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PubMed2001

The gamma subunit determines whether anesthetic-induced leftward shift is altered by a mutation at alpha1S270 in alpha1beta2gamma2L GABA(A) receptors.

BACKGROUND: A major action of volatile anesthetics is enhancement of gamma-aminobutyric acid receptor type A (GABA(A)R) currents. In recombinant GABA(A)Rs consisting of several subunit mixtures, mutating the alpha1 subunit serine at position 270 to isoleucine [alpha1(S270I)] was reported to eliminate anesthetic-induced enhancement at low GABA concentrations. In the absence of studies at high GABA concentrations, it remains unclear whether alpha1(S270I) affects enhancement versus inhibition by volatile anesthetics. Furthermore, the majority of GABA(A)Rs in mammalian brain are thought to consist of alpha1, beta2, and gamma2 subunits, and the alpha1(S270I) mutation has not been studied in the context of this combination. METHODS: Recombinant GABA(A)Rs composed of alpha1beta2 or alpha1beta2gamma2L subunit mixtures were studied electrophysiologically in whole Xenopus oocytes in the voltage clamp configuration. Currents elicited by GABA (0.03 microM to 1 mM) were measured in the absence and presence of isoflurane or halothane. Anesthetic effects on GABA concentration responses were evaluated for individual oocytes. RESULTS: In wild-type alpha1beta2gamma2L GABA(A), anesthetics at approximately 2 minimum alveolar concentration (MAC) shifted GABA concentration response curves to the left approximately threefold, decreased the Hill coefficient, and enhanced currents at all GABA concentrations. The alpha1(S270I) mutation itself rendered the GABA(A)R more sensitive to GABA and reduced the Hill coefficient. At low GABA concentrations (EC5), anesthetic enhancement of peak current was much smaller in alpha1(S270I)beta2gamma2L versus wild-type channels. Paradoxically, the leftward shift of the whole GABA concentration-response relation by anesthetics was the same in both mutant and wild-type channels. At high GABA concentrations, volatile anesthetics reduced currents in alpha1(S270I)beta2gammaL GABA(A)Rs. In parallel studies on alpha1beta2 (gamma-less) GABA(A)Rs, anesthetic-induced leftward shifts in wild-type receptors were more than eightfold at 2 MAC, and the alpha1(S270I) mutation nearly eliminated anesthetic-induced leftward shift. CONCLUSIONS: The results support a role for alpha1S270 in alpha1beta2gamma2L GABA(A)R gating and sensitivity to inhibition by volatile anesthetics. The alpha1S270 locus also modulates anesthetic enhancement in alpha1beta2 GABA(A)R. The presence of the gamma2L subunit reduces anesthetic-induced left shift of wild-type GABA(A)R and nullifies the impact of the alpha1(S2701) mutation on anesthetic modulation. Thus, the gamma2L subunit plays a significant role in GABA(A)R modulation by volatile anesthetic compounds.

Amino Acid Substitution↗
PubMed1997

Filamentous growth of the budding yeast Saccharomyces cerevisiae induced by overexpression of the WHi2 gene.

The WHI2 gene of the budding yeast Saccharomyces cerevisiae is required for the arrest of cell proliferation upon nutrient exhaustion: whi2 mutants carry on dividing and in the absence of growth become abnormally small. It is reported here that overexpression of Whi2 from the GAL1 promoter results in filamentous growth - cells fail to complete cytokinesis, the budding pattern changes from axial to polar, cells become elongated and cell size increases threefold. In many ways, these filaments resemble the pseudohyphae which result from nitrogen-limited growth and the filaments seen during the invasive growth of haploids. However, Whi2-induced filament formation is reduced, but not blocked, by mutations in STE7, STE12 or STE20 which do block pseudohypha formation. Furthermore, pseudohypha formation can still occur in a diploid in which both copies of the WHI2 gene have been deleted. Thus Whi2-induced filament formation and pseudohypha formation must come about through the action of different pathways. Despite this, a mutation in the STE11 gene, which is required for pseudohypha formation, does block Whi2-induced filament formation. Concanavalin A pulse-chase experiments show that new cell wall material is incorporated only into the tips of the apical cells. An extragenic suppressor of the Whi2 allele also results in filamentous growth. These results suggest that Whi2 negatively regulates a function required for the budding mode of cell proliferation including cytokinesis. This function is defined wholly or in part by the fsw1 allele.

Cell Cycle Proteins↗
PubMed2001

Genetic changes induced in human cells in Space Shuttle experiment (STS-95).

BACKGROUND: Results of past space experiments suggest that the biological effect of space radiation could be enhanced under microgravity. To assess the radiation risk for humans during long-term spaceflight, it is very important to clarify whether human cells exhibit a synergistic effect of radiation and microgravity. HYPOTHESIS: If significant synergism occurs in human cells, genetic changes induced during spaceflight may be detected by using human tumor HCT-116 cells which are hypermutable due to a defect in the DNA mismatch repair system. METHODS: Cultured HCT-116 cells were loaded on the Space Shuttle Discovery (STS-95) and grown during the 9-d mission. After landing, many single-cell clones were isolated, microsatellite repetitive sequences in each clone were amplified by PCR, and mutations in the microsatellite loci were detected as changes in the length of PCR fragments. Mutation frequencies of ouabain-resistant phenotype were also analyzed. RESULTS: The frequencies of microsatellite mutations as well as ouabain-resistant mutations in the flight sample were similar to those of the ground control samples. Some cells were treated in space with bleomycin which mimics the action of radiation, but the frequencies of microsatellite mutations were not significantly different between the flight and the ground control samples. CONCLUSION: Under the present flight conditions, neither space radiation (about 20 mSv during this mission) nor microgravity caused excess mutations in human cells.

Bleomycin↗
PubMed1993

Modulation of different K+ currents in Drosophila: a hypothetical role for the Eag subunit in multimeric K+ channels.

We examined the role of the ether a go-go (eag) gene in modulation of K+ currents and the possibility of its protein product Eag as a subunit in the heteromultimeric assembly of K+ channels by voltage-clamp analysis of larval muscle membrane currents. Previous DNA sequence studies indicate that the eag gene codes for a polypeptide homologous to, but distinct from, the Shaker (Sh) K+ channel subunits (Warmke et al., 1991), and electrophysiological recordings revealed allele-specific effects of eag on four identified K+ currents in Drosophila larval muscles (Zhong and Wu, 1991). Further studies of eag alleles indicated that none of the eag mutations, including alleles producing truncated mRNA messages, eliminate any of the four K+ currents, and that the mutational effects exhibit strong temperature dependence. We found that both W7, an antagonist of Ca2+/calmodulin, and cGMP analogs modulated K+ currents and that their actions were altered or even abolished by eag mutations. These results suggest a role of eag in modulation of K+ currents that may subserve integration of signals at a converging site of the two independent modulatory pathways. The Sh locus is known to encode certain subunits of the IA channel in larval muscle. The existence of multiple eag and Sh alleles enabled an independent test of the idea of Eag as a K+ channel subunit by studying IA in different double-mutant combinations. An array of allele-specific interaction between eag and Sh was observed, which reflects a close association between the Sh and eag subunits within the IA channel. Taken together, our data strengthen the possibility that the eag locus provides a subunit common to different K+ channels. The role of the eag subunit for modulating channels, as opposed to that of Sh subunits required for gating, selectivity, and conductance of the channel, suggest a combinatorial genetic framework for generating diversified K+ channels.

Animals↗
PubMed1986

Mutagenic and comutagenic action of 5'-deoxy-5'-(methylthio)adenosine.

5'-Deoxy-5'-(methylthio)adenosine (MTA) alone and in combination with methionine was found to increase frequencies of background HGPRT mutation and SCE in V79 Chinese hamster cells. The same agents exert a comutagenic action on these effects as well as upon mutation induction in Escherichia coli stain AB1157 (but not in its non-adaptable derivative ada-6) following treatment with N-methyl-N-nitrosourea (MNU). MTA plus methionine also enhanced the lethal action of MNU on hamster cells. The effects observed may tentatively be ascribed to hypomethylation due to inhibition of DNA methylase by MTA.

Adenosine↗
PubMed2005

A novel form of short QT syndrome (SQT3) is caused by a mutation in the KCNJ2 gene.

Short QT syndrome (SQTS) leads to an abbreviated QTc interval and predisposes patients to life-threatening arrhythmias. To date, two forms of the disease have been identified: SQT1, caused by a gain of function substitution in the HERG (I(Kr)) channel, and SQT2, caused by a gain of function substitution in the KvLQT1 (I(Ks)) channel. Here we identify a new variant, "SQT3", which has a unique ECG phenotype characterized by asymmetrical T waves, and a defect in the gene coding for the inwardly rectifying Kir2.1 (I(K1)) channel. The affected members of a single family had a G514A substitution in the KCNJ2 gene that resulted in a change from aspartic acid to asparagine at position 172 (D172N). Whole-cell patch-clamp studies of the heterologously expressed human D172N channel demonstrated a larger outward I(K1) than the wild-type (P<0.05) at potentials between -75 mV and -45 mV, with the peak current being shifted in the former with respect to the latter (WT, -75 mV; D172N, -65 mV). Coexpression of WT and mutant channels to mimic the heterozygous condition of the proband yielded an outward current that was intermediate between WT and D172N. In computer simulations using a human ventricular myocyte model the increased outward I(K1) greatly accelerated the final phase of repolarization, and shortened the action potential duration. Hence, unlike the known mutations in the two other SQTS forms (N588K in HERG and V307L in KvLQT1), simulations using the D172N and WT/D172N mutations fully accounted for the ECG phenotype of tall and asymmetrically shaped T waves. Although we were unable to test for inducibility of arrhythmia susceptibility due to lack of patients' consent, our computer simulations predict a steeper steady-state restitution curve for the D172N and WT/D172N mutation, compared with WT or to HERG or KvLQT1 mutations, which may predispose SQT3 patients to a greater risk of reentrant arrhythmias.

Action Potentials↗
PubMed1997

Dominant-negative KvLQT1 mutations underlie the LQT1 form of long QT syndrome.

BACKGROUND: Mutations that map to the KvLQT1 gene on human chromosome 11 account for more than 50% of inherited long QT syndrome (LQTS). It has been discovered recently that the KvLQT1 and minK proteins functionally interact to generate a current with biophysical properties similar to I(Ks), the slowly activating delayed-rectifier cardiac potassium current. Since I(Ks) modulates the repolarization of cardiac action potentials it is reasonable to hypothesize that mutations in KvLQT1 reduce I(Ks), resulting in the prolongation of cardiac action potential duration. METHODS AND RESULTS: We expressed LQTS-associated KvLQT1 mutants in Xenopus oocytes either individually or in combination with wild-type KvLQT1 or in combination with both wild-type KvLQT1 and minK. Substitutions of alanine with proline in the S2-S3 cytoplasmic loop (A177P) or threonine with isoleucine in the highly conserved signature sequence of the pore (T311I) yield inactive channels when expressed individually, whereas substitution of leucine with phenylalanine in the S5 transmembrane domain (L272F) yields a functional channel with reduced macroscopic conductance. However, all these mutants inhibit wild-type KvLQT1 currents in a dominant-negative fashion. CONCLUSIONS: In LQTS-affected individuals these mutations would be predicted to result in a diminution of the cardiac I(Ks) current, subsequent prolongation of cardiac repolarization, and an increased risk of arrhythmias.

Animals↗
PubMed1999

The synergistic effects of vanillin on recombination predominate over its antimutagenic action in relation to MMC-induced lesions in somatic cells of Drosophila melanogaster.

The wing Somatic Mutation And Recombination Test (SMART) in Drosophila melanogaster was used to study the modulating action of vanillin (VA) in combination with the alkylating agents mitomycin C (MMC), methylmethanesulphonate (MMS) and the bifunctional nitrogen mustard (HN2). Two types of treatments with VA and each of the three genotoxins were performed: chronic co-treatments of three-day-old larvae of the standard cross as well as post-treatments after acute exposure with the genotoxins. This allowed the study of the action of VA not only in the steps that precede the induction of DNA lesions but also in the repair processes. The overall findings from the co-treatment series suggest that ingestion of VA with MMS or MMC can lead to significant protection against genotoxicity; but this is not the case with HN2. Antioxidant activity, suppression of metabolic activation or interaction with the active groups of these two alkylating agents could be mechanisms by means of which VA exerts its desmutagenic action. In contrast, when evaluated in the post-treatment procedure, VA causes two antagonistic effects on the genotoxicity of MMC: (i) synergism on recombination (172.8%) and (ii) protection against mutation (79.0%). Consequently, both activities together lead to a considerable increase in mitotic recombination. In spite of being separate events, recombination and gene mutation are correlated during mitosis since the fate of a DNA lesion depends on the repair pathway followed. Our results may suggest that VA is a modifying factor that blocks the mutagenic pathway and consequently directs the MMC-induced lesions into a recombinational repair. Furthermore, VA did not modify the genotoxicity when administered after treatments with HN2 or MMS. Therefore, the major finding of the present study, namely the co-recombinagenic activity of VA on MMC-induced lesions, seems to be related to the type of induced lesion and consequently to the repair processes involved in its correction.

Animals↗
PubMed2002

Discriminative stimulus properties of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(+/-)DOI] in C57BL/6J mice.

RATIONALE: The drug discrimination procedure has proven to be a valuable tool for studying the mechanism of action of psychoactive drugs. Recently, mice with targeted gene mutations have been developed that may also prove useful in evaluating the role of specific receptors in mediating the actions of drugs. We were interested in studying the effects of hallucinogens in genetically modified mice using the drug discrimination procedure. OBJECTIVE: To establish the training procedures and characterize the stimulus properties of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(+/-)DOI] versus saline in wild-type mice. METHODS: Using a two-lever drug discrimination procedure, C57BL/6J mice were trained to discriminate (+/-)DOI (2.5 mg/kg) from saline on a VI 30-s schedule of reinforcement. RESULTS: The acquisition function was orderly and similar to that found previously with rats, although the training dose required for the mice was four times higher (2.5 versus 0.75 mg/kg). The dose-response relationship indicated that percent drug lever responding was dose-dependent. Two other hallucinogens, LSD and (-)DOB, substituted fully for (+/-)DOI. Mice were tested for their ability to discriminate (+/-)DOI following pretreatment with the 5-HT(2A) receptor antagonist MDL 100,907, or with 5-HT(2C) selective antagonists, SB 206,553 or SB 242,084. A dose of 0.25 mg/kg MDL 100,907 essentially completely blocked the discriminative stimulus effects of 2.5 mg/kg (+/-)DOI. Surprisingly, both SB 206,553 and SB 242,084 also attenuated the effect of 2.5 mg/kg (+/-)DOI. The effect of SB 206,553 was surmountable at 5.0 mg/kg (+/-)DOI. CONCLUSIONS: These data agree with the results from studies with rats indicating a prominent role for the 5-HT(2A) receptors in mediating the discriminative stimulus effects of (+/-)DOI but in addition, suggest a small but significant role for the 5-HT(2C) receptor in mice.

DOM 2,5-Dimethoxy-4-Methylamphetamine↗
PubMed2004

Mutation analysis of five candidate genes in Chinese patients with hypospadias.

Hypospadias is the displacement of the urethral meatus from the tip of the glans to the ventral side of the phallus. During fetal development, SRY, SOX9, WT1, SRD5A2 and AR are important at different stages in the differentiation and development of the male genital system. Mutations in these genes impair masculinization and may be associated with hypospadias. In order to explore these possibilities, we employed polymerase chain reaction and direct sequencing to analyze the coding regions of these five genes in 90 Chinese hypospadias patients. We found a total of 16 different mutations in SRD5A2, AR and WT1 in 24 of these 90 patients. Seven mutations are novel. No mutation was found in SRY or SOX9. SNP V89L found in SRD5A2 was statistically significant between patients and controls. Our results indicated that mutations in SRD5A2, AR and WT1 were associated with hypospadias. In conclusion, mutations are frequently found in genes that control androgen action and metabolism, but are seldom found in genes active in the early phase of sex determination and differentiation. Mutations in AR, SRD5A2 or WT1 seem to be associated not only with hypospadias but also with micropenis.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed2004

Estrogen receptor mutations in human disease.

As early as the 1800s, the actions of estrogen have been implicated in the development and progression of breast cancer. The estrogen receptor (ER) was identified in the late 1950s and purified a few years later. However, it was not until the 1980s that the first ER was molecularly cloned, and in the mid 1990s, a second ER was cloned. These two related receptors are now called ERalpha and ERbeta, respectively. Since their discovery, much research has focused on identifying alterations within the coding sequence of these receptors in clinical samples. As a result, a large number of naturally occurring splice variants of both ERalpha and ERbeta have been identified in normal epithelium and diseased or cancerous tissues. In contrast, only a few point mutations have been identified in human patient samples from a variety of disease states, including breast cancer, endometrial cancer, and psychiatric diseases. To elucidate the mechanism of action for these variant isoforms or mutant receptors, experimental mutagenesis has been used to analyze the function of distinct amino acid residues in the ERs. This review will focus on ERalpha and ERbeta alterations in breast cancer.

Alternative Splicing↗
PubMed2003

Prolonged atrial action potential durations and polymorphic atrial tachyarrhythmias in patients with long QT syndrome.

UNLABELLED: Atrial APD and Polymorphic AT in LQTS. INTRODUCTION: Prolongation of the QT interval and torsades de pointes tachycardias due to altered expression or function of repolarizing ion channels are the hallmark of congenital long QT syndrome (LQTS). The same ion channels also contribute to atrial repolarization, and familial atrial fibrillation may be associated with a mutated KVLQT1 gene. We therefore assessed atrial action potential characteristics and atrial arrhythmias in LQTS patients. METHODS AND RESULTS: Monophasic action potentials (MAPs) were simultaneously recorded from the right atrial appendage and the inferolateral right atrium in 10 patients with LQTS (8 with identifiable genotype) and compared to 7 control patients. Atrial arrhythmias also were compared to MAPs recorded in patients with persistent (n = 10) and induced (n = 4) atrial fibrillation. Atrial action potential durations (APD) and effective refractory periods (ERP) were prolonged in LQTS patients at cycle lengths of 300 to 500 msec (APD prolongation 30-41 msec; ERP prolongation 26-52 msec; all P < 0.05). Short episodes of polymorphic atrial tachyarrhythmias (polyAT, duration 4-175 sec) occurred spontaneously or during pauses after pacing in 5 of 10 LQTS patients, but not in controls (P < 0.05). P waves showed undulating axis during polyAT. Cycle lengths of polyAT were longer than during persistent and induced atrial fibrillation. Afterdepolarizations preceded polyAT in 2 patients. The electrical restitution curve was shifted to longer APD in LQTS patients and to even longer APD in LQTS patients with polyAT. CONCLUSION: This group of LQTS patients has altered atrial electrophysiology: action potentials are prolonged, and polyAT occurs. PolyAT appears to be a specific arrhythmia of LQTS reminiscent of an atrial form of "torsades de pointes".

Action Potentials↗
PubMed1981

Long range control circuits within mitochondria and between nucleus and mitochondria. II. Genetic and biochemical analyses of suppressors which selectively alleviate the mitochondrial intron mutations.

In the preceding paper of this series (Dujardin et al. 1980 a) we described general methods of selecting and genetically characterizing suppressor mutations that restore the respiratory capacity of mit- mitochondrial mutations. Two dominant nuclear (NAM1-1 and NAM2-1) and one mitochondrial (mim2-1) suppressors are more extensively studied in this paper. We have analysed the action spectrum of these suppressors on 433 mit- mutations located in various mitochondrial genes and found that they preferentially alleviate the effects of mutations located within intron open reading frames of the cob-box gene. We conclude that these suppressors permit the maturation of cytochrome b mRNA by restoring the synthesis of intron encoded protein(s) catalytically involved in splicing i.e. mRNA-maturase(s) (cf. Lazowska et al. 1980). NAM1-1 is allele specific and gene non-specific; it suppresses mutations located within different introns. NAM2-1 and mim2-1 are intron-specific: they suppress mutations all located in the same (box7) intron of the cob-box gene. Analyses of cytochrome absorption spectra and mitochondrial translation products of cells in which the suppressors are associated with various other mit- mutations show that the suppressors restore cytochrome b and/or cytochrome oxidase (cox I) synthesis, as expected from their growth phenotype. This suppression is, however, only partial: some new polypeptides characteristic of the mit- mutations can be still detected in the presence of suppressor. Interestingly enough when box7 specific suppressors NAM2-1 and mim2-1 are associated with a complete cob-box deletion (leading to a total deficiency of cytochrome b and oxidase) partial restoration of cox I synthesis is observed while cytochrome b is still totally absent. These results show that in strains carrying NAM2-1 or mim2-1 the presence of cytochrome b gene is no longer required for the expression of the oxi3 gene pointing out to the possibility of a mutational switch-on of silent genes, whether mitochondrial, mim2-1, or nuclear, NAM2-1. This switch-on would permit the synthesis of an active maturase acting as a substitute for the box7 maturase in order to splice the cytochrome b and oxidase mRNAs.

Base Sequence↗
PubMed2001

Profiles of accepted mutation: from neutrality in a pseudogene to disease-causing mutation on its homologous gene.

We have compared the substitution pattern of the glucocerebrosidase gene (GBA) and the glucocerebrosidase pseudogene (psGBA), two highly homologous regions under different selective pressures and within the same genomic background. Mutations in GBA may lead to Gaucher disease, an inborn metabolic disorder. Disease-causing mutations and neutral variation in the gene have been compared to neutral variation in the pseudogene. This comparison offers a unique opportunity to better understand the action of purifying selection, since the differences between mutational patterns can be attributed to different selective pressures. A similar frequency of CpG dinucleotides was observed in GBA and in psGBA, and CpG pairs were mutated with the same high frequency in both regions. However, nucleotides not in CpG pairs were more likely to contribute to disease-causing mutation than to accepted polymorphisms. This pattern, which resulted in a lower transition to transversion ratio in the gene, may be due to CpG avoidance on critical regions within exons.

Base Composition↗
PubMed1986

Microcin-E492-insensitive mutants of Escherichia coli K12.

Mutations in three Escherichia coli K12 genes, tonB, exbB and the newly discovered semA, reduce sensitivity to the low Mr polypeptide antibiotic microcin E492. The products of the tonB and exbB genes were previously shown to be involved in the uptake of siderophore-complexed iron and in the action of a number of colicins. Strains mutated at or close to semA (collectively referred to as sem mutations) remained fully sensitive to these colicins, and grew as well as wild-type strains under conditions of iron starvation. Expression of a number of sem-lacZ operon fusions was not affected by iron limitation, and sem mutations did not affect the production of iron-regulated outer membrane proteins which are known or thought to be involved in iron uptake. Hfr conjugation and P1 phage transduction experiments indicated that semA is located close to pabB at 40 min on the E. coli K12 chromosome. This places semA close to the mng locus, wherein mutations result in decreased manganese sensitivity. However, strains carrying the semA mutation exhibited increased manganese sensitivity.

Anti-Bacterial Agents↗
PubMed2000

Allosteric silencing of activating function 1 in the 4-hydroxytamoxifen estrogen receptor complex is induced by substituting glycine for aspartate at amino acid 351.

The active metabolite of tamoxifen, 4-hydroxytamoxifen (4-OHT), is used in the laboratory for mechanistic studies of antiestrogen action. This compound binds to the estrogen receptor alpha (ER) and silences activating function 2 (AF2) in the ligand binding domain, but activating function 1 (AF1) at the other end of the ER remains constitutive and is considered to be ligand independent. Amino acid D351 in the ligand binding domain appears to be critical for interactions with the antiestrogenic side chain of antiestrogens. We have devised an assay to evaluate the biological activity of 351 mutant ERs and antiestrogens at the transforming growth factor alpha (TGFalpha) gene in situ (J. I. MacGregor Schafer et al., Cancer Res., 59: 4308-4313, 1999). The substitution of glycine for aspartate at position 351 results in the conversion of the 4-OHT:ER complex from estrogen-like to completely antiestrogenic. In cells stably expressing D351G ER, the ER retains responsiveness to estradiol (E2) and also retains antiestrogenic responsiveness to both raloxifene and ICI 182,780. The relative binding affinity of E2 for D351G ER (0.77 +/- 0.17 x 10(-9) M) is comparable with wild-type ER (0.42 +/- 0.08 x 10(-9) M). In addition, the D351G ER retains the ability to bind SRC-1 in the presence of E2, thus D351G ER AF2 activity has not been compromised. We also used a cell line stably expressing an ER with a triple mutation in helix 12 (D538A, E542A, and D545A) that ablated AF2 activity, which resulted in decreased effects of E2, suggesting that both AF1 and AF2 activity are required for maximal estrogen activity in MDA-MB-231 cells. Interestingly, the triple mutation also completely reduced the estrogen-like actions of 4-OHT. We propose that a specific mutation at amino acid 351 can allosterically silence AF1 in the 4-OHT:ER complex by either preventing the binding of coactivators or encouraging the binding of a corepressor molecule. We suggest that the 4-OHT-specific site responsible for estrogen-like actions can be referred to as AF2b. This binding site would consist of at least four carboxylic acids at amino acids 351 and 538, 542 and 545 in helix 12 to permit coactivator docking for gene activation. The AF2b site is distinct from AF2 for E2 action. Further studies will provide insight into the estrogen-like actions of tamoxifen in select tissues and breast tumors and identify a significant mechanism of drug resistance to tamoxifen.

Allosteric Regulation↗
PubMed1996

Inherited activated protein C resistance in a patient with familial primary antiphospholipid syndrome.

The mechanism of thrombophilia in patients with antiphospholipid antibodies (aPL) is not clearly understood. A number of contributing factors have been described, but more than one may be operative. It was recently found that aPL may cause the acquired activated protein C resistance phenotype, whereas in familial thrombophilia, activated protein C resistance frequently results from a point mutation in the factor V gene (replacing arginine 506 with a glutamine) that leads into the (R-506-Q), the so-called Leiden mutation, that produces a mutated factor V, resistant to the catalytic action of activated protein C, otherwise normal in its procoagulant properties. We describe one patient heterozygous for the activated protein C resistance genotype with a familial form of primary antiphospholipid syndrome, who had a nephew who died with this disease. In individuals who are heterozygous for the factor V Leiden mutation, the presence of aPL may cause further activated protein C resistance, resulting in increased thrombophilia.

Adult↗
PubMed2004

Directed evolution of human estrogen receptor variants with significantly enhanced androgen specificity and affinity.

Human estrogen receptor alpha (hERalpha) and human androgen receptor exhibit exquisite ligand specificity, which underlies their remarkable ability to effect ligand-regulated gene transcription in a highly distinctive and specific manner. Here we used a directed evolution approach to create hERalpha variants with enhanced androgen specificity and affinity with the goal to better understand the molecular basis of ER ligand specificity and the evolutionary mechanism of nuclear receptors. We developed a sensitive yeast two-hybrid system to screen for hERalpha variants with increased transactivation potency toward testosterone. After two rounds of directed evolution, we identified five hERalpha variants with dramatically improved transactivation potency toward testosterone in both yeast and mammalian cells. These variants showed up to 7,600-fold improvement in the binding affinity for testosterone and only slightly reduced affinity toward 17beta-estradiol. Detailed analysis of these evolved variants and a few site-directed mutants generated de novo led to several unexpected findings including the following. 1) Only two beneficial mutations were needed to create hERalpha variants with near nanomolar affinity for testosterone. 2) Some beneficial mutations were synergistic, context-dependent, or non-additive. 3) Of the five identified beneficial mutations, four of them were not in the ER ligand binding pocket and yet exerted important action on ligand specificity. 4) The single ligand-contacting mutation E353Q plays a dominant role in discriminating androgens and estrogens. These results, viewed in conjunction with the ligand exploitation model of nuclear receptor evolution, suggest that the mutation E353Q may represent a key event in the evolution of androgen receptors from an ancestral estrogen receptor and that ligand promiscuity may play an important role in the creation of new nuclear receptors via divergent evolution.

Amino Acid Sequence↗