Identification of ICAD-derived peptides capable of inhibiting caspase-activated DNase.

The DNA fragmentation factor is a heterodimeric complex that consists of caspase-activated DNase (CAD) and its inhibitor (ICAD). As only partial structural information on this nuclease/inhibitor complex is available, understanding of how its subunits interact on the molecular level remains largely elusive, particularly how CAD inhibition is achieved by ICAD. In this study, we used the SPOT (peptide array) method to identify protein-protein interaction sites in the DNA fragmentation factor complex, focusing on those possibly involved in CAD inhibition. We observed a particularly strong interaction of ICAD with the dimerization (C2) domain of CAD. Additional interactions with the Zn(2+) -binding site close to the catalytic centre and the catalytic centre itself in the C3 domain of CAD were detected, suggesting that prevention of CAD homodimerization and local structural perturbation or blocking of the active site together constitute a dual inhibitory mechanism to effectively inhibit CAD. The results obtained by the SPOT method were validated by performing inhibition assays employing selected soluble ICAD-derived peptides. In these assays, two ICAD-derived peptides were identified that are capable of efficiently and specifically inhibiting CAD activity in solution.

Chemical rescue of active site mutants of S. pneumoniae surface endonuclease EndA and other nucleases of the HNH family by imidazole.

The His-Asn-His (HNH) motif characterizes the active sites of a large number of different nucleases such as homing endonucleases, restriction endonucleases, structure-specific nucleases and, in particular, nonspecific nucleases. Several biochemical studies have revealed an essential catalytic function for the first amino acid of this motif in HNH nucleases. This histidine residue was identified as the general base that activates a water molecule for a nucleophilic attack on the sugar phosphate backbone of nucleic acids. Replacement of histidine by an amino acid such as glycine or alanine, which lack the catalytically active imidazole side chain, leads to decreases of several orders of magnitude in the nucleolytic activities of members of this nuclease family. We were able, however, to restore the activity of HNH nuclease variants (i.e., EndA (Streptococcus pneumoniae), SmaNuc (Serratia marcescens) and NucA (Anabaena sp.)) that had been inactivated by His→Gly or His→Ala substitution by adding excess imidazole to the inactive enzymes in vitro. Imidazole clearly replaces the missing histidine side chain and thereby restores nucleolytic activity. Significantly, this chemical rescue could also be observed in vivo (Escherichia coli). The in vivo assay might be a promising starting point for the development of a high-throughput screening system for functional EndA inhibitors because, unlike the wild-type enzyme, the H160G and H160A variants of EndA can easily be produced in E. coli. A simple viability assay would allow inhibitors of EndA to be identified because these would counteract the toxicities of the chemically rescued EndA variants. Such inhibitors could be used to block the nucleolytic activity of EndA, which as a surface-exposed enzyme in its natural host destroys the DNA scaffolds of neutrophil extracellular traps (NETs) and thereby allows S. pneumoniae to escape the innate immune response.

Apoptosis induced by persistent single-strand breaks in mitochondrial genome: critical role of EXOG (5'-EXO/endonuclease) in their repair.

Reactive oxygen species (ROS), continuously generated as by-products of respiration, inflict more damage on the mitochondrial (mt) than on the nuclear genome because of the nonchromatinized nature and proximity to the ROS source of the mitochondrial genome. Such damage, particularly single-strand breaks (SSBs) with 5'-blocking deoxyribose products generated directly or as repair intermediates for oxidized bases, is repaired via the base excision/SSB repair pathway in both nuclear and mt genomes. Here, we show that EXOG, a 5'-exo/endonuclease and unique to the mitochondria unlike FEN1 or DNA2, which, like EXOG, has been implicated in the removal of the 5'-blocking residue, is required for repairing endogenous SSBs in the mt genome. EXOG depletion induces persistent SSBs in the mtDNA, enhances ROS levels, and causes apoptosis in normal cells but not in mt genome-deficient rho0 cells. Thus, these data show for the first time that persistent SSBs in the mt genome alone could provide the initial trigger for apoptotic signaling in mammalian cells.

Mutational and biochemical analysis of the DNA-entry nuclease EndA from Streptococcus pneumoniae.

EndA is a membrane-attached surface-exposed DNA-entry nuclease previously known to be required for genetic transformation of Streptococcus pneumoniae. More recent studies have shown that the enzyme also plays an important role during the establishment of invasive infections by degrading extracellular chromatin in the form of neutrophil extracellular traps (NETs), enabling streptococci to overcome the innate immune system in mammals. As a virulence factor, EndA has become an interesting target for future drug design. Here we present the first mutational and biochemical analysis of recombinant forms of EndA produced either in a cell-free expression system or in Escherichia coli. We identify His160 and Asn191 to be essential for catalysis and Asn182 to be required for stability of EndA. The role of His160 as the putative general base in the catalytic mechanism is supported by chemical rescue of the H160A variant of EndA with imidazole added in excess. Our study paves the way for the identification and development of protein or low-molecular-weight inhibitors for EndA in future high-throughput screening assays.

Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae.

EndA is a sequence non-specific endonuclease that serves as a virulence factor during Streptococcus pneumoniae infection. Expression of EndA provides a strategy for evasion of the host's neutrophil extracellular traps, digesting the DNA scaffold structure and allowing further invasion by S. pneumoniae. To define mechanisms of catalysis and substrate binding, we solved the structure of EndA at 1.75 Å resolution. The EndA structure reveals a DRGH (Asp-Arg-Gly-His) motif-containing ßßα-metal finger catalytic core augmented by an interesting 'finger-loop' interruption of the active site α-helix. Subsequently, we delineated DNA binding versus catalytic functionality using structure-based alanine substitution mutagenesis. Three mutants, H154A, Q186A and Q192A, exhibited decreased nuclease activity that appears to be independent of substrate binding. Glu205 was found to be crucial for catalysis, while residues Arg127/Lys128 and Arg209/Lys210 contribute to substrate binding. The results presented here provide the molecular foundation for development of specific antibiotic inhibitors for EndA.

Production and characterization of recombinant protein preparations of Endonuclease G-homologs from yeast, C. elegans and humans.

Nuc1p, CPS-6, EndoG and EXOG are evolutionary conserved mitochondrial nucleases from yeast, Caenorhabditis elegans and humans, respectively. These enzymes play an important role in programmed cell death as well as mitochondrial DNA-repair and recombination. Whereas a significant interest has been given to the cell biology of these proteins, in particular their recruitment during caspase-independent apoptosis, determination of their biochemical properties has lagged behind. In part, biochemical as well as structural analysis of mitochondrial nucleases has been hampered by the fact that upon cloning and overexpression in Escherichia coli these enzymes can exert considerable toxicity and tend to aggregate and form inclusion bodies. We have, therefore, established a uniform E. coli expression system allowing us to obtain these four evolutionary related nucleases in active form from the soluble as well as insoluble fractions of E. coli cell lysates. Using preparations of recombinant Nuc1p, CPS-6, EndoG and EXOG we have compared biochemical properties and the substrate specificities of these related nucleases on selected substrates in parallel. Whereas Nuc1p and EXOG in addition to their endonuclease activity exert 5'-3'-exonuclease activity, CPS-6 and EndoG predominantly are endonucleases. These findings allow speculating that the mechanisms of action of these related nucleases in cell death as well as DNA-repair and recombination differ according to their enzyme activities and substrate specificities.

Inhibition of UV-C light-induced apoptosis in liver cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic pollutant ubiquitously present in the environment. Most of the toxic effects of TCDD are believed to be mediated by high-affinity binding to the aryl hydrocarbon receptor (AhR) and subsequent effects on gene transcription. TCDD causes cancer in multiple tissues in different animal species and is classified as a class 1 human carcinogen. In initiation-promotion studies TCDD was shown to be a potent liver tumor promotor. Among other theories it has been hypothesized that TCDD acts as a tumor promotor by preventing initiated cells from undergoing apoptosis. We examined the effects of TCDD on ultraviolet C (UV-C) light-induced apoptosis in primary rat hepatocytes and Huh-7 human hepatoma cells. TCDD inhibits UV-C light-induced apoptosis in both cell types. This effect is seen with chromatin condensation and fragmentation and appears to be mediated by the AhR in rat hepatocytes. Apoptosis induced by UV-C light in these cells is caspase-dependent and is accompanied by alterations in apoptosis-related gene expression such as up-regulation of proapoptotic bcl-2 family genes like bak and bax, and a marked down regulation of the expression of the antiapoptotic bcl-2. TCDD treatment of irradiated hepatocytes induces the expression of some apoptosis-related genes (birc3, dad1, pycard, tnf). Upstream apoptotic events, namely caspase activation and caspase substrate cleavage are not inhibited by TCDD treatment. We hypothesize that TCDD inhibits late-stage apoptotic events that lead to internucleosomal DNA fragmentation, maintaining chromosomal integrity probably in order to sustain metabolic capacity and hepatic elimination of substrates despite of an initiation of apoptosis.

Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells.

TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18((R)):DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.

Mitochondrial protein quality control by the proteasome involves ubiquitination and the protease Omi.

We report here that blocking the activity of the 26 S proteasome results in drastic changes in the morphology of the mitochondria and accumulation of intermembrane space (IMS) proteins. Using endonuclease G (endoG) as a model IMS protein, we found that accumulation of wild-type but to a greater extent mutant endoG leads to changes in the morphology of the mitochondria similar to those observed following proteasomal inhibition. Further, we show that wild-type but to a greater extent mutant endoG is a substrate for ubiquitination, suggesting the presence of a protein quality control. Conversely, we also report that wild-type but not mutant endoG is a substrate for the mitochondrial protease Omi but only upon inhibition of the proteasome. These findings suggest that although elimination of mutant IMS proteins is strictly dependent on ubiquitination, elimination of excess or spontaneously misfolded wild-type IMS proteins is monitored by ubiquitination and as a second checkpoint by Omi cleavage when the proteasome function is deficient. One implication of our finding is that in the context of attenuated proteasomal function, accumulation of IMS proteins would contribute to the collapse of the mitochondrial network such as that observed in neurodegenerative diseases. Another implication is that such collapse could be accelerated either by mutations in IMS proteins or by mutations in Omi itself.

EXOG, a novel paralog of Endonuclease G in higher eukaryotes.

Evolutionary conserved mitochondrial nucleases are involved in programmed cell death and normal cell proliferation in lower and higher eukaryotes. The endo/exonuclease Nuc1p, also termed 'yeast Endonuclease G (EndoG)', is a member of this class of enzymes that differs from mammalian homologs by the presence of a 5'-3' exonuclease activity in addition to its broad spectrum endonuclease activity. However, this exonuclease activity is thought to be essential for a function of the yeast enzyme in DNA recombination and repair. Here we show that higher eukaryotes in addition to EndoG contain its paralog 'EXOG', a novel EndoG-like mitochondrial endo/exonuclease. We find that during metazoan evolution duplication of an ancestral nuclease gene obviously generated the paralogous EndoG- and EXOG-protein subfamilies in higher eukaryotes, thereby maintaining the full endo/exonuclease activity found in mitochondria of lower eukaryotes. We demonstrate that human EXOG is a dimeric mitochondrial enzyme that displays 5'-3' exonuclease activity and further differs from EndoG in substrate specificity. We hypothesize that in higher eukaryotes the complementary enzymatic activities of EndoG and EXOG probably together account for both, the lethal and vital functions of conserved mitochondrial endo/exonucleases.

Human lysosomal DNase IIalpha contains two requisite PLD-signature (HxK) motifs: evidence for a pseudodimeric structure of the active enzyme species.

Lysosomal DNase IIalpha is essential for DNA waste removal and auxiliary apoptotic DNA fragmentation in higher eukaryotes. Despite the key role of this enzyme, little is known about its structure-function relationships. Here, mutational and biochemical analyses were used to characterize human DNase IIalpha variants expressed in mammalian cells. The resulting data strongly support the hypothesis that the enzyme is a monomeric phospholipase D-family member with a pseudodimeric protein fold. According to our results, DNase IIalpha contains two requisite PLD-signature motifs ((113)HTK(115) and (295)HSK(297)) in the N- and C-terminal subdomains, respectively, that together form a single active site. Based on these data, we present an experimentally validated structural model of DNase IIalpha.

The nuclease a-inhibitor complex is characterized by a novel metal ion bridge.

Nonspecific, extracellular nucleases have received enhanced attention recently as a consequence of the critical role that these enzymes can play in infectivity by overcoming the host neutrophil defense system. The activity of the cyanobacterial nuclease NucA, a member of the betabetaalpha Me superfamily, is controlled by the specific nuclease inhibitor, NuiA. Here we report the 2.3-A resolution crystal structure of the NucA-NuiA complex, showing that NucA inhibition by NuiA involves an unusual divalent metal ion bridge that connects the nuclease with its inhibitor. The C-terminal Thr-135(NuiA) hydroxyl oxygen is directly coordinated with the catalytic Mg(2+) of the nuclease active site, and Glu-24(NuiA) also extends into the active site, mimicking the charge of a scissile phosphate. NuiA residues Asp-75 and Trp-76 form a second interaction site, contributing to the strength and specificity of the interaction. The crystallographically defined interface is shown to be consistent with results of studies using site-directed NuiA mutants. This mode of inhibition differs dramatically from the exosite mechanism of inhibition seen with the DNase colicins E7/E9 and from other nuclease-inhibitor complexes that have been studied. The structure of this complex provides valuable insights for the development of inhibitors for related nonspecific nucleases that share the DRGH active site motif such as the Streptococcus pneumoniae nuclease EndA, which mediates infectivity of this pathogen, and mitochondrial EndoG, which is involved in recombination and apoptosis.

Interaction of the membrane-bound GlnK-AmtB complex with the master regulator of nitrogen metabolism TnrA in Bacillus subtilis.

PII proteins are widespread and highly conserved signal transduction proteins occurring in bacteria, Archaea, and plants and play pivotal roles in controlling nitrogen assimilatory metabolism. This study reports on biochemical properties of the PII-homologue GlnK (originally termed NrgB) in Bacillus subtilis (BsGlnK). Like other PII proteins, the native BsGlnK protein has a trimeric structure and readily binds ATP in the absence of divalent cations, whereas 2-oxoglutarate is only weakly bound. In contrast to other PII-like proteins, Mg2+ severely affects its ATP-binding properties. BsGlnK forms a tight complex with the membrane-bound ammonium transporter AmtB (NrgA), from which it can be relieved by millimolar concentrations of ATP. Immunoprecipitation and co-localization experiments identified a novel interaction between the BsGlnK-AmtB complex and the major transcription factor of nitrogen metabolism, TnrA. In vitro in the absence of ATP, TnrA is completely tethered to membrane (AmtB)-bound GlnK, whereas in extracts from BsGlnK- or AmtB-deficient cells, TnrA is entirely soluble. The presence of 4 mm ATP leads to concomitant solubilization of BsGlnK and TnrA. This ATP-dependent membrane re-localization of TnrA by BsGlnK/AmtB may present a novel mechanism to control the global nitrogen-responsive transcription regulator TnrA in B. subtilis under certain physiological conditions.

Structural basis for stable DNA complex formation by the caspase-activated DNase.

We describe a structural model for DNA binding by the caspase-activated DNase (CAD). Results of a mutational analysis and computational modeling suggest that DNA is bound via a positively charged surface with two functionally distinct regions, one being the active site facing the DNA minor groove and the other comprising distal residues close to or directly from helix alpha4, which binds DNA in the major groove. This bipartite protein-DNA interaction is present once in the CAD/inhibitor of CAD heterodimer and repeated twice in the active CAD dimer.

DNase II is a member of the phospholipase D superfamily.

MOTIVATION: DNase II is an endodeoxyribonuclease involved in apoptosis and essential for the mammalian development. Despite the understanding of biochemical properties of this enzyme, its structure and relationships to other protein families remain unknown. RESULTS: Using protein fold-recognition we found that DNase II exhibits a catalytic domain common to the phospholipase D superfamily. Our model explains the available experimental data and provides the first structural platform for sequence-function analyses of this important nuclease.

Structural insights into the mechanism of nuclease A, a betabeta alpha metal nuclease from Anabaena.

Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single- and double-stranded DNA and RNA in the presence of divalent metal ions. We have determined the structure of the delta(2-24),D121A mutant of NucA in the presence of Zn2+ and Mn2+ (PDB code 1ZM8). The mutations were introduced to remove the N-terminal signal peptide and to reduce the activity of the nonspecific nuclease, thereby reducing its toxicity to the Escherichia coli expression system. NucA contains a betabeta alpha metal finger motif and a hydrated Mn2+ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites approximately 26 A apart from the catalytic metal binding site. A structural comparison between NucA and the closest analog for which structural data exist, the Serratia nuclease, indicates several interesting differences. First, NucA is a monomer rather than a dimer. Second, there is an unexpected structural homology between the N-terminal segments despite a poorly conserved sequence, which in Serratia includes a cysteine bridge thought to play a regulatory role. In addition, although a sequence alignment had suggested that NucA lacks a proposed catalytic residue corresponding to Arg57 in Serratia, the structure determined here indicates that Arg93 in NucA is positioned to fulfill this role. Based on comparison with DNA-bound nuclease structures of the betabeta alpha metal finger nuclease family and available mutational data on NucA, we propose that His124 acts as a catalytic base, and Arg93 participates in the catalysis possibly through stabilization of the transition state.

Interaction of DNA fragmentation factor (DFF) with DNA reveals an unprecedented mechanism for nuclease inhibition and suggests that DFF can be activated in a DNA-bound state.

DNA fragmentation factor (DFF) is a complex of the DNase DFF40 (CAD) and its chaperone/inhibitor DFF45 (ICAD-L) that can be activated during apoptosis to induce DNA fragmentation. Here, we demonstrate that DFF directly binds to DNA in vitro without promoting DNA cleavage. DNA binding by DFF is mediated by the nuclease subunit, which can also form stable DNA complexes after release from DFF. Recombinant and reconstituted DFF is catalytically inactive yet proficient in DNA binding, demonstrating that the nuclease subunit in DFF is inhibited in DNA cleavage but not in DNA binding, revealing an unprecedented mode of nuclease inhibition. Activation of DFF in the presence of naked DNA or isolated nuclei stimulates DNA degradation by released DFF40 (CAD). In transfected HeLa cells transiently expressed DFF associates with chromatin, suggesting that DFF could be activated during apoptosis in a DNA-bound state.

Structural and functional characterization of mitochondrial EndoG, a sugar non-specific nuclease which plays an important role during apoptosis.

Combining sequence analysis, structure prediction, and site-directed mutagenesis, we have investigated the mechanism of catalysis and substrate binding by the apoptotic mitochondrial nuclease EndoG, which belongs to the large family of DNA/RNA non-specific betabetaalpha-Me-finger nucleases. Catalysis of phosphodiester bond cleavage involves several highly conserved amino acid residues, namely His143, Asn174, and Glu182 required for water activation and metal ion binding, as well as Arg141 required for proper substrate binding and positioning, respectively. These results indicate that EndoG basically follows a similar mechanism as the Serratia nuclease, the best studied representative of the family of DNA/RNA non-specific nucleases, but that differences are observed for transition state stabilisation. In addition, we have identified two putative DNA/RNA binding residues of bovine EndoG, Arg135 and Arg186, strictly conserved only among mammalian members of the nuclease family, suggesting a similar mode of binding to single and double-stranded nucleic acid substrates by these enzymes. Finally, we demonstrate by ectopic expression of active and inactive variants of bovine EndoG in HeLa and CV1-cells that extramitochondrial active EndoG by itself induces cell death, whereas expression of an enzymatically inactive variant does not.

Experimental evidence for a beta beta alpha-Me-finger nuclease motif to represent the active site of the caspase-activated DNase.

The caspase-activated DNase (CAD) is an important nuclease involved in apoptotic DNA degradation. Results of a sequence comparison of CAD proteins with beta beta alpha-Me-finger nucleases in conjunction with a mutational and chemical modification analysis suggest that CAD proteins constitute a new family of beta beta alpha-Me-finger nucleases. Nucleases of this family have widely different functions but are characterized by a common active-site fold and similar catalytic mechanisms. According to our results and comparisons with related nucleases, the active site of CAD displays features that partly resemble those of the colicin E9 and partly those of the T4 endonuclease VII active sites. We suggest that the catalytic mechanism of CAD involves a conserved histidine residue, acting as a general base, and another histidine as well as an aspartic acid residue required for cofactor binding. Our findings provide a first insight into the likely active-site structure and catalytic mechanism of a nuclease involved in the degradation of chromosomal DNA during programmed cell death.

The effect of ICAD-S on the formation and intracellular distribution of a nucleolytically active caspase-activated DNase.

We show here that co-expression of murine CAD with either ICAD-L or ICAD-S in Escherichia coli as well as mammalian cells leads to a functional DFF complex, which after caspase-3 activation releases a nucleolytically active DNase. The chaperone activity of ICAD-S is between one and two orders of magnitude less effective than that of ICAD-L, as deduced from cleavage experiments with different activated recombinant DFF complexes produced in E.coli. With nucleolytically active EGFP fusion proteins of CAD it is demonstrated that co-expression of ICAD-S, which lacks the C-terminal domain of ICAD-L, including the NLS, leads to a homogeneous intracellular distribution of the DNase in transfected cells, whereas co-expression of human or murine ICAD-L variants lacking the NLS leads to exclusion of EGFP-CAD from the nuclei in approximately 50% of cells. These results attribute a particular importance of the NLS in the long isoform of the inhibitor of CAD for nuclear accumulation of the DFF complex in living cells. It is concluded that ICAD-L and ICAD-S in vivo might function as tissue-specific modulators in the regulation of apoptotic DNA degradation by controlling not only the enzymatic activity but also the amount of CAD available in the nuclei of mammalian cells.