Poly(ADP-ribose) polymerases are involved in many aspects of regulation of cellular functions. Using NAD ؉ as a substrate, they catalyse the covalent transfer of ADP-ribose units onto several acceptor proteins to form a branched ADP-ribose polymer. The best characterised and first discovered member of this multiprotein family is PARP-1. Its catalytic activity is markedly stimulated upon binding to DNA strand interruptions, and the resulting polymer is thought to function in chromatin relaxation as well as in signalling the presence of damage to DNA repair complexes and in regulating enzyme activities. Moderate activation of PARP-1 facilitates the efficient repair of DNA damage arising from monofunctional alkylating agents, reactive oxygen species or ionising radiation, but severe genotoxic stress leads to rapid energy consumption and subsequently to necrotic cell death. The latter aspect of PARP-1 activity has been implicated in the pathogenesis of various clinical conditions such as shock, ischaemia-reperfusion and diabetes. Inhibition of ADP-ribose polymer formation has been shown to be effective, on the one hand, in the treatment of cancer in combination with alkylating agents by suppressing DNA repair and thus driving tumour cells into apoptosis, and on the other hand it appears to be a promising drug target for the treatment of pathologic conditions involving oxidative stress. In view of the existence of several members of the PARP family in mammalian cells, one has to be aware of possible side effects but also of a wide spectrum of potential clinical applications, which calls for the development of more specific inhibitors. © 2004 Wiley-Liss, Inc. Key words: DNA damage; PARP; cancer chemotherapy; radiotherapy; ischaemia-reperfusion damage; diabetes; shock; Parkinson syndromePoly(ADP-ribosyl)ation is a posttranslational modification of proteins in eukaryotic cells performed by a family of NAD ϩ ADP-ribosyl transferases, the poly(ADP-ribose) polymerases (PARPs). NAD ϩ molecules as precursor are cleaved into nicotinamide and ADP-ribose moieties, and the latter are covalently attached to glutamic or aspartic acid residues of proteins, with PARP itself being the major acceptor. Poly(ADP-ribosyl)ation may alter the activity of acceptor protein, leading to inactivation due to the attachment of a highly complex, branched polymer carrying large numbers of negative charges (Fig. 1). The polymer is rapidly degraded by the enzyme poly(ADP-ribose) glycohydrolase (PARG), limiting the half-life of the polymer to approximately 1 min under conditions of DNA breakage. Poly(ADPribosyl)ation was originally discovered as an immediate response of cells to DNA strand-break-inducing agents, i.e., ionising radiation, alkylating agents and oxidants. Polymer formation in living cells after genotoxic stresses is mainly dependent on PARP-1 (encoded by the ADPRT gene), the first-discovered and best-investigated member of the PARP family. After binding of PARP-1 with its 2 zinc-fingers within the N-terminal DNA-binding domain to double...

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Poly(ADP-ribosyl)ation is a posttranslational modification of proteins in eukaryotic cells catalysed by a family of NAD+ ADP-ribosyl transferases, the poly(ADP-ribose) polymerases (PARPs). PARP-encoding genes now constitute a superfamily of at least 18 members encoding proteins that share homology with the catalytic domain of the founding member, PARP-1. Poly(ADP-ribose) metabolism is of central importance in a wide variety of biological processes including maintenance of genomic stability, DNA repair, transcriptional regulation, centromere function, modulation of telomere length, regulation of proteasomal protein degradation, regulation of endosomal vesicle trafficking and apoptosis. The life cycle of poly(ADP-ribose) is discussed in the following section. In addition, an overview of the genes and proteins involved in poly(ADP-ribose) metabolism and their possible cellular function is provided.

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DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase±Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.