Nuclear localization and dominant-negative suppression by a mutant SKCa3 N-terminal channel fragment identified in a patient with schizophrenia

The small conductance calcium-activated K+ channel gene SKCa3/KCNN3 maps to 1q21, a region strongly linked to schizophrenia. Recently, a 4-base pair deletion in SKCa3 was reported in a patient with schizophrenia, which truncates the protein at the end of the N-terminal cytoplasmic region (SKCa3Delta). We generated a green fluorescent protein-SKCa3 N-terminal construct (SKCa3-1/285) that is identical to SKCa3Delta except for the last two residues. Using confocal microscopy we demonstrate that SKCa3-1/285 localizes rapidly and exclusively to the nucleus of mammalian cells like several other pathogenic polyglutamine-containing proteins. This nuclear targeting is mediated in part by two polybasic sequences present at the C-terminal end of SKCa3-1/285. In contrast, full-length SKCa3, SKCa2, and IKCa1 polypeptides are all excluded from the nucleus and express as functional channels. When overexpressed in human Jurkat T cells, SKCa3-1/285 can suppress endogenous SKCa2 currents but not voltage-gated K+ currents. This dominant-negative suppression is most likely mediated through the co-assembly of SKCa3-1/285 with native subunits and the formation of non-functional tetramers. The nuclear localization of SKCa3-1/285 may alter neuronal architecture, and its ability to dominantly suppress endogenous small conductance K(Ca) currents may affect patterns of neuronal firing. Together, these two effects may play a part in the pathogenesis of schizophrenia and other neuropsychiatric disorders.     

accordion, a zebrafish behavioral mutant, has a muscle relaxation defect due to a mutation in the ATPase Ca2+ pump SERCA1

When wild-type zebrafish embryos are touched at 24 hours post-fertilization (hpf), they typically perform two rapid alternating coils of the tail. By contrast, accordion (acc) mutants fail to coil their tails normally but contract the bilateral trunk muscles simultaneously to shorten the trunk, resulting in a pronounced dorsal bend. Electrophysiological recordings from muscles showed that the output from the central nervous system is normal in mutants, suggesting a defect in muscles is responsible. In fact, relaxation in acc muscle is significantly slower than normal. In vivo imaging of muscle Ca2+ transients revealed that cytosolic Ca2+ decay was significantly slower in acc muscle. Thus, it appears that the mutant behavior is caused by a muscle relaxation defect due to the impairment of Ca2+ re-uptake. Indeed, acc mutants carry a mutation in atp2a1 gene that encodes the sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1), a Ca2+ pump found in the muscle sarcoplasmic reticulum (SR) that is responsible for pumping Ca2+ from the cytosol back to the SR. As SERCA1 mutations in humans lead to Brody disease, an exercise-induced muscle relaxation disorder, zebrafish accordion mutants could be a useful animal model for this condition.     

nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction

Summary Eleven green individuals were isolated when 95000 M₂ plants of barley (Hordeum vulgare L.), mutagenised with azide in the M₁, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F₂ progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase. Elevation of NADH-nitrate reductase activity in the nir1 mutant suggests a regulatory perturbation in the expression of the Narl gene.     

Contribution of a tyrosine-based motif to cellular trafficking of wild-type and truncated NPY Y1 receptors

The human NPY Y₁ receptor undergoes fast agonist-induced internalization via clathrin-coated pits then recycles back to the cell membrane. In an attempt to identify the molecular determinants involved in this process, we studied several C-terminal truncation mutants tagged with EFGP. In the absence of agonist, Y₁ receptors lacking the last 32 C-terminal amino acids (Y₁Δ32) are constitutively internalized, unlike full-length Y₁ receptors. At steady state, internalized Y₁Δ32 receptors co-localize with transferrin, a marker of early and recycling endosomes. Inhibition of constitutive internalization of Y₁Δ32 receptors by hypertonic sucrose or by co-expression of Rab5aS34N, a dominant negative form of the small GTPase Rab5a or depletion of all three isoforms of Rab5 indicates the involvement of clathrin-coated pits. In contrast, a truncated receptor lacking the last 42 C-terminal amino acids (Y₁Δ42) does not constitutively internalize, consistent with the possibility that there is a molecular determinant responsible for constitutive internalization located in the last 10 amino acids of Y₁Δ32 receptors. We show that the agonist-independent internalization of Y₁Δ32 receptors involves a tyrosine-based motif YXXΦ. The potential role of this motif in the behaviour of full-length Y₁ receptors has also been explored. Our results indicate that a C-terminal tyrosine-based motif is critical for the constitutive internalization of truncated Y₁Δ32 receptors. We suggest that this motif is masked in full-length Y₁ receptors which do not constitutively internalize in the absence of agonist.     

A mutant with a defect in telomere elongation leads to senescence in yeast

We describe a general assay designed to detect mutants of yeast that are defective for any of several aspects of telomere function. Using this assay, we have isolated a mutant that displays a progressive decrease in telomere length as well as an increased frequency of chromosome loss. This mutation defines a new gene, designated EST1 (for ever shorter telomeres). Null alleles of EST1 are not immediately inviable; instead, they have a senescence phenotype, due to the gradual loss of sequences essential for telomere function, leading to a progressive decrease in chromosomal stability and subsequent cell death.     

A novel approach to assess insulin sensitivity reveals no increased insulin sensitivity in mice with a dominant-negative mutant hepatocyte nuclear factor-1alpha

In phenotype experiments in mice, determination of dynamic insulin sensitivity often uses the insulin tolerance test. However, the interpretation of this test is complicated by the counterregulation occurring at low glucose. To overcome this problem, we determined the dynamic insulin sensitivity after inhibition of endogenous insulin secretion by diazoxide (25 mg/kg) in association with intravenous administration of glucose plus insulin (the DSGIT technique). Estimation of insulin sensitivity index (SI) by this technique showed good correlation to SI from a regular intravenous glucose tolerance test (r = 0.87; P < 0.001; n = 15). With DSGIT, we evaluated dynamic insulin sensitivity in mice with a rat insulin promoter (beta-cell-targeted) dominant-negative mutation of hepatic nuclear factor (HNF)-1alpha [RIP-DN HNF-1alpha (Tg) mice]. When insulin was administered exogenously at the same dose in Tg and wild-type (WT) mice, plasma insulin levels were higher in WT, indicating an increased insulin clearance in Tg mice. When the diazoxide test was used, different doses of insulin were therefore administered (0.1 and 0.15 U/kg in WT and 0.2 and 0.25 U/kg in Tg) to achieve similar insulin levels in the groups. Minimal model analysis showed that SI was the same in the two groups (0.78 +/- 0.21 x 10(-4) min.pmol(-1).l(-1) in WT vs. 0.60 +/- 0.11 in Tg; P = 0.45) as was the glucose elimination rate (P = 0.27). We conclude that 1) the DSGIT technique determines the in vivo dynamic insulin action in mice, 2) insulin clearance is increased in Tg mice, and 3) chronic islet dysfunction in RIP-DN HNF-1alpha mice is not compensated with increased insulin sensitivity.     

DKT1, a novel K+ channel from carrot, forms functional heteromeric channels with KDC1

We report the isolation and characterisation of DKT1, a new carrot K+ channel alpha-subunit belonging to the Shaker-like family. DKT1 is expressed in many tissues of the adult plant, suggesting that it may play important roles in both nutrition and other important physiological processes. During embryo development, DKT1 is expressed at later phases implying the involvement of K+ in embryo maturation. When co-expressed with KDC1 in Xenopus oocytes, DKT1 is able to form functional, heteromeric channels, suggesting that possible interactions between these two ion channels in plant tissues may modulate K+ uptake.     

PC12 cells transfected with human mutant gene causing one of Alzheimer's disease forms have a high sensitivity to oxidative stress

Used in this work are PC12 cells transfected with human gene expressing amyloid precursor protein of beta-peptide and carrying the so-called "Swedish mutation" leading to the appearance of one Alzheimer's disease family forms. It has been shown that the PC12 cells transfected with this mutant gene, at action of various hydrogen peroxide concentrations, die to the significant greater degree than the used for comparison PC12 cells transfected with analogous human gene of the wild type or than vector-transfected cells. It has been found that ganglioside GM1 at micro- or nanomolar concentrations is able to increase viability of the PC12 cells transfected with the mutant gene causing a significant accumulation of endogenous amyloid beta-peptide. The obtained data confirm an important role of oxidative stress in injury and death of brain nerve cells in Alzheimer's disease.     

PC12 cells transfected with human mutant gene causing one of Alzheimer’s disease forms have a high sensitivity to oxidative stress

Used in this work are PC12 cells transfected with human gene expressing amyloid-precursor protein of β-peptide and carrying the so-called “Swedish mutation” leading to the appearance of one of Alzheimer’s disease family forms. It has been shown that the PC12 cells transfected with this mutant gene, at action of various hydrogen peroxide concentrations, die to the significant greater degree than the used for comparison PC12 cells transfected with analogous human gene of the wild type or than vector-transfected cells. It has been found that ganglioside GM1 at micro-or nanomolar concentrations is able to increase viability of the PC12 cells transfected with the mutant gene causing a significant accumulation of endogenous amyloid β-peptide. The obtained data confirm an important role of oxidative stress in injury and death of brain nerve cells in Alzheimer’s disease.     

An enzyme trafficking defect in two patients with primary hyperoxaluria type 1: peroxisomal alanine/glyoxylate aminotransferase rerouted to mitochondria

Most patients with the autosomal recessive disease primary hyperoxaluria type 1 (PH1) have a complete deficiency of alanine/glyoxylate aminotransferase (AGT) enzyme activity and immunoreactive protein. However a few possess significant residual activity and protein. In normal human liver, AGT is entirely peroxisomal, whereas it is entirely mitochondrial in carnivores, and both peroxisomal and mitochondrial in rodents. Using the techniques of isopycnic sucrose and Percoll density gradient centrifugation and quantitative protein A-gold immunoelectron microscopy, we have found that in two PH1 patients, possessing 9 and 27% residual AGT activity, both the enzyme activity and immunoreactive protein were largely mitochondrial and not peroxisomal. In addition, these individuals were more severely affected than expected from the levels of their residual AGT activity. In these patients, the PH1 appears to be due, at least in part, to a unique trafficking defect, in which peroxisomal AGT is diverted to the mitochondria. To our knowledge, this is the first example of a genetic disease caused by such interorganellar rerouting.     

A Chinese hamster ovary cell mutant with a heat-sensitive, conditional- lethal defect in vacuolar function

We describe a mutant derived from Chinese hamster ovary cells that is offt-sensitive for viability and for resistance to certain protein toxins. This mutant, termed G.7.1, grows normally at 34 degrees C but does not grow in Dulbecco's modified Eagle's medium at 39.5 degrees C. However, when this medium is supplemented with FeSO4, the mutant cells will grow at the elevated temperature. At 39.5 degrees C, G.7.1 cells acquire resistance to diphtheria toxin, modeccin, and Pseudomonas aeruginosa exotoxin A, all of which are protein toxins that require endocytosis and exposure to a low pH within vesicles before they can invade the cytosol and kill cells. The properties of mutant G.7.1 could result from a heat-sensitive lesion that impairs vacuolar acidification. We assayed the ATP-stimulated generation of pH gradients across the membrane of vesicles in cell-free preparations from mutant and parental cells by the partitioning of acridine orange into acidic compartments and found that the acidification response of the mutant cells was heat-labile. Altogether the evidence suggests that G.7.1 cells contain a heat-sensitive lesion that impairs vacuolar acidification and that they fail to grow in normal medium at 39.5 degrees C because they cannot extract Fe+3 from transferrin, a process that normally requires exposing transferrin to a low pH within endosomal vesicles.     

A genetic analysis of a mutant of Escherichia coli with a defect in the assembly of ribosomes

Summary The complex phenotype of a mutant of Escherichia coli with a defect in the assembly of ribosomes has been shown to be due to the presence of two mutations, both of which are closely linked to the gene (xyl) that determines the ability to ferment xylose. One of the mutations is responsible for the defect in ribosome synthesis, whilst the other is associated with an altered degree of resistance to streptomycin and a reduced efficiency in the utilisation of the growth substrate.     

A novel response-regulator is able to suppress the nodulation defect of a Bradyrhizobium japonicum nodW mutant

The two-component regulatory system Nod-VW of Bradyrhizobium japonicum is essential for the nodulation of the legume host plants Vigna radiata, V. unguiculata and Macroptilium atropurpureum. The NodV protein shares homology with the sensor-kinases, whereas the NodW protein is a member of the response-regulator class. We report here the identification of a new B. japonicum DNA region that is able to suppress the phenotypic defect of a nodW mutant, provided that this region is expressed from a foreign promoter. The minimal complementing region, which itself is not essential for nodulation in a nodW ⁺ background, consists of one gene designated nwsB (nodW-suppressor). The deduced amino acid sequence of the nwsB gene product shows a high degree of homology to NodW. The nwsB gene is preceded by a long open reading frame, nwsA, whose putative product appears to be a sensor-kinase. Downstream of nwsB, an open reading frame encoding a second putative response-regulator was identified. Interspecies hybridization revealed the presence of nwsAB-like DNA also in other Bradyrhizobium strains. Using nwsB-lacZ fusions, the nwsB gene was found to be expressed rather weakly in B. japonicum. This low level of expression is obviously not sufficient to compensate for a nodW ^– defect, whereas strong overexpression of nwsB is a condition that leads to suppression of the nodW ^– mutation.     

A quantum mechanic/molecular mechanic study of the wild-type and N155S mutant HIV-1 integrase complexed with diketo acid

Integrase (IN) is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. Recently, mutation studies have been reported that have shown that a certain degree of viral resistance to diketo acids (DKAs) appears when some amino acid residues of the IN active site are mutated. Mutations represent a fascinating experimental challenge, and we invite theoretical simulations for the disclosure of still unexplored features of enzyme reactions. The aim of this work is to understand the molecular mechanisms of HIV-1 IN drug resistance, which will be useful for designing anti-HIV inhibitors with unique resistance profiles. In this study, we use molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for wild-type and N155S mutant HIV-1 IN, both complexed with a DKA. This hybrid methodology has the advantage of the inclusion of quantum effects such as ligand polarization upon binding, which can be very important when highly polarizable groups are embedded in anisotropic environments, for example in metal-containing active sites. Furthermore, an energy terms decomposition analysis was performed to determine contributions of individual residues to the enzyme-inhibitor interactions. The results reveal that there is a strong interaction between the Lys-159, Lys-156, and Asn-155 residues and Mg²⁺ cation and the DKA inhibitor. Our calculations show that the binding energy is higher in wild-type than in the N155S mutant, in accordance with the experimental results. The role of the mutated residue has thus been checked as maintaining the structure of the ternary complex formed by the protein, the Mg²⁺ cation, and the inhibitor. These results might be useful to design compounds with more interesting anti-HIV-1 IN activity on the basis of its three-dimensional structure.     

The Get1/2 insertase forms a channel to mediate the insertion of tail-anchored proteins into the ER

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A novel extranuclear mutant of Neurospora with a temperature-sensitive defect in mitochondrial protein synthesis and mitochondrial ATPase

Summary [C93] is a novel, extranuclear mutant of Neurospora crassa which has a normal mitochondrial phenotype when grown at 25°, but which is deficient in cytochromes b and aa ₃ when grown at 37° (Pittenger and West 1979). In the present work, the phenotype of [C93] was characterized in greater detail. When [C93] is grown at 37°, the rate of mitochondrial protein synthesis is decreased to approximately 25% that of wild type; the ratio of mitochondrial small to large ribosomal subunits is decreased to 1:4 and mitochondrial small subunits are deficient in the mitochondrially-synthesized protein, S-5. The mitochondrial ribosome assembly defects in 37°-grown [C93] resemble those in chloramphenicol-treated wild-type cells and could merely be a consequence of the decreased rates of mitochondrial protein synthesis. Analysis of mitochondrial translation products by SDS gel electrophoresis suggests that 37°-grown [C93] is grossly deficient in the 19,000 Mr subunit of the oligomycin-sensitive ATPase relative to other mitochondrially-synthesized proteins. The ATPase defect was not found in other extranuclear or nuclear mutants deficient in mitochondrial protein synthesis. These data and additional evidence suggest that the primary defect in [C93] may be in the assembly of the ATPase complex. The possible connection between the ATPase defect and the deficiency of mitochondrial protein synthesis is discussed.     

Downregulation of MHC class I expression due to interference with p105-NF kappa B1 processing by Ad12E1A.

We have previously demonstrated that expression of major histocompatibility complex (MHC) class I genes is repressed in baby rat kidney cells transformed by early region 1 of oncogenic adenovirus type 12 (Ad12E1). Reduced expression of MHC class I antigens contributes to the escape of Ad12-transformed cells from T-cell-mediated immune surveillance and to tumour induction. In this study, we show that repression of MHC class I expression by Ad12E1A is mediated via the H2TF1 element of the MHC class I promoter. This element binds NF kappa B and KBF1, two factors which play a major role in the regulation of MHC class I expression in vivo. In extracts from Ad12E1-transformed cells, binding of KBF1 and NF kappa B to the H2TF1 element is decreased. This is caused by reduced production of p50-NF kappa B1, the 50 kDa subunit shared by KBF1 and NF kappa B, due to interference with p105-NF kappa B1 processing by Ad12-13S-E1A protein. Overexpression of the p105-NF kappa B1 cDNA, or of a truncated p105-NF kappa B1 cDNA that codes for p50-NF kappa B1, restores MHC class I expression in Ad12E1-transformed cells. These data demonstrate that downregulation of MHC class I expression in Ad12E1-transformed cells is due to interference with processing of p105-NF kappa B1 by the Ad12-13S-E1A protein.     

EPI64 interacts with Slp1/JFC1 to coordinate Rab8a and Arf6 membrane trafficking

Cell function requires the integration of cytoskeletal organization and membrane trafficking. Small GTP-binding proteins are key regulators of these processes. We find that EPI64, an apical microvillar protein with a Tre-2/Bub2/Cdc16 (TBC) domain that stabilizes active Arf6 and has RabGAP activity, regulates Arf6-dependent membrane trafficking. Expression of EPI64 in HeLa cells induces the accumulation of actin-coated vacuoles, a distinctive phenotype seen in cells expressing constitutively active Arf6. Expression of EPI64 with defective RabGAP activity does not induce vacuole formation. Coexpression of Rab8a suppresses the vacuole phenotype induced by EPI64, and EPI64 expression lowers the level of Rab8-GTP in cells, strongly suggesting that EPI64 has GAP activity toward Rab8a. JFC1, an effector for Rab8a, colocalizes with and binds directly to a C-terminal region of EPI64. Together this region and the N-terminal TBC domain of EPI64 are required for the accumulation of vacuoles. Through analysis of mutants that uncouple JFC1 from either EPI64 or from Rab8-GTP, our data suggest a model in which EPI64 binds JFC1 to recruit Rab8a-GTP for deactivation by the RabGAP activity of EPI64. We propose that EPI64 regulates membrane trafficking both by stabilizing Arf6-GTP and by inhibiting the recycling of membrane through the tubular endosome by decreasing Rab8a-GTP levels.     

pZ402, an improved SV40-based shuttle vector containing a T-antigen mutant unable to interact with wild-type p53

Shuttle vectors are useful tools for studying DNA replication and mutagenesis. SV40-based shuttle vectors are popular because of their ease of use and quick results. However, one complication with the use of SV40-based shuttle vectors is the interaction of cellular p53 protein with the T-antigen of SV40. Wild-type, but not mutant, p53 has been shown to be involved in DNA replication and DNA repair. To address this concern, we have modified an SV40-based shuttle vector, pZ189, by exchanging the wt T-antigen for a mutant SV40 T-antigen, which is unable to bind with p53. This shuttle vector, pZ402, provides us with a tool to study DNA replication and genomic instability in cells with varying genetic backgrounds without interference from the interaction of T-antigen with p53.     

In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane-associated defect in protein translocation.

Mutants defective in the ability to translocate proteins across the membrane of the endoplasmic reticulum were selected in Trp- Saccharomyces cerevisiae on the basis of their ability to retain a fusion protein in the cytosol. The fusion comprised the prepro region of prepro-alpha-factor (MF alpha 1) N-terminal to phosphoribosyl anthranilate isomerase (TRP1). The first of the protein translocation mutations, called ptl1, results in temperature-sensitivity of growth and protein translocation. At the non-permissive temperature, precursors to several secretory proteins accumulate in the cytosol. Using this mutant, we demonstrate that the prepro-carboxypeptidase Y that had been accumulated in the cytosol at the non-permissive temperature could be post-translationally translocated into the endoplasmic reticulum when cells were returned to the permissive temperature. This result indicates that post-translational translocation of preproteins across endoplasmic reticulum membranes can occur in vivo. We have also determined that the temperature-sensitive component is membrane-associated in ptl1, and that the membranes derived from this strain show a reversible temperature-sensitive translocation phenotype in vitro.