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Activation of caspase-like activity and poly (ADP-ribose) polymerase degradation during sporulation in Aspergillus
C Thrane1, U Kaufmann, B M Stummann
1Department of Ecology, Section of Genetics and Microbiology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. cht@pdir.dk
Abstract:
Mycelium vacuolization, protein degradation, and as the final stage autolysis, often accompanies developmental changes in fungi and similarities between autolysis and apoptosis have previously been suggested. Caspases are the key executors of apoptosis and in this study caspase-like activities were detected in protein extracts from Aspergillus nidulans during sporulation. This was shown by hydrolysis of the fluorescent DEVD- and IETD-AFC peptide substrates specific for caspase 3- and 8-like activities, respectively. These activities were repressed by the caspase 3 and 8 specific irreversible peptide inhibitors DEVD-fmk and IETD-fmk, but were not affected by the unspecific inhibitor E-64. Isoelectric focusing of protein extracts followed by activity staining revealed the presence of two bands with caspase-like activity. One of the proteins degraded both caspase 3 and caspase 8 specific substrates whereas the other only degraded the caspase 8 substrate. Searches in an A. nidulans genome database revealed two genes encoding metacaspase proteins with predicted sizes of 45 kDa that could be responsible for the measured caspase-like activities. The searches also found a single gene encoding a poly (ADP-ribose) polymerase (PARP) protein with a predicted size of 81 kDa. PARP is one of the known target proteins inactivated by caspase degradation in animal cells. Western blotting of fungal extracts using a bovine PARP antibody confirmed the presence of a fungal PARP-like protein of about 81 kDa. By Western blotting it was shown that this PARP-like protein band was present only at early time points until the start of conidia formation and the accompanying increase in caspase-like activity. Thereafter, a degradation product of about 60 kDa appeared indicating that the degradation of the fungal PARP-like protein was specific. The PARP antibody also recognized an 85 kDa protein band that was not degraded, and which conceivably represents a modified form of the 81 kDa PARP. Fungal extracts high in caspase-like activity could degrade both the fungal 81 kDa PARP and bovine PARP. In the presence of the caspase 3 inhibitor DEVD-fmk this degradation was delayed. Thus, as in animal apoptotic cells, caspase activities are involved in fungal mycelium self-activated proteolysis.
Insights
This study detected caspase-like activities in Aspergillus nidulans during sporulation, indicating a role for these enzymes in fungal cell death and protein degradation, similar to apoptosis in animals.
Area of Science:
- * Biochemistry
- * Molecular Biology
- * Mycology
Background:
- * Fungal development involves processes like vacuolization, protein degradation, and autolysis, with suggested similarities to animal apoptosis.
- * Caspases are key executioners of apoptosis in animals, but their role in fungi has been less understood.
Purpose of the Study:
- * To investigate the presence and function of caspase-like activities in the fungus Aspergillus nidulans during sporulation.
- * To determine if fungal caspases are involved in protein degradation, specifically targeting proteins analogous to animal caspase substrates.
Main Methods:
- * Detection of caspase-like activities using fluorogenic peptide substrates specific for caspase 3 and 8.
- * Inhibition assays using specific caspase inhibitors (DEVD-fmk, IETD-fmk) and an unspecific inhibitor (E-64).
- * Isoelectric focusing and activity staining to identify active caspase-like proteins.
- * Genome database searches for fungal metacaspase and poly (ADP-ribose) polymerase (PARP) genes.
- * Western blotting to detect and analyze fungal PARP-like proteins and their degradation products using a bovine PARP antibody.
Main Results:
- * Caspase-like activities, specific for caspase 3 and 8, were detected in Aspergillus nidulans protein extracts during sporulation.
- * Two distinct caspase-like activities were identified, with one degrading both caspase 3 and 8 substrates and the other degrading only caspase 8 substrates.
- * Genes encoding metacaspase proteins and a PARP-like protein were identified in the A. nidulans genome.
- * A fungal PARP-like protein (81 kDa) was detected, which was specifically degraded into a 60 kDa product during sporulation, coinciding with increased caspase-like activity.
- * Fungal extracts with high caspase-like activity degraded both fungal and bovine PARP, a process delayed by caspase 3 inhibition.
Conclusions:
- * Caspase-like activities are present and functional in Aspergillus nidulans, playing a role in mycelial self-activated proteolysis during sporulation.
- * These fungal caspase activities are involved in the specific degradation of a PARP-like protein, mirroring events in animal apoptosis.
- * The findings provide evidence for conserved molecular mechanisms between fungal cell death pathways and animal apoptosis.
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