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 ~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.     

Site-specific DNA cleavage by Chlorella virus topoisomerase II

The DNA cleavage reaction of topoisomerase II is central to the catalytic activity of the enzyme and is the target for a number of important anticancer drugs. Unfortunately, efforts to characterize this fundamental reaction have been limited by the low levels of DNA breaks normally generated by the enzyme. Recently, however, a type II topoisomerase with an extraordinarily high intrinsic DNA cleavage activity was isolated from Chlorella virus PBCV-1. To further our understanding of this enzyme, the present study characterized the site-specific DNA cleavage reaction of PBCV-1 topoisomerase II. Results indicate that the viral enzyme cleaves DNA at a limited number of sites. The DNA cleavage site utilization of PBCV-1 topoisomerase II is remarkably similar to that of human topoisomerase IIalpha, but the viral enzyme cleaves these sites to a far greater extent. Finally, PBCV-1 topoisomerase II displays a modest sensitivity to anticancer drugs and DNA damage in a site-specific manner. These findings suggest that PBCV-1 topoisomerase II represents a unique model with which to dissect the DNA cleavage reaction of eukaryotic type II topoisomerases.     

Rootletin,a novel coiled-coil protein,is a structural component of the ciliary rootlet

The ciliary rootlet, first recognized over a century ago, is a prominent structure originating from the basal body at the proximal end of a cilium. Despite being the largest cytoskeleton, its structural composition has remained unknown. Here, we report a novel 220-kD protein, designated rootletin, found in the rootlets of ciliated cells. Recombinant rootletin forms detergent-insoluble filaments radiating from the centrioles and resembling rootlets found in vivo. An mAb widely used as a marker for vertebrate rootlets recognizes an epitope in rootletin. Rootletin has a globular head domain and a tail domain consisting of extended coiled-coil structures. Rootletin forms parallel in register homodimers and elongated higher order polymers mediated by the tail domain alone. The head domain may be required for targeting to the basal body and binding to a kinesin light chain. In retinal photoreceptors where rootlets appear particularly robust, rootlets extend from the basal bodies to the synaptic terminals and anchor ER membranes along their length. Our data indicate that rootlets are composed of homopolymeric rootletin protofilaments bundled into variably shaped thick filaments. Thus, rootletin is the long-sought structural component of the ciliary rootlet.     

Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion

Peptides derived from heptad repeat regions adjacent to the fusion peptide and transmembrane domains of many viral fusion proteins form stable helical bundles and inhibit fusion specifically. Paramyxovirus SV5 fusion (F) protein-mediated fusion and its inhibition by the peptides N-1 and C-1 were analyzed. The temperature dependence of fusion by F suggests that thermal energy, destabilizing proline residues and receptor binding by the hemagglutinin-neuraminidase (HN) protein collectively contribute to F activation from a metastable native state. F-mediated fusion was reversibly arrested by low temperature or membrane-incorporated lipids, and the resulting F intermediates were characterized. N-1 inhibited an earlier F intermediate than C-1. Co-expression of HN with F lowered the temperature required to attain the N-1-inhibited intermediate, consistent with HN binding to its receptor stimulating a conformational change in F. C-1 bound and inhibited an intermediate of F that could be detected until a point directly preceding membrane merger. The data are consistent with C-1 binding a pre-hairpin intermediate of F and with helical bundle formation being coupled directly to membrane fusion.     

Involvement of 14-3-3 proteins in the osmotic regulation of H+-ATPase in plant plasma membranes

 Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H⁺-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H⁺ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes; the 14-3-3 content in the membranes increased 2- to 3-fold, while the H⁺-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H⁺-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H⁺ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins. Received: 10 February 2000 / Accepted: 31 March 2000     

Monitoring of Brain Spiking Activity and Autoantibodies to N-Terminus Domain of GluR1 Subunit of AMPA Receptors in Blood Serum of Rats with Cobalt-Induced Epilepsy

Abstract: We have monitored EEG spontaneous spiking activity and analyzed serum from rats with cobalt-induced epilepsy for the presence of autoreactive antibodies to α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) glutamate receptor subunits. The presence and the level of autoantibodies were assessed using immunoblot and ELISA with synthetic peptide specific to the N-terminus domain of the GluR1 subunit of the AMPA receptor. Rats with cobalt-induced epilepsy exhibited strong GluR1 immunoreactivity at the end of the first week after surgery compared with vehicle-treated rats. We showed that GluR1 autoantibodies in blood serum of rats with cobalt-induced epilepsy preceded the spiking activity maximum in the EEG. Levels of autoantibodies to GluR1 detected in blood of these rats remained elevated when EEG spiking activity was significantly reduced and seizures disappeared. The EEG monitoring of spiking activity showed a correlation with accumulation of GluR1 autoantibodies in blood serum of rats with cobalt-induced epilepsy.     

The unique Phe–His dyad of 2-ketopropyl coenzyme M oxidoreductase/carboxylase selectively promotes carboxylation and S–C bond cleavage

The 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) enzyme is the only member of the disulfide oxidoreductase (DSOR) family of enzymes, which are important for reductively cleaving S–S bonds, to have carboxylation activity. 2-KPCC catalyzes the conversion of 2-ketopropyl-coenzyme M to acetoacetate, which is used as a carbon source, in a controlled reaction to exclude protons. A conserved His–Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC, the dyad is substituted by Phe–His. Here, we propose that this difference is important for coupling carboxylation with C–S bond cleavage. We substituted the Phe–His dyad in 2-KPCC to be more DSOR like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E), and solved crystal structures of F501H and the double variant F501H_H506E. We found that F501 protects the enolacetone intermediate from protons and that the F501H variant strongly promotes protonation. We also provided evidence for the involvement of the H506 residue in stabilizing the developing charge during the formation of acetoacetate, which acts as a product inhibitor in the WT but not the H506E variant enzymes. Finally, we determined that the F501H substitution promotes a DSOR-like charge transfer interaction with flavin adenine dinucleotide, eliminating the need for cysteine as an internal base. Taken together, these results indicate that the 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.     

Conformational Switching in PolyGln Amyloid Fibrils Resulting from a Single Amino Acid Insertion

The established correlation between neurodegenerative disorders and intracerebral deposition of polyglutamine aggregates motivates attempts to better understand their fibrillar structure. We designed polyglutamines with a few lysines inserted to overcome the hindrance of extreme insolubility and two D-lysines to limit the lengths of β-strands. One is 33 amino acids long (PolyQKd-33) and the other has one fewer glutamine (PolyQKd-32). Both form well-dispersed fibrils suitable for analysis by electron microscopy. Electron diffraction confirmed cross-β structures in both fibrils. Remarkably, the deletion of just one glutamine residue from the middle of the peptide leads to substantially different amyloid structures. PolyQKd-32 fibrils are consistently 10–20% wider than PolyQKd-33, as measured by negative staining, cryo-electron microscopy, and scanning transmission electron microscopy. Scanning transmission electron microscopy analysis revealed that the PolyQKd-32 fibrils have 50% higher mass-per-length than PolyQKd-33. This distinction can be explained by a superpleated β-structure model for PolyQKd-33 and a model with two β-solenoid protofibrils for PolyQKd-32. These data provide evidence for β-arch-containing structures in polyglutamine fibrils and open future possibilities for structure-based drug design.     

Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner

Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.