Identification and analysis of dominant negative mutants of RAIDD and PIDD

Caspases are cysteine proteases that are essential during the initiation and execution of apoptosis and inflammation. The formation of large oligomeric protein complexes is critical to the activation of caspases in apoptotic and inflammatory signaling pathways. These oligomeric protein complexes function as a platform to recruit caspases, which leads to caspase activation via a proximity-induced mechanism. One well-known oligomeric caspase-activating complex is the PIDDosome for caspase-2 activation, which is composed of 3 protein components, PIDD, RAIDD and Caspase-2. Despite the significant role that caspase-2 activated by PIDDosome plays during genotoxic stress-induced apoptosis, the oligomerization mechanism and the method by which the caspase-activating process is mediated by the formation of PIDDosome is currently not well understood. Here, we show that the assembly mechanism of the core of PIDDosome is time-dependent and salt concentration-dependent. In addition, we demonstrate that point mutations on RAIDD (R147E) and on PIDD (Y814A) exert a dominant negative effect on the formation of the PIDDosome, and that this effect cannot be applied after the PIDDosome has been formed.     

Differential potency of dominant negative connexin43 mutants in oculodentodigital dysplasia

Oculodentodigital dysplasia (ODDD) is a congenital autosomal dominant disorder with phenotypic variability, which has been associated with mutations in the GJA1 gene encoding connexin43 (Cx43). Given that Cx43 mutants are thought to be equally co-expressed with wild-type Cx43 in ODDD patients, it is imperative to examine the consequence of these mutants in model systems that reflect this molar ratio. To that end, we used differential fluorescent protein tagging of mutant and wild-type Cx43 to quantitatively monitor the ratio of mutant/wild-type within the same putative gap junction plaques and co-immunoprecipitation to determine if the mutants interact with wild-type Cx43. Together the fluorescence-based assay was combined with patch clamp analysis to assess the dominant negative potency of Cx43 mutants. Our results revealed that the ODDD-linked Cx43 mutants, G21R and G138R, as well as amino terminus green fluorescent protein-tagged Cx43, were able to co-localize with wild-type Cx43 at the gap junction plaque-like structures and to co-immunoprecipitate with wild-type Cx43. All Cx43 mutants demonstrated dominant negative action on gap junctional conductance of wild-type Cx43 but not that of Cx32. More interestingly, these Cx43 mutants demonstrated different potencies in inhibiting the function of wild-type Cx43 with the G21R mutant being two times more potent than the G138R mutant. The potency difference in the dominant negative properties of ODDD-linked Cx43 mutants may have clinical implications for the various symptoms and disease severity observed in ODDD patients.     

Use of chimeric forms of neuronal nitric-oxide synthase as dominant negative mutants

Because the functional form of neuronal nitric-oxide synthase (nNOS) is a homodimer, we investigated whether we could disrupt dimer formation with inactive nNOS chimeras acting as dominant negative mutants. To test this hypothesis, we either expressed the heme and reductase regions of rat nNOS as single domains or produced fusion proteins between the rat nNOS heme domain and various other electron-shuttling proteins. A dominant negative potential of these constructs was demonstrated by their ability to reduce NOS activity when transfected into a cell line stably expressing rat nNOS. In the presence of these nNOS mutant proteins, cellular levels of inactive nNOS monomers were significantly increased, indicating that their mechanism of action is through the disruption of nNOS dimer formation. These dominant negative mutants should prove valuable in analyzing the role of nNOS in biological systems.     

The interaction of nucleotides and yeast hexokinase

The kinetics of ethenoadenosine triphosphate (?ATP) as the phosphate donor in the phosphoryl transfer reaction of hexokinase were examined to obtain the K_m′s, V's, and K_α's for the nucleotide and sugar. Dissociation constants for eATP and ?ADP with hexokinase were obtained from fluorometric measurements and compared with similar constants obtained kinetically. Other selected nucleoside triphosphates were used as phosphate donors in the hexokinase reaction and their kinetic constants were obtained. Reactions were also performed using two nucleotides simultaneously as phosphorylating substrates for the hexokinase reaction in an attempt to find the individual dissociation constants, K_m′s and K_i′s. These were compared with the K_m′s obtained from using the nucleotides separately in the hexokinase reaction. From these kinetic and fluorescence binding studies, evidence is presented supporting the postulate that the K_m′s are primarily dissociation constants in a random bi-bi mechanism. Analysis of the K_m values provides additional evidence to support the importance of the amino group in position 6 on the purine ring as a hydrogen-bond acceptor during binding. It was found that ?CTP was a much better hexokinase substrate than CTP. These observations suggest that the V for this reaction is highly dependent upon the size of the nucleotide.     

Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA.

The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involved or their biological significance. The endosymbiotic origin of plastids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, suggest that the plastid recombination system is related to its eubacterial counterpart. Therefore, we examined whether dominant negative mutants of the E. coli RecA protein can interfere with the activity of their putative homolog in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. Transformants expressing these mutant RecA proteins showed reduced survival rates when exposed to DNA-damaging agents, deficient repair of chloroplast DNA, and diminished plastid DNA recombination. These results strongly support the existence of a RecA-mediated recombination system in chloroplasts. We also found that the wild-type E. coli RecA protein enhances the frequency of plastid DNA recombination over 15-fold, although it has no effect on DNA repair or cell survival. Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather than by the level of initiating DNA substrates. Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses. This hypothesis could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of their genomes.     

Cyclic nucleotides and development ofMyxococcus xanthus: Analysis of mutants

Approximately 60 developmental mutants ofMyxococcus xanthus M300 were obtained through nitrosoguanidine mutagenesis and placed into three operationally defined categories. Type-I strains exhibited no aggregation or sporulation. Type-II strains were able to aggregate but did not sporulate. A strain classed as a type-III strain was a low-capacity fruiter. Each category displayed defects in cyclic nucleotide behavior that could be predicted from the current model. Most significantly, several aggregationless (type I) mutants lacking cGMP phosphodiesterase aggregated in the presence of externally applied phosphodiesterase. A requirement for cell-cell contact in sporulation has been confirmed. Evidence is presented that suggests the involvement of cAMP phosphodiesterase in sporulation and that sporulation may be a developmental pathway independent of aggregation. These results support a previously published hypothesis of the role of cyclic nucleotides in the development ofM. xanthus.     

Identification and analysis of dominant negative mutants of RIP1 DD that disrupt RIPoptosome core formation

The RIPoptosome, composed of RIP1 and caspase-8, plays an important role in the regulation of apoptosis and necroptosis; however, the mechanism of complex formation by oligomerization and how the caspase-activating process and necroptosis are mediated by the formation of the RIPoptosome is not well-understood. This study revealed that the assembly mechanism of the RIPoptosome core is dependent on salt concentration and not on pH and time. In addition, we demonstrated that three RIP1 mutations, E626K, M637K, and S657K, have dominant negative effects. These dominant negative mutations in RIP1 may have potential applications in therapeutic intervention.     

A distinct class of dominant negative Ras mutants: cytosolic GTP-bound Ras effector domain mutants that inhibit Ras signaling and transformation and enhance cell adhesion.

Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a beta(1)-subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Ras-induced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF- or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first extensive characterization of this functionally distinct class of DN Ras inhibitor proteins.     

Isolation of dominant negative mutants and inhibitory antisense RNA sequences by expression selection of random DNA fragments.

Selective inhibition of specific genes can be accomplished using genetic suppressor elements (GSEs) that encode antisense RNA, dominant negative mutant proteins, or other regulatory products. GSEs may correspond to partial sequences of target genes, usually identified by trial and error. We have used bacteriophage lambda as a model system to test a concept that biologically active GSEs may be generated by random DNA fragmentation and identified by expression selection. Fragments from eleven different regions of lambda genome, encoding specific peptides or antisense RNA sequences, rendered E. coli resistant to the phage. Analysis of these GSEs revealed some previously unknown functions of phage lambda, including suppression of the cellular lambda receptor by an 'accessory' gene of the phage. The random fragment selection strategy provides a general approach to the generation of efficient GSEs and elucidation of novel gene functions.     

Truncated TrkB: beyond a dominant negative receptor

BDNF activates trkB receptors to regulate neuronal survival, differentiation, and proliferation. Mutations in the BDNF gene, altered BDNF expression, and altered trkB expression are associated with degenerative and psychiatric disorders. The full-length trkB receptor (trkB.tk(+)) undergoes autophosphorylation to activate intracellular signaling pathways. The truncated trkB receptor (trkB.t1) is abundantly expressed in the brain but lacks the catalytic tyrosine kinase domain. TrkB.t1 is a dominant-negative receptor that inhibits trkB.tk(+) signaling. While this is an important function of trkB.t1, it is only one of its many functions. TrkB.t1 sequesters and translocate BDNF, induces filopodia and neurite outgrowth, stimulates intracellular signaling cascades, regulates Rho GTPase signaling, and modifies cytoskeletal structures. TrkB.t1 is an active signaling molecule with regulatory effects on neurons and astrocytes.     

The interaction of borate and sulfite with pyridine nucleotides.

The kinetics and equilibria of the borate interaction at ribose with NAD+ and NMN+ have been measured using as a chromophoric probe the perturbation effect borate has on the addition of sulfite to the 4 position of the nicotinamide ring. NAD+ and NMN+ have more favorable borate association constants than do their corresponding sulfite addition complexes. The rate of interaction of the ribose moiety with borate at low borate buffer concentration is dependent on the concentration of both borate and boric acid. At high borate concentration the rate becomes independent of borate concentration, indicating the existence of a two-step process for the interaction of NAD-sulfite with borate with a change of rate-determining step from the interaction of the ribose hydroxyl group with borate at low borate to an elimination of sulfite at high borate concentration. A linear free energy relationship with a slope of 0.94 describes an increased reactivity of the nucleotide for sulfite as the affinity of the nucleotide for sulfite increases.     

The interaction of thrombin and nucleotides: Effects of glycolysis in human platelets

Thrombin stimulated lactate formation in intact, but not disrupted, platelets, an effect inhibited by ADP and ATP. ADP and ATP stimulated lactate formation in disrupted, but not intact, platelets, an effect inhibited by thrombin. Both nucleotides altered the electrophoretic mobility of thrombin in polyacrylamide gel without affecting its molecular weight. Binding of thrombin to nucleotides could not be demonstrated by gel filtration, equilibrium dialysis, or affinity chromatography.     

Rapid generation of rice mutants via the dominant negative suppression of the mismatch repair protein OsPMS1

Mismatch repair (MMR) is a conservative pathway for maintaining the genome integrity of different organisms. Although suppression of MMR has resulted in various mutation phenotypes in Arabidopsis, the use of this strategy for mutation breeding in major crops has not been reported. Here, we overexpressed a truncated version of the OsPMS1 protein in rice; this approach is expected to suppress the rice MMR system through a dominant negative mechanism. We observed a wide spectrum of mutation phenotypes in the progeny of the transgenic plants during seed germination and the plant growth stages. Genomic variations were detected with inter-simple sequence repeat (ISSR), and sequencing of the differential ISSR bands revealed that the mutation occurred as a point mutation or as microsatellite instability at high frequencies. Plant lines with agronomically important traits, such as salt and drought tolerance, various tiller number, and early flowering, were obtained. Furthermore, we obtained mutants with important traits that are free of the transgene. Together, these results demonstrate that MMR suppression can be used as an efficient strategy for mutation breeding in rice.     

The interaction between binocular rivalry and negative afterimages

Afterimage formation, historically attributed to retinal mechanisms, may also involve postretinal process. Consistent with this notion are results from experiments, reported here, investigating the interaction between binocular rivalry and negative afterimages (AIs). In Experiment 1, one eye was exposed to a grating never consciously experienced by the observer because this grating remained suppressed in rivalry throughout induction (the exclusively dominant stimulus was designed to preclude formation of an AI). As expected, the suppressed grating generated a vivid AI whose orientation could be accurately identified; not surprisingly, the strength of this AI varied with induction contrast. Experiment 2 revealed, however, that the strength of this AI produced during suppression was significantly weaker than the AI produced by that same stimulus when it was visible throughout the entire induction period, implying that some component of AI induction is susceptible to interocular suppression. In Experiment 3, AIs of dichoptic, orthogonally oriented gratings were induced in a way ensuring that one of the two gratings was exclusively dominant during the induction period. Dissimilar monocular AIs engaged in rivalry, as expected, but, surprisingly, the AI induced by the suppressed grating initially dominated. We offer two alternative accounts of this counterintuitive finding, both based on differential neural adaptation.     

Dendritic nucleotides: interaction with an aliphatic acid monolayer

Dendrimeric-T and dendrimeric-A with 36 nucleotides were synthesized using phosphoramidite reagents. These dendrimers contain the nucleosides dA and dT, which are the components of the dA-dT base pair. Branching was obtained using a bifurcated and trifurcated reagent. The dendrimers were purified by ion-exchange chromatography. No specific interactions between these dendrimers were observed using atomic force microscopy. However, the dendrimeric-T is able to disrupt monolayers of fatty acids, resulting in a reorientation of the lipids (from head-to-tail to head-to-head) as demonstrated by scanning tunneling microscopy (STM).