Gregor Meiss

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.

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.1 Department of Biochemistry & Molecular Biology, 2 Department of Microbiology & Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1079, USA, 3 Institute of Biochemistry, Faculty of Biology and Chemistry, Justus-LiebigUniversity, Heinrich-Buff-Ring 58, 35392 Giessen, Germany, 4 Department of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute, 5117 Centre Avenue Pittsburgh, PA 15213-1863, USA Running title: Mitochondrial DNA Damage-Induced Apoptosis *Address correspondence to: Bartosz Szczesny, PhD, tel (409) 772-2174; fax (409) 747 8608, email: baszczes@utmb.edu

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.

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.

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.

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 ββα Me superfamily, is controlled by the specific nuclease inhibitor, NuiA. Here we report the 2.3-Å 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-135NuiA hydroxyl oxygen is directly coordinated with the catalytic Mg2+ of the nuclease active site, and Glu-24NuiA 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.

On the Advantage of Being a Dimer, a Case Study Using the DimericSerratia Nuclease and the Monomeric Nuclease fromAnabaena sp. Strain PCC 7120

The extracellular endonucleases fromSerratia marcescens and Anabaena sp. are members of a family of nonspecific endonucleases. In contrast to the monomeric Anabaena nuclease, the Serratianuclease is a dimer of two identical subunits. To find out whether the two active sites of the Serratia nuclease function independently of each other and what the advantage of being a dimer for this enzyme might be, we produced (i) dimers in which the two subunits were cross-linked, (ii) heterodimers consisting of a wild type and an inactive mutant subunit which were also cross-linked, and (iii) monomeric variants which are unable to dimerize. The monomeric H184R variant and the cross-linked S140C variant exhibit the same activity as the wild type enzyme, while the cross-linked heterodimer with one inactive subunit shows only half of the activity of the wild type enzyme, demonstrating functional independence of the two subunits of the Serratia nuclease. On the other hand at low enzyme and substrate concentrations dimeric forms of the Serratianuclease are relatively more active than monomeric forms or the monomeric Anabaena nuclease in cleaving polynucleotides, not, however, oligonucleotides, which is correlated with the ability of dimeric forms of the Serratia nuclease to form large enzyme-substrate networks with high molecular weight DNA and to cleave polynucleotides in a processive manner. We conclude that in the natural habitat of Serratia marcescens where the supply of nutrients may become growth limiting the dimeric nuclease can fulfil its nutritive function more efficiently than a monomeric enzyme.

Genetic engineering, production and characterisation of monomeric variants of the dimeric Serratia marcescens endonuclease

The Serratia nuclease is a non‐specific endonuclease which cleaves single‐ and double‐stranded RNA and DNA. It is a member of a large family of related endonucleases, most of which are dimers of identical subunits, with the notable exception of the Anabaena nuclease which is a monomer. In order to find out whether the dimer state of the Serratia nuclease is essential for its function we have produced variants of this nuclease which based on the crystal structure (Miller, M.D. and Krause, K.L. (1996), Protein Science 5, 24–33) were expected to be unable to dimerise. We demonstrate here that these variants, H184A, H184N, H184T and H184R, are monomers and have the same secondary structure, stability towards chemical denaturation and activity as the wild‐type enzyme. This allows to conclude that the dimeric state is not essential for the catalytic function of the Serratia nuclease. In contrast, the S179C variant which is also a monomer shows little activity, presumably because this amino acid substitution changes the structure of the enzyme.

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

Lysosomal DNase IIα 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 IIα 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 IIα contains two requisite PLD‐signature motifs (113HTK115 and 295HSK297) 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 IIα.

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 α4, 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.