Wilhelm bernhard workshop on the cell nucleus

MODULATION OF NUCLEAR POLY(ADP-RIBOSYLATION) IN APOPTOTIC CELLS

Soldani C.¹, Bottone M.G¹, Biggiogera M.^1,2, Pellicciari C.^1,2, Denegri M²., and Scovassi A.I.²

¹Dipartimento Biologia Animale, University of Pavia and ²Istituto di Genetica Molecolare del CNR, Pavia, Italy
Poly(ADP-ribosylation) is a post-translational modification of proteins playing a crucial role in many processes. The best known poly(ADP-ribosylating) enzime, PARP-1, is a DNA nick sensor and uses NAD⁺ to form polymers of ADP-ribose, which are further degraded by the enzyme poly(ADP-ribose) glycohydrolase (PARG). During apoptosis, PARP-1 is responsible for an early and transient poly(ADP-ribose) synthesis, and is cleaved by caspases. PARP-1 proteolysis produces an 89 kDa C-terminal fragment, with a reduced catalytic activity, and a 24 kDa N-terminal peptide, which retains the DNA binding domains. We monitored the fate of p89 and the synthesis of polymers of ADP-ribose in HeLa cells driven to apoptosis by actinomycin D and etoposide. Tricolor fluorescence techniques were used to detect DNA, poly(ADP-ribose), and phosphatidylserine residues at the cell surface. The polymer synthesis proved to occur at early apoptotic stages, whereas the poly(ADP-ribose) was no longer detectable in late-apoptotic cells. To correlate the absence of polymer accumulation with PARG activity, we analyzed the localization of this enzyme and we demonstrated its presence in the nucleus of early apoptotic cells. The p89 PARP-1 fragment, which was found in the nucleoplasm of early apoptotic cells, migrated from the nucleus into the cytoplasm of cells with advanced chromatin condensation and DNA fragmentation. This demonstrates that p89 is among the several nuclear proteins which are extruded from the nucleus, reach the cytoplasm and are finally released inside apoptotic bodies. Collectively, our data indicate that poly(ADP-ribose) synthesis by activated PARP-1 occurs early during the apoptotic process, as a cellular emergency reaction, and that PARP-1 proteolysis is concomitant with PARP-1 activation. The extrusion of p89 from the nucleus in the late steps of apoptosis could allow a better understanding of the role of nuclear proteins as autoimmunity inducers.