ABSTRACT
Poly(ADP-ribosylation) is a post-translational modification of proteins playing a crucial role in DNA repair, replication, transcription and cell death. In this paper, the main features of this process have been reviewed, focusing on the best known poly(ADP-ribose) polymerizing enzyme, PARP-1, a DNA nick-sensor protein that uses beta-NAD+ to form polymers of ADP-ribose. The modulation of poly(ADP-ribosylation) during apoptosis and the possible effects of its inhibition on cell metabolism are discussed.
Subject(s)
Apoptosis/physiology , Poly Adenosine Diphosphate Ribose/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Animals , DNA Damage/physiology , Enzyme Inhibitors/pharmacology , Humans , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/geneticsABSTRACT
The nuclear metabolism of poly(ADP-ribose) is mainly regulated by poly(ADP-ribose) polymerase-1 (PARP-1) and by poly(ADP-ribose) glycohydrolase (PARG). A PARP-like enzyme, V-PARP, and a PARG isoform are present in the extra-nuclear compartment of mammalian cells, even if poly(ADP-ribose) has never been detected therein. In this work, we demonstrate the ability of post-nuclear extracts from HeLa and HL60 cells to degrade synthetic 32P-polymers of ADP-ribose to ADP-ribose and, further, to AMP. This reaction implies the combined action of PARG and of an ADP-ribose-degrading activity, possibly corresponding to a phosphodiesterase and/or to an ADP-ribose pyrophosphatase. The inhibition of PARG or ADP-ribose-degrading enzymes allowed the demonstration that in vitro synthesized 32P-poly(ADP-ribose) is first digested to ADP-ribose monomers by a typical PARG reaction, and that ADP-ribose is further rapidly converted into AMP by an Mg(2+)-dependent activity. Collectively, our results demonstrate the ability of the human cell post-nuclear fraction to convert synthetic poly(ADP-ribose) into utilizable AMP units by the concerted action of PARG and ADP-ribose-degrading activities.
Subject(s)
Cell Nucleus/metabolism , Nuclear Proteins/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Chromatography, High Pressure Liquid , DNA/analysis , Edetic Acid/pharmacology , Electrophoresis, Polyacrylamide Gel , Glycoside Hydrolases/antagonists & inhibitors , Glycoside Hydrolases/metabolism , HL-60 Cells , HeLa Cells , Humans , Hydrogen-Ion Concentration , Phosphoric Diester Hydrolases/isolation & purification , Phosphoric Diester Hydrolases/metabolism , Poly Adenosine Diphosphate Ribose/antagonists & inhibitors , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/metabolism , Proteins/antagonists & inhibitors , Proteins/metabolism , Time FactorsABSTRACT
Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins typical of most eukaryotic cells. This process participates in DNA replication and repair and is mainly regulated by two enzymes, poly(ADP-ribose) polymerase, which is responsible for the synthesis of polymers of ADP-ribose, and poly(ADP-ribose) glycohydrolase, which performs polymer degradation. The aim of this work was to investigate in the cockroach Periplaneta americana L. (Blattaria: Blattidae) the behaviour of poly(ADP-ribosylation). In particular, we addressed: (i) the possible modulation of poly(ADP-ribosylation) during the embryonic development; (ii) the expression of poly(ADP-ribose) polymerase and glycohydrolase in different tissues; and (iii) the role of poly(ADP-ribosylation) during spermatogenesis. In this work we demonstrated that: (i) as revealed by specific biochemical assays, active poly(ADP-ribose) polymerase and glycohydrolase are present exclusively in P. americana embryos at early stages of development; (ii) an activity carrying out poly(ADP-ribose) synthesis was found in extracts from testes; and (iii) the synthesis of poly(ADP-ribose) occurs preferentially in differentiating spermatids/spermatozoa. Collectively, our results indicate that the poly(ADP-ribosylation) process in P. americana, which is a hemimetabolous insect, displays catalytical and structural features similar to those described in the holometabolous insects and in mammalian cells. Furthermore, this process appears to be modulated during embryonic development and spermatogenesis.
Subject(s)
Glycoside Hydrolases/metabolism , Insect Proteins/metabolism , Nuclear Proteins/metabolism , Periplaneta/genetics , Poly(ADP-ribose) Polymerases/metabolism , Protein Processing, Post-Translational , Spermatogenesis/physiology , Animals , DNA Repair , DNA Replication , Embryo, Nonmammalian/enzymology , Male , Periplaneta/embryologyABSTRACT
In 16-17-day-old embryos of Periplaneta americana, the amnion-serosa penetrates the cavity of the middle intestine, where it forms a cluster of compressed roundish cells. We demonstrated that these cells degenerate throughout apoptosis. The programmed cell death revealed by morphological and biochemical approaches showed all the apoptotic steps: chromatin fragmentation and pyknosis, cytoplasm condensation, karyorrhexis, cytoplasm cleavage. Nevertheless, some ultrastructural peculiarities (atypical heterochromatin arrangement, appearance of nuclear envelope protrusions, absence of nucleolar structures) suggest that the apoptotic expression partially depends on the biological situation (type of organism and inducing factors) in which the programmed cell death takes place. The presence of histiocytic cells internalizing cell debris, of apoptotic and non-apototic derivation, may be correlated with the importance of recycling substances useful for embryo growth.
