Science.gov

Sample records for active site mutation

  1. Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

    PubMed

    Lipski, Alexandra; Watzlawick, Hildegard; Ravaud, Stéphanie; Robert, Xavier; Rhimi, Moez; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2013-02-01

    Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

  2. An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

    PubMed

    Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

    2016-10-01

    Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

  3. A mutational analysis of the active site of human type II inosine 5'-monophosphate dehydrogenase.

    PubMed

    Futer, Olga; Sintchak, Michael D; Caron, Paul R; Nimmesgern, Elmar; DeCenzo, Maureen T; Livingston, David J; Raybuck, Scott A

    2002-01-31

    The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD.

  4. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  5. Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations

    SciTech Connect

    Radom,C.; Banerjee, A.; Verdine, G.

    2007-01-01

    The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5 to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is 'hardwired'. Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F{sup *149}) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F{sup *292}) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

  6. Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters.

    PubMed Central

    Rothmel, R K; LeClerc, J E

    1989-01-01

    Second-site mutations that restored activity to severe lacP1 down-promoter mutants were isolated. This was accomplished by using a bacteriophage f1 vector containing a fusion of the mutant E. coli lac promoters with the structural gene for chloramphenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infected cells to chloramphenicol. Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacP1 activity was a T----G substitution at position -14, a weakly conserved site in E. coli promoters. Three other sequence changes, G----A at -2, A----T at +1, and C----A at +10, activated nascent promoters in the lac regulatory region. The nascent promoters conformed to the consensus rule, that activity is gained by sequence changes toward homology with consensus sequences at the -35 and -10 regions of the promoter. However, the relative activities of some promoters cannot be explained solely by consideration of their conserved sequence elements. Images PMID:2660105

  7. Activation induced deaminase mutational signature overlaps with CpG methylation sites in follicular lymphoma and other cancers

    PubMed Central

    Rogozin, Igor B.; Lada, Artem G.; Goncearenco, Alexander; Green, Michael R.; De, Subhajyoti; Nudelman, German; Panchenko, Anna R.; Koonin, Eugene V.; Pavlov, Youri I.

    2016-01-01

    Follicular lymphoma (FL) is an uncurable cancer characterized by progressive severity of relapses. We analyzed sequence context specificity of mutations in the B cells from a large cohort of FL patients. We revealed substantial excess of mutations within a novel hybrid nucleotide motif: the signature of somatic hypermutation (SHM) enzyme, Activation Induced Deaminase (AID), which overlaps the CpG methylation site. This finding implies that in FL the SHM machinery acts at genomic sites containing methylated cytosine. We identified the prevalence of this hybrid mutational signature in many other types of human cancer, suggesting that AID-mediated, CpG-methylation dependent mutagenesis is a common feature of tumorigenesis. PMID:27924834

  8. Multiple-site mutations of phage Bp7 endolysin improves its activities against target bacteria.

    PubMed

    Zhang, Can; Wang, Yuanchao; Sun, Huzhi; Ren, Huiying

    2015-10-01

    The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7 (Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a pET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated (L99A, M102E) to construct pET28a-Bp7Δe, with pET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.

  9. Mutation of active site residues in synthetic T4-lysozyme gene and their effect on lytic activity.

    PubMed

    Anand, N N; Stephen, E R; Narang, S A

    1988-06-16

    The active site amino acids (Glu11 and Asp20) in T4-lysozyme have been mutated to their isosteric residues Gln or Asn and/or acidic residues such as Glu----Asp or Asp----Glu by the oligonucleotide-replacement method. Out of eight mutants so generated the mutant T4-lysozyme obtained from pTLY.Asp11 retains maximum amount of activity (approximately 16%), pTLY.Asn20 the least (0.9%) whereas pTLY.Gln11 lost completely. A systematic study of the active and inactive mutants thus generated supports the important role of Glu11 and Asp20 in T4-lysozyme activity as predicted in earlier studies.

  10. A Mutational Analysis of the Active Site Loop Residues in cis-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Schroeder, Gottfried K.; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 share 34% sequence identity (54% similarity). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis-isomer. Although the basis for these differences is unknown, a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A 6-residue active site loop in cis-CaaD shows a strikingly different conformation from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. In order to examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to the wild type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover due to the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to

  11. Sulfide-Binding Hemoglobins: Effects of Mutations on Active-Site Flexibility

    PubMed Central

    Fernandez-Alberti, S.; Bacelo, D. E.; Binning, R. C.; Echave, J.; Chergui, M.; Lopez-Garriga, J.

    2006-01-01

    The dynamics of Hemoglobin I (HbI) from the clam Lucina pectinata, from wild-type sperm whale (SW) myoglobin, and from the L29F/H64Q/V68F triple mutant of SW, both unligated and bound to hydrogen sulfide (H2S), have been studied in molecular dynamics simulations. Features that account for differences in H2S affinity among the three have been examined. Our results verify the existence of an unusual heme rocking motion in unligated HbI that can promote the entrance of large ligands such as H2S. The FQF-mutant partially reproduces the amplitude and relative orientation of the motion of HbI's heme group. Therefore, besides introducing favorable electrostatic interactions with H2S, the three mutations in the distal pocket change the dynamic properties of the heme group. The active-site residues Gln-64(E7), Phe-43(CD1), and His-93(F8) are also shown to be more flexible in unligated HbI than in FQF-mutant and SW. Further contributions to H2S affinity come from differences in hydrogen bonding between the heme propionate groups and nearby amino acid residues. PMID:16782787

  12. Mutational analysis of the active site flap (20s loop) of mandelate racemase.

    PubMed

    Bourque, Jennifer R; Bearne, Stephen L

    2008-01-15

    Mandelate racemase from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Residues of the 20s and 50s loops determine, in part, the topology and polarity of the active site and hence the substrate specificity. Previously, we proposed that, during racemization, the phenyl ring of mandelate moves between an S-pocket comprised of residues from the 50s loop and an R-pocket comprised of residues from the 20s loop [Siddiqi, F., Bourque, J. R., Jiang, H., Gardner, M., St. Maurice, M., Blouin, C., and Bearne, S. L. (2005) Biochemistry 44, 9013-9021]. The 20s loop constitutes a mobile beta-meander flap that covers the active site cavity shielding it from solvent and controlling entry and egress of ligands. To understand the role of the 20s loop in catalysis and substrate specificity, we constructed a series of mutants (V22A, V22I, V22F, T24S, A25V, V26A, V26L, V26F, V29A, V29L, V29F, V26A/V29L, and V22I/V29L) in which the sizes of hydrophobic side chains of the loop residues were varied. Catalytic efficiencies (kcat/Km) for all mutants were reduced between 6- and 40-fold with the exception of those of V22I, V26A, V29L, and V22I/V29L which had near wild-type efficiencies with mandelate. Thr 24 and Ala 25, located at the tip of the 20s loop, were particularly sensitive to minor alterations in the size of their hydrophobic side chains; however, most mutations were tolerated quite well, suggesting that flap mobility could compensate for increases in the steric bulk of hydrophobic side chains. With the exception of V29L, with mandelate as the substrate, and V22F and V26A/V29L, with 2-naphthylglycolate (2-NG) as the substrate, the values of kcat and Km were not altered in a manner consistent with steric obstruction of the R-pocket, perhaps due to flap mobility compensating for the increased size of the hydrophobic side chains. Surprisingly, V22I and V29L catalyzed the racemization of the bulkier substrate 2-NG

  13. Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

    PubMed Central

    Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

    2014-01-01

    Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

  14. Platelet-activating factor acetylhydrolase deficiency. A missense mutation near the active site of an anti-inflammatory phospholipase.

    PubMed Central

    Stafforini, D M; Satoh, K; Atkinson, D L; Tjoelker, L W; Eberhardt, C; Yoshida, H; Imaizumi, T; Takamatsu, S; Zimmerman, G A; McIntyre, T M; Gray, P W; Prescott, S M

    1996-01-01

    Deficiency of plasma platelet-activating factor (PAF) acetylhydrolase is an autosomal recessive syndrome that has been associated with severe asthma in Japanese children. Acquired deficiency has been described in several human diseases usually associated with severe inflammation. PAF acetylhydrolase catalyzes the degradation of PAF and related phospholipids, which have proinflammatory, allergic, and prothrombotic properties. Thus, a deficiency in the degradation of these lipids should increase the susceptibility to inflammatory and allergic disorders. Miwa et al. reported that PAF acetylhydrolase activity is absent in 4% of the Japanese population, which suggests that it could be a common factor in such disorders, but the molecular basis of the defect is unknown. We show that inherited deficiency of PAF acetylhydrolase is the result of a point mutation in exon 9 and that this mutation completely abolishes enzymatic activity. This mutation is the cause of the lack of enzymatic activity as expression in E. coli of a construct harboring the mutation results in an inactive protein. This mutation as a heterozygous trait is present in 27% in the Japanese population. This finding will allow rapid identification of subjects predisposed to severe asthma and other PAF-mediated disorders. PMID:8675689

  15. The Role of Distant Mutations and Allosteric Regulation on LovD Active Site Dynamics

    PubMed Central

    Jiménez-Osés, Gonzalo; Osuna, Sílvia; Gao, Xue; Sawaya, Michael R.; Gilson, Lynne; Collier, Steven J.; Huisman, Gjalt W.; Yeates, Todd O.; Tang, Yi; Houk, K. N.

    2014-01-01

    Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester, and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is the first non-patent report of the enzyme currently used for the manufacture of simvastatin, as well as the intermediate evolved variants. Crystal structures and microsecond molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations dramatically altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF. PMID:24727900

  16. Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis▿

    PubMed Central

    Venkatesan, Ranga N.; Treuting, Piper M.; Fuller, Evan D.; Goldsby, Robert E.; Norwood, Thomas H.; Gooley, Ted A.; Ladiges, Warren C.; Preston, Bradley D.; Loeb, Lawrence A.

    2007-01-01

    Mammalian DNA polymerase δ (Pol δ) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol δ. Homozygous Pold1L604G/L604G and Pold1L604K/L604K mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1+/L604G mice did not differ, while that of Pold1+/L604K mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1+/L604K mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase δ reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer. PMID:17785453

  17. Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate† ,‡

    PubMed Central

    Valley, Michael P.; Fitzpatrick, Paul F.

    2006-01-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707]. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C–H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the ΔG‡ for C–H bond cleavage by 1.6–1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase. PMID:12741843

  18. Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

    PubMed

    Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas; Deschamps, Joshua; Freedman, J Cody; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Holman, Theodore

    2016-11-01

    Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors.

  19. Complete androgen insensitivity syndrome caused by a novel splice donor site mutation and activation of a cryptic splice donor site in the androgen receptor gene.

    PubMed

    Infante, Joana B; Alvelos, Maria I; Bastos, Margarida; Carrilho, Francisco; Lemos, Manuel C

    2016-01-01

    The androgen insensitivity syndrome is an X-linked recessive genetic disorder characterized by resistance to the actions of androgens in an individual with a male karyotype. We evaluated a 34-year-old female with primary amenorrhea and a 46,XY karyotype, with normal secondary sex characteristics, absence of uterus and ovaries, intra-abdominal testis, and elevated testosterone levels. Sequence analysis of the androgen receptor (AR) gene revealed a novel splice donor site mutation in intron 4 (c.2173+2T>C). RT-PCR analysis showed that this mutation resulted in the activation of a cryptic splice donor site located in the second half of exon 4 and in the synthesis of a shorter mRNA transcript and an in-frame deletion of 41 amino acids. This novel mutation associated with a rare mechanism of abnormal splicing further expands the spectrum of mutations associated with the androgen insensitivity syndrome and may contribute to the understanding of the molecular mechanisms involved in splicing defects.

  20. Mutational analysis of the active site residues of a D: -psicose 3-epimerase from Agrobacterium tumefaciens.

    PubMed

    Kim, Hye-Jung; Yeom, Soo-Jin; Kim, Kwangsoo; Rhee, Sangkee; Kim, Dooil; Oh, Deok-Kun

    2010-02-01

    D-Psicose 3-epimerase from Agrobacterium tumefacience catalyzes the conversion of D: -fructose to D-psicose. According to mutational analysis, the ring at position 112, the negative charge at position 156, and the positive charge at position 215 were essential components for enzyme activity and for binding fructose and psicose. The surface contact area and distance to the bound substrate by molecular modeling suggest that the positive charge of Arg215 was involved in stabilization of cis-endiol intermediate. The distances between the catalytic residues (Glu150 and Glu244) and Mn(2+) are critical to the catalysis, and the negative charges of the metal-binding residues are important for interaction with metal ion. The kinetic parameters of the D183E and H209A mutants for metal-binding residues with substrate and the near-UV circular dichroism spectra indicate that the metal ion bound to Asp183 and His209 is involved not only in catalysis but also in substrate binding.

  1. Structural Characterization of Mutations at the Oxygen Activation Site in Monomeric Sarcosine Oxidase

    SciTech Connect

    Schuman Jorns, Marilyn; Chen, Zhi-wei; Mathews, F. Scott

    2010-04-30

    Oxygen reduction and sarcosine oxidation in monomeric sarcosine oxidase (MSOX) occur at separate sites above the si- and re-faces, respectively, of the flavin ring. Mutagenesis studies implicate Lys265 as the oxygen activation site. Substitution of Lys265 with a neutral (Met, Gln, or Ala) or basic (Arg) residue results in an {approx}10{sup 4}- or 250-fold decrease, respectively, in the reaction rate. The overall structure of MSOX and residue conformation in the sarcosine binding cavity are unaffected by replacement of Lys265 with Met or Arg. The side chain of Met265 exhibits the same configuration in each molecule of Lys265Met crystals and is nearly congruent with Lys265 in wild-type MSOX. The side chain of Arg265 is, however, dramatically shifted (4-5 {angstrom}) compared with Lys265, points in the opposite direction, and exhibits significant conformational variability between molecules of the same crystal. The major species in solutions of Lys265Arg is likely to contain a 'flipped-out' Arg265 and exhibit negligible oxygen activation, similar to Lys265Met. The 400-fold higher oxygen reactivity observed with Lys265Arg is attributed to a minor (<1%) 'flipped-in' Arg265 conformer whose oxygen reactivity is similar to that of wild-type MSOX. A structural water (WAT1), found above the si-face of the flavin ring in all previously determined MSOX structures, is part of an apparent proton relay system that extends from FAD N(5) to bulk solvent. WAT1 is strikingly absent in Lys265Met and Lys265Arg, a feature that may account for the apparent kinetic stabilization of a reductive half-reaction intermediate that is detectable with the mutants but not wild-type MSOX.

  2. Single Active Site Mutation Causes Serious Resistance of HIV Reverse Transcriptase to Lamivudine: Insight from Multiple Molecular Dynamics Simulations.

    PubMed

    Moonsamy, Suri; Bhakat, Soumendranath; Walker, Ross C; Soliman, Mahmoud E S

    2016-03-01

    Molecular dynamics simulations, binding free energy calculations, principle component analysis (PCA), and residue interaction network analysis were employed in order to investigate the molecular mechanism of M184I single mutation which played pivotal role in making the HIV-1 reverse transcriptase (RT) totally resistant to lamivudine. Results showed that single mutations at residue 184 of RT caused (1) distortion of the orientation of lamivudine in the active site due to the steric conflict between the oxathiolane ring of lamivudine and the side chain of beta-branched amino acids Ile at position 184 which, in turn, perturbs inhibitor binding, (2) decrease in the binding affinity by (~8 kcal/mol) when compared to the wild-type, (3) variation in the overall enzyme motion as evident from the PCA for both systems, and (4) distortion of the hydrogen bonding network and atomic interactions with the inhibitor. The comprehensive analysis presented in this report can provide useful information for understanding the drug resistance mechanism against lamivudine. The results can also provide some potential clues for further design of novel inhibitors that are less susceptible to drug resistance.

  3. Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the "fireman's grip" hypothesis.

    PubMed Central

    Strisovsky, K.; Tessmer, U.; Langner, J.; Konvalinka, J.; Kräusslich, H. G.

    2000-01-01

    Aspartic proteinases share a conserved network of hydrogen bonds (termed "fireman's grip"), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the "fireman's grip," Thr26 of HIV-1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV-1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the "fireman's grip" interaction, was virtually inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer-dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV-1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the "fireman's grip" is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the "fireman's grip" is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme. PMID:11045610

  4. Mutational analysis of active site contact residues in anti-fluorescein monoclonal antibody 4-4-20.

    PubMed

    Denzin, L K; Gulliver, G A; Voss, E W

    1993-10-01

    The contribution to high affinity Fl binding by each crystallographically defined Mab 4-4-20 (Ka = 1.7 x 10(10) M-1; Qmax = 90%) ligand contact residue (L27dHis, L32Tyr, L34Arg, L91Ser, L96Trp and H33Trp) has been determined by site-specific mutagenesis studies. All six antigen contact residues were changed to Ala in the single-chain derivative of Mab 4-4-20 and following expression in E. coli, denaturation, refolding and purification, each SCA mutant was characterized in terms of Fl binding affinity, Qmax, lambda max and idiotype. Results demonstrated that Ala substitutions at each ligand contact residue reduced the binding affinities and quenching maxima for all residues except L27d which retained wild type characteristics. The SCA TyrL32Ala, SerL91Ala and TrpH33Ala mutants exhibited binding affinities that were approximately 1000-fold lower than the wild type value and greatly reduced Qmax values. Additionally, other amino acid substitutions were performed at three of the six antigen contact residues (L91Ser, L96Trp and H33Trp) to further evaluate the role of each in Fl binding. Therefore, the following mutations were constructed and characterized: SerL91Asn, TrpL96Tyr, TrpL96Phe, TrpL96Leu, TrpH33Tyr and TrpH33Phe. Results of site-specific mutagenesis studies are discussed in terms of Mab active site structure and suggest that L32Tyr, L91Ser and H33Trp are important for high affinity Fl binding and efficient Fl quenching.

  5. Phosphorylation of the VP16 transcriptional activator protein during herpes simplex virus infection and mutational analysis of putative phosphorylation sites

    PubMed Central

    Ottosen, Søren; Herrera, Francisco J.; Doroghazi, James R.; Hull, Angela; Mittal, Sheenu; Lane, William S.; Triezenberg, Steven J.

    2006-01-01

    VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the viral immediate-early (IE) genes. We identified four novel VP16 phosphorylation sites (Ser18, Ser353, Ser411, and Ser452) at late times in infection, but found no evidence of phosphorylation of Ser375, a residue reportedly phosphorylated when VP16 is expressed from a transfected plasmid. A virus carrying a S375A mutation of VP16 was viable in cell culture but with a slow growth rate. The association of the mutant VP16 protein with IE gene promoters and subsequent IE gene expression was markedly reduced during infection, consistent with prior transfection and in vitro results. Surprisingly, the association of Oct-1 with IE promoters was also diminished during infection by the mutant strain. We propose that Ser375 is important for the interaction of VP16 with Oct-1, and that the interaction is required to enable both proteins to bind to IE promoters. PMID:16297954

  6. Triazole resistance mediated by mutations of a conserved active site tyrosine in fungal lanosterol 14α-demethylase

    PubMed Central

    Sagatova, Alia A.; Keniya, Mikhail V.; Wilson, Rajni K.; Sabherwal, Manya; Tyndall, Joel D. A.; Monk, Brian C.

    2016-01-01

    Emergence of fungal strains showing resistance to triazole drugs can make treatment of fungal disease problematic. Triazole resistance can arise due to single mutations in the drug target lanosterol 14α-demethylase (Erg11p/CYP51). We have determined how commonly occurring single site mutations in pathogenic fungi affect triazole binding using Saccharomyces cerevisiae Erg11p (ScErg11p) as a target surrogate. The mutations Y140F/H were introduced into full-length hexahistidine-tagged ScErg11p. Phenotypes and high-resolution X-ray crystal structures were determined for the mutant enzymes complexed with short-tailed (fluconazole and voriconazole) or long-tailed (itraconazole and posaconazole) triazoles and wild type enzyme complexed with voriconazole. The mutations disrupted a water-mediated hydrogen bond network involved in binding of short-tailed triazoles, which contain a tertiary hydroxyl not present in long-tailed triazoles. This appears to be the mechanism by which resistance to these short chain azoles occurs. Understanding how these mutations affect drug affinity will aid the design of azoles that overcome resistance. PMID:27188873

  7. Galactosemia caused by a point mutation that activates cryptic donor splice site in the galactose-1-phosphate uridyltransferase gene

    SciTech Connect

    Wadelius, C.; Lagerkvist, A. Uppsala Univ. ); Molin, A.K.; Larsson, A. ); Von Doebeln, U. )

    1993-08-01

    Galactosemia affects 1/84,000 in Sweden and is manifested in infancy when the child is exposed to galactose in the diet. If untreated there is a risk of severe early symptoms and, even with a lactose-free diet, late symptoms such as mental retardation and ovarial dysfunction may develop. In classical galactosemia, galactose-1-phosphate uridyltransferase (GALT) (EC 2.7.7.12) is defective and the normal cDNA sequence of this enzyme has been characterized. Recently eight mutations leading to galactosemia were published. Heparinized venous blood was drawn from a patient with classical galactosemia. In the cDNA from the patient examined, an insertion of 54 bp was found at position 1087. Amplification of the relevant genomic region of the patient's DNA was performed. Exon-intron boundaries and intronic sequences thus determined revealed that the 54-bp insertion was located immediately downstream of exon 10. It was further found that the patient was heterozygous for a point mutation, changing a C to a T (in 5 of 9 clones) at the second base in the intron downstream of the insertion. This alteration creates a sequence which, as well as the ordinary splice site, differs in only two positions from the consensus sequence. It was found that the mutation occurred in only one of the 20 alleles from galactosemic patients and in none of the 200 alleles from normal controls. The mutation is inherited from the mother, who also was found to express the 54-bp-long insertion at the mRNA level. Sequences from the 5[prime] end of the coding region were determined after genomic amplification, revealing a sequence identical to that reported. The mutation on the paternal allele has not been identified. 9 refs., 1 fig.

  8. Mutation of a Shc binding site tyrosine residue in ErbB3/HER3 blocks heregulin-dependent activation of mitogen-activated protein kinase.

    PubMed

    Vijapurkar, U; Cheng, K; Koland, J G

    1998-08-14

    The ErbB2 and ErbB3 proteins together constitute a functional coreceptor for heregulin (neuregulin). Heregulin stimulates the phosphorylation of both coreceptor constituents and initiates a variety of other signaling events, which include phosphorylation of the Shc protein. The role of Shc in heregulin-stimulated signal transduction through the ErbB2.ErbB3 coreceptor was investigated here. Heregulin was found to promote ErbB3/Shc association in NIH-3T3 cells expressing endogenous ErbB2 and recombinant ErbB3. A mutant ErbB3 protein was generated in which Tyr-1325 in a consensus Shc phosphotyrosine-binding domain recognition site was mutated to Phe (ErbB3-Y/F). This mutation abolished the association of Shc with ErbB3 and blocked the activation of mitogen-activated protein kinase by heregulin. Whereas heregulin induced mitogenesis in NIH-3T3 cells transfected with wild-type ErbB3 cDNA, this mitogenic response was markedly attenuated in NIH-3T3 cells transfected with the ErbB3-Y/F cDNA. These results showed a specific interaction of Shc with the ErbB3 receptor protein and demonstrated the importance of this interaction in the activation of mitogenic responses by the ErbB2. ErbB3 heregulin coreceptor complex.

  9. Oxalate decarboxylase and oxalate oxidase activities can be interchanged with a specificity switch of up to 282,000 by mutating an active site lid.

    PubMed

    Burrell, Matthew R; Just, Victoria J; Bowater, Laura; Fairhurst, Shirley A; Requena, Laura; Lawson, David M; Bornemann, Stephen

    2007-10-30

    Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features: a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share common catalytic steps that diverge when a carboxylate radical intermediate becomes protonated. The Bacillus subtilis decarboxylase has two manganese binding sites, and we proposed that Glu162 on a flexible lid is the site 1 general acid. We now demonstrate that a decarboxylase can be converted into an oxidase by mutating amino acids of the lid that include Glu162 with specificity switches of 282,000 (SEN161-3DAS), 275,000 (SENS161-4DSSN), and 225,000 (SENS161-4DASN). The structure of the SENS161-4DSSN mutant showed that site 2 was not affected. The requirement for substitutions other than of Glu162 was, at least in part, due to the need to decrease the Km for dioxygen for the oxidase reaction. Reversion of decarboxylase activity could be achieved by reintroducing Glu162 to the SENS161-4DASN mutant to give a relative specificity switch of 25,600. This provides compelling evidence for the crucial role of Glu162 in the decarboxylase reaction consistent with it being the general acid, for the role of the lid in controlling the Km for dioxygen, and for site 1 being the sole catalytically active site. We also report the trapping of carboxylate radicals produced during turnover of the mutant with the highest oxidase activity. Such radicals were also observed with the wild-type decarboxylase.

  10. The effect of active-site isoleucine to alanine mutation on the DHFR catalyzed hydride-transfer

    PubMed Central

    Stojković, Vanja; Perissinotti, Laura L.; Lee, Jeeyeon; Benkovic, Stephen J.; Kohen, Amnon

    2015-01-01

    Comparison of the nature of hydride transfer in wild-type and active site mutant (I14A) of dihydrofolate reductase suggests that the size of this side chain at position 14 modulates H-tunneling. PMID:20972508

  11. Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid.

    PubMed Central

    Man, W J; Li, Y; O'Connor, C D; Wilton, D C

    1991-01-01

    The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp----Asn and Asp----Gly mutations. The Asp----Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563]. PMID:1684105

  12. Mutations affecting enzymatic activity in liver arginase

    SciTech Connect

    Vockley, J.G.; Tabor, D.E.; Goodman, B.K.

    1994-09-01

    The hydrolysis of arginine to ornithine and urea is catalyzed by arginase in the last step of the urea cycle. We examined a group of arginase deficient patients by PCR-SSCP analysis to characterize the molecular basis of this disorder. A heterogeneous population of nonsense mutations, microdeletions, and missense mutations has been identified in our cohort. Microdeletions which introduce premature stop codons downstream of the deletion and nonsense mutations result in no arginase activity. These mutations occur randomly along the gene. The majority of missense mutations identified appear to occur in regions of high cross-species homology. To test the effect of these missense mutations on arginase activity, site-directed mutagenesis was used to re-create the patient mutations for in vivo expression studies in a prokaryotic fusion-protein expression system. Of 4 different missense mutations identified in 6 individuals, only one was located outside of a conserved region. The three substitution mutations within the conserved regions had a significant effect on enzymatic activity (0-3.1 nmole/30min, normal is 1300-1400 nmoles/30min, as determined by in vitro arginase assay), while the fourth mutation, a T to S substitution, did not. In addition, site-directed mutagenesis was utilized to create mutations not in residues postulated to play a significant role in the enzymatic function or active site formation in manganese-binding proteins such as arginase. We have determined that the substitution of glycine for a histidine residue, located in a very highly conserved region of exon 3, and the substitution of a histidine and an aspartic acid residue within a similarly conserved region in exon 4, totally abolishes enzymatic activity. Mutations substituting glycine for an additional histidine and aspartic acid residue in exon 4 and two aspartic acid residues in exon 7 have also been created. We are currently in the process of characterizing these mutations.

  13. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    PubMed

    Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  14. Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

    PubMed Central

    Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I.; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity. PMID:24278284

  15. Strychnine activates neuronal α7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains

    PubMed Central

    Palma, Eleonora; Fucile, Sergio; Barabino, Benedetta; Miledi, Ricardo; Eusebi, Fabrizio

    1999-01-01

    Recent work has shown that strychnine, the potent and selective antagonist of glycine receptors, is also an antagonist of nicotinic acetylcholine (AcCho) receptors including neuronal homomeric α7 receptors, and that mutating Leu-247 of the α7 nicotinic AcCho receptor-channel domain (L247Tα7; mut1) converts some nicotinic antagonists into agonists. Therefore, a study was made of the effects of strychnine on Xenopus oocytes expressing the chick wild-type α7 or L247Tα7 receptors. In these oocytes, strychnine itself did not elicit appreciable membrane currents but reduced the currents elicited by AcCho in a reversible and dose-dependent manner. In sharp contrast, in oocytes expressing L247Tα7 receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189–190Sα7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker α-bungarotoxin. Single-channel recordings showed that strychnine gated mut2-channels with two conductance levels, 56 pS and 42 pS, and with kinetic properties similar to AcCho-activated channels. We conclude that strychnine is a modulator, as well as an activator, of some homomeric nicotinic α7 receptors. After injecting oocytes with mixtures of cDNAs encoding mut1 and mut2 subunits, the expressed hybrid receptors were activated by strychnine, similar to the mut2, and had a high affinity to AcCho like the mut1. A pentameric symmetrical model yields the striking conclusion that two identical α7 subunits may be sufficient to determine the functional properties of α7 receptors. PMID:10557336

  16. FK506-binding protein mutational analysis: defining the active-site residue contributions to catalysis and the stability of ligand complexes.

    PubMed

    DeCenzo, M T; Park, S T; Jarrett, B P; Aldape, R A; Futer, O; Murcko, M A; Livingston, D J

    1996-02-01

    The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolyl isomerase that binds the macrolides FK506 and rapamycin. We have examined the role of the binding pocket residues of FKBP12 in protein-ligand interactions by making conservative substitutions of 12 of these residues by site-directed mutagenesis. For each mutant FKBP12, we measured the affinity for FK506 and rapamycin and the catalytic efficiency in the cis-frans peptidyl-prolyl isomerase reaction. The mutation of Trp59 or Phe99 generates an FKBP12 with a significantly lower affinity for FK506 than wild-type protein. Tyr26 and Tyr82 mutants are enzymatically active, demonstrating that hydrogen bonding by these residues is not required for catalysis of the cis-trans peptidyl-prolyl isomerase reaction, although these mutations alter the substrate specificity of the enzyme. We conclude that hydrophobic interactions in the active site dominate in the stabilization of FKBP12 binding to macrolide ligands and to the twisted-amide peptidyl-prolyl substrate intermediate.

  17. Concerted action of target-site mutations and high EPSPS activity in glyphosate-resistant junglerice (Echinochloa colona) from California

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glyphosate is the most widely used non-selective herbicide and Echinochloa colona is an annual weed affecting field crops and orchards in California. A population carrying a glyphosate-resistance-endowing mutation in the EPSPS gene was found in the Northern Sacramento Valley. We used selfed lines ...

  18. Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B.

    PubMed Central

    Kim, S W; Joo, S; Choi, G; Cho, H S; Oh, B H; Choi, K Y

    1997-01-01

    In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate. PMID:9401033

  19. Mechanism of the Quorum-Quenching Lactonase (AiiA) from Bacillus thuringiensis. 2. Substrate Modeling and Active Site Mutations

    SciTech Connect

    Momb, Jessica; Wang, Canhui; Liu, Dali; Thomas, Pei W.; Petsko, Gregory A.; Guo, Hua; Ringe, Dagmar; Fast, Walter

    2008-12-02

    The N-acyl-l-homoserine lactone hydrolases (AHL lactonases) have attracted considerable attention because of their ability to quench AHL-mediated quorum-sensing pathways in Gram-negative bacteria and because of their relation to other enzymes in the metallo-{beta}-lactamase superfamily. To elucidate the detailed catalytic mechanism of AHL lactonase, mutations are made on residues that presumably contribute to substrate binding and catalysis. Steady-state kinetic studies are carried out on both the wild-type and mutant enzymes using a spectrum of substrates. Two mutations, Y194F and D108N, present significant effects on the overall catalysis. On the basis of a high-resolution structural model of the enzyme-product complex, a hybrid quantum mechanical/molecular mechanical method is used to model the substrate binding orientation and to probe the effect of the Y194F mutation. Combining all experimental and computational results, we propose a detailed mechanism for the ring-opening hydrolysis of AHL substrates as catalyzed by the AHL lactonase from Bacillus thuringiensis. Several features of the mechanism that are also found in related enzymes are discussed and may help to define an evolutionary thread that connects the hydrolytic enzymes of this mechanistically diverse superfamily.

  20. Mutation profiling of adenoid cystic carcinomas from multiple anatomical sites identifies mutations in the RAS pathway, but no KIT mutations

    PubMed Central

    Wetterskog, Daniel; Wilkerson, Paul M; Rodrigues, Daniel N; Lambros, Maryou B; Fritchie, Karen; Andersson, Mattias K; Natrajan, Rachael; Gauthier, Arnaud; Di Palma, Silvana; Shousha, Sami; Gatalica, Zoran; Töpfer, Chantal; Vukovic, Vesna; A’Hern, Roger; Weigelt, Britta; Vincent-Salomon, Anne; Stenman, Göran; Rubin, Brian P; Reis-Filho, Jorge S

    2016-01-01

    Aims The majority of adenoid cystic carcinomas (AdCCs), regardless of anatomical site, harbour the MYB–NFIB fusion gene. The aim of this study was to characterize the repertoire of somatic genetic events affecting known cancer genes in AdCCs. Methods and results DNA was extracted from 13 microdissected breast AdCCs, and subjected to a mutation survey using the Sequenom OncoCarta Panel v1.0. Genes found to be mutated in any of the breast AdCCs and genes related to the same canonical molecular pathways, as well as KIT, a proto-oncogene whose protein product is expressed in AdCCs, were sequenced in an additional 68 AdCCs from various anatomical sites by Sanger sequencing. Using the Sequenom MassARRAY platform and Sanger sequencing, mutations in BRAF and HRAS were identified in three and one cases, respectively (breast, and head and neck). KIT, which has previously been reported to be mutated in AdCCs, was also investigated, but no mutations were identified. Conclusions Our results demonstrate that mutations in genes pertaining to the canonical RAS pathway are found in a minority of AdCCs, and that activating KIT mutations are either absent or remarkably rare in these cancers, and unlikely to constitute a driver and therapeutic target for patients with AdCC. PMID:23398044

  1. Solvent dielectric effect and side chain mutation on the structural stability of Burkholderia cepacia lipase active site: a quantum mechanical/molecular mechanics study.

    PubMed

    Tahan, A; Monajjemi, M

    2011-12-01

    Quantum mechanical and molecular dynamics methods were used to analyze the structure and stability of neutral and zwitterionic configurations of the extracted active site sequence from a Burkholderia cepacia lipase, histidyl-seryl-glutamin (His86-Ser87-Gln88) and its mutated form, histidyl-cysteyl-glutamin (His86-Cys87-Gln88) in vacuum and different solvents. The effects of solvent dielectric constant, explicit and implicit water molecules and side chain mutation on the structure and stability of this sequence in both neutral and zwitterionic forms are represented. The quantum mechanics computations represent that the relative stability of zwitterionic and neutral configurations depends on the solvent structure and its dielectric constant. Therefore, in vacuum and the considered non-polar solvents, the neutral form of the interested sequences is more stable than the zwitterionic form, while their zwitterionic form is more stable than the neutral form in the aqueous solution and the investigated polar solvents in most cases. However, on the potential energy surfaces calculated, there is a barrier to proton transfer from the positively charged ammonium group to the negatively charged carboxylat group or from the ammonium group to the adjacent carbonyl oxygen and or from side chain oxygen and sulfur to negatively charged carboxylat group. Molecular dynamics simulations (MD) were also performed by using periodic boundary conditions for the zwitterionic configuration of the hydrated molecules in a box of water molecules. The obtained results demonstrated that the presence of explicit water molecules provides the more compact structures of the studied molecules. These simulations also indicated that side chain mutation and replacement of sulfur with oxygen leads to reduction of molecular flexibility and packing.

  2. The Startle Disease Mutation E103K Impairs Activation of Human Homomeric α1 Glycine Receptors by Disrupting an Intersubunit Salt Bridge across the Agonist Binding Site*

    PubMed Central

    Safar, Fatemah; Hurdiss, Elliot; Erotocritou, Marios; Greiner, Timo; Irvine, Mark W.; Fang, Guangyu; Jane, David; Yu, Rilei; Dämgen, Marc A.

    2017-01-01

    Glycine receptors (GlyR) belong to the pentameric ligand-gated ion channel (pLGIC) superfamily and mediate fast inhibitory transmission in the vertebrate CNS. Disruption of glycinergic transmission by inherited mutations produces startle disease in man. Many startle mutations are in GlyRs and provide useful clues to the function of the channel domains. E103K is one of few startle mutations found in the extracellular agonist binding site of the channel, in loop A of the principal side of the subunit interface. Homology modeling shows that the side chain of Glu-103 is close to that of Arg-131, in loop E of the complementary side of the binding site, and may form a salt bridge at the back of the binding site, constraining its size. We investigated this hypothesis in recombinant human α1 GlyR by site-directed mutagenesis and functional measurements of agonist efficacy and potency by whole cell patch clamp and single channel recording. Despite its position near the binding site, E103K causes hyperekplexia by impairing the efficacy of glycine, its ability to gate the channel once bound, which is very high in wild type GlyR. Mutating Glu-103 and Arg-131 caused various degrees of loss-of-function in the action of glycine, whereas mutations in Arg-131 enhanced the efficacy of the slightly bigger partial agonist sarcosine (N-methylglycine). The effects of the single charge-swapping mutations of these two residues were largely rescued in the double mutant, supporting the possibility that they interact via a salt bridge that normally constrains the efficacy of larger agonist molecules. PMID:28174298

  3. The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites.

    PubMed

    Fernández-Calero, Tamara; Flouriot, Gilles; Marín, Mónica

    2016-01-01

    Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene.

  4. Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation.

    PubMed Central

    Engeland, K; Höög, J O; Holmquist, B; Estonius, M; Jörnvall, H; Vallee, B L

    1993-01-01

    The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. The activities of the recombinant native and mutant enzymes toward ethanol are essentially identical, but mutagenesis greatly decreases the kcat/Km values for glutathione-dependent formaldehyde oxidation. The catalytic efficiency for the Asp variant is < 0.1% that of the unmutated enzyme, due to both a higher Km and a lower kcat value. As with the native enzyme, neither mutant can oxidize methanol, be saturated by ethanol, or be inhibited by 4-methylpyrazole; i.e., they retain these class III characteristics. In contrast, however, their activation by fatty acids, another characteristic unique to class III alcohol dehydrogenase, is markedly attenuated. The Ala mutant is activated only slightly, but the Asp mutant is not activated at all. The results strongly indicate that Arg-115 in class III alcohol dehydrogenase is a component of the binding site for activating fatty acids and is critical for the binding of S-hydroxymethylglutathione in glutathione-dependent formaldehyde dehydrogenase activity. PMID:8460164

  5. Functional impact of HIV coreceptor-binding site mutations

    SciTech Connect

    Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.; Baik, Sarah S.W.; Lee, Fang-Hua; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Reeves, Jacqueline D. . E-mail: jreeves@MonogramBio.com

    2006-07-20

    The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.

  6. Mutations in exons 10 and 11 of human glucokinase result in conformational variations in the active site of the structure contributing to poor substrate binding - explains hyperglycemia in type 2 diabetic patients.

    PubMed

    Yellapu, Nandakumar; Mahto, Manoj Kumar; Valasani, Koteswara Rao; Sarma, P V G K; Matcha, Bhaskar

    2015-01-01

    Mutations in the glucokinase (GK) gene play a critical role in the establishment of type 2 diabetes. In our earlier study, R308K mutation in GK in a clinically proven type 2 diabetic patient showed, structural and functional variations that contributed immensely to the hyperglycemic condition. In the extension of this work, a cohort of 30 patients with established type 2 diabetic condition were chosen and the exons 10 and 11 of GK were PCR-amplified and sequenced. The sequence alignment showed A379S, D400Y, E300A, E395A, E395G, H380N, I348N, L301M, M298I, M381G, M402R, R308K, R394P, R397S, and S398R mutations in 12 different patients. The structural analysis of these mutated GKs, showed a variable number of β-α-β units, hairpins, β-bulges, strands, helices, helix-helix interactions, β-turns, and γ-turns along with the RMSD variations when compared to wild-type GK. Molecular modeling studies revealed that the substrate showed variable binding orientations and could not fit into the active site of these mutated structures; moreover, it was expelled out of the conformations. Therefore, these structural variations in GK due to mutations could be one of the strongest reasons for the hyperglycemic levels in these type 2 diabetic patients.

  7. Autosomal recessive hyponatremia due to isolated salt wasting in sweat associated with a mutation in the active site of Carbonic Anhydrase 12.

    PubMed

    Muhammad, Emad; Leventhal, Neta; Parvari, Galit; Hanukoglu, Aaron; Hanukoglu, Israel; Chalifa-Caspi, Vered; Feinstein, Yael; Weinbrand, Jenny; Jacoby, Harel; Manor, Esther; Nagar, Tal; Beck, John C; Sheffield, Val C; Hershkovitz, Eli; Parvari, Ruti

    2011-04-01

    Genetic disorders of excessive salt loss from sweat glands have been observed in pseudohypoaldosteronism type I (PHA) and cystic fibrosis that result from mutations in genes encoding epithelial Na+ channel (ENaC) subunits and the transmembrane conductance regulator (CFTR), respectively. We identified a novel autosomal recessive form of isolated salt wasting in sweat, which leads to severe infantile hyponatremic dehydration. Three affected individuals from a small Bedouin clan presented with failure to thrive, hyponatremic dehydration and hyperkalemia with isolated sweat salt wasting. Using positional cloning, we identified the association of a Glu143Lys mutation in carbonic anhydrase 12 (CA12) with the disease. Carbonic anhydrase is a zinc metalloenzyme that catalyzes the reversible hydration of carbon dioxide to form a bicarbonate anion and a proton. Glu143 in CA12 is essential for zinc coordination in this metalloenzyme and lowering of the protein-metal affinity reduces its catalytic activity. This is the first presentation of an isolated loss of salt from sweat gland mimicking PHA, associated with a mutation in the CA12 gene not previously implicated in human disorders. Our data demonstrate the importance of bicarbonate anion and proton production on salt concentration in sweat and its significance for sodium homeostasis.

  8. Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations

    PubMed Central

    Sun, Zhizeng; Mehta, Shrenik C.; Adamski, Carolyn J.; Gibbs, Richard A.; Palzkill, Timothy

    2016-01-01

    CphA is a Zn2+-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function. PMID:27616327

  9. The roles of active-site residues in the catalytic mechanism of trans-3-chloroacrylic acid dehalogenase: a kinetic, NMR, and mutational analysis.

    PubMed

    Azurmendi, Hugo F; Wang, Susan C; Massiah, Michael A; Poelarends, Gerrit J; Whitman, Christian P; Mildvan, Albert S

    2004-04-13

    trans-3-Chloroacrylic acid dehalogenase (CaaD) converts trans-3-chloroacrylic acid to malonate semialdehyde by the addition of H(2)O to the C-2, C-3 double bond, followed by the loss of HCl from the C-3 position. Sequence similarity between CaaD, an (alphabeta)(3) heterohexamer (molecular weight 47,547), and 4-oxalocrotonate tautomerase (4-OT), an (alpha)(6) homohexamer, distinguishes CaaD from those hydrolytic dehalogenases that form alkyl-enzyme intermediates. The recently solved X-ray structure of CaaD demonstrates that betaPro-1 (i.e., Pro-1 of the beta subunit), alphaArg-8, alphaArg-11, and alphaGlu-52 are at or near the active site, and the >or=10(3.4)-fold decreases in k(cat) on mutating these residues implicate them as mechanistically important. The effect of pH on k(cat)/K(m) indicates a catalytic base with a pK(a) of 7.6 and an acid with a pK(a) of 9.2. NMR titration of (15)N-labeled wild-type CaaD yielded pK(a) values of 9.3 and 11.1 for the N-terminal prolines, while the fully active but unstable alphaP1A mutant showed a pK(a) of 9.7 (for the betaPro-1), implicating betaPro-1 as the acid catalyst, which may protonate C-2 of the substrate. These results provide the first evidence for an amino-terminal proline, conserved in all known tautomerase superfamily members, functioning as a general acid, rather than as a general base as in 4-OT. Hence, a reasonable candidate for the general base in CaaD is the active site residue alphaGlu-52. CaaD has 10 arginine residues, six in the alpha-subunit (Arg-8, Arg-11, Arg-17, Arg-25, Arg-35, and Arg-43), and four in the beta-subunit (Arg-15, Arg-21, Arg-55, and Arg-65). (1)H-(15)N-heteronuclear single quantum coherence (HSQC) spectra of CaaD showed seven to nine Arg-NepsilonH resonances (denoted R(A) to R(I)) depending on the protein concentration and pH. One of these signals (R(D)) disappeared in the spectrum of the largely inactive alphaR11A mutant (deltaH = 7.11 ppm, deltaN = 89.5 ppm), and another one (R

  10. List 9 - Active CERCLIS Sites:

    EPA Pesticide Factsheets

    The List 9 displays the sequence of activities undertaken at active CERCLIS sites. An active site is one at which site assessment, removal, remedial, enforcement, cost recovery, or oversight activities are being planned or conducted.

  11. A single mutation in the hepta-peptide active site of Aspergillus niger PhyA phytase leads to myriad of biochemical changes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The active site motif of proteins belonging to ‘Histidine Acid Phosphatase’ (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs has revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger phyA phytase. However,...

  12. Tryptophanase from Proteus vulgaris: the conformational rearrangement in the active site, induced by the mutation of Tyrosine 72 to phenylalanine, and its mechanistic consequences.

    PubMed

    Kulikova, Vitalia V; Zakomirdina, Ludmila N; Dementieva, Irene S; Phillips, Robert S; Gollnick, Paul D; Demidkina, Tatyana V; Faleev, Nicolai G

    2006-04-01

    Tyr72 is located at the active site of tryptophanase (Trpase) from Proteus vulgaris. For the wild-type Trpase Tyr72 might be considered as the general acid catalyst at the stage of elimination of the leaving groups. The replacement of Tyr72 by Phe leads to a decrease in activity for L-tryptophan by 50,000-fold and to a considerable rearrangement of the active site of Trpase. This rearrangement leads to an increase of room around the alpha-C atom of any bound amino acid, such that covalent binding of alpha-methyl-substituted amino acids becomes possible (which cannot be realized in wild-type Trpase). The changes in reactivities of S-alkyl-L-cysteines provide evidence for an increase of congestion in the proximity of their side groups in the mutant enzyme as compared to wild-type enzyme. The observed alteration of catalytic properties in a large degree originates from a conformational change in the active site. The Y72F Trpase retains significant activity for L-serine, which allowed us to conclude that in the mutant enzyme, some functional group is present which fulfills the role of the general acid catalyst in reactions associated with elimination of small leaving groups.

  13. Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II.

    PubMed Central

    Manak, J R; Prywes, R

    1991-01-01

    Casein kinase II (CKII) phosphorylates the mammalian transcription factor serum response factor (SRF) on a serine residue(s) located within a region of the protein spanning amino acids 70 to 92, thereby enhancing its DNA-binding activity in vitro. We report here that serine 83 appears to be the residue phosphorylated by CKII but that three other serines in this region can also be involved in phosphorylation and the enhancement of DNA-binding activity. A mutant that contained glutamate residues in place of these serines had only low-level binding activity; however, when the serines were replaced with glutamates and further mutations were made that increased the negative charge of the region, the resulting mutant showed a constitutively high level of binding equal to that achieved by phosphorylation of wild-type SRF. We have investigated the mechanism by which phosphorylation of SRF increases its DNA-binding activity. We have ruled out the possibilities that phosphorylation affects SRF dimerization or relieves inhibition due to masking of the DNA-binding domain by an amino-terminal region of the protein. Rather, using partial proteolysis to probe SRF's structure, we find that the conformation of SRF's DNA-binding domain is altered by phosphorylation. Images PMID:2046671

  14. Esophageal melanomas harbor frequent NRAS mutations unlike melanomas of other mucosal sites.

    PubMed

    Sekine, Shigeki; Nakanishi, Yukihiro; Ogawa, Reiko; Kouda, Satoko; Kanai, Yae

    2009-05-01

    Mucosal melanomas have genetic alterations distinct from those in cutaneous melanomas. For example, NRAS- and BRAF-activating mutations occur frequently in cutaneous melanomas, but not in mucosal melanomas. We examined 16 esophageal melanomas for genetic alterations in NRAS, BRAF, and KIT to determine whether they exhibit genetic features common to melanomas arising from other mucosal sites. A sequencing analysis identified NRAS mutations in six cases; notably, four of these mutations were located in exon 1, an uncommon mutation site in cutaneous and other mucosal melanomas. BRAF and KIT mutations were found in one case each. Immunohistochemistry showed KIT expression in four cases, including the tumor with a KIT mutation and two other intramucosal tumors. The low frequency of BRAF mutations and the presence of a KIT mutation-positive case are findings similar to those of mucosal melanomas of other sites, but the prevalence of NRAS mutations was even higher than that of cutaneous melanomas. The present study implies that esophageal melanomas have genetic alterations unique from those observed in other mucosal melanomas.

  15. Androgen receptor exon 1 mutation causes androgen insensitivity by creating phosphorylation site and inhibiting melanoma antigen-A11 activation of NH2- and carboxyl-terminal interaction-dependent transactivation.

    PubMed

    Lagarde, William H; Blackwelder, Amanda J; Minges, John T; Hnat, Andrew T; French, Frank S; Wilson, Elizabeth M

    2012-03-30

    Naturally occurring germ line mutations in the X-linked human androgen receptor (AR) gene cause incomplete masculinization of the external genitalia by disrupting AR function in males with androgen insensitivity syndrome. Almost all AR missense mutations that cause androgen insensitivity syndrome are located in the highly structured DNA and ligand binding domains. In this report we investigate the functional defect associated with an AR exon 1 missense mutation, R405S, that caused partial androgen insensitivity. The 46,XX heterozygous maternal carrier had a wild-type Arg-405 CGC allele but transmitted an AGC mutant allele coding for Ser-405. At birth, the 46,XY proband had a bifid scrotum, hypospadias, and micropenis consistent with clinical stage 3 partial androgen insensitivity. Androgen-dependent transcriptional activity of AR-R405S expressed in CV1 cells was less than wild-type AR and refractory in androgen-dependent AR NH(2)- and carboxyl interaction transcription assays that depend on the coregulator effects of melanoma antigen-A11. This mutation created a Ser-405 phosphorylation site evident by the gel migration of an AR-R405S NH(2)-terminal fragment as a double band that converted to the wild-type single band after treatment with λ-phosphatase. Detrimental effects of the R405S mutation were related to the proximity of the AR WXXLF motif (433)WHTLF(437) required for melanoma antigen-A11 and p300 to stimulate transcriptional activity associated with the AR NH(2)- and carboxyl-terminal interaction. We conclude that the coregulator effects of melanoma antigen-A11 on the AR NH(2)- and carboxyl-terminal interaction amplify the androgen-dependent transcriptional response to p300 required for normal human male sex development in utero.

  16. MUTATION OF THE ACTIVE SITE CARBOXY-LYSINE (K70) OF OXA-1 β-LACTAMASE RESULTS IN A DEACYLATION-DEFICIENT ENZYME†

    PubMed Central

    Schneider, Kyle D.; Bethel, Christopher R.; Distler, Anne M.; Hujer, Andrea M.; Bonomo, Robert A.; Leonard, David A.

    2009-01-01

    Class D β-lactamases hydrolyze β-lactam antibiotics by using an active site serine nucleophile to form a covalent acyl-enzyme intermediate, and subsequently employ water to deacylate the β-lactam and release product. Class D β-lactamases are carboxylated on the ε-amino group of an active site lysine, with the resulting carbamate functional group serving as a general base. We discovered that substitutions of the active site serine and lysine in OXA-1 β-lactamase, a monomeric class D enzyme, significantly disrupt catalytic turnover. Substitution of glycine for the nucleophilic serine (S67G) results in an enzyme that can still bind substrate but is unable to form a covalent acyl-enzyme intermediate. Substitution of the carboxylated lysine (K70), on the other hand, results in enzyme that can be acylated by substrate, but is impaired for deacylation. We employed the fluorescent penicillin BOCILLIN FL™ to show that three different substitutions for K70 (alanine, aspartate and glutamate) accumulate significant acyl-enzyme intermediate. Interestingly, BOCILLIN FL™ deacylation rates vary depending on the identity of the substituting residue, from t1/2 ≈ 60 min for K70A to undetectable deacylation for K70D. Tryptophan fluorescence spectroscopy was used to confirm that these results are applicable to natural (i.e. non-fluorescent) substrates. Deacylation by K70A, but not K70D or K70E, can be partially restored by the addition of short-chain carboxylic acid mimetics of the lysine carbamate. In conclusion, we establish the functional role of the carboxylated lysine in OXA-1 and highlight its specific role in acylation and deacylation. PMID:19485421

  17. Mutations and Binding Sites of Human Transcription Factors

    PubMed Central

    Kamanu, Frederick Kinyua; Medvedeva, Yulia A.; Schaefer, Ulf; Jankovic, Boris R.; Archer, John A. C.; Bajic, Vladimir B.

    2012-01-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, “insertions” are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. PMID:22670148

  18. Activating GNAS mutations in parosteal osteosarcoma.

    PubMed

    Carter, Jodi M; Inwards, Carrie Y; Jin, Long; Evers, Barbara; Wenger, Doris E; Oliveira, Andre M; Fritchie, Karen J

    2014-03-01

    Parosteal osteosarcoma is a surface-based osteosarcoma that often exhibits deceptively bland cytologic features, hindering diagnosis in small biopsies or when correlative radiologic imaging is not readily available. A number of benign and malignant fibro-osseous lesions, including fibrous dysplasia (FD) and low-grade central osteosarcoma, fall within the morphologic differential diagnosis of parosteal osteosarcoma. Somatic mutations in GNAS, encoding the α-subunit of the heterotrimeric G protein complex (Gsα), occur in FD and McCune-Albright syndrome but have not been reported in parosteal osteosarcoma. We evaluated GNAS mutational status in parosteal osteosarcoma and several of its histologic mimics to determine its utility in differentiating these entities. Eleven of 14 (79%) FD cases had GNAS mutations within codon 201 (5 R201C and 6 R201H mutations). GNAS mutations were not detected in any cases of adamantinoma or osteofibrous dysplasia. Direct sequencing of 9 parosteal osteosarcomas, including 3 of low grade and 6 with dedifferentiation, revealed activating GNAS mutations in 5 cases (55%), distributed as 4 R201C-mutated tumors and 1 tumor with an R201H mutation. GNAS codon 227 mutations were not detected in any of the cases. There was no association between GNAS mutational status and patient demographics, histologic dedifferentiation, or clinical outcome. To our knowledge, we report the first series of parosteal osteosarcomas harboring activating GNAS mutations. Our data suggest that GNAS mutational status may have limited utility as an ancillary technique in differentiating benign and malignant fibro-osseous lesions of the bone.

  19. Neurocognitive Profiles in Duchenne Muscular Dystrophy and Gene Mutation Site

    PubMed Central

    D’Angelo, Maria Grazia; Lorusso, Maria Luisa; Civati, Federica; Comi, Giacomo Pietro; Magri, Francesca; Del Bo, Roberto; Guglieri, Michela; Molteni, Massimo; Turconi, Anna Carla; Bresolin, Nereo

    2011-01-01

    The presence of nonprogressive cognitive impairment is recognized as a common feature in a substantial proportion of patients with Duchenne muscular dystrophy. To investigate the possible role of mutations along the dystrophin gene affecting different brain dystrophin isoforms and specific cognitive profiles, 42 school-age children affected with Duchenne muscular dystrophy, subdivided according to sites of mutations along the dystrophin gene, underwent a battery of tests tapping a wide range of intellectual, linguistic, and neuropsychologic functions. Full-scale intelligence quotient was approximately 1 S.D. below the population average in the whole group of dystrophic children. Patients with Duchenne muscular dystrophy and mutations located in the distal portion of the dystrophin gene (involving the 140-kDa brain protein isoform, called Dp140) were generally more severely affected and expressed different patterns of strengths and impairments, compared with patients with Duchenne muscular dystrophy and mutations located in the proximal portion of the dystrophin gene (not involving Dp140). Patients with Duchenne muscular dystrophy and distal mutations demonstrated specific impairments in visuospatial functions and visual memory (which seemed intact in proximally mutated patients) and greater impairment in syntactic processing. PMID:22000308

  20. Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1

    PubMed Central

    Milichovský, Jan; Bárta, František; Schmeiser, Heinz H.; Arlt, Volker M.; Frei, Eva; Stiborová, Marie; Martínek, Václav

    2016-01-01

    Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are NAD(P)H:quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using 32P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast

  1. Effect of Single-Site Mutations on HP Lattice Proteins

    SciTech Connect

    Shi, Guangjie; Vogel, Thomas; Wuest, Thomas; Li, Ying Wai; Landau, David P

    2014-01-01

    We developed a heuristic method for determining the ground-state degeneracy of hydrophobic-polar (HP) lattice proteins, based on Wang-Landau and multicanonical sampling. It is applied during comprehensive studies of single-site mutations in specific HP proteins with different sequences. The effects in which we are interested include structural changes in ground-states, changes of ground-state energy, degeneracy, and thermodynamic properties of the system. With respect to mutations, both extremely sensitive and insensitive positions in the HP sequence have been found. That is, ground state energies and degeneracies, as well as other thermodynamic and structural quantities may be either largely unaffected or may change significantly due to mutation.

  2. A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae.

    PubMed

    Tomberg, Joshua; Temple, Brenda; Fedarovich, Alena; Davies, Christopher; Nicholas, Robert A

    2012-04-03

    Insertion of an aspartate residue at position 345a in penicillin-binding protein 2 (PBP 2), which lowers the rate of penicillin acylation by ~6-fold, is commonly observed in penicillin-resistant strains of Neisseria gonorrhoeae. Here, we show that insertions of other amino acids also lower the penicillin acylation rate of PBP 2, but none supported growth of N. gonorrhoeae, indicating loss of essential transpeptidase activity. The Asp345a mutation likely acts by altering the interaction between its adjacent residue, Asp346, in the β2a-β2d hairpin loop and Ser363, the middle residue of the SXN active site motif. Because the adjacent aspartate creates ambiguity in the position of the insertion, we also examined if insertions at position 346a could confer decreased susceptibility to penicillin. However, only aspartate insertions were identified, indicating that only an Asp-Asp couple can confer resistance and retain transpeptidase function. The importance of the Asp346-Ser363 interaction was assessed by mutation of each residue to Ala. Although both mutants lowered the acylation rate of penicillin G by 5-fold, neither could support growth of N. gonorrhoeae, again indicating loss of transpeptidase function. Interaction between a residue in the equivalent of the β2a-β2d hairpin loop and the middle residue of the SXN motif is observed in crystal structures of other Class B PBPs, and its importance is also supported by multisequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g., by insertion) to lower the acylation rate by β-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved.

  3. Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.

    PubMed

    Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

    2010-10-01

    Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na(+)(Li(+))/H(+) antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na(+)](in). Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na(+) (Li(+))/H(+) antiport.

  4. Mutation of a Conserved Active Site Residue Converts Tyrosyl-DNA Phosphodiesterase l Into a DNA Topoisomerase l-Dependent Poison

    SciTech Connect

    He,X.; van Waardenburg, R.; Babaoglu, K.; Price, A.; Nitiss, K.; Nitiss, J.; Bjornsti, M.; White, S.

    2007-01-01

    Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3' and 5' phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1-DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.0 {angstrom} crystal structure of yeast Tdp1 was determined that is very similar to human Tdp1. Poorly conserved regions of primary structure are peripheral to an essentially identical catalytic core. Enzyme mechanism was also conserved, because the yeast SCAN1 mutant (H{sub 432}R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1-dependent lethality of Tdp1H{sub 432}N was drug-independent, coinciding with increased covalent Top1-DNA and Tdp1-DNA complex formation in vivo. However, both H432 mutants were recessive to wild-type Tdp1. Thus, yeast H{sub 432} acts in the general acid/base catalytic mechanism of Tdp1 to resolve 3' phosphotyrosyl and 3' phosphoamide linkages. However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H{sub 432}N-catalyzed resolution of 3' phospho-adducts.

  5. Mutational Biases Drive Elevated Rates of Substitution at Regulatory Sites across Cancer Types

    PubMed Central

    Semple, Colin A.

    2016-01-01

    Disruption of gene regulation is known to play major roles in carcinogenesis and tumour progression. Here, we comprehensively characterize the mutational profiles of diverse transcription factor binding sites (TFBSs) across 1,574 completely sequenced cancer genomes encompassing 11 tumour types. We assess the relative rates and impact of the mutational burden at the binding sites of 81 transcription factors (TFs), by comparing the abundance and patterns of single base substitutions within putatively functional binding sites to control sites with matched sequence composition. There is a strong (1.43-fold) and significant excess of mutations at functional binding sites across TFs, and the mutations that accumulate in cancers are typically more disruptive than variants tolerated in extant human populations at the same sites. CTCF binding sites suffer an exceptionally high mutational load in cancer (3.31-fold excess) relative to control sites, and we demonstrate for the first time that this effect is seen in essentially all cancer types with sufficient data. The sub-set of CTCF sites involved in higher order chromatin structures has the highest mutational burden, suggesting a widespread breakdown of chromatin organization. However, we find no evidence for selection driving these distinctive patterns of mutation. The mutational load at CTCF-binding sites is substantially determined by replication timing and the mutational signature of the tumor in question, suggesting that selectively neutral processes underlie the unusual mutation patterns. Pervasive hyper-mutation within transcription factor binding sites rewires the regulatory landscape of the cancer genome, but it is dominated by mutational processes rather than selection. PMID:27490693

  6. Identification of a polymorphic site as a mutational site in exon VI of the mouse p53 gene

    SciTech Connect

    Paunesku, T.; Gemmell, M.A.; Crkvenjakov, R.; Woloschak, G.E.

    1993-07-01

    Sequencing by hybridization techniques are being used to analyze the incidence of specific p53 mutations associated with radiation-induced and spontaneous lymphosarcomas in mice. One sequence difference noted as being a mouse strain-specific polymorphism has been identified through these experiments as being a mutational, rather than a polymorphic, site.

  7. Effect of mutations in putative hormone binding sites on V2 vasopressin receptor function.

    PubMed

    Sebti, Y; Rabbani, M; Sadeghi, H Mir Mohammad; Sardari, S; Ghahremani, M H; Innamorati, G

    2015-01-01

    The vasopressin V2 receptor belongs to the large family of the G-protein coupled receptors and is responsible for the antidiuretic effect of the neurohypophyseal hormone arginine vasopressin (AVP). Based on bioinformatic studies it seems that Ala300 and Asp297 of the V2 vasopressin receptor (V2R) are involved in receptor binding. Ala300Glu mutation resulted in lower energy while Asp297Tyr mutation resulted in higher energy in AVP-V2R docked complex rather than the wild type. Therefore we hypothesized that the Ala300Glu mutation results in stronger and Asp297Tyr mutation leads to weaker ligand-receptor binding. Site directed mutagenesis of Asp297Tyr and Ala300Glu was performed using nested polymerase chain reaction. After restriction enzyme digestion, the inserts were ligated into the pcDNA3 vector and Escherichia coli XL1-Blue competent cells were transformed using commercial kit and electroporation methods. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using ESCORT™ IV transfection reagent, the adenylyl cyclase activity assay was performed for functional studies. The cell surface expression of V2R was analyzed by indirect ELISA method. Based on the obtained results, the Ala300Glu mutation of V2R led to reduced levels of cAMP production without a marked effect on the receptor expression and the receptor binding. Effect of Asp297Tyr mutation on cell surface expression of V2R was the same as the wild type receptor. Pretreatment with 1 nM vasopressin showed an increased level of Asp297Tyr mutant receptor internalization as compared to the wild type receptor, while the effect of 100 nM vasopressin was similar in the mutant and wild type receptors. These data suggest that alterations in Asp297 but not Ala300 would affect the hormone receptor binding.

  8. Genetic diagnosis of familial hypercholesterolaemia: the importance of functional analysis of potential splice-site mutations.

    PubMed

    Bourbon, M; Duarte, M A; Alves, A C; Medeiros, A M; Marques, L; Soutar, A K

    2009-05-01

    Familial hypercholesterolemia (FH) results from defective low-density lipoprotein receptor (LDLR) activity, mainly due to LDLR gene defects. Of the many different LDLR mutations found in patients with FH, about 6% of single base substitutions are located near or within introns, and are predicted to result in exon skipping, retention of an intron, or activation of cryptic sites during mRNA splicing. This paper reports on the Portuguese FH Study, which found 10 such mutations, 6 of them novel. For the mutations that have not been described before or those whose effect on function have not been analysed, their effect on splicing was investigated, using reverse transcriptase PCR analysis of LDLR mRNA from freshly isolated blood mononuclear cells. Two of these variants (c.313+6 T-->C, c.2389G-->T (p.V776L)) caused exon skipping, and one caused retention of an intron (c.1359-5C-->G), whereas two others (c.2140+5 G-->A and c.1061-8T-->C) had no apparent effect. Any effect of c.1185G-->C (p.V374V) on splicing could not be determined because it was on an allele with a promoter mutation (-42C-->G) that was probably not transcribed. Variants in four patients lost to follow-up could not be tested experimentally, but they almost certainly affect splicing because they disrupt the invariant AG or GT in acceptor (c.818-2A-->G) or donor (c.1060+1G-->A, c.1845+1delG and c.2547+1G-->A) spice sites. These findings emphasise that care must be taken before reporting the presence or absence of a splice-site mutation in the LDLR gene for diagnostic purposes. The study also shows that relatively simple, quick and inexpensive RNA assays can evaluate putative splicing mutations that are not always predictable by available software, thereby reducing genetic misdiagnosis of patients with FH.

  9. Intra- and Interdomain Effects Due to Mutation of Calcium-binding Sites in Calmodulin*

    PubMed Central

    Xiong, Liang-Wen; Kleerekoper, Quinn K.; Wang, Xu; Putkey, John A.

    2010-01-01

    The IQ-motif protein PEP-19, binds to the C-domain of calmodulin (CaM) with significantly different kon and koff rates in the presence and absence of Ca2+, which could play a role in defining the levels of free CaM during Ca2+ transients. The initial goal of the current study was to determine whether Ca2+ binding to sites III or IV in the C-domain of CaM was responsible for affecting the kinetics of binding PEP-19. EF-hand Ca2+-binding sites were selectively inactivated by the common strategy of changing Asp to Ala at the X-coordination position. Although Ca2+ binding to both sites III and IV appeared necessary for native-like interactions with PEP-19, the data also indicated that the mutations caused undesirable structural alterations as evidenced by significant changes in amide chemical shifts for apoCaM. Mutations in the C-domain also affected chemical shifts in the unmodified N-domain, and altered the Ca2+ binding properties of the N-domain. Conversion of Asp93 to Ala caused the greatest structural perturbations, possibly due to the loss of stabilizing hydrogen bonds between the side chain of Asp93 and backbone amides in apo loop III. Thus, although these mutations inhibit binding of Ca2+, the mutated CaM may not be able to support potentially important native-like activity of the apoprotein. This should be taken into account when designing CaM mutants for expression in cell culture. PMID:20048169

  10. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  11. Identification of putative active site residues of ACAT enzymes.

    PubMed

    Das, Akash; Davis, Matthew A; Rudel, Lawrence L

    2008-08-01

    In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

  12. Bioinformatics study of cancer-related mutations within p53 phosphorylation site motifs.

    PubMed

    Ji, Xiaona; Huang, Qiang; Yu, Long; Nussinov, Ruth; Ma, Buyong

    2014-07-29

    p53 protein has about thirty phosphorylation sites located at the N- and C-termini and in the core domain. The phosphorylation sites are relatively less mutated than other residues in p53. To understand why and how p53 phosphorylation sites are rarely mutated in human cancer, using a bioinformatics approaches, we examined the phosphorylation site and its nearby flanking residues, focusing on the consensus phosphorylation motif pattern, amino-acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer mutations observed within the phosphorylation motifs. Many p53 phosphorylation sites are targets for several kinases. The phosphorylation sites match 17 consensus sequence motifs out of the 29 classified. In addition to proline, which is common in kinase specificity-determining sites, we found high propensity of acidic residues to be adjacent to phosphorylation sites. Analysis of human cancer mutations in the phosphorylation motifs revealed that motifs with adjacent acidic residues generally have fewer mutations, in contrast to phosphorylation sites near proline residues. p53 phosphorylation motifs are mostly disordered. However, human cancer mutations within phosphorylation motifs tend to decrease the disorder propensity. Our results suggest that combination of acidic residues Asp and Glu with phosphorylation sites provide charge redundancy which may safe guard against loss-of-function mutations, and that the natively disordered nature of p53 phosphorylation motifs may help reduce mutational damage. Our results further suggest that engineering acidic amino acids adjacent to potential phosphorylation sites could be a p53 gene therapy strategy.

  13. Alanine-Scanning Mutational Analysis of Durancin GL Reveals Residues Important for Its Antimicrobial Activity.

    PubMed

    Ju, Xingrong; Chen, Xinquan; Du, Lihui; Wu, Xueyou; Liu, Fang; Yuan, Jian

    2015-07-22

    Durancin GL is a novel class IIa bacteriocin with 43 residues produced by Enterococcus durans 41D. This bacteriocin demonstrates narrow inhibition spectrum and potent antimicrobial activity against several Listeria monocytogenes strains, including nisin-resistant L. monocytogenes NR30. A systematic alanine-scanning mutational analysis with site-directed mutagenesis was performed to analyze durancin GL residues important for antimicrobial activity and specificity. Results showed that three mutations lost their antimicrobial activity, ten mutations demonstrated a decreased effect on the activity, and seven mutations exhibited relatively high activity. With regard to inhibitory spectrum, four mutants demonstrated a narrower antimicrobial spectrum than wild-type durancin GL. Another four mutants displayed a broader target cell spectrum and increased potency relative to wild-type durancin GL. These findings broaden our understanding of durancin GL residues important for its antimicrobial activity and contribute to future rational design of variants with increased potency.

  14. Analyses of disease-related GNPTAB mutations define a novel GlcNAc-1-phosphotransferase interaction domain and an alternative site-1 protease cleavage site

    PubMed Central

    Velho, Renata Voltolini; De Pace, Raffaella; Klünder, Sarah; Sperb-Ludwig, Fernanda; Lourenço, Charles Marques; Schwartz, Ida V. D.; Braulke, Thomas; Pohl, Sandra

    2015-01-01

    Mucolipidosis II (MLII) and III alpha/beta are autosomal-recessive diseases of childhood caused by mutations in GNPTAB encoding the α/β-subunit precursor protein of the GlcNAc-1-phosphotransferase complex. This enzyme modifies lysosomal hydrolases with mannose 6-phosphate targeting signals. Upon arrival in the Golgi apparatus, the newly synthesized α/β-subunit precursor is catalytically activated by site-1 protease (S1P). Here we performed comprehensive expression studies of GNPTAB mutations, including two novel mutations T644M and T1223del, identified in Brazilian MLII/MLIII alpha/beta patients. We show that the frameshift E757KfsX1 and the non-sense R587X mutations result in the retention of enzymatically inactive truncated precursor proteins in the endoplasmic reticulum (ER) due to loss of cytosolic ER exit motifs consistent with a severe clinical phenotype in homozygosity. The luminal missense mutations, C505Y, G575R and T644M, partially impaired ER exit and proteolytic activation in accordance with less severe MLIII alpha/beta disease symptoms. Analogous to the previously characterized S399F mutant, we found that the missense mutation I403T led to retention in the ER and loss of catalytic activity. Substitution of further conserved residues in stealth domain 2 (I346 and W357) revealed similar biochemical properties and allowed us to define a putative binding site for accessory proteins required for ER exit of α/β-subunit precursors. Interestingly, the analysis of the Y937_M972del mutant revealed partial Golgi localization and formation of abnormal inactive β-subunits generated by S1P which correlate with a clinical MLII phenotype. Expression analyses of mutations identified in patients underline genotype–phenotype correlations in MLII/MLIII alpha/beta and provide novel insights into structural requirements of proper GlcNAc-1-phosphotransferase activity. PMID:25788519

  15. Mutation of Glycosylation Sites in BST-2 Leads to Its Accumulation at Intracellular CD63-Positive Vesicles without Affecting Its Antiviral Activity against Multivesicular Body-Targeted HIV-1 and Hepatitis B Virus.

    PubMed

    Han, Zhu; Lv, Mingyu; Shi, Ying; Yu, Jinghua; Niu, Junqi; Yu, Xiao-Fang; Zhang, Wenyan

    2016-02-29

    BST-2/tetherin blocks the release of various enveloped viruses including HIV-1 with a "physical tethering" model. The detailed contribution of N-linked glycosylation to this model is controversial. Here, we confirmed that mutation of glycosylation sites exerted an effect of post-translational mis-trafficking, leading to an accumulation of BST-2 at intracellular CD63-positive vesicles. BST-2 with this phenotype potently inhibited the release of multivesicular body-targeted HIV-1 and hepatitis B virus, without affecting the co-localization of BST-2 with EEA1 and LAMP1. These results suggest that N-linked glycosylation of human BST-2 is dispensable for intracellular virion retention and imply that this recently discovered intracellular tethering function may be evolutionarily distinguished from the canonical antiviral function of BST-2 by tethering nascent virions at the cell surface.

  16. Application of natural and amplification created restriction sites for the diagnosis of PKU mutations.

    PubMed Central

    Eiken, H G; Odland, E; Boman, H; Skjelkvåle, L; Engebretsen, L F; Apold, J

    1991-01-01

    PCR amplification, either conventional, or as site directed mutagenesis using primers with mismatched 3'-ends, followed by restriction endonuclease digestion, provides rapid, non-isotope assays of known mutations in the human phenylalanine hydroxylase gene. Such assays were shown to have the potential to detect all of the 18 presently reported phenylketonuria mutations. The practical applicability of this approach was demonstrated for eight mutations in Norwegian phenylketonuria patients, among them the most common ones. Images PMID:1851292

  17. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    SciTech Connect

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E.

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  18. Quantitation of normal CFTR mRNA in CF patients with splice-site mutations

    SciTech Connect

    Zhou, Z.; Olsen, J.C.; Silverman, L.M.

    1994-09-01

    Previously we identified two mutations in introns of the CFTR gene associated with partially active splice sites and unusual clinical phenotypes. One mutation in intron 19 (3849+10 kb C to T) is common in CF patients with normal sweat chloride values; an 84 bp sequence from intron 19, which contains a stop codon, is inserted between exon 19 and exon 20 in most nasal CFTR transcripts. The other mutation in intron 14B (2789+5 G to A) is associated with elevated sweat chloride levels, but mild pulmonary disease; exon 14B (38 bp) is spliced out of most nasal CFTR transcipts. The remaining CFTR cDNA sequences, other than the 84 bp insertion of exon 14B deletion, are identical to the published sequence. To correlate genotype and phenotype, we used quantitative RT-PCR to determine the levels of normally-spliced CFTR mRNA in nasal epithelia from these patients. CFTR cDNA was amplified (25 cycles) by using primers specific for normally-spliced species, {gamma}-actin cDNA was amplified as a standard.

  19. Identification of a splice-site mutation in the human growth hormone-variant gene.

    PubMed Central

    MacLeod, J N; Liebhaber, S A; MacGillivray, M H; Cooke, N E

    1991-01-01

    The human growth-hormone-variant (hGH-V) gene normally expresses two alternatively spliced forms of mRNA--hGH-V and hGH-V2--in the placenta. hGH-V2 mRNA differs from hGH-V rDNA by the retention of intron 4 and represents approximately 15% of transcripts at term. In a survey of hGH-V gene expression in 20 placentas of gestational age 8-40 wk, we detected a single placenta that contained, in addition to the two normal hGH-V mRNA species, a set of two slightly larger hGH-V mRNAs. Sequence analysis of the elongated hGH-V mRNA demonstrated retention of the first 12 bases of intron 2, resulting from both a base substitution at the intron 2 splice-donor dinucleotide (GT----AT) and activation of a cryptic splice-donor site 12 bases downstream. Survey of a total of 60 additional chromosomes failed to reveal additional incidence of this mutation. The mutation, which we have designated hGH-Vintron 2, pos 1 (G----A), represents both an initial example of a nondeletional mutation within the hGH-V gene and corresponding structural alteration in the encoded hGH-V hormone. Images Figure 1 Figure 2 Figure 4 PMID:2035535

  20. Classification of Cancer Primary Sites Using Machine Learning and Somatic Mutations.

    PubMed

    Chen, Yukun; Sun, Jingchun; Huang, Liang-Chin; Xu, Hua; Zhao, Zhongming

    2015-01-01

    An accurate classification of human cancer, including its primary site, is important for better understanding of cancer and effective therapeutic strategies development. The available big data of somatic mutations provides us a great opportunity to investigate cancer classification using machine learning. Here, we explored the patterns of 1,760,846 somatic mutations identified from 230,255 cancer patients along with gene function information using support vector machine. Specifically, we performed a multiclass classification experiment over the 17 tumor sites using the gene symbol, somatic mutation, chromosome, and gene functional pathway as predictors for 6,751 subjects. The performance of the baseline using only gene features is 0.57 in accuracy. It was improved to 0.62 when adding the information of mutation and chromosome. Among the predictable primary tumor sites, the prediction of five primary sites (large intestine, liver, skin, pancreas, and lung) could achieve the performance with more than 0.70 in F-measure. The model of the large intestine ranked the first with 0.87 in F-measure. The results demonstrate that the somatic mutation information is useful for prediction of primary tumor sites with machine learning modeling. To our knowledge, this study is the first investigation of the primary sites classification using machine learning and somatic mutation data.

  1. Tuning calcium biosensors with a single-site mutation: structural dynamics insights from femtosecond Raman spectroscopy.

    PubMed

    Tachibana, Sean R; Tang, Longteng; Wang, Yanli; Zhu, Liangdong; Liu, Weimin; Fang, Chong

    2017-03-08

    Fluorescent protein biosensors are popular reporters for biological processes and life sciences, but their fundamental working mechanisms remain unclear. To characterize the functional fluorescence events on their native timescales, we implemented wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) to shed light on a blue-green emission-ratiometric fluorescent protein based Ca(2+) biosensor with a single Pro377Arg mutation. The transient Raman modes of the embedded chromophore from ca. 1000-1650 cm(-1) exhibit characteristic intensity and frequency dynamics which infer the underlying atomic motions and photochemical reaction stages. Our experimental study reveals the hidden structural inhomogeneity of the protein local environment upon Ca(2+) binding with the mutated arginine residue trapping multiple chromophore subpopulations, which manifest distinct time constants of ∼16 and 90 ps for excited state proton transfer (ESPT) following 400 nm photoexcitation. The altered ESPT reaction pathways and emission properties of the Ca(2+) biosensor represent the foundational step of rationally designing advanced fluorescent protein biosensors to tune their functionalities by site-specifically altering the local environment (e.g., the active site) of the embedded chromophore.

  2. Second-Site Suppression of RNase E Essentiality by Mutation of the deaD RNA Helicase in Escherichia coli

    PubMed Central

    Tamura, Masaru; Kers, Johan A.

    2012-01-01

    Escherichia coli cells normally require RNase E activity to propagate and form colonies. Using random Tn10 insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) to E. coli cells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of the rne mutant phenotype by inactivation of deaD was partial, as rne deaD doubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration by deaD inactivation requires normal expression of the endogenous rng gene in doubly mutant rne deaD cells. Second-site suppression by deaD mutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality. PMID:22328678

  3. Active and regulatory sites of cytosolic 5'-nucleotidase.

    PubMed

    Pesi, Rossana; Allegrini, Simone; Careddu, Maria Giovanna; Filoni, Daniela Nicole; Camici, Marcella; Tozzi, Maria Grazia

    2010-12-01

    Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap₄A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap₄A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation.

  4. Triallelic Population Genomics for Inferring Correlated Fitness Effects of Same Site Nonsynonymous Mutations.

    PubMed

    Ragsdale, Aaron P; Coffman, Alec J; Hsieh, PingHsun; Struck, Travis J; Gutenkunst, Ryan N

    2016-05-01

    The distribution of mutational effects on fitness is central to evolutionary genetics. Typical univariate distributions, however, cannot model the effects of multiple mutations at the same site, so we introduce a model in which mutations at the same site have correlated fitness effects. To infer the strength of that correlation, we developed a diffusion approximation to the triallelic frequency spectrum, which we applied to data from Drosophila melanogaster We found a moderate positive correlation between the fitness effects of nonsynonymous mutations at the same codon, suggesting that both mutation identity and location are important for determining fitness effects in proteins. We validated our approach by comparing it to biochemical mutational scanning experiments, finding strong quantitative agreement, even between different organisms. We also found that the correlation of mutational fitness effects was not affected by protein solvent exposure or structural disorder. Together, our results suggest that the correlation of fitness effects at the same site is a previously overlooked yet fundamental property of protein evolution.

  5. Space environment induced mutations prefer to occur at polymorphic sites of rice genomes

    NASA Astrophysics Data System (ADS)

    Li, Y.; Liu, M.; Cheng, Z.; Sun, Y.

    To explore the genomic characteristics of rice mutants induced by space environment, space-induced mutants 971-5, 972-4, and R955, which acquired new traits after space flight such as increased yield, reduced resistance to rice blast, and semi-dwarfism compared with their on-ground controls, 971ck, 972ck, and Bing95-503, respectively, together with other 8 japonica and 3 indica rice varieties, 17 in total, were analyzed by amplified fragment length polymorphism (AFLP) method. We chose 16 AFLP primer-pairs which generated a total of 1251 sites, of which 745 (59.6%) were polymorphic over all the genotypes. With the 16 pairs of primer combinations, 54 space-induced mutation sites were observed in 971-5, 86 in 972-4, and 5 in R955 compared to their controls, and the mutation rates were 4.3%, 6.9% and 0.4%, respectively. Interestingly, 75.9%, 84.9% and 100% of the mutation sites identified in 971-5, 972-4, and R955 occurred in polymorphic sites. This result suggests that the space environment preferentially induced mutations at polymorphic sites in rice genomes and might share a common mechanism with other types of mutagens. It also implies that polymorphic sites in genomes are potential "hotspots" for mutations induced by the space environment.

  6. 1H NMR study of the influence of mutation on the interaction of the C-terminus with the active site in heme oxygenase from Neisseria meningitidis: implications for product release.

    PubMed

    Peng, Dungeng; Ma, Li-Hua; Ogura, Hiroshi; Yang, En-Che; Zhang, Xuhong; Yoshida, Tadashi; La Mar, Gerd N

    2010-07-20

    The HO from the pathogenic bacterium Neisseria meningitidis, NmHO, possesses C-terminal His207, Arg208, and His209 residues that are undetected in crystal structures. NMR found the C-terminus ordered and interacting with the active site and shown to undergo a spontaneous cleavage of the C-terminal Arg208-His209 bond that affects the product off rate. A preliminary model for the interaction based on the wild-type (WT) NmHO complexes has been presented [Liu, Y., Ma, L.-H., Satterlee, J. D., Zhang, X., Yoshida, T., and La Mar, G. N. (2006) Biochemistry 45, 3875-3886]. Two-dimensional (1)H NMR data of resting-state, azide-inhibited substrate complexes of the three C-terminal truncation mutants (Des-His209-, Des-Arg208His209-, and Des-His207Arg208His209-NmHO) confirm the previous proposed roles for His207 and Arg208 and reveal important additional salt bridges involving the His209 carboxylate and the side chains of both Lys126 and Arg208. Deletion of His209 leads to a qualitatively retained C-terminal geometry, but with increased separation between the C-terminus and active site. Moreover, replacing vinyls with methyls on the substrate leads to a decrease in the separation between the C-terminus and the active site. The expanded model for the C-terminus reveals a less stable His207-Arg208 cis peptide bond, providing a rationalization for its spontaneous cleavage. The rate of this spontaneous cleavage is shown to correlate with the proximity of the C-terminus to the active site, suggesting that the closer interaction leads to increased strain on the already weak His207-Arg208 peptide bond. The relevance of the C-terminus structure for in vitro studies, and the physiological function of product release, is discussed.

  7. Gain-Of-Function Mutational Activation of Human TRNA Synthetase Procytokine

    SciTech Connect

    Yang, X.L.; Kapoor, M.; Otero, F.J.; Slike, B.M.; Tsuruta, H.; Frausto, R.; Bates, A.; Ewalt, K.L.; Cheresh, D.A.; Schimmel, P.; /Scripps Res. Inst. /SLAC, SSRL

    2009-04-30

    Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases.

  8. Predicting changes in protein thermostability brought about by single- or multi-site mutations

    PubMed Central

    2010-01-01

    Background An important aspect of protein design is the ability to predict changes in protein thermostability arising from single- or multi-site mutations. Protein thermostability is reflected in the change in free energy (ΔΔG) of thermal denaturation. Results We have developed predictive software, Prethermut, based on machine learning methods, to predict the effect of single- or multi-site mutations on protein thermostability. The input vector of Prethermut is based on known structural changes and empirical measurements of changes in potential energy due to protein mutations. Using a 10-fold cross validation test on the M-dataset, consisting of 3366 mutants proteins from ProTherm, the classification accuracy of random forests and the regression accuracy of random forest regression were slightly better than support vector machines and support vector regression, whereas the overall accuracy of classification and the Pearson correlation coefficient of regression were 79.2% and 0.72, respectively. Prethermut performs better on proteins containing multi-site mutations than those with single mutations. Conclusions The performance of Prethermut indicates that it is a useful tool for predicting changes in protein thermostability brought about by single- or multi-site mutations and will be valuable in the rational design of proteins. PMID:20598148

  9. Promoter-proximal polyadenylation sites reduce transcription activity

    PubMed Central

    Andersen, Pia K.; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels. PMID:23028143

  10. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

  11. Somatic mosaicism for a newly identified splice-site mutation in a patient with adenosine deaminase-deficient immunodeficiency and spontaneous clinical recovery

    SciTech Connect

    Hirschhorn, R.; Yang, D.R.; Israni, A.; Huie, M.L. ); Ownby, D.R. )

    1994-07-01

    Absent or severely reduced adenosine deaminase (ADA) activity produces inherited immunodeficiency of varying severity, with defects of both cellular and humoral immunity. The authors report somatic mosaicism as the basis for a delayed presentation and unusual course of a currently healthy young adult receiving no therapy. He was diagnosed at age 2[1/2] years because of life-threatening pneumonia, recurrent infections, failure of normal growth, and lymphopenia, but he retained significant cellular immune function. A fibroblast cell line and a B cell line, established at diagnosis, lacked ADA activity and were heteroallelic for a splice-donor-site mutation in IVS 1 (+1GT[yields]CT) and a missense mutation (Arg101Gln). All clones (17/17) isolated from the B cell mRNA carried the missense mutation, indicating that the allele with the splice-site mutation produced unstable mRNA. In striking contrast, a B cell line established at age 16 years expressed 50% of normal ADA; 50% had the missense mutation. Genomic DNA contained the missense mutation but not the splice-site mutation. All three cell lines were identical for multiple polymorphic markers and the presence of a Y chromosome. In vivo somatic mosaicism was demonstrated in genomic DNA from peripheral blood cells obtained at 16 years of age, in that less than half the DNA carried the splice-site mutation (P<.0.02, vs. original B cell line). Consistent with mosaicism, erythrocyte content of the toxic metabolite deoxyATP was only minimally elevated. Somatic mosaicism could have arisen either by somatic mutation or by reversion at the site of mutation. Selection in vivo for ADA normal hematopoietic cells may have played a role in the return to normal health, in the absence of therapy. 57 refs., 4 figs., 2 tabs.

  12. A Novel Intronic Splice Site Tafazzin Gene Mutation Detected Prenatally in a Family with Barth Syndrome

    PubMed Central

    Bakšienė, M; Benušienė, E; Morkūnienė, A; Ambrozaitytė, L; Utkus, A; Kučinskas, V

    2016-01-01

    Abstract Barth syndrome (BTHS) is a rare X-linked disease characterized by dilated cardiomyopathy, proximal skeletal myopathy and cyclic neutropenia. It is caused by various mutations in the tafazzin (TAZ) gene located on Xq28 that results in remodeling of cardiolipin and abnormalities in mitochondria stability and energy production. Here we report on a novel c.285-1G>C splice site mutation in intron 3 of the TAZ gene that was detected prenatally. PMID:28289596

  13. Sec3 Mutations Are Synthetically Lethal with Profilin Mutations and Cause Defects in Diploid-Specific Bud-Site Selection

    PubMed Central

    Haarer, B. K.; Corbett, A.; Kweon, Y.; Petzold, A. S.; Silver, P.; Brown, S. S.

    1996-01-01

    Replacement of the wild-type yeast profilin gene (PFY1) with a mutated form (pfy1-111) that has codon 72 changed to encode glutamate rather than arginine results in defects similar to, but less severe than, those that result from complete deletion of the profilin gene. We have used a colony color-sectoring assay to identify mutations that cause pfy1-111, but not wild-type, cells to be inviable. These profilin synthetic lethal (psl) mutations result in various degrees of abnormal growth, morphology, and temperature sensitivity in PFY1 cells. We have examined psl1 strains in the most detail. Interestingly, these strains display a diploid-specific defect in bud-site selection; haploid strains bud normally, while homozygous diploid strains show a dramatic increase in random budding. We discovered that PSL1 is the late secretory gene, SEC3, and have found that mutations in several other late secretory genes are also synthetically lethal with pfy1-111. Our results are likely to reflect an interdependence between the actin cytoskeleton and secretory processes in directing cell polarity and growth. Moreover, they indicate that the secretory pathway is especially crucial for maintaining budding polarity in diploids. PMID:8889515

  14. Activating mutations in CTNNB1 in aldosterone producing adenomas

    PubMed Central

    Åkerström, Tobias; Maharjan, Rajani; Sven Willenberg, Holger; Cupisti, Kenko; Ip, Julian; Moser, Ana; Stålberg, Peter; Robinson, Bruce; Alexander Iwen, K.; Dralle, Henning; Walz, Martin K.; Lehnert, Hendrik; Sidhu, Stan; Gomez-Sanchez, Celso; Hellman, Per; Björklund, Peyman

    2016-01-01

    Primary aldosteronism (PA) is the most common cause of secondary hypertension with a prevalence of 5–10% in unreferred hypertensive patients. Aldosterone producing adenomas (APAs) constitute a large proportion of PA cases and represent a surgically correctable form of the disease. The WNT signaling pathway is activated in APAs. In other tumors, a frequent cause of aberrant WNT signaling is mutation in the CTNNB1 gene coding for β-catenin. Our objective was to screen for CTNNB1 mutations in a well-characterized cohort of 198 APAs. Somatic CTNNB1 mutations were detected in 5.1% of the tumors, occurring mutually exclusive from mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. All of the observed mutations altered serine/threonine residues in the GSK3β binding domain in exon 3. The mutations were associated with stabilized β-catenin and increased AXIN2 expression, suggesting activation of WNT signaling. By CYP11B2 mRNA expression, CYP11B2 protein expression, and direct measurement of aldosterone in tumor tissue, we confirmed the ability for aldosterone production. This report provides compelling evidence that aberrant WNT signaling caused by mutations in CTNNB1 occur in APAs. This also suggests that other mechanisms that constitutively activate the WNT pathway may be important in APA formation. PMID:26815163

  15. A detailed mutational analysis of the VSG gene expression site promoter.

    PubMed

    Pham, V P; Qi, C C; Gottesdiener, K M

    1996-01-01

    The African trypanosome Trypanosoma brucei is a protozoan parasite that causes the disease African sleeping sickness. The parasite avoids the host's immune response by the process of antigenic variation, or by sequentially expressing antigenically different cell-surface coat proteins. These proteins, called variant surface glycoproteins (VSGs), are expressed from a specific locus, the VSG gene expression site (ES). In an attempt to understand expression of VSG genes, we expanded on earlier investigations of the promoter that controls the large VSG gene expression site transcription unit. We studied VSG ES promoter function both in transient transfection assays, and after stable integration at a chromosomal locus. Analysis of closely spaced deletion mutants showed that the minimum VSG ES promoter fragment that gives full activity is extremely small, and mapped precisely to a fragment that contains no more than -67 bp 5' to the putative transcription initiation site. The promoter lacked an upstream control element, or UCE, an element found at the PARP promoter, and at most eukaryotic Pol I promoters. Furthermore, linker scanning mutagenesis demonstrated that the VSG ES promoter contains at least two essential regulatory elements, including sequences within the region -67/-60 and the region -35/-20, both numbered relative to the initiation site. An altered promoter with mutated nucleotides surrounding the transcription initiation site still directed wild-type levels of expression. In this study, the results were similar for both insect and bloodstream form trypanosomes, suggesting that the same basic machinery for expression from the VSG ES promoter is found in both stages of the parasite.

  16. Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs.

    PubMed

    Suzukawa, Keisuke; Yamagami, Takeshi; Ohnuma, Takayuki; Hirakawa, Hideki; Kuhara, Satoru; Aso, Yoichi; Ishiguro, Masatsune

    2003-02-01

    We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

  17. Palmitoylation of muscarinic acetylcholine receptor m2 subtypes: reduction in their ability to activate G proteins by mutation of a putative palmitoylation site, cysteine 457, in the carboxyl-terminal tail.

    PubMed

    Hayashi, M K; Haga, T

    1997-04-15

    A putative palmitoylation site, Cys457, of muscarinic acetylcholine receptor m2 subtype (m2 receptor) was eliminated by conversion to alanine or stop codon by site-directed mutagenesis. The mutant m2 receptor C457A was not metabolically labeled with [3H] palmitic acid when expressed in Sf9 cells, whereas the wild-type m2 receptor was labeled under the same conditions. These results confirm that the Cys457 is the palmitoylation site. The rate of palmitoylation was markedly accelerated by addition of agonist, indicating that the palmitoylation reaction is affected by conformational changes of the receptor induced by agonist binding. The m2 receptor mutants without palmitoylation were purified and reconstituted with G proteins into phospholipid vesicles. Both mutants were good substrates of G protein-coupled receptor kinase 2 and the phosphorylation was stimulated by agonist and G protein beta gamma subunits, as was the case for wild-type receptors. The mutant receptors interacted with and activate Gi2 and G(o). However, the rate of [35S] GTP gamma S binding to Gi2 was half as much for the mutants as that for the wild type, and the proportion of guanine nucleotide-sensitive high-affinity agonist binding sites was significantly less for mutants (42-42%) compared to wild type (62%). These results indicate that the palmitoylation of m2 receptors is not an absolute requirement for their interaction with G proteins but enhances the ability of the receptors to interact with G proteins.

  18. Splice-site mutations: a novel genetic mechanism of Crigler-Najjar syndrome type 1.

    PubMed Central

    Gantla, S; Bakker, C T; Deocharan, B; Thummala, N R; Zweiner, J; Sinaasappel, M; Roy Chowdhury, J; Bosma, P J; Roy Chowdhury, N

    1998-01-01

    Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from deficiency of the hepatic enzyme bilirubin-UDP-glucuronosyltransferase. In all CN-1 patients studied, structural mutations in one of the five exons of the gene (UGT1A1) encoding the uridinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirubin-UGT1 were implicated in the absence or inactivation of the enzyme. We report two patients in whom CN-1 is caused, instead, by mutations in the noncoding intronic region of the UGT1A1 gene. One patient (A) was homozygous for a G-->C mutation at the splice-donor site in the intron, between exon 1 and exon 2. The other patient (B) was heterozygous for an A-->G shift at the splice-acceptor site in intron 3, and in the second allele a premature translation-termination codon in exon 1 was identified. Bilirubin-UGT1 mRNA is difficult to obtain, since it is expressed in the liver only. To determine the effects of these splice-junction mutations, we amplified genomic DNA of the relevant splice junctions. The amplicons were expressed in COS-7 cells, and the expressed mRNAs were analyzed. In both cases, splice-site mutations led to the use of cryptic splice sites, with consequent deletions in the processed mRNA. This is the first report of intronic mutations causing CN-1 and of the determination of the consequences of these mutations on mRNA structure, by ex vivo expression. PMID:9497253

  19. Rice tungro spherical virus polyprotein processing: identification of a virus-encoded protease and mutational analysis of putative cleavage sites.

    PubMed

    Thole, V; Hull, R

    1998-07-20

    Rice tungro spherical virus encodes a large polyprotein containing motifs with sequence similarity to viral serine-like proteases and RNA polymerases. Polyclonal antisera raised against domains of the putative protease and polymerase in fusion with glutathione S-transferase detected a protein of about 35 kDa and, in very low amounts, a protein of about 70 kDa, respectively, in extracts from infected plants. In in vitro transcription/translation systems and in Escherichia coli we demonstrated a proteolytic activity in the C-terminal region of the polyprotein. This protease rapidly cleaved its polyprotein precursors in vitro. Mutating a potential cleavage site located N-terminal to the protease domain, Gln2526-Asp2527, diminished processing. The transversion mutation at the putative C-terminal cleavage site of the protease, at Gln2852-Ala2853, led to a delayed and partial processing.

  20. Enhancing human spermine synthase activity by engineered mutations.

    PubMed

    Zhang, Zhe; Zheng, Yueli; Petukh, Margo; Pegg, Anthony; Ikeguchi, Yoshihiko; Alexov, Emil

    2013-01-01

    Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing.

  1. TERT promoter mutations and monoallelic activation of TERT in cancer

    PubMed Central

    Huang, F W; Bielski, C M; Rinne, M L; Hahn, W C; Sellers, W R; Stegmeier, F; Garraway, L A; Kryukov, G V

    2015-01-01

    Here we report that promoter mutations in telomerase (TERT), the most common noncoding mutations in cancer, give rise to monoallelic expression of TERT. Through deep RNA sequencing, we find that TERT activation in human cancer cell lines can occur in either mono- or biallelic manner. Without exception, hotspot TERT promoter mutations lead to the re-expression of only one allele, accounting for approximately half of the observed cases of monoallelic TERT expression. Furthermore, we show that monoallelic TERT expression is highly prevalent in certain tumor types and widespread across a broad spectrum of cancers. Taken together, these observations provide insights into the mechanisms of TERT activation and the ramifications of noncoding mutations in cancer. PMID:26657580

  2. mutLBSgeneDB: mutated ligand binding site gene DataBase

    PubMed Central

    Kim, Pora; Zhao, Junfei; Lu, Pinyi; Zhao, Zhongming

    2017-01-01

    Mutations at the ligand binding sites (LBSs) can influence protein structure stability, binding affinity with small molecules, and drug resistance in cancer patients. Our recent analysis revealed that ligand binding residues had a significantly higher mutation rate than other parts of the protein. Here, we built mutLBSgeneDB (mutated Ligand Binding Site gene DataBase) available at http://zhaobioinfo.org/mutLBSgeneDB. We collected and curated over 2300 genes (mutLBSgenes) having ∼12 000 somatic mutations at ∼10 000 LBSs across 16 cancer types and selected 744 drug targetable genes (targetable_mutLBSgenes) by incorporating kinases, transcription factors, pharmacological genes, and cancer driver genes. We analyzed LBS mutation information, differential gene expression network, drug response correlation with gene expression, and protein stability changes for all mutLBSgenes using integrated genetic, genomic, transcriptomic, proteomic, network and functional information. We calculated and compared the binding affinities of 20 carefully selected genes with their drugs in wild type and mutant forms. mutLBSgeneDB provides a user-friendly web interface for searching and browsing through seven categories of annotations: Gene summary, Mutated information, Protein structure related information, Differential gene expression and gene-gene network, Phenotype information, Pharmacological information, and Conservation information. mutLBSgeneDB provides a useful resource for functional genomics, protein structure, drug and disease research communities. PMID:27907895

  3. KRAS-mutation incidence and prognostic value are metastatic site-specific in lung adenocarcinoma: poor prognosis in patients with KRAS mutation and bone metastasis

    PubMed Central

    Lohinai, Zoltan; Klikovits, Thomas; Moldvay, Judit; Ostoros, Gyula; Raso, Erzsebet; Timar, Jozsef; Fabian, Katalin; Kovalszky, Ilona; Kenessey, István; Aigner, Clemens; Renyi-Vamos, Ferenc; Klepetko, Walter; Dome, Balazs; Hegedus, Balazs

    2017-01-01

    Current guidelines lack comprehensive information on the metastatic site-specific role of KRAS mutation in lung adenocarcinoma (LADC). We investigated the effect of KRAS mutation on overall survival (OS) in this setting. In our retrospective study, 500 consecutive Caucasian metastatic LADC patients with known KRAS mutational status were analyzed after excluding 32 patients with EGFR mutations. KRAS mutation incidence was 28.6%. The most frequent metastatic sites were lung (45.6%), bone (26.2%), adrenal gland (17.4%), brain (16.8%), pleura (15.6%) and liver (11%). Patients with intrapulmonary metastasis had significantly increased KRAS mutation frequency compared to those with extrapulmonary metastases (35% vs 26.5%, p = 0.0125). In contrast, pleural dissemination and liver involvement were associated with significantly decreased KRAS mutation incidence (vs all other metastatic sites; 17% (p < 0.001) and 16% (p = 0.02) vs 33%, respectively). Strikingly, we found a significant prognostic effect of KRAS status only in the bone metastatic subcohort (KRAS-wild-type vs KRAS-mutant; median OS 9.7 v 3.7 months; HR, 0.49; 95% CI, 0.31 to 0.79; p  = 0.003). Our study suggests that KRAS mutation frequency in LADC patients shows a metastatic site dependent variation and, moreover, that the presence of KRAS mutation is associated with significantly worse outcome in bone metastatic cases. PMID:28051122

  4. Sites of preferential induction of cyclobutane pyrimidine dimers in the nontranscribed strand of lacI correspond with sites of UV-induced mutation in Escherichia coli

    SciTech Connect

    Koehler, D.R.; Awadallah, S.S.; Glickman, B.W. )

    1991-06-25

    An approach utilizing fluorescence-activated DNA sequencing technology was used to study the position and frequency of UV-induced lesions in the lacI gene of Escherichia coli. The spectrum of sites of UV damage in the NC+ region of the gene was compared with a published spectrum of UV-induced mutation in lacI . On average, the frequency of UV-induced lesions in the nontranscribed strand was higher than that in the transcribed strand in the region analyzed. A large fraction of mutations occurs at sites of UV-induced lesions in the nontranscribed strand, but not in the transcribed strand. This bias is reduced in an excision repair deficient (UvrB-) strain. In addition, mutations occur overwhelmingly at sites where a dipyrimidine sequence is present in the nontranscribed strand. This bias is also markedly reduced in the UvrB- strain. In light of recent work Mellon and Hanawalt describing the preferential removal of cyclobutane dimers from the transcribed strand of the expressed lacZ gene in E. coli, our data suggest that preferential strand repair may have a significant effect on mutagenesis.

  5. Introduction of Site-Specific Mutations Into the Genome of Influenza Virus

    NASA Astrophysics Data System (ADS)

    Enami, Masayoshi; Luytjes, Willem; Krystal, Mark; Palese, Peter

    1990-05-01

    We succeeded in rescuing infectious influenza virus by transfecting cells with RNAs derived from specific recombinant DNAs. RNA corresponding to the neuraminidase (NA) gene of influenza A/WSN/33 (WSN) virus was transcribed in vitro from plasmid DNA and, following the addition of purified influenza virus RNA polymerase complex, was transfected into MDBK cells. Superinfection with helper virus lacking the WSN NA gene resulted in the release of virus containing the WSN NA gene. We then introduced five point mutations into the WSN NA gene by cassette mutagenesis of the plasmid DNA. Sequence analysis of the rescued virus revealed that the genome contained all five mutations present in the mutated plasmid. The ability to create viruses with site-specific mutations will allow the engineering of influenza viruses with defined biological properties.

  6. A Novel ECM1 Splice Site Mutation in Lipoid Proteinosis: Case Report plus Review of the Literature

    PubMed Central

    Rey, Linda K.; Kohlhase, Jürgen; Möllenhoff, Katrin; Dekomien, Gabriele; Epplen, Jörg T.; Hoffjan, Sabine

    2016-01-01

    Lipoid proteinosis (LP) is an autosomal recessive genodermatosis known to be caused by mutations in ECM1. Nonsense and missense mutations are the most common variations in LP. Up to date, only 6 splice site mutations have been observed. We report on a 26-year-old female LP patient from a Turkish consanguineous family carrying a novel homozygous splice site mutation in intron 8 of the ECM1 gene and summarize the current knowledge on ECM1 mutations and possible genotype-phenotype correlations. PMID:27194970

  7. De novo SCN2A splice site mutation in a boy with Autism spectrum disorder

    PubMed Central

    2014-01-01

    Background SCN2A is a gene that codes for the alpha subunit of voltage-gated, type II sodium channels, and is highly expressed in the brain. Sodium channel disruptions, such as mutations in SCN2A, may play an important role in psychiatric disorders. Recently, de novo SCN2A mutations in autism spectrum disorder (ASD) have been identified. The current study characterizes a de novo splice site mutation in SCN2A that alters mRNA and protein products. Case presentation We describe results from clinical and genetic characterizations of a seven-year-old boy with ASD. Psychiatric interview and gold standard autism diagnostic instruments (ADOS and ADI-R) were used to confirm ASD diagnosis, in addition to performing standardized cognitive and adaptive functioning assessments (Leiter-R and Vineland Adaptive Behavior Scale), and sensory reactivity assessments (Sensory Profile and Sensory Processing Scales). Genetic testing by whole exome sequencing revealed four de novo events, including a splice site mutation c.476 + 1G > A in SCN2A, a missense mutation (c.2263G > A) causing a p.V755I change in the TLE1 gene, and two synonymous mutations (c.2943A > G in the BUB1 gene, and c.1254 T > A in C10orf68 gene). The de novo SCN2A splice site mutation produced a stop codon 10 amino acids downstream, possibly resulting in a truncated protein and/or a nonsense-mediated mRNA decay. The participant met new DSM-5 criteria for ASD, presenting with social and communication impairment, repetitive behaviors, and sensory reactivity issues. The participant’s adaptive and cognitive skills fell in the low range of functioning. Conclusion This report indicates that a splice site mutation in SCN2A might be contributing to the risk of ASD. Describing the specific phenotype associated with SCN2A mutations might help to reduce heterogeneity seen in ASD. PMID:24650168

  8. Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A.

    PubMed

    Tagawa, K; Taya, C; Hayashi, Y; Nakagawa, M; Ono, Y; Fukuda, R; Karasuyama, H; Toyama-Sorimachi, N; Katsui, Y; Hata, S; Ishiura, S; Nonaka, I; Seyama, Y; Arahata, K; Yonekawa, H; Sorimachi, H; Suzuki, K

    2000-05-22

    A defect of the gene for p94 (calpain 3), a skeletal muscle-specific calpain, is responsible for limb girdle muscular dystrophy type 2A (LGMD2A), or 'calpainopathy', which is an autosomal recessive and progressive neuromuscular disorder. To study the relationships between the physiological functions of p94 and the etiology of LGMD2A, we created transgenic mice that express an inactive mutant of p94, in which the active site Cys129 is replaced by Ser (p94:C129S). Three lines of transgenic mice expressing p94:C129S mRNA at various levels showed significantly decreased grip strength. Sections of soleus and extensor digitorum longus (EDL) muscles of the aged transgenic mice showed increased numbers of lobulated and split fibers, respectively, which are often observed in limb girdle muscular dystrophy muscles. Centrally placed nuclei were also frequently found in the EDL muscle of the transgenic mice, whereas wild-type mice of the same age had almost none. There was more p94 protein produced in aged transgenic mice muscles and it showed significantly less autolytic degradation activity than that of wild-type mice. Although no necrotic-regenerative fibers were observed, the age and p94:C129S expression dependence of the phenotypes strongly suggest that accumulation of p94:C129S protein causes these myopathy phenotypes. The p94:C129S transgenic mice could provide us with crucial information on the molecular mech-anism of LGMD2A.

  9. Potential sites of CFTR activation by tyrosine kinases

    PubMed Central

    Billet, Arnaud; Jia, Yanlin; Jensen, Timothy J.; Hou, Yue-Xian; Chang, Xiu-Bao; Riordan, John R.; Hanrahan, John W.

    2016-01-01

    ABSTRACT The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation. PMID:26645934

  10. Glucocerebrosidase activity in Parkinson's disease with and without GBA mutations.

    PubMed

    Alcalay, Roy N; Levy, Oren A; Waters, Cheryl C; Fahn, Stanley; Ford, Blair; Kuo, Sheng-Han; Mazzoni, Pietro; Pauciulo, Michael W; Nichols, William C; Gan-Or, Ziv; Rouleau, Guy A; Chung, Wendy K; Wolf, Pavlina; Oliva, Petra; Keutzer, Joan; Marder, Karen; Zhang, Xiaokui

    2015-09-01

    Glucocerebrosidase (GBA) mutations have been associated with Parkinson's disease in numerous studies. However, it is unknown whether the increased risk of Parkinson's disease in GBA carriers is due to a loss of glucocerebrosidase enzymatic activity. We measured glucocerebrosidase enzymatic activity in dried blood spots in patients with Parkinson's disease (n = 517) and controls (n = 252) with and without GBA mutations. Participants were recruited from Columbia University, New York, and fully sequenced for GBA mutations and genotyped for the LRRK2 G2019S mutation, the most common autosomal dominant mutation in the Ashkenazi Jewish population. Glucocerebrosidase enzymatic activity in dried blood spots was measured by a mass spectrometry-based assay and compared among participants categorized by GBA mutation status and Parkinson's disease diagnosis. Parkinson's disease patients were more likely than controls to carry the LRRK2 G2019S mutation (n = 39, 7.5% versus n = 2, 0.8%, P < 0.001) and GBA mutations or variants (seven homozygotes and compound heterozygotes and 81 heterozygotes, 17.0% versus 17 heterozygotes, 6.7%, P < 0.001). GBA homozygotes/compound heterozygotes had lower enzymatic activity than GBA heterozygotes (0.85 µmol/l/h versus 7.88 µmol/l/h, P < 0.001), and GBA heterozygotes had lower enzymatic activity than GBA and LRRK2 non-carriers (7.88 µmol/l/h versus 11.93 µmol/l/h, P < 0.001). Glucocerebrosidase activity was reduced in heterozygotes compared to non-carriers when each mutation was compared independently (N370S, P < 0.001; L444P, P < 0.001; 84GG, P = 0.003; R496H, P = 0.018) and also reduced in GBA variants associated with Parkinson's risk but not with Gaucher disease (E326K, P = 0.009; T369M, P < 0.001). When all patients with Parkinson's disease were considered, they had lower mean glucocerebrosidase enzymatic activity than controls (11.14 µmol/l/h versus 11.85 µmol/l/h, P = 0.011). Difference compared to controls persisted in patients with

  11. Activating Somatic FGFR2 Mutations in Breast Cancer

    PubMed Central

    Reintjes, Nadine; Li, Yun; Becker, Alexandra; Rohmann, Edyta; Schmutzler, Rita; Wollnik, Bernd

    2013-01-01

    It is known that FGFR2 gene variations confer a risk for breast cancer. FGFR2 and FGF10, the main ligand of FGFR2, are both overexpressed in 5–10% of breast tumors. In our study, we sequenced the most important coding regions of FGFR2 in somatic tumor tissue of 140 sporadic breast cancer patients and performed MLPA analysis to detect copy number variations in FGFR2 and FGF10. We identified one somatic heterozygous missense mutation, p.K660N (c.1980G>C), within the tyrosine kinase domain of FGFR2 in tumor tissue of a sporadic breast cancer patient, which is likely mediated by the FGFR2-IIIb isoform. The presence of wild type and mutated alleles in equal quantities suggests that the mutation has driven clonal amplification of mutant cells. We have analyzed the tyrosine kinase activity of p.K660N and another recently described somatic breast cancer mutation in FGFR2, p.R203C, after expression in HEK293 cells and demonstrated that the intrinsic tyrosine kinase activity of both mutant proteins is strongly increased resulting in elevated phosphorylation and activity of downstream effectors. To our knowledge, this is the first report of functional analysis of somatic breast cancer mutations in FGFR2 providing evidence for the activating nature of FGFR2-mediated signalling in the pathogenesis of breast cancer. PMID:23527311

  12. Mutational Analysis of the Putative High-Affinity Propofol Binding Site in Human β3 Homomeric GABAA Receptors

    PubMed Central

    Eaton, Megan M.; Cao, Lily Q.; Chen, Ziwei; Franks, Nicholas P.; Evers, Alex S.

    2015-01-01

    Propofol is a sedative and anesthetic agent that can both activate GABAA receptors and potentiate receptor activation elicited by submaximal concentrations of the transmitter. A recent modeling study of the β3 homomeric GABAA receptor postulated a high-affinity propofol binding site in a hydrophobic pocket in the middle of a triangular cleft lined by the M1 and M2 membrane-spanning domains of one subunit and the M2 domain of the neighboring subunit. The goal of the present study was to gain functional evidence for the involvement of this pocket in the actions of propofol. Human β3 and α1β3 receptors were expressed in Xenopus oocytes, and the effects of substitutions of selected residues were probed on channel activation by propofol and pentobarbital. The data demonstrate the vital role of the β3(Y143), β3(F221), β3(Q224), and β3(T266) residues in the actions of propofol but not pentobarbital in β3 receptors. The effects of β3(Y143W) and β3(Q224W) on activation by propofol are likely steric because propofol analogs with less bulky ortho substituents activated both wild-type and mutant receptors. The T266W mutation removed activation by propofol in β3 homomeric receptors; however, this mutation alone or in combination with a homologous mutation (I271W) in the α1 subunit had almost no effect on activation properties in α1β3 heteromeric receptors. We hypothesize that heteromeric α1β3 receptors can be activated by propofol interactions with β3–β3, α1–β3, and β3–α1 interfaces, but the exact locations of the binding site and/or nature of interactions vary in different classes of interfaces. PMID:26206487

  13. Mutational Analysis of the Putative High-Affinity Propofol Binding Site in Human β3 Homomeric GABAA Receptors.

    PubMed

    Eaton, Megan M; Cao, Lily Q; Chen, Ziwei; Franks, Nicholas P; Evers, Alex S; Akk, Gustav

    2015-10-01

    Propofol is a sedative and anesthetic agent that can both activate GABA(A) receptors and potentiate receptor activation elicited by submaximal concentrations of the transmitter. A recent modeling study of the β3 homomeric GABA(A) receptor postulated a high-affinity propofol binding site in a hydrophobic pocket in the middle of a triangular cleft lined by the M1 and M2 membrane-spanning domains of one subunit and the M2 domain of the neighboring subunit. The goal of the present study was to gain functional evidence for the involvement of this pocket in the actions of propofol. Human β3 and α1β3 receptors were expressed in Xenopus oocytes, and the effects of substitutions of selected residues were probed on channel activation by propofol and pentobarbital. The data demonstrate the vital role of the β3(Y143), β3(F221), β3(Q224), and β3(T266) residues in the actions of propofol but not pentobarbital in β3 receptors. The effects of β3(Y143W) and β3(Q224W) on activation by propofol are likely steric because propofol analogs with less bulky ortho substituents activated both wild-type and mutant receptors. The T266W mutation removed activation by propofol in β3 homomeric receptors; however, this mutation alone or in combination with a homologous mutation (I271W) in the α1 subunit had almost no effect on activation properties in α1β3 heteromeric receptors. We hypothesize that heteromeric α1β3 receptors can be activated by propofol interactions with β3-β3, α1-β3, and β3-α1 interfaces, but the exact locations of the binding site and/or nature of interactions vary in different classes of interfaces.

  14. Revealing the function of a novel splice-site mutation of CHD7 in CHARGE syndrome.

    PubMed

    Lee, Byeonghyeon; Duz, Mehmet Bugrahan; Sagong, Borum; Koparir, Asuman; Lee, Kyu-Yup; Choi, Jae Young; Seven, Mehmet; Yuksel, Adnan; Kim, Un-Kyung; Ozen, Mustafa

    2016-02-01

    Most cases of CHARGE syndrome are sporadic and autosomal dominant. CHD7 is a major causative gene of CHARGE syndrome. In this study, we screened CHD7 in two Turkish patients demonstrating symptoms of CHARGE syndrome such as coloboma, heart defect, choanal atresia, retarded growth, genital abnomalities and ear anomalies. Two mutations of CHD7 were identified including a novel splice-site mutation (c.2443-2A>G) and a previously known frameshift mutation (c.2504_2508delATCTT). We performed exon trapping analysis to determine the effect of the c.2443-2A>G mutation at the transcriptional level, and found that it caused a complete skip of exon 7 and splicing at a cryptic splice acceptor site. Our current study is the second study demonstrating an exon 7 deficit in CHD7. Results of previous studies suggest that the c.2443-2A>G mutation affects the formation of nasal tissues and the neural retina during early development, resulting in choanal atresia and coloboma, respectively. The findings of the present study will improve our understanding of the genetic causes of CHARGE syndrome.

  15. A mutational analysis of the acetylcholine receptor channel transmitter binding site.

    PubMed Central

    Akk, G; Zhou, M; Auerbach, A

    1999-01-01

    Mutagenesis and single-channel kinetic analysis were used to investigate the roles of four acetylcholine receptor channel (AChR) residues that are candidates for interacting directly with the agonist. The EC50 of the ACh dose-response curve was increased following alpha-subunit mutations Y93F and Y198F and epsilon-subunit mutations D175N and E184Q. Single-channel kinetic modeling indicates that the increase was caused mainly by a reduced gating equilibrium constant (Theta) in alphaY198F and epsilonD175N, by an increase in the equilibrium dissociation constant for ACh (KD) and a reduction in Theta in alphaY93F, and only by a reduction in KD in epsilonE184Q. This mutation altered the affinity of only one of the two binding sites and was the only mutation that reduced competition by extracellular K+. Additional mutations of epsilonE184 showed that K+ competition was unaltered in epsilonE184D and was virtually eliminated in epsilonE184K, but that neither of these mutations altered the intrinsic affinity for ACh. Thus there is an apparent electrostatic interaction between the epsilonE184 side chain and K+ ( approximately 1.7kBT), but not ACh+. The results are discussed in terms of multisite and induced-fit models of ligand binding to the AChR. PMID:9876135

  16. DNA fingerprinting reveals elevated mutation rates in herring gulls inhabiting a genotoxically contaminated site

    SciTech Connect

    Yauk, C.L.; Quinn, J.S.

    1995-12-31

    The authors used multi-locus DNA fingerprinting to examine families of herring gulls (Larus argentatus) from a genotoxically contaminated site (Hamilton Harbour) and from a pristine location (Kent Island, Bay of Fundy) to show significant differences in mutation rates between the locations. Overall the authors identified 17 mutant bands from 15 individuals of the 35 examined from Hamilton Harbour, and 7 mutant fragments from 7 individuals, of the 43 examined from Kent Island; a mutation frequency of 0.429 per nestling for Hamilton Harbour and 0.163 for Kent Island. The total number of individuals with mutant bands was significantly higher at Hamilton Harbour than at Kent Island (X{sup 2}=6.734; df = 1; P < 0.01). Ongoing analysis of other less contaminated sites also reveals lower mutation rates than those seen in Hamilton Harbour. With multi-locus DNA fingerprinting many regions of the genome can be surveyed simultaneously. The tandemly repeated arrays of nucleotides examined with DNA fingerprinting are known to have elevated rates of mutation. Furthermore, the mutations seen with DNA fingerprinting are predominantly heritable. Other biomarkers currently used in situ are not able to monitor direct and heritable DNA mutation, or measure biological endpoints that frequently result in spontaneous abortion creating difficulty in observing significantly elevated levels in viable offspring. The authors suggest that multilocus DNA fingerprinting can be used as a biomarker to identify potentially heritable risks before the onset of other types of ecological damage. This approach provides a direct measure of mutation in situ and in vivo in a vertebrate species under ambient conditions.

  17. In silico analysis of the thermodynamic stability changes of psychrophilic and mesophilic alpha-amylases upon exhaustive single-site mutations.

    PubMed

    Gilis, Dimitri

    2006-01-01

    Identifying sequence modifications that distinguish psychrophilic from mesophilic proteins is important for designing enzymes with different thermodynamic stabilities and to understand the underlying mechanisms. The PoPMuSiC algorithm is used to introduce, in silico, all the single-site mutations in four mesophilic and one psychrophilic chloride-dependent alpha-amylases and to evaluate the changes in thermodynamic stability. The analysis of the distribution of the sequence positions that could be stabilized upon mutation shows a clear difference between the three domains of psychrophilic and mesophilic alpha-amylases. Most of the mutations stabilizing the psychrophilic enzyme are found in domains B and C, contrary to the mesophilic proteins where they are preferentially situated in the catalytic domain A. Moreover, the calculations show that the environment of some residues responsible for the activity of the psychrophilic protein has evolved to reinforce favorable interactions with these residues. In the second part, these results are exploited to propose rationally designed mutations that are predicted to confer to the psychrophilic enzyme mesophilic-like thermodynamic properties. Interestingly, most of the mutations found in domain C strengthen the interactions with domain A, in agreement with suggestions made on the basis of structural analyses. Although this study focuses on single-site mutations, the thermodynamic effects of the recommended mutations should be additive if the mutated residues are not close in space.

  18. Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII

    PubMed Central

    Wei, Wei; Zheng, Chunlei; Zhu, Min; Zhu, Xiaofan; Yang, Renchi; Misra, Saurav; Zhang, Bin

    2017-01-01

    Missense mutation is the most common mutation type in hemophilia. However, the majority of missense mutations remain uncharacterized. Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain (N582) of FVIII affect protein conformation and intracellular trafficking. N582 is located in the middle of a short 310-helical turn (D580-S584), in which most amino acids have multiple hemophilia mutations. All 14 missense mutations found in this 310-helix reduced secretion levels of the A2 domain and full-length FVIII. Secreted mutants have decreased activities relative to WT FVIII. Selected mutations also lead to partial glycosylation of N582, suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII. Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant. Most of the mutants interact with the ER chaperone BiP, while only mutants with aberrant glycosylation interact with calreticulin. Our results show that the short 310-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII, and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A. PMID:28327546

  19. Mutations Outside the Anisomycin-Binding Site Can Make Ribosomes Drug-Resistant

    SciTech Connect

    Blaha,G.; Gurel, G.; Schroeder, S.; Moore, P.; Steitz, T.

    2008-01-01

    Eleven mutations that make Haloarcula marismortui resistant to anisomycin, an antibiotic that competes with the amino acid side chains of aminoacyl tRNAs for binding to the A-site cleft of the large ribosomal unit, have been identified in 23S rRNA. The correlation observed between the sensitivity of H. marismortui to anisomycin and the affinity of its large ribosomal subunits for the drug indicates that its response to anisomycin is determined primarily by the binding of the drug to its large ribosomal subunit. The structures of large ribosomal subunits containing resistance mutations show that these mutations can be divided into two classes: (1) those that interfere with specific drug-ribosome interactions and (2) those that stabilize the apo conformation of the A-site cleft of the ribosome relative to its drug-bound conformation. The conformational effects of some mutations of the second kind propagate through the ribosome for considerable distances and are reversed when A-site substrates bind to the ribosome.

  20. Whole Exome Sequencing Reveals Novel PHEX Splice Site Mutations in Patients with Hypophosphatemic Rickets

    PubMed Central

    Gillies, Christopher; Sampson, Matthew G.; Kher, Vijay; Sethi, Sidharth K.; Otto, Edgar A.

    2015-01-01

    Objective Hypophosphatemic rickets (HR) is a heterogeneous genetic phosphate wasting disorder. The disease is most commonly caused by mutations in the PHEX gene located on the X-chromosome or by mutations in CLCN5, DMP1, ENPP1, FGF23, and SLC34A3. The aims of this study were to perform molecular diagnostics for four patients with HR of Indian origin (two independent families) and to describe their clinical features. Methods We performed whole exome sequencing (WES) for the affected mother of two boys who also displayed the typical features of HR, including bone malformations and phosphate wasting. B-lymphoblast cell lines were established by EBV transformation and subsequent RT-PCR to investigate an uncommon splice site variant found by WES. An in silico analysis was done to obtain accurate nucleotide frequency occurrences of consensus splice positions other than the canonical sites of all human exons. Additionally, we applied direct Sanger sequencing for all exons and exon/intron boundaries of the PHEX gene for an affected girl from an independent second Indian family. Results WES revealed a novel PHEX splice acceptor mutation in intron 9 (c.1080-3C>A) in a family with 3 affected individuals with HR. The effect on splicing of this mutation was further investigated by RT-PCR using RNA obtained from a patient’s EBV-transformed lymphoblast cell line. RT-PCR revealed an aberrant splice transcript skipping exons 10-14 which was not observed in control samples, confirming the diagnosis of X-linked dominant hypophosphatemia (XLH). The in silico analysis of all human splice sites adjacent to all 327,293 exons across 81,814 transcripts among 20,345 human genes revealed that cytosine is, with 64.3%, the most frequent nucleobase at the minus 3 splice acceptor position, followed by thymidine with 28.7%, adenine with 6.3%, and guanine with 0.8%. We generated frequency tables and pictograms for the extended donor and acceptor splice consensus regions by analyzing all human

  1. Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites.

    PubMed Central

    Chakravarti, D; Pelling, J C; Cavalieri, E L; Rogan, E G

    1995-01-01

    Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin. PMID:7479797

  2. Effect of single-site mutations on hydrophobic-polar lattice proteins.

    PubMed

    Shi, Guangjie; Vogel, Thomas; Wüst, Thomas; Li, Ying Wai; Landau, David P

    2014-09-01

    We developed a heuristic method for determining the ground-state degeneracy of hydrophobic-polar (HP) lattice proteins, based on Wang-Landau and multicanonical sampling. It is applied during comprehensive studies of single-site mutations in specific HP proteins with different sequences. The effects in which we are interested include structural changes in ground states, changes of ground-state energy, degeneracy, and thermodynamic properties of the system. With respect to mutations, both extremely sensitive and insensitive positions in the HP sequence have been found. That is, ground-state energies and degeneracies, as well as other thermodynamic and structural quantities, may be either largely unaffected or may change significantly due to mutation.

  3. Effect of single-site mutations on hydrophobic-polar lattice proteins

    NASA Astrophysics Data System (ADS)

    Shi, Guangjie; Vogel, Thomas; Wüst, Thomas; Li, Ying Wai; Landau, David P.

    2014-09-01

    We developed a heuristic method for determining the ground-state degeneracy of hydrophobic-polar (HP) lattice proteins, based on Wang-Landau and multicanonical sampling. It is applied during comprehensive studies of single-site mutations in specific HP proteins with different sequences. The effects in which we are interested include structural changes in ground states, changes of ground-state energy, degeneracy, and thermodynamic properties of the system. With respect to mutations, both extremely sensitive and insensitive positions in the HP sequence have been found. That is, ground-state energies and degeneracies, as well as other thermodynamic and structural quantities, may be either largely unaffected or may change significantly due to mutation.

  4. A de novo splice site mutation in CASK causes FG syndrome-4 and congenital nystagmus.

    PubMed

    Dunn, P; Prigatano, G P; Szelinger, S; Roth, J; Siniard, A L; Claasen, A M; Richholt, R F; De Both, M; Corneveaux, J J; Moskowitz, A M; Balak, C; Piras, I S; Russell, M; Courtright, A L; Belnap, N; Rangasamy, S; Ramsey, K; Opitz, J M; Craig, D W; Narayanan, V; Huentelman, M J; Schrauwen, I

    2017-03-01

    Mutations in CASK cause X-linked intellectual disability, microcephaly with pontine and cerebellar hypoplasia, optic atrophy, nystagmus, feeding difficulties, GI hypomotility, and seizures. Here we present a patient with a de novo carboxyl-terminus splice site mutation in CASK (c.2521-2A>G) and clinical features of the rare FG syndrome-4 (FGS4). We provide further characterization of genotype-phenotype correlations in CASK mutations and the presentation of nystagmus and the FGS4 phenotype. There is considerable variability in clinical phenotype among patients with a CASK mutation, even among variants predicted to have similar functionality. Our patient presented with developmental delay, nystagmus, and severe gastrointestinal and gastroesophageal complications. From a cognitive and neuropsychological perspective, language skills and IQ are within normal range, although visual-motor, motor development, behavior, and working memory were impaired. The c.2521-2A>G splice mutation leads to skipping of exon 26 and a 9 base-pair deletion associated with a cryptic splice site, leading to a 28-AA and a 3-AA in-frame deletion, respectively (p.Ala841_Lys843del and p.Ala841_Glu868del). The predominant mutant transcripts contain an aberrant guanylate kinase domain and thus are predicted to degrade CASK's ability to interact with important neuronal and ocular development proteins, including FRMD7. Upregulation of CASK as well as dysregulation among a number of interactors is also evident by RNA-seq. This is the second CASK mutation known to us as cause of FGS4. © 2017 Wiley Periodicals, Inc.

  5. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  6. Distributions of selectively constrained sites and deleterious mutation rates in the hominid and murid genomes.

    PubMed

    Eory, Lél; Halligan, Daniel L; Keightley, Peter D

    2010-01-01

    Protein-coding sequences make up only about 1% of the mammalian genome. Much of the remaining 99% has been long assumed to be junk DNA, with little or no functional significance. Here, we show that in hominids, a group with historically low effective population sizes, all classes of noncoding DNA evolve more slowly than ancestral transposable elements and so appear to be subject to significant evolutionary constraints. Under the nearly neutral theory, we expected to see lower levels of selective constraints on most sequence types in hominids than murids, a group that is thought to have a higher effective population size. We found that this is the case for many sequence types examined, the most extreme example being 5'UTRs, for which constraint in hominids is only about one-third that of murids. Surprisingly, however, we observed higher constraints for some sequence types in hominids, notably 4-fold sites, where constraint is more than twice as high as in murids. This implies that more than about one-fifth of mutations at 4-fold sites are effectively selected against in hominids. The higher constraint at 4-fold sites in hominids suggests a more complex protein-coding gene structure than murids and indicates that methods for detecting selection on protein-coding sequences (e.g., using the d(N)/d(S) ratio), with 4-fold sites as a neutral standard, may lead to biased estimates, particularly in hominids. Our constraint estimates imply that 5.4% of nucleotide sites in the human genome are subject to effective negative selection and that there are three times as many constrained sites within noncoding sequences as within protein-coding sequences. Including coding and noncoding sites, we estimate that the genomic deleterious mutation rate U = 4.2. The mutational load predicted under a multiplicative model is therefore about 99% in hominids.

  7. Crystallization and preliminary crystallographic studies of human septin 1 with site-directed mutations

    SciTech Connect

    Hu, Hao; Yu, Wen-bo; Li, Shu-xing; Ding, Xiang-ming; Yu, Long; Bi, Ru-Chang

    2006-02-01

    The homogeneity of septin 1 has been improved by site-directed mutation of serine residues and only a small alteration in the secondary structure is observed to arise from the mutations. Crystals of the septin 1 mutant were grown and diffraction data were collected to 2.5 Å resolution. Septin 1 is a member of an evolutionarily conserved family of GTP-binding and filament-forming proteins named septins, which function in diverse processes including cytokinasis, vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration and neoplasia. Human septin 1 has been expressed and purified, but suffers from severe aggregation. Studies have shown that septin 1 with site-directed mutations of five serine residues (Ser19, Ser206, Ser307, Ser312 and Ser315) has a much lower degree of aggregation and better structural homogeneity and that the mutations cause only slight perturbations in the secondary structure of septin 1. This septin 1 mutant was crystallized and diffraction data were collected to 2.5 Å resolution. The space group is P422, with unit-cell parameters a = b = 106.028, c = 137.852 Å.

  8. Validated ligand mapping of ACE active site

    NASA Astrophysics Data System (ADS)

    Kuster, Daniel J.; Marshall, Garland R.

    2005-08-01

    Crystal structures of angiotensin-converting enzyme (ACE) complexed with three inhibitors (lisinopril, captopril, enalapril) provided experimental data for testing the validity of a prior active site model predicting the bound conformation of the inhibitors. The ACE active site model - predicted over 18 years ago using a series of potent ACE inhibitors of diverse chemical structure - was recreated using published data and commercial software. Comparison between the predicted structures of the three inhibitors bound to the active site of ACE and those determined experimentally yielded root mean square deviation (RMSD) values of 0.43-0.81 Å, among the distances defining the active site map. The bound conformations of the chemically relevant atoms were accurately deduced from the geometry of ligands, applying the assumption that the geometry of the active site groups responsible for binding and catalysis of amide hydrolysis was constrained. The mapping of bound inhibitors at the ACE active site was validated for known experimental compounds, so that the constrained conformational search methodology may be applied with confidence when no experimentally determined structure of the enzyme yet exists, but potent, diverse inhibitors are available.

  9. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

    PubMed Central

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity. PMID:25706379

  10. A Novel Splice-Site Mutation in the GJB2 Gene Causing Mild Postlingual Hearing Impairment

    PubMed Central

    Gandía, Marta; del Castillo, Francisco J.; Rodríguez-Álvarez, Francisco J.; Garrido, Gema; Villamar, Manuela; Calderón, Manuela; Moreno-Pelayo, Miguel A.; Moreno, Felipe; del Castillo, Ignacio

    2013-01-01

    The DFNB1 subtype of autosomal recessive, nonsyndromic hearing impairment, caused by mutations affecting the GJB2 (connection-26) gene, is highly prevalent in most populations worldwide. DFNB1 hearing impairment is mostly severe or profound and usually appears before the acquisition of speech (prelingual onset), though a small number of hypomorphic missense mutations result in mild or moderate deafness of postlingual onset. We identified a novel GJB2 splice-site mutation, c. -22-2A>C, in three siblings with mild postlingual hearing impairment that were compound heterozygous for c. -22-2A>C and c.35delG. Reverse transcriptase-PCR experiments performed on total RNA extracted from saliva samples from one of these siblings confirmed that c. -22-2A>C abolished the acceptor splice site of the single GJB2 intron, resulting in the absence of normally processed transcripts from this allele. However, we did isolate transcripts from the c. -22-2A>C allele that keep an intact GJB2 coding region and that were generated by use of an alternative acceptor splice site previously unknown. The residual expression of wild-type connection-26 encoded by these transcripts probably underlies the mild severity and late onset of the hearing impairment of these subjects. PMID:24039984

  11. Antisense suppression of donor splice site mutations in the dystrophin gene transcript

    PubMed Central

    Fletcher, Sue; Meloni, Penny L; Johnsen, Russell D; Wong, Brenda L; Muntoni, Francesco; Wilton, Stephen D

    2013-01-01

    We describe two donor splice site mutations, affecting dystrophin exons 16 and 45 that led to Duchenne muscular dystrophy (DMD), through catastrophic inactivation of the mRNA. These gene lesions unexpectedly resulted in the retention of the downstream introns, thereby increasing the length of the dystrophin mRNA by 20.2 and 36 kb, respectively. Splice-switching antisense oligomers targeted to exon 16 excised this in-frame exon and the following intron from the patient dystrophin transcript very efficiently in vitro, thereby restoring the reading frame and allowing synthesis of near-normal levels of a putatively functional dystrophin isoform. In contrast, targeting splice-switching oligomers to exon 45 in patient cells promoted only modest levels of an out-of-frame dystrophin transcript after transfection at high oligomer concentrations, whereas dual targeting of exons 44 and 45 or 45 and 46 resulted in more efficient exon skipping, with concomitant removal of intron 45. The splice site mutations reported here appear highly amenable to antisense oligomer intervention. We suggest that other splice site mutations may need to be evaluated for oligomer interventions on a case-by-case basis. PMID:24498612

  12. The Thrombopoietin Receptor: Structural Basis of Traffic and Activation by Ligand, Mutations, Agonists, and Mutated Calreticulin

    PubMed Central

    Varghese, Leila N.; Defour, Jean-Philippe; Pecquet, Christian; Constantinescu, Stefan N.

    2017-01-01

    A well-functioning hematopoietic system requires a certain robustness and flexibility to maintain appropriate quantities of functional mature blood cells, such as red blood cells and platelets. This review focuses on the cytokine receptor that plays a significant role in thrombopoiesis: the receptor for thrombopoietin (TPO-R; also known as MPL). Here, we survey the work to date to understand how this receptor functions at a molecular level throughout its lifecycle, from traffic to the cell surface, dimerization and binding cognate cytokine via its extracellular domain, through to its subsequent activation of associated Janus kinases and initiation of downstream signaling pathways, as well as the regulation of these processes. Atomic level resolution structures of TPO-R have remained elusive. The identification of disease-causing mutations in the receptor has, however, offered some insight into structure and function relationships, as has artificial means of receptor activation, through TPO mimetics, transmembrane-targeting receptor agonists, and engineering in dimerization domains. More recently, a novel activation mechanism was identified whereby mutated forms of calreticulin form complexes with TPO-R via its extracellular N-glycosylated domain. Such complexes traffic pathologically in the cell and persistently activate JAK2, downstream signal transducers and activators of transcription (STATs), and other pathways. This pathologic TPO-R activation is associated with a large fraction of human myeloproliferative neoplasms.

  13. Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma.

    PubMed

    Taylor, Kathryn R; Mackay, Alan; Truffaux, Nathalène; Butterfield, Yaron S; Morozova, Olena; Philippe, Cathy; Castel, David; Grasso, Catherine S; Vinci, Maria; Carvalho, Diana; Carcaboso, Angel M; de Torres, Carmen; Cruz, Ofelia; Mora, Jaume; Entz-Werle, Natacha; Ingram, Wendy J; Monje, Michelle; Hargrave, Darren; Bullock, Alex N; Puget, Stéphanie; Yip, Stephen; Jones, Chris; Grill, Jacques

    2014-05-01

    Diffuse intrinsic pontine gliomas (DIPGs) are highly infiltrative malignant glial neoplasms of the ventral pons that, due to their location within the brain, are unsuitable for surgical resection and consequently have a universally dismal clinical outcome. The median survival time is 9-12 months, with neither chemotherapeutic nor targeted agents showing substantial survival benefit in clinical trials in children with these tumors. We report the identification of recurrent activating mutations in the ACVR1 gene, which encodes a type I activin receptor serine/threonine kinase, in 21% of DIPG samples. Strikingly, these somatic mutations (encoding p.Arg206His, p.Arg258Gly, p.Gly328Glu, p.Gly328Val, p.Gly328Trp and p.Gly356Asp substitutions) have not been reported previously in cancer but are identical to mutations found in the germ line of individuals with the congenital childhood developmental disorder fibrodysplasia ossificans progressiva (FOP) and have been shown to constitutively activate the BMP-TGF-β signaling pathway. These mutations represent new targets for therapeutic intervention in this otherwise incurable disease.

  14. Novel splice site mutation in the growth hormone receptor gene in Turkish patients with Laron-type dwarfism.

    PubMed

    Arman, Ahmet; Ozon, Alev; Isguven, Pinar S; Coker, Ajda; Peker, Ismail; Yordam, Nursen

    2008-01-01

    Growth hormone (GH) is involved in growth, and fat and carbohydrate metabolism. Interaction of GH with the GH receptor (GHR) is necessary for systemic and local production of insulin-like growth factor-I (IGF-I) which mediates GH actions. Mutations in the GHR cause severe postnatal growth failure; the disorder is an autosomal recessive genetic disease resulting in GH insensitivity, called Laron syndrome. It is characterized by dwarfism with elevated serum GH and low levels of IGF-I. We analyzed the GHR gene for mutations and polymorphisms in eight patients with Laron-type dwarfism from six families. We found three missense mutations (S40L, V125A, I526L), one nonsense mutation (W157X), and one splice site mutation in the extracellular domain of GHR. Furthermore, G168G and exon 3 deletion polymorphisms were detected in patients with Laron syndrome. The splice site mutation, which is a novel mutation, was located at the donor splice site of exon 2/ intron 2 within GHR. Although this mutation changed the highly conserved donor splice site consensus sequence GT to GGT by insertion of a G residue, the intron splicing between exon 2 and exon 3 was detected in the patient. These results imply that the splicing occurs arthe GT site in intron 2, leaving the extra inserted G residue at the end of exon 2, thus changing the open reading frame of GHR resulting in a premature termination codon in exon 3.

  15. XPD Helicase Structures And Activities: Insights Into the Cancer And Aging Phenotypes From XPD Mutations

    SciTech Connect

    Fan, L.; Fuss, J.O.; Cheng, Q.J.; Arvai, A.S.; Hammel, M.; Roberts, V.A.; Cooper, P.K.; Tainer, J.A.

    2009-05-18

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  16. XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations

    SciTech Connect

    Tainer, John; Fan, Li; Fuss, Jill O.; Cheng, Quen J.; Arvai, Andrew S.; Hammel, Michal; Roberts, Victoria A.; Cooper, Priscilla K.; Tainer, John A.

    2008-06-02

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  17. Effect of lysine to alanine mutations on the phosphate activation and BPTES inhibition of glutaminase.

    PubMed

    McDonald, Charles J; Acheff, Eric; Kennedy, Ryan; Taylor, Lynn; Curthoys, Norman P

    2015-09-01

    The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme. These two segments also contain the binding sites for bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide (BPTES), a highly specific and potent uncompetitive inhibitor of this glutaminase. BPTES is also the lead compound for development of novel cancer chemotherapeutic agents. To provide a preliminary assessment of the potential effects of N-acetylation, the corresponding lysine to alanine mutations were constructed in the hGACΔ1 plasmid. The wild type and mutated proteins were purified by Ni(+)-affinity chromatography and their phosphate activation and BPTES inhibition profiles were analyzed. Two of the alanine substitutions in the loop segment (K311A and K328A) and the one in the dimer:dimer interface (K396A) form enzymes that require greater concentrations of phosphate to produce half-maximal activation and exhibit greater sensitivity to BPTES inhibition. By contrast, the K320A mutation results in a glutaminase that exhibits near maximal activity in the absence of phosphate and is not inhibited by BPTES. Thus, lysine N-acetylation may contribute to the acute regulation of glutaminase activity in various tissues and alter the efficacy of BPTES-type inhibitors.

  18. Four novel mutations in mucopolysaccharidosis type VII including a unique base substitution in exon 10 of the beta-glucuronidase gene that creates a novel 5'-splice site.

    PubMed

    Yamada, S; Tomatsu, S; Sly, W S; Islam, R; Wenger, D A; Fukuda, S; Sukegawa, K; Orii, T

    1995-04-01

    Mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, is a lysosomal storage disorder caused by a deficiency in the enzyme beta-glucuronidase. Various clinical phenotypes of this autosomal recessively inherited disease have been described. Recent isolation and characterization of human beta-glucuronidase cDNA and the genomic sequences facilitate analysis of molecular defects underlying the different phenotypes, and eight mutations in the beta-glucuronidase gene have been described. This report summarizes studies characterizing four new mutations in two Caucasian patients with a severe form of MPS VII. Three are point mutations, resulting in two missense and one nonsense change, and one is a 38 bp deletion. The first patient was a compound heterozygote having P148S and Y495C alleles. The second patient was a compound heterozygote of W507X and a 38 bp deletion at position 1642-1679 in exon 10(1642 delta 38nt). The 38 bp deletion was caused by a single base change mutation in exon 10 that generates a new, premature 5' splice site. Expression of mutant cDNAs encoding each of the four mutations showed that all four resulted in a severe reduction of beta-glucuronidase activity, indicating that these mutations are responsible for the reduced enzyme activity in patient cells. These four previously undescribed mutations provide further evidence for the broad molecular heterogeneity in Sly syndrome.

  19. An abnormal mRNA produced by a novel PMP22 splice site mutation associated with HNPP.

    PubMed

    Bellone, E; Balestra, P; Ribizzi, G; Schenone, A; Zocchi, G; Di Maria, E; Ajmar, F; Mandich, P

    2006-04-01

    Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant, demyelinating neuropathy. Point mutations in the PMP22 gene are a rare cause of HNPP. A novel PMP22 splice site mutation (c.179+1 G-->C) is reported in an HNPP family. By reverse transcriptase-polymerase chain reaction experiments, this mutation was shown to cause the synthesis of an abnormal mRNA in which a premature stop codon probably produces a truncated non-functional protein.

  20. Combining Natural Sequence Variation with High Throughput Mutational Data to Reveal Protein Interaction Sites

    PubMed Central

    Melamed, Daniel; Young, David L.; Miller, Christina R.; Fields, Stanley

    2015-01-01

    Many protein interactions are conserved among organisms despite changes in the amino acid sequences that comprise their contact sites, a property that has been used to infer the location of these sites from protein homology. In an inter-species complementation experiment, a sequence present in a homologue is substituted into a protein and tested for its ability to support function. Therefore, substitutions that inhibit function can identify interaction sites that changed over evolution. However, most of the sequence differences within a protein family remain unexplored because of the small-scale nature of these complementation approaches. Here we use existing high throughput mutational data on the in vivo function of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein, Pab1, to analyze its sites of interaction. Of 197 single amino acid differences in 52 Pab1 homologues, 17 reduce the function of Pab1 when substituted into the yeast protein. The majority of these deleterious mutations interfere with the binding of the RRM2 domain to eIF4G1 and eIF4G2, isoforms of a translation initiation factor. A large-scale mutational analysis of the RRM2 domain in a two-hybrid assay for eIF4G1 binding supports these findings and identifies peripheral residues that make a smaller contribution to eIF4G1 binding. Three single amino acid substitutions in yeast Pab1 corresponding to residues from the human orthologue are deleterious and eliminate binding to the yeast eIF4G isoforms. We create a triple mutant that carries these substitutions and other humanizing substitutions that collectively support a switch in binding specificity of RRM2 from the yeast eIF4G1 to its human orthologue. Finally, we map other deleterious substitutions in Pab1 to inter-domain (RRM2–RRM1) or protein-RNA (RRM2–poly(A)) interaction sites. Thus, the combined approach of large-scale mutational data and evolutionary conservation can be used to characterize interaction sites at single

  1. Mutation-selection models of coding sequence evolution with site-heterogeneous amino acid fitness profiles

    PubMed Central

    Rodrigue, Nicolas; Philippe, Hervé; Lartillot, Nicolas

    2010-01-01

    Modeling the interplay between mutation and selection at the molecular level is key to evolutionary studies. To this end, codon-based evolutionary models have been proposed as pertinent means of studying long-range evolutionary patterns and are widely used. However, these approaches have not yet consolidated results from amino acid level phylogenetic studies showing that selection acting on proteins displays strong site-specific effects, which translate into heterogeneous amino acid propensities across the columns of alignments; related codon-level studies have instead focused on either modeling a single selective context for all codon columns, or a separate selective context for each codon column, with the former strategy deemed too simplistic and the latter deemed overparameterized. Here, we integrate recent developments in nonparametric statistical approaches to propose a probabilistic model that accounts for the heterogeneity of amino acid fitness profiles across the coding positions of a gene. We apply the model to a dozen real protein-coding gene alignments and find it to produce biologically plausible inferences, for instance, as pertaining to site-specific amino acid constraints, as well as distributions of scaled selection coefficients. In their account of mutational features as well as the heterogeneous regimes of selection at the amino acid level, the modeling approaches studied here can form a backdrop for several extensions, accounting for other selective features, for variable population size, or for subtleties of mutational features, all with parameterizations couched within population-genetic theory. PMID:20176949

  2. A novel ARH splice site mutation in a Mexican kindred with autosomal recessive hypercholesterolemia.

    PubMed

    Canizales-Quinteros, Samuel; Aguilar-Salinas, Carlos A; Huertas-Vázquez, Adriana; Ordóñez-Sánchez, María L; Rodríguez-Torres, Maribel; Venturas-Gallegos, José L; Riba, Laura; Ramírez-Jimenez, Salvador; Salas-Montiel, Rocío; Medina-Palacios, Giovani; Robles-Osorio, Ludivina; Miliar-García, Angel; Rosales-León, Luis; Ruiz-Ordaz, Blanca H; Zentella-Dehesa, Alejandro; Ferré-D'Amare, Adrian; Gómez-Pérez, Francisco J; Tusié-Luna, Ma Teresa

    2005-01-01

    Autosomal recessive hypercholesterolemia (ARH) is characterized by elevated LDL serum levels, xanthomatosis, and premature coronary artery disease. Three loci have been described for this condition (1p35, 15q25-q26 and 13q). Recently, the responsible gene at the 1p35 locus, encoding an LDL receptor adaptor protein (ARH) has been identified. We studied a Mexican ARH family with two affected siblings. Sequence analysis of the ARH gene (1p35 locus) revealed that the affected siblings are homozygous for a novel mutation (IVS4+2T>G) affecting the donor splice site in intron 4, whereas both the parents and an unaffected sister are heterozygous for this mutation. The IVS4+2T>G mutation results in a major alternative transcript derived from a cryptic splice site, which carries an in-frame deletion of 78 nucleotides in the mature mRNA. The translation of this mRNA yields a mutant protein product (ARH-26) lacking 26 amino acids, resulting in the loss of beta-strands beta6 and beta7 from the PTB domain. This is the first case where a naturally occurring mutant with an altered PTB domain has been identified.

  3. Effects of the pathological Q212P mutation on human prion protein non-octarepeat copper-binding site.

    PubMed

    D'Angelo, Paola; Della Longa, Stefano; Arcovito, Alessandro; Mancini, Giordano; Zitolo, Andrea; Chillemi, Giovanni; Giachin, Gabriele; Legname, Giuseppe; Benetti, Federico

    2012-08-07

    Prion diseases are a class of fatal neurodegenerative disorders characterized by brain spongiosis, synaptic degeneration, microglia and astrocytes activation, neuronal loss and altered redox control. These maladies can be sporadic, iatrogenic and genetic. The etiological agent is the prion, a misfolded form of the cellular prion protein, PrP(C). PrP(C) interacts with metal ions, in particular copper and zinc, through the octarepeat and non-octarepeat binding sites. The physiological implication of this interaction is still unclear, as is the role of metals in the conversion. Since prion diseases present metal dyshomeostasis and increased oxidative stress, we described the copper-binding site located in the human C-terminal domain of PrP-HuPrP(90-231), both in the wild-type protein and in the protein carrying the pathological mutation Q212P. We used the synchrotron-based X-ray absorption fine structure technique to study the Cu(II) and Cu(I) coordination geometries in the mutant, and we compared them with those obtained using the wild-type protein. By analyzing the extended X-ray absorption fine structure and the X-ray absorption near-edge structure, we highlighted changes in copper coordination induced by the point mutation Q212P in both oxidation states. While in the wild-type protein the copper-binding site has the same structure for both Cu(II) and Cu(I), in the mutant the coordination site changes drastically from the oxidized to the reduced form of the copper ion. Copper-binding sites in the mutant resemble those obtained using peptides, confirming the loss of short- and long-range interactions. These changes probably cause alterations in copper homeostasis and, consequently, in redox control.

  4. Omenn syndrome associated with a functional reversion due to a somatic second-site mutation in CARD11 deficiency

    PubMed Central

    Fuchs, Sebastian; Rensing-Ehl, Anne; Pannicke, Ulrich; Lorenz, Myriam R.; Fisch, Paul; Jeelall, Yogesh; Rohr, Jan; Speckmann, Carsten; Vraetz, Thomas; Farmand, Susan; Schmitt-Graeff, Annette; Krüger, Marcus; Strahm, Brigitte; Henneke, Philipp; Enders, Anselm; Horikawa, Keisuke; Goodnow, Christopher; Schwarz, Klaus

    2015-01-01

    Omenn syndrome (OS) is a severe immunodeficiency associated with erythroderma, lymphoproliferation, elevated IgE, and hyperactive oligoclonal T cells. A restricted T-cell repertoire caused by defective thymic T-cell development and selection, lymphopenia with homeostatic proliferation, and lack of regulatory T cells are considered key factors in OS pathogenesis. We report 2 siblings presenting with cytomegalovirus (CMV) and Pneumocystis jirovecii infections and recurrent sepsis; one developed all clinical features of OS. Both carried homozygous germline mutations in CARD11 (p.Cys150*), impairing NF-κB signaling and IL-2 production. A somatic second-site mutation reverting the stop codon to a missense mutation (p.Cys150Leu) was detected in tissue-infiltrating T cells of the OS patient. Expression of p.Cys150Leu in CARD11-deficient T cells largely reconstituted NF-κB signaling. The reversion likely occurred in a prethymic T-cell precursor, leading to a chimeric T-cell repertoire. We speculate that in our patient the functional advantage of the revertant T cells in the context of persistent CMV infection, combined with lack of regulatory T cells, may have been sufficient to favor OS. This first observation of OS in a patient with a T-cell activation defect suggests that severely defective T-cell development or homeostatic proliferation in a lymphopenic environment are not required for this severe immunopathology. PMID:26289640

  5. The degree of intratumor mutational heterogeneity varies by primary tumor sub-site

    PubMed Central

    Eterovic, Agda Karina; Wick, Jo; Chen, Ken; Zhao, Hao; Tazi, Loubna; Manna, Pradip; Kerley, Spencer; Joshi, Radhika; Wang, Lin; Chiosea, Simion I.; Garnett, James David; Tsue, Terance Ted; Chien, Jeremy; Mills, Gordon B.; Grandis, Jennifer Rubin; Thomas, Sufi Mary

    2016-01-01

    In an era where mutational profiles inform treatment options, it is critical to know the extent to which tumor biopsies represent the molecular profile of the primary and metastatic tumor. Head and neck squamous cell carcinoma (HNSCC) arise primarily in the mucosal lining of oral cavity and oropharynx. Despite aggressive therapy the 5-year survival rate is at 50%. The primary objective of this study is to characterize the degree of intratumor mutational heterogeneity in HNSCC. We used multi-region sequencing of paired primary and metastatic tumor DNA of 24 spatially distinct samples from seven patients with HNSCC of larynx, floor of the mouth (FOM) or oral tongue. Full length, in-depth sequencing of 202 genes implicated in cancer was carried out. Larynx and FOM tumors had more than 69.2% unique SNVs between the paired primary and metastatic lesions. In contrast, the oral tongue HNSCC had only 33.3% unique SNVs across multiple sites. In addition, HNSCC of the oral tongue had fewer mutations than larynx and FOM tumors. These findings were validated on the Affymetrix whole genome 6.0 array platform and were consistent with data from The Cancer Genome Atlas (TCGA). This is the first report demonstrating differences in mutational heterogeneity varying by subsite in HNSCC. The heterogeneity within laryngeal tumor specimens may lead to an underestimation of the genetic abnormalities within tumors and may foster resistance to standard treatment protocols. These findings are relevant to investigators and clinicians developing personalized cancer treatments based on identification of specific mutations in tumor biopsies. PMID:27034009

  6. Mosaic Activating Mutations in FGFR1 Cause Encephalocraniocutaneous Lipomatosis

    PubMed Central

    Bennett, James T.; Tan, Tiong Yang; Alcantara, Diana; Tétrault, Martine; Timms, Andrew E.; Jensen, Dana; Collins, Sarah; Nowaczyk, Malgorzata J.M.; Lindhurst, Marjorie J.; Christensen, Katherine M.; Braddock, Stephen R.; Brandling-Bennett, Heather; Hennekam, Raoul C.M.; Chung, Brian; Lehman, Anna; Su, John; Ng, SuYuen; Amor, David J.; Majewski, Jacek; Biesecker, Les G.; Boycott, Kym M.; Dobyns, William B.; O’Driscoll, Mark; Moog, Ute; McDonell, Laura M.

    2016-01-01

    Encephalocraniocutaneous lipomatosis (ECCL) is a sporadic condition characterized by ocular, cutaneous, and central nervous system anomalies. Key clinical features include a well-demarcated hairless fatty nevus on the scalp, benign ocular tumors, and central nervous system lipomas. Seizures, spasticity, and intellectual disability can be present, although affected individuals without seizures and with normal intellect have also been reported. Given the patchy and asymmetric nature of the malformations, ECCL has been hypothesized to be due to a post-zygotic, mosaic mutation. Despite phenotypic overlap with several other disorders associated with mutations in the RAS-MAPK and PI3K-AKT pathways, the molecular etiology of ECCL remains unknown. Using exome sequencing of DNA from multiple affected tissues from five unrelated individuals with ECCL, we identified two mosaic mutations, c.1638C>A (p.Asn546Lys) and c.1966A>G (p.Lys656Glu) within the tyrosine kinase domain of FGFR1, in two affected individuals each. These two residues are the most commonly mutated residues in FGFR1 in human cancers and are associated primarily with CNS tumors. Targeted resequencing of FGFR1 in multiple tissues from an independent cohort of individuals with ECCL identified one additional individual with a c.1638C>A (p.Asn546Lys) mutation in FGFR1. Functional studies of ECCL fibroblast cell lines show increased levels of phosphorylated FGFRs and phosphorylated FRS2, a direct substrate of FGFR1, as well as constitutive activation of RAS-MAPK signaling. In addition to identifying the molecular etiology of ECCL, our results support the emerging overlap between mosaic developmental disorders and tumorigenesis. PMID:26942290

  7. Loss-of-function mutation in the X-linked TBX22 promoter disrupts an ETS-1 binding site and leads to cleft palate.

    PubMed

    Fu, Xiazhou; Cheng, Yibin; Yuan, Jia; Huang, Chunhua; Cheng, Hanhua; Zhou, Rongjia

    2015-02-01

    The cleft palate only (CPO) is a common congenital defect with complex etiology in humans. The molecular etiology of the CPO remains unknown. Here, we report a loss-of-function mutation in X-linked TBX22 gene (T-box 22) in a six-generation family of the CPO with obvious phenotypes of both cleft palate and hyper-nasal speech. We identify a functional -73G>A mutation in the promoter of TBX22, which is located at the core-binding site of transcription factor ETS-1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1). Phylogenetic analysis showed that the sequence around the -73G>A mutation site is specific in primates. The mutation was detected in all five affected male members cosegregating with the affected phenotype and heterozygote occurred only in some unaffected females of the family, suggesting an X-linked transmission of the mutation in the family. The -73G>A variant is a novel single nucleotide mutation. Cell co-transfections indicated that ETS-1 could activate the TBX22 promoter. Moreover, EMSA and ChIP assays demonstrated that the allele A disrupts the binding site of ETS-1, thus markedly decreases the activity of the TBX22 promoter, which is likely to lead to the birth defect of the CPO without ankyloglossia. These results suggest that a loss-of-function mutation in the X-linked TBX22 promoter may cause the cleft palate through disruption of TBX22-ETS-1 pathway.

  8. NMR and Mutational Identification of the Collagen-Binding Site of the Chaperone Hsp47

    PubMed Central

    Yagi-Utsumi, Maho; Yoshikawa, Sumi; Yamaguchi, Yoshiki; Nishi, Yohei; Kurimoto, Eiji; Ishida, Yoshihito; Homma, Takayuki; Hoseki, Jun; Nishikawa, Yoshimi; Koide, Takaki; Nagata, Kazuhiro; Kato, Koichi

    2012-01-01

    Heat shock protein 47 (Hsp47) acts as a client-specific chaperone for collagen and plays a vital role in collagen maturation and the consequent embryonic development. In addition, this protein can be a potential target for the treatment of fibrosis. Despite its physiological and pathological importance, little is currently known about the collagen-binding mode of Hsp47 from a structural aspect. Here, we describe an NMR study that was conducted to identify the collagen-binding site of Hsp47. We used chicken Hsp47, which has higher solubility than its human counterpart, and applied a selective 15N-labeling method targeting its tryptophan and histidine residues. Spectral assignments were made based on site-directed mutagenesis of the individual residues. By inspecting the spectral changes that were observed upon interaction with a trimeric collagen peptide and the mutational data, we successfully mapped the collagen-binding site in the B/C β-barrel domain and a nearby loop in a 3D-homology model based upon a serpin fold. This conclusion was confirmed by mutational analysis. Our findings provide a molecular basis for the design of compounds that target the interaction between Hsp47 and procollagen as therapeutics for fibrotic diseases. PMID:23049894

  9. A novel donor splice-site mutation of major intrinsic protein gene associated with congenital cataract in a Chinese family

    PubMed Central

    Zeng, Lu; Liu, Wenqiang; Feng, Wenguo; Wang, Xing; Dang, Hui; Gao, Luna; Yao, Jing

    2013-01-01

    Purpose To identify the disease-causing gene in a Chinese family with autosomal dominant congenital cataract. Methods Clinical and ophthalmologic examinations were performed on all members of a Chinese family with congenital cataract. Nine genes associated with congenital cataract were screened using direct DNA sequencing. Mutations were confirmed using restriction fragment length polymorphism (RFLP) analysis. The mutated major intrinsic protein (MIP) minigene, which carries the disease-causing splice-site mutation, and the wild-type (WT) MIP minigene were constructed using the pcDNA3.1 expression vector. Wild-type and mutant MIP minigene constructs were transiently transfected into HeLa cells. After 48 h of incubation at 37 °C, total RNA isolation and reverse transcription (RT)–PCR analysis were performed, and PCR products were separated and confirmed with sequencing. Results Direct DNA sequence analysis identified a novel splice-site mutation in intron 3 (c.606+1 G>A) of the MIP gene. To investigate the manner in which the splice donor mutation could affect mRNA splicing, WT and mutant MIP minigenes were inserted in the pcDNA3.1 (+) vector. Constructs were transfected into HeLa cells. RT–PCR analysis showed that the donor splice site mutation led to deletion of exon 3 in the mRNA encoded by the MIP gene. Conclusions The present study identified a novel donor splice-site mutation (c.606+1G>A) in the MIP gene in a Chinese family with congenital cataract. In vitro RT–PCR analysis showed that this splice-site mutation resulted in the deletion of exon 3 from mRNA encoded by the MIP gene. This is the first report to show that donor splice-site mutation in MIP gene can cause autosomal dominant congenital cataract. PMID:24319327

  10. Three faces of recombination activating gene 1 (RAG1) mutations.

    PubMed

    Patiroglu, Turkan; Akar, Himmet Haluk; Van Der Burg, Mirjam

    2015-12-01

    Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation.

  11. [Mechanism of arginine deiminase activity by site-directed mutagenesis].

    PubMed

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

  12. LYN-activating mutations mediate antiestrogen resistance in estrogen receptor–positive breast cancer

    PubMed Central

    Schwarz, Luis J.; Fox, Emily M.; Balko, Justin M.; Garrett, Joan T.; Kuba, María Gabriela; Estrada, Mónica Valeria; González-Angulo, Ana María; Mills, Gordon B.; Red-Brewer, Monica; Mayer, Ingrid A.; Abramson, Vandana; Rizzo, Monica; Kelley, Mark C.; Meszoely, Ingrid M.; Arteaga, Carlos L.

    2014-01-01

    Estrogen receptor–positive (ER+) breast cancers adapt to hormone deprivation and become resistant to antiestrogen therapy. Here, we performed deep sequencing on ER+ tumors that remained highly proliferative after treatment with the aromatase inhibitor letrozole and identified a D189Y mutation in the inhibitory SH2 domain of the SRC family kinase (SFK) LYN. Evaluation of 463 breast tumors in The Cancer Genome Atlas revealed four LYN mutations, two of which affected the SH2 domain. In addition, LYN was upregulated in multiple ER+ breast cancer lines resistant to long-term estrogen deprivation (LTED). An RNAi-based kinome screen revealed that LYN is required for growth of ER+ LTED breast cancer cells. Kinase assays and immunoblot analyses of SRC substrates in transfected cells indicated that LYND189Y has higher catalytic activity than WT protein. Further, LYND189Y exhibited reduced phosphorylation at the inhibitory Y507 site compared with LYNWT. Other SH2 domain LYN mutants, E159K and K209N, also exhibited higher catalytic activity and reduced inhibitory site phosphorylation. LYND189Y overexpression abrogated growth inhibition by fulvestrant and/or the PI3K inhibitor BKM120 in 3 ER+ breast cancer cell lines. The SFK inhibitor dasatinib enhanced the antitumor effect of BKM120 and fulvestrant against estrogen-deprived ER+ xenografts but not LYND189Y-expressing xenografts. These results suggest that LYN mutations mediate escape from antiestrogens in a subset of ER+ breast cancers. PMID:25401474

  13. Disease Mutations in Rab7 Result in Unregulated Nucleotide Exchange and Inappropriate Activation

    SciTech Connect

    B McCray; E Skordalakes; J Taylor

    2011-12-31

    Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 A crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.

  14. Signal transducer and activator of transcription 3 mutation with invasive eosinophilic disease

    PubMed Central

    Swender, David; Chernin, Leah; Hafez-Khayyata, Said; Ochs, Hans; Tcheurekdjian, Haig; Hostoffer, Robert

    2012-01-01

    Hyper-IgE syndrome (HIES), or Jobs disease, is a rare immunologic disorder characterized by the triad of staphylococcal abscesses, pneumonia with pneumatocele formation, and elevated IgE. It has been shown to have multiple modes of inheritance, autosomal dominant being more common than autosomal recessive, with sporadic cases as well. A mutation in signal transducer and activator of transcription 3 (STAT3) gene has been linked to the development of the sporadic and dominant forms of HIES. Peripheral eosinophilia, typically greater than two standard deviations from the normal population, is often seen in association with HIES. Despite these elevated levels of blood eosinophils, there have been no reported cases of invasive eosinophilic disease, such as eosonophilic esophagitic. Here we report the first description, to our knowledge, of a patient with HIES with a STAT3 mutation involving exon 12, Thr389Ile, and invasive eosinophilic disease of the esophagus. STAT3 modulates the expression of several genes that control central cell processes such as growth and death in response to external soluble stimuli. A mutation in the STAT3 molecule may affect the eosinophil's response to IL-5 and thus reduce the chemotaxic ability of those cells to migrate into tissues. This may then explain the paucity of eosinophilic infiltrative disease in patients with STAT3 mutations. The level of eosinophilic involvement may be related to the site or type of mutation within the STAT3 molecule. As more data are collected, we may be able to assess whether certain mutations dictate different clinical outcomes, which could prove helpful in directing therapy. PMID:23342295

  15. Exon 10 skipping in ACAT1 caused by a novel c.949G>A mutation located at an exonic splice enhancer site.

    PubMed

    Otsuka, Hiroki; Sasai, Hideo; Nakama, Mina; Aoyama, Yuka; Abdelkreem, Elsayed; Ohnishi, Hidenori; Konstantopoulou, Vassiliki; Sass, Jörn Oliver; Fukao, Toshiyuki

    2016-11-01

    Beta-ketothiolase deficiency, also known as mitochondrial acetoacetyl-CoA thiolase (T2) deficiency, is an autosomal recessive disease caused by mutations in the acetyl‑CoA acetyltransferase 1 (ACAT1) gene. A German T2‑deficient patient that developed a severe ketoacidotic episode at the age of 11 months, was revealed to be a compound heterozygote of a previously reported null mutation, c.472A>G (p.N158D) and a novel mutation, c.949G>A (p.D317N), in ACAT1. The c.949G>A mutation was suspected to cause aberrant splicing as it is located within an exonic splicing enhancer sequence (c. 947CTGACGC) that is a potential binding site for serine/arginine‑rich splicing factor 1. A mutation in this sequence, c.951C>T, results in exon 10 skipping. A minigene construct was synthesized that included exon 9‑truncated intron 9‑exon 10‑truncated intron 10‑exon 11, and the splicing of this minigene revealed that the c.949G>A mutant construct caused exon 10 skipping in a proportion of the transcripts. Furthermore, additional substitution of G for C at the first nucleotide of exon 10 (c.941G>C) abolished the effect of the c.949G>A mutation. Transient expression analysis of the c.949G>A mutant cDNA revealed no residual T2 activity in the mutated D317N enzyme. Therefore, c.949G>A (D317N) is a pathogenic missense mutation, and diminishes the effect of an exonic splicing enhancer and causes exon 10 skipping. The present study demonstrates that a missense mutation, or even a synonymous substitution, may disrupt enzyme function by interference with splicing.

  16. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  17. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  18. Mutations within potential glycosylation sites in the capsid protein of hepatitis E virus prevent the formation of infectious virus particles.

    PubMed

    Graff, Judith; Zhou, Yi-Hua; Torian, Udana; Nguyen, Hanh; St Claire, Marisa; Yu, Claro; Purcell, Robert H; Emerson, Suzanne U

    2008-02-01

    Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.

  19. Dynamics and Mechanism of Efficient DNA Repair Reviewed by Active-Site Mutants

    NASA Astrophysics Data System (ADS)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

    2010-06-01

    Photolyases repair the UV-induced pyrimidine dimers in damage DNA via a photoreaction which includes a series of light-driven electron transfers between the two-electron-reduced flavin cofactor FADH^- and the dimer. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of several active-site residues. With femtosecond resolution, we observed the significant change in the forward electron transfer from the excited FADH^- to the dimer and the back electron transfer from the repaired thymines by mutation of E274A, R226A, R342A, N378S and N378C. We also found that the mutation of E274A accelerates the bond-breaking of the thymine dimer. The dynamics changes are consistent with the quantum yield study of these mutants. These results suggest that the active-site residues play a significant role, structurally and chemically, in the DNA repair photocycle.

  20. MSK1 activity is controlled by multiple phosphorylation sites

    PubMed Central

    McCOY, Claire E.; Campbell, David G.; Deak, Maria; Bloomberg, Graham B.; Arthur, J. Simon C.

    2004-01-01

    MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2. PMID:15568999

  1. A gripping tale of ribosomal frameshifting: extragenic suppressors of frameshift mutations spotlight P-site realignment.

    PubMed

    Atkins, John F; Björk, Glenn R

    2009-03-01

    Mutants of translation components which compensate for both -1 and +1 frameshift mutations showed the first evidence for framing malleability. Those compensatory mutants isolated in bacteria and yeast with altered tRNA or protein factors are reviewed here and are considered to primarily cause altered P-site realignment and not altered translocation. Though the first sequenced tRNA mutant which suppressed a +1 frameshift mutation had an extra base in its anticodon loop and led to a textbook "yardstick" model in which the number of anticodon bases determines codon size, this model has long been discounted, although not by all. Accordingly, the reviewed data suggest that reading frame maintenance and translocation are two distinct features of the ribosome. None of the -1 tRNA suppressors have anticodon loops with fewer than the standard seven nucleotides. Many of the tRNA mutants potentially affect tRNA bending and/or stability and can be used for functional assays, and one has the conserved C74 of the 3' CCA substituted. The effect of tRNA modification deficiencies on framing has been particularly informative. The properties of some mutants suggest the use of alternative tRNA anticodon loop stack conformations by individual tRNAs in one translation cycle. The mutant proteins range from defective release factors with delayed decoding of A-site stop codons facilitating P-site frameshifting to altered EF-Tu/EF1alpha to mutant ribosomal large- and small-subunit proteins L9 and S9. Their study is revealing how mRNA slippage is restrained except where it is programmed to occur and be utilized.

  2. Mutations in the p53 and Ki-ras genes, microsatellite instability and site of tumor origin in colorectal cancer.

    PubMed

    Catalano, Teresa; Curia, Maria Cristina; Aceto, Gitana; Verginelli, Fabio; Cascinu, Stefano; Cama, Alessandro; Mariani-Costantini, Renato; Teti, Diana; Battista, Pasquale

    2005-09-01

    Using PCR-SSCP screening and direct sequencing we analyzed a series of 28 colorectal carcinomas for mutations in p53 (exons 5-8) and Ki-ras (codons 12, 13 and 61), and for micro-satellite instability (MSI) at BAT25 and BAT26, supplementing data with the analysis of the IARC colorectal cancer p53 mutation database. Mutations were correlated with the site of tumor origin (proximal or distal to the splenic flexure). We identified 19 mutations in p53, 9 in Ki-ras, and 4 MSI-positive cases in a total of 20 tumors. Only 6/20 cases (30%) carried mutations in both p53 and Ki-ras. Mutations in p53 were detected at similar frequencies in proximal and distal tumors, while IARC data pointed to a strong association of p53 mutations with distal cancers. Ki-ras mutations were more frequent in proximal tumors, and MSI occurred at similar frequencies in proximal and distal tumors and was associated with mutations in p53 or Ki-ras. The p53 mutations detected in the series analyzed, as well as those retrieved from the IARC database, were predominantly transitions, with no preferential sequence localization or nucleotide position. Ki-ras mutations were predominantly transversions in position 2 at codon 12. MSI-H occurred at similar frequencies in proximal and distal tumors and was associated with either p53 or Ki-ras mutations. Overall these data suggest that distinct mutagenic factors target p53 and Ki-ras in colorectal epithelium irrespective of MSI-H status.

  3. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation

    PubMed Central

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease. PMID:27570485

  4. Investigating the Role of Loop C Hydrophilic Residue ‘T244’ in the Binding Site of ρ1 GABAC Receptors via Site Mutation and Partial Agonism

    PubMed Central

    Naffaa, Moawiah M.; Absalom, Nathan; Solomon, V. Raja; Chebib, Mary; Hibbs, David E.; Hanrahan, Jane R.

    2016-01-01

    The loop C hydrophilic residue, threonine 244 lines the orthosteric binding site of ρ1 GABAC receptors was studied by point mutation into serine, alanine and cysteine, and tested with GABA, some representative partial agonists and antagonists. Thr244 has a hydroxyl group essential for GABA activity that is constrained by the threonine methyl group, orienting it toward the binding site. Significant decreases in activation effects of the studied ligands at ρ1 T244S mutant receptors, suggests a critical role for this residue. Results of aliphatic and heteroaromatic partial agonists demonstrate different pharmacological effects at ρ1 T244S mutant receptors when co-applied with GABA EC50 responses. ρ1 T244A and ρ1 T244C mutant receptors have minimal sensitivity to GABA at high mM concentrations, whereas, the ρ1 WT partial agonists, β-alanine and MTSEA demonstrate more efficacy and potency, respectively, than GABA at these mutant receptors. This study explores the role of Thr244 in the binding of agonists as an initial step during channel gating by moving loop C towards the ligand. PMID:27244450

  5. Structural characterization of single nucleotide variants at ligand binding sites and enzyme active sites of human proteins

    PubMed Central

    Yamada, Kazunori D.; Nishi, Hafumi; Nakata, Junichi; Kinoshita, Kengo

    2016-01-01

    Functional sites on proteins play an important role in various molecular interactions and reactions between proteins and other molecules. Thus, mutations in functional sites can severely affect the overall phenotype. Progress of genome sequencing projects has yielded a wealth of information on single nucleotide variants (SNVs), especially those with less than 1% minor allele frequency (rare variants). To understand the functional influence of genetic variants at a protein level, we investigated the relationship between SNVs and protein functional sites in terms of minor allele frequency and the structural position of variants. As a result, we observed that SNVs were less abundant at ligand binding sites, which is consistent with a previous study on SNVs and protein interaction sites. Additionally, we found that non-rare variants tended to be located slightly apart from enzyme active sites. Examination of non-rare variants revealed that most of the mutations resulted in moderate changes of the physico-chemical properties of amino acids, suggesting the existence of functional constraints. In conclusion, this study shows that the mapping of genetic variants on protein structures could be a powerful approach to evaluate the functional impact of rare genetic variations. PMID:27924270

  6. Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel.

    PubMed

    Tobelaim, William Sam; Dvir, Meidan; Lebel, Guy; Cui, Meng; Buki, Tal; Peretz, Asher; Marom, Milit; Haitin, Yoni; Logothetis, Diomedes E; Hirsch, Joel Alan; Attali, Bernard

    2017-01-31

    Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current IKS that repolarizes the cardiac action potential. The physiological importance of the IKS channel is underscored by the existence of mutations in human Kv7.1 and KCNE1 genes, which cause cardiac arrhythmias, such as the long-QT syndrome (LQT) and atrial fibrillation. The proximal Kv7.1 C terminus (CT) binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2), but the role of CaM in channel function is still unclear, and its possible interaction with PIP2 is unknown. Our recent crystallographic study showed that CaM embraces helices A and B with the apo C lobe and calcified N lobe, respectively. Here, we reveal the competition of PIP2 and the calcified CaM N lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor an LQT mutation. Protein pulldown, molecular docking, molecular dynamics simulations, and patch-clamp recordings indicate that residues K526 and K527 in Kv7.1 helix B form a critical site where CaM competes with PIP2 to stabilize the channel open state. Data indicate that both PIP2 and Ca(2+)-CaM perform the same function on IKS channel gating by producing a left shift in the voltage dependence of activation. The LQT mutant K526E revealed a severely impaired channel function with a right shift in the voltage dependence of activation, a reduced current density, and insensitivity to gating modulation by Ca(2+)-CaM. The results suggest that, after receptor-mediated PIP2 depletion and increased cytosolic Ca(2+), calcified CaM N lobe interacts with helix B in place of PIP2 to limit excessive IKS current inhibition.

  7. Mutations in the BRCT binding site of BRCA1 result in hyper-recombination.

    PubMed

    Dever, Seth M; Golding, Sarah E; Rosenberg, Elizabeth; Adams, Bret R; Idowu, Michael O; Quillin, John M; Valerie, Nicholas; Xu, Bo; Povirk, Lawrence F; Valerie, Kristoffer

    2011-05-01

    We introduced a K1702M mutation in the BRCA1 BRCT domain known to prevent the binding of proteins harboring pS-X-X-F motifs such as Abraxas-RAP80, BRIP1, and CtIP. Surprisingly, rather than impairing homologous recombination repair (HRR), expression of K1702M resulted in hyper-recombination coinciding with an accumulation of cells in S-G2 and no effect on nonhomologous end-joining. These cells also showed increased RAD51 and RPA nuclear staining. More pronounced effects were seen with a naturally occurring BRCT mutant (M1775R) that also produced elevated levels of ssDNA, in part co-localizing with RPA, in line with excessive DNA resection. M1775R induced unusual, thread-like promyelocytic leukemia (PML) nuclear bodies and clustered RPA foci rather than the typical juxtaposed RPA-PML foci seen with wild-type BRCA1. Interestingly, K1702M hyper-recombination diminished with a second mutation in the BRCA1 RING domain (I26A) known to reduce BRCA1 ubiquitin-ligase activity. Thesein vitro findings correlated with elevated nuclear RAD51 and RPA staining of breast cancer tissue from a patient with the M1775R mutation. Altogether, the disruption of BRCA1 (BRCT)-pS-X-X-F protein binding results in ubiquitination-dependent hyper-recombination via excessive DNA resection and the appearance of atypical PML-NBs. Thus, certain BRCA1 mutations that cause hyper-recombination instead of reduced DSB repair might lead to breast cancer.

  8. Novel splice-site mutation in TTLL5 causes cone dystrophy in a consanguineous family

    PubMed Central

    Dias, Miguel de Sousa; Hamel, Christian P.; Meunier, Isabelle; Varin, Juliette; Blanchard, Steven; Boyard, Fiona; Sahel, José-Alain

    2017-01-01

    Purpose To report the clinical and genetic findings of one family with autosomal recessive cone dystrophy (CD) and to identify the causative mutation. Methods An institutional study of three family members from two generations. The clinical examination included best-corrected Snellen visual acuity measurement, fundoscopy, the Farnsworth D-15 color vision test, a full-field electroretinogram (ERG) that incorporated the International Society for Clinical Electrophysiology of Vision standards and methodology, fundus autofluorescence (FAF) and infrared (IR), and spectral-domain optical coherence tomography (SD-OCT). Genetic findings were achieved with DNA analysis using whole exome sequencing (WES) and Sanger sequencing. Results The proband, a 9-year-old boy, presented with a condition that appeared to be congenital and stationary. The clinical presentation initially reflected incomplete congenital stationary night blindness (icCSNB) because of myopia, a decrease in visual acuity, abnormal oscillatory potentials, and reduced amplitudes on the 30 Hz flicker ERG but was atypical because there were no clear electronegative responses. However, no disease-causing mutations in the genes underlying icCSNB were identified. Following WES analysis of family members, a homozygous splice-site mutation in intron 3 of TTLL5 (c.182–3_182–1delinsAA) was found cosegregating within the phenotype in the family. Conclusions The distinction between icCSNB and CD phenotypes is not always straightforward in young patients. The patient was quite young, which most likely explains why the progression of the CD was not obvious. WES analysis provided prompt diagnosis for this family; thus, the use of this technique to refine the diagnosis is highlighted in this study. PMID:28356705

  9. Characterization of Active Site Residues of Nitroalkane Oxidase†

    PubMed Central

    Valley, Michael P.; Fenny, Nana S.; Ali, Shah R.; Fitzpatrick, Paul F.

    2010-01-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitrolkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Serl71 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by ~5-fold and decreases in the rate constant for product release of ~2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  10. Identification of MGMT promoter methylation sites correlating with gene expression and IDH1 mutation in gliomas.

    PubMed

    Zhang, Jie; Yang, Jian-Hui; Quan, Jia; Kang, Xing; Wang, Hui-Juan; Dai, Peng-Gao

    2016-10-01

    O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation was reported to be an independent prognostic and predictive factor in glioma patients who received temozolomide treatment. However, the predictive value of MGMT methylation was recently questioned by several large clinical studies. The purpose of this study is to identify MGMT gene promoter CpG sites or region whose methylation were closely correlated with its gene expression to elucidate this contradictory clinical observations. The methylation status for all CpG dinucleotides in MGMT promoter and first exon region were determined in 42 Chinese glioma patients, which were then correlated with MGMT gene expression, IDH1 mutation, and tumor grade. In whole 87 CpG dinucleotides analyzed, three distinct CpG regions covering 28 CpG dinucleotides were significantly correlated with MGMT gene expression; 10 CpG dinucleotides were significantly correlated with glioma classification (p < 0.05). Isocitrate dehydrogenase 1 (IDH1) mutation and MGMT gene hypermethylation significantly co-existed, but not for MGMT gene expression. The validation cohort of gliomas treated with standard of care and comparison of the CpGs we identified with the current CpGs used in clinical setting will be very important for gliomas individual medicine in the future.

  11. Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

    PubMed

    Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed.

  12. Prenatal diagnosis and a donor splice site mutation in fibrillin in a family with Marfan syndrome

    SciTech Connect

    Godfrey, M.; Vandemark, N.; Wang, M.; Han, J.; Rao, V.H. ); Velinov, M.; Tsipouras, P. ); Wargowski, D.; Becker, J.; Robertson, W.; Droste, S. )

    1993-08-01

    The Marfan syndrome, an autosomal dominant connective tissue disorder, is manifested by abnormalities in the cardiovascular, skeletal, and ocular systems. Recently, fibrillin, an elastic-associated microfibrillar glycoprotein, has been linked to the Marfan syndrome, and fibrillin mutations in affected individuals have been documented. In this study, genetic linkage analysis with fibrillin-specific markers was used to establish the prenatal diagnosis in an 11-wk-gestation fetus in a four-generation Marfan kindred. At birth, skeletal changes suggestive of the Marfan syndrome were observed. Reverse transcription-PCR amplification of the fibrillin gene mRNA detected a deletion of 123 bp in one allele in affected relatives. This deletion corresponds to an exon encoding an epidermal growth factor-like motif. Examination of genomic DNA showed a G[yields]C transversion at the +1 consensus donor splice site. 45 refs., 7 figs.

  13. Generation of rodent malaria parasites with a high mutation rate by destructing proofreading activity of DNA polymerase δ.

    PubMed

    Honma, Hajime; Hirai, Makoto; Nakamura, Shota; Hakimi, Hassan; Kawazu, Shin-Ichiro; Palacpac, Nirianne M Q; Hisaeda, Hajime; Matsuoka, Hiroyuki; Kawai, Satoru; Endo, Hiroyoshi; Yasunaga, Teruo; Ohashi, Jun; Mita, Toshihiro; Horii, Toshihiro; Furusawa, Mitsuru; Tanabe, Kazuyuki

    2014-08-01

    Plasmodium falciparum malaria imposes a serious public health concern throughout the tropics. Although genetic tools are principally important to fully investigate malaria parasites, currently available forward and reverse tools are fairly limited. It is expected that parasites with a high mutation rate can readily acquire novel phenotypes/traits; however, they remain an untapped tool for malaria biology. Here, we generated a mutator malaria parasite (hereinafter called a 'malaria mutator'), using site-directed mutagenesis and gene transfection techniques. A mutator Plasmodium berghei line with a defective proofreading 3' → 5' exonuclease activity in DNA polymerase δ (referred to as PbMut) and a control P. berghei line with wild-type DNA polymerase δ (referred to as PbCtl) were maintained by weekly passage in ddY mice for 122 weeks. High-throughput genome sequencing analysis revealed that two PbMut lines had 175-178 mutations and a 86- to 90-fold higher mutation rate than that of a PbCtl line. PbMut, PbCtl, and their parent strain, PbWT, showed similar course of infection. Interestingly, PbMut lost the ability to form gametocytes during serial passages. We believe that the malaria mutator system could provide a novel and useful tool to investigate malaria biology.

  14. Development and validation of a clinical trial patient stratification assay that interrogates 27 mutation sites in MAPK pathway genes.

    PubMed

    Chang, Ken C N; Galuska, Stefan; Weiner, Russell; Marton, Matthew J

    2013-01-01

    Somatic mutations identified on genes related to the cancer-developing signaling pathways have drawn attention in the field of personalized medicine in recent years. Treatments developed to target a specific signaling pathway may not be effective when tumor activating mutations occur downstream of the target and bypass the targeted mechanism. For instance, mutations detected in KRAS/BRAF/NRAS genes can lead to EGFR-independent intracellular signaling pathway activation. Most patients with these mutations do not respond well to anti-EGFR treatment. In an effort to detect various mutations in FFPE tissue samples among multiple solid tumor types for patient stratification many mutation assays were evaluated. Since there were more than 30 specific mutations among three targeted RAS/RAF oncogenes that could activate MAPK pathway genes, a custom designed Single Nucleotide Primer Extension (SNPE) multiplexing mutation assay was developed and analytically validated as a clinical trial assay. Throughout the process of developing and validating the assay we overcame many technical challenges which include: the designing of PCR primers for FFPE tumor tissue samples versus normal blood samples, designing of probes for detecting consecutive nucleotide double mutations, the kinetics and thermodynamics aspects of probes competition among themselves and against target PCR templates, as well as validating an assay when positive control tumor tissue or cell lines with specific mutations are not available. We used Next Generation sequencing to resolve discordant calls between the SNPE mutation assay and Sanger sequencing. We also applied a triplicate rule to reduce potential false positives and false negatives, and proposed special considerations including pre-define a cut-off percentage for detecting very low mutant copies in the wild-type DNA background.

  15. Mutational Studies on Resurrected Ancestral Proteins Reveal Conservation of Site-Specific Amino Acid Preferences throughout Evolutionary History

    PubMed Central

    Risso, Valeria A.; Manssour-Triedo, Fadia; Delgado-Delgado, Asunción; Arco, Rocio; Barroso-delJesus, Alicia; Ingles-Prieto, Alvaro; Godoy-Ruiz, Raquel; Gavira, Jose A.; Gaucher, Eric A.; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2015-01-01

    Local protein interactions (“molecular context” effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations. PMID:25392342

  16. Wobble Pairs of the HDV Ribozyme Play Specific Roles in Stabilization of Active Site Dynamics

    PubMed Central

    Sripathi, Kamali N.; Banáš, Pavel; Reblova, Kamila; Šponer, Jiři; Otyepka, Michal

    2015-01-01

    The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5′) hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5′) general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5′) hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs. PMID:25631765

  17. Effects of point mutation on enzymatic activity: correlation between protein electronic structure and motion in chorismate mutase reaction.

    PubMed

    Ishida, Toyokazu

    2010-05-26

    Assignment of particular roles to catalytic residues is an important requirement in clearly understanding enzyme functions. Therefore, predicting the catalytic activities of mutant variants is a fundamental challenge in computational biochemistry. Although site-directed mutagenesis is widely used for studying enzymatic activities and other important classes of protein function, interpreting mutation experiments is usually difficult mainly due to side effects induced by point mutations. Because steric and, in many cases, electrostatic effects may affect the local, fine geometries conserved in wild-type proteins that are usually believed to be thermodynamically stable, simply reducing a loss in catalytic activity into clear elements is difficult. To address these important but difficult issues, we performed a systematic ab initio QM/MM computational analysis combined with MD-FEP simulations and all-electron QM calculations for the entire protein matrix. We selected chorismate mutase, one of the simplest and well-known enzymes, to discuss the details of mutational effects on the enzymatic reaction process. On the basis of the reliable free energy profiles of the wild-type enzyme and several mutant variants, we analyzed the effects of point mutations relative to electronic structure and protein dynamics. In general, changes in geometrical parameters introduced by a mutation were usually limited to the local mutational site. However, this local structural modification could affect the global protein dynamics through correlated motions of particular amino acid residues even far from the mutation site. Even for mutant reactions with low catalytic activity, transition state stabilization was observed as a result of conformational modifications and reorganization around the active site. As for the electrostatic effect created by the polar protein environment, the wild-type enzyme was most effectively designed to stabilize the transition state of the reactive substrate, and

  18. βI-tubulin mutations in the laulimalide/peloruside binding site mediate drug sensitivity by altering drug-tubulin interactions and microtubule stability.

    PubMed

    Kanakkanthara, Arun; Rowe, Matthew R; Field, Jessica J; Northcote, Peter T; Teesdale-Spittle, Paul H; Miller, John H

    2015-09-01

    Peloruside A (PLA) and laulimalide (LAU) are potent microtubule-stabilizing natural products that are effective against a broad spectrum of cancer cells. The interactions of PLA and LAU with tubulin have attracted a great deal of attention, mainly because they bind to β-tubulin at a site that is different from the classical taxoid site. Multiple βI-tubulin amino acid residues have been predicted by computer modelling studies and more recently by protein crystallography to participate in the binding of PLA and LAU to tubulin. The relevance of these residues in determining cellular sensitivity to the compounds, however, remains largely uncertain. To determine the role of four binding site residues, Q291, D295, V333, and N337 on PLA and LAU activity, we introduced single mutations to these sites by site-directed mutagenesis and transfected each mutant tubulin separately into HEK and/or HeLa cells. We found that a Q291M βI-tubulin mutation increased sensitivity of the cells to PLA, but not to LAU, paclitaxel (PTX), or vinblastine (VBL). In contrast, V333W and N337L mutations led to less stable microtubules, with the V333W causing resistance to PLA and PTX, but not LAU, and the N337L causing resistance to PLA, LAU, and PTX. Moreover, cells expressing either W333 or L337 were hypersensitive to the microtubule-destabilizing agent, VBL. The D295I mutation conferred resistance to both PLA and LAU without affecting microtubule stability or sensitivity to PTX or ixabepilone (IXB). This study identifies the first mammalian βI-tubulin mutation that specifically increases sensitivity to PLA, and reports mutations at PLA and LAU binding site residues that can either reduce microtubule stability or impair drug-tubulin binding, conferring resistance to these microtubule-stabilizing agents. This information provides insights on β-tubulin residues important for maintaining microtubule structural integrity and for sensitivity to microtubule-targeting agents, and suggests novel

  19. Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors.

    PubMed

    Rayes, Diego; De Rosa, María José; Sine, Steven M; Bouzat, Cecilia

    2009-05-06

    Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.

  20. New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly, Trafficking, and Activation Mechanisms*

    PubMed Central

    Bode, Anna; Wood, Sian-Elin; Mullins, Jonathan G. L.; Keramidas, Angelo; Cushion, Thomas D.; Thomas, Rhys H.; Pickrell, William O.; Drew, Cheney J. G.; Masri, Amira; Jones, Elizabeth A.; Vassallo, Grace; Born, Alfred P.; Alehan, Fusun; Aharoni, Sharon; Bannasch, Gerald; Bartsch, Marius; Kara, Bulent; Krause, Amanda; Karam, Elie G.; Matta, Stephanie; Jain, Vivek; Mandel, Hanna; Freilinger, Michael; Graham, Gail E.; Hobson, Emma; Chatfield, Sue; Vincent-Delorme, Catherine; Rahme, Jubran E.; Afawi, Zaid; Berkovic, Samuel F.; Howell, Owain W.; Vanbellinghen, Jean-François; Rees, Mark I.; Chung, Seo-Kyung; Lynch, Joseph W.

    2013-01-01

    Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated that the dominant mutations p.Q226E, p.V280M, and p.R414H induced spontaneous channel activity, indicating that this is a recurring mechanism in hGlyR pathophysiology. p.Q226E, at the top of TM1, most likely induced tonic activation via an enhanced electrostatic attraction to p.R271 at the top of TM2, suggesting a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated into functional hGlyRs together with unmutated α1 or α1 plus β subunits. These aberrant receptors exhibit significantly reduced glycine sensitivity. To our knowledge, this is the first suggestion that subunits lacking TM4 domains might be incorporated into functional pentameric ligand-gated ion channel receptors. PMID:24108130

  1. Pathways of H2 toward the Active Site of [NiFe]-Hydrogenase

    PubMed Central

    Teixeira, Vitor H.; Baptista, António M.; Soares, Cláudio M.

    2006-01-01

    Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H2), but little is known about the diffusion of H2 toward the active site. Here we analyze pathways for H2 permeation using molecular dynamics (MD) simulations in explicit solvent. Various MD simulation replicates were done, to improve the sampling of the system states. H2 easily permeates hydrogenase in every simulation and it moves preferentially in channels. All H2 molecules that reach the active site made their approach from the side of the Ni ion. H2 is able to reach distances of <4 Å from the active site, although after 6 Å permeation is difficult. In this region we mutated Val-67 into alanine and perform new MD simulations. These simulations show an increase of H2 inside the protein and at lower distances from the active site. This valine can be a control point in the H2 access to the active center. PMID:16731562

  2. Transcriptionally Silenced Transgenes in Maize Are Activated by Three Mutations Defective in Paramutation

    PubMed Central

    McGinnis, Karen M.; Springer, Catherine; Lin, Yan; Carey, Charles C.; Chandler, Vicki

    2006-01-01

    Plants with mutations in one of three maize genes, mop1, rmr1, and rmr2, are defective in paramutation, an allele-specific interaction that leads to meiotically heritable chromatin changes. Experiments reported here demonstrate that these genes are required to maintain the transcriptional silencing of two different transgenes, suggesting that paramutation and transcriptional silencing of transgenes share mechanisms. We hypothesize that the transgenes are silenced through an RNA-directed chromatin mechanism, because mop1 encodes an RNA-dependent RNA polymerase. In all the mutants, DNA methylation was reduced in the active transgenes relative to the silent transgenes at all of the CNG sites monitored within the transgene promoter. However, asymmetrical methylation persisted at one site within the reactivated transgene in the rmr1-1 mutant. With that one mutant, rmr1-1, the transgene was efficiently resilenced upon outcrossing to reintroduce the wild-type protein. In contrast, with the mop1-1 and rmr2-1 mutants, the transgene remained active in a subset of progeny even after the wild-type proteins were reintroduced by outcrossing. Interestingly, this immunity to silencing increased as the generations progressed, consistent with a heritable chromatin state being formed at the transgene in plants carrying the mop1-1 and rmr2-1 mutations that becomes more resistant to silencing in subsequent generations. PMID:16702420

  3. A novel splice site mutation in a Becker muscular dystrophy patient.

    PubMed

    Bartolo, C; Papp, A C; Snyder, P J; Sedra, M S; Burghes, A H; Hall, C D; Mendell, J R; Prior, T W

    1996-04-01

    A Becker muscular dystrophy patient was found to have a single base substitution at the 5' end of intron 54. This single base substitution disrupts the invariant GT dinucleotide within the 5' donor splice site and was shown to cause an out of frame deletion of exon 54 during mRNA processing. This is predicted to produce a truncated dystrophin protein which is more consistent with a DMD phenotype. However, small quantities of normal mRNA are also transcribed and these are sufficient to produce a reduced amount of normal molecular weight dystrophin and give rise to a milder BMD phenotype. This indicates that a single base substitution at an invariant dinucleotide of the splice site consensus sequence may still allow read through of the message and allow the production of some normal protein. This shows that there are a greater number of possible intronic mutations that can lead to a mild phenotype and it also underlines the importance of performing cDNA analysis when screening for small gene alterations in the BMD patient population.

  4. A novel splice site mutation in a Becker muscular dystrophy patient.

    PubMed Central

    Bartolo, C; Papp, A C; Snyder, P J; Sedra, M S; Burghes, A H; Hall, C D; Mendell, J R; Prior, T W

    1996-01-01

    A Becker muscular dystrophy patient was found to have a single base substitution at the 5' end of intron 54. This single base substitution disrupts the invariant GT dinucleotide within the 5' donor splice site and was shown to cause an out of frame deletion of exon 54 during mRNA processing. This is predicted to produce a truncated dystrophin protein which is more consistent with a DMD phenotype. However, small quantities of normal mRNA are also transcribed and these are sufficient to produce a reduced amount of normal molecular weight dystrophin and give rise to a milder BMD phenotype. This indicates that a single base substitution at an invariant dinucleotide of the splice site consensus sequence may still allow read through of the message and allow the production of some normal protein. This shows that there are a greater number of possible intronic mutations that can lead to a mild phenotype and it also underlines the importance of performing cDNA analysis when screening for small gene alterations in the BMD patient population. Images PMID:8730289

  5. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  6. Molecular dynamics explorations of active site structure in designed and evolved enzymes.

    PubMed

    Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

    2015-04-21

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

  7. A distinct mutation on the alternative splice site of APC exon 9 results in attenuated familial adenomatous polyposis phenotype.

    PubMed

    Fostira, Florentia; Yannoukakos, Drakoulis

    2010-09-01

    A subset of APC mutation carriers shows a milder familial adenomatous polyposis phenotype (attenuated FAP) developing smaller number of polyps and colorectal cancer at an older age. It seems that a different mechanism to carcinogenesis is initiated according to the initial site of the germline mutation. The APC gene of a female patient with AFAP phenotypic features was analysed. A novel mutation located on the alternatively splice site of exon 9 was identified. This is the first reported mutation in the specific site. Transcripts characterization revealed disruption of splicing occurring within exon 9, resulting in the expression of a shorter mRNA transcript, which surprisingly does not affect the ratio between the two wild type transcripts, as well as the production of wild type short isoform by the mutant allele. The short wild type isoform, produced by the mutant allele, needs to be inactivated, on top of the wild type allele, for colorectal cancer to develop. These observations enhance the 'three hit hypothesis' and indicate that a distinct mechanism for the adenoma to carcinoma sequence should be followed, for truncated mutations taking place on the borderline of the alternatively spliced exon 9 of the APC gene, as well.

  8. Mutations in KIT occur at low frequency in melanomas arising from anatomical sites associated with chronic and intermittent sun exposure.

    PubMed

    Handolias, Despina; Salemi, Renato; Murray, William; Tan, Angela; Liu, Wendy; Viros, Amaya; Dobrovic, Alexander; Kelly, John; McArthur, Grant A

    2010-04-01

    In melanoma, mutations in KIT are most frequent in acral and mucosal subtypes and rarely reported in cutaneous melanomas particularly those associated with intermittent UV exposure. Conversely melanomas arising within chronic sun damaged skin are considered to harbour KIT mutations at higher rates. To characterize the frequency of KIT mutations in a representative melanoma population, 261 patients from two Australian melanoma centres were prospectively screened for mutations in exons 11, 13 and 17 of the KIT gene. A total of 257 patients had cutaneous melanoma arising from non-acral sites and four were acral melanomas. No mucosal or ocular melanomas were analysed. KIT mutations were identified in five tumours (2% of the entire cohort) including two acral melanomas. Two of the three non-acral melanomas with KIT mutations were associated with markers of chronic sun damage as assessed by the degree of skin elastosis. In the remaining cohort, 43% had chronically sun damaged skin. This report confirms that within an Australian population, KIT mutations are infrequent in cutaneous melanomas associated with both intermittent and chronic sun exposed skin.

  9. Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregation.

    PubMed

    Sant'Anna, Ricardo; Almeida, Maria Rosário; Varejāo, Nathalia; Gallego, Pablo; Esperante, Sebastian; Ferreira, Priscila; Pereira-Henriques, Alda; Palhano, Fernando L; de Carvalho, Mamede; Foguel, Debora; Reverter, David; Saraiva, Maria João; Ventura, Salvador

    2017-03-24

    More than a hundred different Transthyretin (TTR) mutations are associated with fatal systemic amyloidoses. They destabilize the protein tetrameric structure and promote the extracellular deposition of TTR as pathological amyloid fibrils. So far, only mutations R104H and T119M have been shown to stabilize significantly TTR, acting as disease suppressors. We describe a novel A108V non-pathogenic mutation found in a Portuguese subject. This variant is more stable than wild type TTR both in vitro and in human plasma, a feature that prevents its aggregation. The crystal structure of A108V reveals that this stabilization comes from novel intra and inter subunit contacts involving the thyroxine (T4) binding site. Exploiting this observation, we engineered a A108I mutation that fills the T4 binding cavity, as evidenced in the crystal structure. This synthetic protein becomes one of the most stable TTR variants described so far, with potential application in gene and protein replacement therapies.

  10. Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Alter the Choice of a 3′ Splice Site In Vivo

    PubMed Central

    Ma, Long; Horvitz, H. Robert

    2009-01-01

    The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential for animal survival has limited the study of the in vivo regulation of splicing. From a screen for suppressors of the Caenorhabditis elegans unc-93(e1500) rubberband Unc phenotype, we identified mutations in genes that encode the C. elegans orthologs of two splicing factors, the U2AF large subunit (UAF-1) and SF1/BBP (SFA-1). The uaf-1(n4588) mutation resulted in temperature-sensitive lethality and caused the unc-93 RNA transcript to be spliced using a cryptic 3′ splice site generated by the unc-93(e1500) missense mutation. The sfa-1(n4562) mutation did not cause the utilization of this cryptic 3′ splice site. We isolated four uaf-1(n4588) intragenic suppressors that restored the viability of uaf-1 mutants at 25°C. These suppressors differentially affected the recognition of the cryptic 3′ splice site and implicated a small region of UAF-1 between the U2AF small subunit-interaction domain and the first RNA recognition motif in affecting the choice of 3′ splice site. We constructed a reporter for unc-93 splicing and using site-directed mutagenesis found that the position of the cryptic splice site affects its recognition. We also identified nucleotides of the endogenous 3′ splice site important for recognition by wild-type UAF-1. Our genetic and molecular analyses suggested that the phenotypic suppression of the unc-93(e1500) Unc phenotype by uaf-1(n4588) and sfa-1(n4562) was likely caused by altered splicing of an unknown gene. Our observations provide in vivo evidence that UAF-1 can act in regulating 3′ splice-site choice and establish a system that can be used to investigate the in vivo regulation of RNA splicing in C. elegans. PMID:19893607

  11. [Structural regularities in activated cleavage sites of thrombin receptors].

    PubMed

    Mikhaĭlik, I V; Verevka, S V

    1999-01-01

    Comparison of thrombin receptors activation splitting sites sequences testifies to their similarity both in activation splitting sites of protein precursors and protein proteinase inhibitors reactive sites. In all these sites corresponded to effectory sites P2'-positions are placed by hydrophobic amino-acids only. The regularity defined conforms with previous thesis about the role of effectory S2'-site in regulation of the processes mediated by serine proteinases.

  12. LYN-activating mutations mediate antiestrogen resistance in estrogen receptor-positive breast cancer.

    PubMed

    Schwarz, Luis J; Fox, Emily M; Balko, Justin M; Garrett, Joan T; Kuba, María Gabriela; Estrada, Mónica Valeria; González-Angulo, Ana María; Mills, Gordon B; Red-Brewer, Monica; Mayer, Ingrid A; Abramson, Vandana; Rizzo, Monica; Kelley, Mark C; Meszoely, Ingrid M; Arteaga, Carlos L

    2014-12-01

    Estrogen receptor-positive (ER(+)) breast cancers adapt to hormone deprivation and become resistant to antiestrogen therapy. Here, we performed deep sequencing on ER(+) tumors that remained highly proliferative after treatment with the aromatase inhibitor letrozole and identified a D189Y mutation in the inhibitory SH2 domain of the SRC family kinase (SFK) LYN. Evaluation of 463 breast tumors in The Cancer Genome Atlas revealed four LYN mutations, two of which affected the SH2 domain. In addition, LYN was upregulated in multiple ER(+) breast cancer lines resistant to long-term estrogen deprivation (LTED). An RNAi-based kinome screen revealed that LYN is required for growth of ER(+) LTED breast cancer cells. Kinase assays and immunoblot analyses of SRC substrates in transfected cells indicated that LYN(D189Y) has higher catalytic activity than WT protein. Further, LYN(D189Y) exhibited reduced phosphorylation at the inhibitory Y507 site compared with LYN(WT). Other SH2 domain LYN mutants, E159K and K209N, also exhibited higher catalytic activity and reduced inhibitory site phosphorylation. LYN(D189Y) overexpression abrogated growth inhibition by fulvestrant and/or the PI3K inhibitor BKM120 in 3 ER(+) breast cancer cell lines. The SFK inhibitor dasatinib enhanced the antitumor effect of BKM120 and fulvestrant against estrogen-deprived ER(+) xenografts but not LYN(D189Y)-expressing xenografts. These results suggest that LYN mutations mediate escape from antiestrogens in a subset of ER(+) breast cancers.

  13. COL1 C-propeptide Cleavage Site Mutations Cause High Bone Mass Osteogenesis Imperfecta

    PubMed Central

    Lindahl, Katarina; Barnes, Aileen M.; Fratzl-Zelman, Nadja; Whyte, Michael P.; Hefferan, Theresa E.; Makareeva, Elena; Brusel, Marina; Yaszemski, Michael J.; Rubin, Carl-Johan; Kindmark, Andreas; Roschger, Paul; Klaushofer, Klaus; McAlister, William H.; Mumm, Steven; Leikin, Sergey; Kessler, Efrat; Boskey, Adele L.; Ljunggren, Östen; Marini, Joan C.

    2011-01-01

    Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA z-score was +3.9 and pQCT vBMD was +3.1; Patient 2 had L1-L4 DXA z-score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FT-IR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2’s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization. PMID:21344539

  14. CAPN5 mutation in hereditary uveitis: the R243L mutation increases calpain catalytic activity and triggers intraocular inflammation in a mouse model

    PubMed Central

    Wert, Katherine J.; Bassuk, Alexander G.; Wu, Wen-Hsuan; Gakhar, Lokesh; Coglan, Diana; Mahajan, MaryAnn; Wu, Shu; Yang, Jing; Lin, Chyuan-Sheng; Tsang, Stephen H.; Mahajan, Vinit B.

    2015-01-01

    A single amino acid mutation near the active site of the CAPN5 protease was linked to the inherited blinding disorder, autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). In homology modeling with other calpains, this R243L CAPN5 mutation was situated in a mobile loop that gates substrate access to the calcium-regulated active site. In in vitro activity assays, the mutation increased calpain protease activity and made it far more active at low concentrations of calcium. To test whether the disease allele could yield an animal model of ADNIV, we created transgenic mice expressing human (h) CAPN5R243L only in the retina. The resulting hCAPN5R243L transgenic mice developed a phenotype consistent with human uveitis and ADNIV, at the clinical, histological and molecular levels. The fundus of hCAPN5R243L mice showed enhanced autofluorescence (AF) and pigment changes indicative of reactive retinal pigment epithelial cells and photoreceptor degeneration. Electroretinography showed mutant mouse eyes had a selective loss of the b-wave indicating an inner-retina signaling defect. Histological analysis of mutant mouse eyes showed protein extravasation from dilated vessels into the anterior chamber and vitreous, vitreous inflammation, vitreous and retinal fibrosis and retinal degeneration. Analysis of gene expression changes in the hCAPN5R243L mouse retina showed upregulation of several markers, including members of the Toll-like receptor pathway, chemokines and cytokines, indicative of both an innate and adaptive immune response. Since many forms of uveitis share phenotypic characteristics of ADNIV, this mouse offers a model with therapeutic testing utility for ADNIV and uveitis patients. PMID:25994508

  15. Saccharomyces cerevisiae-based mutational analysis of the bc1 complex Qo site residue 279 to study the trade-off between atovaquone resistance and function.

    PubMed

    Song, Zehua; Clain, Jérôme; Iorga, Bogdan I; Yi, Zhou; Fisher, Nicholas; Meunier, Brigitte

    2015-07-01

    The bc1 complex is central to mitochondrial bioenergetics and the target of the antimalarial drug atovaquone that binds in the quinol oxidation (Qo) site of the complex. Structural analysis has shown that the Qo site residue Y279 (Y268 in Plasmodium falciparum) is key for atovaquone binding. Consequently, atovaquone resistance can be acquired by mutation of that residue. In addition to the probability of amino acid substitution, the level of atovaquone resistance and the loss of bc1 complex activity that are associated with the novel amino acid would restrict the nature of resistance-driven mutations occurring on atovaquone exposure in native parasite populations. Using the yeast model, we characterized the effect of all the amino acid replacements resulting from a single nucleotide substitution at codon 279: Y279C, Y279D, Y279F, Y279H, Y279N, and Y279S (Y279C, D, F, H, N, and S). Two residue changes that required a double nucleotide substitution, Y279A and W, were added to the series. We found that mutations Y279A, C, and S conferred high atovaquone resistance but decreased the catalytic activity. Y279F had wild-type enzymatic activity and sensitivity to atovaquone, while the other substitutions caused a dramatic respiratory defect. The results obtained with the yeast model were examined in regard to atomic structure and compared to the reported data on the evolution of acquired atovaquone resistance in P. falciparum.

  16. Mutations activating human c-Ha-ras1 protooncogene (HRAS1) induced by chemical carcinogens and depurination.

    PubMed Central

    Vousden, K H; Bos, J L; Marshall, C J; Phillips, D H

    1986-01-01

    In vitro modification of plasmids containing the human c-Ha-ras1 protooncogene (HRAS1) with the ultimate carcinogens N-acetoxy-2-acetylaminofluorene and r-7, t-8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (anti-BPDE) generated a transforming oncogene when the modified DNA was transfected into NIH 3T3 cells. The protooncogene was also activated by heating the plasmid at 70 degrees C, pH 4, to generate apurinic/apyrimidinic sites in the DNA. DNA isolated from transformed foci was analyzed by hybridization with 20-mer oligonucleotides designed to detect single point mutations within two regions of the gene commonly found to be mutated in tumor DNA. Of 23 transformants studied, 7 contained a mutation in the region of the 12th codon, whereas the remaining 16 were mutated in the 61st codon. Of the codon-61 mutants, 6 were mutated at the first base position (C X G), 5 at the second (A X T), and 5 at the third (G X C). The point mutations induced by anti-BPDE were predominantly G X C----T X A and A X T----T X A base substitutions, whereas four N-acetoxy-2-acetylaminofluorene-induced mutations were all G X C----T X A, and a single depurination-induced activation that was analyzed contained an A X T----T X A transversion. Together, these methods provide a useful means of determining point mutations produced by DNA-damaging agents in mammalian cells. Images PMID:3513171

  17. Roles of s3 site residues of nattokinase on its activity and substrate specificity.

    PubMed

    Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong

    2007-09-01

    Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.

  18. Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

    PubMed Central

    Petrosino, J F; Palzkill, T

    1996-01-01

    Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants. PMID:8606154

  19. Mutations That Extend the Specificity of the Endonuclease Activity of λ Terminase

    PubMed Central

    Arens, Jean Sippy; Hang, Qi; Hwang, Young; Tuma, Bill; Max, Sara; Feiss, Mike

    1999-01-01

    Terminase, an enzyme encoded by the Nu1 and A genes of bacteriophage lambda, is crucial for packaging concatemeric DNA into virions. cosN, a 22-bp segment, is the site on the virus chromosome where terminase introduces staggered nicks to cut the concatemer to generate unit-length virion chromosomes. Although cosN is rotationally symmetric, mutations in cosN have asymmetric effects. The cosN G2C mutation (a G-to-C change at position 2) in the left half of cosN reduces the phage yield 10-fold, whereas the symmetric mutation cosN C11G, in the right half of cosN, does not affect the burst size. The reduction in phage yield caused by cosN G2C is correlated with a defect in cos cleavage. Three suppressors of the cosN G2C mutation, A-E515G, A-N509K, and A-R504C, have been isolated that restore the yield of λ cosN G2C to the wild-type level. The suppressors are missense mutations that alter amino acids located near an ATPase domain of gpA. λ A-E515G, A-N509K, and A-R504C phages, which are cosN+, also had wild-type burst sizes. In vitro cos cleavage experiments on cosN G2C C11G DNA showed that the rate of cleavage for A-E515G terminase is three- to fourfold higher than for wild-type terminase. The A-E515G mutation changes residue 515 of gpA from glutamic acid to glycine. Uncharged polar and hydrophobic residues at position 515 suppressed the growth defect of λ cosN G2C C11G. In contrast, basic (K, R) and acidic (E, D) residues at position 515 failed to suppress the growth defect of λ cosN G2C C11G. In a λ cosN+ background, all amino acids tested at position 515 were functional. These results suggest that A-E515G plays an indirect role in extending the specificity of the endonuclease activity of λ terminase. PMID:9864333

  20. Altered intrinsic brain activity in patients with paroxysmal kinesigenic dyskinesia by PRRT2 mutation: altered brain activity by PRRT2 mutation.

    PubMed

    Luo, ChunYan; Chen, Yongping; Song, Wei; Chen, Qin; Gong, QiYong; Shang, Hui-Fang

    2013-11-01

    The proline-rich transmembrane protein 2 (PRRT2) gene has been recently identified as a causative gene of paroxysmal kinesigenic dyskinesia (PKD), with an insertion mutation c.649_650insC (p.P217fsX7) reported as the most common mutation. However, the pathogenic mechanism of the mutation of PRRT2 remains largely unknown. Resting-state functional magnetic resonance imaging is a promising approach to assess cerebral function and reveals underlying functional changes. Resting-state functional magnetic resonance imaging was performed in 4 Chinese PKD patients with p.P217fsX7 mutation, 6 Chinese PKD patients without the mutation, and 10 healthy control subjects. Voxel-based analysis was used to characterize alterations in the amplitude of low-frequency fluctuation (ALFF). When compared with the healthy control subjects, both groups of PKD patients showed alterations in spontaneous brain activities within cortical-basal ganglia circuitry. Besides, the group of patients with p.P217fsX7 mutation also exhibited increased ALFF in the right postcenral gyrus and right rolandic operculum area, while the alteration of ALFF in group of patients without the mutation additionally involved the middle orbitofrontal cortex. Direct comparative analysis between these two patient groups revealed significantly increased ALFF in the right postcentral gyrus in the group with p.P217fsX7 mutation. Increased spontaneous brain activity in the cortical-basal ganglia circuitry, especially in the motor preparation areas, is a common pathophysiology in PKD. Differences in the spatial patterns of increased ALFF between patients with and those without the mutation might reflect the distinct pathological mechanism resulting from PRRT2 mutation.

  1. Structured Regions of Alpha-synuclein Fibrils Include the Early Onset Parkinson's Disease Mutation Sites

    SciTech Connect

    Comellas Canal, Gemma; Lemkau, Luisel R.; Nieuwkoop, Andrew J.; Kloepper, Kathryn D.; Ladror, Daniel T.; Ebisu, Reika; Woods, Wendy S.; Lipton, Andrew S.; George, Julia M.; Rienstra, Chad M.

    2011-08-26

    Alpha-Synuclein (AS) fibrils constitute the major proteinaceous component of Lewy bodies (LBs), the pathological hallmark of Parkinson’s disease (PD) and other neurodegenerative diseases. Three single point mutations in the AS gene, as well as multiplication of the wild-type (WT) AS allele, have been previously identified in families with early-onset PD. Although AS fibrils have been the subject of intense study, critical details about their structure including the precise location of the B-strands and the extent of the core, the three-dimensional structure and the effects of the mutations—remain unknown. Here, we have used magic-angle spinning solid-state NMR spectroscopy to present a detailed characterization of the full-length WT AS fibrils. With improved sample preparations, isotopic labeling patterns and NMR experiments, we have confidently assigned more than 90% of the 13C and 15N backbone and sidechain chemical shifts of the detected residues from residue 39 to 97, and quantified the conformational dynamics throughout this region. Our results demonstrate that the core of AS fibrils extends with a repeated motif and that residues 30, 46 and 53-the early-onset PD mutant sites-are located in structured regions of AS fibrils.

  2. A novel splice site mutation of CRYBA3/A1 gene associated with congenital cataract in a Chinese family

    PubMed Central

    Wu, Meng-Han; Yu, Yin-Hui; Hao, Qin-Long; Gong, Xiao-Hua; Yao, Ke

    2017-01-01

    AIM To identify the disease-causing mutation responsible for the presence of congenital cataract in a Chinese family. METHODS The study recruited a four-generation Chinese pedigree affected by autosomal dominant congenital cataract (ADCC). Family history and the history of cataract extraction were recorded. Blood samples were collected from individuals for DNA extraction. Direct sequencing of congenital cataract-associated genes was performed. Single-strand conformational polymorphism and bioinformatic analysis were conducted to further study the mutation. RESULTS Direct sequencing revealed a novel splice site mutation of c.30-2 A>G in the CRYBA3/A1 gene. The mutation co-segregated within all affected individuals in the family and was not found in unaffected members or 100 unrelated normal controls. These results were further confirmed by single-strand conformational polymorphism and bioinformatic analysis using the Human Splicing Finder and MaxEnt online software and Annovar computer software. CONCLUSION c.30-2 A>G mutation of CRYBA3/A1 gene is a novel mutation and broadens the genetic spectrum of ADCC. PMID:28149769

  3. FBN1 gene mutation defines the profibrillin to fibrillin processing site and segregates with tall stature in a family

    SciTech Connect

    Grossfield, J.; Cao, S.; Milewicz, D.

    1994-09-01

    Dermal fibroblasts from a 13-year-old boy with skeletal features of the Marfan syndrome were used to study fibrillin synthesis and processing. Synthesis and secretion of profibrillin was normal but only half of the secreted profibrillin was converted to fibrillin, an extracellular proteolytic processing that removes a 20 kDa fragment from the protein. All the secreted profibrillin was processed to fibrillin in control cells. Only the processed form of fibrillin was deposited into the extracellular matrix in both the proband`s and the control cells. Electron microscopic examination of rotary shadowed microfibrils made by the proband`s fibroblasts were indistinguishable from control cells. Screening exons in the 3{prime} end of the FBN1 gene revealed a heterozygous C to T transition at nucleotide 5482 of the FBN1 cDNA changing R 1828 to W. This mutation disrupts a known consensus sequence recognized by a cellular protease and is located in the carboxy terminus at a site predicted to remove a 19 kD fragment. The proband and his 22-year-old brother, also heterozygous for the mutation, have had normal echocardiograms and ophthalmologic exams. The mutation segregated in the proband`s three generation family with autosomal dominant inheritance of height (> 90th percentile) and no known cardiovascular or ocular problems, including the 67-year-old grandmother (exams pending). The mutation was not found in 90 chromosomes from unrelated individuals. In summary, (1) the mutation identifies the cleavage site for the conversion of profibrillin to fibrillin; (2) the characterized mutation segregates in the family with tall stature without known cardiovascular or ocular problems; (3) this mutation potentially defines the phenotype associated with a {open_quotes}null{close_quotes} allele for the FBN1 gene.

  4. Activating mutations and senescence secretome: new insights into HER2 activation, drug sensitivity and metastatic progression.

    PubMed

    Acharyya, Swarnali

    2013-04-23

    HER2 amplification and overexpression is observed in approximately 20% of breast cancers and is strongly associated with poor prognosis and therapeutic responsiveness to HER2 targeted agents. A recent study by Bose and colleagues suggests that another subset of breast cancer patients without HER2 amplification but with activating HER2 mutation might also benefit from existing HER2-targeted agents and the authors functionally characterize these somatic mutations in experimental models. In a second study on HER2-driven breast cancer, Angelini and colleagues investigate how the constitutively active, truncated carboxy-terminal fragment of HER2, p95HER2, promotes metastatic progression through non-cellautonomous secretion of factors from senescent cells. These new findings advance our understanding of HER2 biology in the context of HER2 activation as well as offer new insights into our understanding of drug sensitivity and metastatic progression.

  5. Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1

    PubMed Central

    2016-01-01

    Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates. PMID:28026940

  6. Divergent Activity Profiles of Type 1 Ryanodine Receptor Channels Carrying Malignant Hyperthermia and Central Core Disease Mutations in the Amino-Terminal Region.

    PubMed

    Murayama, Takashi; Kurebayashi, Nagomi; Yamazawa, Toshiko; Oyamada, Hideto; Suzuki, Junji; Kanemaru, Kazunori; Oguchi, Katsuji; Iino, Masamitsu; Sakurai, Takashi

    2015-01-01

    The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in several diseases, including malignant hyperthermia (MH) and central core disease (CCD). Most MH and CCD mutations cause accelerated Ca2+ release, resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, how specific mutations affect the channel to produce different phenotypes is not well understood. In this study, we have investigated 11 mutations at 7 different positions in the amino (N)-terminal region of RyR1 (9 MH and 2 MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging at room temperature (~25 °C), cells expressing mutant channels exhibited alterations in Ca2+ homeostasis, i.e., an enhanced sensitivity to caffeine, a depletion of Ca2+ in the ER and an increase in resting cytoplasmic Ca2+. RyR1 channel activity was quantitatively evaluated by [3H]ryanodine binding and three parameters (sensitivity to activating Ca2+, sensitivity to inactivating Ca2+ and attainable maximum activity, i.e., gain) were obtained by fitting analysis. The mutations increased the gain and the sensitivity to activating Ca2+ in a site-specific manner. The gain was consistently higher in both MH and MH/CCD mutations. Sensitivity to activating Ca2+ was markedly enhanced in MH/CCD mutations. The channel activity estimated from the three parameters provides a reasonable explanation to the pathological phenotype assessed by Ca2+ homeostasis. These properties were also observed at higher temperatures (~37 °C). Our data suggest that divergent activity profiles may cause varied disease phenotypes by specific mutations. This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.

  7. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  8. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  9. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    PubMed

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

  10. Dominance and interloci interactions in transcriptional activation cascades: models explaining compensatory mutations and inheritance patterns.

    PubMed

    Bost, Bruno; Veitia, Reiner A

    2014-01-01

    Mutations in human genes encoding transcription factors are often dominant because one active allele cannot ensure a normal phenotype (haploinsufficiency). In other instances, heterozygous mutations of two genes are required for a phenotype to appear (combined haploinsufficiency). Here, we explore with models (i) the basis of haploinsufficiency and combined haploinsufficiency owing to mutations in transcription activators, and (ii) how the effects of such mutations can be amplified or buffered by subsequent steps in a transcription cascade. We propose that the non-linear (sigmoidal) response of transcription to the concentration of activators can explain haploinsufficiency. We further show that the sigmoidal character of the output of a cascade increases with the number of steps involved, the settings of which will determine the buffering or enhancement of the effects of a decreased concentration of an upstream activator. This exploration provides insights into the bases of compensatory mutations and on interloci interactions underlying oligogenic inheritance patterns.

  11. A novel biallelic splice site mutation of TECTA causes moderate to severe hearing impairment in an Algerian family.

    PubMed

    Behlouli, Asma; Bonnet, Crystel; Abdi, Samia; Hasbellaoui, Mokhtar; Boudjenah, Farid; Hardelin, Jean-Pierre; Louha, Malek; Makrelouf, Mohamed; Ammar-Khodja, Fatima; Zenati, Akila; Petit, Christine

    2016-08-01

    Congenital deafness is certainly one of the most common monogenic diseases in humans, but it is also one of the most genetically heterogeneous, which makes molecular diagnosis challenging in most cases. Whole-exome sequencing in two out of three Algerian siblings affected by recessively-inherited, moderate to severe sensorineural deafness allowed us to identify a novel splice donor site mutation (c.5272+1G > A) in the gene encoding α-tectorin, a major component of the cochlear tectorial membrane. The mutation was present at the homozygous state in the three affected siblings, and at the heterozygous state in their unaffected, consanguineous parents. To our knowledge, this is the first reported TECTA mutation leading to the DFNB21 form of hearing impairment among Maghrebian individuals suffering from congenital hearing impairment, which further illustrates the diversity of the genes involved in congenital deafness in the Maghreb.

  12. Identification of a novel splice-site mutation in the Lebercilin (LCA5) gene causing Leber congenital amaurosis

    PubMed Central

    Ramprasad, Vedam Lakshmi; Soumittra, Nagasamy; Nancarrow, Derek; Sen, Parveen; McKibbin, Martin; Williams, Grange A; Arokiasamy, Tharigopala; Lakshmipathy, Praveena; Inglehearn, Chris F

    2008-01-01

    Purpose Leber congenital amaurosis (LCA) is one of the most common causes of hereditary blindness in infants. To date, mutations in 13 known genes and at two other loci have been implicated in LCA causation. An examination of the known genes highlights several processes which, when defective, cause LCA, including photoreceptor development and maintenance, phototransduction, vitamin A metabolism, and protein trafficking. In addition, it has been known for some time that defects in sensory cilia can cause syndromes involving hereditary blindness. More recently evidence has come to light that non-syndromic LCA can also be a “ciliopathy.” Methods Here we present a homozygosity mapping analysis in a consanguineous sibship that led to the identification of a mutation in the recently discovered LCA5 gene. Homozygosity mapping was done using Affymetrix 10K Xba I Gene Chip and a 24.5cM region on chromosome 6 (6q12- q16.3) was identified to be significantly homozygous. The LCA5 gene on this region was sequenced and cDNA sequencing also done to characterize the mutation. Results A c.955G>A missense mutation in the last base of exon 6 causing disruption of the splice donor site was identified in both the affected sibs. Since there is a second consensus splice donor sequence 5 bp into the adjacent intron, this mutation results in a transcript with a 5 bp insertion of intronic sequence, leading to a frameshift and premature truncation. Conclusions We report a missense mutation functionally altering the splice donor site and leading to a truncated protein. This is the second report of LCA5 mutations causing LCA. It may also be significant that one affected child died at eleven months of age due to asphyxia during sleep. To date the only phenotype unambiguously associated with mutations in this gene is LCA. However the LCA5 gene is known to be expressed in nasopharynx, trachea and lungs and was originally identified in the proteome of bronchial epithelium ciliary axonemes. The

  13. Computational Identification of Amino-Acid Mutations that Further Improve the Activity of a Chalcone–Flavonone Isomerase from Glycine max

    PubMed Central

    Yuan, Hui; Wu, Jiaqi; Wang, Xiaoqiang; Chen, Jiakuan; Zhong, Yang; Huang, Qiang; Nan, Peng

    2017-01-01

    Protein design for improving enzymatic activity remains a challenge in biochemistry, especially to identify target amino-acid sites for mutagenesis and to design beneficial mutations for those sites. Here, we employ a computational approach that combines multiple sequence alignment, positive selection detection, and molecular docking to identify and design beneficial amino-acid mutations that further improve the intramolecular-cyclization activity of a chalcone–flavonone isomerase from Glycine max (GmCHI). By this approach, two GmCHI mutants with higher activities were predicted and verified. The results demonstrate that this approach could determine the beneficial amino-acid mutations for improving the enzymatic activity, and may find more applications in engineering of enzymes. PMID:28286513

  14. Activating HER2 mutations in HER2 gene amplification negative breast cancer

    PubMed Central

    Bose, Ron; Kavuri, Shyam M.; Searleman, Adam C.; Shen, Wei; Shen, Dong; Koboldt, Daniel C.; Monsey, John; Goel, Nicholas; Aronson, Adam B.; Li, Shunqiang; Ma, Cynthia X.; Ding, Li; Mardis, Elaine R.; Ellis, Matthew J.

    2012-01-01

    Data from eight breast cancer genome sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized thirteen HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGFR exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings demonstrate that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment. PMID:23220880

  15. Suppressor Mutations for Presenilin 1 Familial Alzheimer Disease Mutants Modulate γ-Secretase Activities.

    PubMed

    Futai, Eugene; Osawa, Satoko; Cai, Tetsuo; Fujisawa, Tomoya; Ishiura, Shoichi; Tomita, Taisuke

    2016-01-01

    γ-Secretase is a multisubunit membrane protein complex containing presenilin (PS1) as a catalytic subunit. Familial Alzheimer disease (FAD) mutations within PS1 were analyzed in yeast cells artificially expressing membrane-bound substrate, amyloid precursor protein, or Notch fused to Gal4 transcriptional activator. The FAD mutations, L166P and G384A (Leu-166 to Pro and Gly-384 to Ala substitution, respectively), were loss-of-function in yeast. We identified five amino acid substitutions that suppress the FAD mutations. The cleavage of amyloid precursor protein or Notch was recovered by the secondary mutations. We also found that secondary mutations alone activated the γ-secretase activity. FAD mutants with suppressor mutations, L432M or S438P within TMD9 together with a missense mutation in the second or sixth loops, regained γ-secretase activity when introduced into presenilin null mouse fibroblasts. Notably, the cells with suppressor mutants produced a decreased amount of Aβ42, which is responsible for Alzheimer disease. These results indicate that the yeast system is useful to screen for mutations and chemicals that modulate γ-secretase activity.

  16. HotSpot Wizard 2.0: automated design of site-specific mutations and smart libraries in protein engineering

    PubMed Central

    Bendl, Jaroslav; Stourac, Jan; Sebestova, Eva; Vavra, Ondrej; Musil, Milos; Brezovsky, Jan; Damborsky, Jiri

    2016-01-01

    HotSpot Wizard 2.0 is a web server for automated identification of hot spots and design of smart libraries for engineering proteins’ stability, catalytic activity, substrate specificity and enantioselectivity. The server integrates sequence, structural and evolutionary information obtained from 3 databases and 20 computational tools. Users are guided through the processes of selecting hot spots using four different protein engineering strategies and optimizing the resulting library's size by narrowing down a set of substitutions at individual randomized positions. The only required input is a query protein structure. The results of the calculations are mapped onto the protein's structure and visualized with a JSmol applet. HotSpot Wizard lists annotated residues suitable for mutagenesis and can automatically design appropriate codons for each implemented strategy. Overall, HotSpot Wizard provides comprehensive annotations of protein structures and assists protein engineers with the rational design of site-specific mutations and focused libraries. It is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard. PMID:27174934

  17. Missense point mutations of tau to segregate with FTDP-17 exhibit site-specific effects on microtubule structure in COS cells: a novel action of R406W mutation.

    PubMed

    Sahara, N; Tomiyama, T; Mori, H

    2000-05-01

    Missense and splicing point mutations have been found in the tau gene in families with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Of these mutations, we examined four exonic missense point mutations (G272V, P301L, V337M and R406W) in 3-repeat or 4-repeat tau isoform on the transfection experiment. The effects of two mutations (G272V or P301L) on microtubules were subtle whereas those of two other mutations (V337M or R406W) were dramatically significant when these two mutations were constructed into 3-repeat tau but not into 4-repeat tau. The R406W mutation induced an alternation of microtubules to form dotted or fragmented forms retaining colocalization of tau with tubulin whereas the V337M mutation predominantly disrupted microtubule networks and diminished colocalization of tau and tubulin. The effect of the mutations on microtubules were thus site-dependent and isoform-dependent. Tau with R406W mutation was found to be colocalized with tubulin without filamentous structures on confocal views, suggesting that the carboxyl region of tau played a different role from tubulin-binding domain on microtubule assemble. Another abnormal property was identified in tau with R406W mutation that failed to suffer phosphorylation. Thus, diverse effects of tau mutations on microtubules may explain the various clinicopathologies of FTDP-17 and related tauopathies.

  18. Cancer-associated mutations are preferentially distributed in protein kinase functional sites.

    PubMed

    Izarzugaza, Jose M G; Redfern, Oliver C; Orengo, Christine A; Valencia, Alfonso

    2009-12-01

    Protein kinases are a superfamily involved in many crucial cellular processes, including signal transmission and regulation of cell cycle. As a consequence of this role, kinases have been reported to be associated with many types of cancer and are considered as potential therapeutic targets. We analyzed the distribution of pathogenic somatic point mutations (drivers) in the protein kinase superfamily with respect to their location in the protein, such as in structural, evolutionary, and functionally relevant regions. We find these driver mutations are more clearly associated with key protein features than other somatic mutations (passengers) that have not been directly linked to tumor progression. This observation fits well with the expected implication of the alterations in protein kinase function in cancer pathogenicity. To explain the relevance of the detected association of cancer driver mutations at the molecular level in the human kinome, we compare these with genetically inherited mutations (SNPs). We find that the subset of nonsynonymous SNPs that are associated to disease, but sufficiently mild to the point of being widespread in the population, tend to avoid those key protein regions, where they could be more detrimental for protein function. This tendency contrasts with the one detected for cancer associated-driver-mutations, which seems to be more directly implicated in the alteration of protein function. The detailed analysis of protein kinase groups and a number of relevant examples, confirm the relation between cancer associated-driver-mutations and key regions for protein kinase structure and function.

  19. Interaction of aspartic acid-104 and proline-287 with the active site of m-calpain.

    PubMed Central

    Arthur, J S; Elce, J S

    1996-01-01

    In an ongoing study of the mechanisms of calpain catalysis and Ca(2+)-induced activation, the effects of Asp-104-->Ser and Pro-287-->Ser large subunit mutations on m-calpain activity, the pH-activity profile, Ca(2+)-sensitivity, and autolysis were measured. The importance of these positions was suggested by sequence comparisons between the calpain and papain families of cysteine proteinases. Asp-104 is adjacent to the active-site Cys-105, and Pro-287 is adjacent to the active-site Asn-286 and probably to the active-site His-262; both Asp-104 and Pro-287 are absolutely conserved in the known calpains, but are replaced by highly conserved serine residues in the papains. The single mutants had approx. 10-15% of wild-type activity, due mainly to a decrease in kcat, since Km was only slightly increased. The Pro-287-->Ser mutation appeared to cause a local perturbation of the catalytic Cys-105/His-262 catalytic ion pair, reducing its efficiency without major effect on the conformation and stability of the enzyme. The Asp-104-->Ser mutation caused a marked narrowing of the pH-activity curve, a 9-fold increase in Ca2+ requirement, and an acceleration of autolysis, when compared with the wild-type enzyme. The results indicated that Asp-104 alters the nature of its interaction with the catalytic ion pair during Ca(2+)-induced conformational change in calpain. This interaction may be direct or indirect, but is important in activation of the enzyme. PMID:8912692

  20. Alternating Hemiplegia of Childhood mutations have a differential effect on Na(+),K(+)-ATPase activity and ouabain binding.

    PubMed

    Weigand, Karl M; Messchaert, Muriël; Swarts, Herman G P; Russel, Frans G M; Koenderink, Jan B

    2014-07-01

    De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na(+),K(+)-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na(+),K(+)-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K(+)/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na(+),K(+)-ATPase. Functional impairment of Na(+),K(+)-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.

  1. Clinical activity of regorafenib in PDGFRA-mutated gastrointestinal stromal tumor.

    PubMed

    Grellety, Thomas; Kind, Michèle; Coindre, Jean-Michel; Italiano, Antoine

    2015-11-01

    Gastrointestinal stromal tumor (GIST) is the most frequent mesenchymal tumor of the gastrointestinal tract and one of the most frequent sarcoma. Mutually exclusive KIT and PDGFRA mutations are central events in GIST pathogenesis, and their understanding is crucial because specific treatment targeting oncogenic KIT and PDGFRA activation (especially imatinib) has become available. The most frequent PDGFRA mutation (D842V) is associated with primary resistance to imatinib. Data related to regorafenib efficacy in PDGFRA-mutated GIST are lacking. We report here a case report of a prolonged response with regorafenib in a patient with a PDGFRA-mutated GIST.

  2. A targeted mutation at the known collagenase cleavage site in mouse type I collagen impairs tissue remodeling

    PubMed Central

    1995-01-01

    Degradation of type I collagen, the most abundant collagen, is initiated by collagenase cleavage at a highly conserved site between Gly775 and Ile776 of the alpha 1 (I) chain. Mutations at or around this site render type I collagen resistant to collagenase digestion in vitro. We show here that mice carrying a collagenase-resistant mutant Col1a-1 transgene die late in embryo-genesis, ascribable to overexpression of the transgene, since the same mutation introduced into the endogenous Col1a-1 gene by gene targeting permitted normal development of mutant mice to young adulthood. With increasing age, animals carrying the targeted mutation developed marked fibrosis of the dermis similar to that in human scleroderma. Postpartum involution of the uterus in the mutant mice was also impaired, with persistence of collagenous nodules in the uterine wall. Although type I collagen from the homozygous mutant mice was resistant to cleavage by human or rat fibroblast collagenases at the helical site, only the rat collagenase cleaved collagen trimers at an additional, novel site in the nonhelical N-telopeptide domain. Our results suggest that cleavage by murine collagenase at the N-telopeptide site could account for resorption of type I collagen during embryonic and early adult life. During intense collagen resorption, however, such as in the immediate postpartum uterus and in the dermis later in life, cleavage at the helical site is essential for normal collagen turnover. Thus, type I collagen is degraded by at least two differentially controlled mechanisms involving collagenases with distinct, but overlapping, substrate specificities. PMID:7790374

  3. Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation.

    PubMed

    Kawarai, Toshitaka; Miyamoto, Ryosuke; Mori, Atsuko; Oki, Ryosuke; Tsukamoto-Miyashiro, Ai; Matsui, Naoko; Miyazaki, Yoshimichi; Orlacchio, Antonio; Izumi, Yuishin; Nishida, Yoshihiko; Kaji, Ryuji

    2015-12-15

    We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis.

  4. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  5. The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors.

    PubMed

    Chatzidaki, Anna; D'Oyley, Jarryl M; Gill-Thind, JasKiran K; Sheppard, Tom D; Millar, Neil S

    2015-10-01

    Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding at an extracellular orthosteric site. Previous studies have described several positive allosteric modulators (PAMs) that are selective for homomeric α7 nAChRs. These include type I PAMs, which exert little or no effect on the rate of receptor desensitisation, and type II PAMs, which cause a dramatic loss of agonist-induced desensitisation. Here we report evidence that transmembrane mutations in α7 nAChRs have diverse effects on receptor activation and desensitisation by allosteric ligands. It has been reported previously that the L247T mutation, located toward the middle of the second transmembrane domain (at the 9' position), confers reduced levels of desensitisation. In contrast, the M260L mutation, located higher up in the TM2 domain (at the 22' position), does not show any difference in desensitisation compared to wild-type receptors. We have found that in receptors containing the L247T mutation, both type I PAMs and type II PAMs are converted into non-desensitising agonists. In contrast, in receptors containing the M260L mutation, this effect is seen only with type II PAMs. These findings, indicating that the M260L mutation has a selective effect on type II PAMs, have been confirmed both with previously described PAMs and also with a series of novel α7-selective PAMs. The novel PAMs examined in this study have close chemical similarity but diverse pharmacological properties. For example, they include compounds displaying effects on receptor desensitisation that are typical of classical type I and type II PAMs but, in addition, they include compounds with intermediate properties.

  6. Gene mutation in microRNA target sites of CFTR gene: a novel pathogenetic mechanism in cystic fibrosis?

    PubMed

    Amato, Felice; Seia, Manuela; Giordano, Sonia; Elce, Ausilia; Zarrilli, Federica; Castaldo, Giuseppe; Tomaiuolo, Rossella

    2013-01-01

    Cystic fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians. It depends on alterations of a chloride channel expressed by most epithelial cells and encoded by CFTR gene. Also using scanning techniques to analyze the whole coding regions of CFTR gene, mutations are not identified in up to 10% of CF alleles, and such figure increases in CFTR-related disorders (CFTR-RD). Other gene regions may be the site of causing-disease mutations. We searched for genetic variants in the 1500 bp of CFTR 3' untranslated region, typical target of microRNA (miRNA) posttranscriptional gene regulation, in either CF patients with the F508del homozygous genotype and different clinical expression (n = 20), CF (n = 32) and CFTR-RD (n = 43) patients with one or none mutation after CFTR scanning and in controls (n = 50). We identified three SNPs, one of which, the c.*1043A>C, was located in a region predicted to bind miR-433 and miR-509-3p. Such mutation was peculiar of a CFTR-RD patient that had Congenital Bilateral Absence of Vas Deferens (CBAVD), diffuse bronchiectasis, a borderline sweat chloride test and the heterozygous severe F508del mutation on the other allele. The expression analysis demonstrated that the c.*1043A>C increases the affinity for miR-509-3p and slightly decreases that for the miR-433. Both miRNAs cause in vitro a reduced expression of CFTR protein. Thus, the c.*1043A>C may act as a mild CFTR mutation enhancing the affinity for inhibitory miRNAs as a novel pathogenetic mechanism in CF.

  7. Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels*

    PubMed Central

    Tang, Qiong-Yao; Larry, Trevor; Hendra, Kalen; Yamamoto, Erica; Bell, Jessica; Cui, Meng; Logothetis, Diomedes E.; Boland, Linda M.

    2015-01-01

    All vertebrate inwardly rectifying potassium (Kir) channels are activated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Logothetis, D. E., Petrou, V. I., Zhang, M., Mahajan, R., Meng, X. Y., Adney, S. K., Cui, M., and Baki, L. (2015) Annu. Rev. Physiol. 77, 81–104; Fürst, O., Mondou, B., and D'Avanzo, N. (2014) Front. Physiol. 4, 404–404). Structural components of a PIP2-binding site are conserved in vertebrate Kir channels but not in distantly related animals such as sponges and sea anemones. To expand our understanding of the structure-function relationships of PIP2 regulation of Kir channels, we studied AqKir, which was cloned from the marine sponge Amphimedon queenslandica, an animal that represents the phylogenetically oldest metazoans. A requirement for PIP2 in the maintenance of AqKir activity was examined in intact oocytes by activation of a co-expressed voltage-sensing phosphatase, application of wortmannin (at micromolar concentrations), and activation of a co-expressed muscarinic acetylcholine receptor. All three mechanisms to reduce the availability of PIP2 resulted in inhibition of AqKir current. However, time-dependent rundown of AqKir currents in inside-out patches could not be re-activated by direct application to the inside membrane surface of water-soluble dioctanoyl PIP2, and the current was incompletely re-activated by the more hydrophobic arachidonyl stearyl PIP2. When we introduced mutations to AqKir to restore two positive charges within the vertebrate PIP2-binding site, both forms of PIP2 strongly re-activated the mutant sponge channels in inside-out patches. Molecular dynamics simulations validate the additional hydrogen bonding potential of the sponge channel mutants. Thus, nature's mutations conferred a high affinity activation of vertebrate Kir channels by PIP2, and this is a more recent evolutionary development than the structures that explain ion channel selectivity and inward rectification. PMID:25957411

  8. The Ribotoxin Restrictocin Recognizes Its RNA Substrate by Selective Engagement of Active Site Residues

    PubMed Central

    2011-01-01

    Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of ∼1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein−RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes. PMID:21417210

  9. The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions.

    PubMed

    Backus, Keriann M; Dolan, Michael A; Barry, Conor S; Joe, Maju; McPhie, Peter; Boshoff, Helena I M; Lowary, Todd L; Davis, Benjamin G; Barry, Clifton E

    2014-09-05

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro(216)-Phe(228) loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.

  10. Alternative Selection of β-Site APP-Cleaving Enzyme 1 (BACE1) Cleavage Sites in Amyloid β-Protein Precursor (APP) Harboring Protective and Pathogenic Mutations within the Aβ Sequence.

    PubMed

    Kimura, Ayano; Hata, Saori; Suzuki, Toshiharu

    2016-11-11

    β-Site APP-cleaving enzyme 1 (BACE1) cleaves amyloid β-protein precursor (APP) at the bond between Met(671) and Asp(672) (β-site) to generate the carboxyl-terminal fragment (CTFβ/C99). BACE1 also cleaves APP at another bond between Thr(681) and Gln(682) (β'-site), yielding CTFβ'/C89. Cleavage of CTFβ/C99 by γ-secretase generates Aβ(1-XX), whereas cleavage of CTFβ'/C89 generates Aβ(11-XX). Thus, β'-site cleavage by BACE1 is amyloidolytic rather than amyloidogenic. β' cleavage of mouse APP is more common than the corresponding cleavage of human APP. We found that the H684R substitution within human Aβ, which replaces the histidine in the human protein with the arginine found at the corresponding position in mouse, facilitated β' cleavage irrespective of the species origin of BACE1, thereby significantly increasing the level of Aβ(11-XX) and decreasing the level of Aβ(1-XX). Thus, amino acid substitutions within the Aβ sequence influenced the selectivity of alternative β- or β'-site cleavage of APP by BACE1. In familial Alzheimer's disease (FAD), the APP gene harbors pathogenic variations such as the Swedish (K670N/M671L), Leuven (E682K), and A673V mutations, all of which decrease Aβ(11-40) generation, whereas the protective Icelandic mutation (A673T) increases generation of Aβ(11-40). Thus, A673T promotes β' cleavage of APP and protects subjects against AD. In addition, CTFβ/C99 was cleaved by excess BACE1 activity to generate CTFβ'/C89, followed by Aβ(11-40), even if APP harbored pathogenic mutations. The resultant Aβ(11-40) was more metabolically labile in vivo than Aβ(1-40). Our analysis suggests that some FAD mutations in APP are amyloidogenic and/or amyloidolytic via selection of alternative BACE1 cleavage sites.

  11. Novel Mutation Sites in the Development of Vancomycin- Intermediate Resistance in Staphylococcus aureus

    PubMed Central

    Wang, Yubing; Li, Xiaoli; Jiang, Libo; Han, Wentao; Xie, Xiangming; Jin, Yi; He, Xiaoqing; Wu, Rongling

    2017-01-01

    Increased use of vancomycin has led to the emergence of vancomycin-intermediate Staphylococcus aureus (VISA). To investigate the mechanism of VISA development, 39 methicillin-susceptible strains and 3 MRSA strains were treated with vancomycin to induce non-susceptibility, and mutations in six genes were analyzed. All the strains were treated with vancomycin in vitro for 60 days. MICs were determined by the agar dilution and E-test methods. Vancomycin was then removed to assess the stability of VISA strains and mutations. Following 60 days of vancomycin treatment in vitro, 29/42 VISA strains were generated. The complete sequences of rpoB, vraS, graR, graS, walK, and walR were compared with those in the parental strains. Seven missense mutations including four novel mutations (L466S in rpoB, R232K in graS, I594M in walk, and A111T in walR) were detected frequently in strains with vancomycin MIC ≥ 12 μg/mL. Jonckheere-Terpstra trend test indicated these mutations might play an important role during VISA evolution. After the vancomycin treatment, strains were passaged to vancomycin-free medium for another 60 days, and the MICs of all strains decreased. Our results suggest that rpoB, graS, walk, and walR are more important than vraS and graR in VISA development. PMID:28119680

  12. Characterisation of three novel splice site mutations in introns 11, 18 and 30 of the NF-1 gene

    SciTech Connect

    Purandare, S.M.; Lanyon, W.G.; Arngrimsson, R.

    1994-09-01

    Identification and characterization of germline mutations within the NF-1 gene was carried out in 25 unrelated NF-1 patients, in whom we have detected three splice site mutations which cause exon skipping. Our detection strategy incorporated both RNA and DNA as templates for PCR, chemical mismatch cleavage and direct sequencing. The first mutation was detected in the splice donor sequence of intron 11 (1721+3A{r_arrow}G), which results in the skipping of exon 11 and causes a shift in the translational reading frame and the creation of a premature stop codon at position 560. This is predicted to result in the synthesis of a shorter protein product of 559 amino acids instead of 2818, with loss of the NF-1 GAP related domain. The patient is a familial case of NF-1 with neurological complications and no evidence of malignancy. She has an affected son who has inherited the same mutation and has skeletal complications. The second mutation was detected at the splice donor site in intron 18 (3113+1G{r_arrow}A) and caused the skipping of exon 18. This did not cause a shift in the reading frame but resulted in the exclusion of 41 amino acids from the predicted protein product and was seen in a familial case of NF-1 with neurological complications. The third mutation, at the splice donor site in intron 30 (5749+2T{r_arrow}G), caused the skipping of exon 30, shifting the translational reading frame and creating a premature stop codon at position 1851. The predicted protein product is reduced from the normal 2818 to 1850 amino acids. This patient is a sporadic case of NF-1, has neurological and skeletal complications and no evidence of malignancy. Thus in our analysis of 25 patients, the strategy of using RT-PCR to amplify the NF-1 cDNA greatly facilitated the detection of these errors of splicing, each of which is predicted to cause a major distruption of the protein product neurofibromin.

  13. Mutations in the Drosophila alphaPS2 integrin subunit uncover new features of adhesion site assembly.

    PubMed

    Devenport, Danelle; Bunch, Thomas A; Bloor, James W; Brower, Danny L; Brown, Nicholas H

    2007-08-15

    The Drosophila alphaPS2betaPS integrin is required for diverse development events, including muscle attachment. We characterized six unusual mutations in the alphaPS2 gene that cause a subset of the null phenotype. One mutation changes a residue in alphaPS2 that is equivalent to the residue in alphaV that contacts the arginine of RGD. This change severely reduced alphaPS2betaPS affinity for soluble ligand, abolished the ability of the integrin to recruit laminin to muscle attachment sites in the embryo and caused detachment of integrins and talin from the ECM. Three mutations that alter different parts of the alphaPS2 beta-propeller, plus a fourth that eliminated a late phase of alphaPS2 expression, all led to a strong decrease in alphaPS2betaPS at muscle ends, but, surprisingly, normal levels of talin were recruited. Thus, although talin recruitment requires alphaPS2betaPS, talin levels are not simply specified by the amount of integrin at the adhesive junction. These mutations caused detachment of talin and actin from integrins, suggesting that the integrin-talin link is weaker than the ECM-integrin link.

  14. Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregation

    PubMed Central

    Sant’Anna, Ricardo; Almeida, Maria Rosário; Varejāo, Nathalia; Gallego, Pablo; Esperante, Sebastian; Ferreira, Priscila; Pereira-Henriques, Alda; Palhano, Fernando L.; de Carvalho, Mamede; Foguel, Debora; Reverter, David; Saraiva, Maria João; Ventura, Salvador

    2017-01-01

    More than a hundred different Transthyretin (TTR) mutations are associated with fatal systemic amyloidoses. They destabilize the protein tetrameric structure and promote the extracellular deposition of TTR as pathological amyloid fibrils. So far, only mutations R104H and T119M have been shown to stabilize significantly TTR, acting as disease suppressors. We describe a novel A108V non-pathogenic mutation found in a Portuguese subject. This variant is more stable than wild type TTR both in vitro and in human plasma, a feature that prevents its aggregation. The crystal structure of A108V reveals that this stabilization comes from novel intra and inter subunit contacts involving the thyroxine (T4) binding site. Exploiting this observation, we engineered a A108I mutation that fills the T4 binding cavity, as evidenced in the crystal structure. This synthetic protein becomes one of the most stable TTR variants described so far, with potential application in gene and protein replacement therapies. PMID:28338000

  15. Clonal analysis of NRAS activating mutations in KIT-D816V systemic mastocytosis

    PubMed Central

    Wilson, Todd M.; Maric, Irina; Simakova, Olga; Bai, Yun; Ching Chan, Eunice; Olivares, Nicolas; Carter, Melody; Maric, Dragan; Robyn, Jamie; Metcalfe, Dean D.

    2011-01-01

    Cooperating genetic events are likely to contribute to the phenotypic diversity of KIT-D816V systemic mastocytosis. In this study, 44 patients with KIT-D816V systemic mastocytosis were evaluated for coexisting NRAS, KRAS, HRAS or MRAS mutations. Activating NRAS mutations were identified in 2 of 8 patients with advanced disease. NRAS mutations were not found in patients with indolent systemic mastocytosis. To better understand the clonal evolution of mastocytosis, we evaluated the cell compartments impacted by the NRAS and KIT mutations. Clonal mast cells harbored both mutations. KIT-D816V was not detected in bone marrow CD34+ progenitors, whereas the NRAS mutation was present. These findings suggest that NRAS mutations may have the potential to precede KIT-D816V in clonal development. Unlike other mature lineages, mast cell survival is dependent on KIT and the presence of these two activating mutations may have a greater impact on the expansion of this cell compartment and in resultant disease severity. (Clinicaltrials.gov identifier: NCT00044122, NCT00001756) PMID:21134978

  16. A single splice site mutation in human-specific ARHGAP11B causes basal progenitor amplification

    PubMed Central

    Florio, Marta; Namba, Takashi; Pääbo, Svante; Hiller, Michael; Huttner, Wieland B.

    2016-01-01

    The gene ARHGAP11B promotes basal progenitor amplification and is implicated in neocortex expansion. It arose on the human evolutionary lineage by partial duplication of ARHGAP11A, which encodes a Rho guanosine triphosphatase–activating protein (RhoGAP). However, a lack of 55 nucleotides in ARHGAP11B mRNA leads to loss of RhoGAP activity by GAP domain truncation and addition of a human-specific carboxy-terminal amino acid sequence. We show that these 55 nucleotides are deleted by mRNA splicing due to a single C→G substitution that creates a novel splice donor site. We reconstructed an ancestral ARHGAP11B complementary DNA without this substitution. Ancestral ARHGAP11B exhibits RhoGAP activity but has no ability to increase basal progenitors during neocortex development. Hence, a single nucleotide substitution underlies the specific properties of ARHGAP11B that likely contributed to the evolutionary expansion of the human neocortex. PMID:27957544

  17. Balancing protein stability and activity in cancer: a new approach for identifying driver mutations affecting CBL ubiquitin ligase activation

    PubMed Central

    Li, Minghui; Kales, Stephen C.; Ma, Ke; Shoemaker, Benjamin A.; Crespo-Barreto, Juan; Cangelosi, Andrew L.; Lipkowitz, Stanley; Panchenko, Anna R.

    2015-01-01

    Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved depicting the protein at different stages of its activation cycle and thus provide mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than did random non-cancer mutations. We further tested the ability of these computational models assessing the changes in CBL stability and its binding to ubiquitin conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two-thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL. PMID:26676746

  18. Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction.

    PubMed

    Chen, Shirui; Gendelman, Hannah K; Roche, John P; Alsharif, Peter; Graf, Ethan R

    2015-01-01

    At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.

  19. Characterization of the Glycosylation Site of Human PSA Prompted by Missense Mutation using LC-MS/MS.

    PubMed

    Song, Ehwang; Hu, Yunli; Hussein, Ahmed; Yu, Chuan-Yih; Tang, Haixu; Mechref, Yehia

    2015-07-02

    Prostate specific antigen (PSA) is currently used as a diagnostic biomarker for prostate cancer. It is a glycoprotein possessing a single glycosylation site at N69. During our previous study of PSA N69 glycosylation, additional glycopeptides were observed in the PSA sample that were not previously reported and did not match glycopeptides of impure glycoproteins existing in the sample. This extra glycosylation site of PSA is associated with a mutation in KLK3 genes. Among single nucleotide polymorphisms (SNPs) of KLKs families, the rs61752561 in KLK3 genes is an unusual missense mutation resulting in the conversion of D102 to N in PSA amino acid sequence. Accordingly, a new N-linked glycosylation site is created with an N102MS motif. Here we report the first qualitative and quantitative glycoproteomic study of PSA N102 glycosylation site by LC-MS/MS. We successfully applied tandem MS to verify the amino acid sequence possessing N102 glycosylation site and associated glycoforms of PSA samples acquired from different suppliers. Among the three PSA samples, HexNAc2Hex5 was the predominant glycoform at N102, while HexNAc4Hex5Fuc1NeuAc1 or HexNAc4Hex5Fuc1NeuAc2 was the primary glycoforms at N69. D102 is the first amino acid of "kallikrein loop", which is close to a zinc-binding site and catalytic triad. The different glycosylation of N102 relative to N69 might be influenced by the close vicinity of N102 to these functional sites and steric hindrance.

  20. Familial Alzheimer’s mutations within APPTM increase Aβ42 production by enhancing accessibility of ɛ-cleavage site

    NASA Astrophysics Data System (ADS)

    Chen, Wen; Gamache, Eric; Rosenman, David J.; Xie, Jian; Lopez, Maria M.; Li, Yue-Ming; Wang, Chunyu

    2014-01-01

    The high Aβ42/Aβ40 production ratio is a hallmark of familial Alzheimer’s disease, which can be caused by mutations in the amyloid precursor protein (APP). The C-terminus of Aβ is generated by γ-secretase cleavage within the transmembrane domain of APP (APPTM), a process that is primed by an initial ɛ-cleavage at either T48 or L49, resulting in subsequent production of Aβ42 or Aβ40, respectively. Here we solve the dimer structures of wild-type APPTM (AAPTM WT) and mutant APPTM (FAD mutants V44M) with solution NMR. The right-handed APPTM helical dimer is mediated by GXXXA motif. From the NMR structural and dynamic data, we show that the V44M and V44A mutations can selectively expose the T48 site by weakening helical hydrogen bonds and increasing hydrogen-deuterium exchange rate (kex). We propose a structural model in which FAD mutations (V44M and V44A) can open the T48 site γ-secretase for the initial ɛ-cleavage, and consequently shift cleavage preference towards Aβ42.

  1. A novel splicing site mutation of the GPR143 gene in a Chinese X-linked ocular albinism pedigree.

    PubMed

    Cai, C Y; Zhu, H; Shi, W; Su, L; Shi, O; Cai, C Q; Ling, C; Li, W D

    2013-11-18

    Ocular albinism is an X-linked inherited disease characterized by hypopigmentation of the iris and nystagmus. To identify a new disease-causing mutation of ocular albinism, we collected a Han Chinese pedigree with 7 male congenital nystagmus patients over 3 generations. Slit-lamp photography and optical coherence tomography were performed for the proband. Genomic DNA was extracted from a whole blood sample from the proband using the high-salt method. Polymerase chain reaction (PCR) sequencing was carried out for GPR143 and FRMD7 genes. The three-dimensional structures of the wild-type and mutant GPR143 proteins were determined using SWISS-MODEL. The transmission of the disease in the pedigree clearly followed an X-linked pattern. The proband had significant iris and fundus hypopigmentation. Optical coherence tomography showed severe foveal hypoplasias in both eyes of the proband. A novel splicing site (G/C) mutation was found on the boundary of the 6th intron and the 7th exon of the GPR143 gene, resulting in a 9-amino-acid deletion (codons 257-265) in the 6th transmembrane domain of the GPR143 protein. In conclusion, a novel splicing site mutation of the GPR143 gene was found in a Han Chinese congenital ocular albinism pedigree.

  2. A presenilin-1 mutation identified in familial Alzheimer disease with cotton wool plaques causes a nearly complete loss of gamma-secretase activity.

    PubMed

    Heilig, Elizabeth A; Xia, Weiming; Shen, Jie; Kelleher, Raymond J

    2010-07-16

    Mutations in presenilin-1 and presenilin-2 (PS1 and PS2) are the most common cause of familial Alzheimer disease. PS1 and PS2 are the presumptive catalytic components of the multisubunit gamma-secretase complex, which proteolyzes a number of type I transmembrane proteins, including the amyloid precursor protein (APP) and Notch. APP processing by gamma-secretase produces beta-amyloid peptides (Abeta40 and Abeta42) that accumulate in the Alzheimer disease brain. Here we identify a pathogenic L435F mutation in PS1 in two affected siblings with early-onset familial Alzheimer disease characterized by deposition of cerebral cotton wool plaques. The L435F mutation resides in a conserved C-terminal PAL sequence implicated in active site conformation and catalytic activity. The impact of PS1 mutations in and around the PAL motif on gamma-secretase activity was assessed by expression of mutant PS1 in mouse embryo fibroblasts lacking endogenous PS1 and PS2. Surprisingly, the L435F mutation caused a nearly complete loss of gamma-secretase activity, including >90% reductions in the generation of Abeta40, Abeta42, and the APP and Notch intracellular domains. Two nonpathogenic PS1 mutations, P433L and L435R, caused essentially complete loss of gamma-secretase activity, whereas two previously identified pathogenic PS1 mutations, P436Q and P436S, caused partial loss of function with substantial reductions in production of Abeta40, Abeta42, and the APP and Notch intracellular domains. These results argue against overproduction of Abeta42 as an essential property of presenilin proteins bearing pathogenic mutations. Rather, our findings provide support for the hypothesis that pathogenic mutations cause a general loss of presenilin function.

  3. A Presenilin-1 Mutation Identified in Familial Alzheimer Disease with Cotton Wool Plaques Causes a Nearly Complete Loss of γ-Secretase Activity*

    PubMed Central

    Heilig, Elizabeth A.; Xia, Weiming; Shen, Jie; Kelleher, Raymond J.

    2010-01-01

    Mutations in presenilin-1 and presenilin-2 (PS1 and PS2) are the most common cause of familial Alzheimer disease. PS1 and PS2 are the presumptive catalytic components of the multisubunit γ-secretase complex, which proteolyzes a number of type I transmembrane proteins, including the amyloid precursor protein (APP) and Notch. APP processing by γ-secretase produces β-amyloid peptides (Aβ40 and Aβ42) that accumulate in the Alzheimer disease brain. Here we identify a pathogenic L435F mutation in PS1 in two affected siblings with early-onset familial Alzheimer disease characterized by deposition of cerebral cotton wool plaques. The L435F mutation resides in a conserved C-terminal PAL sequence implicated in active site conformation and catalytic activity. The impact of PS1 mutations in and around the PAL motif on γ-secretase activity was assessed by expression of mutant PS1 in mouse embryo fibroblasts lacking endogenous PS1 and PS2. Surprisingly, the L435F mutation caused a nearly complete loss of γ-secretase activity, including >90% reductions in the generation of Aβ40, Aβ42, and the APP and Notch intracellular domains. Two nonpathogenic PS1 mutations, P433L and L435R, caused essentially complete loss of γ-secretase activity, whereas two previously identified pathogenic PS1 mutations, P436Q and P436S, caused partial loss of function with substantial reductions in production of Aβ40, Aβ42, and the APP and Notch intracellular domains. These results argue against overproduction of Aβ42 as an essential property of presenilin proteins bearing pathogenic mutations. Rather, our findings provide support for the hypothesis that pathogenic mutations cause a general loss of presenilin function. PMID:20460383

  4. recA mutations that reduce the constitutive coprotease activity of the RecA1202(Prtc) protein: possible involvement of interfilament association in proteolytic and recombination activities.

    PubMed Central

    Liu, S K; Eisen, J A; Hanawalt, P C; Tessman, I

    1993-01-01

    Twenty-eight recA mutants, isolated after spontaneous mutagenesis generated by the combined action of RecA1202(Prtc) and UmuDC proteins, were characterized and sequenced. The mutations are intragenic suppressors of the recA1202 allele and were detected by the reduced coprotease activity of the gene product. Twenty distinct mutation sites were found, among which two mutations, recA1620 (V-275-->D) and recA1631 (I-284-->N), were mapped in the C-terminal portion of the interfilament contact region (IFCR) in the RecA crystal. An interaction of this region with the part of the IFCR in which the recA1202 mutation (Q-184-->K) is mapped could occur only intermolecularly. Thus, altered IFCR and the likely resulting change in interfilament association appear to be important aspects of the formation of a constitutively active RecA coprotease. This observation is consistent with the filament-bundle theory (R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 335:318-325, 1992). Furthermore, we found that among the 20 suppressor mutations, 3 missense mutations that lead to recombination-defective (Rec-) phenotypes also mapped in the IFCR, suggesting that the IFCR, with its putative function in interfilament association, is required for the recombinase activity of RecA. We propose that RecA-DNA complexes may form bundles analogous to the RecA bundles (lacking DNA) described by Story et al. and that these RecA-DNA bundles play a role in homologous recombination. Images PMID:8407828

  5. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  6. DOE site performance assessment activities. Radioactive Waste Technical Support Program

    SciTech Connect

    Not Available

    1990-07-01

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions.

  7. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D&D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities.

  8. Mutations Conferring Resistance to SCH6, a Novel Hepatitis C Virus NS3/4A Protease Inhibitor: Reduced DNA Replication Fitness and Partial Rescue by Second-Site Mutations

    SciTech Connect

    Yi, MinKyung; Tong, Xiao; Skelton, Angela; Chase, Robert; Chen, Tong; Prongay, Andrew; Bogen, Stephane L.; Saksena, Anil K.; Njoroge, F. George; Veselenak, Ronald L.; Pyles, Richard B.; Bourne, Nigel; Malcolm, Bruce A.; Lemon, Stanley M.

    2008-06-30

    Drug resistance is a major issue in the development and use of specific antiviral therapies. Here we report the isolation and characterization of hepatitis C virus RNA replicons resistant to a novel ketoamide inhibitor of the NS3/4A protease, SCH6 (originally SCH446211). Resistant replicon RNAs were generated by G418 selection in the presence of SCH6 in a dose-dependent fashion, with the emergence of resistance reduced at higher SCH6 concentrations. Sequencing demonstrated remarkable consistency in the mutations conferring SCH6 resistance in genotype 1b replicons derived from two different strains of hepatitis C virus, A156T/A156V and R109K. R109K, a novel mutation not reported previously to cause resistance to NS3/4A inhibitors, conferred moderate resistance only to SCH6. Structural analysis indicated that this reflects unique interactions of SCH6 with P{prime}-side residues in the protease active site. In contrast, A156T conferred high level resistance to SCH6 and a related ketoamide, SCH503034, as well as BILN 2061 and VX-950. Unlike R109K, which had minimal impact on NS3/4A enzymatic function, A156T significantly reduced NS3/4A catalytic efficiency, polyprotein processing, and replicon fitness. However, three separate second-site mutations, P89L, Q86R, and G162R, were capable of partially reversing A156T-associated defects in polyprotein processing and/or replicon fitness, without significantly reducing resistance to the protease inhibitor.

  9. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.

  10. Outline structure of the human L1 cell adhesion molecule and the sites where mutations cause neurological disorders.

    PubMed Central

    Bateman, A; Jouet, M; MacFarlane, J; Du, J S; Kenwrick, S; Chothia, C

    1996-01-01

    The L1 cell adhesion molecule has six domains homologous to members of the immunoglobulin superfamily and five homologous to fibronectin type III domains. We determined the outline structure of the L1 domains by showing that they have, at the key sites that determine conformation, residues similar to those in proteins of known structure. The outline structure describes the relative positions of residues, the major secondary structures and residue solvent accessibility. We use the outline structure to investigate the likely effects of 22 mutations that cause neurological diseases. The mutations are not randomly distributed but cluster in a few regions of the structure. They can be divided into those that act mainly by changing conformation or denaturing their domain and those that alter its surface properties. Images PMID:8947027

  11. Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex

    PubMed Central

    Lim, Sung In; Yang, Byungseop; Jung, Younghan; Cha, Jaehyun; Cho, Jinhwan; Choi, Eun-Sil; Kim, Yong Hwan; Kwon, Inchan

    2016-01-01

    Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other. PMID:28004799

  12. Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

    PubMed Central

    Moomaw, Ellen W.; Hoffer, Eric; Moussatche, Patricia; Salerno, John C.; Grant, Morgan; Immelman, Bridget; Uberto, Richard; Ozarowski, Andrew; Angerhofer, Alexander

    2013-01-01

    Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site. PMID:23469254

  13. Mutation of a Single Envelope N-Linked Glycosylation Site Enhances the Pathogenicity of Bovine Leukemia Virus

    PubMed Central

    Bouzar, Amel Baya; Jacques, Jean-Rock; Cosse, Jean-Philippe; Gillet, Nicolas; Callebaut, Isabelle; Reichert, Michal

    2015-01-01

    ABSTRACT Viruses have coevolved with their host to ensure efficient replication and transmission without inducing excessive pathogenicity that would indirectly impair their persistence. This is exemplified by the bovine leukemia virus (BLV) system in which lymphoproliferative disorders develop in ruminants after latency periods of several years. In principle, the equilibrium reached between the virus and its host could be disrupted by emergence of more pathogenic strains. Intriguingly but fortunately, such a hyperpathogenic BLV strain was never observed in the field or designed in vitro. In this study, we sought to understand the role of envelope N-linked glycosylation with the hypothesis that this posttranslational modification could either favor BLV infection by allowing viral entry or allow immune escape by using glycans as a shield. Using reverse genetics of an infectious molecular provirus, we identified a N-linked envelope glycosylation site (N230) that limits viral replication and pathogenicity. Indeed, mutation N230E unexpectedly leads to enhanced fusogenicity and protein stability. IMPORTANCE Infection by retroviruses requires the interaction of the viral envelope protein (SU) with a membrane-associated receptor allowing fusion and release of the viral genomic RNA into the cell. We show that N-linked glycosylation of the bovine leukemia virus (BLV) SU protein is, as expected, essential for cell infection in vitro. Consistently, mutation of all glycosylation sites of a BLV provirus destroys infectivity in vivo. However, single mutations do not significantly modify replication in vivo. Instead, a particular mutation at SU codon 230 increases replication and accelerates pathogenesis. This unexpected observation has important consequences in terms of disease control and managing. PMID:26085161

  14. Characterization of the Dielectric Constant in the Trichoderma reesei Cel7B Active Site.

    PubMed

    Song, Xiangfei; Wang, Yefei; Zhang, Shujun; Yan, Shihai; Li, Tong; Yao, Lishan

    2015-07-27

    An attempt is made to evaluate the dielectric constant of the Trichoderma reesei Cel7B active site. Through kinetic measurements, the pKa value of the catalytic acid E201 is determined. Mutations (away from E201) with net charge changes are introduced to perturb the E201 pKa. It is shown that the mutation with a +1 charge change (including G225R, G230R, and A335R) decreases the pKa of E201, whereas the mutation with a -1 charge change (including Q149E, A222D, G225D, and G230D) increases the pKa. This effect is consistent with the electrostatic interaction between the changed charge and the E201 side chain. The fitting of the experimental data yields an apparent dielectric constant of 25-80. Molecular dynamics simulations with explicit water molecules indicate that the high solvent accessibility of the active site contributes largely to the high dielectric constant. ONIOM calculations show that high dielectric constant benefits the catalysis through decreasing the energy of the transition state relative to that of the enzyme substrate complex.

  15. Selectivity of fungal sesquiterpene synthases: role of the active site's H-1 alpha loop in catalysis.

    PubMed

    López-Gallego, Fernando; Wawrzyn, Grayson T; Schmidt-Dannert, Claudia

    2010-12-01

    Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-α1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-α1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-α1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-α1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-α1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases.

  16. Effect of the G375C and G346E achondroplasia mutations on FGFR3 activation.

    PubMed

    He, Lijuan; Serrano, Christopher; Niphadkar, Nitish; Shobnam, Nadia; Hristova, Kalina

    2012-01-01

    Two mutations in FGFR3, G380R and G375C are known to cause achondroplasia, the most common form of human dwarfism. The G380R mutation accounts for 98% of the achondroplasia cases, and thus has been studied extensively. Here we study the effect of the G375C mutation on the phosphorylation and the cross-linking propensity of full-length FGFR3 in HEK 293 cells, and we compare the results to previously published results for the G380R mutant. We observe identical behavior of the two achondroplasia mutants in these experiments, a finding which supports a direct link between the severity of dwarfism phenotypes and the level and mechanism of FGFR3 over-activation. The mutations do not increase the cross-linking propensity of FGFR3, contrary to previous expectations that the achondroplasia mutations stabilize the FGFR3 dimers. Instead, the phosphorylation efficiency within un-liganded FGFR3 dimers is increased, and this increase is likely the underlying cause for pathogenesis in achondroplasia. We further investigate the G346E mutation, which has been reported to cause achondroplasia in one case. We find that this mutation does not increase FGFR3 phosphorylation and decreases FGFR3 cross-linking propensity, a finding which raises questions whether this mutation is indeed a genetic cause for human dwarfism.

  17. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    PubMed Central

    Lin, Xiaofeng; Ayrapetov, Marina K; Sun, Gongqin

    2005-01-01

    Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1) binds to ATP, and the other (M2) acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator. PMID:16305747

  18. Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells.

    PubMed Central

    Maruyama, K; MacLennan, D H

    1988-01-01

    Full-length cDNAs encoding neonatal and adult isoforms of the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum were expressed transiently in COS-1 cells. The microsomal fraction isolated from transfected COS-1 cells contained immunoreactive Ca2+-ATPase and catalyzed Ca2+ transport at rates at least 15-fold above controls. No differences were observed in either the rates or Ca2+ dependency of Ca2+ transport catalyzed by the two isoforms. Aspartic acid-351, the site of formation of the catalytic acyl phosphate in the enzyme, was mutated to asparagine, glutamic acid, serine, threonine, histidine, or alanine. In every case, Ca2+ transport activity and Ca2+-dependent phosphorylation were eliminated. Ca2+ transport was also eliminated by mutation of lysine-352 to arginine, glutamine, or glutamic acid or by mutation of Asp351-Lys352 to Lys351-Asp352. Mutation of lysine-515, the site of fluorescein isothiocyanate modification in the enzyme, resulted in diminished Ca2+ transport activity as follows: arginine, 60%; glutamine, 25%; glutamic acid, 5%. These results demonstrate the absolute requirement of acylphosphate formation for the Ca2+ transport function and define a residue important for ATP binding. They also demonstrate the feasibility of a thorough analysis of active sites in the Ca2+-ATPase by expression and site-specific mutagenesis. Images PMID:2966962

  19. Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene

    PubMed Central

    Känsäkoski, Johanna; Jääskeläinen, Jarmo; Jääskeläinen, Tiina; Tommiska, Johanna; Saarinen, Lilli; Lehtonen, Rainer; Hautaniemi, Sampsa; Frilander, Mikko J.; Palvimo, Jorma J.; Toppari, Jorma; Raivio, Taneli

    2016-01-01

    Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation. PMID:27609317

  20. Substrate shuttling between active sites of uroporphyrinogen decarboxylase is not required to generate coproporphyrinogen

    PubMed Central

    Phillips, John D.; Warby, Christy A.; Whitby, Frank G.; Kushner, James P.; Hill, Christopher P.

    2009-01-01

    Summary Uroporphyrinogen Decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of the four acetate side chains on the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer with the active site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single chain protein (scURO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposible with wild-type activity and have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of scURO-D resulted in approximately half of wild-type activity. The distribution of reaction intermediates was the same for mutant and wild-type sequences, and was unaltered in a competition experiment using the I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function, and suggest that the dimeric structure of URO-D is required to achieve conformational stability and create a large active site cleft. PMID:19362562

  1. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    SciTech Connect

    Phillips, J.; Warby, C; Whitby, F; Kushner, J; Hill, C

    2009-01-01

    Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.

  2. Characterization of sulfonylurea-resistant Schoenoplectus juncoides having a target-site Asp(376)Glu mutation in the acetolactate synthase.

    PubMed

    Sada, Yoshinao; Ikeda, Hajime; Yamato, Seiji; Kizawa, Satoru

    2013-09-01

    Schoenoplectus juncoides, a noxious weed for paddy rice, is known to become resistant to sulfonylurea (SU) herbicides by a target-site mutation in either of the two acetolactate synthase (ALS) genes (ALS1 and ALS2). SU-resistant S. juncoides plants having an Asp376Glu mutation in ALS2 were found from a paddy rice field in Japan, but their resistance profile has not been quantitatively investigated. In this study, dose-response of the SU-resistant accession was compared with that of a SU-susceptible accession at in vivo whole-plant level as well as at in vitro enzymatic level. In whole-plant tests, resistance factors (RFs) based on 50% growth reduction (GR50) for imazosulfuron (ISF), bensulfuron-methyl (BSM), metsulfuron-methyl (MSM), bispyribac-sodium (BPS), and imazaquin (IMQ) were 176, 40, 14, 5.2 and 1.5, respectively. Thus, the accession having an Asp376Glu mutation in ALS2 was highly resistant to the three SU herbicides and moderately resistant to BPS, but was not substantially resistant to IMQ. This is slightly different from the earlier results reported from other weeds with an Asp376Glu mutation, in which the mutation confers resistance to broadly all the chemical classes of ALS-inhibiting herbicides. In enzymatic tests, ALS2 of S. juncoides was expressed in E. coli; the resultant ALS2 was subjected to an in vitro assay. RFs of the mutated ALS2 based on 50% enzymatic inhibition (I50) for ISF, BSM, MSM, BPS, and IMQ were 3699, 2438, 322, 80, and 4.8, respectively. The RFs of ALS2 were highly correlated with those of the whole-plant; this suggests that the Asp376Glu mutation in ALS2 is a molecular basis for the whole-plant resistance. The presence of two ALS genes in S. juncoides can at least partially explain why the whole-plant RFs were less than those of the expressed ALS2 enzymes.

  3. Recurrent mTORC1-activating RRAGC mutations in follicular lymphoma

    PubMed Central

    Wang, Jun; Araf, Shamzah; Wilkins, Lucy; Castellano, Brian M.; Escudero-Ibarz, Leire; Al Seraihi, Ahad Fahad; Richter, Julia; Bernhart, Stephan H.; Efeyan, Alejo; Iqbal, Sameena; Matthews, Janet; Clear, Andrew; Guerra-Assunção, José Afonso; Bödör, Csaba; Quentmeier, Hilmar; Mansbridge, Christopher; Johnson, Peter; Davies, Andrew; Strefford, Jonathan C.; Packham, Graham; Barrans, Sharon; Jack, Andrew; Du, Ming-Qing; Calaminici, Maria; Lister, T. Andrew; Auer, Rebecca; Montoto, Silvia; Gribben, John G.; Siebert, Reiner; Chelala, Claude; Zoncu, Roberto; Sabatini, David M.; Fitzgibbon, Jude

    2015-01-01

    Follicular lymphoma is an incurable B-cell malignancy1 characterized by the t(14;18) and mutations in one or more components of the epigenome2,3. Whilst frequent gene mutations in signaling pathways, including JAK-STAT, NOTCH and NF-κB, have also been defined2-7, the spectrum of these mutations typically overlap with the closely-related diffuse large B cell lymphoma (DLBCL)6-13. A combination of discovery exome and extended targeted sequencing revealed recurrent somatic mutations in RRAGC uniquely enriched in FL patients (17%). More than half of the mutations preferentially co-occurred with ATP6V1B2 and ATP6AP1 mutations, components of the vacuolar H+-adenosine triphosphate ATPase (v-ATPase) known to be necessary for amino acid-induced mTORC1 activation. The RagC mutants increased raptor binding whilst rendering mTORC1 signaling resistant to amino acid deprivation. Collectively, the activating nature of the RRAGC mutations, their existence within the dominant clone and stability during disease progression supports their potential as an excellent candidate to be therapeutically exploited. PMID:26691987

  4. Perturbed Length–Dependent Activation in Human Hypertrophic Cardiomyopathy With Missense Sarcomeric Gene Mutations

    PubMed Central

    Sequeira, Vasco; Wijnker, Paul J.M.; Nijenkamp, Louise L.A.M.; Kuster, Diederik W.D.; Najafi, Aref; Witjas-Paalberends, E. Rosalie; Regan, Jessica A.; Boontje, Nicky; ten Cate, Folkert J.; Germans, Tjeerd; Carrier, Lucie; Sadayappan, Sakthivel; van Slegtenhorst, Marjon A.; Zaremba, Ruud; Foster, D. Brian; Murphy, Anne M.; Poggesi, Corrado; dos Remedios, Cris; Stienen, Ger J.M.; Ho, Carolyn Y.; Michels, Michelle; van der Velden, Jolanda

    2013-01-01

    Rationale High-myofilament Ca2+-sensitivity has been proposed as trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) based on in vitro and transgenic mice studies. However, myofilament Ca2+-sensitivity depends on protein phosphorylation and muscle length, and at present, data in human are scarce. Objective To investigate whether high-myofilament Ca2+-sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick- and thin-filament proteins. Methods and Results Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca2+-sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA)-targets compared with donors. After exogenous PKA treatment, myofilament Ca2+-sensitivity was either similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations, but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values. Conclusions High-myofilament Ca2+-sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA-targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via post-translational modifications other than PKA-hypophosphorylation or altered protein–protein interactions, and represents a

  5. Perspective: On the active site model in computational catalyst screening

    NASA Astrophysics Data System (ADS)

    Reuter, Karsten; Plaisance, Craig P.; Oberhofer, Harald; Andersen, Mie

    2017-01-01

    First-principles screening approaches exploiting energy trends in surface adsorption represent an unparalleled success story in recent computational catalysis research. Here we argue that our still limited understanding of the structure of active sites is one of the major bottlenecks towards an ever extended and reliable use of such computational screening for catalyst discovery. For low-index transition metal surfaces, the prevalently chosen high-symmetry (terrace and step) sites offered by the nominal bulk-truncated crystal lattice might be justified. For more complex surfaces and composite catalyst materials, computational screening studies will need to actively embrace a considerable uncertainty with respect to what truly are the active sites. By systematically exploring the space of possible active site motifs, such studies might eventually contribute towards a targeted design of optimized sites in future catalysts.

  6. Diffusional correlations among multiple active sites in a single enzyme.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  7. Trimer Enhancement Mutation Effects on HIV-1 Matrix Protein Binding Activities

    PubMed Central

    Alfadhli, Ayna; Mack, Andrew; Ritchie, Christopher; Cylinder, Isabel; Harper, Logan; Tedbury, Philip R.; Freed, Eric O.

    2016-01-01

    ABSTRACT The HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein cytoplasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA mutations that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus particles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation. IMPORTANCE The HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of Env CT domains with the MA domains of the viral precursor Gag proteins. Despite this knowledge, little is known about the mechanisms by which MA facilitates the virion incorporation of Env proteins. To help elucidate this process, we examined the binding activities of an MA mutant that stabilizes MA trimers. We found that the mutant proteins organized similarly to WT proteins on membranes, and that mutant and WT proteins revealed only slight differences in their binding to RNAs or lipids. However, the mutant proteins showed

  8. An Activating Mutation in STAT3 Results in Neonatal Diabetes Through Reduced Insulin Synthesis.

    PubMed

    Velayos, Teresa; Martínez, Rosa; Alonso, Milagros; Garcia-Etxebarria, Koldo; Aguayo, Anibal; Camarero, Cristina; Urrutia, Inés; Martínez de LaPiscina, Idoia; Barrio, Raquel; Santin, Izortze; Castaño, Luis

    2017-04-01

    Neonatal diabetes mellitus (NDM) is a rare form of diabetes diagnosed within the first 6 months of life. Genetic studies have allowed the identification of several genes linked to the development of NDM; however, genetic causes for ∼20% of the cases remain to be clarified. Most cases of NDM involve isolated diabetes, but sometimes NDM appears in association with other pathological conditions, including autoimmune diseases. Recent reports have linked activating mutations in STAT3 with early-onset autoimmune disorders that include diabetes of autoimmune origin, but the functional impact of STAT3-activating mutations have not been characterized at the pancreatic β-cell level. By using whole-exome sequencing, we identified a novel missense mutation in the binding domain of the STAT3 protein in a patient with NDM. The functional analyses showed that the mutation results in an aberrant activation of STAT3, leading to deleterious downstream effects in pancreatic β-cells. The identified mutation leads to hyperinhibition of the transcription factor Isl-1 and, consequently, to a decrease in insulin expression. These findings represent the first functional indication of a direct link between an NDM-linked activating mutation in STAT3 and pancreatic β-cell dysfunction.

  9. Use of human tissue to assess the oncogenic activity of melanoma-associated mutations.

    PubMed

    Chudnovsky, Yakov; Adams, Amy E; Robbins, Paul B; Lin, Qun; Khavari, Paul A

    2005-07-01

    Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Induction of Ras and Raf can be caused by active N-Ras and B-Raf mutants as well as by gene amplification. Activation of PI3K pathway components occurs by PTEN loss and by AKT3 amplification. Melanomas also commonly show impairment of the p16(INK4A)-CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16(INK4A) and ARF protein loss. Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification. In addition to ARF deletion, p53 pathway disruption can result from dominant negative TP53 mutations. TERT amplification also occurs in melanoma. The extent to which these mutations can induce human melanocytic neoplasia is unknown. Here we characterize pathways sufficient to generate human melanocytic neoplasia and show that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.

  10. Neurologic syndrome associated with homozygous mutation at MAG sialic acid binding site.

    PubMed

    Roda, Ricardo H; FitzGibbon, Edmond J; Boucekkine, Houda; Schindler, Alice B; Blackstone, Craig

    2016-08-01

    The MAG gene encodes myelin-associated glycoprotein (MAG), an abundant protein involved in axon-glial interactions and myelination during nerve regeneration. Several members of a consanguineous family with a clinical syndrome reminiscent of Pelizaeus-Merzbacher disease and demyelinating leukodystrophy on brain MRI were recently found to harbor a homozygous missense p.Ser133Arg MAG mutation. Here, we report two brothers from a nonconsanguineous family afflicted with progressive cognitive impairment, neuropathy, ataxia, nystagmus, and gait disorder. Exome sequencing revealed the homozygous missense mutation p.Arg118His in MAG. This Arg118 residue in immunoglobulin domain 1 is critical for sialic acid binding, providing a compelling mechanistic basis for disease pathogenesis.

  11. Robotics at Savannah River site: activity report

    SciTech Connect

    Byrd, J.S.

    1984-09-01

    The objectives of the Robotics Technology Group at the Savannah River Laboratory are to employ modern industrial robots and to develop unique automation and robotic systems to enhance process operations at the Savannah River site (SRP and SRL). The incentives are to improve safety, reduce personnel radiation exposure, improve product quality and productivity, and to reduce operating costs. During the past year robotic systems have been installed to fill chemical dilution vials in a SRP laboratory at 772-F and remove radioactive waste materials in the SRL Californium Production Facility at 773-A. A robotic system to lubricate an extrusion press has been developed and demonstrated in the SRL robotics laboratory and is scheduled for installation at the 321-M fuel fabrication area. A mobile robot was employed by SRP for a radiation monitoring task at a waste tank top in H-Area. Several other robots are installed in the SRL robotics laboratories and application development programs are underway. The status of these applications is presented in this report.

  12. Response to everolimus is seen in TSC-associated SEGAs and angiomyolipomas independent of mutation type and site in TSC1 and TSC2.

    PubMed

    Kwiatkowski, David J; Palmer, Michael R; Jozwiak, Sergiusz; Bissler, John; Franz, David; Segal, Scott; Chen, David; Sampson, Julian R

    2015-12-01

    Tuberous sclerosis complex is an autosomal dominant disorder that occurs owing to inactivating mutations in either TSC1 or TSC2. Tuberous sclerosis complex-related tumors in the brain, such as subependymal giant cell astrocytoma, and in the kidney, such as angiomyolipoma, can cause significant morbidity and mortality. Recently, randomized clinical trials (EXIST-1 and EXIST-2) of everolimus for each of these tuberous sclerosis complex-associated tumors demonstrated the benefit of this drug, which blocks activated mammalian target of rapamycin complex 1. Here we report on the spectrum of mutations seen in patients treated during these trials and the association between mutation and response. TSC2 mutations were predominant among patients in both trials and were present in nearly all subjects with angiomyolipoma in whom a mutation was identified (97%), whereas TSC1 mutations were rare in those subjects (3%). The spectrum of mutations seen in each gene was similar to those previously reported. In both trials, there was no apparent association between mutation type or location within each gene and response to everolimus. Everolimus responses were also seen at a similar frequency for the 16-18% of patients in each trial in whom no mutation in either gene was identified. These observations confirm the strong association between TSC2 mutation and angiomyolipoma burden seen in previous studies, and they indicate that everolimus response occurs regardless of mutation type or location or when no mutation in TSC1 or TSC2 has been identified.

  13. SQSTM1 splice site mutation in distal myopathy with rimmed vacuoles

    PubMed Central

    Bucelli, Robert C.; Arhzaouy, Khalid; Pestronk, Alan; Pittman, Sara K.; Rojas, Luisa; Sue, Carolyn M.; Evilä, Anni; Hackman, Peter; Udd, Bjarne; Harms, Matthew B.

    2015-01-01

    Objective: To identify the genetic etiology and characterize the clinicopathologic features of a novel distal myopathy. Methods: We performed whole-exome sequencing on a family with an autosomal dominant distal myopathy and targeted exome sequencing in 1 patient with sporadic distal myopathy, both with rimmed vacuolar pathology. We also evaluated the pathogenicity of identified mutations using immunohistochemistry, Western blot analysis, and expression studies. Results: Sequencing identified a likely pathogenic c.1165+1 G>A splice donor variant in SQSTM1 in the affected members of 1 family and in an unrelated patient with sporadic distal myopathy. Affected patients had late-onset distal lower extremity weakness, myopathic features on EMG, and muscle pathology demonstrating rimmed vacuoles with both TAR DNA-binding protein 43 and SQSTM1 inclusions. The c.1165+1 G>A SQSTM1 variant results in the expression of 2 alternatively spliced SQSTM1 proteins: 1 lacking the C-terminal PEST2 domain and another lacking the C-terminal ubiquitin-associated (UBA) domain, both of which have distinct patterns of cellular and skeletal muscle localization. Conclusions: SQSTM1 is an autophagic adaptor that shuttles aggregated and ubiquitinated proteins to the autophagosome for degradation via its C-terminal UBA domain. Similar to mutations in VCP, dominantly inherited mutations in SQSTM1 are now associated with rimmed vacuolar myopathy, Paget disease of bone, amyotrophic lateral sclerosis, and frontotemporal dementia. Our data further suggest a pathogenic connection between the disparate phenotypes. PMID:26208961

  14. Mutations increasing exposure of a receptor binding site epitope in the soluble and oligomeric forms of the caprine arthritis-encephalitis lentivirus envelope glycoprotein

    SciTech Connect

    Hoetzel, Isidro . E-mail: ihotzel@gene.com; Cheevers, William P.

    2005-09-01

    The caprine arthritis-encephalitis (CAEV) and ovine maedi-visna (MVV) viruses are resistant to antibody neutralization, a feature shared with all other lentiviruses. Whether the CAEV gp135 receptor binding site(s) (RBS) in the functional surface envelope glycoprotein (Env) is protected from antibody binding, allowing the virus to resist neutralization, is not known. Two CAEV gp135 regions were identified by extrapolating a gp135 structural model that could affect binding of antibodies to the RBS: the V1 region and a short sequence analogous in position to the human immunodeficiency virus type 1 gp120 loop B postulated to be located between two major domains of CAEV gp135. Mutation of isoleucine-166 to alanine in the putative loop B of gp135 increased the affinity of soluble gp135 for the CAEV receptor(s) and goat monoclonal antibody (Mab) F7-299 which recognizes an epitope overlapping the gp135 RBS. The I166A mutation also stabilized or exposed the F7-299 epitope in anionic detergent buffers, indicating that the I166A mutation induces conformational changes and stabilizes the RBS of soluble gp135 and enhances Mab F7-299 binding. In contrast, the affinity of a V1 deletion mutant of gp135 for the receptor and Mab F7-299 and its structural stability did not differ from that of the wild-type gp135. However, both the I166A mutation and the V1 deletion of gp135 increased cell-to-cell fusion activity and binding of Mab F7-299 to the oligomeric Env. Therefore, the CAEV gp135 RBS is protected from antibody binding by mechanisms both dependent and independent of Env oligomerization which are disrupted by the V1 deletion and the I166A mutation, respectively. In addition, we found a correlation between side-chain {beta}-branching at amino acid position 166 and binding of Mab F7-299 to oligomeric Env and cell-to-cell fusion, suggesting local secondary structure constraints in the region around isoleucine-166 as one determinant of gp135 RBS exposure and antibody binding.

  15. Effect of acidic amino acids engineered into the active site cleft of Thermopolyspora flexuosa GH11 xylanase.

    PubMed

    Li, He; Turunen, Ossi

    2015-01-01

    Thermopolyspora flexuosa GH11 xylanase (XYN11A) shows optimal activity at pH 6-7 and 75-80 °C. We studied how mutation to aspartic acid (N46D and V48D) in the vicinity of the catalytic acid/base affects the pH activity of highly thermophilic GH11 xylanase. Both mutations shifted the pH activity profile toward acidic pH. In general, the Km values were lower at pH 4-5 than at pH 6, and in line with this, the rate of hydrolysis of xylotetraose was slightly faster at pH 4 than at pH 6. The N46D mutation and also lower pH in XYN11A increased the hydrolysis of xylotriose. The Km value increased remarkably (from 2.5 to 11.6 mg/mL) because of V48D, which indicates the weakening of binding affinity of the substrate to the active site. Xylotetraose functioned well as a substrate for other enzymes, but with lowered reaction rate for V48D. Both N46D and V48D increased the enzyme inactivation by ionic liquid [emim]OAc. In conclusion, the pH activity profile could be shifted to acidic pH due to an effect from two different directions, but the tightly packed GH11 active site can cause steric problems for the mutations.

  16. An Effective Molecular Target Site in Hepatitis B Virus S Gene for Cas9 Cleavage and Mutational Inactivation

    PubMed Central

    Li, Hao; Sheng, Chunyu; Liu, Hongbo; Liu, Guangze; Du, Xinying; Du, Juan; Zhan, Linsheng; Li, Peng; Yang, Chaojie; Qi, Lihua; Wang, Jian; Yang, Xiaoxia; Jia, Leili; Xie, Jing; Wang, Ligui; Hao, Rongzhang; Xu, Dongping; Tong, Yigang; Zhou, Yusen; Zhou, Jianjun; Sun, Yansong; Li, Qiao; Qiu, Shaofu; Song, Hongbin

    2016-01-01

    Chronic hepatitis B infection remains incurable because HBV cccDNA can persist indefinitely in patients recovering from acute HBV infection. Given the incidence of HBV infection and the shortcomings of current therapeutic options, a novel antiviral strategy is urgently needed. To inactivate HBV replication and destroy the HBV genome, we employed genome editing tool CRISPR/Cas9. Specifically, we found a CRISPR/Cas9 system (gRNA-S4) that effectively targeted the HBsAg region and could suppress efficiently viral replication with minimal off-target effects and impact on cell viability. The mutation mediated by CRISPR/Cas9 in HBV DNA both in a stable HBV-producing cell line and in HBV transgenic mice had been confirmed and evaluated using deep sequencing. In addition, we demonstrated the reduction of HBV replication was caused by the mutation of S4 site through three S4 region-mutated monoclonal cells. Besides, the gRNA-S4 system could also reduce serum surface-antigen levels by 99.91 ± 0.05% and lowered serum HBV DNA level below the negative threshold in the HBV hydrodynamics mouse model. Together, these findings indicate that the S4 region may be an ideal target for the development of innovative therapies against HBV infection using CRISPR/Cas9. PMID:27570484

  17. L1198F Mutation Resensitizes Crizotinib to ALK by Altering the Conformation of Inhibitor and ATP Binding Sites

    PubMed Central

    Li, Jian; Sun, Rong; Wu, Yuehong; Song, Mingzhu; Li, Jia; Yang, Qianye; Chen, Xiaoyi; Bao, Jinku; Zhao, Qi

    2017-01-01

    The efficacy of anaplastic lymphoma kinase (ALK) positive non-small-cell lung cancer (NSCLC) treatment with small molecule inhibitors is greatly challenged by acquired resistance. A recent study reported the newest generation inhibitor resistant mutation L1198F led to the resensitization to crizotinib, which is the first Food and Drug Administration (FDA) approved drug for the treatment of ALK-positive NSCLC. It is of great importance to understand how this extremely rare event occurred for the purpose of overcoming the acquired resistance of such inhibitors. In this study, we exploited molecular dynamics (MD) simulation to dissect the molecular mechanisms. Our MD results revealed that L1198F mutation of ALK resulted in the conformational change at the inhibitor site and altered the binding affinity of ALK to crizotinib and lorlatinib. L1198F mutation also affected the autoactivation of ALK as supported by the identification of His1124 and Tyr1278 as critical amino acids involved in ATP binding and phosphorylation. Our findings are valuable for designing more specific and potent inhibitors for the treatment of ALK-positive NSCLC and other types of cancer. PMID:28245558

  18. Mutation of Tyr697, a GRB2-binding site, and Tyr721, a PI 3-kinase binding site, abrogates signal transduction by the murine CSF-1 receptor expressed in Rat-2 fibroblasts.

    PubMed Central

    van der Geer, P; Hunter, T

    1993-01-01

    The receptor for the myeloid cell growth factor colony stimulating factor 1 (CSF-1) is a protein tyrosine kinase that is closely related to the PDGF receptor. Ligand binding results in kinase activation and autophosphorylation. Three autophosphorylation sites, Tyr697, Tyr706 and Tyr721, have been mapped to the kinase insert domain. Deletion of the entire kinase insert domain completely abrogates signal transduction by the CSF-1 receptor expressed in Rat-2 fibroblasts. To investigate the function of individual phosphorylation sites present in the CSF-1 receptor kinase insert domain, a number of phosphorylation site mutants were expressed in Rat-2 fibroblasts. Mutation of either Tyr697 or Tyr721 compromised signal transduction by the CSF-1 receptor. A mutant receptor, in which both Tyr697 and Tyr721 were replaced by phenylalanine, has lost all ability to induce changes in morphology or to increase cell growth rate in response to CSF-1. Tyr721 has been identified recently as the binding site for PI 3-kinase. Here we report that GRB2 associates with the CSF-1 receptor upon ligand binding. The phosphorylation on tyrosine of SHC and several other GRB2-associated proteins increased upon stimulation with CSF-1. Tyr697 was identified as a binding site for GRB2. We suggest that PI 3-kinase, GRB2 and some of the GRB2-associated proteins could play an important role in signal transduction by the CSF-1 receptor. Images PMID:8262059

  19. Prediction of P53 Mutants (Multiple Sites) Transcriptional Activity Based on Structural (2D&3D) Properties

    PubMed Central

    Geetha Ramani, R.; Jacob, Shomona Gracia

    2013-01-01

    Prediction of secondary site mutations that reinstate mutated p53 to normalcy has been the focus of intense research in the recent past owing to the fact that p53 mutants have been implicated in more than half of all human cancers and restoration of p53 causes tumor regression. However laboratory investigations are more often laborious and resource intensive but computational techniques could well surmount these drawbacks. In view of this, we formulated a novel approach utilizing computational techniques to predict the transcriptional activity of multiple site (one-site to five-site) p53 mutants. The optimal MCC obtained by the proposed approach on prediction of one-site, two-site, three-site, four-site and five-site mutants were 0.775,0.341,0.784,0.916 and 0.655 respectively, the highest reported thus far in literature. We have also demonstrated that 2D and 3D features generate higher prediction accuracy of p53 activity and our findings revealed the optimal results for prediction of p53 status, reported till date. We believe detection of the secondary site mutations that suppress tumor growth may facilitate better understanding of the relationship between p53 structure and function and further knowledge on the molecular mechanisms and biological activity of p53, a targeted source for cancer therapy. We expect that our prediction methods and reported results may provide useful insights on p53 functional mechanisms and generate more avenues for utilizing computational techniques in biological data analysis. PMID:23468845

  20. Active sites of thioredoxin reductases: why selenoproteins?

    PubMed

    Gromer, Stephan; Johansson, Linda; Bauer, Holger; Arscott, L David; Rauch, Susanne; Ballou, David P; Williams, Charles H; Schirmer, R Heiner; Arnér, Elias S J

    2003-10-28

    Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

  1. Genome-Wide Estimates of Mutation Rates and Spectrum in Schizosaccharomyces pombe Indicate CpG Sites are Highly Mutagenic Despite the Absence of DNA Methylation.

    PubMed

    Behringer, Megan G; Hall, David W

    2015-11-12

    We accumulated mutations for 1952 generations in 79 initially identical, haploid lines of the fission yeast Schizosaccharomyces pombe, and then performed whole-genome sequencing to determine the mutation rates and spectrum. We captured 696 spontaneous mutations across the 79 mutation accumulation (MA) lines. We compared the mutation spectrum and rate to a recently published equivalent experiment on the same species, and to another model ascomycetous yeast, the budding yeast Saccharomyces cerevisiae. While the two species are approximately 600 million years diverged from each other, they share similar life histories, genome size and genomic G/C content. We found that Sc. pombe and S. cerevisiae have similar mutation rates, but Sc. pombe exhibits a stronger insertion bias. Intriguingly, we observed an increased mutation rate at cytosine nucleotides, specifically CpG nucleotides, which is also seen in S. cerevisiae. However, the absence of methylation in Sc. pombe and the pattern of mutation at these sites, primarily C → A as opposed to C → T, strongly suggest that the increased mutation rate is not caused by deamination of methylated cytosines. This result implies that the high mutability of CpG dinucleotides in other species may be caused in part by a methylation-independent mechanism. Many of our findings mirror those seen in the recent study, despite the use of different passaging conditions, indicating that MA is a reliable method for estimating mutation rates and spectra.

  2. Severe fluoropyrimidine toxicity due to novel and rare DPYD missense mutations, deletion and genomic amplification affecting DPD activity and mRNA splicing.

    PubMed

    van Kuilenburg, André B P; Meijer, Judith; Maurer, Dirk; Dobritzsch, Doreen; Meinsma, Rutger; Los, Maartje; Knegt, Lia C; Zoetekouw, Lida; Jansen, Rob L H; Dezentjé, Vincent; van Huis-Tanja, Lieke H; van Kampen, Roel J W; Hertz, Jens Michael; Hennekam, Raoul C M

    2017-03-01

    Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). Genetic variations in DPD have emerged as predictive risk factors for severe fluoropyrimidine toxicity. Here, we report novel and rare genetic variants underlying DPD deficiency in 9 cancer patients presenting with severe fluoropyrimidine-associated toxicity. All patients possessed a strongly reduced DPD activity, ranging from 9 to 53% of controls. Analysis of the DPD gene (DPYD) showed the presence of 21 variable sites including 4 novel and 4 very rare aberrations: 3 missense mutations, 2 splice-site mutations, 1 intronic mutation, a deletion of 21 nucleotides and a genomic amplification of exons 9-12. Two novel/rare variants (c.2843T>C, c.321+1G>A) were present in multiple, unrelated patients. Functional analysis of recombinantly-expressed DPD mutants carrying the p.I948T and p.G284V mutation showed residual DPD activities of 30% and 0.5%, respectively. Analysis of a DPD homology model indicated that the p.I948T and p.G284V mutations may affect electron transfer and the binding of FAD, respectively. cDNA analysis showed that the c.321+1G>A mutation in DPYD leads to skipping of exon 4 immediately upstream of the mutated splice-donor site in the process of DPD pre-mRNA splicing. A lethal toxicity in two DPD patients suggests that fluoropyrimidines combined with other therapies such as radiotherapy might be particularly toxic for DPD deficient patients. Our study advocates a more comprehensive genotyping approach combined with phenotyping strategies for upfront screening for DPD deficiency to ensure the safe administration of fluoropyrimidines.

  3. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    PubMed

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS.

  4. Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERT expression in multiple cancers

    PubMed Central

    Stern, Josh Lewis; Theodorescu, Dan; Vogelstein, Bert; Papadopoulos, Nickolas; Cech, Thomas R.

    2015-01-01

    Somatic mutations in the promoter of the gene for telomerase reverse transcriptase (TERT) are the most common noncoding mutations in cancer. They are thought to activate telomerase, contributing to proliferative immortality, but the molecular events driving TERT activation are largely unknown. We observed in multiple cancer cell lines that mutant TERT promoters exhibit the H3K4me2/3 mark of active chromatin and recruit the GABPA/B1 transcription factor, while the wild-type allele retains the H3K27me3 mark of epigenetic silencing; only the mutant promoters are transcriptionally active. These results suggest how a single-base-pair mutation can cause a dramatic epigenetic switch and monoallelic expression. PMID:26515115

  5. Mutations in the substrate entrance region of β-glucosidase from Trichoderma reesei improve enzyme activity and thermostability.

    PubMed

    Lee, Hsiao-Lin; Chang, Chih-Kang; Jeng, Wen-Yih; Wang, Andrew H-J; Liang, Po-Huang

    2012-11-01

    β-Glucosidase (EC 3.2.1.21) plays an essential role in biofuel production since it can cleave β-1,4-glycosidic bond to convert cellobiose into fermentable glucose. Based on the structure of Trichoderma reesei β-glucosidase 2 (TrBgl2) we solved, the amino acids in the outer channel of active site were mutated in this study. Mutants P172L and P172L/F250A showed the most enhanced k(cat)/K(m) and k(cat) values by 5.3- and 6.9-fold, respectively, compared to the wild type (WT) toward 4-nitrophenyl-β-D-glucopyranoside (p-NPG) substrate at 40°C. L167W and P172L/F250A mutations resulted in shift of optimal temperature to 50°C, at which WT was almost inactive. However, thin-layer chromatography analysis revealed that mutant L167W had the best synergism with T. reesei cellulases on degrading cellulosic substrates into glucose. This study enhances our understanding on the roles of amino acids in the substrate entrance region away from the active site and provides engineered T. reesei β-glucosidases with better activity and/or thermostability to hydrolyze cellobiose.

  6. Activating mutations for the Met tyrosine kinase receptor in human cancer

    PubMed Central

    Jeffers, Michael; Schmidt, Laura; Nakaigawa, Noboru; Webb, Craig P.; Weirich, Gregor; Kishida, Takeshi; Zbar, Berton; Vande Woude, George F.

    1997-01-01

    Recently, mutations in the Met tyrosine kinase receptor have been identified in both hereditary and sporadic forms of papillary renal carcinoma. We have introduced the corresponding mutations into the met cDNA and examined the effect of each mutation in biochemical and biological assays. We find that the Met mutants exhibit increased levels of tyrosine phosphorylation and enhanced kinase activity toward an exogenous substrate when compared with wild-type Met. Moreover, NIH 3T3 cells expressing mutant Met molecules form foci in vitro and are tumorigenic in nude mice. Enzymatic and biological differences were evident among the various mutants examined, and the somatic mutations were generally more active than those of germ-line origin. A strong correlation between the enzymatic and biological activity of the mutants was observed, indicating that tumorigenesis by Met is quantitatively related to its level of activation. These results demonstrate that the Met mutants originally identified in human papillary renal carcinoma are oncogenic and thus are likely to play a determinant role in this disease, and these results raise the possibility that activating Met mutations also may contribute to other human malignancies. PMID:9326629

  7. Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation

    PubMed Central

    Van Gorp, Hanne; Saavedra, Pedro H. V.; de Vasconcelos, Nathalia M.; Van Opdenbosch, Nina; Vande Walle, Lieselotte; Matusiak, Magdalena; Prencipe, Giusi; Insalaco, Antonella; Van Hauwermeiren, Filip; Demon, Dieter; Bogaert, Delfien J.; Dullaers, Melissa; De Baere, Elfride; Hochepied, Tino; Dehoorne, Joke; Vermaelen, Karim Y.; Haerynck, Filomeen; De Benedetti, Fabrizio; Lamkanfi, Mohamed

    2016-01-01

    Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1β and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations. PMID:27911804

  8. Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site

    PubMed Central

    Ma, Yi; Zhang, Beilei; Wang, Meng; Ou, Yanghui

    2016-01-01

    The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas. PMID:27981047

  9. Intron retention resulting from a silent mutation in the VWF gene that structurally influences the 5′ splice site

    PubMed Central

    Yadegari, Hamideh; Biswas, Arijit; Akhter, Mohammad Suhail; Driesen, Julia; Ivaskevicius, Vytautas; Marquardt, Natascha

    2016-01-01

    Disease-associated silent mutations are considered to affect the accurate pre–messenger RNA (mRNA) splicing either by influencing regulatory elements, leading to exon skipping, or by creating a new cryptic splice site. This study describes a new molecular pathological mechanism by which a silent mutation inhibits splicing and leads to intron retention. We identified a heterozygous silent mutation, c.7464C>T, in exon 44 of the von Willebrand factor (VWF) gene in a family with type 1 von Willebrand disease. In vivo and ex vivo transcript analysis revealed an aberrantly spliced transcript, with intron 44 retained in the mRNA, implying disruption of the first catalytic step of splicing at the 5′ splice site (5′ss). The abnormal transcript with the retained intronic region coded a truncated protein that lacked the carboxy-terminal end of the VWF protein. Confocal immunofluorescence characterizations of blood outgrowth endothelial cells derived from the patient confirmed the presence of the truncated protein by demonstrating accumulation of VWF in the endoplasmic reticulum. In silico pre-mRNA secondary and tertiary structure analysis revealed that this substitution, despite its distal position from the 5′ss (85 bp downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 5′ss. This hairpin sequesters the 5′ss residues involved in U1 small nuclear RNA interactions, thereby inhibiting excision of the pre-mRNA intronic region. This study is the first to show the allosteric-like/far-reaching effect of an exonic variation on pre-mRNA splicing that is mediated by structural changes in the pre-mRNA. PMID:27543438

  10. Community Update on Site Activities, July 19, 2013

    EPA Pesticide Factsheets

    In an effort to engage and inform community members interested in the New Bedford Harbor Superfund Site cleanup, EPA will be issuing periodic topic-based fact sheets that will provide background information and updates about ongoing activities.

  11. Frequencies, Laboratory Features, and Granulocyte Activation in Chinese Patients with CALR-Mutated Myeloproliferative Neoplasms

    PubMed Central

    Tian, Ruiyuan; Chang, Jianmei; Li, Jianlan; Tan, Yanhong; Xu, Zhifang; Ren, Fanggang; Zhao, Junxia; Pan, Jie; Zhang, Na; Wang, Xiaojuan; He, Jianxia; Yang, Wanfang; Wang, Hongwei

    2015-01-01

    Somatic mutations in the CALR gene have been recently identified as acquired alterations in myeloproliferative neoplasms (MPNs). In this study, we evaluated mutation frequencies, laboratory features, and granulocyte activation in Chinese patients with MPNs. A combination of qualitative allele-specific polymerase chain reaction and Sanger sequencing was used to detect three driver mutations (i.e., CALR, JAK2V617F, and MPL). CALR mutations were identified in 8.4% of cases with essential thrombocythemia (ET) and 5.3% of cases with primary myelofibrosis (PMF). Moreover, 25% of polycythemia vera, 29.5% of ET, and 48.1% of PMF were negative for all three mutations (JAK2V617F, MPL, and CALR). Compared with those patients with JAK2V617F mutation, CALR-mutated ET patients displayed unique hematological phenotypes, including higher platelet counts, and lower leukocyte counts and hemoglobin levels. Significant differences were not found between Chinese PMF patients with mutants CALR and JAK2V617F in terms of laboratory features. Interestingly, patients with CALR mutations showed markedly decreased levels of leukocyte alkaline phosphatase (LAP) expression, whereas those with JAK2V617F mutation presented with elevated levels. Overall, a lower mutant rate of CALR gene and a higher triple-negative rate were identified in the cohort of Chinese patients with MPNs. This result indicates that an undiscovered mutant gene may have a significant role in these patients. Moreover, these pathological features further imply that the disease biology varies considerably between mutants CALR and JAK2V617F. PMID:26375990

  12. Activation of Antibiotic Production in Bacillus spp. by Cumulative Drug Resistance Mutations.

    PubMed

    Tojo, Shigeo; Tanaka, Yukinori; Ochi, Kozo

    2015-12-01

    Bacillus subtilis strains produce a wide range of antibiotics, including ribosomal and nonribosomal peptide antibiotics, as well as bacilysocin and neotrehalosadiamine. Mutations in B. subtilis strain 168 that conferred resistance to drugs such as streptomycin and rifampin resulted in overproduction of the dipeptide antibiotic bacilysin. Cumulative drug resistance mutations, such as mutations in the mthA and rpsL genes, which confer low- and high-level resistance, respectively, to streptomycin, and mutations in rpoB, which confer resistance to rifampin, resulted in cells that overproduced bacilysin. Transcriptional analysis demonstrated that the enhanced transcription of biosynthesis genes was responsible for the overproduction of bacilysin. This approach was effective also in activating the cryptic genes of Bacillus amyloliquefaciens, leading to actual production of antibiotic(s).

  13. Activation of Antibiotic Production in Bacillus spp. by Cumulative Drug Resistance Mutations

    PubMed Central

    Tojo, Shigeo; Tanaka, Yukinori

    2015-01-01

    Bacillus subtilis strains produce a wide range of antibiotics, including ribosomal and nonribosomal peptide antibiotics, as well as bacilysocin and neotrehalosadiamine. Mutations in B. subtilis strain 168 that conferred resistance to drugs such as streptomycin and rifampin resulted in overproduction of the dipeptide antibiotic bacilysin. Cumulative drug resistance mutations, such as mutations in the mthA and rpsL genes, which confer low- and high-level resistance, respectively, to streptomycin, and mutations in rpoB, which confer resistance to rifampin, resulted in cells that overproduced bacilysin. Transcriptional analysis demonstrated that the enhanced transcription of biosynthesis genes was responsible for the overproduction of bacilysin. This approach was effective also in activating the cryptic genes of Bacillus amyloliquefaciens, leading to actual production of antibiotic(s). PMID:26369962

  14. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    PubMed

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates.

  15. Mutations affecting export and activity of cytolysin A from Escherichia coli.

    PubMed

    Ludwig, Albrecht; Völkerink, Guido; von Rhein, Christine; Bauer, Susanne; Maier, Elke; Bergmann, Birgit; Goebel, Werner; Benz, Roland

    2010-08-01

    Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several Escherichia coli and Salmonella enterica strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the E. coli ClyA protein for export and activity. The data indicate that ClyA translocation to the periplasm requires several protein segments located closely adjacent to each other in the "tail" domain of the ClyA monomer, namely, the N- and C-terminal regions and the hydrophobic sequence ranging from residues 89 to 101. Deletion of most of the "head" domain of the monomer (residues 181 to 203), on the other hand, did not strongly affect ClyA secretion, suggesting that the tail domain plays a particular role in export. Furthermore, we found that the N-terminal amphipathic helix alphaA1 of ClyA is crucial for the formation and the properties of the transmembrane channel, and hence for hemolytic activity. Several mutations affecting the C-terminal helix alphaG, the "beta-tongue" region in the head domain, or the hydrophobic region in the tail domain of the ClyA monomer strongly impaired the hemolytic activity and reduced the activity toward planar lipid bilayer membranes but did not totally prevent formation of wild-type-like channels in these artificial membranes. The latter regions thus apparently promote membrane interaction without being directly required for pore formation in a lipid bilayer.

  16. Antitumor effects and molecular mechanisms of ponatinib on endometrial cancer cells harboring activating FGFR2 mutations

    PubMed Central

    Kim, Do-Hee; Kwak, Yeonui; Kim, Nam Doo; Sim, Taebo

    2016-01-01

    ABSTRACT Aberrant mutational activation of FGFR2 is associated with endometrial cancers (ECs). AP24534 (ponatinib) currently undergoing clinical trials has been known to be an orally available multi-targeted tyrosine kinase inhibitor. Our biochemical kinase assay showed that AP24534 is potent against wild-type FGFR1-4 and 5 mutant FGFRs (V561M-FGFR1, N549H-FGFR2, K650E-FGFR3, G697C-FGFR3, N535K-FGFR4) and possesses the strongest kinase-inhibitory activity on N549H-FGFR2 (IC50 of 0.5 nM) among all FGFRs tested. We therefore investigated the effects of AP24534 on endometrial cancer cells harboring activating FGFR2 mutations and explored the underlying molecular mechanisms. AP24534 significantly inhibited the proliferation of endometrial cancer cells bearing activating FGFR2 mutations (N549K, K310R/N549K, S252W) and mainly induced G1/S cell cycle arrest leading to apoptosis. AP24534 also diminished the kinase activity of immunoprecipitated FGFR2 derived from MFE-296 and MFE-280 cells and reduced the phosphorylation of FGFR2 and FRS2 on MFE-296 and AN3CA cells. AP24534 caused substantial reductions in ERK phosphorylation, PLCγ signaling and STAT5 signal transduction on ECs bearing FGFR2 activating mutations. Akt signaling pathway was also deactivated by AP24534. AP24534 causes the chemotherapeutic effect through mainly the blockade of ERK, PLCγ and STAT5 signal transduction on ECs. Moreover, AP24534 inhibited migration and invasion of endometrial cancer cells with FGFR2 mutations. In addition, AP24534 significantly blocked anchorage-independent growth of endometrial cancer cells. We, for the first time, report the molecular mechanisms by which AP24534 exerts antitumor effects on ECs with FGFR2 activating mutations, which would provide mechanistic insight into ongoing clinical investigations of AP24534 for ECs. PMID:26574622

  17. Global control of aberrant splice-site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition.

    PubMed

    Královicová, Jana; Vorechovsky, Igor

    2007-01-01

    Auxiliary splicing signals play a major role in the regulation of constitutive and alternative pre-mRNA splicing, but their relative importance in selection of mutation-induced cryptic or de novo splice sites is poorly understood. Here, we show that exonic sequences between authentic and aberrant splice sites that were activated by splice-site mutations in human disease genes have lower frequencies of splicing enhancers and higher frequencies of splicing silencers than average exons. Conversely, sequences between authentic and intronic aberrant splice sites have more enhancers and less silencers than average introns. Exons that were skipped as a result of splice-site mutations were smaller, had lower SF2/ASF motif scores, a decreased availability of decoy splice sites and a higher density of silencers than exons in which splice-site mutation activated cryptic splice sites. These four variables were the strongest predictors of the two aberrant splicing events in a logistic regression model. Elimination or weakening of predicted silencers in two reporters consistently promoted use of intron-proximal splice sites if these elements were maintained at their original positions, with their modular combinations producing expected modification of splicing. Together, these results show the existence of a gradient in exon and intron definition at the level of pre-mRNA splicing and provide a basis for the development of computational tools that predict aberrant splicing outcomes.

  18. EGFR-activating mutations, DNA copy number abundance of ErbB family, and prognosis in lung adenocarcinoma.

    PubMed

    Chen, Hsuan-Yu; Liu, Chia-Hsin; Chang, Ya-Hsuan; Yu, Sung-Liang; Ho, Bing-Ching; Hsu, Chung-Ping; Yang, Tsung-Ying; Chen, Kun-Chieh; Hsu, Kuo-Hsuan; Tseng, Jeng-Sen; Hsia, Jiun-Yi; Chuang, Cheng-Yen; Chang, Chi-Sheng; Li, Yu-Cheng; Li, Ker-Chau; Chang, Gee-Chen; Yang, Pan-Chyr

    2016-02-23

    In this study, EGFR-activating mutation status and DNA copy number abundances of members of ErbB family were measured in 261 lung adenocarcinomas. The associations between DNA copy number abundances of ErbB family, EGFR-activating mutation status, and prognosis were explored. Results showed that DNA copy number abundances of EGFR, ERBB2, ERBB3, and ERBB4 had associations with overall survival in lung adenocarcinoma with EGFR-activating mutations. In the stratification analysis, only ERBB2 showed significant discrepancy in patients carrying wild type EGFR and other members of ErbB family in patients carrying EGFR-activating mutation. This indicated that CNAs of ErbB family had effect modifications of EGFR-activating mutation status. Findings of this study demonstrate potential molecular guidance of patient management of lung adenocarcinoma with or without EGFR-activating mutations.

  19. The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites.

    PubMed

    Martin, K; Huo, L; Schleif, R F

    1986-06-01

    Two sets of experiments have been performed to test the DNA loop model of repression of the araBAD operon of Escherichia coli. First, dimethyl sulfate methylation protection measurements on normally growing cells show that the AraC regulatory protein occupies the araI site in the presence and absence of the inducer arabinose. Similarly, the araO2 site is shown to be occupied by AraC protein in the presence and absence of arabinose; however, its occupancy by AraC is greatly reduced when araI and adjacent sequences are deleted. Thus, AraC protein binds to araO2 cooperatively with some other component of the ara system located at least 60 base pairs away. Second, the mutational analysis presented here shows that the DNA components required for repression of araBAD are araI, araO2, and perhaps the araBAD operon RNA polymerase binding site.

  20. Site-directed mutations and kinetic studies show key residues involved in alkylammonium interactions and reveal two sites for phosphorylcholine in Pseudomonas aeruginosa phosphorylcholine phosphatase.

    PubMed

    Beassoni, Paola R; Otero, Lisandro H; Boetsch, Cristhian; Domenech, Carlos E; González-Nilo, Fernado D; Lisa, Angela T

    2011-07-01

    Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pcho) to produce choline and inorganic phosphate. PchP belongs to the haloacid dehalogenase superfamily (HAD) and possesses the three characteristic motifs of this family: motif I ((31)D and (33)D), motif II ((166)S), and motif III ((242)K, (261)G, (262)D and (267)D), which fold to form the catalytic site that binds the metal ion and the phosphate moiety of Pcho. Based on comparisons to the PHOSPHO1 and PHOSPHO2 human enzymes and the choline-binding proteins of Gram-(+) bacteria, we selected residues (42)E and (43)E and the aromatic triplet (82)YYY(84) for site-directed mutagenesis to study the interactions with Pcho and p-nitrophenylphosphate as substrates of PchP. Because mutations in (42)E, (43)E and the three tyrosine residues affect both the substrate affinity and the inhibitory effect produced by high Pcho concentrations, we postulate that two sites, one catalytic and one inhibitory, are present in PchP and that they are adjacent and share residues.

  1. Site-directed mutagenesis of an alkaline phytase: influencing specificity, activity and stability in acidic milieu.

    PubMed

    Tran, Thuy T; Mamo, Gashaw; Búxo, Laura; Le, Nhi N; Gaber, Yasser; Mattiasson, Bo; Hatti-Kaul, Rajni

    2011-07-10

    Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.

  2. Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation

    PubMed Central

    Gao, Wenqing; Yang, Jieling; Liu, Wang; Wang, Yupeng; Shao, Feng

    2016-01-01

    Pyrin, encoded by the MEFV gene, is best known for its gain-of-function mutations causing familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a caspase-1–activating inflammasome in response to inactivating modifications of Rho GTPases by various bacterial toxins or effectors. Pyrin-mediated innate immunity is unique in that it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Here we show that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. We identified Ser-205 and Ser-241 in mouse Pyrin whose phosphorylation resulted in inhibitory binding by cellular 14-3-3 proteins. The two serines underwent dephosphorylation upon toxin stimulation or bacterial infection, triggering 14-3-3 dissociation, which correlated with Pyrin inflammasome activation. We developed antibodies specific for phosphorylated Ser-205 and Ser-241, which confirmed the stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses indicated that both phosphorylation and signal-induced dephosphorylation of Ser-205/241 are important for Pyrin activation. Moreover, microtubule drugs, including colchicine, commonly used to treat FMF, effectively blocked activation of the Pyrin inflammasome. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated apoptosis-associated Speck-like protein containing CARD (ASC) aggregation. Our study reveals that site-specific (de)phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity. PMID:27482109

  3. Mutations in the GM1 Binding Site of Simian Virus 40 VP1 Alter Receptor Usage and Cell Tropism

    PubMed Central

    Magaldi, Thomas G.; Buch, Michael H. C.; Murata, Haruhiko; Erickson, Kimberly D.; Neu, Ursula; Garcea, Robert L.; Peden, Keith; Stehle, Thilo

    2012-01-01

    Polyomaviruses are nonenveloped viruses with capsids composed primarily of 72 pentamers of the viral VP1 protein, which forms the outer shell of the capsid and binds to cell surface oligosaccharide receptors. Highly conserved VP1 proteins from closely related polyomaviruses recognize different oligosaccharides. To determine whether amino acid changes restricted to the oligosaccharide binding site are sufficient to determine receptor specificity and how changes in receptor usage affect tropism, we studied the primate polyomavirus simian virus 40 (SV40), which uses the ganglioside GM1 as a receptor that mediates cell binding and entry. Here, we used two sequential genetic screens to isolate and characterize viable SV40 mutants with mutations in the VP1 GM1 binding site. Two of these mutants were completely resistant to GM1 neutralization, were no longer stimulated by incorporation of GM1 into cell membranes, and were unable to bind to GM1 on the cell surface. In addition, these mutant viruses displayed an infection defect in monkey cells with high levels of cell surface GM1. Interestingly, one mutant infected cells with low cell surface GM1 more efficiently than wild-type virus, apparently by utilizing a different ganglioside receptor. Our results indicate that a small number of mutations in the GM1 binding site are sufficient to alter ganglioside usage and change tropism, and they suggest that VP1 divergence is driven primarily by a requirement to accommodate specific receptors. In addition, our results suggest that GM1 binding is required for vacuole formation in permissive monkey CV-1 cells. Further study of these mutants will provide new insight into polyomavirus entry, pathogenesis, and evolution. PMID:22514351

  4. A novel phosphorylation site mutation in profilin 1 revealed in a large screen of US, Nordic, and German amyotrophic lateral sclerosis/frontotemporal dementia cohorts.

    PubMed

    Ingre, Caroline; Landers, John E; Rizik, Naji; Volk, Alexander E; Akimoto, Chizuru; Birve, Anna; Hübers, Annemarie; Keagle, Pamela J; Piotrowska, Katarzyna; Press, Rayomand; Andersen, Peter Munch; Ludolph, Albert C; Weishaupt, Jochen H

    2013-06-01

    Profilin 1 is a central regulator of actin dynamics. Mutations in the gene profilin 1 (PFN1) have very recently been shown to be the cause of a subgroup of amyotrophic lateral sclerosis (ALS). Here, we performed a large screen of US, Nordic, and German familial and sporadic ALS and frontotemporal dementia (FTLD) patients for PFN1 mutations to get further insight into the spectrum and pathogenic relevance of this gene for the complete ALS/FTLD continuum. Four hundred twelve familial and 260 sporadic ALS cases and 16 ALS/FTLD cases from Germany, the Nordic countries, and the United States were screened for PFN1 mutations. Phenotypes of patients carrying PFN1 mutations were studied. In a German ALS family we identified the novel heterozygous PFN1 mutation p.Thr109Met, which was absent in controls. This novel mutation abrogates a phosphorylation site in profilin 1. The recently described p.Gln117Gly sequence variant was found in another familial ALS patient from the United States. The ALS patients with mutations in PFN1 displayed spinal onset motor neuron disease without overt cognitive involvement. PFN1 mutations were absent in patients with motor neuron disease and dementia, and in patients with only FTLD. We provide further evidence that PFN1 mutations can cause ALS as a Mendelian dominant trait. Patients carrying PFN1 mutations reported so far represent the "classic" ALS end of the ALS-FTLD spectrum. The novel p.Thr109Met mutation provides additional proof-of-principle that mutant proteins involved in the regulation of cytoskeletal dynamics can cause motor neuron degeneration. Moreover, this new mutation suggests that fine-tuning of actin polymerization by phosphorylation of profilin 1 might be necessary for motor neuron survival.

  5. Structural Motif-Based Homology Modeling of CYP27A1 and Site-Directed Mutational Analyses Affecting Vitamin D Hydroxylation

    PubMed Central

    Prosser, David E.; Guo, YuDing; Jia, Zongchao; Jones, Glenville

    2006-01-01

    Human CYP27A1 is a mitochondrial cytochrome P450, which is principally found in the liver and plays important roles in the biological activation of vitamin D3 and in the biosynthesis of bile acids. We have applied a systematic analysis of hydrogen bonding patterns in 11 prokaryotic and mammalian CYP crystal structures to construct a homology-based model of CYP27A1. Docking of vitamin D3 structures into the active site of this model identified potential substrate contact residues in the F-helix, the β-3 sheet, and the β-5 sheet. Site-directed mutagenesis and expression in COS-1 cells confirmed that these positions affect enzymatic activity, in some cases shifting metabolism of 1α-hydroxyvitamin D3 to favor 25- or 27-hydroxylation. The results suggest that conserved hydrophobic residues in the β-5 hairpin help define the shape of the substrate binding cavity and that this structure interacts with Phe-248 in the F-helix. Mutations directed toward the β-3a strand suggested a possible heme-binding interaction centered on Asn-403 and a structural role for substrate contact residues Thr-402 and Ser-404. PMID:16500955

  6. Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors

    PubMed Central

    Goh, Gerald; Scholl, Ute I.; Healy, James M.; Choi, Murim; Prasad, Manju L.; Nelson-Williams, Carol; Kuntsman, John W.; Korah, Reju; Suttorp, Anna-Carinna; Dietrich, Dimo; Haase, Matthias; Willenberg, Holger S.; Stålberg, Peter; Hellman, Per; Åkerström, Göran; Björklund, Peyman; Carling, Tobias; Lifton, Richard P.

    2014-01-01

    Adrenal tumors autonomously producing cortisol cause Cushing syndrome1–4. Exome sequencing of 25 tumor-normal pairs revealed two groups. Eight tumors (including 3 carcinomas) had many somatic copy number variants (CNV+) with frequent deletion of CDC42 and CDKN2A, amplification of 5q31.2, and protein-altering mutations in TP53 and RB1. Seventeen (all adenomas) had no CNVs (CNV-), TP53 or RB1 mutations. Six of these had known gain of function mutations in CTNNB15,6 (beta-catenin) or GNAS7,8 (Gαs), Six others had somatic p.Leu206Arg mutations in PRKACA (protein kinase A (PKA) catalytic subunit). Further sequencing identified this mutation in 13 of 63 tumors (35% of adenomas with overt CS). PRKACA, GNAS and CTNNB1 mutations were mutually exclusive. Leu206 directly interacts with PKA’s regulatory subunit, PRKAR1A9,10. PRKACAL206R loses PRKAR1A binding, increasing phosphorylation of downstream targets. PKA activity induces cortisol production and cell proliferation11–15, providing a mechanism for tumor development. These findings define distinct mechanisms underlying adrenal cortisol-producing tumors. PMID:24747643

  7. SCN10A Mutation in a Patient with Erythromelalgia Enhances C-Fiber Activity Dependent Slowing

    PubMed Central

    Neacsu, Cristian; Eberhardt, Esther; Schmidt, Roland; Lunden, Lars Kristian; Ørstavik, Kristin; Kaluza, Luisa; Meents, Jannis; Zhang, Zhiping; Carr, Thomas Hedley; Salter, Hugh; Malinowsky, David; Wollberg, Patrik; Krupp, Johannes; Kleggetveit, Inge Petter; Schmelz, Martin; Jørum, Ellen; Namer, Barbara

    2016-01-01

    Gain-of-function mutations in the tetrodotoxin (TTX) sensitive voltage-gated sodium channel (Nav) Nav1.7 have been identified as a key mechanism underlying chronic pain in inherited erythromelalgia. Mutations in TTX resistant channels, such as Nav1.8 or Nav1.9, were recently connected with inherited chronic pain syndromes. Here, we investigated the effects of the p.M650K mutation in Nav1.8 in a 53 year old patient with erythromelalgia by microneurography and patch-clamp techniques. Recordings of the patient’s peripheral nerve fibers showed increased activity dependent slowing (ADS) in CMi and less spontaneous firing compared to a control group of erythromelalgia patients without Nav mutations. To evaluate the impact of the p.M650K mutation on neuronal firing and channel gating, we performed current and voltage-clamp recordings on transfected sensory neurons (DRGs) and neuroblastoma cells. The p.M650K mutation shifted steady-state fast inactivation of Nav1.8 to more hyperpolarized potentials and did not significantly alter any other tested gating behaviors. The AP half-width was significantly broader and the stimulated action potential firing rate was reduced for M650K transfected DRGs compared to WT. We discuss the potential link between enhanced steady state fast inactivation, broader action potential width and the potential physiological consequences. PMID:27598514

  8. Mutation of Asn28 Disrupts the Dimerization and Enzymatic Activity of SARS 3CL

    SciTech Connect

    Barrila, J.; Gabelli, S; Bacha, U; Amzel, M; Freire, E

    2010-01-01

    Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL{sup pro}, is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL{sup pro} and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 {angstrom} from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CLpro. Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K{sub d}. The crystallographic structure of the N28A mutant determined at 2.35 {angstrom} resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL{sup pro}.

  9. Mutations in the 'Fingers' subdomain of the deubiquitinase USP1 modulate its function and activity.

    PubMed

    Olazabal-Herrero, Anne; García-Santisteban, Iraia; Rodríguez, Jose Antonio

    2016-03-01

    Ubiquitin-specific protease (USP)1 is a member of the USP family of deubiquitinating enzymes. Efficient USP1 activity requires binding to its cofactor USP1-associated factor 1 (UAF1), and the USP1-UAF1 deubiquitinase complex has important roles in regulating DNA damage-related processes. USPs show common folding of their catalytic domain, with three subdomains termed Thumb, Palm, and Fingers. The Fingers subdomain appears to be the primary site for ubiquitin binding. In USP1, the Fingers subdomain also mediates its interaction with UAF1, and thus represents a crucial, but poorly characterized, motif in USP1. To explore the role of USP1-UAF1 in ubiquitin-dependent nuclear processes, we tested the effect of modulating USP1-UAF1 activity on the level and/or localization of conjugated ubiquitin and the DNA damage-related proteins phosphorylated histone H2AX, Lys56-acetylated histone H3, and p53-binding protein 1 (53BP1). Small interfering RNA-mediated USP1 knockdown or treatment with the novel USP1-UAF1 inhibitor ML323 increased the recruitment of conjugated ubiquitin and 53BP1 into nuclear foci. Strikingly, ectopic coexpression of USP1 and UAF1 depleted conjugated ubiquitin in the nucleus and blocked the recruitment of 53BP1 to DNA damage foci. In a direct comparison with other overexpressed USPs, USP1-UAF1 behaved as a relatively promiscuous deubiquitinase. Experimental and cancer-related mutations in the USP1 The Fingers subdomain abrogated substrate deubiquitination without interfering with other USP1 activities, such as UAF1 binding or autocleavage. These results provide new insights into the function and regulation of the USP1-UAF1 complex.

  10. Impact of Proximal and Distal Pocket Site-Directed Mutations on the Ferric/Ferrous Heme Redox Potential of Yeast Cytochrome-c-Peroxidase

    PubMed Central

    Goodin, D. B.

    2012-01-01

    Cytochrome-c-peroxidase (CCP) contains a five-coordinate heme active site. The reduction potential for the ferric to ferrous couple in CCP is anomalously low and pH dependent (Eo = ~−180 mV vs. S.H.E. at pH 7). The contribution of the protein environment to the tuning of the redox potential of this couple is evaluated using site directed mutants of several amino acid residues in the environment of the heme. These include proximal pocket mutation to residues Asp-235, Trp-191, Phe-202 and His-175, distal pocket mutation to residues Trp-51, His-52, and Arg-48; and a heme edge mutation to Ala-147. Where unknown, the structural changes resulting from the amino acid substitution have been studied by X-ray crystallography. In most cases, ostensibly polar or charged residues are replaced by large hydrophobic groups or alternatively by Ala or Gly. These latter have been shown to generate large, solvent filled cavities. Reduction potentials are measured as a function of pH by spectroelectrochemistry. Starting with the X-ray derived structures of CCP and the mutants, or with predicted structures generated by Molecular Dynamics (MD), predictions of redox potential changes are modeled using the Protein Dipoles Langevin Dipoles (PDLD) method. These calculations serve to model an electrostatic assessment of the redox potential change with simplified assumptions about heme iron chemistry, with the balance of the experimentally observed shifts in redox potential being thence attributed to changes in the ligand set and heme coordination chemistry, and/or other changes in the structure not directly evident in the X-ray structures (e.g. ionization states, specific roles played by solvent species, or conformationally flexible portions of the protein). Agreement between theory and experiment is good for all mutant proteins with the exception of the mutation Arg 48 to Ala, and His 52 to Ala. In the former case, the influence of phosphate buffer is adduced to account for the discrepancy

  11. Insights into the Activity Change of Spore Photoproduct Lyase Induced by Mutations at a Peripheral Glycine Residue

    PubMed Central

    Yang, Linlin; Li, Lei

    2017-01-01

    UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e., the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. This mechanism is consistent with the recently solved SPL structure that shows all three residues are located in proximity and thus able to participate in the radical transfer process during the enzyme catalysis. In contrast, an earlier in vivo mutational study identified a glycine to arginine mutation at the position 168 on the B. subtilis SPL that is >15 Å away from the enzyme active site. This mutation appears to abolish the enzyme activity because endospores carrying this mutant were sensitive to UV light. To understand the molecular basis for this rendered enzyme activity, we constructed two SPL mutations G168A and G168R, examined their repair of dinucleotide SP TpT, and found that both mutants exhibit reduced enzyme activity. Comparing with the wildtype (WT) SPL enzyme, the G168A mutant slows down the SP TpT repair by 3~4-fold while the G168R mutant by ~ 80-fold. Both mutants exhibit a smaller apparent (DV) kinetic isotope effect (KIE) but a bigger competitive (DV/K) KIE than that by the WT SPL. Moreover, the G168R mutant also produces a large portion of the abortive repair product TpT-SO2−; the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate. All these data imply that the mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process.

  12. Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

    PubMed Central

    Wu, Hong; Zeng, Hong; Dong, Aiping; Li, Fengling; He, Hao; Senisterra, Guillermo; Seitova, Alma; Duan, Shili; Brown, Peter J.; Vedadi, Masoud; Arrowsmith, Cheryl H.; Schapira, Matthieu

    2013-01-01

    Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation. PMID:24367611

  13. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  14. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide.

  15. Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry.

    PubMed

    Greig, Michael J; Niessen, Sherry; Weinrich, Scott L; Feng, Jun Li; Shi, Manli; Johnson, Ted O

    2015-01-01

    Rapid mutations of proteins that are targeted in cancer therapy often lead to drug resistance. Often, the mutation directly affects a drug's binding site, effectively blocking binding of the drug, but these mutations can have other effects such as changing the protein turnover half-life. Utilizing SILAC MS, we measured the cellular turnover rates of an important non-small cell lung cancer target, epidermal growth factor receptor (EGFR). Wild-type (WT) EGFR, EGFR with a single activating mutant (Del 746-750 or L858R), and the drug-resistant double mutant (L858R/T790M) EGFR were analyzed. In non-small cell lung cancer cell lines, EGFR turnover rates ranged from 28 hours in A431 cells (WT) to 7.5 hours in the PC-9 cells (Del 746-750 mutant). The measurement of EGFR turnover rate in PC-9 cells dosed with irreversible inhibitors has additional complexity due to inhibitor effects on cell viability and results were reported as a range. Finally, essential amino acid recycling (K and R) was measured in different cell lines. The recycling was different in each cell line, but the overall inclusion of the effect of amino acid recycling on calculating EGFR turnover rates resulted in a 10-20% reduction in rates.

  16. Factor IX Amagasaki: A new mutation in the catalytic domain resulting in the loss of both coagulant and esterase activities

    SciTech Connect

    Miyata, Toshiyuki; Iwanaga, Sadaaki ); Sakai, Toshiyuki; Sugimoto, Mitsuhiko; Naka, Hiroyuki; Yamamoto, Kazukuni; Yoshioka, Akira; Fukui, Hiromu ); Mitsui, Kotoko; Kamiya, Kensyu; Umeyama, Hideaki )

    1991-11-26

    Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. The authors identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate for factor X in the presence of factor VIII, phospholipids, and Ca{sup 2+}, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311 {yields} Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.

  17. Correct splicing despite mutation of the invariant first nucleotide of a 5[prime] splice site: A possible basis for disparate clinical phenotypes in siblings with adenosine deaminase deficiency

    SciTech Connect

    Arredondo-Vega, F.X.; Santisteban, I.; Kelly, S.; Hershfield, M.S. ); Umetsu, D.T. ); Schlossman, C.M.

    1994-05-01

    Adenosine deaminase (ADA) deficiency usually causes severe combined immune deficiency in infancy. Milder phenotypes also occur and are associated with less severely impaired deoxyadenosine (dAdo) catabolism. The authors have characterized the mutations responsible for ADA deficiency in siblings with disparity in clinical phenotype. Erythrocyte dAdo nucleotide pool size, which reflects total residual ADA activity, was lower in the older, more mildly affected sib (RG) than in her younger, more severely affected sister (EG). Cultured T cells, fibroblasts, and B lymphoblasts of RG had detectable residual ADA activity, while cells of EG did not. ADA mRNA was undetectable by northern analysis in cells of both patients. Both sibs were found to be compound heterozygotes for the following novel splicing defects: (1) a G[sup +1][yields]A substitution at the 5' splice site of IVS 2 and (2) a complex 17-bp rearrangement of the 3' splice site of IVS 8, which inserted a run of seven purines into the polypyrimidine tract and altered the reading frame of exon 9. PCR-amplified ADA cDNA clones with premature translation stop codons arising from aberrant pre-mRNA splicing were identified, which were consistent with these mutations. However, some cDNA clones from T cells of both patients and from fibroblasts and Epstein-Barr virus (EBV)-transformed B cells of RG, were normally spliced at both the exon 2/3 and exon 8/9 junctions. A normal coding sequence was documented for clones from both sibs. The normal cDNA clones did not appear to arise from either contamination or PCR artifact, and mosaicism seems unlikely to have been involved. These findings suggest (1) that a low level of normal pre-mRNA splicing may occur despite mutation of the invariant first nucleotide of the 5' splice sequence and (2) that differences in efficiency of such splicing may account for the difference in residual ADA activity, immune dysfunction, and clinical severity in these siblings. 66 refs., 6 figs., 1 tab.

  18. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    PubMed Central

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr890, Ser893, Thr894 and Thr899, differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  19. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase.

    PubMed

    Irani, Seema; Yogesha, S D; Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L; Nie, Grace; Prescott, Nicholas A; Zhang, Yan Jessie

    2016-03-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.

  20. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

    PubMed Central

    Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L.; Nie, Grace; Prescott, Nicholas A.; Zhang, Yan Jessie

    2016-01-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families. PMID:26933063

  1. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity

    PubMed Central

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H.

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

  2. Novel JAK3-Activating Mutations in Extranodal NK/T-cell Lymphoma, Nasal Type.

    PubMed

    Sim, Sung H; Kim, Soyeon; Kim, Tae M; Jeon, Yoon K; Nam, Soo J; Ahn, Yong-Oon; Keam, Bhumsuk; Park, Hyun H; Kim, Dong-Wan; Kim, Chul W; Heo, Dae S

    2017-03-08

    Inhibition of the Janus kinase (JAK)-STAT pathway has been implicated as a treatment option for extranodal natural killer/T-cell lymphoma, nasal type (NTCL). However, JAK-STAT pathway alterations in NTCL are variable, and the efficacy of JAK-STAT pathway inhibition has been poorly evaluated. JAK3 mutation and STAT3 genetic alterations were investigated by direct sequencing and immunohistochemistry in 84 patients with newly diagnosed NTCL. Five of 71 patients with NTCL (7.0%) had JAK3 mutations in the pseudokinase domain: two JAK3(A573V), two JAK3(H583Y), and one JAK3(G589D) mutation. Proliferation of Ba/F3 cells transduced with novel JAK3 mutations (JAK3(H583Y) and JAK3(G589D)) was independent of IL-3 and was inhibited by the JAK3 inhibitor tofacitinib (means ± SD drug concentration causing a 50% inhibition of the desired activity, 85 ± 10 nmol/L and 54 ± 9 nmol/L). Ribbon diagrams revealed that these JAK3 pseudokinase domain mutations were located at the pseudokinase-kinase domain interface. Although phosphorylated STAT3 was overexpressed in 35 of 68 patients with NTCL (51.4%), a STAT3 mutation (p.Tyr640Phe; STAT3(Y640F)) at the SRC homology 2 domain was detected in 1 of the 63 patients (1.5%). A STAT3 inhibitor was active against STAT3-mutant SNK-6 and YT cells. Novel JAK3 mutations are oncogenic and druggable in NTCL. The JAK3 or STAT3 signal was altered in NTCL, and pathway inhibition might be a therapeutic option for patients with JAK3- or STAT3-mutant NTCL.

  3. Synergistic and compensatory effects of two point mutations conferring target-site resistance to fipronil in the insect GABA receptor RDL

    PubMed Central

    Zhang, Yixi; Meng, Xiangkun; Yang, Yuanxue; Li, Hong; Wang, Xin; Yang, Baojun; Zhang, Jianhua; Li, Chunrui; Millar, Neil S.; Liu, Zewen

    2016-01-01

    Insecticide resistance can arise from a variety of mechanisms, including changes to the target site, but is often associated with substantial fitness costs to insects. Here we describe two resistance-associated target-site mutations that have synergistic and compensatory effects that combine to produce high and persistent levels of resistance to fipronil, an insecticide targeting on γ-aminobytyric acid (GABA) receptors. In Nilaparvata lugens, a major pest of rice crops in many parts of Asia, we have identified a single point mutation (A302S) in the GABA receptor RDL that has been identified previously in other species and which confers low levels of resistance to fipronil (23-fold) in N. lugans. In addition, we have identified a second resistance-associated RDL mutation (R300Q) that, in combination with A302S, is associated with much higher levels of resistance (237-fold). The R300Q mutation has not been detected in the absence of A302S in either laboratory-selected or field populations, presumably due to the high fitness cost associated with this mutation. Significantly, it appears that the A302S mutation is able to compensate for deleterious effects of R300Q mutation on fitness cost. These findings identify a novel resistance mechanism and may have important implications for the spread of insecticide resistance. PMID:27557781

  4. Effects of active site cleft residues on oligosaccharide binding, hydrolysis, and glycosynthase activities of rice BGlu1 and its mutants.

    PubMed

    Pengthaisong, Salila; Ketudat Cairns, James R

    2014-12-01

    Rice BGlu1 (Os3BGlu7) is a glycoside hydrolase family 1 β-glucosidase that hydrolyzes cellooligosaccharides with increasing efficiency as the degree of polymerization (DP) increases from 2 to 6, indicating six subsites for glucosyl residue binding. Five subsites have been identified in X-ray crystal structures of cellooligosaccharide complexes with its E176Q acid-base and E386G nucleophile mutants. X-ray crystal structures indicate that cellotetraose binds in a similar mode in BGlu1 E176Q and E386G, but in a different mode in the BGlu1 E386G/Y341A variant, in which glucosyl residue 4 (Glc4) interacts with Q187 instead of the eliminated phenolic group of Y341. Here, we found that the Q187A mutation has little effect on BGlu1 cellooligosaccharide hydrolysis activity or oligosaccharide binding in BGlu1 E176Q, and only slight effects on BGlu1 E386G glycosynthase activity. X-ray crystal structures showed that cellotetraose binds in a different position in BGlu1 E176Q/Y341A, in which it interacts directly with R178 and W337, and the Q187A mutation had little effect on cellotetraose binding. Mutations of R178 and W337 to A had significant and nonadditive effects on oligosaccharide hydrolysis by BGlu1, pNPGlc cleavage and cellooligosaccharide inhibition of BGlu1 E176Q and BGlu1 E386G glycosynthase activity. Hydrolysis activity was partially rescued by Y341 for longer substrates, suggesting stacking of Glc4 on Y341 stabilizes binding of cellooligosaccharides in the optimal position for hydrolysis. This analysis indicates that complex interactions between active site cleft residues modulate substrate binding and hydrolysis.

  5. Second site mutation in the virus envelope expands the host range of a cytopathic variant of Moloney murine leukemia virus.

    PubMed

    Ferrarone, John; Knoper, Ryan C; Li, Randolph; Kozak, Christine A

    2012-11-10

    Spl574 MLV (murine leukemia virus) is a variant of Moloney ecotropic MLV (MoMLV) that is cytopathic in Mus dunni cells and restricted by other mouse cells. Its host range and cytopathicity are due to a mutation, S82F, at a site critical for binding to the CAT-1 receptor. To identify residues that affect affinity for receptor variants, virus with S82F was passed in restrictive cells. The env genes of the adapted viruses contained 18 novel mutations, including one, E114G, present in 6 of 30 sequenced envs. MoMLV-E114G efficiently infected all mouse cells as well as ecotropic MLV resistant Chinese hamster cells. Virus with E114G and S82F induced large multinucleated syncytia in NIH 3T3 and SC-1 cells as well as M. dunni cells. Inoculation of Mo-S82F,E114G into mice produced lymphomas typical of MoMLV. Residues at env position 114 are thus important determinants of host range, and E114G suppresses host range restriction due to S82F, but does not affect S82F-governed cytopathicity.

  6. Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma

    PubMed Central

    Moore, Amanda R; Ceraudo, Emilie; Sher, Jessica J; Guan, Youxin; Shoushtari, Alexander N; Chang, Matthew T; Zhang, Jenny Q; Walczak, Edward G; Kazmi, Manija A; Taylor, Barry S; Huber, Thomas; Chi, Ping; Sakmar, Thomas P; Chen, Yu

    2016-01-01

    Uveal melanomas are molecularly distinct from cutaneous melanomas and lack mutations in BRAF, NRAS, KIT, and NF1. Instead, they are characterized by activating mutations in GNAQ and GNA11, two highly homologous α subunits of Gαq/11 heterotrimeric G proteins, and in PLCB4 (phospholipase C β4), the downstream effector of Gαq signaling 1–3. We analyzed genomics data from 136 uveal melanoma samples and found a recurrent mutation in CYSLTR2 (cysteinyl leukotriene receptor 2) encoding a p.Leu129Gln substitution in 4 of 9 samples that lacked mutations in GNAQ, GNA11, and PLCB4 but in 0 of 127 samples that harbored mutations in these genes. The Leu129Gln CysLT2R mutant protein constitutively activates endogenous Gαq and is unresponsive to stimulation by leukotriene. Expression of Leu129Gln CysLT2R in melanocytes enforces expression of a melanocyte-lineage signature, drives phorbol ester–independent growth in vitro, and promotes tumorigenesis in vivo. Our findings implicate CYSLTR2 as a uveal melanoma oncogene and highlight the critical role of Gαq signaling in uveal melanoma pathogenesis. PMID:27089179

  7. Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii.

    PubMed

    Leitgeb, Stefan; Straganz, Grit D; Nidetzky, Bernd

    2009-03-01

    beta-diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1protomer adopts a single-domain beta-barrel fold characteristic of the cupin superfamily of proteins and features a mononuclear non-haem Fe2+ centre where a triad of histidine residues, His-62, His-64 and His-104, co-ordinate the catalytic metal. To provide structure-function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp binding site for non-haem Fe2+,we replaced each histidine residue individually with glutamate and asparagine and compared binding of Fe2+ and four non-native catalytically inactive metals with purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe2+) and fluorescence titration (Fe2+, Cu2+, Ni2+, Mn2+ and Zn2+) experiments revealed the presence of two broadly specific metal-binding sites in native Dke1 that bind Fe2+ with a dissociation constant (Kd) of 5 microM (site I) and approximately 0.3 mM (site II). Each mutation, except for the substitution of asparagine for His-104, disrupted binding of Fe2+, but not that of the other bivalent metal ions, at site I,while leaving metal binding at site II largely unaffected. Dke1 mutants harbouring glutamate substitutions were completely inactive and not functionally complemented by external Fe2+.The Fe2+ catalytic centre activity (kcat) of mutants with asparagine substitution of His-62 and His-104 was decreased 140- and 220-fold respectively, compared with the kcat value of 8.5 s(-1) for the wild-type enzyme in the reaction with pentane-2,4-dione.The H64N mutant was not catalytically competent, except in the presence of external Fe2+ (1 mM) which elicited about 1/1000 of wild-type activity. Therefore co-ordination of Fe2+ by Dke1 requires an uncharged metallocentre, and three histidine ligands are needed for the assembly of a fully functional catalytic site. Oxidative inactivation of Dke

  8. Probing the pigment binding sites in LHCII with resonance Raman spectroscopy: The effect of mutations at S123.

    PubMed

    Kish, Elizabeth; Wang, Ke; Llansola-Portoles, Manuel J; Ilioaia, Cristian; Pascal, Andrew A; Robert, Bruno; Yang, Chunhong

    2016-09-01

    Resonance Raman spectroscopy was used to evaluate the structure of light-harvesting chlorophyll (Chl) a/b complexes of photosystem II (LHCII), reconstituted from wild-type (WT) and mutant apoproteins over-expressed in Escherichia coli. The point mutations involved residue S123, exchanged for either P (S123P) or G (S123G). In all reconstituted proteins, lutein 2 displayed a distorted conformation, as it does in purified LHCII trimers. Reconstituted WT and S123G also exhibited a conformation of bound neoxanthin (Nx) molecules identical to the native protein, while the S123P mutation was found to induce a change in Nx conformation. This structural change of neoxanthin is accompanied by a blue shift of the absorption of this carotenoid molecule. The interactions assumed by (and thus the structure of the binding sites of) the bound Chls b were found identical in all the reconstituted proteins, and only marginally perturbed as compared to purified LHCII. The interactions assumed by bound Chls a were also identical in purified LHCII and the reconstituted WT. However, the keto carbonyl group of one Chl a, originally free-from-interactions in WT LHCII, becomes involved in a strong H-bond with its environment in LHCII reconstituted from the S123P apoprotein. As the absorption in the Qy region of this protein is identical to that of the LHCII reconstituted from the WT apoprotein, we conclude that the interaction state of the keto carbonyl of Chl a does not play a significant role in tuning the binding site energy of these molecules.

  9. Effect of mutations in the cyclic AMP receptor protein-binding site on araBAD and araC expression.

    PubMed

    Stoltzfus, L; Wilcox, G

    1989-02-01

    Maximum expression of the adjacent but divergently transcribed araBAD operon and araC gene requires the presence of cyclic AMP (cAMP) and the cAMP receptor protein (CRP). DNase I protection studies have previously revealed a high-affinity CRP-binding site in the ara regulatory region. Deletion mutations introduced into this site resulted in reduced expression of araBAD and araC. However, other experiments have demonstrated that spacing changes in the ara regulatory region may have multiple effects due to disruption of a DNA loop. Thus, the deletions could have destroyed the CRP-binding site, the ability to form a loop, or both. In the present study, substitution mutations were introduced into the CRP site in order to avoid creating spacing changes. We found that a 3-base-pair substitution resulted in a 30% reduction in araBAD expression, whereas a 6-base-pair substitution resulted in an 80% reduction. Both of these substitution mutations reduced araC expression threefold. We conclude that CRP bound to this site regulates expression in both directions. We found that a spacing change in the CRP site does not alter araBAD expression any more than does a substitution mutation.

  10. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined.

  11. Active site proton delivery and the lyase activity of human CYP17A1

    SciTech Connect

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G.

    2014-01-03

    equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.

  12. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  13. Mutational activation of ErbB2 reveals a new protein kinase autoinhibition mechanism.

    PubMed

    Fan, Ying-Xin; Wong, Lily; Ding, Jinhui; Spiridonov, Nikolay A; Johnson, Richard C; Johnson, Gibbes R

    2008-01-18

    Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the alphaC helix and beta4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to approximately 10- and approximately 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the alphaC-beta4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the alphaC-beta4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the alphaC-beta4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of alphaC-beta4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.

  14. 8-Methoxypsoralen induced mutations are highly targeted at crosslinkable sites of photoaddition on the non-transcribed strand of a mammalian chromosomal gene.

    PubMed Central

    Sage, E; Drobetsky, E A; Moustacchi, E

    1993-01-01

    We have determined the mutational specificity of 8-methoxypsoralen photoaddition at the endogenous adenine phosphoribosyltransferase gene of Chinese hamster ovary cells hemizygous for this locus. In addition, the distribution of 8-methoxypsoralen photo-adducts was resolved in vitro at the DNA sequence level, and compared with the observed site specificity for mutation. Among 27 mutants characterized, all were single base changes at AT base pairs: 16 A:T-->T:A, six A:T-->C:G, four A:T-->G:C and one -T frameshift. All these vents were targeted to potential sites of photoaddition. The vast majority of these sites were also detectable in vitro, suggesting that 8-methoxypsoralen plus UVA-induced mutational hotspots may be damage hotspots. Furthermore 26/27 mutations occurred at crosslinkable 5'TpA sites, supporting the notion that 8-methoxypsoralen biadducts rather than monoadducts are major premutagenic lesions in mammalian cells. Since 90% of our mutation collection could have resulted from damage on the non-transcribed strand, it appears that photoadducted thymine residues on the transcribed strand of the adenine phosphoribosyltransferase gene may be preferentially repaired. We therefore suggest a model for mutagenesis, induced by psoralen biadducts, based on the preferential incision of biadducts followed by translesion synthesis past modified T bases persisting on the non-transcribed strand. Images PMID:8440233

  15. Asp120Asn mutation impairs the catalytic activity of NDM-1 metallo-β-lactamase: experimental and computational study.

    PubMed

    Chen, Jiao; Chen, Hui; Zhu, Tong; Zhou, Dandan; Zhang, Fang; Lao, Xingzhen; Zheng, Heng

    2014-04-14

    New Delhi metallo-β-lactamase-1 (NDM-1) has attracted extensive attention in recent years for its high activity for hydrolyzing almost all β-lactam antibiotics. Like other metallo-β-lactamases (MβLs), NDM-1 features an invariant Asp120 that ligates the zinc ion (ZN2) in the active site. Previous studies showed that substitutions of Asp120 with residues such as Ala, Ser, Asn and Glu dramatically impaired the MβL (BcII, IMP-1, L1) activity, but no consensus about the exact role of Asp120 has reached. Here we constructed D120N mutant of NDM-1 by site-directed mutagenesis. The replacement of Asp120 with Asn, which has much weaker metal ligating capabilities than Asp, severely impaired the lactamase activity without abolishing the ZN2 site. Molecular dynamics simulations suggested that the ZN1-ZN2 distance increased because of mutation, leading to a rearrangement of the active site, including the bridging OH(-). Thereby, the Mulliken charges of ZN1 and ZN2 redistributed, especially for ZN2, which might be the major cause of the impaired activity. Reducing the point charges of Asp120 carboxyl oxygens weakened the ionic interactions between Asp120 and ZN2, and the positions of the zinc ions were also changed as a result. It is proposed that Asp120 acts as a strong ZN2 ligand, positioning ZN2 for catalytically important interactions with the substrate, stabilizing the negatively charged amide nitrogen of the hydrolyzed intermediate, and more importantly, orienting the ZN-bound OH(-) for nucleophilic attacks and protonation. These functions are of general importance for catalyzing β-lactam antibiotics by NDM-1 as well as other MβLs.

  16. Genetic characterization of seasonal influenza A (H3N2) viruses in Ontario during 2010–2011 influenza season: high prevalence of mutations at antigenic sites

    PubMed Central

    Eshaghi, AliReza; Duvvuri, Venkata R; Li, Aimin; Patel, Samir N; Bastien, Nathalie; Li, Yan; Low, Donald E; Gubbay, Jonathan B

    2014-01-01

    Background The direct effect of antigenic site mutations in influenza viruses on antigenic drift and vaccine effectiveness is poorly understood. Objective To investigate the genetic and antigenic characteristics of human influenza A (H3N2) viruses circulating in Ontario during the early 2010–2011 winter season. Study design We sequenced the hemagglutinin (HA) and neuraminidase (NA) genes from 41 A(H3N2) viruses detected in nasopharyngeal specimens. Strain typing was performed by hemagglutination inhibition (HI) assay. Molecular and phylogenetic tree analyses were conducted. Results HA and NA genes showed high similarity to the 2010–2011 vaccine strain, A/Perth/16/2009 (H3N2)-like virus (97·7–98·5% and 98·7–99·5% amino acid (AA) identity, respectively). Compared to A/Perth/16/2009 strain, HA gene mutations were documented at 28 different AA positions across all five H3 antigenic sites, with a range of 5–11 mutations in individual viruses. Thirty-six (88%) viruses had 8 AA substitutions in common; none of these had reduced HI titer. Among Ontario isolates, 11 antigenic site AAs were positively selected with an increase in glycosylation sites. Conclusion The presence of antigenic site mutations with high frequency among 2010–2011 influenza H3N2 isolates confirms ongoing adaptive H3N2 evolution. These may represent early phylogenetic changes that could cause antigenic drift with further mutations. Clinical relevance of antigenic site mutations not causing drift in HI assays is unknown and requires further investigation. In addition, viral sequencing information will assist with vaccine strain planning and may facilitate early detection of vaccine escape. PMID:24313991

  17. The prognostic IDH1( R132 ) mutation is associated with reduced NADP+-dependent IDH activity in glioblastoma.

    PubMed

    Bleeker, Fonnet E; Atai, Nadia A; Lamba, Simona; Jonker, Ard; Rijkeboer, Denise; Bosch, Klazien S; Tigchelaar, Wikky; Troost, Dirk; Vandertop, W Peter; Bardelli, Alberto; Van Noorden, Cornelis J F

    2010-04-01

    Somatic mutations in the isocitrate dehydrogenase 1 gene (IDH1) occur at high frequency in gliomas and seem to be a prognostic factor for survival in glioblastoma patients. In our set of 98 glioblastoma patients, IDH1 ( R132 ) mutations were associated with improved survival of 1 year on average, after correcting for age and other variables with Cox proportional hazards models. Patients with IDH1 mutations were on average 17 years younger than patients without mutation. Mutated IDH1 has a gain of function to produce 2-hydroxyglutarate by NADPH-dependent reduction of alpha-ketoglutarate, but it is unknown whether NADPH production in gliomas is affected by IDH1 mutations. We assessed the effect of IDH1 (R132 ) mutations on IDH-mediated NADPH production in glioblastomas in situ. Metabolic mapping and image analysis was applied to 51 glioblastoma samples of which 16 carried an IDH1 (R132 ) mutation. NADP+-dependent IDH activity was determined in comparison with activity of NAD+-dependent IDH and all other NADPH-producing dehydrogenases, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, and hexose-6-phosphate dehydrogenase. The occurrence of IDH1 mutations correlated with approx. twofold diminished NADP+-dependent IDH activity, whereas activity of NAD+-dependent IDH and the other NADP+-dependent dehydrogenases was not affected in situ in glioblastoma. The total NADPH production capacity in glioblastoma was provided for 65% by IDH activity and the occurrence of IDH1 (R132 ) mutation reduced this capacity by 38%. It is concluded that NADPH production is hampered in glioblastoma with IDH1 (R132 ) mutation. Moreover, mutated IDH1 consumes rather than produces NADPH, thus likely lowering NADPH levels even further. The low NADPH levels may sensitize glioblastoma to irradiation and chemotherapy, thus explaining the prolonged survival of patients with mutated glioblastoma.

  18. Instability of buried hydration sites increases protein subdomains fluctuations in the human prion protein by the pathogenic mutation T188R

    NASA Astrophysics Data System (ADS)

    Tomobe, Katsufumi; Yamamoto, Eiji; Akimoto, Takuma; Yasui, Masato; Yasuoka, Kenji

    2016-05-01

    The conformational change from the cellular prion protein (PrPc) to scrapie prion protein (PrPsc) is a key process in prion diseases. The prion protein has buried water molecules which significantly contribute to the stability of the protein; however, there has been no report investigating the influence on the buried hydration sites by a pathogenic mutation not adjacent to the buried hydration sites. Here, we perform molecular dynamics simulations of wild type (WT) PrPc and pathogenic point mutant T188R to investigate conformational changes and the buried hydration sites. In WT-PrPc, four buried hydration sites are identified by residence time and rotational relaxation analysis. However, there are no stable buried hydration sites in one of T188R simulations, which indicates that T188R sometimes makes the buried hydration sites fragile. We also find that fluctuations of subdomains S1-H1-S2 and H1-H2 increase in T188R when the buried hydration sites become unstable. Since the side chain of arginine which is replaced from threonine in T188R is larger than of threonine, the side chain cannot be embedded in the protein, which is one of the causes of the instability of subdomains. These results show correlations between the buried hydration sites and the mutation which is far from them, and provide a possible explanation for the instability by mutation.

  19. An important site in PBP2x of penicillin-resistant clinical isolates of Streptococcus pneumoniae: mutational analysis of Thr338.

    PubMed

    Zerfass, Ilka; Hakenbeck, Regine; Denapaite, Dalia

    2009-03-01

    Penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae represents a primary resistance determinant for beta-lactams, and low-affinity PBP2x variants can easily be selected with cefotaxime. Penicillin-resistant clinical isolates of S. pneumoniae frequently contain in their mosaic PBP2x the mutation T338A adjacent to the active site S337, and T338P as well as T338G substitutions are also known. Site-directed mutagenesis has now documented that a single point mutation at position T338 confers selectable levels of beta-lactam resistance preferentially to oxacillin. Despite the moderate impact on beta-lactam susceptibility, the function of the PBP2x mutants appears to be impaired, as can be documented in the absence of a functional CiaRH regulatory system, resulting in growth defects and morphological changes. The combination of low-affinity PBP2x and PBP1a encoded by mosaic genes is known to result in high cefotaxime resistance. In contrast, introduction of a mosaic pbp1a into the PBP2x(T338G) mutant did not lead to increased resistance. However, the mosaic PBP1a gene apparently complemented the PBP2x(T338G) defect, since Cia mutant derivatives grew normally. The data support the view that PBP2x and PBP1a interact with each other on some level and that alterations of both PBPs in resistant clinical isolates have evolved to ensure cooperation between both proteins.

  20. Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30.

    PubMed

    Narumi, I; Satoh, K; Kikuchi, M; Funayama, T; Kitayama, S; Yanagisawa, T; Watanabe, H; Yamamoto, K

    1999-12-07

    Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.

  1. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses.

    PubMed

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F X; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A; Roffler, Stefan

    2016-09-07

    DNA (class 2) transposons are mobile genetic elements which move within their 'host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind.

  2. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses

    PubMed Central

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F. X.; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A.; Roffler, Stefan

    2016-01-01

    DNA (class 2) transposons are mobile genetic elements which move within their ‘host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind. PMID:27599761

  3. BAX Activation is Initiated at a Novel Interaction Site

    PubMed Central

    Gavathiotis, Evripidis; Suzuki, Motoshi; Davis, Marguerite L.; Pitter, Kenneth; Bird, Gregory H.; Katz, Samuel G.; Tu, Ho-Chou; Kim, Hyungjin; Cheng, Emily H.-Y.; Tjandra, Nico; Walensky, Loren D.

    2008-01-01

    BAX is a pro-apoptotic protein of the BCL-2 family stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the BIM SAHB-BAX interaction is highlighted by point mutagenesis that abrogates functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis. PMID:18948948

  4. Five novel mutations of the protein S active gene (PROS 1) in 8 Norman families.

    PubMed

    Duchemin, J; Borg, J Y; Borgel, D; Vasse, M; Lévèque, H; Aiach, M; Gandrille, S

    1996-03-01

    To further elucidate the molecular basis for hereditary thrombophilia, we screened the protein S active gene in 11 families with type I deficiency, using a strategy based on denaturing gradient gel electrophoresis (DGGE) of all the coding sequences. Fragments with an abnormal DGGE pattern were sequenced, and 5 novel mutations were identified in 8 families. The mutations were a 7-nucleotide deletion in exon II, a 4-nucleotide deletion in exon III, a T insertion in exon VII, a C to T transition transforming Leu 259 into Pro and a T to C transition transforming Cys 625 into Arg in 4 families. These mutations were the only sequence variations found in the propositus' gene exons and co-segregated with the plasma phenotype. A total of 28 members of these 8 families were heterozygous for one of the 5 mutations. Twenty-four (58,5%) of the 41 deficient subjects over 18 years of age had clinical thrombophilia, whereas the 13 subjects under 18 were asymptomatic. Of the 28 subjects, 6 (21,5%) were also found to bear the factor V Arg 506 Gln mutation.

  5. Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency.

    PubMed

    Schlotawa, Lars; Steinfeld, Robert; von Figura, Kurt; Dierks, Thomas; Gärtner, Jutta

    2008-01-01

    Multiple Sulfatase Deficiency (MSD) is a rare inborn autosomal-recessive disorder, which mainly combines clinical features of metachromatic leukodystrophy, mucopolysaccharidosis and X-linked ichthyosis. The clinical course ranges from neonatal severe to mild juvenile cases. MSD is caused by mutations in the SUMF1 gene encoding the formylglycine-generating enzyme (FGE). FGE posttranslationally activates sulfatases by generating formylglycine in their catalytic sites. We analyzed the functional consequences of missense mutations p.A177P, p.W179S, p.A279V and p.R349W with regard to FGE's subcellular localization, enzymatic activity, protein stability, intracellular retention and resulting sulfatase activities. All four mutations did not affect localization of FGE in the endoplasmic reticulum of MSD fibroblasts. However, they decreased its specific enzymatic activity to less than 1% (p.A177P and p.R349W), 3% (p.W179S) or 23% (p.A279V). Protein stability was severely decreased for p.A279V and p.R349W, and almost comparable to wild type for p.A177P and p.W179S. The patient with the mildest clinical phenotype carries the mutation p.A279V leading to decreased FGE protein stability, but high residual enzymatic activity and only slightly reduced sulfatase activities. In contrast, the most severely affected patient carries the mutation p.R349W leading to drastically decreased protein stability, very low residual enzymatic activity and considerably reduced sulfatase activities. Our functional studies provide novel insight into the molecular defect underlying MSD and reveal that both residual enzyme activity and protein stability of FGE contribute to the clinical phenotype. The application of improved functional assays to determine these two molecular parameters of FGE mutants may enable the prediction of the clinical outcome in the future.

  6. Identification of a novel K311 ubiquitination site critical for androgen receptor transcriptional activity.

    PubMed

    McClurg, Urszula L; Cork, David M W; Darby, Steven; Ryan-Munden, Claudia A; Nakjang, Sirintra; Mendes Côrtes, Leticia; Treumann, Achim; Gaughan, Luke; Robson, Craig N

    2016-11-29

    The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway.

  7. Somatic activating mutations in Pik3ca cause sporadic venous malformations in mice and humans.

    PubMed

    Castillo, Sandra D; Tzouanacou, Elena; Zaw-Thin, May; Berenjeno, Inma M; Parker, Victoria E R; Chivite, Iñigo; Milà-Guasch, Maria; Pearce, Wayne; Solomon, Isabelle; Angulo-Urarte, Ana; Figueiredo, Ana M; Dewhurst, Robert E; Knox, Rachel G; Clark, Graeme R; Scudamore, Cheryl L; Badar, Adam; Kalber, Tammy L; Foster, Julie; Stuckey, Daniel J; David, Anna L; Phillips, Wayne A; Lythgoe, Mark F; Wilson, Valerie; Semple, Robert K; Sebire, Neil J; Kinsler, Veronica A; Graupera, Mariona; Vanhaesebroeck, Bart

    2016-03-30

    Venous malformations (VMs) are painful and deforming vascular lesions composed of dilated vascular channels, which are present from birth. Mutations in the TEK gene, encoding the tyrosine kinase receptor TIE2, are found in about half of sporadic (nonfamilial) VMs, and the causes of the remaining cases are unknown. Sclerotherapy, widely accepted as first-line treatment, is not fully efficient, and targeted therapy for this disease remains underexplored. We have generated a mouse model that faithfully mirrors human VM through mosaic expression of Pik3ca(H1047R), a constitutively active mutant of the p110α isoform of phosphatidylinositol 3-kinase (PI3K), in the embryonic mesoderm. Endothelial expression of Pik3ca(H1047R)resulted in endothelial cell (EC) hyperproliferation, reduction in pericyte coverage of blood vessels, and decreased expression of arteriovenous specification markers. PI3K pathway inhibition with rapamycin normalized EC hyperproliferation and pericyte coverage in postnatal retinas and stimulated VM regression in vivo. In line with the mouse data, we also report the presence of activating PIK3CA mutations in human VMs, mutually exclusive with TEK mutations. Our data demonstrate a causal relationship between activating Pik3ca mutations and the genesis of VMs, provide a genetic model that faithfully mirrors the normal etiology and development of this human disease, and establish the basis for the use of PI3K-targeted therapies in VMs.

  8. The Arabidopsis AtPP2CA Protein Phosphatase Inhibits the GORK K+ Efflux Channel and Exerts a Dominant Suppressive Effect on Phosphomimetic-activating Mutations*

    PubMed Central

    Lefoulon, Cécile; Boeglin, Martin; Moreau, Bertrand; Véry, Anne-Aliénor; Szponarski, Wojciech; Dauzat, Myriam; Michard, Erwan; Gaillard, Isabelle; Chérel, Isabelle

    2016-01-01

    The regulation of the GORK (Guard Cell Outward Rectifying) Shaker channel mediating a massive K+ efflux in Arabidopsis guard cells by the phosphatase AtPP2CA was investigated. Unlike the gork mutant, the atpp2ca mutants displayed a phenotype of reduced transpiration. We found that AtPP2CA interacts physically with GORK and inhibits GORK activity in Xenopus oocytes. Several amino acid substitutions in the AtPP2CA active site, including the dominant interfering G145D mutation, disrupted the GORK-AtPP2CA interaction, meaning that the native conformation of the AtPP2CA active site is required for the GORK-AtPP2CA interaction. Furthermore, two serines in the GORK ankyrin domain that mimic phosphorylation (Ser to Glu) or dephosphorylation (Ser to Ala) were mutated. Mutations mimicking phosphorylation led to a significant increase in GORK activity, whereas mutations mimicking dephosphorylation had no effect on GORK. In Xenopus oocytes, the interaction of AtPP2CA with “phosphorylated” or “dephosphorylated” GORK systematically led to inhibition of the channel to the same baseline level. Single-channel recordings indicated that the GORK S722E mutation increases the open probability of the channel in the absence, but not in the presence, of AtPP2CA. The dephosphorylation-independent inactivation mechanism of GORK by AtPP2CA is discussed in relation with well known conformational changes in animal Shaker-like channels that lead to channel opening and closing. In plants, PP2C activity would control the stomatal aperture by regulating both GORK and SLAC1, the two main channels required for stomatal closure. PMID:26801610

  9. Involvement of novel autophosphorylation sites in ATM activation.

    PubMed

    Kozlov, Sergei V; Graham, Mark E; Peng, Cheng; Chen, Philip; Robinson, Phillip J; Lavin, Martin F

    2006-08-09

    ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

  10. Resonant active sites in catalytic ammonia synthesis: A structural model

    NASA Astrophysics Data System (ADS)

    Cholach, Alexander R.; Bryliakova, Anna A.; Matveev, Andrey V.; Bulgakov, Nikolai N.

    2016-03-01

    Adsorption sites Mn consisted of n adjacent atoms M, each bound to the adsorbed species, are considered within a realistic model. The sum of bonds Σ lost by atoms in a site in comparison with the bulk atoms was used for evaluation of the local surface imperfection, while the reaction enthalpy at that site was used as a measure of activity. The comparative study of Mn sites (n = 1-5) at basal planes of Pt, Rh, Ir, Fe, Re and Ru with respect to heat of N2 dissociative adsorption QN and heat of Nad + Had → NHad reaction QNH was performed using semi-empirical calculations. Linear QN(Σ) increase and QNH(Σ) decrease allowed to specify the resonant Σ for each surface in catalytic ammonia synthesis at equilibrium Nad coverage. Optimal Σ are realizable for Ru2, Re2 and Ir4 only, whereas other centers meet steric inhibition or unreal crystal structure. Relative activity of the most active sites in proportion 5.0 × 10- 5: 4.5 × 10- 3: 1: 2.5: 3.0: 1080: 2270 for a sequence of Pt4, Rh4, Fe4(fcc), Ir4, Fe2-5(bcc), Ru2, Re2, respectively, is in agreement with relevant experimental data. Similar approach can be applied to other adsorption or catalytic processes exhibiting structure sensitivity.

  11. Effect of point mutations on Herbaspirillum seropedicae NifA activity.

    PubMed

    Aquino, B; Stefanello, A A; Oliveira, M A S; Pedrosa, F O; Souza, E M; Monteiro, R A; Chubatsu, L S

    2015-08-01

    NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

  12. Point mutations at the catalytic site of PCSK9 inhibit folding, autoprocessing, and interaction with the LDL receptor.

    PubMed

    Garvie, Colin W; Fraley, Cara V; Elowe, Nadine H; Culyba, Elizabeth K; Lemke, Christopher T; Hubbard, Brian K; Kaushik, Virendar K; Daniels, Douglas S

    2016-11-01

    Circulating low-density lipoprotein cholesterol (LDLc) is regulated by membrane-bound LDL receptor (LDLr). Upon LDLc and LDLr interaction the complex is internalized by the cell, leading to LDLc degradation and LDLr recycling back to the cell surface. The proprotein convertase subtilisin/kexin type 9 (PCSK9) protein regulates this cycling. PCSK9 is secreted from the cell and binds LDLr. When the complex is internalized, PCSK9 prevents LDLr from shuttling back to the surface and instead targets it for degradation. PCSK9 is a serine protease expressed as a zymogen that undergoes autoproteolysis, though the two resulting protein domains remain stably associated as a heterodimer. This PCSK9 autoprocessing is required for the protein to be secreted from the cell. To date, direct analysis of PCSK9 autoprocessing has proven challenging, as no catalytically active zymogen has been isolated. A PCSK9 loss-of-function point mutation (Q152H) that reduces LDLc levels two-fold was identified in a patient population. LDLc reduction was attributed to a lack of PCSK9(Q152H) autoprocessing preventing secretion of the protein. We have isolated a zymogen form of PCSK9, PCSK9(Q152H), and a related mutation (Q152N), that can undergo slow autoproteolysis. We show that the point mutation prevents the formation of the mature form of PCSK9 by hindering folding, reducing the rate of autoproteolysis, and destabilizing the heterodimeric form of the protein. In addition, we show that the zymogen form of PCSK9 adopts a structure that is distinct from the processed form and is unable to bind a mimetic peptide based on the EGF-A domain of the LDLr.

  13. Branchio-Oto-Renal Syndrome (BOR) associated with focal glomerulosclerosis in a patient with a novel EYA1 splice site mutation

    PubMed Central

    2013-01-01

    Background Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial, ear, and renal anomalies. The most common gene mutated in BOR patients is EYA1, the human homolog of the Drosophila eyes absent gene, while mutations in SIX1 gene, the human homolog of sine oculis, encoding a DNA binding protein interacting with EYA1, have been reported less frequently. Recently, mutations in another SIX family member, SIX5, have been described in BOR patients, however, this association has not been confirmed by other groups. Case presentation In this study, we have clinically and genetically characterized a proband that displayed hearing loss, pre-auricular pits, branchial fistulae, hypoplasia of the left kidney, bilateral mild hydronephrosis, progressive proteinuria and focal glomerulosclerosis. Mutational analysis of EYA1 gene revealed a novel splice site mutation, c.1475 + 1G > C, that affects EYA1 splicing and produces an aberrant mRNA transcript, lacking exon 15, which is predicted to encode a truncated protein of 456 aa. Conclusion This report provided the functional description of a novel EYA1 splice site mutation and described for the first time a case of BOR syndrome associated with the atypical renal finding of focal glomerulosclerosis, highlighting the importance of molecular testing and detailed clinical evaluation to provide accurate diagnosis and appropriate genetic counselling. PMID:23506628

  14. A transforming mutation enhances the activity of the c-Kit soluble tyrosine kinase domain.

    PubMed Central

    Lam, L P; Chow, R Y; Berger, S A

    1999-01-01

    An activating mutation (DY814) located in the catalytic domain of the c-Kit receptor has been found in mastocytomas from human, mouse and rat. We evaluated the enzymic properties of purified wild-type (WT) and DY814 tyrosine kinase domains expressed in Pichia pastoris. A linker encoding the Flag epitope was fused to c-Kit cDNA species, enabling affinity purification of the proteins with anti-Flag antibodies. Yeast lysates expressing DY814 contained multiple tyrosine-phosphorylated proteins, whereas WT lysates had no detectable tyrosine phosphorylation. Purification of the WT and mutant kinases in the presence of vanadate demonstrated that both enzymes undergo autophosphorylation. Kinetic analyses of WT and DY814 kinases indicated that at 20 nM enzyme concentration the mutation increases the specific activity 10-fold and decreases the apparent Km for ATP 9-fold. WT activity displayed a hyperbolic dependence on enzyme concentration, consistent with a requirement for dimerization or aggregation for activity. This activity was also enhanced by anti-Flag antibodies. In contrast, the dependence of DY814 activity on enzyme concentration was primarily linear and only marginally enhanced by anti-Flag antibodies. Gel-filtration analysis showed that the WT kinase migrated as a monomer, whereas the DY814 mutant migrated as a dimer. These results indicate that this point mutation promotes dimerization of the c-Kit kinase, potentially contributing to its transforming potential in mast cells. PMID:9931308

  15. Chemical Modification of Papain and Subtilisin: An Active Site Comparison

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

    An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

  16. Spectroscopic studies of the active site of galactose oxidase

    SciTech Connect

    Knowles, P.F.; Brown, R.D. III; Koenig, S.H.

    1995-07-19

    X-ray absorption and EPR spectroscopy have been used to probe the copper site structure in galactose oxidase at pH 4.5 and 7.0. the results suggest that there are no major differences in the structure of the tetragonal Cu(II) site at these pH values. Analysis of the extended X-ray absorption fine structure (EXAFS) indicates that four N,O scatterers are present at approximately 2 {Angstrom}; these are presumably the equatorial ligands. In addition, the EXAFS data establish that oxidative activation to produce the active-site tyrosine radical does not cause major changes in the copper coordination environment. Therefore results obtained on the one-electron reduced enzyme, containing Cu(II) but not the tyrosine radical, probably also apply to the catalytically active Cu(II)/tyrosine radical state. Solvent water exchange, inhibitor binding, and substrate binding have been probed via nuclear magnetic relaxation dispersion (NMRD) measurements. The NMRD profile of galactose oxidase is quantitatively consistent with the rapid exchange of a single, equatorial water ligand with a Cu(II)-O separation of about 2.4 {Angstrom}. Azide and cyanide displace this coordinated water. The binding of azide and the substrate dihydroxyacetone produce very similar effects on the NMRD profile of galactose oxidase, indicating that substrates also bind to the active site Cu(II) in an equatorial position.

  17. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  18. Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3' Splice Site Selection through Use of a Different Branch Point.

    PubMed

    Darman, Rachel B; Seiler, Michael; Agrawal, Anant A; Lim, Kian H; Peng, Shouyong; Aird, Daniel; Bailey, Suzanna L; Bhavsar, Erica B; Chan, Betty; Colla, Simona; Corson, Laura; Feala, Jacob; Fekkes, Peter; Ichikawa, Kana; Keaney, Gregg F; Lee, Linda; Kumar, Pavan; Kunii, Kaiko; MacKenzie, Crystal; Matijevic, Mark; Mizui, Yoshiharu; Myint, Khin; Park, Eun Sun; Puyang, Xiaoling; Selvaraj, Anand; Thomas, Michael P; Tsai, Jennifer; Wang, John Y; Warmuth, Markus; Yang, Hui; Zhu, Ping; Garcia-Manero, Guillermo; Furman, Richard R; Yu, Lihua; Smith, Peter G; Buonamici, Silvia

    2015-11-03

    Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.

  19. Promiscuous Mutations Activate the Non-Canonical NF-kB Pathway in Multiple Myeloma

    PubMed Central

    Keats, Jonathan J.; Fonseca, Rafael; Chesi, Marta; Schop, Roelandt; Baker, Angela; Chng, Wee-Joo; Van Wier, Scott; Tiedemann, Rodger; Shi, Chang-Xin; Sebag, Michael; Braggio, Esteban; Henry, Travis; Zhu, Yuan-Xiao; Fogle, Homer; Price-Troska, Tammy; Ahmann, Gregory; Mancini, Catherine; Brents, Leslie A.; Kumar, Shaji; Greipp, Philip; Dispenzieri, Angela; Bryant, Barb; Mulligan, George; Bruhn, Laurakay; Barrett, Michael; Valdez, Riccardo; Trent, Jeff; Stewart, A. Keith; Carpten, John; Bergsagel, P. Leif

    2007-01-01

    Summary Activation of NF-kB has been noted in many tumor types, however only rarely has this been linked to an underlying genetic mutation. An integrated analysis of high-density oligonucleotide array CGH and gene expression profiling data from 155 multiple myeloma samples identified a promiscuous array of abnormalities contributing to the dysregulation of NF-kB in approximately 20% of patients. We report mutations in ten genes causing the inactivation of TRAF2, TRAF3, CYLD, cIAP1/cIAP2, and activation of NFKB1, NFKB2, CD40, LTBR, TACI, and NIK that result primarily in constitutive activation of the non-canonical NF-kB pathway, with the single most common abnormality being inactivation of TRAF3. These results highlight the critical importance of the NF-kB pathway in the pathogenesis of multiple myeloma. PMID:17692805

  20. A novel TMPRSS6 mutation that prevents protease auto-activation causes IRIDA

    PubMed Central

    Altamura, Sandro; D'Alessio, Flavia; Selle, Barbara; Muckenthaler, Martina U.

    2010-01-01

    IRIDA (iron-refractory iron-deficiency anaemia) is a rare autosomal-recessive disorder hallmarked by hypochromic microcytic anaemia, low transferrin saturation and high levels of the iron-regulated hormone hepcidin. The disease is caused by mutations in the transmembrane serine protease TMPRSS6 (transmembrane protease serine 6) that prevent inactivation of HJV (haemojuvelin), an activator of hepcidin transcription. In the present paper, we describe a patient with IRIDA who carries a novel mutation (Y141C) in the SEA domain of the TMPRSS6 gene. Functional characterization of the TMPRSS6(Y141C) mutant protein in cultured cells showed that it localizes to similar subcellular compartments as wild-type TMPRSS6 and binds HJV, but fails to auto-catalytically activate itself. As a consequence, hepcidin mRNA expression is increased, causing the clinical symptoms observed in this IRIDA patient. The present study provides important mechanistic insight into how TMPRSS6 is activated. PMID:20704562

  1. An activating Pik3ca mutation coupled with Pten loss is sufficient to initiate ovarian tumorigenesis in mice.

    PubMed

    Kinross, Kathryn M; Montgomery, Karen G; Kleinschmidt, Margarete; Waring, Paul; Ivetac, Ivan; Tikoo, Anjali; Saad, Mirette; Hare, Lauren; Roh, Vincent; Mantamadiotis, Theo; Sheppard, Karen E; Ryland, Georgina L; Campbell, Ian G; Gorringe, Kylie L; Christensen, James G; Cullinane, Carleen; Hicks, Rodney J; Pearson, Richard B; Johnstone, Ricky W; McArthur, Grant A; Phillips, Wayne A

    2012-02-01

    Mutations in the gene encoding the p110α subunit of PI3K (PIK3CA) that result in enhanced PI3K activity are frequently observed in human cancers. To better understand the role of mutant PIK3CA in the initiation or progression of tumorigenesis, we generated mice in which a PIK3CA mutation commonly detected in human cancers (the H1047R mutation) could be conditionally knocked into the endogenous Pik3ca locus. Activation of this mutation in the mouse ovary revealed that alone, Pik3caH1047R induced premalignant hyperplasia of the ovarian surface epithelium but no tumors. Concomitantly, we analyzed several human ovarian cancers and found PIK3CA mutations coexistent with KRAS and/or PTEN mutations, raising the possibility that a secondary defect in a co-regulator of PI3K activity may be required for mutant PIK3CA to promote transformation. Consistent with this notion, we found that Pik3caH1047R mutation plus Pten deletion in the mouse ovary led to the development of ovarian serous adenocarcinomas and granulosa cell tumors. Both mutational events were required for early, robust Akt activation. Pharmacological inhibition of PI3K/mTOR in these mice delayed tumor growth and prolonged survival. These results demonstrate that the Pik3caH1047R mutation with loss of Pten is enough to promote ovarian cell transformation and that we have developed a model system for studying possible therapies.

  2. Clinical features reflect exon sites of EGFR mutations in patients with resected non-small-cell lung cancer.

    PubMed

    Na, Im Il; Rho, Jin Kyung; Choi, Yun Jung; Kim, Cheol Hyeon; Koh, Jae Soo; Ryoo, Baek-Yeol; Yang, Sung Hyun; Lee, Jae Cheol

    2007-06-01

    The aim of the current study was to determine the clinical significance according to the subtypes of epidermal growth factor receptor (EGFR) mutations and presence of KRAS mutations in operable non-small-cell lung cancer (NSCLC). We sequenced exons 18-21 of the EGFR tyrosine kinase domain and examined mutations in codons 12 and 13 of KRAS in tissues of patients with NSCLC who had undergone surgical resection. EGFR mutations were more frequent in never-smokers than smokers (33% vs. 14%, respectively; p=0.009) and in females than in males (31% vs. 16%, respectively; p=0.036). Mutations in exon 18-19 and 20-21 were found in 10 and 22 patients, respectively. Never-smokers and broncho-alveolar cell carcinoma features were positively associated with a mutation in exon 18-19 (p=0.027 and 0.016, respectively). The five-year survival rate in patients with a mutation in exons 18-19 (100%) was higher than that in patients without such mutation (47%; p=0.021). KRAS mutations were found in 16 patients (12%) and were not related to the overall survival (p=0.742). Patients with an EGFR mutation in exons 18-19 had better survival than patients without such mutation. Subtypes of EGFR mutations may be prognostic factors in patients undergoing curative resection.

  3. A 3' splice site mutation in the thyroglobulin gene responsible for congenital goiter with hypothyroidism.

    PubMed Central

    Ieiri, T; Cochaux, P; Targovnik, H M; Suzuki, M; Shimoda, S; Perret, J; Vassart, G

    1991-01-01

    A case of congenital goiter with defective thyroglobulin synthesis has been studied in molecular terms. The patient is the fifth of a kindred of six, three of which have a goiter. The parents are first cousins. Segregation of thyroglobulin alleles in the family was studied by Southern blotting with a probe revealing a diallelic restriction fragment length polymorphism (RFLP). The results demonstrated that the three affected siblings were homozygous for the RFLP. Northern blotting analysis of the goiter RNA with a thyroglobulin probe suggested that thyroglobulin mRNA size was slightly reduced. Polymerase chain reaction amplification of the 8.5-kb thyroglobulin mRNA as overlapping cDNA fragments demonstrated that a 200-bp segment was missing from the 5' region of the goiter mRNA. Subcloning and sequencing of the cDNA fragments, and of the patient genomic DNA amplified from this region, revealed that exon 4 is missing from the major thyroglobulin transcript in the goiter, and that this aberrant splicing is due to a C to G transversion at position minus 3 in the acceptor splice site of intron 3. The presence in exon 4 of a putative donor tyrosine residue (Tyrosine nr 130) involved in thyroid hormone formation provides a coherent explanation to the hypothyroid status of the patient. Images PMID:1752952

  4. Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity

    PubMed Central

    West, Andrew B.; Moore, Darren J.; Biskup, Saskia; Bugayenko, Artem; Smith, Wanli W.; Ross, Christopher A.; Dawson, Valina L.; Dawson, Ted M.

    2005-01-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) cause late-onset Parkinson's disease (PD) with a clinical appearance indistinguishable from idiopathic PD. Initial studies suggest that LRRK2 mutations are the most common yet identified determinant of PD susceptibility, transmitted in an autosomal-dominant mode of inheritance. Herein, we characterize the LRRK2 gene and transcript in human brain and subclone the predominant ORF. Exogenously expressed LRRK2 protein migrates at ≈280 kDa and is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Familial-linked mutations G2019S or R1441C do not have an obvious effect on protein steady-state levels, turnover, or localization. However, in vitro kinase assays using full-length recombinant LRRK2 reveal an increase in activity caused by familial-linked mutations in both autophosphorylation and the phosphorylation of a generic substrate. These results suggest a gain-of-function mechanism for LRRK2-linked disease with a central role for kinase activity in the development of PD. PMID:16269541

  5. Erythromelalgia mutation Q875E Stabilizes the activated state of sodium channel Nav1.7.

    PubMed

    Stadler, Theresa; O'Reilly, Andrias O; Lampert, Angelika

    2015-03-06

    The human voltage-gated sodium channel Nav1.7 plays a crucial role in transmission of noxious stimuli. The inherited pain disorder erythromelalgia (IEM) has been linked to Nav1.7 gain-of-function mutations. Here we show that the IEM-associated Q875E mutation located on the pore module of Nav1.7 produces a large hyperpolarizing shift (-18 mV) in the voltage dependence of activation. Three-dimensional homology modeling indicates that the side chains of Gln-875 and the gating charge Arg-214 of the domain I voltage sensor are spatially close in the activated conformation of the channel. We verified this proximity by using an engineered disulfide bridge approach. The Q875E mutation introduces a negative charge that may modify the local electrical field experienced by the voltage sensor and, upon activation, interact directly via a salt bridge with the Arg-214 gating charge residue. Together these processes could promote transition to, and stabilization of, the domain I voltage sensor in the activated conformation and thus produce the observed gain of function. In support of this hypothesis, an increase in the extracellular concentration of Ca(2+) or Mg(2+) reverted the voltage dependence of activation of the IEM mutant to near WT values, suggesting a cation-mediated electrostatic screening of the proposed interaction between Q875E and Arg-214.

  6. Activation of initiation factor 2 by ligands and mutations for rapid docking of ribosomal subunits

    PubMed Central

    Pavlov, Michael Y; Zorzet, Anna; Andersson, Dan I; Ehrenberg, Måns

    2011-01-01

    We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNAi). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNAi, implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNAi and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10−4 for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases. PMID:21151095

  7. Improving Polymerase Activity with Unnatural Substrates by Sampling Mutations in Homologous Protein Architectures.

    PubMed

    Dunn, Matthew R; Otto, Carine; Fenton, Kathryn E; Chaput, John C

    2016-05-20

    The ability to synthesize and propagate genetic information encoded in the framework of xeno-nucleic acid (XNA) polymers would inform a wide range of topics from the origins of life to synthetic biology. While directed evolution has produced examples of engineered polymerases that can accept XNA substrates, these enzymes function with reduced activity relative to their natural counterparts. Here, we describe a biochemical strategy that enables the discovery of engineered polymerases with improved activity for a given unnatural polymerase function. Our approach involves identifying specificity determining residues (SDRs) that control polymerase activity, screening mutations at SDR positions in a model polymerase scaffold, and assaying key gain-of-function mutations in orthologous protein architectures. By transferring beneficial mutations between homologous protein structures, we show that new polymerases can be identified that function with superior activity relative to their starting donor scaffold. This concept, which we call scaffold sampling, was used to generate engineered DNA polymerases that can faithfully synthesize RNA and TNA (threose nucleic acid), respectively, on a DNA template with high primer-extension efficiency and low template sequence bias. We suggest that the ability to combine phenotypes from different donor and recipient scaffolds provides a new paradigm in polymerase engineering where natural structural diversity can be used to refine the catalytic activity of synthetic enzymes.

  8. Site specific mutation of the Zic2 locus by microinjection of TALEN mRNA in mouse CD1, C3H and C57BL/6J oocytes.

    PubMed

    Davies, Benjamin; Davies, Graham; Preece, Christopher; Puliyadi, Rathi; Szumska, Dorota; Bhattacharya, Shoumo

    2013-01-01

    Transcription Activator-Like Effector Nucleases (TALENs) consist of a nuclease domain fused to a DNA binding domain which is engineered to bind to any genomic sequence. These chimeric enzymes can be used to introduce a double strand break at a specific genomic site which then can become the substrate for error-prone non-homologous end joining (NHEJ), generating mutations at the site of cleavage. In this report we investigate the feasibility of achieving targeted mutagenesis by microinjection of TALEN mRNA within the mouse oocyte. We achieved high rates of mutagenesis of the mouse Zic2 gene in all backgrounds examined including outbred CD1 and inbred C3H and C57BL/6J. Founder mutant Zic2 mice (eight independent alleles, with frameshift and deletion mutations) were created in C3H and C57BL/6J backgrounds. These mice transmitted the mutant alleles to the progeny with 100% efficiency, allowing the creation of inbred lines. Mutant mice display a curly tail phenotype consistent with Zic2 loss-of-function. The efficiency of site-specific germline mutation in the mouse confirm TALEN mediated mutagenesis in the oocyte to be a viable alternative to conventional gene targeting in embryonic stem cells where simple loss-of-function alleles are required. This technology enables allelic series of mutations to be generated quickly and efficiently in diverse genetic backgrounds and will be a valuable approach to rapidly create mutations in mice already bearing one or more mutant alleles at other genetic loci without the need for lengthy backcrossing.

  9. Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20.

    PubMed

    Denzin, L K; Whitlow, M; Voss, E W

    1991-07-25

    Previous crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (Ka = 1.7 x 10(10) M-1) complexed with fluorescyl ligand resolved active site contact residues involved in binding. For better definition of the relative roles of three light chain antigen contact residues (L27dhis, L32tyr and L34arg), four site-specific mutations (L27dhis to L27lys, L32tyr to L32phe, and L34arg to L34lys and L34his) were generated and expressed in single-chain antigen binding derivatives of monoclonal antibody 4-4-20 containing two different polypeptide linkers (SCA 4-4-20/205c, 25 amino acids and SCA 4-4-20/212, 14 amino acids). Results showed that L27dhis and L32tyr were necessary for wild type binding affinities, however, were not required for near-wild type Qmax values (where Qmax is the maximum fluoroscein fluorescence quenching expressed as percent). Tyrosine L32 which hydrogen bonds with ligand was also characterized at the haptenic level through the use of 9-hydroxyphenylfluoron which lacks the carboxyl group to which L32 tyrosine forms a hydrogen bond. Results demonstrated that wild type SCA and mutant L32phe possessed similar HPF binding characteristics. Active site contact residue L34arg was important for fluorescein quenching maxima and binding affinity (L34his mutant), however, substitution of lysine for arginine at L34 did not have a significant effect on observed Qmax value. In addition, substitutions had no effect on structural and topological characteristics, since all mutants retained similar idiotypic and metatypic properties. Finally, two linkers were comparatively examined to determine relative contributions to mutant binding properties and stability. No linker effects were observed. Collectively, these results verified the importance of these light chain fluorescein contact residues in the binding pocket of monoclonal antibody 4-4-20.

  10. Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus kandleri.

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

    Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. Several aaRSs are multimeric, and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric aaRSs. Questions remained unanswered pertaining to the dynamics of the active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time-dependent changes at the reaction center, which are essential for facilitating the nucleophilic attack, and interactions at the interface of neighboring monomers. In the present work, we carried out all-atom molecular dynamics simulation of dimeric (mk)SerRS from Methanopyrus kandleri bound with tRNA using an explicit solvent system. Two dimeric states of seryl tRNA synthetase (open, substrate bound, and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound tRNA. The aim is to understand the conformational dynamics of (mk)SerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of (mk)SerRS, they corroborate with the results from the time-averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center, which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the

  11. Effects of tobacco ethylene receptor mutations on receptor kinase activity, plant growth and stress responses.

    PubMed

    Chen, Tao; Liu, Jun; Lei, Gang; Liu, Yun-Feng; Li, Zhi-Gang; Tao, Jian-Jun; Hao, Yu-Jun; Cao, Yang-Rong; Lin, Qing; Zhang, Wan-Ke; Ma, Biao; Chen, Shou-Yi; Zhang, Jin-Song

    2009-09-01

    Ethylene receptor is the first component of ethylene signaling that regulates plant growth, development and stress responses. Previously, we have demonstrated that tobacco subfamily 2 ethylene receptor NTHK1 had Ser/Thr kinase activity, and overexpression of NTHK1 caused large rosette, reduced ethylene sensitivity, and increased salt sensitivity in transgenic Arabidopsis plants. Here we found that N-box mutation in the NTHK1 kinase domain abolished the kinase activity and led to disruption of NTHK1 roles in conferring reduced ethylene sensitivity and salt sensitive response in transgenic Arabidopsis plants. However, N-box mutation had partial effects on NTHK1 regulation of rosette growth and expression of salt- and ethylene-responsive genes AtNAC2, AtERF1 and AtCor6.6. Mutation of conserved residues in the H box did not affect kinase activity, seedling growth, ethylene sensitivity or salt-induced epinasty in transgenic plants but did influence NTHK1 function in control of specific salt- and ethylene-responsive gene expression. Compared with NTHK1, the tobacco subfamily 1 ethylene receptor NtETR1 had His kinase activity and played a weak role in regulation of rosette growth, triple response and salt response. Mutation of the conserved His residue in the NtETR1 H box eliminated phosphorylation and altered the effect of Ntetr1-1 on reporter gene activity. These results imply that the Ser/Thr kinase activity of NTHK1 is differentially required for various responses, and NTHK1 plays a larger role than NtETR1.

  12. Effect of Mutations on the Binding of Kanamycin-B to RNA Hairpins Derived from the Mycobacterium tuberculosis Ribosomal A-Site.

    PubMed

    Truitt, Amber R; Choi, Bok-Eum; Li, Jenny; Soto, Ana Maria

    2015-12-29

    Kanamycin is an aminoglycoside antibiotic used in the treatment of drug-resistant tuberculosis. Mutations at the rRNA A-site have been associated with kanamycin resistance in Mycobacterium tuberculosis clinical isolates. Understanding the effect of these mutations on the conformation of the M. tuberculosis A-site is critical for understanding the mechanisms of antibiotic resistance in M. tuberculosis. In this work, we have studied RNA hairpins derived from the M. tuberculosis A-site, the wild type and three mutants at the following positions (M. tuberculosis/Escherichia coli numbering): A1400/1408 → G, C1401/1409 → U, and the double mutant G1483/1491 C1401/1409 → UA. Specifically, we used circular dichroism, ultraviolet spectroscopy, and fluorescence spectroscopy to characterize the conformation, stability, and binding affinity of kanamycin-B and other aminoglycoside antibiotics for these RNA hairpins. Our results show that the mutations affect the conformation of the decoding site, with the mutations at position 1401/1409 resulting in significant destabilizations. Interestingly, the mutants bind paromomycin with weaker affinity than the wild type, but they bind kanamycin-B with similar affinity than the wild type. The results suggest that the presence of mutations does not prevent kanamycin-B from binding. Instead, kanamycin may promote different interactions with a third partner in the mutants compared to the wild type. Furthermore, our results with longer and shorter hairpins suggest that the region of the A-site that varies among organisms may have modulating effects on the binding and interactions of the A-site.

  13. Identification of an AR Mutation-Negative Class of Androgen Insensitivity by Determining Endogenous AR Activity

    PubMed Central

    Ukat, M.; Schweikert, H. U.; Hiort, O.; Werner, R.; Drop, S. L. S.; Cools, M.; Hughes, I. A.; Audi, L.; Ahmed, S. F.; Demiri, J.; Rodens, P.; Worch, L.; Wehner, G.; Kulle, A. E.; Dunstheimer, D.; Müller-Roßberg, E.; Reinehr, T.; Hadidi, A. T.; Eckstein, A. K.; van der Horst, C.; Seif, C.; Siebert, R.; Ammerpohl, O.; Holterhus, P.-M.

    2016-01-01

    Context: Only approximately 85% of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30% with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene. Objective: The objective of the study was to clarify this discrepancy by in vitro determination of AR transcriptional activity in individuals with disorders of sex development (DSD) and male controls. Design: Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D (APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus. Setting: The study was conducted at a university hospital endocrine research laboratory. Patients: GFs from 169 individuals were studied encompassing control males (n = 68), molecular defined DSD other than androgen insensitivity syndrome (AIS; n = 18), AR mutation-positive AIS (n = 37), and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n = 46). Intervention(s): There were no interventions. Main Outcome Measure(s): DHT-dependent APOD expression in cultured GF and AR mutation status in 169 individuals was measured. Results: The APOD assay clearly separated control individuals (healthy males and molecular defined DSD patients other than AIS) from genetically proven AIS (cutoff < 2.3-fold APOD-induction; 100% sensitivity, 93.3% specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling. Conclusions: AR mutation-positive AIS can be reliably identified by the APOD assay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high

  14. Kinetic and structural evaluation of selected active site mutants of the Aspergillus fumigatus KDNase (sialidase).

    PubMed

    Yeung, Juliana H F; Telford, Judith C; Shidmoossavee, Fahimeh S; Bennet, Andrew J; Taylor, Garry L; Moore, Margo M

    2013-12-23

    Aspergillus fumigatus is an airborne fungal pathogen. We previously cloned and characterized an exo-sialidase from A. fumigatus and showed that it preferred 2-keto-3-deoxynononic acid (KDN) as a substrate to N-acetylneuraminic acid (Neu5Ac). The purpose of this study was to investigate the structure-function relationships of critical catalytic site residues. Site-directed mutagenesis was used to create three mutant recombinant enzymes: the catalytic nucleophile (Y358H), the general acid/base catalyst (D84A), and an enlargement of the binding pocket to attempt to accommodate the N-acetyl group of Neu5Ac (R171L). Crystal structures for all enzymes were determined. The D84A mutation had an effect in decreasing the activity of AfKDNase that was stronger than that of the same mutation in the structurally similar sialidase from the bacterium Micromonospora viridifaciens. These data suggest that the catalytic acid is more important in the reaction of AfKDNase and that catalysis is less dependent on nucleophilic or electrostatic stabilization of the developing positive charge at the transition state for hydrolysis. Removal of the catalytic nucleophile (Y358H) significantly lowered the activity of the enzyme, but this mutant remained a retaining glycosidase as demonstrated by nuclear magnetic resonance spectroscopic analysis. This is a novel finding that has not been shown with other sialidases. Kinetic activity measured at pH 5.2 revealed that R171L had higher activity on a Neu5Ac-based substrate than wild-type KDNase; hence, leucine in place of arginine in the binding pocket improved catalysis toward Neu5Ac substrates. Hence, whether a sialidase is primarily a KDNase or a neuraminidase is due in part to the presence of an amino acid that creates a steric clash with the N-acetyl group.

  15. Detection of T lymphocytes with a second-site mutation in skin lesions of atypical X-linked severe combined immunodeficiency mimicking Omenn syndrome.

    PubMed

    Wada, Taizo; Yasui, Masahiro; Toma, Tomoko; Nakayama, Yuko; Nishida, Mika; Shimizu, Masaki; Okajima, Michiko; Kasahara, Yoshihito; Koizumi, Shoichi; Inoue, Masami; Kawa, Keisei; Yachie, Akihiro

    2008-09-01

    X-linked severe combined immunodeficiency (XSCID) is caused by mutations of the common gamma chain (gammac) and usually characterized by the absence of T and natural killer (NK) cells. Here, we report an atypical case of XSCID presenting with autologous T and NK cells and Omenn syndrome-like manifestations. The patient carried a splice-site mutation (IVS1+5G>A) that caused most of the mRNA to be incorrectly spliced but produced normally spliced transcript in lesser amount, leading to residual gammac expression and development of T and NK cells. The skin biopsy specimen showed massive infiltration of revertant T cells. Those T cells were found to have a second-site mutation and result in complete restoration of correct splicing. These findings suggest that the clinical spectrum of XSCID is quite broad and includes atypical cases mimicking Omenn syndrome, and highlight the importance of revertant mosaicism as a possible cause for variable phenotypic expression.

  16. Changes in active site histidine hydrogen bonding trigger cryptochrome activation.

    PubMed

    Ganguly, Abir; Manahan, Craig C; Top, Deniz; Yee, Estella F; Lin, Changfan; Young, Michael W; Thiel, Walter; Crane, Brian R

    2016-09-06

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.

  17. Changes in active site histidine hydrogen bonding trigger cryptochrome activation

    PubMed Central

    Ganguly, Abir; Manahan, Craig C.; Top, Deniz; Yee, Estella F.; Lin, Changfan; Young, Michael W.; Thiel, Walter; Crane, Brian R.

    2016-01-01

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa. Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions. PMID:27551082

  18. CO2 phase mutation by fluctuating water table in the vadose zone over a CCS site

    NASA Astrophysics Data System (ADS)

    Joun, W.; Ha, S. W.; Kim, H. H.; Kim, T. W.; Lee, S. S.; Lee, K. K.

    2015-12-01

    Geological sequestration of carbon dioxide (CO2) is one of the feasible plans to control greenhouse gas emissions. In order to be more perfect, the plan has to prove that the injected CO2 gas will not be leaking. Even if CO2 leaking happens, we should possess a technique which provides information on specific aquifer system before critical effect to ground and subsurface environments. Many parameters have been utilized for early detection before risk to environments by sensing CO2 gas concentration, electric conductivity, pH, and ion analysis. However, these are not enough to all CCS sites for leakage detection. For example, the importance of gas leaking path is emphasized because finding the dominant gas flow path can reduce risk and provide a quick estimation. Herein, we investigate dissolved solute degassing and vertical flow from saturated zone to unsaturated zone in shallow depth aquifer. Especially we focused on the water table fluctuation effect. Based on field data and basic parameters, we perform a pilot scale gas injection test and calculate gas flow saturation with STOMP simulator. The CO2 gas concentrations at different depth levels according to amount of injected CO2 infused water, CO2 gas saturation in vadose zone have different concentration values. If we estimate this phenomenon in vadose zone by using CO2 gas detection method, we could presume that the CO2 dissolved in shallow groundwater is degassing and flow upward into vadose zone. However, the concentration level and change patterns are not same and will be changed according to the pattern of water table fluctuation. This study could be usefully applied to strategic CCS environmental monitoring of CO2 leakage.Acknowledgement: Financial support was provided by the "R&D Project on Environmental Management of Geologic CO2 Storage" from the KEITI (Project Number: 2014001810003).

  19. Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification.

    PubMed

    Daniellou, Richard; Zheng, Hongyan; Langill, David M; Sanders, David A R; Palmer, David R J

    2007-06-26

    The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant.

  20. Accurate detection for a wide range of mutation and editing sites of microRNAs from small RNA high-throughput sequencing profiles

    PubMed Central

    Zheng, Yun; Ji, Bo; Song, Renhua; Wang, Shengpeng; Li, Ting; Zhang, Xiaotuo; Chen, Kun; Li, Tianqing; Li, Jinyan

    2016-01-01

    Various types of mutation and editing (M/E) events in microRNAs (miRNAs) can change the stabilities of pre-miRNAs and/or complementarities between miRNAs and their targets. Small RNA (sRNA) high-throughput sequencing (HTS) profiles can contain many mutated and edited miRNAs. Systematic detection of miRNA mutation and editing sites from the huge volume of sRNA HTS profiles is computationally difficult, as high sensitivity and low false positive rate (FPR) are both required. We propose a novel method (named MiRME) for an accurate and fast detection of miRNA M/E sites using a progressive sequence alignment approach which refines sensitivity and improves FPR step-by-step. From 70 sRNA HTS profiles with over 1.3 billion reads, MiRME has detected thousands of statistically significant M/E sites, including 3′-editing sites, 57 A-to-I editing sites (of which 32 are novel), as well as some putative non-canonical editing sites. We demonstrated that a few non-canonical editing sites were not resulted from mutations in genome by integrating the analysis of genome HTS profiles of two human cell lines, suggesting the existence of new editing types to further diversify the functions of miRNAs. Compared with six existing studies or methods, MiRME has shown much superior performance for the identification and visualization of the M/E sites of miRNAs from the ever-increasing sRNA HTS profiles. PMID:27229138

  1. The active site structure and mechanism of phosphoenolpyruvate utilizing enzymes

    SciTech Connect

    Cheng, K.C.

    1989-01-01

    Arginine specific reagents showed irreversible inhibition of avian liver mitochondrial phosphoenolpyruvate carboxykinase. Potent protection against modification was elicited by CO{sub 2} or CO{sub 2} in the presence of other substrates. Labeling of enzyme with (7-{sup 14}C) phenylglyoxal showed that 1 or 2 arginines are involved in CO{sub 2} binding and activation. Peptide map studies showed this active site arginine residues is located at position 289. Histidine specific reagents showed pseudo first order inhibition of avian mitochondrial phosphoenolpyruvate carboxykinase activity. The best protection against modification was elicited by IDP or IDP and Mn{sup +2}. One histidine residue is at or near the phosphoenolpyruvate binding site as demonstrated in the increased absorbance at 240 nm and proton relaxation rate studies. Circular dichroism studies reveal that enzyme structure was perturbed by diethylpyrocarbonate modification. Metal binding studies suggest that this enzyme has only one metal binding site. The putative binding sites from several GTP and phosphoenolpyruvate utilizing enzymes are observed in P-enolpyruvate carboxykinase from different species.

  2. Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites.

    PubMed

    Straniero, Letizia; Rimoldi, Valeria; Soldà, Giulia; Mauri, Lucia; Manfredini, Emanuela; Andreucci, Elena; Bargiacchi, Sara; Penco, Silvana; Gesu, Giovanni P; Del Longo, Alessandra; Piozzi, Elena; Asselta, Rosanna; Primignani, Paola

    2015-09-01

    Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type IV (OCA4) is one of the four commonly recognized forms of albinism, and is determined by mutation in the SLC45A2 gene. Here, we investigated the genetic basis of OCA4 in an Italian child. The mutational screening of the SLC45A2 gene identified two novel potentially pathogenic splicing mutations: a synonymous transition (c.888G>A) involving the last nucleotide of exon 3 and a single-nucleotide insertion (c.1156+2dupT) within the consensus sequence of the donor splice site of intron 5. As computer-assisted analysis for mutant splice-site prediction was not conclusive, we investigated the effects on pre-mRNA splicing of these two variants by using an in vitro minigene approach. Production of mutant transcripts in HeLa cells demonstrated that both mutations cause the almost complete abolishment of the physiologic donor splice site, with the concomitant unmasking of cryptic donor splice sites. To our knowledge, this work represents the first in-depth molecular characterization of splicing defects in a OCA4 patient.

  3. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    PubMed

    Ledoux, Sarah; Guthrie, Christine

    2016-06-03

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices.

  4. Site-directed mutagenesis of the Anabaena sp. strain PCC 7120 nitrogenase active site to increase photobiological hydrogen production.

    PubMed

    Masukawa, Hajime; Inoue, Kazuhito; Sakurai, Hidehiro; Wolk, C Peter; Hausinger, Robert P

    2010-10-01

    Cyanobacteria use sunlight and water to produce hydrogen gas (H₂), which is potentially useful as a clean and renewable biofuel. Photobiological H₂ arises primarily as an inevitable by-product of N₂ fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H₂ production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N₂. Most of the 49 variants examined were deficient in N₂-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N₂ atmosphere significantly increased their in vivo rates of H₂ production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H₂ compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H₂ in an aerobic, nitrogen-containing environment.

  5. Exploring functionally related enzymes using radially distributed properties of active sites around the reacting points of bound ligands

    PubMed Central

    2012-01-01

    Background Structural genomics approaches, particularly those solving the 3D structures of many proteins with unknown functions, have increased the desire for structure-based function predictions. However, prediction of enzyme function is difficult because one member of a superfamily may catalyze a different reaction than other members, whereas members of different superfamilies can catalyze the same reaction. In addition, conformational changes, mutations or the absence of a particular catalytic residue can prevent inference of the mechanism by which catalytic residues stabilize and promote the elementary reaction. A major hurdle for alignment-based methods for prediction of function is the absence (despite its importance) of a measure of similarity of the physicochemical properties of catalytic sites. To solve this problem, the physicochemical features radially distributed around catalytic sites should be considered in addition to structural and sequence similarities. Results We showed that radial distribution functions (RDFs), which are associated with the local structural and physicochemical properties of catalytic active sites, are capable of clustering oxidoreductases and transferases by function. The catalytic sites of these enzymes were also characterized using the RDFs. The RDFs provided a measure of the similarity among the catalytic sites, detecting conformational changes caused by mutation of catalytic residues. Furthermore, the RDFs reinforced the classification of enzyme functions based on conventional sequence and structural alignments. Conclusions Our results demonstrate that the application of RDFs provides advantages in the functional classification of enzymes by providing information about catalytic sites. PMID:22536854

  6. The BALB/c secondary response to the Sb site of influenza virus hemagglutinin. Nonrandom silent mutation and unequal numbers of VH and Vk mutations.

    PubMed

    Clarke, S; Rickert, R; Wloch, M K; Staudt, L; Gerhard, W; Weigert, M

    1990-10-01

    We have determined the nucleotide sequences of the expressed VH and Vk genes from 13 secondary (2 degrees) hemagglutinin (HA) (Sb) specific hybridomas derived from a single mouse. These antibodies share an Id, H37-68 (68Id) that dominates the 2 degrees HA(Sb) response in this mouse, but is rare or absent from 2 degrees responses of other mice. We find that these antibodies derive from five clones. The H chains of these antibodies are encoded by a single VH gene joined to a variety of DH and JH genes. The length of complementarity-determining region (CDR) 3 and sequence of the D-J junction are restricted, suggesting selection on CDR3 of the H chain. The L chains are more diverse. In the presented examples, they are encoded by the Vk21C and Vk21E genes and a Vk9 gene, and are joined to Jk1, 2, or 4. Each antibody is extensively mutated. The nature and distribution of the mutations suggests that 68Id-producing cells have been selected by Ag, although there are differences regarding the domain (VH, Vk, or both) in which mutations were selected. The implications of these findings on the idiosyncratic nature of the 68Id antibody response to HA(Sb) are discussed. There are two unusual characteristics regarding somatic mutation in these hybridomas. Whereas the expressed VH and Vk21 genes appear to have accumulated mutations at a high rate (1 to 1.5 x 10(-3)/base pairs/division, the expressed Vk9 genes appear to have accumulated mutations at a 5 to 15-fold lower rate than the expressed VH genes in the same cells. There is also a surprisingly high number of parallel silent somatic mutations in the VH genes, of which all but one are clustered to a 28-bp region in framework region 2 and CDR 2-encoding segments. The probability that this could have occurred by a random mutational process is essentially zero.

  7. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    PubMed

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.

  8. Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP).

    PubMed

    Zhao, Hong-Yan; Wu, Li-Ying; Liu, Gang; Feng, Hong

    2016-12-01

    To engineer dehairing alkaline protease (DHAP) variants to improve cold activity and increase thermostability so these variants are suitable for the leather processing industry. Based on previous studies with bacterial alkaline proteases, double-site mutations (W106K/V149I and W106K/M124L) were introduced into the DHAP from Bacillus pumilus. Compared with the wild-type DHAP hydrolytic activity, the double-site variant W106K/V149I showed an increase in specific hydrolytic activity at 15 °C by 2.3-fold toward casein in terms of hydrolytic rate and 2.7-fold toward the synthetic peptide AAPF-pN by means of kcat/Km value. The thermostability of the variant (W106K/V149I) was improved with the half-life at 60 and 70 °C increased by 2.7- and 5.0-fold, respectively, when compared with the thermostability of the wild-type DHAP. Conclusively, an increase in the cold activity and thermostability of a bacterial alkaline protease was achieved by protein engineering.

  9. Reverse evolution leads to genotypic incompatibility despite functional and active site convergence

    PubMed Central

    Kaltenbach, Miriam; Jackson, Colin J; Campbell, Eleanor C; Hollfelder, Florian; Tokuriki, Nobuhiko

    2015-01-01

    Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>104-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peaks. DOI: http://dx.doi.org/10.7554/eLife.06492.001 PMID:26274563

  10. Compensatory signals associated with the activation of human GC 5' splice sites.

    PubMed

    Kralovicova, Jana; Hwang, Gyulin; Asplund, A Charlotta; Churbanov, Alexander; Smith, C I Edvard; Vorechovsky, Igor

    2011-09-01

    GC 5' splice sites (5'ss) are present in ∼1% of human introns, but factors promoting their efficient selection are poorly understood. Here, we describe a case of X-linked agammaglobulinemia resulting from a GC 5'ss activated by a mutation in BTK intron 3. This GC 5'ss was intrinsically weak, yet it was selected in >90% primary transcripts in the presence of a strong and intact natural GT counterpart. We show that efficient selection of this GC 5'ss required a high density of GAA/CAA-containing splicing enhancers in the exonized segment and was promoted by SR proteins 9G8, Tra2β and SC35. The GC 5'ss was efficiently inhibited by splice-switching oligonucleotides targeting either the GC 5'ss itself or the enhancer. Comprehensive analysis of natural GC-AG introns and previously reported pathogenic GC 5'ss showed that their efficient activation was facilitated by higher densities of splicing enhancers and lower densities of silencers than their GT 5'ss equivalents. Removal of the GC-AG introns was promoted to a minor extent by the splice-site strength of adjacent exons and inhibited by flanking Alu repeats, with the first downstream Alus located on average at a longer distance from the GC 5'ss than other transposable elements. These results provide new insights into the splicing code that governs selection of noncanonical splice sites.

  11. Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity

    PubMed Central

    Baradaran-Heravi, Alireza; Niesser, Jürgen; Balgi, Aruna D.; Choi, Kunho; Zimmerman, Carla; South, Andrew P.; Anderson, Hilary J.; Strynadka, Natalie C.; Bally, Marcel B.; Roberge, Michel

    2017-01-01

    Nonsense mutations underlie about 10% of rare genetic disease cases. They introduce a premature termination codon (PTC) and prevent the formation of full-length protein. Pharmaceutical gentamicin, a mixture of several related aminoglycosides, is a frequently used antibiotic in humans that can induce PTC readthrough and suppress nonsense mutations at high concentrations. However, testing of gentamicin in clinical trials has shown that safe doses of this drug produce weak and variable readthrough activity that is insufficient for use as therapy. In this study we show that the major components of pharmaceutical gentamicin lack PTC readthrough activity but the minor component gentamicin B1 (B1) is a potent readthrough inducer. Molecular dynamics simulations reveal the importance of ring I of B1 in establishing a ribosome configuration that permits pairing of a near-cognate complex at a PTC. B1 induced readthrough at all three nonsense codons in cultured cancer cells with TP53 (tumor protein p53) mutations, in cells from patients with nonsense mutations in the TPP1 (tripeptidyl peptidase 1), DMD (dystrophin), SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), and COL7A1 (collagen type VII alpha 1 chain) genes, and in an in vivo tumor xenograft model. The B1 content of pharmaceutical gentamicin is highly variable and major gentamicins suppress the PTC readthrough activity of B1. Purified B1 provides a consistent and effective source of PTC readthrough activity to study the potential of nonsense suppression for treatment of rare genetic disorders. PMID:28289221

  12. Activating JAK1 mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma.

    PubMed

    Yang, Shuqun; Luo, Chonglin; Gu, Qingyang; Xu, Qiang; Wang, Guan; Sun, Hongye; Qian, Ziliang; Tan, Yexiong; Qin, Yuxin; Shen, Yuhong; Xu, Xiaowei; Chen, Shu-Hui; Chan, Chi-Chung; Wang, Hongyang; Mao, Mao; Fang, Douglas D

    2016-02-02

    Hepatocellular carcinoma (HCC) is the fifth most common type of cancers worldwide. However, current therapeutic approaches for this epidemic disease are limited, and its 5-year survival rate hasn't been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited protein-altering mutations in JAK1 (Janus Kinase 1) gene. To explore the transforming capability, these mutations were then introduced into HEK293FT and Ba/F3 cells. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore,the sensitivity to the treatment of a JAK1/2 inhibitor, ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other non-activating mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly suppressed. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo, and JAK-STAT pathway might represent a new therapeutic approach for HCC treatment. Monotherapy using a more potent and specific JAK1 inhibitor and combinatory therapy should be further explored in JAK1 mutant PDX models.

  13. Novel Mutations in the Transcriptional Activator Domain of the Human TBX20 in Patients with Atrial Septal Defect

    PubMed Central

    Monroy-Muñoz, Irma Eloisa; Rodríguez-Pérez, José Manuel; Muñoz-Medina, José Esteban; Angeles-Martínez, Javier; García-Trejo, José J.; Morales-Ríos, Edgar; Massó, Felipe; Sandoval-Jones, Juan Pablo; Cervantes-Salazar, Jorge; García-Montes, José Antonio; Calderón-Colmenero, Juan; Vargas-Alarcón, Gilberto

    2015-01-01

    Background. The relevance of TBX20 gene in heart development has been demonstrated in many animal models, but there are few works that try to elucidate the effect of TBX20 mutations in human congenital heart diseases. In these studies, all missense mutations associated with atrial septal defect (ASD) were found in the DNA-binding T-box domain, none in the transcriptional activator domain. Methods. We search for TBX20 mutations in a group of patients with ASD or ventricular septal defect (VSD) using the High Resolution Melting (HRM) method and DNA sequencing. Results. We report three missense mutations (Y309D, T370O, and M395R) within the transcriptional activator domain of human TBX20 that were associated with ASD. Conclusions. This is the first association of TBX20 transcriptional activator domain missense mutations with ASD. These findings could have implications for diagnosis, genetic screening, and patient follow-up. PMID:25834824

  14. Identification of Ice Nucleation Active Sites on Silicate Dust Particles

    NASA Astrophysics Data System (ADS)

    Zolles, Tobias; Burkart, Julia; Häusler, Thomas; Pummer, Bernhard; Hitzenberger, Regina; Grothe, Hinrich

    2015-04-01

    Mineral dusts originating from Earth's crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts [1-3]. Nevertheless, among those structures K-feldspar showed by far the highest ice nucleation activity. In this study, the reasons for its activity and the difference in the activity of the different feldspars were investigated in closer details. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. We give a potential explanation of the increased ice nucleation activity of K-feldspar. The ice nucleating sites are very much dependent on the alkali ion present by altering the water structure and the feldspar surface. The higher activity of K-feldspar can be attributed to the presence of potassium ions on the surface and surface bilayer. The alkali-ions have different hydration shells and thus an influence on the ice nucleation activity of feldspar. Chaotropic behavior of Calcium and Sodium ions are lowering the ice nucleation potential of the other feldspars, while kosmotropic Potassium has a neutral or even positive effect. Furthermore we investigated the influence of milling onto the ice nucleation of quartz particles. The ice nucleation activity can be increased by mechanical milling, by introducing more molecular, nucleation active defects to the particle surface. This effect is larger than expected by plane surface increase. [1] Atkinson et al. The Importance of Feldspar for Ice Nucleation by Mineral Dust in Mixed-Phase Clouds. Nature 2013, 498, 355-358. [2] Yakobi-Hancock et al.. Feldspar Minerals as Efficient Deposition Ice Nuclei. Atmos. Chem. Phys. 2013, 13, 11175-11185. [3] Zolles et al. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles. J. Phys. Chem. A 2015 accepted.

  15. Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence.

    PubMed

    Drayer, A L; Meima, M E; Derks, M W; Tuik, R; van Haastert, P J

    1995-10-15

    Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.

  16. Face the Edges: Catalytic Active Sites of Nanomaterials

    PubMed Central

    Ni, Bing

    2015-01-01

    Edges are special sites in nanomaterials. The atoms residing on the edges have different environments compared to those in other parts of a nanomaterial and, therefore, they may have different properties. Here, recent progress in nanomaterial fields is summarized from the viewpoint of the edges. Typically, edge sites in MoS2 or metals, other than surface atoms, can perform as active centers for catalytic reactions, so the method to enhance performance lies in the optimization of the edge structures. The edges of multicomponent interfaces present even more possibilities to enhance the activities of nanomaterials. Nanoframes and ultrathin nanowires have similarities to conventional edges of nanoparticles, the application of which as catalysts can help to reduce the use of costly materials. Looking beyond this, the edge structures of graphene are also essential for their properties. In short, the edge structure can influence many properties of materials. PMID:27980960

  17. The effects of expression of an activated rasG mutation on the differentiation of Dictyostelium.

    PubMed

    Thiery, R; Robbins, S; Khosla, M; Spiegelman, G B; Weeks, G

    1992-01-01

    Dictyostelium discoideum contains two ras genes, rasG and rasD, that are expressed during growth and differentiation, respectively. It was shown previously that Dictyostelium transformants expressing an activated rasD gene (a mutation producing a change in amino acid 12 from glycine to threonine) developed abnormally. When developed on filters these transformants formed multitipped aggregates, which did not go on to produce final fruiting bodies, but in a submerged culture assay on a plastic surface they either formed small aggregates or did not aggregate. In this study we transformed cells with the rasG gene, mutated to change amino acid 12 from glycine to threonine. The resulting transformants developed normally on filters, but aggregation under other conditions was impaired. In particular, in submerged culture on a plastic surface they either produced very small aggregates or did not aggregate, one of the phenotypes exhibited by the activated rasD transformants. Molecular analysis of the transformants revealed the presence of high copy numbers of the mutated rasG gene, but the level of expression of the mutant gene never exceeded the level of expression of the endogenous gene. These results indicate a powerful dominant effect of a relatively small amount of the activated RasG protein in Dictyostelium.

  18. Antihelminth compound niclosamide downregulates Wnt signaling and elicits antitumor responses in tumors with activating APC mutations.

    PubMed

    Osada, Takuya; Chen, Minyong; Yang, Xiao Yi; Spasojevic, Ivan; Vandeusen, Jeffrey B; Hsu, David; Clary, Bryan M; Clay, Timothy M; Chen, Wei; Morse, Michael A; Lyerly, H Kim

    2011-06-15

    Wnt/β-catenin pathway activation caused by adenomatous polyposis coli (APC) mutations occurs in approximately 80% of sporadic colorectal cancers (CRC). The antihelminth compound niclosamide downregulates components of the Wnt pathway, specifically Dishevelled-2 (Dvl2) expression, resulting in diminished downstream β-catenin signaling. In this study, we determined whether niclosamide could inhibit the Wnt/β-catenin pathway in human CRCs and whether its inhibition might elicit antitumor effects in the presence of APC mutations. We found that niclosamide inhibited Wnt/β-catenin pathway activation, downregulated Dvl2, decreased downstream β-catenin signaling, and exerted antiproliferative effects in human colon cancer cell lines and CRC cells isolated by surgical resection of metastatic disease, regardless of mutations in APC. In contrast, inhibition of NF-κB or mTOR did not exert similar antiproliferative effects in these CRC model systems. In mice implanted with human CRC xenografts, orally administered niclosamide was well tolerated, achieved plasma and tumor levels associated with biologic activity, and led to tumor control. Our findings support clinical explorations to reposition niclosamide for the treatment of CRC.

  19. A case of colorectal cancer with double-activating epidermal growth factor receptor mutations.

    PubMed

    Rai, Kammei; Fujiwara, Keiichi; Tsushima, Mizuho; Kudo, Kenichiro; Mizuta, Makoto; Matsuo, Kiyoshi; Yonei, Toshiro; Yamadori, Ichiro; Kiura, Katsuyuki; Sato, Toshio

    2011-09-01

    We describe the case of a 72-year-old woman with locally advanced lung tumor mimicking primary lung cancer. She was diagnosed with rectal cancer at the age of 65 years and was initially treated with platinum-based chemotherapy and thoracic irradiation as a treatment for primary lung cancer. One year later, a thyroid tumor was detected in her right thyroid lobe and was confirmed to have metastasized from rectal cancer based on pathological findings. Therefore, we suspected that she had metachronous double cancers and treated her with conventional chemotherapy for colorectal cancer. However, new life-threatening multiple lung metastases appeared. We treated her with the drug erlotinib because additional genetic analysis against primary lung tumor revealed typical double-activating epidermal growth factor receptor mutations. Histological review by immunostaining concluded that the primary lung tumor was composed of metastatic tumors from rectal cancer. In addition, genetic analysis revealed that the primary rectal cancer contained nearly the same types of double-activating epidermal growth factor receptor mutations as were present in the lung tumor. This is the first report of a case of rectal adenocarcinoma with double-activating epidermal growth factor receptor mutations.

  20. Further linkage evidence for localization of mutational sites for nonsyndromic types of X-linked mental retardation at pericentromeric region

    SciTech Connect

    Robledo, R.; Melis, P.; Siniscalco, M.

    1996-07-12

    We used several microsatellite markers scattered along the X chromosome to search for linkage relationships in a large Sardinian pedigree segregating for nonspecific X-linked mental retardation (MRX). Markers DXS573 and AR, located at chromosomal subregions Xp11.4-p11.22 and Xq11.2-q12, respectively, were found to segregate in full concordance with the disease, leading to a LOD score of 4.21 at zero recombination value. Recombination with the disease was found with markers MAOB and DXS454 located at Xp11.4-p11.3 and Xq21.1-q22, respectively; accordingly, markers distal to Xp11.4 and Xq22 also segregated independently of the disease. These findings provide strong linkage evidence in favor of the localization of one MRX mutational site in the pericentromeric region of the human X chromosome, justifying the assignment of a new symbol (MRX26) to our pedigree. Finally, on the basis of the recombinational events observed in the Xq21-q22 region, we have been able to refine the assignment of marker DXS456 to Xq21.33-q22. 26 refs., 3 figs., 1 tab.

  1. Bovine papillomavirus with a mutation in the E2 serine 301 phosphorylation site replicates at a high copy number.

    PubMed Central

    McBride, A A; Howley, P M

    1991-01-01

    The E2 open reading frame of bovine papillomavirus type 1 (BPV-1) encodes at least three proteins with transcriptional regulatory properties. The full-length E2 open reading frame encodes a transcriptional transactivator, and the 3' region encodes two smaller polypeptides that repress E2-mediated transactivation. The full-length gene product is also required for viral DNA replication. We have demonstrated that the BPV-1 E2 polypeptides are phosphorylated primarily on two serine residues at a site adjacent to the carboxy-terminal DNA binding domain, which is common to all three E2 proteins (A. A. McBride, J. B. Bolen, and P. M. Howley, J. Virol. 63:5076-5085, 1989). These serine residues, at amino acid positions 298 and 301, were substituted with alanine residues in the context of the entire BPV-1 genome. The mutated BPV-1 genomes were introduced into rodent cell lines and assayed for focus formation, viral gene expression, and extrachromosomal viral DNA replication. Viral DNAs containing the E2 serine-to-alanine substitution mutants transformed C127 cells with efficiencies comparable to that of wild-type BPV-1. However, the viral genome containing the serine-to-alanine substitution at position 301 of the E2 polypeptide replicated to a copy number 20-fold higher than that of wild-type DNA. Images PMID:1658358

  2. Characterization of structural changes in vimentin bearing an epidermolysis bullosa simplex-like mutation using site-directed spin labeling and electron paramagnetic resonance.

    PubMed

    Hess, John F; Budamagunta, Madhu S; FitzGerald, Paul G; Voss, John C

    2005-01-21

    Mutations in intermediate filament protein genes are responsible for a number of inherited genetic diseases including skin blistering diseases, corneal opacities, and neurological degenerations. Mutation of the arginine (Arg) residue of the highly conserved LNDR motif has been shown to be causative in inherited disorders in at least four different intermediate filament (IF) proteins found in skin, cornea, and the central nervous system. Thus this residue appears to be broadly important to IF assembly and/or function. While the genetic basis for these diseases has been clearly defined, the inability to determine crystal structure for IFs has precluded a determination of how these mutations affect assembly/structure/function of IFs. To investigate the impact of mutation at this site in IFs, we have mutated the LNDR to LNDS in vimentin, a Type III intermediate filament protein, and have examined the impact of this change on assembly using electron paramagnetic resonance. Compared with wild type vimentin, the mutant shows normal formation of the coiled coil dimer, with a slight reduction in the stability of the dimer in rod domain 1. Probing the dimer-dimer interactions shows the formation of normal dimer centered on residue 191 but a failure of dimerization at residue 348 in rod domain 2. These data point toward a specific stage of assembly at which a common disease-causing mutation in IF proteins interrupts assembly.

  3. Systematic Design of Trypsin Cleavage Site Mutated Exendin4-Cysteine 1, an Orally Bioavailable Glucagon-Like Peptide-1 Receptor Agonist

    PubMed Central

    Sai, Wenbo; Tian, Hong; Yang, Kangmin; Tang, Daoqi; Bao, Jinxiao; Ge, Yang; Song, Xiaoda; Zhang, Yu; Luo, Cheng; Gao, Xiangdong; Yao, Wenbing

    2017-01-01

    Exendin-4 is a strong therapeutic candidate for the treatment of metabolic syndrome. Related receptor agonist drugs have been on the market since 2005. However, technical limitations and the pain caused by subcutaneous injection have severely limited patient compliance. The goal of the study is to investigate a biologically active exendin-4 analog could be administered orally. Using intraperitoneal glucose tolerance tests, we discovered that exendin4-cysteine administered by oral gavage had a distinct hypoglycemic effect in C57BL/6J mice. Using Rosetta Design and Amber, we designed and screened a series of exendin4-cysteine analogs to identify those that retained biological activity while resisting trypsin digestion. Trypsin Cleavage Site Mutated Exendin4-cysteine 1 (TSME-1), an analog whose bioactivity was similar to exendin-4 and was almost completely resistant to trypsin, was screened out. In addition, TSME-1 significantly normalized the blood glucose levels and the availability of TSME-1 was significantly higher than that of exendin-4 and exendin4-cysteine. Collectively orally administered TSME-1, a trypsin-resistant exendin-4 analog obtained by the system, is a strong candidate for future treatments of type 2 diabetes. PMID:28282854

  4. Mutational analysis of the catalytic and feedback sites of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli.

    PubMed Central

    Ray, J M; Yanofsky, C; Bauerle, R

    1988-01-01

    The nucleotide sequence of aroH, the structural gene for the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [DAHPS(Trp)], is presented, and the deduced amino acid sequence of AroH is compared with that of the tyrosine-sensitive (AroF) and phenylalanine-sensitive (AroG) DAHPS isoenzymes. The high degree of sequence similarity among the three isoenzymes strongly indicates that they have a common evolutionary origin. In vitro chemical mutagenesis of the cloned aroH gene was used to identify residues and regions of the polypeptide essential for catalytic activity and for tryptophan feedback regulation. Missense mutations leading either to loss of catalytic activity or to feedback resistance were found interspersed throughout the polypeptide, suggesting overlapping catalytic and regulatory sites in DAHPS(Trp). We conclude that the specificity of feedback regulation of the isoenzymes was probably acquired by the duplication and divergent evolution of an ancestral gene, rather than by domain recruitment. PMID:2903857

  5. Unequal prognostic potentials of p53 gain-of-function mutations in human cancers associate with drug-metabolizing activity.

    PubMed

    Xu, J; Wang, J; Hu, Y; Qian, J; Xu, B; Chen, H; Zou, W; Fang, J-Y

    2014-03-06

    Mutation of p53 is the most common genetic change in human cancer, causing complex effects including not only loss of wild-type function but also gain of novel oncogenic functions (GOF). It is increasingly likely that p53-hotspot mutations may confer different types and magnitudes of GOF, but the evidences are mainly supported by cellular and transgenic animal models. Here we combine large-scale cancer genomic data to characterize the prognostic significance of different p53 mutations in human cancers. Unexpectedly, only mutations on the Arg248 and Arg282 positions displayed significant association with shorter patient survival, but such association was not evident for other hotspot GOF mutations. Gene set enrichment analysis on these mutations revealed higher activity of drug-metabolizing enzymes, including the CYP3A4 cytochrome P450. Ectopic expression of p53 mutant R282W in H1299 and SaOS2 cells significantly upregulated CYP3A4 mRNA and protein levels, and cancer cell lines bearing mortality-associated p53 mutations display higher CYP3A4 expression and resistance to several CYP3A4-metabolized chemotherapeutic drugs. Our results suggest that p53 mutations have unequal GOF activities in human cancers, and future evaluation of p53 as a cancer biomarker should consider which mutation is present in the tumor, rather than having comparison between wild-type and mutant genotypes.

  6. Active sites in char gasification: Final technical report

    SciTech Connect

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  7. Growth-regulatory human galectin-1: crystallographic characterisation of the structural changes induced by single-site mutations and their impact on the thermodynamics of ligand binding.

    PubMed

    López-Lucendo, María F; Solís, Dolores; André, Sabine; Hirabayashi, Jun; Kasai, Ken-ichi; Kaltner, Herbert; Gabius, Hans-Joachim; Romero, Antonio

    2004-10-29

    Human galectin-1 is a potent multifunctional effector that participates in specific protein-carbohydrate and protein-protein (lipid) interactions. By determining its X-ray structure, we provide the basis to define the structure of its ligand-binding pocket and to perform rational drug design. We have also analysed whether single-site mutations introduced at some distance from the carbohydrate recognition domain can affect the lectin fold and influence sugar binding. Both the substitutions introduced in the C2S and R111H mutants altered the presentation of the loop, harbouring Asp123 in the common "jelly-roll" fold. The orientation of the side-chain was inverted 180 degrees and the positions of two key residues in the sugar-binding site of the R111H mutant were notably shifted, i.e. His52 and Trp68. Titration calorimetry was used to define the decrease in ligand affinity in both mutants and a significant increase in the entropic penalty was found to outweigh a slight enhancement of the enthalpic contribution. The position of the SH-groups in the galectin appeared to considerably restrict the potential to form intramolecular disulphide bridges and was assumed to be the reason for the unstable lectin activity in the absence of reducing agent. However, this offers no obvious explanation for the improved stability of the C2S mutant under oxidative conditions. The noted long-range effects in single-site mutants are relevant for the functional divergence of closely related galectins and in more general terms, the functionality definition of distinct amino acids.

  8. Nest predation increases with parental activity: Separating nest site and parental activity effects

    USGS Publications Warehouse

    Martin, T.E.; Scott, J.; Menge, C.

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection.

  9. Nest predation increases with parental activity: separating nest site and parental activity effects.

    PubMed Central

    Martin, T E; Scott, J; Menge, C

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection. PMID:11413645

  10. Active site amino acid sequence of human factor D.

    PubMed

    Davis, A E

    1980-08-01

    Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However, complete definition of the degree of relationship between factor D and other proteases will require determination of the remainder of the primary structure.

  11. Brownian aggregation rate of colloid particles with several active sites

    SciTech Connect

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V.; Polshchitsin, Alexey A.; Yakovleva, Galina E.; Maltsev, Valeri P.

    2014-08-14

    We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.

  12. Reconstructing a missing link in the evolution of a recently diverged phosphotriesterase by active-site loop remodeling.

    PubMed

    Afriat-Jurnou, Livnat; Jackson, Colin J; Tawfik, Dan S

    2012-08-07

    Only decades after the introduction of organophosphate pesticides, bacterial phosphotriesterases (PTEs) have evolved to catalyze their degradation with remarkable efficiency. Their closest known relatives, lactonases, with promiscuous phosphotriasterase activity, dubbed PTE-like lactonases (PLLs), share only 30% sequence identity and also differ in the configuration of their active-site loops. PTE was therefore presumed to have evolved from a yet unknown PLL whose primary activity was the hydrolysis of quorum sensing homoserine lactones (HSLs) (Afriat et al. (2006) Biochemistry 45, 13677-13686). However, how PTEs diverged from this presumed PLL remains a mystery. In this study we investigated loop remodeling as a means of reconstructing a homoserine lactonase ancestor that relates to PTE by few mutational steps. Although, in nature, loop remodeling is a common mechanism of divergence of enzymatic functions, reproducing this process in the laboratory is a challenge. Structural and phylogenetic analyses enabled us to remodel one of PTE's active-site loops into a PLL-like configuration. A deletion in loop 7, combined with an adjacent, highly epistatic, point mutation led to the emergence of an HSLase activity that is undetectable in PTE (k(cat)/K(M) values of up to 2 × 10(4)). The appearance of the HSLase activity was accompanied by only a minor decrease in PTE's paraoxonase activity. This specificity change demonstrates the potential role of bifunctional intermediates in the divergence of new enzymatic functions and highlights the critical contribution of loop remodeling to the rapid divergence of new enzyme functions.

  13. Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation

    PubMed Central

    Svensson, Lena; Howarth, Kimberley; McDowall, Alison; Patzak, Irene; Evans, Rachel; Ussar, Siegfried; Moser, Markus; Metin, Ayse; Fried, Mike; Tomlinson, Ian; Hogg, Nancy

    2009-01-01

    Integrins are the major adhesion receptors of leukocytes and platelets. β1 and β2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin αIIbβ3 initiates the process of blood clotting through binding fibrinogen1-3. Integrins on circulating cells bind poorly to their ligands but become active after ‘inside-out’ signaling through other membrane receptors4,5. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express β2 integrins because of mutations in the gene specifying the β2 subunit, and they suffer recurrent bacterial infections6,7. Mutations affecting αIIbβ3 integrin cause the bleeding disorder termed Glanzmann’s thrombasthenia3. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann’s thrombasthenia. Their hematopoietically-derived cells express β1, β2 and β3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems8. The LAD-III lesion has been attributed to a C→A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein9, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects’ lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration. PMID:19234463

  14. Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

    SciTech Connect

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.; Elsen, Nathaniel L.; Phillips, Jr., George N.; Fox, Brian G.

    2014-10-02

    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential

  15. Constitutive activation of NF-κB signaling by NOTCH1 mutations in chronic lymphocytic leukemia.

    PubMed

    Xu, Zhen-Shu; Zhang, Ju-Shun; Zhang, Jing-Yan; Wu, Shun-Quan; Xiong, Dong-Lian; Chen, Hui-Jun; Chen, Zhi-Zhe; Zhan, Rong

    2015-04-01

    NOTCH1 mutations occur in approximately 10% of patients with chronic lymphocytic leukemia (CLL). However, the relationship between the genetic aberrations and tumor cell drug resistance or disease progression remains unclear. Frameshift deletions were detected by gene sequencing in the NOTCH1 PEST domain in three naive CLL patients. These mutations were associated with chromosomal abnormalities including trisomy 12 or 13q deletion. Of note, one of the patients developed Richter's transformation during FCR treatment. Immunofluorescent and western blot analyses revealed a markedly higher intracellular domain of NOTCH (ICN) expression in the mutated cells compared with their unmutated counterparts and normal CD19+ B lymphocytes (P<0.01 and P<0.001, respectively). In addition, strong DNA-κB binding activities were observed in the mutant cells by gel shift assays. RT-PCR analysis revealed elevated RelA mRNA expression in the mutant cells, while RelB levels were variable. Reduced levels of RelA and RelB mRNA were observed in unmutated CLL and normal B cells. Compared to unmutated CLL and normal B cells, increased apoptosis occurred in the mutant cells in the presence of GSI (ICN inhibitor) and PDTC (NF-κB inhibitor), particularly under the synergistic effects of the two drugs (P=0.03). Moreover, IKKα and IKKβ, the active components in the NF-κB pathway, were markedly inhibited following prolonged treatment with GSI and PDTC. These results suggested that NOTCH1 mutations constitutively activate the NF-κB signaling pathway in CLL, which is likely related to ICN overexpression, indicating NOTCH1 and NF-κB as potential therapeutic targets in the treatment of CLL.

  16. Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase.

    PubMed Central

    Stoughton, D M; Zapata, G; Picone, R; Vann, W F

    1999-01-01

    Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12-->Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in k(cat), K(m) and K(s) for CTP, which implicates Arg-12 in catalysis and substrate binding. PMID:10510306

  17. TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia

    PubMed Central

    Yamazaki, Jumpei; Jelinek, Jaroslav; Lu, Yue; Cesaroni, Matteo; Madzo, Jozef; Neumann, Frank; He, Rong; Taby, Rodolphe; Vasanthakumar, Aparna; Macrae, Trisha; Ostler, Kelly R.; Kantarjian, Hagop M.; Liang, Shoudan; Estecio, Marcos R.; Godley, Lucy A.; Issa, Jean-Pierre J.

    2015-01-01

    TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently-modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here we report on DNA methylomes in TET2 wild type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites (28,114 sites in CpG islands (CGIs) and 57,020 in non-CpG islands (NCGIs)). TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. By contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and non-promoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1, and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor binding sites. PMID:25972343

  18. Prosaposin Deficiency and Saposin B Deficiency (Activator-Deficient Metachromatic Leukodystrophy): Report on Two Patients Detected by Analysis of Urinary Sphingolipids and Carrying Novel PSAP Gene Mutations

    PubMed Central

    Kuchař, Ladislav; Ledvinová, Jana; Hřebíček, Martin; Myšková, Helena; Dvořáková, Lenka; Berná, Linda; Chrastina, Petr; Asfaw, Befekadu; Elleder, Milan; Petermöller, Margret; Mayrhofer, Heidi; Staudt, Martin; Krägeloh-Mann, Ingeborg; Paton, Barbara C; Harzer, Klaus

    2009-01-01

    Prosaposin deficiency (pSap-d) and saposin B deficiency (SapB-d) are both lipid storage disorders caused by mutations in the PSAP gene that codes for the 65–70 kDa prosaposin protein, which is the precursor for four sphingolipid activator proteins, saposins A–D. We report on two new patients with PSAP gene defects; one, with pSap-d, who had a severe neurovisceral dystrophy and died as a neonate, and the other with SapB-d, who presented with a metachromatic leukodystrophy-like disorder but had normal arylsulfatase activity. Screening for urinary sphingolipids was crucial to the diagnosis of both patients, with electrospray ionization tandem mass spectrometry also providing quantification. The pSap-d patient is the first case with this condition where urinary sphingolipids have been investigated. Multiple sphingolipids were elevated, with globotriaosylceramide showing the greatest increase. Both patients had novel mutations in the PSAP gene. The pSap-d patient was homozygous for a splice-acceptor site mutation two bases upstream of exon 10. This mutation led to a premature stop codon and yielded low levels of transcript. The SapB-d patient was a compound heterozygote with a splice-acceptor site variant exclusively affecting the SapB domain on one allele, and a 2 bp deletion leading to a null, that is, pSap-d mutation, on the other allele. Phenotypically, pSap-d is a relatively uniform disease of the neonate, whereas SapB-d is heterogeneous with a spectrum similar to that in metachromatic leukodystrophy. The possible existence of genotypes and phenotypes intermediate between those of pSap-d and the single saposin deficiencies is speculated. © 2009 Wiley-Liss, Inc. PMID:19267410

  19. Genetic polymorphisms and expression of minisatellite mutations in a 3-generation population around the Semipalatinsk nuclear explosion test-site, Kazakhstan.

    PubMed

    Bolegenova, N K; Bekmanov, B O; Djansugurova, L B; Bersimbaev, R I; Salama, S A; Au, W W

    2009-11-01

    We have reported previously that a population near the Semipalatinsk nuclear explosion test site had significantly increased minisatellite mutations (MM), suggesting increased germ-line mutation rates from the exposure in 3 generations. We hypothesize that the MM can be used as a surrogate biomarker for functional genetic alterations, e.g. gene mutations and chromosome aberrations. Therefore, we have investigated the influence of polymorphisms in genes on the expression of MM in the same two populations (247 and 172 individuals, for exposed and control, respectively, in 3 generations), and their relationships with radiation exposure. We have chosen the analyses of three polymorphic DNA - repair genes (XRCC1, XRCC1 and XRCC3) and two xenobiotic detoxification genes (GSTT1 and GSTM1). Among the exposed and in comparison with the wild-type gene, the functionally active XRCC1 Arg194Trp was significantly associated with low MM and over-represented in the exposed compared with the control populations. In a similar analysis, the functionally deficient XRCC1 Arg399Glu and XRCC3 Trp241Met were associated with increased and significantly reduced MM, respectively, but these variant genes were under-represented in the exposed population. Both GSTT1 and GSTM1 nulls were significantly associated with increased MM. The former was under-represented but the latter was significantly over-represented in the exposed compared with the control populations. In summary, the data indicate that the expected enzymatic functions of the polymorphic genes are consistent with the MM expression, except the XRCC1 Arg399Glu variant gene. In addition, the variant genes were retained in the three generations in association with their useful function, except for the GSTM1 null. However, the MM frequencies in the exposed were not consistently and significantly higher than those in the control populations, radiation exposure may therefore not have been the only cause for the high MM frequency among the

  20. Overexpression of the yeast transcriptional activator ADR1 induces mutation of the mitochondrial genome.

    PubMed

    Cherry, J R; Denis, C L

    1989-05-01

    It was previously observed that increased dosages of the ADR1 gene, which encodes a yeast transcriptional activator required for alcohol dehydrogenase II (ADH II) expression, cause a decreased rate of growth in medium containing ethanol as the carbon source. Here we show that observed reduction in growth rate is mediated by the ADR1 protein which, when overexpressed, increases the frequency of cytoplasmic petites. Unlike previously characterized mutations known to potentiate petite formation, the ADR1 effect is dominant, with the petite frequency rising concomitantly with increasing ADR1 dosage. The ability of ADR1 to increase the frequency of mitochondrial mutation is correlated with its ability to activate ADH II transcription but is independent of the level of ADH II being expressed. Based on restoration tests using characterized mit- strains, ADR1 appears to cause non-specific deletions within the mitochondrial genome to produce rho- petites. Pedigree analysis of ADR1-overproducing strains indicates that only daughter cells become petite. This pattern is analogous to that observed for petite induction by growth at elevated temperature and by treatment with the acridine dye euflavine. One strain resistant to ADR1-induced petite formation displayed cross-resistance to petite mutation by growth at elevated temperature and euflavine treatment, yet was susceptible to petite induction by ethidium bromide. These results suggest that ADR1 overexpression disrupts the fidelity of mitochondrial DNA replication or repair.

  1. Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

    PubMed

    Zhong, Franklin L; Mamaï, Ons; Sborgi, Lorenzo; Boussofara, Lobna; Hopkins, Richard; Robinson, Kim; Szeverényi, Ildikó; Takeichi, Takuya; Balaji, Reshmaa; Lau, Aristotle; Tye, Hazel; Roy, Keya; Bonnard, Carine; Ahl, Patricia J; Jones, Leigh Ann; Baker, Paul; Lacina, Lukas; Otsuka, Atsushi; Fournie, Pierre R; Malecaze, François; Lane, E Birgitte; Akiyama, Masashi; Kabashima, Kenji; Connolly, John E; Masters, Seth L; Soler, Vincent J; Omar, Salma Samir; McGrath, John A; Nedelcu, Roxana; Gribaa, Moez; Denguezli, Mohamed; Saad, Ali; Hiller, Sebastian; Reversade, Bruno

    2016-09-22

    Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

  2. Activation of diverse signaling pathways by oncogenic PIK3CA mutations

    PubMed Central

    Wu, Xinyan; Renuse, Santosh; Sahasrabuddhe, Nandini A.; Zahari, Muhammad Saddiq; Chaerkady, Raghothama; Kim, Min-Sik; Nirujogi, Raja S.; Mohseni, Morassa; Kumar, Praveen; Raju, Rajesh; Zhong, Jun; Yang, Jian; Neiswinger, Johnathan; Jeong, Jun-Seop; Newman, Robert; Powers, Maureen A.; Somani, Babu Lal; Gabrielson, Edward; Sukumar, Saraswati; Stearns, Vered; Qian, Jiang; Zhu, Heng; Vogelstein, Bert; Park, Ben Ho; Pandey, Akhilesh

    2014-01-01

    The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing ‘driver’ oncogenic mutations of PIK3CA to dissect the signaling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signaling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signaling events and for discovering novel signaling molecules as readouts of pathway activation or potential therapeutic targets. PMID:25247763

  3. Contribution of active-site glutamine to rate enhancement in ubiquitin carboxy terminal hydrolases

    PubMed Central

    Boudreaux, David; Chaney, Joseph; Maiti, Tushar K.; Das, Chittaranjan

    2012-01-01

    Ubiquitin carboxy terminal hydrolases (UCHs) are cysteine proteases featuring a classical cysteine-histidine-aspartate catalytic triad, also a highly conserved glutamine thought to be a part of the oxyanion hole. However, the contribution of this side chain to the catalysis by UCH enzymes is not known. Herein, we demonstrate that the glutamine side chain contributes to rate enhancement in UCHL1, UCHL3 and UCHL5. Mutation of the glutamine to alanine in these enzymes impairs the catalytic efficiency mainly due to a 16 to 30-fold reduction in kcat, which is consistent with a loss of approximately 2 kcal/mol in transition-state stabilization. However, the contribution to transition-state stabilization observed here is rather modest for the side chain’s role in oxyanion stabilization. Interestingly, we discovered that the carbonyl oxygen of this side chain is engaged in a C—H•••O hydrogen-bonding contact with the CεH group of the catalytic histidine. Upon further analysis, we found that this interaction is a common active-site structural feature in most cysteine proteases, including papain, belonging to families with the QCH(N/D) type of active-site configuration. It is possible that removal of the glutamine side chain might have abolished the C—H•••O interaction, which typically accounts for 2 kcal/mol of stabilization, leading to the effect on catalysis observed here. Additional studies performed on UCHL3 by mutating the glutamine to glutamate (strong C—H•••O acceptor but oxyanion destabilizer) and to lysine (strong oxyanion stabilizer but lacking C—H•••O hydrogen-bonding property) suggest that the C—H•••O hydrogen bond could contribute to catalysis. PMID:22284438

  4. Role of a strictly conserved active site tyrosine in cofactor genesis in the copper amine oxidase from Hansenula polymorpha.

    PubMed