (ii) at 40 h a low level of peroxisome degradation is detected which is not seen in hyphae treated solely with methylglyoxal and (iii) at 60 h the tendency of GFP to localise to vacuoles is similar to samples treated with methylglyoxal alone. The phenotype at 40 h indicates that methylglyoxal cannot halt the induction of peroxisome degradation by rapamycin treatment.Fig. 2


Penicillium chrysogenum as a model system for studying cellular effects of methylglyoxal.

Scheckhuber CQ - BMC Microbiol. (2015)

Localisation of GFP-SKL in Ws54-1255 (GFP-SKL) treated with methylglyoxal and/or rapamycin. At the indicated times cultures grown on starvation pads supplemented with or without methylglyoxal and/or rapamycin were analysed using fluorescence microscopy. Representative images are shown for each time point. a control; b + 0.05 % (v/v) methylglyoxal; c + 1 μM rapamycin; d + 0.05 % (v/v) methylglyoxal/+ 1 μM rapamycin. Corresponding bright field areas are shown below each fluorescence channel image. White arrows: GFP-SKL localised to vacuoles. Scale bars: 10 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4496818&req=5

Fig2: Localisation of GFP-SKL in Ws54-1255 (GFP-SKL) treated with methylglyoxal and/or rapamycin. At the indicated times cultures grown on starvation pads supplemented with or without methylglyoxal and/or rapamycin were analysed using fluorescence microscopy. Representative images are shown for each time point. a control; b + 0.05 % (v/v) methylglyoxal; c + 1 μM rapamycin; d + 0.05 % (v/v) methylglyoxal/+ 1 μM rapamycin. Corresponding bright field areas are shown below each fluorescence channel image. White arrows: GFP-SKL localised to vacuoles. Scale bars: 10 μm
Mentions: The observed differences in mycelium growth in the presence or absence of methylglyoxal could be accompanied by a delayed entry into stationary phase. In filamentous fungi, autophagy induction is a marker for stationary phase entry. Therefore degradation of GFP-SKL labelled peroxisomes as an autophagy marker was analysed by fluorescence microscopy (Fig. 2, Table 1). The utilised assay is based on the principle that GFP is not efficiently degraded by proteases localized in vacuoles [28]. Therefore the presence of GFP in vacuoles is a measure for peroxisomal degradation in the Ws54-1255 (GFP-SKL) reporter strain. In untreated samples vacuoles filled with GFP become visible after 40 h of incubation and these are also present after 60 h (Fig. 2a). At the later time point GFP localized to the vacuole lumen is observed in ca. 50 % of hyphae (Table 1). By contrast, in samples subjected to 0.05 % (v/v) methylglyoxal no GFP-labelled vacuoles are observed up to 40 h (Fig. 2b, Table 1) indicating that these mycelia have not entered stationary phase by this time point. At a later time point (60 h) GFP-labelled vacuoles become visible (Fig. 2b) but only in approximately 25 % of hyphae (Table 1). After 40 h pronounced vacuolation of hyphae is observed when 1 μM rapamycin was added to the samples (Fig. 2c, Table 1). These vacuoles contain GFP suggesting peroxisomal degradation by autophagy (pexophagy). At 60 h almost all hyphae contain GFP-labelled vacuoles (Table 1) which are differing in morphology from vacuoles observed at 40 h (their structure seems less regular and they are filled with granulate matter which might be due to the high level of degradation occurring in strains that are subjected to both starvation and rapamycin). When rapamycin and methylglyoxal are synergistically applied the following observations are made (Fig. 2d): (i) at 20 h, there is no apparent difference to the untreated control and exclusive methylglyoxal treatment, (ii) at 40 h a low level of peroxisome degradation is detected which is not seen in hyphae treated solely with methylglyoxal and (iii) at 60 h the tendency of GFP to localise to vacuoles is similar to samples treated with methylglyoxal alone. The phenotype at 40 h indicates that methylglyoxal cannot halt the induction of peroxisome degradation by rapamycin treatment.Fig. 2

Bottom Line: The biologically most important compound of this class, methylglyoxal, results from spontaneous phosphate elimination from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate which are intermediate glycolysis products.Methylglyoxal leads to growth rate reduction of this fungus so that the entry into the stationary phase is delayed.Furthermore, three proteins are identified that are present in lower abundance when methylglyoxal is added to the growth medium (aldo-keto reductase [Pc22g04850], 5-methyl-tetrahydropteroyl-triglutamate-homocysteine S-methyltransferase [Pc22g18630] and NAD-dependent formate dehydrogenase [Pc12g04310]).

View Article: PubMed Central - PubMed

Affiliation: Senckenberg Research Institute, LOEWE Excellence Cluster for Integrative Fungal Research (IPF), Georg-Voigt-Str. 14-16, D-60325, Frankfurt am Main, Germany. Christian.Scheckhuber@senckenberg.de.

ABSTRACT

Background: α-oxoaldehydes are formed as toxic by-products during metabolic activity. The biologically most important compound of this class, methylglyoxal, results from spontaneous phosphate elimination from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate which are intermediate glycolysis products. Methylglyoxal-mediated modification of lipids, nucleic acids and proteins is known to lead to the formation of advanced glycation end products. These modifications contribute to the aetiology of severe diseases like diabetes and neurodegenerative disorders. By using simple model organisms it is possible to conveniently study the effects of methylglyoxal on cellular processes. Here, results are presented on the effects of methylglyoxal on mycelium growth, stationary phase entry (monitored by autophagy induction), mitochondrial morphology and protein composition in the filamentous fungus Penicillium chrysogenum.

Results: Methylglyoxal leads to growth rate reduction of this fungus so that the entry into the stationary phase is delayed. Mitochondrial morphology is not changed by methylglyoxal. However, rapamycin-mediated fragmentation of mitochondria is prevented by methylglyoxal. Furthermore, three proteins are identified that are present in lower abundance when methylglyoxal is added to the growth medium (aldo-keto reductase [Pc22g04850], 5-methyl-tetrahydropteroyl-triglutamate-homocysteine S-methyltransferase [Pc22g18630] and NAD-dependent formate dehydrogenase [Pc12g04310]).

Conclusions: The presented results contribute to the understanding of cellular pathways and mechanisms that are affected by the ubiquitous α-oxoaldehyde methylglyoxal.

No MeSH data available.


Related in: MedlinePlus