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PubMed2001

G protein beta3 subunit variant: tendency of increasing susceptibility to hypertension in Japanese.

The mechanism of human G beta mutation, G beta3-s, to produce a "gain-of-function" G-protein signaling abnormality remains to be elucidated. Both the enhanced Gi-mediated signalings and the expression of G beta3-s have been demonstrated to be associated with hypertension, together with the finding that the T825 variant might be associated with the occurrence of a splice variant GNB3-s in a white population. The aim of the present study was to reveal a key role of G beta by confirming the association between this polymorphism and susceptibility of hypertension in Japanese. Genotype analysis of 180 normotensive and 179 hypertensive subjects suggests that the T allele tends to be related to the prevalence of hypertension. When age, sex and body mass index were controlled by multiple logistic regression, odds ratios for hypertension associated with the T allele were 1.77 (TT vs CC; 95% confidence interval (CI) 0.97-3.21; p = 0.06), 1.13 (TC vs CC; 95% CI 0.62-2.05; p = 0.69) and 1.40 (TT + TC vs CC; 95% CI 0.81-2.40; p = 0.23). The T allele frequency was found to be significantly higher in hypertensives than in normotensives (0.63 vs 0.56; chi2 = 4.27; p = 0.04), both of which are considerably higher than the frequency observed in Whites. Higher T allele frequencies in Japanese may represent one of the ethnic differences: a larger subgroup whose hypertension is increased by excessive salt diet. Confirming the association between the T allele and hypertension by further investigation and then utilizing the various intermediate features or phenotypes, such as Na+/H+ exchanger activity and renin levels, may help to unravel the active role of the beta subunit.

Adult↗
PubMed2004

Molecular population genetics of redundant floral-regulatory genes in Arabidopsis thaliana.

Functional redundancy between duplicated genes is predicted to be transitory, as one gene either loses its function or gains a new function, or both genes accrue degenerative, yet complimentary mutations. Yet there are many examples where functional redundancy has been maintained between gene duplicates. To determine whether selection is acting on functionally redundant gene duplicates, we performed molecular evolution and population genetic analyses between two pairs of functionally redundant MADS-box genes from the model plant Arabidopsis thaliana: SEPALLATA1 (SEP1) and SEPALLATA2 (SEP2), involved in floral organ identity, and SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2), involved in seed shattering. We found evidence for purifying selection acting to constrain functional divergence between paralogous genes. The protein evolution of both pairs of duplicate genes is functionally constrained, as evidenced by Ka/Ks ratios of 0.16 between paralogs. This functional constraint is stronger in the highly conserved DNA-binding and protein-binding MIK region than in the C-terminal region. We also assayed the evolutionary forces acting between orthologs of the SEP and SHP genes in A. thaliana and the closely related species, Arabidopsis lyrata. Heterogeneity analyses of the polymorphism-to-divergence ratio indicate selective sweeps have occurred within the transcriptional unit of SHP1 and the promoter of SHP2 in the A. thaliana lineage. Similar analyses identified a significant reduction in polymorphism within the SEP1 locus, spanning the 3' region of intron 1 to exon 3, that may represent an intragenic sweep within the SEP1 locus. We discuss whether the evolutionary forces acting on SEP1 and SEP2 versus SHP1 and SHP2 vary according to their position in the floral developmental pathway, as found with other floral-regulatory genes.

Arabidopsis↗
PubMed2004

Rme1 is necessary for Mi-1-mediated resistance and acts early in the resistance pathway.

The tomato gene Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphid, and whitefly. Using genetic screens, we have isolated a mutant, rme1 (resistance to Meloidogyne spp.), compromised in resistance to M. javanica and potato aphid. Here, we show that the rme1 mutant is also compromised in resistance to M. incognita, M. arenaria, and whitefly. In addition, using an Agrobacterium-mediated transient assay in leaves to express constitutive gain-of-function mutant Pto(L205D), we demonstrated that the rme1 mutation is not compromised in Pto-mediated hypersensitive response. Moreover, the mutation in rme1 does not result in increased virulence of pathogenic Pseudomonas syringae or Mi-1-virulent M. incognita. Using a chimeric Mi-1 construct, Mi-DS4, which confers constitutive cell death phenotype and A. rhizogenes root transformation, we showed that the Mi-1-mediated cell death pathway is intact in this mutant. Our results indicate that Rme1 is required for Mi-1-mediated resistance and acts either at the same step in the signal transduction pathway as Mi-1 or upstream of Mi-1.

Animals↗
PubMed1995

Positional signaling by hedgehog in Drosophila imaginal disc development.

We describe a dominant gain-of-function allele of the segment polarity gene hedgehog. This mutation causes ectopic expression of hedgehog mRNA in the anterior compartment of wing discs, leading to overgrowth of tissue in the anterior of the wing and partial duplication of distal wing structures. The posterior compartment of the wing is unaffected. Other imaginal derivatives are affected, resulting in duplications of legs and antennae and malformations of eyes. In mutant imaginal wing discs, expression of the decapentaplegic gene, which is implicated in the hedgehog signaling pathway, is also perturbed. The results suggest that hedgehog protein acts in the wing as a signal to instruct neighboring cells to adopt fates appropriate to the region of the wing just anterior to the compartmental boundary.

Alleles↗
PubMed1994

The dominant Drop eye mutations of Drosophila melanogaster define two loci implicated in normal eye development.

The three existing dominant gain-of-function Drop alleles, Dr1, DrMio and DrWe, previously assumed to define a single locus, severely disrupt eye development. Genetic analysis of ethylmethanesulphonate (EMS) and irradiation-induced revertants revealed that the Drop mutations define two loci: the Drop locus, which is defined by the Dr1 and DrMio mutants, and a separate locus defined by the DrWe mutation, which has been renamed Wedge. The majority of the Dr1 and DrMio revertants are embryonic lethal in trans, mutant embryos exhibiting trachea that fail to join the Filzkörper, thus revealing a role for the Drop gene in embryogenesis. Clonal analysis of lethal revertant alleles suggests a role for both genes in eye development. In the Drop homozygous mutant clones, the outer photoreceptor cells R1-R6 develop aberrantly. Wedge, however, is not required by the developing photoreceptor cells but its absence does disrupt normal ommatidial alignment. Although the Drop and nearby string loci were shown to be genetically distinct, both Dr1 and DrMio were found to interact in trans with lesions at the string locus, causing loss and derangement of bristles and loss of neuromuscular coordination.

Alleles↗
PubMed2004

Identification of two novel mutations in the Cu/Zn superoxide dismutase gene with familial amyotrophic lateral sclerosis: mass spectrometric and genomic analyses.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons. The majority of patients are sporadic cases, while 5-10% of the patients have a family history of ALS (fALS). Mutations in the gene that encodes cytoplasmic Cu/Zn superoxide dismutase (SOD1) have been identified in about 25% of fALS cases. Although the precise pathogenesis of ALS is still unknown, experimental studies including animal models suggest that fALS is caused by the toxic gain-of-function of the SOD1 mutant. We have analyzed not only SOD1 gene mutation by genomic sequencing, but also SOD1 mutant protein by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). We analyzed 33 fALS patients and found 10 mutations in SOD1 gene, in which two were novel: Asp101His substitution in exon 4 and Gly141Glu substitution in exon 5. Here, we present their mass spectrometric protein analyses and clinical features.

Age of Onset↗
PubMed2003

Hyperthermostabilization of Bacillus licheniformis alpha-amylase and modulation of its stability over a 50 degrees C temperature range.

Bacillus licheniformis alpha-amylase (BLA) is a highly thermostable starch-degrading enzyme that has been extensively studied in both academic and industrial laboratories. For over a decade, we have investigated BLA thermal properties and identified amino acid substitutions that significantly increase or decrease the thermostability. This paper describes the cumulative effect of some of the most beneficial point mutations identified in BLA. Remarkably, the Q264S-N265Y double mutation led to a rather limited gain in stability but significantly improved the amylolytic function. The most hyperthermostable variants combined seven amino acid substitutions and inactivated over 100 times more slowly and at temperatures up to 23 degrees C higher than the wild-type enzyme. In addition, two highly destabilizing mutations were introduced in the metal binding site and resulted in a decrease of 25 degrees C in the half-inactivation temperature of the double mutant enzyme compared with wild-type. These mutational effects were analysed by protein modelling based on the recently determined crystal structure of a hyperthermostable BLA variant. Our engineering work on BLA shows that the thermostability of an already naturally highly thermostable enzyme can be substantially improved and modulated over a temperature range of 50 degrees C through a few point mutations.

Amino Acid Substitution↗
PubMed2006

Mutant caveolin-3 induces persistent late sodium current and is associated with long-QT syndrome.

BACKGROUND: Congenital long-QT syndrome (LQTS) is a primary arrhythmogenic syndrome stemming from perturbed cardiac repolarization. LQTS, which affects approximately 1 in 3000 persons, is 1 of the most common causes of autopsy-negative sudden death in the young. Since the sentinel discovery of cardiac channel gene mutations in LQTS in 1995, hundreds of mutations in 8 LQTS susceptibility genes have been identified. All 8 LQTS genotypes represent primary cardiac channel defects (ie, ion channelopathy) except LQT4, which is a functional channelopathy because of mutations in ankyrin-B. Approximately 25% of LQTS remains unexplained pathogenetically. We have pursued a "final common pathway" hypothesis to elicit novel LQTS-susceptibility genes. With the recent observation that the LQT3-associated, SCN5A-encoded cardiac sodium channel localizes in caveolae, which are known membrane microdomains whose major component in the striated muscle is caveolin-3, we hypothesized that mutations in caveolin-3 may represent a novel pathogenetic mechanism for LQTS. METHODS AND RESULTS: Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, we performed open reading frame/splice site mutational analysis on CAV3 in 905 unrelated patients referred for LQTS genetic testing. CAV3 mutations were engineered by site-directed mutagenesis and the molecular phenotype determined by transient heterologous expression into cell lines that stably express the cardiac sodium channel hNa(v)1.5. We identified 4 novel mutations in CAV3-encoded caveolin-3 that were absent in >1000 control alleles. Electrophysiological analysis of sodium current in HEK293 cells stably expressing hNa(v)1.5 and transiently transfected with wild-type and mutant caveolin-3 demonstrated that mutant caveolin-3 results in a 2- to 3-fold increase in late sodium current compared with wild-type caveolin-3. Our observations are similar to the increased late sodium current associated with LQT3-associated SCN5A mutations. CONCLUSIONS: The present study reports the first CAV3 mutations in subjects with LQTS, and we provide functional data demonstrating a gain-of-function increase in late sodium current.

Amino Acid Sequence↗
PubMed1999

Overproduction of SecA suppresses the export defect caused by a mutation in the gene encoding the Escherichia coli export chaperone secB.

SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins in Escherichia coli. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the secA locus that compensate for the export defect caused by the secB missense mutation secBL75Q were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.

Adenosine Triphosphatases↗
PubMed1994

p53 expression in Barrett's oesophagus, dysplasia, and adenocarcinoma using antibody DO-7.

Barrett's oesophagus has a well-recognized association with oesophageal adenocarcinoma, with phenotypic progression through dysplasia to malignancy. The nuclear phosphoprotein p53 is a putative tumour suppressor with mutations resulting in both loss of negative growth regulatory function and possible gain of oncogene function. Many mutant forms have a prolonged half-life and are demonstrable with immunohistochemical techniques. We examined 62 endoscopic oesophageal biopsies and 36 oesophageal resections for p53 overexpression using the monoclonal antibody DO-7 on paraffin-embedded tissue. The series included 40 cases of Barrett's metaplasia, 13 cases of dysplasia, and 81 cases of adenocarcinoma. None of the cases of metaplasia was p53-positive, compared with 4/13 cases of dysplasia and 52/81 cases of adenocarcinoma. There was no association between the degree of dysplasia and p53 expression, although a trend emerged of increasing p53 expression with higher tumour grade. We conclude that p53 overexpression is frequent in oesophageal adenocarcinoma and may be related to tumour grade. p53 overexpression is not restricted to neoplastic lesions and mutation of this tumour suppressor may occur early in the malignant progression of Barrett's oesophagus.

Adenocarcinoma↗
PubMed1991

Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal-ventral polarity in the Drosophila embryo.

The asymmetry of the dorsal-ventral pattern of the Drosophila embryo appears to depend on the ventral activation of the transmembrane Toll protein. The Toll protein is found around the entire dorsal-ventral circumference of the embryo, and it appears to act as a receptor for a ventral, extracellular signal and to then relay that signal to the cytoplasm in ventral regions of the embryo. Three of five recessive loss-of-function alleles of Toll are caused by point mutations in the region of the cytoplasmic domain of Toll that is similar to the mammalian interleukin-1 receptor, supporting the hypothesis that Toll acts as a signal-transducing receptor. Nine dominant gain-of-function alleles that cause Toll to be active in dorsal, as well as ventral, regions of the embryo are caused by mutations in the extracellular domain. Three of the dominant alleles appear to cause the protein to be constitutively active and are caused by cysteine-to-tyrosine changes immediately outside the transmembrane domain. All six of the remaining dominant alleles require the presence of a wild-type transmembrane Toll protein for their ventralizing effect and all encode truncated proteins that lack the transmembrane and cytoplasmic domains.

Alleles↗
PubMed1993

Effect of type IIB von Willebrand disease mutation Arg(545)Cys on platelet glycoprotein Ib binding--studies with recombinant von Willebrand factor.

Type IIB von Willebrand disease (vWD) is characterized by a selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma due to their abnormally enhanced reactivity with platelets. Several missense mutations in the platelet glycoprotein Ib (GPIb) binding domain of vWF were recently characterized that cause type IIB vWD. The effect of type IIB mutation Arg(545)Cys on vWF binding to platelet GPIb was studied using recombinant wild type (rvWFWT) and mutant rvWFR545C expressed in COS-7 cells. In the absence of ristocetin, 50% of rvWFR545C bound spontaneously to platelet GPIb and the binding increased to 70% in the presence of 0.2 mg/ml ristocetin; rvWFWT did not bind significantly under either condition. Botrocetin-induced binding of rvWFR545C was only slightly increased compared to rvWFWT. These data demonstrate that the Arg(545)Cys mutation increases the affinity of vWF for GPIb, resulting in the characteristics gain-of-function type IIB vWD phenotype.

Arginine↗
PubMed1998

[Recent progress in clinical aspects of receptor research].

Clinical receptology encompasses broad areas, including receptor or postreceptor defects due to mutations of receptor or other genes, abnormalities due to receptor antibodies and secondary changes of receptors under various pathological conditions. Recent progress in molecular biology has succeeded in cloning genes of receptors, G-proteins and other cellular proteins that are involved in the signal transduction and clarified their germ-line and somatic mutations. It is of importance that mutations of receptors and G-proteins do not necessarily cause loss of function but sometimes cause gain of function of receptors or G-proteins, thus leading to hyperfunction. Molecular basis that causes either loss or gain of function has been studied but is not completely understood. Some examples of gain of function mutatious of G-protein coupled receptors, tyrosin kinase-type receptors and G alpha protein are shown. Another important aspect in receptor research is that mutation of a single receptor gene sometimes result in different phenotypes and even different modes of inheritance. For example, mutations of rhodopsin (a G-protein coupled receptor) gene cause retinitis pigmentosa of autosomal dominant type and autosomal recessive type and also cause congenital stationary night blindness. Exact mechanisms responsible for such differences are not completely understood. There are polymorphisms in some genes that may be involved in some diseases. An example is a polymorphism in beta 3-adrenergic receptor that is claimed but not clearly demonstrated to be a cause of obesity or type II diabetes. Such polymorphism is possibly a gene in polygenic diseases. Receptology is important for elucidating pathogenesis of complex diseases.

Endocrine System Diseases↗
PubMed1997

Increased 3-nitrotyrosine and oxidative damage in mice with a human copper/zinc superoxide dismutase mutation.

Mutations in copper/zinc superoxide dismutase (SOD1) cause a subset of cases of autosomal dominant familial amyotrophic lateral sclerosis (FALS). Transgenic mice that express these point mutations develop progressive paralysis and motor neuron loss thought to be caused by a gain-of-function of the enzyme. The gain-of-function may be an enhanced ability of the mutant SOD1 to generate .OH radicals or to facilitate peroxynitrite-mediated nitration of proteins. We found significant increases in concentrations of 3-nitrotyrosine, a marker of peroxynitrite-mediated nitration, in upper and lower spinal cord and in cerebral cortex of transgenic mice with the FALS-associated G93A mutation. Malondialdehyde, a marker of lipid peroxidation, was increased in cerebral cortex. 3-Nitrotyrosine-, heme oxygenase-1-, and malondialdehyde-modified protein immunoreactivities were increased throughout SOD1 transgenic mice spinal cord but particularly within motor neurons. These results suggest that the gain-of-function of at least one mutant SOD1 associated with FALS involves increased protein nitration and oxidative damage, which may play a role in neuronal degeneration.

Analysis of Variance↗
PubMed2000

Oncogenic mutations of the p53 tumor suppressor: the demons of the guardian of the genome.

The p53 guardian of the genome is inactivated in the majority of cancers, mostly through missense mutations that cause single residue changes in the DNA binding core domain of the protein. Not only do such mutations result in the abrogation of wild-type p53 activity, but the expressed p53 mutant proteins also tend to gain oncogenic functions, such as interference with wild-type p53-independent apoptosis. Because p53 mutants are highly expressed in cancer cells and not in normal cells, their reactivation to wild-type p53 function may eliminate the cancer by apoptosis or another p53-dependent mechanism. Several studies that embarked on this quest for reactivation have succeeded in restoring wildtype p53 activity to several p53 mutants. However, mutants with more extensive structural changes in the DNA binding core domain may be refractory to reactivation to the wild-type p53 phenotype. Therefore, understanding the structure and functions of oncogenic p53 mutants may lead to more potent reactivation modalities or to the ability to eliminate mutant p53 gain of function.

Animals↗
PubMed2006

Inactivating mutations of G protein-coupled receptors and diseases: structure-function insights and therapeutic implications.

Since the discovery of the first rhodopsin mutation that causes retinitis pigmentosa in 1990, significant progresses have been made in elucidating the pathophysiology of diseases caused by inactivating mutations of G protein-coupled receptors (GPCRs). This review aims to compile the compelling evidence accumulated during the past 15 years demonstrating the etiologies of more than a dozen diseases caused by inactivating GPCR mutations. A generalized classification scheme, based on the life cycle of GPCRs, is proposed. Insights gained through detailed studies of these naturally occurring mutations into the structure-function relationship of these receptors are reviewed. Therapeutic approaches directed against the different classes of mutants are being developed. Since intracellular retention emerges as the most common defect, recent progresses aimed at correcting this defect through membrane permeable pharmacological chaperones are highlighted.

Animals↗
PubMed2003

Domain organization of the MscS mechanosensitive channel of Escherichia coli.

The major structural features of the Escherichia coli MscS mechanosensitive channel protein have been explored using alkaline phosphatase (PhoA) fusions, precise deletions and site-directed mutations. PhoA protein fusion data, combined with the positive-inside rule, strongly support a model in which MscS crosses the membrane three times, adopting an N(out)-C(in) configuration. Deletion data suggest that the C-terminal domain of the protein is essential for the stability of the MscS channel, whereas the protein will tolerate small deletions at the N-terminus. Four mutants that exhibit either gain-of-function (GOF) or loss-of-function have been identified: a double mutation I48D/S49P inactivates MscS, whereas the MscS mutants T93R, A102P and L109S cause a strong GOF phenotype. The similarity of MscS to the last two domains of MscK (formerly KefA) is reinforced by the demonstration that expression of a truncated MscK protein can substitute for MscL and MscS in downshock survival assays. The data derived from studies of the organization, conservation and the influence of mutations provide significant insights into the structure of the MscS channel.

Alkaline Phosphatase↗
PubMed1998

Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator.

In order to gain a better insight into the structure and function of the regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, 19 RD missense mutations that had been identified in patients were functionally characterized. Nine of these (I601F, L610S, A613T, D614G, I618T, L619S, H620P, G628R and L633P) resulted in aberrant processing. No or a very small number of functional CFTR proteins will therefore appear at the cell membrane in cells expressing these mutants. These mutations were clustered in the N-terminal part of the RD, suggesting that this subdomain has a folding pattern that is very sensitive to amino acid changes. Mutations that caused no aberrant processing were further characterized at the electrophysiological level. First, they were studied at the whole cell level in Xenopus laevis oocytes. Mutants that induced a whole cell current that was significantly different from wild-type CFTR were subsequently analysed at the single channel level in COS1 cells transiently expressing the different mutant and wild-type proteins. Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Finally, T665S and E826K CFTR had single channel properties not significantly different from wild-type CFTR.

Amino Acid Sequence↗