title: Poly(ADP-ribose) polymerase is a substrate recognized by two metacaspases of Podospora anserina.
abstract: The two metacaspases MCA1 and MCA2 of the fungal aging model organism Podospora anserina (PaMCA1 and PaMCA2, respectively) have previously been demonstrated to be involved in the control of programmed cell death (PCD) and life span. In order to identify specific pathways and components which are controlled by the activity of these enzymes, we set out to characterize them further. Heterologous overexpression in Escherichia coli of the two metacaspase genes resulted in the production of proteins which aggregate and form inclusion bodies from which the active protein has been recovered via refolding in appropriate buffers. The renaturated proteins are characterized by an arginine-specific activity and are active in caspase-like self-maturation leading to the generation of characteristic small protein fragments. Both activities are dependent on the presence of calcium. Incubation of the two metacaspases with recombinant poly(ADP-ribose) polymerase (PARP), a known substrate of mammalian caspases, led to the identification of PARP as a substrate of the two P. anserina proteases. Using double mutants in which P. anserina Parp (PaParp) is overexpressed and PaMca1 is either overexpressed or deleted, we provide evidence for in vivo degradation of PaPARP by PaMCA1. These results support the idea that the substrate profiles of caspases and metacaspases are at least partially overlapping. Moreover, they link PCD and DNA maintenance in the complex network of molecular pathways involved in aging and life span control.
authors.affiliation: Department of Biosciences, Institute of Molecular Biosciences and Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Frankfurt am Main, Germany.
authors.forename: Ingmar
authors.initials: I
authors.lastname: Strobel
authors.forename: Heinz D
authors.initials: HD
authors.lastname: Osiewacz
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics* / Q000378:metabolism; D020169:Caspases / Q000235:genetics* / Q000378:metabolism; D016923:Cell Death / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D002479:Inclusion Bodies / Q000737:chemistry; D007527:Isoenzymes; D008969:Molecular Sequence Data; D009154:Mutation; D048468:Podospora / Q000235:genetics* / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics* / Q000378:metabolism; D059748:Proteolysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D005656:Fungal Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D001120:Arginine; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases