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Posttranslational modifications are used by cells from all kingdoms of life to control enzymatic activity and to regulate protein function. For many cellular processes, including DNA repair, spindle function, and apoptosis, reversible mono- and polyADP-ribosylation constitutes a very important regulatory mechanism. Moreover, many pathogenic bacteria secrete toxins which ADP-ribosylate human proteins, causing diseases such as whooping cough, cholera, and diphtheria. Whereas the 3D structures of numerous ADP-ribosylating toxins and related mammalian enzymes have been elucidated, virtually nothing is known about the structure of protein de-ADP-ribosylating enzymes. Here, we report the 3D structure of human ADP-ribosylhydrolase 3 (hARH3). The molecular architecture of hARH3 constitutes the archetype of an all-α-helical protein fold and provides insights into the reversibility of protein ADP-ribosylation. Two magnesium ions flanked by highly con- served amino acids pinpoint the active-site crevice. Recombinant hARH3 binds free ADP-ribose with micromolar affinity and efficiently de-ADP-ribosylates poly- but not monoADP-ribosylated proteins. Docking experiments indicate a possible binding mode for ADP-ribose polymers and suggest a reaction mechanism. Our results underscore the importance of endogenous ADP-ribosylation cycles and provide a basis for structure-based design of ADP-ribosylhydrolase inhibitors. [ABSTRACT FROM AUTHOR]

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is induced as part of the IFN response in mammals and acts to shut down protein synthesis by the phosphorylation of eukaryotk initiation factor 2α (eIF2α). In fish, a PKR-like kinase activity has been detected, but the enzyme responsible has eluded characterization. Here, we describe a PKR-like kinase from zebrafish. Phylogenetic analysis shows that the C-terminal kinase domain is more closely related to the kinase domain of PKR than to any of the other three known eIF2α kinases. Surprisingly, instead of the two dsRNA binding domains found at the N terminus of PKR, there are two Zα domains Zα domains specifically bind dsDNA and RNA in the left-handed Z conformation, often with high affinity. They have been found previously in two other IFN-inducible proteins, the dsRNA editing enzyme, ADARI, and Z-DNA binding protein 1 (ZBP1), as well as in the poxvirus virulence factor, E3L. This previously undescribed kinase, designated PKZ (protein kinase containing Z-DNA binding domains), is transcribed constitutively at low levels and is highly induced after injection of poly(inosinic)- poly(cytidylic) acid, which simulates viral infection. Binding of Z-DNA by the Za domain of PKZ was demonstrated by circular dichroism. PKZ inhibits translation in transfected cells; site-directed mutagenesis indicates that this inhibition depends on its catalytic activity. Identification of a gene combining Za domains with a PKR-like kinase domain strengthens the hypothesis that the ability to bind left-handed nucleic acid plays a role in the host response to viruses. [ABSTRACT FROM AUTHOR]

Examines the role of a polymorphic dinucleotide repeat in the formation of Z-DNA in the rat nucleolin gene. Inhibition of Z-DNA activity in the promoter region; Use of real time PCR in measuring Z-DNA formation; Factors contributing to genetic variation.