Sixth Mutant Research Articles

Molecular mechanisms underlying angiotensin II‐induced angiotensin converting enzyme 2 internalization and degradation

Angiotensin converting enzyme II (ACE2), the newest identified member of renin‐angiotensin system (RAS), catalyses the conversion of angiotensin (ang)‐II to ang‐(1‐7) which plays a compensatory role through Mas1 receptor in the cardiovascular system. We previously reported that decreased ACE2 expression and activity leads to the development of ang‐II‐mediated hypertension in mice, that ang‐II stimulated ACE2 internalization and degradation in lysosomes and that inhibition of this process prevents the hypertensive actions of ang‐II. On the other hand, severe acute respiratory syndrome coronavirus is internalized together with ACE2, clearly indicating a regulated endocytotic mechanisms for this enzyme. However, the molecular mechanisms controlling this event remain largely unknown and the aim of the present study was to clarify these aspects.We first performed a proteomic study in HEK293T cells co‐transfected with ACE2 and AT1R and found that ang‐II treatment (100 nM, 4 h) significantly changed the interactions of ACE2 only with fascin, an actin‐bundling protein. Confocal microscopy experiments confirmed that ang‐II strongly enhanced the co‐localization of the two proteins in the perinuclear space. In addition, we demonstrated that ang‐II treatment augmented ubiquitination of ACE2 and amino acid analysis indicated that in the C‐terminus the enzyme has only five putative lysine ubiquitination sites. Six ACE2 mutants were constructed, five in which each individual ubiquitination site was replaced by arginine, and the sixth mutant (ACE2‐L5R) included all five mutations. In basal conditions in HEK293T cells co‐transfected with AT1R, ACE2 wild‐type activity was 3536.88 ±331.63FU/min/μg protein. Ang‐II treatment (100 nM, 4h) significantly attenuated enzymatic activity (3298.60±255.89). Remarkably, every mutant displayed enhanced enzymatic activity in basal conditions of at least 195±45%, with the highest increase observed in the case of ACE2‐L5R mutant being 285±61%. Furthermore, none of these ACE2 mutants displayed decreased enzymatic activity after ang‐II treatment. The present results demonstrate that ACE2 is constitutively ubiquinated and that this posttranslational modification is augmented by increased levels of ang‐II, contributing to lysosomal degradation of the enzyme in fascin‐dependent manner and overall diminishing its compensatory role against overactive RAS. These data indicate that interfering with the mechanisms contributing to ang‐II‐mediated ACE2 degradation may constitute an alternative therapy in cardiovascular diseases.Support or Funding InformationSupported by NIH‐P20‐GM103514 (EL, JJG and CMF), R01‐HL093178 (EL), and startup funds from Howard University (CMF)

The Core Oligosaccharide and Thioredoxin of Vibrio cholerae Are Necessary for Binding and Propagation of Its Typing Phage VP3

VP3 is a T7-like phage and was used as one of the typing phages in a phage-biotyping scheme that has been used for the typing of Vibrio cholerae O1 biotype El Tor. Here, we studied the receptor and other host genes of V. cholerae necessary for the lytic propagation of VP3. Six mutants resistant to VP3 infection were obtained from the random transposon insertion mutant bank of the sensitive strain N16961. The genes VC0229 and VC0231, which belong to the wav gene cluster encoding the core oligosaccharide (OS) region of lipopolysaccharide, were found to be interrupted by the transposon in five mutants, and the sixth mutant had the transposon inserted between the genes rhlB and trxA, which encode the ATP-dependent RNA helicase RhlB and thioredoxin, respectively. Gene complementation, transcription analysis, and the loss of VP3 sensitivity by the gene deletion mutants confirmed the relationship between VP3 resistance and VC0229, VC0231, and trxA mutation. The product of VP3 gene 44 (gp44) was predicted to be a tail fiber protein. gp44 could bind to the sensitive wild-type strain and the trxA mutant, but not to VC0229 and VC0231 mutants. The results showed that OS is a VP3 receptor on the surface of N16961, thioredoxin of the host strain is involved in the propagation of the phage, and gp44 is the tail fiber protein of VP3. This revealed the first step in the infection mechanism of the T7-like phage VP3 in V. cholerae.

The Vacuolar-ATPase Complex Regulates Retinoblast Proliferation and Survival, Photoreceptor Morphogenesis, and Pigmentation in the Zebrafish Eye

The vacuolar (v)-ATPase complex is a key regulator of the acidification of endosomes, lysosomes, and the luminal compartments of several cell types, tissues, and organs; however, little is know about the in vivo function of the v-ATPase complex or its roles during eye development. This study was conducted to characterize ocular defects in five zebrafish mutants in which core components of the v-ATPase complex were affected (atp6v1h, atp6v1f, atp6v1e1, atp6v0c, and atp6v0d1), as well as a sixth mutant in which a v-ATPase associated protein (atp6ap1) was affected. v-ATPase mutant zebrafish were characterized by histologic, molecular, and ultrastructural analyses. v-ATPase mutant zebrafish were oculocutaneous albinos and presented with defects in the formation and/or survival of melanosomes and with malformations in the retinal pigmented epithelium (RPE) that compromised melanosome distribution. They were microphthalmic, and BrdU incorporation assays indicated that retinoblast cell cycle exit and sustained proliferation in the ciliary marginal zone (CMZ) were compromised. v-ATPase mutants also possessed elevated levels of apoptotic neurons within their retinas and brains. Photoreceptor outer segment morphology was abnormal in the mutant eye with rosette structures forming adjacent to the affected regions of the RPE. Ultrastructural analyses indicate that RPE cells in v-ATPase mutants possess numerous membrane-bounded vacuoles containing undigested outer segment material. In situ hybridization analyses localized v-ATPase subunit transcripts within the RPE. These results demonstrate that the v-ATPase complex plays several critical roles during vertebrate eye development and maintenance, and they suggest that defects in v-ATPase complex function could possibly underlie human ocular disorders that affect the RPE.

Isolation and Characterization of a Genetically Tractable Photoautotrophic Fe(II)-Oxidizing Bacterium, Rhodopseudomonas palustris Strain TIE-1

We report the isolation and characterization of a phototrophic ferrous iron [Fe(II)]-oxidizing bacterium named TIE-1 that differs from other Fe(II)-oxidizing phototrophs in that it is genetically tractable. Under anaerobic conditions, TIE-1 grows photoautotrophically with Fe(II), H2, or thiosulfate as the electron donor and photoheterotrophically with a variety of organic carbon sources. TIE-1 also grows chemoheterotrophically in the dark. This isolate appears to be a new strain of the purple nonsulfur bacterial species Rhodopseudomonas palustris, based on physiological and phylogenetic analysis. Fe(II) oxidation is optimal at pH 6.5 to 6.9. The mineral products of Fe(II) oxidation are pH dependent: below pH 7.0 goethite (alpha-FeOOH) forms, and above pH 7.2 magnetite (Fe3O4) forms. TIE-1 forms colonies on agar plates and is sensitive to a variety of antibiotics. A hyperactive mariner transposon is capable of random insertion into the chromosome with a transposition frequency of approximately 10(-5). To identify components involved in phototrophic Fe(II) oxidation, mutants of TIE-1 were generated by transposon mutagenesis and screened for defects in Fe(II) oxidation in a cell suspension assay. Among approximately 12,000 mutants screened, 6 were identified that are specifically impaired in Fe(II) oxidation. Five of these mutants have independent disruptions in a gene that is predicted to encode an integral membrane protein that appears to be part of an ABC transport system; the sixth mutant has an insertion in a gene that is a homolog of CobS, an enzyme involved in cobalamin (vitamin B12) biosynthesis.

The function of conserved amino acid residues adjacent to the effector domain in elongation factor G

Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino acid positions necessary for EF-G functions, a series of mutations were constructed in the EF-G structural gene (fusA) of Escherichia coli, specifically at positions flanking the effector domain. A mutated allele was isolated in which the wild-type sequence from codons 29 to 47 ("EFG2947") was replaced with a sequence encoding 28 amino acids from ribosomal protein S7. This mutated gene was unable to complement a fusAts strain when supplied in trans at the nonpermissive temperature. In vitro biochemical analysis demonstrated that nucleotide crosslinking was unaffected in EFG2947, while ribosome binding appeared to be completely abolished. A series of point mutations created within this region, encoding L30A, Y32A, H37A, and K38A were shown to give rise to fully functional proteins, suggesting that side chains of these individual residues are not essential for EF-G function. A sixth mutant, E41A, was found to inefficiently rescue growth in a fusAts background, and was also unable to bind ribosomes normally in vitro. In contrast E41Q could restore growth at the nonpermissive temperature. These results can be explained within the context of a three-dimensional model for the effector region of EF-G. This model indicates that the effector domain contains a negative potential field that may be important for ribosome binding.

Evaluation of the role of His447 in the reaction catalyzed by cholesterol oxidase.

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that His447 acts as the general base catalyst for oxidation, and that the resulting imidazolium ion formed acts as an electrophile for isomerization. In this work, we undertook an assessment of the proposed dual roles of His447 in the oxidation and isomerization reactions. To test its role, we constructed five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hydrogen bond donors and acceptors and carboxylate bases at this position, and a sixth mutant, E361Q, to test the interplay between His447 and Glu361. These mutants were characterized using steady-state kinetics and deuterium substrate and solvent isotope effects. For those mutants that catalyze either oxidation of cholesterol or isomerization of cholest-5-en-3-one, the Km's vary no more than 3-fold relative to wild type. H447K is inactive in both oxidation (> 100,000-fold reduced) and isomerization assays (> 10,000-fold reduced). H447E and H447D do not catalyze oxidation (> 100,000-fold reduced), but do catalyze isomerization, 10(4) times slower than wild type. The k(cat) for H447Q is 120-fold lower than wild type for oxidation, and the same as wild type for isomerization. The k(cat) for H447N is 4400-fold lower than wild type for oxidation, and is 30-fold lower than wild type for isomerization. E361Q does not catalyze isomerization (> 10,000-fold reduced), and the k(cat) for oxidation is 30-fold lower than wild type. The substrate deuterium kinetic isotope effects for the wild-type and mutant-catalyzed oxidation reactions suggest that mutation of His447 to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl deprotonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electrophile to catalyze formation of a dienolic intermediate. Moreover, consideration of kinetic and structural results together suggests that a hydrogen bonding network involving His447, Glu361 and Asn485, Wat541, and substrate serves to position the substrate and coordinate general base and electrophilic catalysis. That is, in addition to its previously demonstrated role as base for deprotonation of carbon-4 during isomerization, Glu361 has a structural role and may act as a general base during oxidation. The His447, Asn485, Glu361, and Wat541 residues are conserved in other GMC oxidoreductases. Observation of this catalytic tetrad in flavoproteins of unknown function may be diagnostic for an ability to oxidize unactivated alcohols.

Behavioral analysis ofDrosophila mutants displaying abnormal male courtship

We describe six recessive autosomal male sterile mutations in Drosophila, generated by mobilization of single P-elements, exhibiting abnormal male courtship behavior. Detailed analysis of courtship behavior elicited by virgin wild type females indicated that five of the six mutants are affected in the early steps of courtship. The sixth mutant is blocked at the step of attempted copulation which occurs later in the courtship sequence. All of the mutants have normal olfactory responses and normal locomotor activity. No defect in the visual modality has been observed for the five mutants affected in the initiation of courtship. The mutant blocked at attempted copulation lacks the 'on' and 'off' transients, but this appears to be due to genetic background rather than the mutation itself. Abnormal spermatogenesis was observed in five of the mutants. Spermatogenic defects vary and include lesions in the proliferation of the germline, in meiosis, and in the differentiation and maturation of the spermatids into motile sperm.

Importance of the content and localization of tyrosine residues for thyroxine formation within the N-terminal part of human thyroglobulin

Thyroxine (T4) is formed by coupling of iodinated tyrosine residues within thyroglobulin (TG). In mature TG, some iodinated tyrosine residues are involved preferentially in T4 formation. In order to investigate the specific role of various tyrosine residues in T4 formation, N-terminal TG fragments with mutated tyrosine residues were constructed. An N-terminal TG fragment 198 amino acids in size and containing seven tyrosine residues at amino acid positions 5, 29, 89, 97, 107, 130 and 192 was expressed in a baculovirus system. Using site-directed mutagenesis, eight mutant TG fragments were constructed in which different tyrosine residues were replaced by phenylalanine. In the first four TG mutants, one single tyrosine residue (5, 89, 97 or 130) was mutated. In the mutant Y(5,89,97,130)F all of these four tyrosine residues were replaced. The sixth mutant Y(29,89,107,130,192)F contained only tyrosine residues 5 and 97 and the seventh (Y(29,89,97,192)F) contained only tyrosine residues 5, 107 and 130. A TG fragment (Y(5,29,89,97,107,130,192)F) in which all tyrosine residues were replaced by phenylalanine was used as a negative control. After in vitro iodination with lactoperoxidase, specific T4 formation was established in the non-mutated wild-type N-terminal TG fragment. In general the T4 formation in the mutant TG constructs decreased when the total number of tyrosine residues in the 198 amino acid fragment decreased, except fragment Y(29,89,97,192) containing three tyrosine residues, two of them being 5 and 130. Although the rate of T4 formation in this mutated N-terminal TG fragment was lower, the ultimate T4 generation was the same as in the wild-type fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Complementation of Virus Movement in Transgenic Tobacco Expressing the Cucumber Mosaic Virus 3a Gene

Tobacco plants were transformed with the cucumber mosaic cucumovirus (CMV) 3a gene and the in planta-expressed 3a protein was detected immunologically. The 3a protein was predominantly localized in a subcellular fraction corresponding to the cytosol. Two frameshift and four deletion mutants were created within the 3a open reading frame of CMV RNA 3. Five of these mutants, containing an N-terminal, large central, or C-terminal 70-amino-acid deletion could not infect nontransformed tobacco plants, but could infect the 3a transgenic tobacco plants, and generally accumulated to wild-type levels. The sixth mutant, lacking the C-terminal 43 amino acids of the 3a protein, was able to infect nontransformed tobacco plants. A delay in accumulation of viral RNA in both the inoculated and the systemically infected leaves was demonstrated for one of the mutants. Thus, the CMV 3a protein is a virus movement protein, the functions of which can be complemented in a transgenic plant. The CMV 3a transgenic plants were able to complement the long-distance movement of a pseudorecombinant cucumovirus defective for this function in tobacco, as well as the cell-to-cell, but not the long-distance, movement of two other related viruses. However, these transgenic plants were unable to complement the long-distance movement of viruses from several other taxonomic groups that could move cell to cell but not long distance in tobacco.

Characterization of Mutants ofCercospora nicotianaeSensitive to the Toxin Cercosporin

Six mutants of Cercospora nicotianae that were isolated for sensitivity to the photoactivated toxin cercosporin were characterized for cercosporin sensitivity and production, presence of protective compounds, and pathogenicity. The mutants fell into two classes. Five of the mutants (CS2, CS6, CS7, CS8, and CS9) were inhibited when grown on medium amended with cercosporin concentrations as low as 0.1 μM. Cercosporin sensitivity of these mutants increased markedly with increasing light intensity from 3 to 40 μE m -2 s -1 . These mutants showed no general light sensitivity in the absence of cercosporin. These mutants were capable of synthesizing cercosporin even though their growth was inhibited when cercosporin was produced. These five mutants were protected against cercosporin toxicity by exogenously added ascorbate, cysteine, and reduced glutathione. The sixth mutant (CS10) was not inhibited by cercosporin concentrations below 10 μM and showed little change in cercosporin sensitivity under increasing light intensity. CS10 also synthesized cercosporin. However, growth of this mutant was unaffected under cercosporin-producing conditions. CS10 was protected against high concentrations of cercosporin by exogenously added cysteine and reduced glutathione, but not by ascorbate. None of the six mutants were altered in endogenous levels of β-carotene, ascorbate, cysteine, reduced glutathione, or total soluble or protein thiols. CS2, CS8, and CS10 were compared to wild-type for ability to produce lesions on tobacco. CS2 and CS8 produced significantly fewer lesions than did wild-type; CS10 was intermediate.

Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display

A single-chain variable fragment (Fv) version of a murine monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-polysaccharide, was used as a model system for testing monovalent phage display as a route for enhancing the relatively low affinities that typify anti-carbohydrate antibodies. Random single-chain Fv mutant libraries generated by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a randomly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, five of which showed wild type activity by enzyme immunoassay. Two of these were apparently selected on the basis of better functional single-chain Fv yield in Escherichia coli. A heavy chain mutation (Ile77-->Thr) in one mutant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic product. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, by enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding properties of 4B2 were attributable to a higher association rate constant and interaction with an epitope that is larger than the trisaccharide recognized by the wild type. None of the mutations involved known trisaccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Examination of the structure indicated that two mutations in the heavy chain CDR2 provided improved surface complementarity between the protein and the extended epitope encompassing 2 additional hexose residues. However, introduction of only the CDR2 mutations into the wild type structure failed to confer the improved binding properties of 4B2, indicating an indirect effect by the more distant mutations. Panning of randomly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or binding properties.

Detection of recombinational mutations in cultured human cells by Southern blot analysis with minisatellite DNA probes

Using the human acute monocytic leukemia cell line, THP-1, a hypermutability of minisatellite loci was demonstrated in cell culture by Southern blot analysis with minisatellite DNA probes. DNA was isolated from 98 subclones and hybridized to a panel of minisatellite probes consisting of three multilocus minisatellite probes (ML probes) and seven locus-specific minisatellite probes (LS probes). The Southern blot patterns of the hybridized subclones were compared with those of the parental THP-1. Four mutated bands with two ML probes and two mutated bands with two LS probes were detected. The mutation frequency was estimated roughly at 0.1% based on the total number of bands analyzed, and it was much higher than that expected for other DNA regions. Four of these mutations were thought to be alterations of repetitions caused by insertion or deletion of tandem repeats, and one mutant lost a complete minisatellite allele. The nature of the sixth mutant was unclear. Because of the hypermutability of minisatellite DNA, Southern blot analysis using minisatellite DNA probes can be used as a mutation assay system directly based on the DNA.

Isolation and characterization of visible light-sensitive mutants of Escherichia coli K12

Six mutants of Escherichia coli K12 that are sensitive to visible light have been isolated. Five of them, including an amber mutant, are defective in a gene that maps near 11 minutes on the linkage map of the chromosome, and this gene has been designated visA. The sixth mutant, which was isolated from bacteria that carried the visA + visA + diploid allele, is defective in a gene that maps near 63 minutes on the linkage map, which has been designated visB. These mutant strains of bacteria are killed by illumination with visible light. The effective wavelength of the light is around 460 nm. The nucleotide sequence of the visA gene was determined. As a result of a search for homologous products, we found that visA may be identical to hemH, the structural gene for ferrochelatase which catalyzes a final step in the biosynthesis of heme. A possible mechanism for the killing of the visA mutant bacteria is discussed.

Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): Genes controlling movement of single cells

M. xanthus is a gliding bacterium whose motility is subject to intercellular control. Strain DK101 of M. xanthus gives rise to 6 distinct types of nonmotile mutants and transduction of motility between mutants, mediated by the generalized transducing phage Mx8, identifies the gene loci that underlie the six types. Five of the types, B, C, D, E, and F, are contitional mutants that can be stimulated to move by wild-type cells or by cells of a different mutant type. Mutants of each stimulation type lie in separate and distinct loci, cglB, cglC, cglD, cglE and cglF. The sixth mutant type can stimulate any of the five other types to move, never moves itself, and is produced by mutations in at least 17 loci.

Genetic Studies of Induced Mutants in Melilotus alba. II. Inheritance and Complementation of Chlorophyll‐deficient Mutants1

Six ethyl methanesulfonate‐induced mutants of Melilotus alba Desr. were studied. Five of the mutants behaved as monogenic recessives. In the sixth mutant, two independent recessive alleles were responsible for the observed phenotype. Of these two genes, one had a phenotypic effect similar to the five single‐gene mutants. The other (the veined gene) caused a chlorophyll deficiency in which the leaf veins were darker in color than the tissue between the veins. Complementation analysis revealed that five of the seven genes detected in the mutants were nonallelic. Suggested designations for the five genes are ch4, ch5, ch6, ch7, and chv.

Isolation of nutritional mutants in Arabidopsis thaliana.

An accumulation method was devised for the isolation of nutritional mutants of the CruciferArabidopsis thaliana. Six mutants were obtained, in all of which the synthesis of thiamine was blocked. The mutants showed chlorophyll deficiencies and were more or less depressed in growth. Thiamine, when added to the substrate, fully restored normal development. Three of the mutants also grew on a substrate containing the pyrimidine moiety of the vitamin molecule, one on a medium with the thiazole part, one on a substrate containing a mixture of both compounds, whereas the sixth mutant required the complete thiamine molecule for normal growth. Two of the three “pyrimidine”-less mutants were allelic, the third, when crossed with each of the other two, yielded F1's showing wild type growth in their youth but deficiency symptoms in later stages. In each of the other mutants different loci were involved. The relation between the method employed and the type of mutant isolated is discussed.