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Sample records for atpase domain mutation

  1. MSH2 ATPase domain mutation affects CTG*CAG repeat instability in transgenic mice.

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    Stéphanie Tomé

    2009-05-01

    Full Text Available Myotonic dystrophy type 1 (DM1 is associated with one of the most highly unstable CTG*CAG repeat expansions. The formation of further repeat expansions in transgenic mice carrying expanded CTG*CAG tracts requires the mismatch repair (MMR proteins MSH2 and MSH3, forming the MutSbeta complex. It has been proposed that binding of MutSbeta to CAG hairpins blocks its ATPase activity compromising hairpin repair, thereby causing expansions. This would suggest that binding, but not ATP hydrolysis, by MutSbeta is critical for trinucleotide expansions. However, it is unknown if the MSH2 ATPase activity is dispensible for instability. To get insight into the mechanism by which MSH2 generates trinucleotide expansions, we crossed DM1 transgenic mice carrying a highly unstable >(CTG(300 repeat tract with mice carrying the G674A mutation in the MSH2 ATPase domain. This mutation impairs MSH2 ATPase activity and ablates base-base MMR, but does not affect the ability of MSH2 (associated with MSH6 to bind DNA mismatches. We found that the ATPase domain mutation of MSH2 strongly affects the formation of CTG expansions and leads instead to transmitted contractions, similar to a Msh2-null or Msh3-null deficiency. While a decrease in MSH2 protein level was observed in tissues from Msh2(G674 mice, the dramatic reduction of expansions suggests that the expansion-biased trinucleotide repeat instability requires a functional MSH2 ATPase domain and probably a functional MMR system.

  2. P160L mutation in the Ca(2+) ATPase 2A domain in a patient with severe Darier disease.

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    Godic, Aleksandar; Glavac, Damjan; Korosec, Branka; Miljković, Jovan; Potocnik, Marko; Kansky, Aleksej

    2004-01-01

    Darier disease (DD) is caused by mutations of the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2). The mutations affect protein expression, degradation and activity. We report a patient with severe sporadic DD, who did not respond adequately to repeated courses of orally administered acitretin and isotretinoin. He was found to harbor the missense P160L mutation of the ATP2A2 gene in a heterozygous state in the A domain of SERCA2 and polymorphism in intron 18 (2741 + 54 G --> A). The A domain plays a key role in translocation of Ca(2+) from cytoplasm to endoplasmic reticulum lumen, thus establishing a low intracellular Ca(2+) concentration.

  3. Familial hemiplegic migraine mutations affect Na,K-ATPase domain interactions

    National Research Council Canada - National Science Library

    Swarts, H.G.P; Weigand, K.M; Venselaar, H; Maagdenberg, A.M. van den; Russel, F.G.M; Koenderink, J.B

    2013-01-01

    Familial hemiplegic migraine (FHM) is a monogenic variant of migraine with aura. One of the three known causative genes, ATP1A2, which encodes the alpha2 isoform of Na,K-ATPase, causes FHM type 2 (FHM2...

  4. Conformational alterations resulting from mutations in cytoplasmic domains of the alpha subunit of the Na,K-ATPase

    DEFF Research Database (Denmark)

    Blostein, R; Daly, S E; MacAulay, Nanna;

    1998-01-01

    , and decreased sensitivity to inhibition by vanadate. The striking changes observed with alpha 1M32E233K suggests interactions between the N-terminus, the beta-strand in the M2-M3 loop and the catalytic phosphorylation site. The behavior of these mutants contrasts with that of least one mutant involving......This paper summarizes experiments concerned with the functional consequences of mutations in cytoplasmic regions of the alpha 1 subunit of the Na,K-ATPase, in particular the amino terminus, the first cytoplasmic loop between transmembrane segments M2 and M3, and the major cytoplasmic loop between M......4 and M5. In the first mutation (alpha 1M32), 32 residues were removed from the N-terminus. The second mutation (E233K) was in the putative beta strand of M2-M3 loop and the third, comprised the replacement of the amino terminal half of loop M4-M5 of the Na,K-ATPase with the homologous segment...

  5. Conformational alterations resulting from mutations in cytoplasmic domains of the alpha subunit of the Na,K-ATPase

    DEFF Research Database (Denmark)

    Blostein, R; Daly, S E; MacAulay, Nanna

    1998-01-01

    substitution of a residue in the putative cation binding pocket, namely S775A in the fifth transmembrane segment (Arguello, J.M., & Lingrel, J. B. J. Biol. Chem. 270: 22764-22771, 1995). Although its K+/ATP antagonism resembles that of the foregoing cytoplasmic mutants, its vanadate sensitivity is unaltered...... in the presence or absence of Na+ and at low versus high pH indicate a marked alteration in cation affinity and/or selectivity. This results suggests coupling of an alteration in the large M4-M5 cytoplasmic domain to cation binding in, presumably, the juxtapositioned transmembrane domain....

  6. ATPase domain and interdomain linker play a key role in aggregation of mitochondrial Hsp70 chaperone Ssc1.

    Science.gov (United States)

    Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

    2010-02-12

    The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

  7. Investigation of the Mitochondrial ATPase 6/8 and tRNALys Genes Mutations in Autism

    OpenAIRE

    Sepideh Dadgar; Zahra-Soheila Soheili; Omid Aryani; Massoud Houshmand; Fahimeh Piryaei

    2012-01-01

    Objective: Autism results from developmental factors that affect many or all functional brain systems. Brain is one of tissues which are crucially in need of adenosine triphosphate (ATP). Autism is noticeably affected by mitochondrial dysfunction which impairs energy metabolism. Considering mutations within ATPase 6, ATPase 8 and tRNALys genes, associated with different neural diseases, and the main role of ATPase 6/8 in energy generation, we decided to investigate mutations on these mtDNA-en...

  8. Investigation of the Mitochondrial ATPase 6/8 and tRNALys Genes Mutations in Autism

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    Sepideh Dadgar

    2012-01-01

    Full Text Available Objective: Autism results from developmental factors that affect many or all functional brain systems. Brain is one of tissues which are crucially in need of adenosine triphosphate (ATP. Autism is noticeably affected by mitochondrial dysfunction which impairs energy metabolism. Considering mutations within ATPase 6, ATPase 8 and tRNALys genes, associated with different neural diseases, and the main role of ATPase 6/8 in energy generation, we decided to investigate mutations on these mtDNA-encoded genes to reveal their roles in autism pathogenesis.Materials and Methods: In this experimental study, mutation analysis for the mentioned genes were performed in a cohort of 24 unrelated patients with idiopathic autism by employing amplicon sequencing of mtDNA fragments.Results: In this study, 12 patients (50% showed point mutations that represent a significant correlation between autism and mtDNA variations. Most of the identified substitutions (55.55% were observed on MT-ATP6, altering some conserved amino acids to other ones which could potentially affect ATPase 6 function. Mutations causing amino acid replacement denote involvement of mtDNA genes, especially ATPase 6 in autism pathogenesis.Conclusion: MtDNA mutations in relation with autism could be remarkable to realize an understandable mechanism of pathogenesis in order to achieve therapeutic solutions.

  9. Regulation of plant plasma membrane H+- and Ca2+-ATPases by terminal domains

    DEFF Research Database (Denmark)

    Bækgaard, Lone; Fuglsang, Anja Thoe; Palmgren, Michael Gjedde

    2005-01-01

    In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H(+)-and Ca(2+)-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulator...... mechanisms of these pumps. In plant plasma membrane H(+)- and Ca(2+)-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed....

  10. Roles of long-range electrostatic domain interactions and K+ in phosphoenzyme transition of Ca2+-ATPase.

    Science.gov (United States)

    Yamasaki, Kazuo; Daiho, Takashi; Danko, Stefania; Suzuki, Hiroshi

    2013-07-12

    Sarcoplasmic reticulum Ca(2+)-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca(2+) transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 m inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K(+) is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.

  11. Biochemical Characterization of P4-ATPase Mutations Associated with Intrahepatic Cholestatic Disease

    DEFF Research Database (Denmark)

    Gantzel, Rasmus; Vestergaard, Anna Lindeløv; Mikkelsen, Stine;

    The cholestatic disorders progressive familial intrahepatic cholestasis type 1 (PFIC1, also referred to as Byler’s disease) and benign recurrent intrahepatic cholestasis type 1 (BRIC1) are caused by mutation of the P4-ATPase ATP8B1. The substrate of ATP8B1 is very likely to be phosphatidylserine...... families have been investigated, and more than 50 distinct disease mutations have been identified, with roughly half being missense mutations. In this project we try to answer the question whether PFIC1 mutations are generally more disturbing than BRIC1 mutations with respect to expression, structural...... stability and function. We investigate the mutations in our well functioning system of ATP8A2, being expressed in mammalian HEK293T cells, affinity-purified, and reconstituted in lipid vesicles. Well-known mutations from both groups of patients have been selected for study. I91P in ATP8A2 (L127P in ATP8B1...

  12. Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.

    Science.gov (United States)

    Laurent, Clémentine; Lekeux, Gilles; Ukuwela, Ashwinie A; Xiao, Zhiguang; Charlier, Jean-Benoit; Bosman, Bernard; Carnol, Monique; Motte, Patrick; Damblon, Christian; Galleni, Moreno; Hanikenne, Marc

    2016-03-01

    PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases.

  13. Neurological disease mutations compromise a C-terminal ion pathway in the Na(+)/K(+)-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Khandelia, Himanshu; Morth, J Preben

    2010-01-01

    The Na(+)/K(+)-ATPase pumps three sodium ions out of and two potassium ions into the cell for each ATP molecule that is split, thereby generating the chemical and electrical gradients across the plasma membrane that are essential in, for example, signalling, secondary transport and volume...... potassium is released the proton will also return to the cytoplasm, thus allowing an overall asymmetric stoichiometry of the transported ions. The C terminus controls the gate to the pathway. Its structure is crucial for pump function, as demonstrated by at least eight mutations in the region that cause...... severe neurological diseases. This novel model for ion transport by the Na(+)/K(+)-ATPase is established by electrophysiological studies of C-terminal mutations in familial hemiplegic migraine 2 (FHM2) and is further substantiated by molecular dynamics simulations. A similar ion regulation is likely...

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

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    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.

  15. Multivalent Chromatin Engagement and Inter-domain Crosstalk Regulate MORC3 ATPase

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    Forest H. Andrews

    2016-09-01

    Full Text Available MORC3 is linked to inflammatory myopathies and cancer; however, the precise role of MORC3 in normal cell physiology and disease remains poorly understood. Here, we present detailed genetic, biochemical, and structural analyses of MORC3. We demonstrate that MORC3 is significantly upregulated in Down syndrome and that genetic abnormalities in MORC3 are associated with cancer. The CW domain of MORC3 binds to the methylated histone H3K4 tail, and this interaction is essential for recruitment of MORC3 to chromatin and accumulation in nuclear bodies. We show that MORC3 possesses intrinsic ATPase activity that requires DNA, but it is negatively regulated by the CW domain, which interacts with the ATPase domain. Natively linked CW impedes binding of the ATPase domain to DNA, resulting in a decrease in the DNA-stimulated enzymatic activity. Collectively, our studies provide a molecular framework detailing MORC3 functions and suggest that its modulation may contribute to human disease.

  16. Further examination of seventeen mutations in Escherichia coli F1-ATPase beta-subunit.

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    Senior, A E; al-Shawi, M K

    1992-10-25

    Seventeen mutations in beta-subunit of Escherichia coli F1-ATPase which had previously been characterized in strain AN1272 (Mu-induced mutant) were expressed in strain JP17 (beta-subunit gene deletion). Six showed unchanged behavior, namely: C137Y; G142D; G146S; G207D; Y297F; and Y354F. Five failed to assemble F1F0 correctly, namely: G149I; G154I; G149I,G154I; G223D; and P403S,G415D. Six assembled F1F0 correctly, but with membrane ATPase lower than in AN1272, namely: K155Q; K155E; E181Q; E192Q; D242N; and D242V. AN1272 was shown to unexpectedly produce a small amount of wild-type beta-subunit; F1-ATPase activities reported previously in AN1272 were referable to hybrid enzymes containing both mutant and wild-type beta-subunits. Purified F1 was obtained from K155Q; K155E; E181Q; E192Q; and D242N mutants in JP17. Vmax ATPase values were lower, and unisite catalysis rate and equilibrium constants were perturbed to greater extent, than in AN1272. However, general patterns of perturbation revealed by difference energy diagrams were similar to those seen previously, and the new data correlated well in linear free energy relationships for reaction steps of unisite catalysis. Correlation between multisite and unisite ATPase activity was seen in the new enzymes. Overall, the data give strong support to previously proposed mechanisms of unisite catalysis, steady-state catalysis, and energy coupling in F1-ATPases (Al-Shawi, M. K., Parsonage, D. and Senior, A. E. (1990) J. Biol. Chem. 265, 4402-4410). The K155Q, K155E, D242N, and E181Q mutations caused 5000-fold, 4000-fold, 1800-fold, and 700-fold decrease, respectively, in Vmax ATPase, implying possibly direct roles for these residues in catalysis. Experiments with the D242N mutant suggested a role for residue beta D242 in catalytic site Mg2+ binding.

  17. Biochemical characterization of P4-ATPase mutations associated with Intrahepatic Cholestatic Disease

    DEFF Research Database (Denmark)

    Gantzel, Rasmus; Vestergaard, Anna Lindeløv; Mikkelsen, Stine

    Progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1) are caused by mutation of the P4-ATPase ATP8B1 that flips phospholipid from the exoplasmic leaflet to the cytoplasmic leaflet of canalicular membranes. It is hypothesized...... that PFIC1 mutations are the most disturbing with respect to expression, structural stability and/or function. Although recent data indicates that the specific phospholipid substrate of ATP8B1 is phosphatidylcholine (PC) [1] whereas ATP8A2 flips phosphatidylserine (PS) and phosphatidylethanolamine (PE......), there may be several mechanistic similarities between ATP8B1 and ATP8A2, and here we investigate known disease mutations using our well-functioning methodology for expression, affinity purification and assay of the partial reactions of ATP8A2. Mutations I91P (L127P in ATP8B1) and L308F (I344F) are located...

  18. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

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    Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

    2017-01-01

    Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

  19. Diverse functional consequences of mutations in the Na+/K+-ATPase alpha2-subunit causing familial hemiplegic migraine type 2.

    NARCIS (Netherlands)

    Tavraz, N.N.; Friedrich, T.; Durr, K.L.; Koenderink, J.B.; Bamberg, E.; Freilinger, T.; Dichgans, M.

    2008-01-01

    Mutations in ATP1A2, the gene coding for the Na(+)/K(+)-ATPase alpha(2)-subunit, are associated with both familial hemiplegic migraine and sporadic cases of hemiplegic migraine. In this study, we examined the functional properties of 11 ATP1A2 mutations associated with familial or sporadic hemiplegi

  20. Identification of Domains within the V-ATPase Accessory Subunit Ac45 Involved in V-ATPase Transport and Ca2+-dependent Exocytosis

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    Jansen, Eric J. R.; van Bakel, Nick. H. M.; Loohuis, Nikkie F. M. Olde; Hafmans, Theo G. M.; Arentsen, Tim; Coenen, Anthon J. M.; Scheenen, Wim J. J. M.; Martens, Gerard J. M.

    2012-01-01

    The vacuolar (H+)-ATPase (V-ATPase) is crucial for maintenance of the acidic microenvironment in intracellular organelles, whereas its membrane-bound V0-sector is involved in Ca2+-dependent membrane fusion. In the secretory pathway, the V-ATPase is regulated by its type I transmembrane and V0-associated accessory subunit Ac45. To execute its function, the intact-Ac45 protein is proteolytically processed to cleaved-Ac45 thereby releasing its N-terminal domain. Here, we searched for the functional domains within Ac45 by analyzing a set of deletion mutants close to the in vivo situation, namely in transgenic Xenopus intermediate pituitary melanotrope cells. Intact-Ac45 was poorly processed and accumulated in the endoplasmic reticulum of the transgenic melanotrope cells. In contrast, cleaved-Ac45 was efficiently transported through the secretory pathway, caused an accumulation of the V-ATPase at the plasma membrane and reduced dopaminergic inhibition of Ca2+-dependent peptide secretion. Surprisingly, removal of the C-tail from intact-Ac45 caused cellular phenotypes also found for cleaved-Ac45, whereas C-tail removal from cleaved-Ac45 still allowed its transport to the plasma membrane, but abolished V-ATPase recruitment into the secretory pathway and left dopaminergic inhibition of the cells unaffected. We conclude that domains located in the N- and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca2+-dependent-regulated exocytosis. PMID:22736765

  1. Identification of domains within the V-ATPase accessory subunit Ac45 involved in V-ATPase transport and Ca2+-dependent exocytosis.

    Science.gov (United States)

    Jansen, Eric J R; van Bakel, Nick H M; Olde Loohuis, Nikkie F M; Hafmans, Theo G M; Arentsen, Tim; Coenen, Anthon J M; Scheenen, Wim J J M; Martens, Gerard J M

    2012-08-10

    The vacuolar (H(+))-ATPase (V-ATPase) is crucial for maintenance of the acidic microenvironment in intracellular organelles, whereas its membrane-bound V(0)-sector is involved in Ca(2+)-dependent membrane fusion. In the secretory pathway, the V-ATPase is regulated by its type I transmembrane and V(0)-associated accessory subunit Ac45. To execute its function, the intact-Ac45 protein is proteolytically processed to cleaved-Ac45 thereby releasing its N-terminal domain. Here, we searched for the functional domains within Ac45 by analyzing a set of deletion mutants close to the in vivo situation, namely in transgenic Xenopus intermediate pituitary melanotrope cells. Intact-Ac45 was poorly processed and accumulated in the endoplasmic reticulum of the transgenic melanotrope cells. In contrast, cleaved-Ac45 was efficiently transported through the secretory pathway, caused an accumulation of the V-ATPase at the plasma membrane and reduced dopaminergic inhibition of Ca(2+)-dependent peptide secretion. Surprisingly, removal of the C-tail from intact-Ac45 caused cellular phenotypes also found for cleaved-Ac45, whereas C-tail removal from cleaved-Ac45 still allowed its transport to the plasma membrane, but abolished V-ATPase recruitment into the secretory pathway and left dopaminergic inhibition of the cells unaffected. We conclude that domains located in the N- and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca(2+)-dependent-regulated exocytosis.

  2. A conserved sequence extending motif III of the motor domain in the Snf2-family DNA translocase Rad54 is critical for ATPase activity.

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    Xiao-Ping Zhang

    Full Text Available Rad54 is a dsDNA-dependent ATPase that translocates on duplex DNA. Its ATPase function is essential for homologous recombination, a pathway critical for meiotic chromosome segregation, repair of complex DNA damage, and recovery of stalled or broken replication forks. In recombination, Rad54 cooperates with Rad51 protein and is required to dissociate Rad51 from heteroduplex DNA to allow access by DNA polymerases for recombination-associated DNA synthesis. Sequence analysis revealed that Rad54 contains a perfect match to the consensus PIP box sequence, a widely spread PCNA interaction motif. Indeed, Rad54 interacts directly with PCNA, but this interaction is not mediated by the Rad54 PIP box-like sequence. This sequence is located as an extension of motif III of the Rad54 motor domain and is essential for full Rad54 ATPase activity. Mutations in this motif render Rad54 non-functional in vivo and severely compromise its activities in vitro. Further analysis demonstrated that such mutations affect dsDNA binding, consistent with the location of this sequence motif on the surface of the cleft formed by two RecA-like domains, which likely forms the dsDNA binding site of Rad54. Our study identified a novel sequence motif critical for Rad54 function and showed that even perfect matches to the PIP box consensus may not necessarily identify PCNA interaction sites.

  3. Synthetic null-cysteine phospholamban analogue and the corresponding transmembrane domain inhibit the Ca-ATPase.

    Science.gov (United States)

    Karim, C B; Marquardt, C G; Stamm, J D; Barany, G; Thomas, D D

    2000-09-05

    Chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) have been used to analyze the structure and function of phospholamban (PLB), a 52-residue integral membrane protein that regulates the calcium pump (Ca-ATPase) in cardiac sarcoplasmic reticulum (SR). PLB exists in equilibrium between monomeric and pentameric forms, as observed by SDS-PAGE, EPR, and fluorescence. It has been proposed that inhibition of the pump is due primarily to the monomeric form, with both pentameric stability and inhibition dependent primarily on the transmembrane (TM) domain. To test these hypotheses, we have studied the physical and functional properties of a synthetic null-cysteine PLB analogue that is entirely monomeric on SDS-PAGE, and compared it with the synthetic null-cysteine TM domain (residues 26-52). The TM domain was found to be primarily oligomeric on SDS-PAGE, and boundary lipid spin label analysis in lipid bilayers verified that the isolated TM domain is more oligomeric than the full-length parent molecule. These results indicate that the stability of the PLB pentamer is due primarily to attractive interactions between hydrophobic TM domains, overcoming the repulsive electrostatic interactions between the cationic cytoplasmic domains (residues 1-25). When reconstituted into liposomes containing the Ca-ATPase, the null-cysteine TM domain had the same inhibitory function as that of the full-length parent molecule. We conclude that the TM domain of PLB is sufficient for inhibitory function, the oligomeric stability of PLB does not determine its inhibitory activity, and the three Cys residues in the TM domain are not required for inhibitory function.

  4. Domain stability in the AAA+ ATPase ClpB from Escherichia coli.

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    Nagy, Maria; Akoev, Vladimir; Zolkiewski, Michal

    2006-09-01

    ClpB is a heat-shock protein that reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the family of AAA+ ATPases and forms ring-shaped oligomers: heptamers in the absence of nucleotides and hexamers in the presence of nucleotides. We investigated the thermodynamic stability of ClpB in its monomeric and oligomeric forms. ClpB contains six distinct structural domains: the N-terminal domain involved in substrate binding, two AAA+ ATP-binding modules, each consisting of two domains, and a coiled-coil domain inserted between the AAA+ modules. We produced seven variants of ClpB, each containing a single Trp located in each of the ClpB domains and measured the changes in Trp fluorescence during the equilibrium urea-induced unfolding of ClpB. We found that two structural domains: the small domain of the C-terminal AAA+ module and the coiled-coil domain were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation and/or interactions during formation of the ClpB rings.

  5. The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination.

    Directory of Open Access Journals (Sweden)

    Rebecca C Burgess

    Full Text Available Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR. Although its activity has been implicated in several steps of HR, its exact role(s at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA. This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box of Rad54 (Rad54-AA. Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

  6. Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase.

    Science.gov (United States)

    Song, Qiujing; Pallikkuth, Sandeep; Bossuyt, Julie; Bers, Donald M; Robia, Seth L

    2011-03-18

    Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.

  7. Phosphomimetic Mutations Enhance Oligomerization of Phospholemman and Modulate Its Interaction with the Na/K-ATPase*

    Science.gov (United States)

    Song, Qiujing; Pallikkuth, Sandeep; Bossuyt, Julie; Bers, Donald M.; Robia, Seth L.

    2011-01-01

    Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex. PMID:21220422

  8. Mutation of domain III and domain VI in L gene conserved domain of Nipah virus

    Science.gov (United States)

    Jalani, Siti Aishah; Ibrahim, Nazlina

    2016-11-01

    Nipah virus (NiV) is the etiologic agent responsible for the respiratory illness and causes fatal encephalitis in human. NiV L protein subunit is thought to be responsible for the majority of enzymatic activities involved in viral transcription and replication. The L protein which is the viral RNA dependent RNA polymerase has high sequence homology among negative sense RNA viruses. In negative stranded RNA viruses, based on sequence alignment six conserved domain (domain I-IV) have been determined. Each domain is separated on variable regions that suggest the structure to consist concatenated functional domain. To directly address the roles of domains III and VI, site-directed mutations were constructed by the substitution of bases at sequences 2497, 2500, 5528 and 5532. Each mutated L gene can be used in future studies to test the ability for expression on in vitro translation.

  9. The DNA maturation domain of gpA, the DNA packaging motor protein of bacteriophage lambda, contains an ATPase site associated with endonuclease activity.

    Science.gov (United States)

    Ortega, Marcos E; Gaussier, Hélène; Catalano, Carlos E

    2007-11-02

    Terminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in Escherichia coli. Biochemical characterization of gpA-DeltaN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A "P-loop" sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme, DNA maturation and DNA packaging, are discussed.

  10. Phospholamban Modulates the Functional Coupling between Nucleotide Domains in Ca-ATPase Oligomeric Complexes in Cardiac Sarcoplasmic Reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Chen, L.; Yao, Qing; Soares, Thereza A.; Squier, Thomas C.; Bigelow, Diana J.

    2009-03-24

    Oligomeric interactions between Ca-ATPase polypeptide chains and their modulation by phospholamban (PLB) were measured in native cardiac sarcoplasmic reticulum (SR) microsomes. Progressive modification of Lys514 with fluorescein-5-isothiocyanate (FITC), which physically blocks access to the nucleotide binding site by ATP, demonstrates that Ca-ATPase active sites function independently of one another prior to the phosphorylation of PLB. However, upon PKA-dependent phosphorylation of PLB, a second-order dependence between enzyme activity and the fraction of active sites is observed, consistent with a dimeric functional complex. Complementary distance measurements were made using FITC or 5-iodoacetamido-fluorescein (IAF) bound to Cys674 within the N- or P-domains respectively, to detect structural coupling within oligomeric complexes. Accompanying the phosphorylation of PLB, neighboring Ca-ATPase polypeptide chains exhibit a 4 ± 2 Å decrease in the proximity between FITC sites within the N-domain and a 9 ± 3 Å increase in the proximity between IAF sites within P-domains. Thus, the phosphorylation of PLB induces spatial rearrangements between the N- and P-domain elements of proximal Ca-ATPase polypeptide chains which restore functional interactions between neighboring polypeptide chains and, in turn, result in increased rates of catalytic turnover. These results are interpreted in terms of a structural model, calculated through optimization of shape complementarity, desolvation, and electrostatic energies, which suggests a dimeric arrangement of Ca-ATPase polypeptide chains through the proximal association of N-domains. We suggest that the phosphorylation of PLB acts to release constraints involving interdomain subunit interactions that enhance catalytically important N-domain motions.

  11. A docking approach to the study of copper trafficking proteins: interaction between metallochaperones and soluble domains of copper ATPases

    NARCIS (Netherlands)

    Arnesano, F.; Banci, L.; Bertini, I.; Bonvin, A.M.J.J.

    2004-01-01

    A structural model of the transient complex between the yeast copper chaperone Atx1 and the first soluble domain of the copper transporting ATPase Ccc2 was obtained with HADDOCK, combining NMR chemical shift mapping information with in silico docking. These two proteins are involved in copper traffi

  12. Novel ATPase Cu(2+ transporting beta polypeptide mutations in Chinese families with Wilson's disease.

    Directory of Open Access Journals (Sweden)

    Shaojuan Gu

    Full Text Available Wilson's disease (WD is an autosomal recessive inherited disorder caused by mutations in the ATPase Cu(2+ transporting beta polypeptide gene (ATP7B. The detailed metabolism of copper-induced pathology in WD is still unknown. Gene mutations as well as the possible pathways involved in the ATP7B deficiency were documented. The ATP7B gene was analyzed for mutations in 18 Chinese Han families with WD by direct sequencing. Cell viability and apoptosis analysis of ATP7B small interfering RNA (siRNA-treated human liver carcinoma (HepG2 cells were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT assay and Hoechst 33342 staining. Finally, the expression of B-cell CLL/lymphoma 2 (BCL2, BCL2-associated X protein (BAX, sterol regulatory element binding protein 1 (SREBP1, and minichromosome maintenance protein 7 (MCM7 of ATP7B siRNA-treated cells were tested by real-time polymerase chain reaction (real-time PCR and Western blot analysis. Twenty different mutations including four novel mutations (p.Val145Phe, p.Glu388X, p.Thr498Ser and p.Gly837X in the ATP7B gene were identified in our families. Haplotype analysis revealed that founder effects for four mutations (p.Arg778Leu, p.Pro992Leu, p.Ile1148Thr and p.Ala1295Val existed in these families. Transfection of HepG2 cells with ATP7B siRNA resulted in decreased mRNA expression by 86.3%, 93.1% and 90.8%, and decreased protein levels by 58.5%, 85.5% and 82.1% at 24, 48 and 72 hours, respectively (All P<0.01. In vitro study revealed that the apoptotic, cell cycle and lipid metabolism pathway may be involved in the mechanism of WD. Our results revealed that the genetic cause of 18 Chinese families with WD and ATP7B deficiency-induce apoptosis may result from imbalance in cell cycle and lipid metabolism pathway.

  13. Novel ATPase Cu(2+) transporting beta polypeptide mutations in Chinese families with Wilson's disease.

    Science.gov (United States)

    Gu, Shaojuan; Yang, Huarong; Qi, Yong; Deng, Xiong; Zhang, Le; Guo, Yi; Huang, Qing; Li, Jing; Shi, Xiaoliu; Song, Zhi; Deng, Hao

    2013-01-01

    Wilson's disease (WD) is an autosomal recessive inherited disorder caused by mutations in the ATPase Cu(2+) transporting beta polypeptide gene (ATP7B). The detailed metabolism of copper-induced pathology in WD is still unknown. Gene mutations as well as the possible pathways involved in the ATP7B deficiency were documented. The ATP7B gene was analyzed for mutations in 18 Chinese Han families with WD by direct sequencing. Cell viability and apoptosis analysis of ATP7B small interfering RNA (siRNA)-treated human liver carcinoma (HepG2) cells were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst 33342 staining. Finally, the expression of B-cell CLL/lymphoma 2 (BCL2), BCL2-associated X protein (BAX), sterol regulatory element binding protein 1 (SREBP1), and minichromosome maintenance protein 7 (MCM7) of ATP7B siRNA-treated cells were tested by real-time polymerase chain reaction (real-time PCR) and Western blot analysis. Twenty different mutations including four novel mutations (p.Val145Phe, p.Glu388X, p.Thr498Ser and p.Gly837X) in the ATP7B gene were identified in our families. Haplotype analysis revealed that founder effects for four mutations (p.Arg778Leu, p.Pro992Leu, p.Ile1148Thr and p.Ala1295Val) existed in these families. Transfection of HepG2 cells with ATP7B siRNA resulted in decreased mRNA expression by 86.3%, 93.1% and 90.8%, and decreased protein levels by 58.5%, 85.5% and 82.1% at 24, 48 and 72 hours, respectively (All Pmechanism of WD. Our results revealed that the genetic cause of 18 Chinese families with WD and ATP7B deficiency-induce apoptosis may result from imbalance in cell cycle and lipid metabolism pathway.

  14. Short and long range functions of amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase. A mutational study.

    Science.gov (United States)

    Chen, L; Sumbilla, C; Lewis, D; Zhong, L; Strock, C; Kirtley, M E; Inesi, G

    1996-05-01

    Mutational analysis of several amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase was performed by expressing wild type ATPase and 32 site-directed mutants in COS-1 cells followed by functional characterization of the microsomal fraction. Four different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type ATPase; (b) Ca2+ transport inhibited to a greater extent than ATPase hydrolytic activity; (c) inhibition of transport and hydrolytic activity in the presence of high levels of phosphorylated enzyme intermediate; and (d) total inhibition of ATP utilization by the enzyme while retaining the ability to form phosphoenzyme by utilization of P(i). Analysis of experimental observations and molecular models revealed short and long range functions of several amino acids within the transmembrane region. Short range functions include: (a) direct involvement of five amino acids in Ca2+ binding within a channel formed by clustered transmembrane helices M4, M5, M6, and M8; (b) roles of several amino acids in structural stabilization of the helical cluster for optimal channel function; and (c) a specific role of Lys297 in sealing the distal end of the channel, suggesting that the M4 helix rotates to allow vectorial flux of Ca2+ upon enzyme phosphorylation. Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions with ATP or P(i), transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+.

  15. The role of individual domains and the significance of shedding of ATP6AP2/(prorenin receptor in vacuolar H(+-ATPase biogenesis.

    Directory of Open Access Journals (Sweden)

    Kenichiro Kinouchi

    Full Text Available The ATPase 6 accessory protein 2 (ATP6AP2/(prorenin receptor (PRR is essential for the biogenesis of active vacuolar H(+-ATPase (V-ATPase. Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD and transmembrane domain (TM of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M and causes X-linked mental retardation Hedera type (MRXSH and X-linked parkinsonism with spasticity (XPDS in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

  16. The role of individual domains and the significance of shedding of ATP6AP2/(pro)renin receptor in vacuolar H(+)-ATPase biogenesis.

    Science.gov (United States)

    Kinouchi, Kenichiro; Ichihara, Atsuhiro; Sano, Motoaki; Sun-Wada, Ge-Hong; Wada, Yoh; Ochi, Hiroki; Fukuda, Toru; Bokuda, Kanako; Kurosawa, Hideaki; Yoshida, Naohiro; Takeda, Shu; Fukuda, Keiichi; Itoh, Hiroshi

    2013-01-01

    The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H(+)-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

  17. Function and Subunit Interactions of the N-terminal Domain of Subunit a (Vph1p) of the Yeast V-ATPase*

    OpenAIRE

    Qi, Jie; Forgac, Michael

    2008-01-01

    The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that operate by a rotary mechanism in which ATP hydrolysis drives rotation of a ring of proteolipid subunits relative to subunit a within the integral V0 domain. In vivo dissociation of the V-ATPase (an important regulatory mechanism) generates a V0 domain that does not passively conduct protons. EM analysis indicates that the N-terminal domain of subunit a approaches the rotary subunits in free V0, ...

  18. Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle.

    Science.gov (United States)

    Deng, Maoxian; Boopathi, Ettickan; Hypolite, Joseph A; Raabe, Tobias; Chang, Shaohua; Zderic, Stephen; Wein, Alan J; Chacko, Samuel

    2013-11-15

    Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

  19. A common Greenlandic Inuit BRCA1 RING domain founder mutation

    DEFF Research Database (Denmark)

    Hansen, T.v.O.; Ejlertsen, B.; Albrechtsen, Anders;

    2009-01-01

    Germ-line mutations in the tumour suppressor proteins BRCA1 and BRCA2 predispose to breast and ovarian cancer. We examined 32 breast and/or ovarian cancer patients from Greenland for mutations in BRCA1 and BRCA2. Whereas no mutations were identified in 19 families, 13 families exhibited a BRCA1...... exon 3 nucleotide 234 T > G mutation, which has not previously been reported in the breast cancer information core (BIC) database. The mutation changes a conserved cysteine 39 to a glycine in the Zn(2+) site II of the RING domain, which is essential for BRCA1 ubiquitin ligase activity. Eight...... of the families had members with ovarian cancer, suggesting that the RING domain may be an ovarian cancer hotspot. By SNP array analysis, we find that all 13 families share a 4.5 Mb genomic fragment containing the BRCA1 gene, showing that the mutation originates from a founder. Finally, analysis of 1152 Inuit...

  20. Insights into the molecular evolution of HslU ATPase through biochemical and mutational analyses.

    Directory of Open Access Journals (Sweden)

    Kwang Hoon Sung

    Full Text Available The ATP-dependent HslVU complexes are found in all three biological kingdoms. A single HslV protease exists in each species of prokaryotes, archaea, and eukaryotes, but two HslUs (HslU1 and HslU2 are present in the mitochondria of eukaryotes. Previously, a tyrosine residue at the C-terminal tail of HslU2 has been identified as a key determinant of HslV activation in Trypanosoma brucei and a phenylalanine at the equivalent position to E. coli HslU is found in T. brucei HslU1. Unexpectedly, we found that an F441Y mutation in HslU enhanced the peptidase and caseinolytic activity of HslV in E. coli but it showed partially reduced ATPase and SulA degradation activity. Previously, only the C-terminal tail of HslU has been the focus of HslV activation studies. However, the Pro315 residue interacting with Phe441 in free HslU has also been found to be critical for HslV activation. Hence, our current biochemical analyses explore the importance of the loop region just before Pro315 for HslVU complex functionality. The proline and phenylalanine pair in prokaryotic HslU was replaced with the threonine and tyrosine pair from the functional eukaryotic HslU2. Sequence comparisons between multiple HslUs from three different biological kingdoms in combination with biochemical analysis of E. coli mutants have uncovered important new insights into the molecular evolutionary pathway of HslU.

  1. MutationAligner: a resource of recurrent mutation hotspots in protein domains in cancer.

    Science.gov (United States)

    Gauthier, Nicholas Paul; Reznik, Ed; Gao, Jianjiong; Sumer, Selcuk Onur; Schultz, Nikolaus; Sander, Chris; Miller, Martin L

    2016-01-04

    The MutationAligner web resource, available at http://www.mutationaligner.org, enables discovery and exploration of somatic mutation hotspots identified in protein domains in currently (mid-2015) more than 5000 cancer patient samples across 22 different tumor types. Using multiple sequence alignments of protein domains in the human genome, we extend the principle of recurrence analysis by aggregating mutations in homologous positions across sets of paralogous genes. Protein domain analysis enhances the statistical power to detect cancer-relevant mutations and links mutations to the specific biological functions encoded in domains. We illustrate how the MutationAligner database and interactive web tool can be used to explore, visualize and analyze mutation hotspots in protein domains across genes and tumor types. We believe that MutationAligner will be an important resource for the cancer research community by providing detailed clues for the functional importance of particular mutations, as well as for the design of functional genomics experiments and for decision support in precision medicine. MutationAligner is slated to be periodically updated to incorporate additional analyses and new data from cancer genomics projects. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Concerted but Noncooperative Activation of Nucleotide and Actuator Domains of the Ca-ATPase Upon Calcium Binding

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baowei; Mahaney, James E.; Mayer, M. Uljana; Bigelow, Diana J.; Squier, Thomas C.

    2008-11-25

    Calcium-dependent domain movements of the nucleotide (N) and actuator (A) domains of the SERCA2a isoform of the Ca-ATPase were assessed using constructs containing engineered tetracysteine binding motifs, which were expressed in insect High-Five cells and subsequently labeled with the biarsenical fluorophore 4’,5’-bis(1,3,2-dithoarsolan-2-yl)fluorescein (FlAsH-EDT2). Maximum catalytic function is retained in microsomes isolated from High-Five cells and labeled with FlAsH-EDT2. Distance measurements using the nucleotide analog TNP-ATP, which acts as a fluorescence resonance energy transfer (FRET) acceptor from FlAsH, identify a 2.4 Å increase in the spatial separation between the N- and A-domains induced by high-affinity calcium binding; this structural change is comparable to that observed in crystal structures. No significant distance changes occur across the N-domain between FlAsH and TNP-ATP, indicating that calcium activation induces rigid body domain movements rather than intradomain conformational changes. Calcium-dependent decreases in the fluorescence of FlAsH bound respectively to either the N- or A-domains indicate coordinated and noncooperative domain movements, where both N- and A-domains domains display virtually identical calcium dependencies (i.e., Kd = 4.8 ± 0.4 μM). We suggest that occupancy of a single high-affinity calcium binding site induces the rearrangement of the A- and N-domains of the Ca-ATPase to form an intermediate state, which facilitates ATP utilization upon occupancy of the second high-affinity calcium site to enhance transport efficiency.

  3. Rescue of Na+ and H+ binding in Na+,K+-ATPase M8 aspartate mutants by secondary mutation

    DEFF Research Database (Denmark)

    Holm, Rikke; Einholm, Anja P.; Andersen, Jens Peter

    A mutation replacing the aspartate in transmembrane segment M8 in the a3-isoform of Na,K-ATPase with asparagine has been found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood. This aspartate may be a critical Na+ coordinating residue, but the crystal......H optimum of Na,K-ATPase activity was left-shifted, again with D928N being most disruptive. The reduced affinity for activating Na+ and for inhibitory protons, caused by D928N and D928A mutations, could be rescued by introduction of an additional mutation of a glutamate located far away from D928....

  4. Two novel functional mutations in the Na+,K+-ATPase alpha2-subunit ATP1A2 gene in patients with familial hemiplegic migraine and associated neurological phenotypes.

    NARCIS (Netherlands)

    Castro, M.J.; Nunes, B.; Vries, B. de; Lemos, C.; Molkot, K.R. van; Heuvel, J.J.M.W. van den; Temudo, T.; Barros, J.; Sequeiros, J.; Frants, R.R.; Koenderink, J.B.; Pereira-Monteiro, J.M.; Maagdenberg, A.M. van den

    2008-01-01

    Mutations in the ATP1A2 gene, encoding the alpha2-subunit of the Na+,K+-ATPase, are associated with familial hemiplegic migraine type 2. The majority of ATP1A2 mutations were reported in patients with hemiplegic migraine without any additional neurological findings. Here, we report on two novel ATP1

  5. The medaka mutation tintachina sheds light on the evolution of V-ATPase B subunits in vertebrates

    Science.gov (United States)

    Müller, Claudia; Maeso, Ignacio; Wittbrodt, Joachim; Martínez-Morales, Juan R.

    2013-11-01

    Vacuolar-type H+ ATPases (V-ATPases) are multimeric protein complexes that play a universal role in the acidification of intracellular compartments in eukaryotic cells. We have isolated the recessive medaka mutation tintachina (tch), which carries an inactivating modification of the conserved glycine residue (G75R) of the proton pump subunit atp6v1Ba/vatB1. Mutant embryos show penetrant pigmentation defects, massive brain apoptosis and lethality before hatching. Strikingly, an equivalent mutation in atp6v1B1 (G78R) has been reported in a family of patients suffering from distal renal tubular acidosis (dRTA), a hereditary disease that causes metabolic acidosis due to impaired kidney function. This poses the question as to how molecularly identical mutations result in markedly different phenotypes in two vertebrate species. Our work offers an explanation for this phenomenon. We propose that, after successive rounds of whole-genome duplication, the emergence of paralogous copies allowed the divergence of the atp6v1B cis-regulatory control in different vertebrate groups.

  6. Differential sensitivity of ERBB2 kinase domain mutations towards lapatinib.

    Directory of Open Access Journals (Sweden)

    Rama Krishna Kancha

    Full Text Available BACKGROUND: Overexpression of the ERBB2 kinase is observed in about one-third of breast cancer patients and the dual ERBB1/ERBB2 kinase inhibitor lapatinib was recently approved for the treatment of advanced ERBB2-positive breast cancer. Mutations in the ERBB2 receptor have recently been reported in breast cancer at diagnosis and also in gastric, colorectal and lung cancer. These mutations may have an impact on the clinical responses achieved with lapatinib in breast cancer and may also have a potential impact on the use of lapatinib in other solid cancers. However, the sensitivity of lapatinib towards clinically observed ERBB2 mutations is not known. METHODOLOGY/PRINCIPAL FINDINGS: We cloned a panel of 8 clinically observed ERBB2 mutations, established stable cell lines and characterized their sensitivity towards lapatinib and alternative ERBB2 inhibitors. Both lapatinib-sensitive and lapatinib-resistant ERBB2 mutations were observed. Interestingly, we were able to generate lapatinib resistance mutations in wt-ERBB2 cells incubated with lapatinib for prolonged periods of time. This indicates that these resistance mutations may also cause secondary resistance in lapatinib-treated patients. Lapatinib-resistant ERBB2 mutations were found to be highly resistant towards AEE788 treatment but remained sensitive towards the dual irreversible inhibitors CL-387785 and WZ-4002. CONCLUSIONS/SIGNIFICANCE: Patients harbouring certain ERBB2 kinase domain mutations at diagnosis may not benefit from lapatinib treatment. Moreover, secondary lapatinib resistance may develop due to kinase domain mutations. Irreversible ERBB2 inhibitors may offer alternative treatment options for breast cancer and other solid tumor patients harbouring lapatinib resistance mutations. In addition, these inhibitors may be of interest in the scenario of secondary lapatinib resistance.

  7. Mutational analysis of a ras catalytic domain

    DEFF Research Database (Denmark)

    Willumsen, B M; Papageorge, A G; Kung, H F

    1986-01-01

    domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence...... homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane...

  8. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    DEFF Research Database (Denmark)

    Tidow, Henning; Hein, Kim Langmach; Bækgaard, Lone

    2010-01-01

    -bound calmodulin (Ca(2+)-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca(2+)-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space......Plasma-membrane Ca(2+)-ATPases (PMCAs) are calcium pumps that expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. They are of major physiological importance, with different isoforms being essential, for example...

  9. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    DEFF Research Database (Denmark)

    Tidow, Henning; Hein, Kim Langmach; Palmgren, Michael Broberg

    2010-01-01

    -bound calmodulin (Ca2+-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space......Plasma-membrane Ca2+-ATPases (PMCAs) are calcium pumps that expel Ca2+ from eukaryotic cells to maintain overall Ca2+ homoeostasis and to provide local control of intracellular Ca2+ signalling. They are of major physiological importance, with different isoforms being essential, for example...

  10. A triple mutation in the a subunit of the Escherichia coli/Propionigenium modestum F1Fo ATPase hybrid causes a switch from Na+ stimulation to Na+ inhibition.

    Science.gov (United States)

    Kaim, G; Dimroth, P

    1998-03-31

    Previously we have shown that the Na+-translocating Escherichia coli (F1-delta)/Propionigenium modestum (Fo+delta) hybrid ATPase acquires a Na+-independent phenotype by the c subunit double mutation F84L, L87V that is reflected by Na+-independent growth of the mutant strain MPC8487 on succinate [Kaim, G., and Dimroth, P. (1995) J. Mol. Biol. 253, 726-738]. Here we describe a new class of mutants that were obtained by random mutagenesis and screening for Na+-independent growth on succinate. All six mutants of the new class contained four mutations in the a subunit (S89P, K220R, V264E, I278N). Results from site-specific mutagenesis revealed that the substitutions K220R, V264E, and I278N were sufficient to create the new phenotype. The resulting E. coli mutant strain MPA762 could only grow in the absence but not in the presence of Na+ ions on succinate minimal medium. This effect of Na+ ions on growth correlated with a Na+-specific inhibition of the mutant ATPase. The Ki for NaCl was 1. 5 mM at pH 6.5, similar to the Km for NaCl in activating the parent hybrid ATPase at this pH. On the other hand, activation by Li+ ions was retained in the new mutant ATPase. In the absence of Na+ or Li+, the mutant enzyme had the same pH optimum at pH 6.5 and twice the specific activity as the parent hybrid ATPase. In accordance with the kinetic data, the reconstituted mutant ATPase catalyzed H+ or Li+ transport but no Na+ transport. These results show for the first time that the coupling ion selectivity of F1Fo ATPases is determined by structural elements not only of the c subunit but also of the a subunit.

  11. Functional analysis of human Na~+/K~+-ATPase familial or sporadic hemiplegic migraine mutations expressed in Xenopus oocytes

    Institute of Scientific and Technical Information of China (English)

    Susan; Spiller; Thomas; Friedrich

    2014-01-01

    AIM: Functional characterization of ATP1A2 mutations that are related to familial or sporadic hemiplegic migraine(FHM2, SHM). METHODS: cRNA of human Na+/K+-ATPase α2- and β1-subunits were injected in Xenopus laevis oocytes. FHM2 or SHM mutations of residues located in putative α/β interaction sites or in the α2-subunit’s C-terminal region were investigated. Mutants were analyzed by the twoelectrode voltage-clamp(TEVC) technique on Xenopus oocytes. Stationary K+-induced Na+/K+ pump currents were measured, and the voltage dependence of apparent K+ affinity was investigated. Transient currents were recorded as ouabain-sensitive currents in Na+ buffers to analyze kinetics and voltage-dependent presteady state charge translocations. The expression of constructs was verified by preparation of plasma membrane and total membrane fractions of cRNA-injected oocytes. RESULTS: Compared to the wild-type enzyme, the mutants G900R and E902K showed no significant dif-ferences in the voltage dependence of K+-induced currents, and analysis of the transient currents indicated that the extracellular Na+ affinity was not affected. Mutant G855R showed no pump activity detectable by TEVC. Also for L994del and Y1009X, pump currents could not be recorded. Analysis of the plasma and total membrane fractions showed that the expressed proteins were not or only minimally targeted to the plasma membrane. Whereas the mutation K1003E had no impact on K+ interaction, D999H affected the voltage dependence of K+-induced currents. Furthermore, kinetics of the transient currents was altered compared to the wild-type enzyme, and the apparent affinity for extracellular Na+ was reduced. CONCLUSION: The investigated FHM2/SHM mutations influence protein function differently depending on the structural impact of the mutated residue.

  12. Impact of the [delta]F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B.; Hunt, John F.; Emtage, Spencer (SG); (Columbia); (JHU)

    2010-07-19

    Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

  13. Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin Subfragment 1.

    Directory of Open Access Journals (Sweden)

    Daria S Logvinova

    Full Text Available Myosin head (myosin subfragment 1, S1 consists of two major structural domains, the motor (or catalytic domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx that mimic S1 ATPase intermediate states S1**-ADP-Pi and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1, we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform, which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle.

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

    Science.gov (United States)

    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

  15. Two novel functional mutations in the Na+,K+-ATPase alpha2-subunit ATP1A2 gene in patients with familial hemiplegic migraine and associated neurological phenotypes.

    Science.gov (United States)

    Castro, M-J; Nunes, B; de Vries, B; Lemos, C; Vanmolkot, K R J; van den Heuvel, J J M W; Temudo, T; Barros, J; Sequeiros, J; Frants, R R; Koenderink, J B; Pereira-Monteiro, J M; van den Maagdenberg, A M J M

    2008-01-01

    Mutations in the ATP1A2 gene, encoding the alpha2-subunit of the Na+,K+-ATPase, are associated with familial hemiplegic migraine type 2. The majority of ATP1A2 mutations were reported in patients with hemiplegic migraine without any additional neurological findings. Here, we report on two novel ATP1A2 mutations that were identified in two Portuguese probands with hemiplegic migraine and interesting additional clinical features. The proband's of family 1 (with a V362E mutation) had mood alterations, classified as a borderline personality. The proband in family 2 (with a P796S mutation) had mild mental impairment, in addition to hemiplegic migraine; more severe mental retardation was observed in his brother, who also had hemiplegic migraine and carried the same mutation. Cell-survival assays clearly showed abnormal functioning of mutant Na+,K+-ATPase, indicating that both ATP1A2 mutants are disease causing. Additionally, our results suggest a possible causal relationship of the ATP1A2 mutations with the complex clinical phenotypes observed in the probands.

  16. Functional significance of SRJ domain mutations in CITED2.

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    Chiann-mun Chen

    Full Text Available CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ that is highly conserved in placental mammals. Loss of Cited2 in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 in vitro at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance in vivo, we generated a T166N mutation of mouse Cited2. We also used PhiC31 integrase-mediated cassette exchange to generate a Cited2 knock-in allele replacing the mouse Cited2 coding sequence with human CITED2 and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using in vivo models and that predictions based on structural conservation and in vitro functional assays, or even in vivo global loss of function models, may be

  17. Mutation in the α2 isoform of Na,K-ATPase associated Familial Hemiplegic Migraine type 2 (FHM2) leads to elevated contractility and vasodilatation of cerebral arteries in mice

    DEFF Research Database (Denmark)

    Hangaard, Lise; Lykke-Hartmann, Karin; Xie, Zijian;

    is associated with few point mutations in the α2 isoform Na,K-ATPase. Mice bearing a mutation corresponding to the inherited mutation in FHM2 patients (G301R) were used in functional studies of middle cerebral arteries. Middle cerebral arteries from heterozygote G301R mice were not different in total α2 Na...

  18. Modeling and Docking Studies on Novel Mutants (K71L and T204V of the ATPase Domain of Human Heat Shock 70 kDa Protein 1

    Directory of Open Access Journals (Sweden)

    Asita Elengoe

    2014-04-01

    Full Text Available The purpose of exploring protein interactions between human adenovirus and heat shock protein 70 is to exploit a potentially synergistic interaction to enhance anti-tumoral efficacy and decrease toxicity in cancer treatment. However, the protein interaction of Hsp70 with E1A32 kDa of human adenovirus serotype 5 remains to be elucidated. In this study, two residues of ATPase domain of human heat shock 70 kDa protein 1 (PDB: 1 HJO were mutated. 3D mutant models (K71L and T204V using PyMol software were then constructed. The structures were evaluated by PROCHECK, ProQ, ERRAT, Verify 3D and ProSA modules. All evidence suggests that all protein models are acceptable and of good quality. The E1A32 kDa motif was retrieved from UniProt (P03255, as well as subjected to docking interaction with NBD, K71L and T204V, using the Autodock 4.2 program. The best lowest binding energy value of −9.09 kcal/mol was selected for novel T204V. Moreover, the protein-ligand complex structures were validated by RMSD, RMSF, hydrogen bonds and salt bridge analysis. This revealed that the T204V-E1A32 kDa motif complex was the most stable among all three complex structures. This study provides information about the interaction between Hsp70 and the E1A32 kDa motif, which emphasizes future perspectives to design rational drugs and vaccines in cancer therapy.

  19. Human mitochondrial Hsp70 (mortalin): shedding light on ATPase activity, interaction with adenosine nucleotides, solution structure and domain organization.

    Science.gov (United States)

    Dores-Silva, Paulo R; Barbosa, Leandro R S; Ramos, Carlos H I; Borges, Júlio C

    2015-01-01

    The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with age-related diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor compound screenings.

  20. Human mitochondrial Hsp70 (mortalin: shedding light on ATPase activity, interaction with adenosine nucleotides, solution structure and domain organization.

    Directory of Open Access Journals (Sweden)

    Paulo R Dores-Silva

    Full Text Available The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with age-related diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor

  1. Structural Insights into the Nucleotide-Binding Domains of the P1B-type ATPases HMA6 and HMA8 from Arabidopsis thaliana

    Science.gov (United States)

    Mayerhofer, Hubert; Sautron, Emeline; Rolland, Norbert; Catty, Patrice; Seigneurin-Berny, Daphné; Pebay-Peyroula, Eva; Ravaud, Stéphanie

    2016-01-01

    Copper is a crucial ion in cells, but needs to be closely controlled due to its toxic potential and ability to catalyse the formation of radicals. In chloroplasts, an important step for the proper functioning of the photosynthetic electron transfer chain is the delivery of copper to plastocyanin in the thylakoid lumen. The main route for copper transport to the thylakoid lumen is driven by two PIB-type ATPases, Heavy Metal ATPase 6 (HMA6) and HMA8, located in the inner membrane of the chloroplast envelope and in the thylakoid membrane, respectively. Here, the crystal structures of the nucleotide binding domain of HMA6 and HMA8 from Arabidopsis thaliana are reported at 1.5Å and 1.75Å resolution, respectively, providing the first structural information on plants Cu+-ATPases. The structures reveal a compact domain, with two short helices on both sides of a twisted beta-sheet. A double mutant, aiding in the crystallization, provides a new crystal contact, but also avoids an internal clash highlighting the benefits of construct modifications. Finally, the histidine in the HP motif of the isolated domains, unable to bind ATP, shows a side chain conformation distinct from nucleotide bound structures. PMID:27802305

  2. Mutations affecting the high affinity ATPase center of gpA, the large subunit of bacteriophage lambda terminase, inactivate the endonuclease activity of terminase.

    Science.gov (United States)

    Hwang, Y; Feiss, M

    1996-08-30

    Phage lambda terminase carries out the cos cleavage reaction that generates mature chromosomes from immature concatemeric DNA. The ATP-stimulated endonuclease activity of terminase is located in gpA, the large terminase subunit. There is a high affinity ATPase center in gpA, and a match to the conserved P-loop of known ATPases is found starting near residue 490. Changing the conserved P-loop lysine at residue 497 of gpA affects the high affinity ATPase activity of terminase. In the present work, mutations causing the gpA changes K497A and K497D were found to be lethal, and phages carrying these mutations were defective in cos cleavage, in vivo. Purified K497A and K497D enzymes cleaved cos in vitro at rates reduced from the wild-type rate by factors of 1000 and 2000, respectively. The strong defects in cos cleavage are sufficient to explain the lethality of the K497A and K497D defects. In in vitro packaging studies using mature (cleaved) phage DNA, the K497A enzyme was indistinguishable from the wild-type enzyme, and the K497D enzyme showed a mild packaging defect under limiting terminase conditions. In a purified DNA packaging system, the wild-type and K497D enzymes showed similar packaging activities that were stimulated to half-maximal levels at about 3 microM ATP, indicating that the K497D change does not affect DNA translocation. In sum, the work indicates that the high affinity ATPase center of gpA is involved in stimulation of the endonuclease activity of terminase.

  3. Crystal Structure of the Cytoplasmic N-Terminal Domain of Subunit I, a Homolog of Subunit a, of V-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Srinivasan, Sankaranarayanan; Vyas, Nand K.; Baker, Matthew L.; Quiocho, Florante A. (Baylor)

    2012-02-27

    Subunit 'a' is associated with the membrane-bound (VO) complex of eukaryotic vacuolar H{sup +}-ATPase acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit 'I' homolog of subunit a. The structure is composed of a curved long central {alpha}-helix bundle capped on both ends by two lobes with similar {alpha}/{beta} architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstructions from electron microscopy of prokaryotic and eukaryotic vacuolar H{sup +}-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.

  4. Migraine- and dystonia-related disease-mutations of Na+/K+-ATPases: Relevance of behavioral studies in mice to disease symptoms and neurological manifestations in humans

    DEFF Research Database (Denmark)

    Bøttger, Pernille; Doganli, Canan; Lykke-Hartmann, Karin

    2012-01-01

    The two autosomal dominantly inherited neurological diseases: familial hemiplegic migraine type 2 (FHM2) and familial rapid-onset of dystonia-parkinsonism (Familial RDP) are caused by in vivo mutations of specific alpha subunits of the sodium–potassium pump (Na+/K+-ATPase). Intriguingly, patients...... patient symptoms and manifestations. Thus, it is interesting that mouse models targeting a specific -isoform cause different, although still comparable, phenotypes consistent with classical symptoms and other manifestations observed in FHM2 and RDP patients. This review highlights that use of mouse models...

  5. A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1

    NARCIS (Netherlands)

    Prattes, M.; Loibl, M.; Zisser, G.; Luschnig, D.; Kappel, L.; Rossler, I.; Grassegger, M.; Hromic, A.; Krieger, E.; Gruber, K.; Pertschy, B.; Bergler, H.

    2017-01-01

    AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP

  6. Tyrosine kinase domain mutations of EGFR gene in head and neck squamous cell carcinoma

    Science.gov (United States)

    Vatte, Chittibabu; Al Amri, Ali M; Cyrus, Cyril; Chathoth, Shahanas; Acharya, Sadananda; Hashim, Tariq Mohammad; Al Ali, Zhara; Alshreadah, Saleh Tawfeeq; Alsayyah, Ahmed; Al-Ali, Amein K

    2017-01-01

    Background Epidermal growth factor receptor (EGFR) is a commonly altered gene that is identified in various cancers, including head and neck squamous cell carcinoma (HNSCC). Therefore, EGFR is a promising molecular marker targeted by monoclonal antibodies and small molecule inhibitors targeting the tyrosine kinase (TK) domain. Objective The objective of this study was to investigate the spectrum of mutations in exons 18, 19, 20, and 21 of the EGFR gene in HNSCC patients. Materials and methods This retrospective study included 47 confirmed HNSCC cases. Mutations in the TK domain, exons 18, 19, 20, and 21 of the EGFR gene, were detected by Scorpion® chemistry and ARMS® technologies on Rotor-Gene Q real-time polymerase chain reaction. Results The tumors exhibited EGFR-TK domain mutations in 57% of cases. Four cases of T790M mutations were reported for the first time among HNSCC patients. Out of the total mutations, L861Q (exon 21), exon 20 insertions and deletions of exon 19 accounted for the majority of mutations (21%, 19%, and 17%, respectively). EGFR mutation status was correlated with the higher grade (P=0.026) and advanced stage (P=0.034) of HNSCC tumors. Conclusion Higher frequency of EGFR-TK domain mutations together with the presence of the T790M mutation suggests that identification of these mutations might streamline the therapy and provide a better prognosis in HNSCC cases. PMID:28352186

  7. Site-directed mutagenesis of cation coordinating residues in the gastric H,K-ATPase.

    Science.gov (United States)

    Rulli, S J; Louneva, N M; Skripnikova, E V; Rabon, E C

    2001-03-01

    Site-mutations were introduced into putative cation binding site 1 of the H,K-ATPase at glu-797, thr-825, and glu-938. The side chain oxygen of each was not essential but the mutations produced different activation and inhibition kinetics. Site mutations thr-825 (ala, leu) and glu-938 (ala, gln) modestly decreased the apparent affinity to K+, while glu-797 (gln) was equivalent to wild type. As expected of competitive inhibition, mutations of thr-825 and glu-938 that decreased the apparent affinity for K+ also increased the apparent affinity for SCH28080. This is consistent with the participation of thr-825 and glu-938 in a cation binding domain. The sidechain geometry, but not the sidechain charge of glu-797, is essential to ATPase function as the site mutant glu-797 (gly) inactivated the H,K-ATPase, while glu-797 (gln) was active but the apparent affinity to SCH 28080 was decreased by four-fold. Lys-793, a unique residue of the H,K-ATPase, was essential for ATPase function. Since this residue is adjacent to site 1, the result suggests that charge pairing between lys-793 and residues at or near this site may be essential to ATPase function.

  8. Vfa1 binds to the N-terminal microtubule-interacting and trafficking (MIT) domain of Vps4 and stimulates its ATPase activity.

    Science.gov (United States)

    Vild, Cody J; Xu, Zhaohui

    2014-04-11

    The endosomal sorting complexes required for transport (ESCRT) are responsible for multivesicular body biogenesis, membrane abscission during cytokinesis, and retroviral budding. They function as transiently assembled molecular complexes on the membrane, and their disassembly requires the action of the AAA-ATPase Vps4. Vps4 is regulated by a multitude of ESCRT and ESCRT-related proteins. Binding of these proteins to Vps4 is often mediated via the microtubule-interacting and trafficking (MIT) domain of Vps4. Recently, a new Vps4-binding protein Vfa1 was identified in a yeast genetic screen, where overexpression of Vfa1 caused defects in vacuolar morphology. However, the function of Vfa1 and its role in vacuolar biology were largely unknown. Here, we provide the first detailed biochemical and biophysical study of Vps4-Vfa1 interaction. The MIT domain of Vps4 binds to the C-terminal 17 residues of Vfa1. This interaction is of high affinity and greatly stimulates the ATPase activity of Vps4. The crystal structure of the Vps4-Vfa1 complex shows that Vfa1 adopts a canonical MIT-interacting motif 2 structure that has been observed previously in other Vps4-ESCRT interactions. These findings suggest that Vfa1 is a novel positive regulator of Vps4 function.

  9. Vfa1 Binds to the N-terminal Microtubule-interacting and Trafficking (MIT) Domain of Vps4 and Stimulates Its ATPase Activity*

    Science.gov (United States)

    Vild, Cody J.; Xu, Zhaohui

    2014-01-01

    The endosomal sorting complexes required for transport (ESCRT) are responsible for multivesicular body biogenesis, membrane abscission during cytokinesis, and retroviral budding. They function as transiently assembled molecular complexes on the membrane, and their disassembly requires the action of the AAA-ATPase Vps4. Vps4 is regulated by a multitude of ESCRT and ESCRT-related proteins. Binding of these proteins to Vps4 is often mediated via the microtubule-interacting and trafficking (MIT) domain of Vps4. Recently, a new Vps4-binding protein Vfa1 was identified in a yeast genetic screen, where overexpression of Vfa1 caused defects in vacuolar morphology. However, the function of Vfa1 and its role in vacuolar biology were largely unknown. Here, we provide the first detailed biochemical and biophysical study of Vps4-Vfa1 interaction. The MIT domain of Vps4 binds to the C-terminal 17 residues of Vfa1. This interaction is of high affinity and greatly stimulates the ATPase activity of Vps4. The crystal structure of the Vps4-Vfa1 complex shows that Vfa1 adopts a canonical MIT-interacting motif 2 structure that has been observed previously in other Vps4-ESCRT interactions. These findings suggest that Vfa1 is a novel positive regulator of Vps4 function. PMID:24567329

  10. Common pathogenic effects of missense mutations in the P-type ATPase ATP13A2 (PARK9 associated with early-onset parkinsonism.

    Directory of Open Access Journals (Sweden)

    Agata Podhajska

    Full Text Available Mutations in the ATP13A2 gene (PARK9 cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome (KRS, a neurodegenerative disease characterized by parkinsonism. KRS mutations produce truncated forms of ATP13A2 with impaired protein stability resulting in a loss-of-function. Recently, homozygous and heterozygous missense mutations in ATP13A2 have been identified in subjects with early-onset parkinsonism. The mechanism(s by which missense mutations potentially cause parkinsonism are not understood at present. Here, we demonstrate that homozygous F182L, G504R and G877R missense mutations commonly impair the protein stability of ATP13A2 leading to its enhanced degradation by the proteasome. ATP13A2 normally localizes to endosomal and lysosomal membranes in neurons and the F182L and G504R mutations disrupt this vesicular localization and promote the mislocalization of ATP13A2 to the endoplasmic reticulum. Heterozygous T12M, G533R and A746T mutations do not obviously alter protein stability or subcellular localization but instead impair the ATPase activity of microsomal ATP13A2 whereas homozygous missense mutations disrupt the microsomal localization of ATP13A2. The overexpression of ATP13A2 missense mutants in SH-SY5Y neural cells does not compromise cellular viability suggesting that these mutant proteins lack intrinsic toxicity. However, the overexpression of wild-type ATP13A2 may impair neuronal integrity as it causes a trend of reduced neurite outgrowth of primary cortical neurons, whereas the majority of disease-associated missense mutations lack this ability. Finally, ATP13A2 overexpression sensitizes cortical neurons to neurite shortening induced by exposure to cadmium or nickel ions, supporting a functional interaction between ATP13A2 and heavy metals in post-mitotic neurons, whereas missense mutations influence this sensitizing effect. Collectively, our study provides support for common loss-of-function effects of homozygous and

  11. Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage Chi.

    Science.gov (United States)

    Hwang, Y; Catalano, C E; Feiss, M

    1996-02-27

    Terminase the DNA packaging enzyme of bacteriophage chi, is a heteromultimer of gpNul (21 kDa) and gpA (74 kDa) subunits, encoded by the chi Nul and A genes, respectively. Sequence comparisons indicate that both gpNu1 and gpA have a match to the P-loop motif of ATPase centers, which is a glycine-rich segment followed by a lysine. By site-specific mutagenesis, we changed the lysines of the putative P-loops of gpNul (k35) and gpA (K497) to arginine, alanine, or aspartic acid, and studied the mutant enzymes by kinetic analysis and photochemical cross-linking with 8-azido-ATP. Both the gpNul and gpA subunits of wild-type terminase were covalently modified with 8-N3[32P] ATP in the presence of UV light. Saturation occurred with apparent dissociation constants of 508 and 3.5 microM for gpNul and gpA, resepctively. ATPase assays showed two activities: a low-affinity activity (Km=469 microM), and a high-affinity activity (Km=4.6 microM). The gpNul K35A and gpNul K35D mutant terminases showed decreased activity in the low-affinity ATPase activity. The reduced activities of these enzymes were recovered when 10 times more DNA was added, suggesting that the primary defect of the enzymes is alteration of the nonspecific, double-stranded DNA binding activity of terminase. ATPase assays and photolabeling of the gpA K497A and gpA K497D mutant terminases showed reduced affinity for ATP at the high-affinity site which was not restored by increased DNA. In summary, the results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA.

  12. Mutation in the α2 isoform of Na,K-ATPase associated Familial Hemiplegic Migraine type 2 (FHM2) leads to elevated contractility and vasodilatation of cerebral arteries in mice

    DEFF Research Database (Denmark)

    Hangaard, Lise; Lykke-Hartmann, Karin; Xie, Zijian

    ,K-ATPase mRNA in comparison with WT, but 51±11% of their mRNA contained G301R mutation. G301R mice had elevated blood pressure and unchanged heart rate. Inner diameter of cerebral arteries from G301R mice was significantly larger than in WT. G301R arteries were more sensitive and had higher maximal...

  13. Fluorescence competition assay for the assessment of ATP binding to an isolated domain of Na+, K(+)-ATPase.

    Science.gov (United States)

    Kubala, M; Plásek, J; Amler, E

    2004-01-01

    An equation allowing estimation of the dissociation constant for binding of a non-fluorescent ligand to the enzyme is presented that is based on the competitive replacement of the ligand by its fluorescent analog. We derived an explicit formula for the probe fluorescence intensity, which is suitable for nonlinear least-squares analysis. We used this formula to evaluate the binding of ATP to the large cytoplasmic loop of Na+,K(+)-ATPase. The estimated value of KD (6.2+/- 0.7 mM) is comparable with the results from other laboratories for similar constructs obtained by a different method.

  14. A single point mutation in a group I WW domain shifts its specificity to that of group II WW domains.

    Science.gov (United States)

    Espanel, X; Sudol, M

    1999-06-11

    WW domains can be divided into three groups based on their binding specificity. By random mutagenesis, we switched the specificity of the Yes-associated protein (YAP) WW1 domain, a Group I WW domain, to that of the FE65 WW domain, which belongs to Group II. We showed that a single mutation, leucine 190 (betaB5) to tryptophan, is required to switch from Group I to Group II. Although this single substitution in YAP WW1 domain is sufficient to precipitate the two protein isoforms of Mena, an in vivo ligand of FE65, we showed that an additional substitution, histidine 192 (betaB7) to glycine, significantly increased the ability of YAP to mimic FE65. This double mutant (L190W/H192G) precipitates eight of the nine protein bands that FE65 pulls down from rat brain protein lysates. Based on both our data and a sequence comparison between Group I and Group II WW domains, we propose that a block of three consecutive aromatic amino acids within the second beta-sheet of the domain is required, but not always sufficient, for a WW domain to belong to Group II. These data deepen our understanding of WW domain binding specificity and provide a basis for the rational design of modified WW domains with potential therapeutic applications.

  15. Characterization of the P4-ATPase ATP8A2: Critical Roles of Key Residues in the Fourth Transmembrane Segment in Aminophospholipid Transport

    DEFF Research Database (Denmark)

    Vestergaard, Anna Lindeløv; Coleman, Jonathan A.; Molday, Robert S.;

    intermediate at the conserved aspartate (Asp416) in the P-type ATPase signature sequence and exists in E1P and E2P forms, similar to Na+,K+-ATPase and Ca2+-ATPase. The mechanism of ATP8A2 resembles that of the well-characterized cation transporting P-type ATPases, as transported aminophospholipids activate...... the dephosphorylation directly, similar to K+ activation of dephosphorylation in Na+,K+-ATPase. By sequence alignment with well-characterized P-type ATPases, we have identified and mutated a series of strategically placed residues in the membrane domain of ATP8A2, which could be speculated to be involved...... in phospholipid binding. We have used the properties of mutant phosphoenzymes to examine the partial transport cycle reaction steps to elucidate the roles of these conserved residues, focusing on the fourth transmembrane segment M4. Here, Ile364 of ATP8A2 is a conserved hydrophobic flippase residue that aligns...

  16. Mutational analysis of the PRYSPRY domain of pyrin and implications for familial mediterranean fever (FMF).

    Science.gov (United States)

    Goulielmos, G N; Fragouli, E; Aksentijevich, I; Sidiropoulos, P; Boumpas, D T; Eliopoulos, E

    2006-07-14

    Familial Mediterranean fever (FMF) is an autosomal, recessively inherited disease, characterized by recurrent fever and serositis that affects mainly patients of the Mediterranean basin. The gene responsible for FMF, named MEFV, was cloned and several missense mutations were found to be responsible for the disease. Based on a recent molecular analysis of MEFV gene mutations in 43 patients from Crete aiming to correlate specific genotypes and clinical manifestations of FMF, we were prompted to construct a three-dimensional model (3-D model) of the PRYSPRY domain of pyrin. The majority of the known MEFV mutations located on this domain have been classified, according to disease severity, and localized on this 3-D model. The functional consequences of these mutations and their implications on disease severity are discussed. Moreover, we report a putative novel missense mutation, S702C, which we identified in exon 10 of the MEFV gene and localized on the constructed 3-D model.

  17. The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

    Directory of Open Access Journals (Sweden)

    Vitali Bialevich

    2017-01-01

    Full Text Available Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

  18. The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

    Science.gov (United States)

    Shamayeva, Katsiaryna; Guzanova, Alena; Řeha, David; Csefalvay, Eva; Carey, Jannette; Weiserova, Marie

    2017-01-01

    Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested. PMID:28133570

  19. COMP mutations: domain-dependent relationship between abnormal chondrocyte trafficking and clinical PSACH and MED phenotypes.

    Science.gov (United States)

    Chen, Tung-Ling L; Posey, Karen L; Hecht, Jacqueline T; Vertel, Barbara M

    2008-02-15

    Mutations in cartilage oligomeric matrix protein (COMP) produce clinical phenotypes ranging from the severe end of the spectrum, pseudoachondroplasia (PSACH), which is a dwarfing condition, to a mild condition, multiple epiphyseal dysplasia (MED). Patient chondrocytes have a unique morphology characterized by distended rER cisternae containing lamellar deposits of COMP and other extracellular matrix proteins. It has been difficult to determine why different mutations give rise to variable clinical phenotypes. Using our in vitro cell system, we previously demonstrated that the most common PSACH mutation, D469del, severely impedes trafficking of COMP and type IX collagen in chondrocytic cells, consistent with observations from patient cells. Here, we hypothesize that PSACH and MED mutations variably affect the cellular trafficking behavior of COMP and that the extent of defective trafficking correlates with clinical phenotype. Twelve different recombinant COMP mutations were expressed in rat chondrosarcoma cells and the percent cells with ER-retained COMP was assessed. For mutations in type 3 (T3) repeats, trafficking defects correlated with clinical phenotype; PSACH mutations had more cells retaining mutant COMP, while MED mutations had fewer. In contrast, the cellular trafficking pattern observed for mutations in the C-terminal globular domain (CTD) was not predictive of clinical phenotype. The results demonstrate that different COMP mutations in the T3 repeat domain have variable effects on intracellular transport, which correlate with clinical severity, while CTD mutations do not show such a correlation. These findings suggest that other unidentified factors contribute to the effect of the CTD mutations. J. Cell. Biochem. 103: 778-787, 2008. (c) 2007 Wiley-Liss, Inc. Copyright 2007 Wiley-Liss, Inc.

  20. The intact CFTR protein mediates ATPase rather than adenylate kinase activity.

    Science.gov (United States)

    Ramjeesingh, Mohabir; Ugwu, Francisca; Stratford, Fiona L L; Huan, Ling-Jun; Li, Canhui; Bear, Christine E

    2008-06-01

    The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

  1. Evolution of plant P-type ATPases

    Directory of Open Access Journals (Sweden)

    Christian N.S. Pedersen

    2012-02-01

    Full Text Available Five organisms having completely sequenced genomes and belonging to all major branches of green plants (Viridiplantae were analyzed with respect to their content of P-type ATPases encoding genes. These were the chlorophytes Ostreococcus tauria and Chlamydomonas reinhardtii, and the streptophytes Physcomitrella patens (a moss, Selaginella moellendorffii (a primitive vascular plant, and Arabidopsis thaliana (a model flowering plant. Each organism contained sequences for all five subfamilies of P-type ATPases. Our analysis demonstrates when specific subgroups of P-type ATPases disappeared in the evolution of Angiosperms. Na/K-pump related P2C ATPases were lost with the evolution of streptophytes whereas Na+ or K+ pumping P2D ATPases and secretory pathway Ca2+-ATPases remained until mosses. An N-terminally located calmodulin binding domain in P2B ATPases can only be detected in pumps from Streptophytae, whereas, like in animals, a C-terminally localized calmodulin binding domain might be present in chlorophyte P2B Ca2+-ATPases. Chlorophyte genomes encode P3A ATPases resembling protist plasma membrane H+-ATPases and a C-terminal regulatory domain is missing. The complete inventory of P-type ATPases in the major branches of Viridiplantae is an important starting point for elucidating the evolution in plants of these important pumps.

  2. Disease-causing mutations in the XIAP BIR2 domain impair NOD2-dependent immune signalling

    DEFF Research Database (Denmark)

    Damgaard, Rune Busk; Fiil, Berthe Katrine; Speckmann, Carsten;

    2013-01-01

    X-linked Inhibitor of Apoptosis (XIAP) is an essential ubiquitin ligase for pro-inflammatory signalling downstream of the nucleotide-binding oligomerization domain containing (NOD)-1 and -2 pattern recognition receptors. Mutations in XIAP cause X-linked lymphoproliferative syndrome type-2 (XLP2......), an immunodeficiency associated with a potentially fatal deregulation of the immune system, whose aetiology is not well understood. Here, we identify the XIAP baculovirus IAP repeat (BIR)2 domain as a hotspot for missense mutations in XLP2. We demonstrate that XLP2-BIR2 mutations severely impair NOD1/2-dependent...... immune signalling in primary cells from XLP2 patients and in reconstituted XIAP-deficient cell lines. XLP2-BIR2 mutations abolish the XIAP-RIPK2 interaction resulting in impaired ubiquitylation of RIPK2 and recruitment of linear ubiquitin chain assembly complex (LUBAC) to the NOD2-complex. We show...

  3. Kinetic characterization of the ATPase and actin-activated ATPase activities of Acanthamoeba castellanii myosin-2.

    Science.gov (United States)

    Heissler, Sarah M; Liu, Xiong; Korn, Edward D; Sellers, James R

    2013-09-13

    Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

  4. Mutation Pattern of Paired Immunoglobulin Heavy and Light Variable Domains in Chronic Lymphocytic Leukemia B Cells

    KAUST Repository

    Ghiotto, Fabio

    2011-01-01

    B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.

  5. Mutations in the consensus helicase domains of the Werner syndrome gene

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Chang-En; Oshima, Junko; Wijsman, E.M. [Univ. of Washington, Seattle, WA (United States)] [and others

    1997-02-01

    Werner syndrome (WS) is an autosomal recessive disease with a complex phenotype that is suggestive of accelerated aging. WS is caused by mutations in a gene, WRN, that encodes a predicted 1,432-amino-acid protein with homology to DNA and RNA helicases. Previous work identified four WS mutations in the 3{prime} end of the gene, which resulted in predicted truncated protein products of 1,060-1,247 amino acids but did not disrupt the helicase domain region (amino acids 569-859). Here, additional WS subjects were screened for mutations, and the intron-exon structure of the gene was determined. A total of 35 exons were defined, with the coding sequences beginning in the second exon. Five new WS mutations were identified: two nonsense mutations at codons 369 and 889; a mutation at a splice-junction site, resulting in a predicted truncated protein of 760 amino acids; a 1-bp deletion causing a frameshift; and a predicted truncated protein of 391 amino acids. Another deletion is >15 kb of genomic DNA, including exons 19-23; the predicted protein is 1,186 amino acids long. Four of these new mutations either partially disrupt the helicase domain region or result in predicted protein products completely missing the helicase region. These results confirm that mutations in the WRN gene are responsible for WS. Also, the location of the mutations indicates that the presence or absence of the helicase domain does not influence the WS phenotype and suggests that WS is the result of complete loss of function of the WRN gene product. 63 refs., 1 fig., 5 tabs.

  6. Molecular mechanism of Na+,K+-ATPase malfunction in mutations characteristic of adrenal hypertension

    DEFF Research Database (Denmark)

    Kopec, Wojciech; Loubet, Bastien; Poulsen, Hanne

    2014-01-01

    the causes of hypertension. We used molecular dynamics simulations to investigate the structural consequences of these mutations, namely, Leu97 substitution by Arg (L97R), Val325 substitution by Gly (V325G), deletion of residues 93-97 (Del93-97), and deletion-substitution of residues 953-956 by Ser (EETA956S...... the sodium leak measured with the mutant. The last mutant, EETA956S, opens an additional water pathway near the C-terminus, affecting the III sodium-specific binding site. The results are in excellent agreement with recent electrophysiology measurements and suggest how three mutations prevent the occlusion...

  7. Dynamin GTPase Regulation is Altered by PH Domain Mutations Found in Centronuclear Myopathy Patients

    Energy Technology Data Exchange (ETDEWEB)

    Kenniston, J.; Lemmon, M

    2010-01-01

    The large GTPase dynamin has an important membrane scission function in receptor-mediated endocytosis and other cellular processes. Self-assembly on phosphoinositide-containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin-homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C-terminal {alpha}-helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either 'sensitize' dynamin to lipid stimulation or elevate basal GTPase rates by promoting self-assembly and thus rendering dynamin no longer lipid responsive. We also describe a low-resolution structure of dimeric dynamin from small-angle X-ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self-assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.

  8. Mutations in the 1A domain of keratin 9 in patients with epidermolytic palmoplantar keratoderma.

    Science.gov (United States)

    Rothnagel, J A; Wojcik, S; Liefer, K M; Dominey, A M; Huber, M; Hohl, D; Roop, D R

    1995-03-01

    Epidermolytic palmoplantar keratoderma is an autosomal dominant skin disorder characterized by hyperkeratosis of the palms and soles. Ultrastructurally the disease exhibits abnormal keratin filament networks and tonofilament clumping like that found in the keratin disorders of epidermolysis bullosa simplex and epidermolytic hyperkeratosis. The disease has been mapped to chromosome 17q11-q23 in the region of the type 1 keratin gene locus and more recently mutations have been found in the palmoplantar specific keratin, keratin 9. We have analyzed six unrelated incidences of epidermolytic palmoplantar keratoderma for mutations in their keratin 9 genes. In two of these, we have identified mutations that alter critical residues within the highly conserved helix initiation motif at the beginning of the rod domain of keratin 9. In a three-generation Middle Eastern kindred we found a C to T transition at codon 162 that results in an arginine to tryptophan substitution at position 10 of the 1A alpha-helical domain, thus confirming this codon as a hot spot for mutation in keratin 9. The other mutation found involves a T to C transition at codon 167 that results in the expression of a serine residue in place of the normal leucine at position 15 of the 1A segment and is the first documentation of this mutation in this gene. The identification of these substitutions extends the current catalog of disease causing mutations in keratin 9.

  9. Tyrosine kinase domain mutations of EGFR gene in head and neck squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Vatte C

    2017-03-01

    Full Text Available Chittibabu Vatte,1 Ali M Al Amri,2 Cyril Cyrus,1 Shahanas Chathoth,1 Sadananda Acharya,3 Tariq Mohammad Hashim,4 Zhara Al Ali,2 Saleh Tawfeeq Alshreadah,2 Ahmed Alsayyah,4 Amein K Al-Ali5 1Department of Genetic Research, Institute for Research and Medical Consultation, University of Dammam, Dammam, 2Department of Internal Medicine, King Fahd Hospital of the University, University of Dammam, Al-Khobar, 3Department of Stemcell Research, Institute for Research and Medical Consultation, 4Department of Pathology, King Fahd Hospital of the University, University of Dammam, Al-Khobar, 5Department of Biochemistry, College of Medicine, University of Dammam, Dammam, Kingdom of Saudi Arabia Background: Epidermal growth factor receptor (EGFR is a commonly altered gene that is identified in various cancers, including head and neck squamous cell carcinoma (HNSCC. Therefore, EGFR is a promising molecular marker targeted by monoclonal antibodies and small molecule inhibitors targeting the tyrosine kinase (TK domain. Objective: The objective of this study was to investigate the spectrum of mutations in exons 18, 19, 20, and 21 of the EGFR gene in HNSCC patients. Materials and methods: This retrospective study included 47 confirmed HNSCC cases. Mutations in the TK domain, exons 18, 19, 20, and 21 of the EGFR gene, were detected by Scorpion® chemistry and ARMS® technologies on Rotor-Gene Q real-time polymerase chain reaction.Results: The tumors exhibited EGFR-TK domain mutations in 57% of cases. Four cases of T790M mutations were reported for the first time among HNSCC patients. Out of the total mutations, L861Q (exon 21, exon 20 insertions and deletions of exon 19 accounted for the majority of mutations (21%, 19%, and 17%, respectively. EGFR mutation status was correlated with the higher grade (P=0.026 and advanced stage (P=0.034 of HNSCC tumors.Conclusion: Higher frequency of EGFR-TK domain mutations together with the presence of the T790M mutation suggests

  10. A Point Mutation in a Domain of Gamma Interferon Receptor 1 Provokes Severe Immunodeficiency

    Science.gov (United States)

    Allende, Luis M.; López-Goyanes, Alberto; Paz-Artal, Estela; Corell, Alfredo; García-Pérez, M. Angel; Varela, Pilar; Scarpellini, Atilio; Negreira, Sagrario; Palenque, Elia; Arnaiz-Villena, Antonio

    2001-01-01

    Gamma interferon (IFN-γ) and the cellular responses induced by it are essential for controlling mycobacterial infections. Most patients bearing an IFN-γ receptor ligand-binding chain (IFN-γR1) deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome. In this report a new mutation that affects the IFN-γR1 ligand-binding domain in a Spanish patient with mycobacterial disseminated infection and multifocal osteomyelitis is characterized. The mutation generates an amino acid change that does not abrogate protein expression on the cellular surface but that severely impairs responses after the binding of IFN-γ (CD64 and HLA class II induction and tumor necrosis factor alpha and interleukin-12 production). A patient's younger brother, who was also probably homozygous for the mutation, died from meningitis due to Mycobacterium bovis. These findings suggest that a point mutation may be fatal when it affects functionally important domains of the receptor and that the severity is not directly related to a lack of IFN-γ receptor expression. Future research on these nontruncating mutations will make it possible to develop new therapeutical alternatives in this group of patients. PMID:11139207

  11. A point mutation in a domain of gamma interferon receptor 1 provokes severe immunodeficiency.

    Science.gov (United States)

    Allende, L M; López-Goyanes, A; Paz-Artal, E; Corell, A; García-Pérez, M A; Varela, P; Scarpellini, A; Negreira, S; Palenque, E; Arnaiz-Villena, A

    2001-01-01

    Gamma interferon (IFN-gamma) and the cellular responses induced by it are essential for controlling mycobacterial infections. Most patients bearing an IFN-gamma receptor ligand-binding chain (IFN-gammaR1) deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome. In this report a new mutation that affects the IFN-gammaR1 ligand-binding domain in a Spanish patient with mycobacterial disseminated infection and multifocal osteomyelitis is characterized. The mutation generates an amino acid change that does not abrogate protein expression on the cellular surface but that severely impairs responses after the binding of IFN-gamma (CD64 and HLA class II induction and tumor necrosis factor alpha and interleukin-12 production). A patient's younger brother, who was also probably homozygous for the mutation, died from meningitis due to Mycobacterium bovis. These findings suggest that a point mutation may be fatal when it affects functionally important domains of the receptor and that the severity is not directly related to a lack of IFN-gamma receptor expression. Future research on these nontruncating mutations will make it possible to develop new therapeutical alternatives in this group of patients.

  12. The Ubiquitin Regulatory X (UBX) Domain-containing Protein TUG Regulates the p97 ATPase and Resides at the Endoplasmic Reticulum-Golgi Intermediate Compartment*

    Science.gov (United States)

    Orme, Charisse M.; Bogan, Jonathan S.

    2012-01-01

    p97/VCP is a hexameric ATPase that is coupled to diverse cellular processes, such as membrane fusion and proteolysis. How p97 activity is regulated is not fully understood. Here we studied the potential role of TUG, a widely expressed protein containing a UBX domain, to control mammalian p97. In HEK293 cells, the vast majority of TUG was bound to p97. Surprisingly, the TUG UBX domain was neither necessary nor sufficient for this interaction. Rather, an extended sequence, comprising three regions of TUG, bound to the p97 N-terminal domain. The TUG C terminus resembled the Arabidopsis protein PUX1. Similar to the previously described action of PUX1 on AtCDC48, TUG caused the conversion of p97 hexamers into monomers. Hexamer disassembly was stoichiometric rather than catalytic and was not greatly affected by the p97 ATP-binding state or by TUG N-terminal regions in vitro. In HeLa cells, TUG localized to the endoplasmic reticulum-to-Golgi intermediate compartment and endoplasmic reticulum exit sites. Although siRNA-mediated TUG depletion had no marked effect on total ubiquitylated proteins or p97 localization, TUG overexpression caused an accumulation of ubiquitylated substrates and targeted both TUG and p97 to the nucleus. A physiologic role of TUG was revealed by siRNA-mediated depletion, which showed that TUG is required for efficient reassembly of the Golgi complex after brefeldin A removal. Together, these data support a model in which TUG controls p97 oligomeric status at a particular location in the early secretory pathway and in which this process regulates membrane trafficking in various cell types. PMID:22207755

  13. Meier-Gorlin syndrome mutations disrupt an Orc1 CDK inhibitory domain and cause centrosome reduplication.

    Science.gov (United States)

    Hossain, Manzar; Stillman, Bruce

    2012-08-15

    Like DNA replication, centrosomes are licensed to duplicate once per cell division cycle to ensure genetic stability. In addition to regulating DNA replication, the Orc1 subunit of the human origin recognition complex controls centriole and centrosome copy number. Here we report that Orc1 harbors a PACT centrosome-targeting domain and a separate domain that differentially inhibits the protein kinase activities of Cyclin E-CDK2 and Cyclin A-CDK2. A cyclin-binding motif (Cy motif) is required for Orc1 to bind Cyclin A and inhibit Cyclin A-CDK2 kinase activity but has no effect on Cyclin E-CDK2 kinase activity. In contrast, Orc1 inhibition of Cyclin E-CDK2 kinase activity occurs by a different mechanism that is affected by Orc1 mutations identified in Meier-Gorlin syndrome patients. The cyclin/CDK2 kinase inhibitory domain of Orc1, when tethered to the PACT domain, localizes to centrosomes and blocks centrosome reduplication. Meier-Gorlin syndrome mutations that disrupt Cyclin E-CDK2 kinase inhibition also allow centrosome reduplication. Thus, Orc1 contains distinct domains that control centrosome copy number and DNA replication. We suggest that the Orc1 mutations present in some Meier-Gorlin syndrome patients contribute to the pronounced microcephaly and dwarfism observed in these individuals by altering centrosome duplication in addition to DNA replication defects.

  14. Comparable rate of EGFR kinase domain mutation in lung adenocarcinomas from Chinese male and female never-smokers

    Institute of Scientific and Technical Information of China (English)

    Yi-hua SUN; Rong FANG; Bin GAO; Xiang-kun HAN; Jun-hua ZHANG; William PAO; Hai-quan CHEN; Hong-bin JI

    2010-01-01

    @@ Lung cancer patients with the epidermal growth factor receptor (EGFR) kinase domain mutations frequently show good responses to small molecule tyrosine kinase inhibitors, including Iressa and Tarceva, in clinical studies[1-3]. Previous studies have demonstrated that EGFR kinase domain mutations are commonly observed in lung adenocarcinomas, never-smokers,East Asian, and females[4-8].

  15. Discovery of a novel azaindole class of antibacterial agents targeting the ATPase domains of DNA gyrase and Topoisomerase IV.

    Science.gov (United States)

    Manchester, John I; Dussault, Daemian D; Rose, Jonathan A; Boriack-Sjodin, P Ann; Uria-Nickelsen, Maria; Ioannidis, Georgine; Bist, Shanta; Fleming, Paul; Hull, Kenneth G

    2012-08-01

    We present the discovery and optimization of a novel series of bacterial topoisomerase inhibitors. Starting from a virtual screening hit, activity was optimized through a combination of structure-based design and physical property optimization. Synthesis of fewer than a dozen compounds was required to achieve inhibition of the growth of methicillin-resistant Staphyloccus aureus (MRSA) at compound concentrations of 1.56 μM. These compounds simultaneously inhibit DNA gyrase and Topoisomerase IV at similar nanomolar concentrations, reducing the likelihood of the spontaneous occurrence of target-based mutations resulting in antibiotic resistance, an increasing threat in the treatment of serious infections.

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

    Science.gov (United States)

    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.

  17. The external domains of the HIV-1 envelope are a mutational cold spot

    Science.gov (United States)

    Geller, Ron; Domingo-Calap, Pilar; Cuevas, José M.; Rossolillo, Paola; Negroni, Matteo; Sanjuán, Rafael

    2015-01-01

    In RNA viruses, mutations occur fast and have large fitness effects. While this affords remarkable adaptability, it can also endanger viral survival due to the accumulation of deleterious mutations. How RNA viruses reconcile these two opposed facets of mutation is still unknown. Here we show that, in human immunodeficiency virus (HIV-1), spontaneous mutations are not randomly located along the viral genome. We find that the viral mutation rate experiences a threefold reduction in the region encoding the most external domains of the viral envelope, which are strongly targeted by neutralizing antibodies. This contrasts with the hypermutation mechanisms deployed by other, more slowly mutating pathogens such as DNA viruses and bacteria, in response to immune pressure. We show that downregulation of the mutation rate in HIV-1 is exerted by the template RNA through changes in sequence context and secondary structure, which control the activity of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (A3)-mediated cytidine deamination and the fidelity of the viral reverse transcriptase. PMID:26450412

  18. The external domains of the HIV-1 envelope are a mutational cold spot.

    Science.gov (United States)

    Geller, Ron; Domingo-Calap, Pilar; Cuevas, José M; Rossolillo, Paola; Negroni, Matteo; Sanjuán, Rafael

    2015-10-09

    In RNA viruses, mutations occur fast and have large fitness effects. While this affords remarkable adaptability, it can also endanger viral survival due to the accumulation of deleterious mutations. How RNA viruses reconcile these two opposed facets of mutation is still unknown. Here we show that, in human immunodeficiency virus (HIV-1), spontaneous mutations are not randomly located along the viral genome. We find that the viral mutation rate experiences a threefold reduction in the region encoding the most external domains of the viral envelope, which are strongly targeted by neutralizing antibodies. This contrasts with the hypermutation mechanisms deployed by other, more slowly mutating pathogens such as DNA viruses and bacteria, in response to immune pressure. We show that downregulation of the mutation rate in HIV-1 is exerted by the template RNA through changes in sequence context and secondary structure, which control the activity of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (A3)-mediated cytidine deamination and the fidelity of the viral reverse transcriptase.

  19. The helicase and ATPase activities of RECQL4 are compromised by mutations reported in three human patients

    DEFF Research Database (Denmark)

    Jensen, Martin Borch; Dunn, Christopher A; Keijzers, Guido

    2012-01-01

    RECQL4 is one of five members of the human RecQ helicase family, and is implicated in three syndromes displaying accelerating aging, developmental abnormalities and a predisposition to cancer. In this study, we purified three variants of RECQL4 carrying previously reported patient mutations....... These three mutant proteins were analyzed for the known biochemical activities of RECQL4: DNA binding, unwinding of duplex DNA, ATP hydrolysis and annealing of simplex DNA. Further, the mutant proteins were evaluated for stability and recruitment to sites of laser-induced DNA damage. One mutant was helicase...... a consistent pattern of functional deficiency and provide further support for a helicase-dependent cellular function of RECQL4 in addition to its N-terminus-dependent role in initiation of replication, a function that may underlie the phenotype of RECQL4-linked disease....

  20. Point mutations in firefly luciferase C-domain demonstrate its significance in green color of bioluminescence.

    Science.gov (United States)

    Modestova, Yulia; Koksharov, Mikhail I; Ugarova, Natalia N

    2014-09-01

    Firefly luciferase is a two-domain enzyme that catalyzes the bioluminescent reaction of firefly luciferin oxidation. Color of the emitted light depends on the structure of the enzyme, yet the exact color-tuning mechanism remains unknown by now, and the role of the C-domain in it is rarely discussed, because a very few color-shifting mutations in the C-domain were described. Recently we reported a strong red-shifting mutation E457K in the C-domain; the bioluminescence spectra of this enzyme were independent of temperature or pH. In the present study we investigated the role of the residue E457 in the enzyme using the Luciola mingrelica luciferase with a thermostabilized N-domain as a parent enzyme for site-directed mutagenesis. We obtained a set of mutants and studied their catalytic properties, thermal stability and bioluminescence spectra. Experimental spectra were represented as a sum of two components (bioluminescence spectra of putative "red" and "green" emitters); λmax of these components were constant for all the mutants, but the ratio of these emitters was defined by temperature and mutations in the C-domain. We suggest that each emitter is stabilized by a specific conformation of the active site; thus, enzymes with two forms of the active site coexist in the reactive media. The rigid structure of the C-domain is crucial for maintaining the conformation corresponding to the "green" emitter. We presume that the emitters are the keto- and enol forms of oxyluciferin.

  1. Disease Mutations in the Ryanodine Receptor Central Region: Crystal Structures of a Phosphorylation Hot Spot Domain

    Energy Technology Data Exchange (ETDEWEB)

    Yuchi, Zhiguang; Lau, Kelvin; Van Petegem, Filip (UBC)

    2015-02-09

    Ryanodine Receptors (RyRs) are huge Ca{sup 2+} release channels in the endoplasmic reticulum membrane and form targets for phosphorylation and disease mutations. We present crystal structures of a domain in three RyR isoforms, containing the Ser2843 (RyR1) and Ser2808/Ser2814 (RyR2) phosphorylation sites. The RyR1 domain is the target for 11 disease mutations. Several of these are clustered near the phosphorylation sites, suggesting that phosphorylation and disease mutations may affect the same interface. The L2867G mutation causes a drastic thermal destabilization and aggregation at room temperature. Crystal structures for other disease mutants show that they affect surface properties and intradomain salt bridges. In vitro phosphorylation experiments show that up to five residues in one long loop of RyR2 can be phosphorylated by PKA or CaMKII. Docking into cryo-electron microscopy maps suggests a putative location in the clamp region, implying that mutations and phosphorylation may affect the allosteric motions within this area.

  2. Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain.

    Directory of Open Access Journals (Sweden)

    Jeffrey C Lee

    2006-12-01

    Full Text Available BACKGROUND: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. METHODS AND FINDINGS: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132 of glioblastomas and 12.5% (1/8 of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. CONCLUSIONS: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

  3. Analysis of a mutation affecting the specificity domain for prohead binding of the bacteriophage lambda terminase.

    OpenAIRE

    1992-01-01

    Genetic studies have identified a specificity domain for prohead binding in the C-terminal 32 amino acids of gpA, the large subunit of bacteriophage lambda terminase (S. Frackman, D. A. Siegele, and M. Feiss, J. Mol. Biol. 180:283-300, 1984). In the present work, an amber mutation, Aam42, in the fifth-to-last codon of the A gene was found to be lethal in nonsuppressing hosts. The mutation, expected to generate gpA lacking the last five amino acids, caused the production of a terminase that cu...

  4. Crystal structure of the full-length ATPase GspE from the Vibrio vulnificus type II secretion system in complex with the cytoplasmic domain of GspL.

    Science.gov (United States)

    Lu, Connie; Korotkov, Konstantin V; Hol, Wim G J

    2014-09-01

    The type II secretion system (T2SS) is present in many Gram-negative bacteria and is responsible for secreting a large number of folded proteins, including major virulence factors, across the outer membrane. The T2SS consists of 11-15 different proteins most of which are present in multiple copies in the assembled secretion machinery. The ATPase GspE, essential for the functioning of the T2SS, contains three domains (N1E, N2E and CTE) of which the N1E domain is associated with the cytoplasmic domain of the inner membrane protein GspL. Here we describe and analyze the structure of the GspE•cyto-GspL complex from Vibrio vulnificus in the presence of an ATP analog, AMPPNP. There are three such ∼83 kDa complexes per asymmetric unit with essentially the same structure. The N2E and CTE domains of a single V. vulnificus GspE subunit adopt a mutual orientation that has not been seen before in any of the previous GspE structures, neither in structures of related ATPases from other secretion systems. This underlines the tremendous conformational flexibility of the T2SS secretion ATPase. Cyto-GspL interacts not only with the N1E domain, but also with the CTE domain and is even in contact with AMPPNP. Moreover, the cyto-GspL domains engage in two types of mutual interactions, resulting in two essentially identical, but crystallographically independent, "cyto-GspL rods" that run throughout the crystal. Very similar rods are present in previous crystals of cyto-GspL and of the N1E•cyto-GspL complex. This arrangement, now seen four times in three entirely different crystal forms, involves contacts between highly conserved residues suggesting a role in the biogenesis or the secretion mechanism or both of the T2SS. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Novel Mutations in the Transcriptional Activator Domain of the Human TBX20 in Patients with Atrial Septal Defect

    Directory of Open Access Journals (Sweden)

    Irma Eloisa Monroy-Muñoz

    2015-01-01

    Full Text Available 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.

  6. Rotary ATPases

    Science.gov (United States)

    Stewart, Alastair G.; Sobti, Meghna; Harvey, Richard P.; Stock, Daniela

    2013-01-01

    Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual “machine elements” to the requirement of the right “fuel” and “oil” for different types of motors. PMID:23369889

  7. MLLT1 YEATS domain mutations in clinically distinctive Favourable Histology Wilms tumours | Office of Cancer Genomics

    Science.gov (United States)

    Wilms tumour is an embryonal tumour of childhood that closely resembles the developing kidney. Genomic changes responsible for the development of the majority of Wilms tumours remain largely unknown. Here we identify recurrent mutations within Wilms tumours that involve the highly conserved YEATS domain of MLLT1 (ENL), a gene known to be involved in transcriptional elongation during early development. The mutant MLLT1 protein shows altered binding to acetylated histone tails.

  8. A comprehensive computational study on pathogenic mis-sense mutations spanning the RING2 and REP domains of Parkin protein.

    Science.gov (United States)

    Biswas, Ria; Bagchi, Angshuman

    2017-04-30

    Various mutations in PARK2 gene, which encodes the protein parkin, are significantly associated with the onset of autosomal recessive juvenile Parkinson (ARJP) in neuronal cells. Parkin is a multi domain protein, the N-terminal part contains the Ubl and the C-terminal part consists of four zinc coordinating domains, viz., RING0, RING1, in between ring (IBR) and RING2. Disease mutations are spread over all the domains of Parkin, although mutations in some regions may affect the functionality of Parkin more adversely. The mutations in the RING2 domain are seen to abolish the neuroprotective E3 ligase activity of Parkin. In this current work, we carried out detailed in silico analysis to study the extent of pathogenicity of mutations spanning the Parkin RING2 domain and the adjoining REP region by SIFT, Mutation Accessor, PolyPhen2, SNPs and GO, GV/GD and I-mutant. To study the structural and functional implications of these mutations on RING2-REP domain of Parkin, we studied the solvent accessibility (SASA/RSA), hydrophobicity, intra-molecular hydrogen bonding profile and domain analysis by various computational tools. Finally, we analysed the interaction energy profiles of the mutants and compared them to the wild type protein using Discovery studio 2.5. By comparing the various analyses it could be safely concluded that except P437L and A379V mutations, all other mutations were potentially deleterious affecting various structural aspects of RING2 domain architecture. This study is based purely on computational approach which has the potential to identify disease mutations and the information could further be used in treatment of diseases and prognosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Structural and mutational analysis of the nonribosomal peptide synthetase heterocyclization domain provides insight into catalysis.

    Science.gov (United States)

    Bloudoff, Kristjan; Fage, Christopher D; Marahiel, Mohamed A; Schmeing, T Martin

    2017-01-03

    Nonribosomal peptide synthetases (NRPSs) are a family of multidomain, multimodule enzymes that synthesize structurally and functionally diverse peptides, many of which are of great therapeutic or commercial value. The central chemical step of peptide synthesis is amide bond formation, which is typically catalyzed by the condensation (C) domain. In many NRPS modules, the C domain is replaced by the heterocyclization (Cy) domain, a homologous domain that performs two consecutive reactions by using hitherto unknown catalytic mechanisms. It first catalyzes amide bond formation, and then the intramolecular cyclodehydration between a Cys, Ser, or Thr side chain and the backbone carbonyl carbon to form a thiazoline, oxazoline, or methyloxazoline ring. The rings are important for the form and function of the peptide product. We present the crystal structure of an NRPS Cy domain, Cy2 of bacillamide synthetase, at a resolution of 2.3 Å. Despite sharing the same fold, the active sites of C and Cy domains have important differences. The structure allowed us to probe the roles of active-site residues by using mutational analyses in a peptide synthesis assay with intact bacillamide synthetase. The drastically different effects of these mutants, interpreted by using our structural and bioinformatic results, provide insight into the catalytic mechanisms of the Cy domain and implicate a previously unexamined Asp-Thr dyad in catalysis of the cyclodehydration reaction.

  10. Crystal structure of the yeast TSC1 core domain and implications for tuberous sclerosis pathological mutations.

    Science.gov (United States)

    Sun, Wei; Zhu, Ye Julia; Wang, Zhizhi; Zhong, Qiang; Gao, Feng; Lou, Jizhong; Gong, Weimin; Xu, Wenqing

    2013-01-01

    Tuberous sclerosis complex is a disease caused by mutations in two tumor-suppressor genes, TSC1 and TSC2. The TSC1 protein, also known as hamartin, has a critical role in controlling mTOR signalling. TSC1 does not bear apparent sequence homology with other proteins. Here we show that the N-terminal half of yeast TSC1 forms a protease-resistant domain, which is evolutionarily conserved. The crystal structure of this yeast TSC1 core domain shows that it contains a pseudo-HEAT repeat fold with its C-terminal end capped by a helical subdomain. This allows us to model the three-dimensional structure of the human TSC1 N-terminal domain (TSC1-NTD), which anchors essentially all pathogenic TSC1 missense mutations found in tuberous sclerosis patients. Interestingly, most pathogenic mutations map inside of the folded TSC1-NTD structure, whereas most non-pathogenic variants are on the structural surface. This indicates that the disruption of the TSC1-NTD globular structure is a major cause of tuberous sclerosis.

  11. Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum

    Science.gov (United States)

    Ferguson, Scott A.; Cook, Gregory M.; Montgomery, Martin G.; Leslie, Andrew G. W.

    2016-01-01

    The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a “down” state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an “up” state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme’s hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual “open” conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis. PMID:27621435

  12. Coupled ATPase-adenylate kinase activity in ABC transporters

    Science.gov (United States)

    Kaur, Hundeep; Lakatos-Karoly, Andrea; Vogel, Ramona; Nöll, Anne; Tampé, Robert; Glaubitz, Clemens

    2016-01-01

    ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters. PMID:28004795

  13. A mutational analysis of the endophilin-A N-BAR domain performed in living flies.

    Directory of Open Access Journals (Sweden)

    Anita G Jung

    Full Text Available BACKGROUND: Endophilin is a cytoplasmic protein with an important function in clathrin-dependent endocytosis at synapses and elsewhere. Endophilin has a BAR (Bin/Amphiphysin/Rvs-homology domain, which is implicated in the sensing and induction of membrane curvature. Previous structure-function studies of the endophilin-A BAR domain have almost exclusively been made in reduced systems, either in vitro or ex vivo in cultured cells. To extend and complement this work, we have analyzed the role played by the structural features of the endophilin-A BAR domain in Drosophila in vivo. METHODOLOGY/PRINCIPAL FINDINGS: The study is based on genetic rescue of endophilin-A (endoA null mutants with wild type or mutated endoA transgenes. We evaluated the viability of the rescuants, the locomotor behavior in adult flies and the neurotransmission at the larval neuromuscular junction. Whereas mutating the endophilin BAR domain clearly affected adult flies, larval endophilin function was surprisingly resistant to mutagenesis. Previous reports have stressed the importance of a central appendage on the convex BAR surface, which forms a hydrophobic ridge able to directly insert into the lipid bilayer. We found that the charge-negative substitution A66D, which targets the hydrophobic ridge and was reported to completely disrupt the ability of endophilin-BAR to tubulate liposomes in vitro, rescued viability and neurotransmission with the same efficiency as wild type endoA transgenes, even in adults. A similar discrepancy was found for the hydrophilic substitutions A63S/A66S and A63S/A66S/M70Q. The A66W mutation, which introduces a bulky hydrophobic side chain and induces massive vesiculation of liposomes in vitro, strongly impeded eye development, even in presence of the endogenous endoA gene. Substantial residual function was observed in larvae rescued with the EndoA(Arf transgene, which encodes a form of endophilin-A that completely lacks the central appendage

  14. A mutational analysis of the endophilin-A N-BAR domain performed in living flies.

    Science.gov (United States)

    Jung, Anita G; Labarrera, Christina; Labarerra, Christina; Jansen, Anna M; Qvortrup, Klaus; Wild, Klemens; Kjaerulff, Ole

    2010-03-03

    Endophilin is a cytoplasmic protein with an important function in clathrin-dependent endocytosis at synapses and elsewhere. Endophilin has a BAR (Bin/Amphiphysin/Rvs-homology) domain, which is implicated in the sensing and induction of membrane curvature. Previous structure-function studies of the endophilin-A BAR domain have almost exclusively been made in reduced systems, either in vitro or ex vivo in cultured cells. To extend and complement this work, we have analyzed the role played by the structural features of the endophilin-A BAR domain in Drosophila in vivo. The study is based on genetic rescue of endophilin-A (endoA) null mutants with wild type or mutated endoA transgenes. We evaluated the viability of the rescuants, the locomotor behavior in adult flies and the neurotransmission at the larval neuromuscular junction. Whereas mutating the endophilin BAR domain clearly affected adult flies, larval endophilin function was surprisingly resistant to mutagenesis. Previous reports have stressed the importance of a central appendage on the convex BAR surface, which forms a hydrophobic ridge able to directly insert into the lipid bilayer. We found that the charge-negative substitution A66D, which targets the hydrophobic ridge and was reported to completely disrupt the ability of endophilin-BAR to tubulate liposomes in vitro, rescued viability and neurotransmission with the same efficiency as wild type endoA transgenes, even in adults. A similar discrepancy was found for the hydrophilic substitutions A63S/A66S and A63S/A66S/M70Q. The A66W mutation, which introduces a bulky hydrophobic side chain and induces massive vesiculation of liposomes in vitro, strongly impeded eye development, even in presence of the endogenous endoA gene. Substantial residual function was observed in larvae rescued with the EndoA(Arf) transgene, which encodes a form of endophilin-A that completely lacks the central appendage. Whereas a mutation (D151P) designed to increase the BAR curvature

  15. The subunit delta-subunit b domain of the Escherichia coli F1F0 ATPase. The B subunits interact with F1 as a dimer and through the delta subunit.

    Science.gov (United States)

    Rodgers, A J; Wilkens, S; Aggeler, R; Morris, M B; Howitt, S M; Capaldi, R A

    1997-12-05

    The delta and b subunits are both involved in binding the F1 to the F0 part in the Escherichia coli ATP synthase (ECF1F0). The interaction of the purified delta subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified delta binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1, i.e. in the presence of ECF1-delta, delta, and bsol interact strongly, and a complex of ECF1.bsol can be isolated by native gel electrophoresis. Both delta subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the delta subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of delta as delta(1-134) cannot replace intact delta in the binding experiments. As purified, bsol is a stable dimer with 80% alpha helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem. 271, 7038-7042). Monomeric bsol has less alpha helix, i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to delta in ECF1. To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124-131 likely forming a pair of parallel alpha helices.

  16. MECP2 missense mutations outside the canonical MBD and TRD domains in males with intellectual disability

    Science.gov (United States)

    Failla, Pinella; Di Marco, Chiara; Grozeva, Detelina; Mencarelli, Maria Antonietta; Spiga, Ottavia; Mari, Francesca; Meloni, Ilaria; Raymond, Lucy; Renieri, Alessandra; Romano, Corrado; Ariani, Francesca

    2015-01-01

    Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein highly expressed in neurons that is involved in transcriptional modulation and chromatin remodeling. Mutations in MECP2 in females are associated with Rett syndrome, a neurological disorder characterized by a normal neonatal period, followed by the arrest of development and regression of acquired skills. Although it was initially thought that MECP2 pathogenic mutations in males were not compatible with life, starting from 1999 about 60 male patients have been identified and their phenotype varies from severe neonatal encephalopathy to mild intellectual disability. Targeted Next Generation Sequencing of a panel of intellectual disability related genes was performed on two unrelated male patients, and two missense variants in MECP2 were identified (p.Gly185Val and p.Arg167Trp). These variants lie outside the canonical MBD and TRD domains, where the pathogenicity of missense variants is more difficult to establish. In both families, variants were found in all affected siblings and were inherited from the asymptomatic mother, showing skewed X-chromosome inactivation. We report here the first missense variant located in AT-hook domain 1 and we underline the importance of MECP2 substitutions outside the canonical MeCP2 domains in X-linked intellectual disability. PMID:26490184

  17. Correlation analysis for protein evolutionary family based on amino acid position mutations and application in PDZ domain.

    Directory of Open Access Journals (Sweden)

    Qi-Shi Du

    Full Text Available BACKGROUND: It has been widely recognized that the mutations at specific directions are caused by the functional constraints in protein family and the directional mutations at certain positions control the evolutionary direction of the protein family. The mutations at different positions, even distantly separated, are mutually coupled and form an evolutionary network. Finding the controlling mutative positions and the mutative network among residues are firstly important for protein rational design and enzyme engineering. METHODOLOGY: A computational approach, namely amino acid position conservation-mutation correlation analysis (CMCA, is developed to predict mutually mutative positions and find the evolutionary network in protein family. The amino acid position mutative function, which is the foundational equation of CMCA measuring the mutation of a residue at a position, is derived from the MSA (multiple structure alignment database of protein evolutionary family. Then the position conservation correlation matrix and position mutation correlation matrix is constructed from the amino acid position mutative equation. Unlike traditional SCA (statistical coupling analysis approach, which is based on the statistical analysis of position conservations, the CMCA focuses on the correlation analysis of position mutations. CONCLUSIONS: As an example the CMCA approach is used to study the PDZ domain of protein family, and the results well illustrate the distantly allosteric mechanism in PDZ protein family, and find the functional mutative network among residues. We expect that the CMCA approach may find applications in protein engineering study, and suggest new strategy to improve bioactivities and physicochemical properties of enzymes.

  18. MLLT1 YEATS domain mutations in clinically distinctive Favourable Histology Wilms tumours

    KAUST Repository

    Perlman, Elizabeth J.

    2015-12-04

    Wilms tumour is an embryonal tumour of childhood that closely resembles the developing kidney. Genomic changes responsible for the development of the majority of Wilms tumours remain largely unknown. Here we identify recurrent mutations within Wilms tumours that involve the highly conserved YEATS domain of MLLT1 (ENL), a gene known to be involved in transcriptional elongation during early development. The mutant MLLT1 protein shows altered binding to acetylated histone tails. Moreover, MLLT1-mutant tumours show an increase in MYC gene expression and HOX dysregulation. Patients with MLLT1-mutant tumours present at a younger age and have a high prevalence of precursor intralobar nephrogenic rests. These data support a model whereby activating MLLT1 mutations early in renal development result in the development of Wilms tumour.

  19. SMC6 is an essential gene in mice, but a hypomorphic mutant in the ATPase domain has a mild phenotype with a range of subtle abnormalities.

    Science.gov (United States)

    Ju, Limei; Wing, Jonathan; Taylor, Elaine; Brandt, Renata; Slijepcevic, Predrag; Horsch, Marion; Rathkolb, Birgit; Rácz, Ildikó; Becker, Lore; Hans, Wolfgang; Adler, Thure; Beckers, Johannes; Rozman, Jan; Klingenspor, Martin; Wolf, Eckhard; Zimmer, Andreas; Klopstock, Thomas; Busch, Dirk H; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; van der Horst, Gilbertus; Lehmann, Alan R

    2013-05-01

    Smc5-6 is a highly conserved protein complex related to cohesin and condensin involved in the structural maintenance of chromosomes. In yeasts the Smc5-6 complex is essential for proliferation and is involved in DNA repair and homologous recombination. siRNA depletion of genes involved in the Smc5-6 complex in cultured mammalian cells results in sensitivity to some DNA damaging agents. In order to gain further insight into its role in mammals we have generated mice mutated in the Smc6 gene. A complete knockout resulted in early embryonic lethality, demonstrating that this gene is essential in mammals. However, mutation of the highly conserved serine-994 to alanine in the ATP hydrolysis motif in the SMC6 C-terminal domain, resulted in mice with a surprisingly mild phenotype. With the neo gene selection marker in the intron following the mutation, resulting in reduced expression of the SMC6 gene, the mice were reduced in size, but fertile and had normal lifespans. When the neo gene was removed, the mice had normal size, but detailed phenotypic analysis revealed minor abnormalities in glucose tolerance, haematopoiesis, nociception and global gene expression patterns. Embryonic fibroblasts derived from the ser994 mutant mice were not sensitive to killing by a range of DNA damaging agents, but they were sensitive to the induction of sister chromatid exchanges induced by ultraviolet light or mitomycin C. They also accumulated more oxidative damage than wild-type cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Novel Mutations of the Tetratricopeptide Repeat Domain 7A Gene and Phenotype/Genotype Comparison

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    Reyin Lien

    2017-09-01

    Full Text Available The gastrointestinal tract contains the largest lymphoid organ to react with pathogenic microorganisms and suppress excess inflammation. Patients with primary immunodeficiency diseases (PIDs can suffer from refractory diarrhea. In this study, we present two siblings who began to suffer from refractory diarrhea with a poor response to aggressive antibiotic and immunosuppressive treatment after surgical release of neonatal intestinal obstruction. Their lymphocyte proliferation was low, but superoxide production and IL-10 signaling were normal. Candidate genetic approach targeted to genes involved in PIDs with inflammatory bowel disease (IBD-like manifestation was unrevealing. Whole-genome sequencing revealed novel heterozygous mutations Glu75Lys and nucleotide 520–521 CT deletion in the tetratricopeptide repeat domain 7A (TTC7A gene. A Medline search identified 49 patients with TTC7A mutations, of whom 20 survived. Their phenotypes included both multiple intestinal atresia (MIA and combined T and/or B immunodeficiency (CID in 16, both IBD and CID in 14, isolated MIA in 8, MIA, IBD, and CID complex in 8, and isolated IBD in 3. Of these 98 mutant alleles over-through the coding region clustering on exon 2 (40 alleles, exon 7 (12 alleles, and exon 20 (10 alleles, 2 common hotspot mutations were c.211 G>A (p.E71K in exon 2 in 26 alleles and AAGT deletion in exon 7 (+3 in 10 alleles. Kaplan–Meier analysis showed that those with biallelic missense mutations (p = 0.0168, unaffected tetratricopeptide repeat domains (p = 0.0311, and developing autoimmune disorders (p = 0.001 had a relatively better prognosis. Hematopoietic stem cell transplantation (HSCT restored immunity and seemed to decrease the frequency of infections; however, refractory diarrhea persisted. Clinical improvement was reported upon intestinal and liver transplantation in a child with CID and MIA of unknown genetic etiology. In conclusion, patients with TTC7A mutations

  1. Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity.

    Science.gov (United States)

    Lorenz, Robin; Moon, Eui-Whan; Kim, Jeong Joo; Schmidt, Sven H; Sankaran, Banumathi; Pavlidis, Ioannis V; Kim, Choel; Herberg, Friedrich W

    2017-07-06

    Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type Iα (RIα) reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here, we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP specificity. Binding data with the generic purine nucleotide 3',5'-cyclic inosine monophosphate (cIMP) reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding and the molecular basis of cyclic nucleotide specificity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  2. Designing Inhibitors against HOX domain mutations of PDX-1 and studying its association in Diabetes

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    Allam Appa Rao

    2012-03-01

    Full Text Available Type 1 diabetes mellitus was formally known as IDDM, type I, or juvenile onset diabetes. Type 1 DM can occur at any age. In this study,we analyzed the involvement of HOX domain of PDX-1 protein.The homeodomain transcription factor, pancreas duodenum homeobox (PDX-1, encoded by PDX-1 gene, which is a transcriptional activator of several genes, including insulin, somatostatin, glucokinase, islet amyloid polypeptide, and glucose transporter type 2 and essential for pancreas development, insulin production, and glucose homeostasis.[1,13]. HOX domain has a length of 63aa and control developmental patterns and cell differentiation in vertebrates by acting positive or negative regulators[4,9,16]. Different approached had been applied to identify the mutational hot spot region of HOX domain and calculate mutational frequency of the amino acids which resides in the hotspot region. Binding site of the domain had been identified and found that THR208, GLN246 ,VAL247, ASN253 involved in interaction with ligand. Potential Inhibitors had been screened on the basis of various criteria and bioactivity score had been calculated. Energy optimization was done by applying AMBER force field and steepest descent method. Docking was performed by CCDC GOLD, Molegro, HEX, and Argus lab to find the best potent inhibitor and increase the accuracy of the docking process. Sitagliptin showed satisfactory result on both docking and bioactivity analysis. It showed a GOLD fitness score of 49.8386 and had a moldock score of -122.919 with a ligand efficiency -4.33692. Compound had a bioactivity score of 0.56 for protease inhibitor. Sitagliptin showed good binding affinity to the target, which helps to work the pancreas in proper way and to secret insulin.

  3. Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore.

    Science.gov (United States)

    Zhang, Zhifen; Park, Minyoung; Tam, John; Auger, Anick; Beilhartz, Greg L; Lacy, D Borden; Melnyk, Roman A

    2014-03-11

    Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues--clustered between amino acids 1,035 and 1,107--that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming "hotspots" of TcdB and the well-characterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.

  4. Specific Mutations in Mammalian P4-ATPase ATP8A2 Catalytic Subunit Entail Differential Glycosylation of the Accessory CDC50A Subunit

    DEFF Research Database (Denmark)

    Vestergaard, Anna L.; Mikkelsen, Stine A.; Coleman, Jonathan A.;

    2015-01-01

    P4-ATPases, or flippases, translocate phospholipids between the two leaflets of eukaryotic biological membranes. They are essential to the physiologically crucial phospholipid asymmetry and involved in severe diseases, but their molecular structure and mechanism are still unresolved. Here, we sho...

  5. Mutations in the KDM5C ARID Domain and Their Plausible Association with Syndromic Claes-Jensen-Type Disease

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    Yunhui Peng

    2015-11-01

    Full Text Available Mutations in KDM5C gene are linked to X-linked mental retardation, the syndromic Claes-Jensen-type disease. This study focuses on non-synonymous mutations in the KDM5C ARID domain and evaluates the effects of two disease-associated missense mutations (A77T and D87G and three not-yet-classified missense mutations (R108W, N142S, and R179H. We predict the ARID domain’s folding and binding free energy changes due to mutations, and also study the effects of mutations on protein dynamics. Our computational results indicate that A77T and D87G mutants have minimal effect on the KDM5C ARID domain stability and DNA binding. In parallel, the change in the free energy unfolding caused by the mutants A77T and D87G were experimentally measured by urea-induced unfolding experiments and were shown to be similar to the in silico predictions. The evolutionary conservation analysis shows that the disease-associated mutations are located in a highly-conserved part of the ARID structure (N-terminal domain, indicating their importance for the KDM5C function. N-terminal residues’ high conservation suggests that either the ARID domain utilizes the N-terminal to interact with other KDM5C domains or the N-terminal is involved in some yet unknown function. The analysis indicates that, among the non-classified mutations, R108W is possibly a disease-associated mutation, while N142S and R179H are probably harmless.

  6. Mutational analysis of EGFR and related signaling pathway genes in lung adenocarcinomas identifies a novel somatic kinase domain mutation in FGFR4.

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    Jenifer L Marks

    Full Text Available BACKGROUND: Fifty percent of lung adenocarcinomas harbor somatic mutations in six genes that encode proteins in the EGFR signaling pathway, i.e., EGFR, HER2/ERBB2, HER4/ERBB4, PIK3CA, BRAF, and KRAS. We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this signaling pathway that could contribute to lung tumorigenesis. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed genomic DNA from a total of 261 resected, clinically annotated non-small cell lung cancer (NSCLC specimens. The coding sequences of 39 genes were screened for somatic mutations via high-throughput dideoxynucleotide sequencing of PCR-amplified gene products. Mutations were considered to be somatic only if they were found in an independent tumor-derived PCR product but not in matched normal tissue. Sequencing of 9MB of tumor sequence identified 239 putative genetic variants. We further examined 22 variants found in RAS family genes and 135 variants localized to exons encoding the kinase domain of respective proteins. We identified a total of 37 non-synonymous somatic mutations; 36 were found collectively in EGFR, KRAS, BRAF, and PIK3CA. One somatic mutation was a previously unreported mutation in the kinase domain (exon 16 of FGFR4 (Glu681Lys, identified in 1 of 158 tumors. The FGFR4 mutation is analogous to a reported tumor-specific somatic mutation in ERBB2 and is located in the same exon as a previously reported kinase domain mutation in FGFR4 (Pro712Thr in a lung adenocarcinoma cell line. CONCLUSIONS/SIGNIFICANCE: This study is one of the first comprehensive mutational analyses of major genes in a specific signaling pathway in a sizeable cohort of lung adenocarcinomas. Our results suggest the majority of gain-of-function mutations within kinase genes in the EGFR signaling pathway have already been identified. Our findings also implicate FGFR4 in the pathogenesis of a subset of lung adenocarcinomas.

  7. Analysis of a mutation affecting the specificity domain for prohead binding of the bacteriophage lambda terminase.

    Science.gov (United States)

    Sippy, J; Feiss, M

    1992-02-01

    Genetic studies have identified a specificity domain for prohead binding in the C-terminal 32 amino acids of gpA, the large subunit of bacteriophage lambda terminase (S. Frackman, D. A. Siegele, and M. Feiss, J. Mol. Biol. 180:283-300, 1984). In the present work, an amber mutation, Aam42, in the fifth-to-last codon of the A gene was found to be lethal in nonsuppressing hosts. The mutation, expected to generate gpA lacking the last five amino acids, caused the production of a terminase that cut cos efficiently both in vivo and in vitro but was defective in DNA packaging. lambda Aam42 lysates contained unused proheads, consistent with a defect in prohead binding. Aam42 terminase was more strongly dependent than wild-type terminase on gpFI, the catalyst of prohead binding. Like wild-type terminase, Aam42 terminase did not cut cos in vivo when prohead assembly was blocked by a mutation in one of the genes encoding the prohead.

  8. The polar domain of the b subunit of Escherichia coli F1F0-ATPase forms an elongated dimer that interacts with the F1 sector.

    Science.gov (United States)

    Dunn, S D

    1992-04-15

    A soluble form of the b subunit of the F0 sector of the F1F0-ATPase of Escherichia coli has been produced, purified, and characterized. In this form of the protein, designated bsol, residues 25-146 (the carboxyl terminus) of b have been fused to an amino-terminal octapeptide extension derived from the vector pUC8. The inferred subunit molecular weight of bsol is 15,459. bsol protein was expressed in E. coli as a soluble cytoplasmic protein and was readily purified to homogeneity by conventional methods. The molecular weight of bsol, determined by sedimentation equilibrium, was 31,200, indicating that the protein is dimeric. Chemical cross-linking studies supported this conclusion. However, bsol sedimented with a coefficient of just 1.8 S and behaved on size exclusion chromatography with an apparent molecular weight of 80,000-85,000. These results indicate that the protein exists in solution as a highly elongated dimer. The circular dichroism spectrum indicated that bsol is highly alpha-helical. Binding of bsol to F1-ATPase was directly demonstrated by size exclusion chromatography. bsol also inhibited the binding of F1-ATPase to F1-depleted membrane vesicles, as measured by reconstitution of energy-dependent quinacrine fluorescence quenching. This result implies that bsol and F0 compete for binding to the same site on F1. The apparently normal interaction of bsol with F1-ATPase strongly suggests that the recombinant protein assumes the correct structure. No substantial effects of bsol on the ATPase activity of purified F1 were observed.

  9. Graph-Theoretic Models of Mutations in the Nucleotide Binding Domain 1 of the Cystic Fibrosis Transmembrane Conductance Regulator

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    Debra J. Knisley

    2013-01-01

    Full Text Available Cystic fibrosis is one of the most common inherited diseases and is caused by a mutation in a membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR. This protein serves as a chloride channel and regulates the viscosity of mucus lining the ducts of a number of organs. Although much has been learned about the consequences of mutations on the energy landscape and the resulting disrupted folding pathway of CFTR, a level of understanding needed to correct the misfolding has not been achieved. The most common mutations of CFTR are located in one of two nucleotide binding domains, namely, the nucleotide binding domain 1 (NBD1. We model NBD1 using a nested graph model. The vertices in the lowest layer each represent an atom in the structure of an amino acid residue, while the vertices in the mid layer each represent the residue. The vertices in the top layer each represent a subdomain of the nucleotide binding domain. We use this model to quantify the effects of a single point mutation on the protein domain. We compare the wildtype structure with eight of the most common mutations. The graph-theoretic model provides insight into how a single point mutation can have such profound structural consequences.

  10. DNA polymerase ε and δ exonuclease domain mutations in endometrial cancer.

    Science.gov (United States)

    Church, David N; Briggs, Sarah E W; Palles, Claire; Domingo, Enric; Kearsey, Stephen J; Grimes, Jonathon M; Gorman, Maggie; Martin, Lynn; Howarth, Kimberley M; Hodgson, Shirley V; Kaur, Kulvinder; Taylor, Jenny; Tomlinson, Ian P M

    2013-07-15

    Accurate duplication of DNA prior to cell division is essential to suppress mutagenesis and tumour development. The high fidelity of eukaryotic DNA replication is due to a combination of accurate incorporation of nucleotides into the nascent DNA strand by DNA polymerases, the recognition and removal of mispaired nucleotides (proofreading) by the exonuclease activity of DNA polymerases δ and ε, and post-replication surveillance and repair of newly synthesized DNA by the mismatch repair (MMR) apparatus. While the contribution of defective MMR to neoplasia is well recognized, evidence that faulty DNA polymerase activity is important in cancer development has been limited. We have recently shown that germline POLE and POLD1 exonuclease domain mutations (EDMs) predispose to colorectal cancer (CRC) and, in the latter case, to endometrial cancer (EC). Somatic POLE mutations also occur in 5-10% of sporadic CRCs and underlie a hypermutator, microsatellite-stable molecular phenotype. We hypothesized that sporadic ECs might also acquire somatic POLE and/or POLD1 mutations. Here, we have found that missense POLE EDMs with good evidence of pathogenic effects are present in 7% of a set of 173 endometrial cancers, although POLD1 EDMs are uncommon. The POLE mutations localized to highly conserved residues and were strongly predicted to affect proofreading. Consistent with this, POLE-mutant tumours were hypermutated, with a high frequency of base substitutions, and an especially large relative excess of G:C>T:A transversions. All POLE EDM tumours were microsatellite stable, suggesting that defects in either DNA proofreading or MMR provide alternative mechanisms to achieve genomic instability and tumourigenesis.

  11. DNA polymerase ɛ and δ exonuclease domain mutations in endometrial cancer

    Science.gov (United States)

    Church, David N.; Briggs, Sarah E.W.; Palles, Claire; Domingo, Enric; Kearsey, Stephen J.; Grimes, Jonathon M.; Gorman, Maggie; Martin, Lynn; Howarth, Kimberley M.; Hodgson, Shirley V.; Kaur, Kulvinder; Taylor, Jenny; Tomlinson, Ian P.M.

    2013-01-01

    Accurate duplication of DNA prior to cell division is essential to suppress mutagenesis and tumour development. The high fidelity of eukaryotic DNA replication is due to a combination of accurate incorporation of nucleotides into the nascent DNA strand by DNA polymerases, the recognition and removal of mispaired nucleotides (proofreading) by the exonuclease activity of DNA polymerases δ and ɛ, and post-replication surveillance and repair of newly synthesized DNA by the mismatch repair (MMR) apparatus. While the contribution of defective MMR to neoplasia is well recognized, evidence that faulty DNA polymerase activity is important in cancer development has been limited. We have recently shown that germline POLE and POLD1 exonuclease domain mutations (EDMs) predispose to colorectal cancer (CRC) and, in the latter case, to endometrial cancer (EC). Somatic POLE mutations also occur in 5–10% of sporadic CRCs and underlie a hypermutator, microsatellite-stable molecular phenotype. We hypothesized that sporadic ECs might also acquire somatic POLE and/or POLD1 mutations. Here, we have found that missense POLE EDMs with good evidence of pathogenic effects are present in 7% of a set of 173 endometrial cancers, although POLD1 EDMs are uncommon. The POLE mutations localized to highly conserved residues and were strongly predicted to affect proofreading. Consistent with this, POLE-mutant tumours were hypermutated, with a high frequency of base substitutions, and an especially large relative excess of G:C>T:A transversions. All POLE EDM tumours were microsatellite stable, suggesting that defects in either DNA proofreading or MMR provide alternative mechanisms to achieve genomic instability and tumourigenesis. PMID:23528559

  12. Long-range effects and functional consequences of stabilizing mutations in the ankyrin repeat domain of IκBα.

    Science.gov (United States)

    Cervantes, Carla F; Handley, Lindsey D; Sue, Shih-Che; Dyson, H Jane; Komives, Elizabeth A

    2013-03-11

    Protein domains containing three or more ankyrin repeats (ARs) are ubiquitous in all phyla. Sequence alignments previously identified certain conserved positions, which have been shown to stabilize AR domains and promote their folding. Consensus mutations [Y254L/T257A (YLTA) and C186P/A220P (CPAP)] stabilize the naturally occuring AR domain of human IκBα to denaturation; however, only the YLTA mutations stabilize the protein to proteasomal degradation. We present results from NMR experiments designed to probe the roles of these consensus mutations in IκBα. According to residual dipolar coupling analysis, the gross structures of the AR domains of both mutants appear to be similar to the wild type (WT). Comparison of chemical shifts of mutant and WT proteins reveals that the YLTA and CPAP consensus mutations cause unexpected long-range effects throughout the AR domains. Backbone dynamics experiments reveal that the YLTA mutations in the sixth AR order the C-terminal PEST sequence on the picosecond-to-nanosecond timescale, compared to either the WT or the CPAP mutant IκBαs. This property is likely the mechanism by which the half-life of YLTA IκBα is extended in vivo.

  13. The SMAD-binding domain of SKI: a hotspot for de novo mutations causing Shprintzen-Goldberg syndrome.

    Science.gov (United States)

    Schepers, Dorien; Doyle, Alexander J; Oswald, Gretchen; Sparks, Elizabeth; Myers, Loretha; Willems, Patrick J; Mansour, Sahar; Simpson, Michael A; Frysira, Helena; Maat-Kievit, Anneke; Van Minkelen, Rick; Hoogeboom, Jeanette M; Mortier, Geert R; Titheradge, Hannah; Brueton, Louise; Starr, Lois; Stark, Zornitza; Ockeloen, Charlotte; Lourenco, Charles Marques; Blair, Ed; Hobson, Emma; Hurst, Jane; Maystadt, Isabelle; Destrée, Anne; Girisha, Katta M; Miller, Michelle; Dietz, Harry C; Loeys, Bart; Van Laer, Lut

    2015-02-01

    Shprintzen-Goldberg syndrome (SGS) is a rare, systemic connective tissue disorder characterized by craniofacial, skeletal, and cardiovascular manifestations that show a significant overlap with the features observed in the Marfan (MFS) and Loeys-Dietz syndrome (LDS). A distinguishing observation in SGS patients is the presence of intellectual disability, although not all patients in this series present this finding. Recently, SGS was shown to be due to mutations in the SKI gene, encoding the oncoprotein SKI, a repressor of TGFβ activity. Here, we report eight recurrent and three novel SKI mutations in eleven SGS patients. All were heterozygous missense mutations located in the R-SMAD binding domain, except for one novel in-frame deletion affecting the DHD domain. Adding our new findings to the existing data clearly reveals a mutational hotspot, with 73% (24 out of 33) of the hitherto described unrelated patients having mutations in a stretch of five SKI residues (from p.(Ser31) to p.(Pro35)). This implicates that the initial molecular testing could be focused on mutation analysis of the first half of exon 1 of SKI. As the majority of the known mutations are located in the R-SMAD binding domain of SKI, our study further emphasizes the importance of TGFβ signaling in the pathogenesis of SGS.

  14. Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

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    Alessandra Pasquo

    Full Text Available Protein tyrosine phosphatase ρ (PTPρ belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

  15. Therapeutic efficacy of artemisinin combination therapies and prevalence of S769N mutation in PfATPase6 gene of Plasmodium falciparum in Kolkata, India

    Institute of Scientific and Technical Information of China (English)

    Pabitra Saha; Shrabanee Mullick; Krishnangshu Ray; Ardhendu K Maji; Arindam Naskar; Swagata Ganguly; Sonali Das; Subhasish K Guha; Asit Biswas; Dilip K Bera; Pratip K Kundu; Madhusudan Das

    2013-01-01

    Objective: To study the in vivo efficacy of these two ACTs in the treatment of Plasmodium falciparum (P. falciparum malaria) in Kolkata and to determine the prevalence of mutant S769N codon of the PfATPase6 gene among field isolates of P. falciparum collected from the study area.Methods:A total of 207 P. falciparum positive cases were enrolled randomly in two study arms and followed up for 42 days as per WHO (2009) protocol. A portion of PfATPase6 gene spanning codon S769N was amplified and sequenced by direct sequencing method. Results: It was observed that the efficacy of both the ACT regimens were highly effective in the study area and no mutant S769N was detected from any isolate. Conclusions: The used, combination AS+SP is effective and the other combination AM+LF might be an alternative, if needed.

  16. Conservation patterns of HIV-1 RT connection and RNase H domains: identification of new mutations in NRTI-treated patients.

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    André F A Santos

    Full Text Available BACKGROUND: Although extensive HIV drug resistance information is available for the first 400 amino acids of its reverse transcriptase, the impact of antiretroviral treatment in C-terminal domains of Pol (thumb, connection and RNase H is poorly understood. METHODS AND FINDINGS: We wanted to characterize conserved regions in RT C-terminal domains among HIV-1 group M subtypes and CRF. Additionally, we wished to identify NRTI-related mutations in HIV-1 RT C-terminal domains. We sequenced 118 RNase H domains from clinical viral isolates in Brazil, and analyzed 510 thumb and connection domain and 450 RNase H domain sequences collected from public HIV sequence databases, together with their treatment status and histories. Drug-naïve and NRTI-treated datasets were compared for intra- and inter-group conservation, and differences were determined using Fisher's exact tests. One third of RT C-terminal residues were found to be conserved among group M variants. Three mutations were found exclusively in NRTI-treated isolates. Nine mutations in the connection and 6 mutations in the RNase H were associated with NRTI treatment in subtype B. Some of them lay in or close to amino acid residues which contact nucleic acid or near the RNase H active site. Several of the residues pointed out herein have been recently associated to NRTI exposure or increase drug resistance to NRTI. CONCLUSIONS: This is the first comprehensive genotypic analysis of a large sequence dataset that describes NRTI-related mutations in HIV-1 RT C-terminal domains in vivo. The findings into the conservation of RT C-terminal domains may pave the way to more rational drug design initiatives targeting those regions.

  17. Migraine- and dystonia-related disease-mutations of Na+/K+-ATPases: Relevance of behavioral studies in mice to disease symptoms and neurological manifestations in humans

    DEFF Research Database (Denmark)

    Bøttger, Pernille; Doganli, Canan; Lykke-Hartmann, Karin

    2012-01-01

    with classical FHM2 and RDP symptoms additionally suffer from other manifestations, such as epilepsy/seizures and developmental disabilities. Recent studies of FHM2 and RDP mouse models provide valuable tools for dissecting the vital roles of the Na+/K+-ATPases, and we discuss their relevance to the complex...... have broad potentials for future research concerning migraine and dystonia-related diseases, which will contribute towards understanding the, yet unknown, pathophysiologies...

  18. Mutations in the ligand-binding domain of the androgen receptor gene cluster in two regions of the gene.

    Science.gov (United States)

    McPhaul, M J; Marcelli, M; Zoppi, S; Wilson, C M; Griffin, J E; Wilson, J D

    1992-11-01

    We have analyzed the nucleotide sequence of the androgen receptor from 22 unrelated subjects with substitution mutations of the hormone-binding domain. Eleven had the phenotype of complete testicular feminization, four had incomplete testicular feminization, and seven had Reifenstein syndrome. The underlying functional defect in cultured skin fibroblasts included individuals with absent, qualitative, or quantitative defects in ligand binding. 19 of the 21 substitution mutations (90%) cluster in two regions that account for approximately 35% of the hormone-binding domain, namely, between amino acids 726 and 772 and between amino acids 826 and 864. The fact that one of these regions is homologous to a region of the human thyroid hormone receptor (hTR-beta) which is a known cluster site for mutations that cause thyroid hormone resistance implies that this localization of mutations is not a coincidence. These regions of the androgen receptor may be of particular importance for the formation and function of the hormone-receptor complex.

  19. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain.

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    William Pao

    2005-03-01

    Full Text Available BACKGROUND: Lung adenocarcinomas from patients who respond to the tyrosine kinase inhibitors gefitinib (Iressa or erlotinib (Tarceva usually harbor somatic gain-of-function mutations in exons encoding the kinase domain of the epidermal growth factor receptor (EGFR. Despite initial responses, patients eventually progress by unknown mechanisms of "acquired" resistance. METHODS AND FINDINGS: We show that in two of five patients with acquired resistance to gefitinib or erlotinib, progressing tumors contain, in addition to a primary drug-sensitive mutation in EGFR, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M in the kinase domain. Tumor cells from a sixth patient with a drug-sensitive EGFR mutation whose tumor progressed on adjuvant gefitinib after complete resection also contained the T790M mutation. This mutation was not detected in untreated tumor samples. Moreover, no tumors with acquired resistance had KRAS mutations, which have been associated with primary resistance to these drugs. Biochemical analyses of transfected cells and growth inhibition studies with lung cancer cell lines demonstrate that the T790M mutation confers resistance to EGFR mutants usually sensitive to either gefitinib or erlotinib. Interestingly, a mutation analogous to T790M has been observed in other kinases with acquired resistance to another kinase inhibitor, imatinib (Gleevec. CONCLUSION: In patients with tumors bearing gefitinib- or erlotinib-sensitive EGFR mutations, resistant subclones containing an additional EGFR mutation emerge in the presence of drug. This observation should help guide the search for more effective therapy against a specific subset of lung cancers.

  20. Mutation of the CH1 Domain in the Histone Acetyltransferase CREBBP Results in Autism-Relevant Behaviors in Mice.

    Science.gov (United States)

    Zheng, Fei; Kasper, Lawryn H; Bedford, David C; Lerach, Stephanie; Teubner, Brett J W; Brindle, Paul K

    2016-01-01

    Autism spectrum disorders (ASDs) are a group of neurodevelopmental afflictions characterized by repetitive behaviors, deficits in social interaction, and impaired communication skills. For most ASD patients, the underlying causes are unknown. Genetic mutations have been identified in about 25 percent of ASD cases, including mutations in epigenetic regulators, suggesting that dysregulated chromatin or DNA function is a critical component of ASD. Mutations in the histone acetyltransferase CREB binding protein (CBP, CREBBP) cause Rubinstein-Taybi Syndrome (RTS), a developmental disorder that includes ASD-like symptoms. Recently, genomic studies involving large numbers of ASD patient families have theoretically modeled CBP and its paralog p300 (EP300) as critical hubs in ASD-associated protein and gene interaction networks, and have identified de novo missense mutations in highly conserved residues of the CBP acetyltransferase and CH1 domains. Here we provide animal model evidence that supports this notion that CBP and its CH1 domain are relevant to autism. We show that mice with a deletion mutation in the CBP CH1 (TAZ1) domain (CBPΔCH1/ΔCH1) have an RTS-like phenotype that includes ASD-relevant repetitive behaviors, hyperactivity, social interaction deficits, motor dysfunction, impaired recognition memory, and abnormal synaptic plasticity. Our results therefore indicate that loss of CBP CH1 domain function contributes to RTS, and possibly ASD, and that this domain plays an essential role in normal motor function, cognition and social behavior. Although the key physiological functions affected by ASD-associated mutation of epigenetic regulators have been enigmatic, our findings are consistent with theoretical models involving CBP and p300 in ASD, and with a causative role for recently described ASD-associated CBP mutations.

  1. Modeling and experimental assessment of a buried Leu–Ile mutation in dengue envelope domain III

    Energy Technology Data Exchange (ETDEWEB)

    Kulkarni, Manjiri R. [Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Nakamachi, Koganei-shi, Tokyo, 184-8588 (Japan); Numoto, Nobutaka; Ito, Nobutoshi [Department of Structural Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima Bunkyo-ku, Tokyo, 113-8510 (Japan); Kuroda, Yutaka, E-mail: ykuroda@cc.tuat.ac.jp [Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Nakamachi, Koganei-shi, Tokyo, 184-8588 (Japan)

    2016-02-26

    Envelope protein domain III (ED3) of the dengue virus is important for both antibody binding and host cell interaction. Here, we focused on how a L387I mutation in the protein core could take place in DEN4 ED3, but cannot be accommodated in DEN3 ED3 without destabilizing its structure. To this end, we modeled a DEN4-L387I structure using the Penultimate Rotamer Library and taking the DEN4 ED3 main-chain as a fixed template. We found that three out of seven Ile{sup 387} conformers fit in DEN4 ED3 without introducing the severe atomic clashes that are observed when DEN3 serotype’s ED3 is used as a template. A more extensive search using 273 side-chain rotamers of the residues surrounding Ile{sup 387} confirmed this prediction. In order to assess the prediction, we determined the crystal structure of DEN4-L387I at 2 Å resolution. Ile{sup 387} indeed adopted one of the three predicted rotamers. Altogether, this study demonstrates that the effects of single mutations are to a large extent successfully predicted by systematically modeling the side-chain structures of the mutated as well as those of its surrounding residues using fixed main-chain structures and assessing inter-atomic steric clashes. More accurate and reliable predictions require considering sub-angstrom main-chain deformation, which remains a challenging task. - Highlights: • We mutated L387I of DEN4 ED3 and examined its effects on structure and stability. • We modeled the side-chain of Ile{sup 387} using DEN4 ED3's structure as a template. • We determined the crystal structure of DEN4-L387I and confirmed the modeling. • Side-chain repacking occurring around Ile{sup 387} involved >3 inter-connected residues. • These results explained why L387I mutation in DEN4 ED3 conserves thermostability.

  2. Mutations in the ligand-binding domain of the androgen receptor gene cluster in two regions of the gene.

    OpenAIRE

    McPhaul, M J; Marcelli, M; Zoppi, S; Wilson, C. M.; Griffin, J E; Wilson, J. D.

    1992-01-01

    We have analyzed the nucleotide sequence of the androgen receptor from 22 unrelated subjects with substitution mutations of the hormone-binding domain. Eleven had the phenotype of complete testicular feminization, four had incomplete testicular feminization, and seven had Reifenstein syndrome. The underlying functional defect in cultured skin fibroblasts included individuals with absent, qualitative, or quantitative defects in ligand binding. 19 of the 21 substitution mutations (90%) cluster ...

  3. Mutational and haplotype analyses of families with familial partial lipodystrophy (Dunnigan variety) reveal recurrent missense mutations in the globular C-terminal domain of lamin A/C.

    Science.gov (United States)

    Speckman, R A; Garg, A; Du, F; Bennett, L; Veile, R; Arioglu, E; Taylor, S I; Lovett, M; Bowcock, A M

    2000-04-01

    Familial partial lipodystrophy (FPLD), Dunnigan variety, is an autosomal dominant disorder characterized by marked loss of subcutaneous adipose tissue from the extremities and trunk but by excess fat deposition in the head and neck. The disease is frequently associated with profound insulin resistance, dyslipidemia, and diabetes. We have localized a gene for FPLD to chromosome 1q21-q23, and it has recently been proposed that nuclear lamin A/C is altered in FPLD, on the basis of a novel missense mutation (R482Q) in five Canadian probands. This gene had previously been shown to be altered in autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD) and in dilated cardiomyopathy and conduction-system disease. We examined 15 families with FPLD for mutations in lamin A/C. Five families harbored the R482Q alteration that segregated with the disease phenotype. Seven families harbored an R482W alteration, and one family harbored a G465D alteration. All these mutations lie within exon 8 of the lamin A/C gene-an exon that has also been shown to harbor different missense mutations that are responsible for EDMD-AD. Mutations could not be detected in lamin A/C in one FPLD family in which there was linkage to chromosome 1q21-q23. One family with atypical FPLD harbored an R582H alteration in exon 11 of lamin A. This exon does not comprise part of the lamin C coding region. All mutations in FPLD affect the globular C-terminal domain of the lamin A/C protein. In contrast, mutations responsible for dilated cardiomyopathy and conduction-system disease are observed in the rod domain of the protein. The FPLD mutations R482Q and R482W occurred on different haplotypes, indicating that they are likely to have arisen more than once.

  4. Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.

    Science.gov (United States)

    Takezaki, Shunichiro; Yamada, Masafumi; Kato, Masahiko; Park, Myoung-Ja; Maruyama, Kenichi; Yamazaki, Yasuhiro; Chida, Natsuko; Ohara, Osamu; Kobayashi, Ichiro; Ariga, Tadashi

    2012-08-01

    Chronic mucocutaneous candidiasis (CMC) is a heterogeneous group of primary immunodeficiency diseases characterized by chronic and recurrent Candida infections of the skin, nails, and oropharynx. Gain-of-function mutations in STAT1 were very recently shown to be responsible for autosomal-dominant or sporadic cases of CMC. The reported mutations have been exclusively localized in the coiled-coil domain, resulting in impaired dephosphorylation of STAT1. However, recent crystallographic analysis and direct mutagenesis experiments indicate that mutations affecting the DNA-binding domain of STAT1 could also lead to persistent phosphorylation of STAT1. To our knowledge, this study shows for the first time that a DNA-binding domain mutation of c.1153C>T in exon 14 (p.T385M) is the genetic cause of sporadic CMC in two unrelated Japanese patients. The underlying mechanisms involve a gain of STAT1 function due to impaired dephosphorylation as observed in the coiled-coil domain mutations.

  5. HER family kinase domain mutations promote tumor progression and can predict response to treatment in human breast cancer

    KAUST Repository

    Boulbes, Delphine R.

    2014-11-11

    Resistance to HER2-targeted therapies remains a major obstacle in the treatment of HER2-overexpressing breast cancer. Understanding the molecular pathways that contribute to the development of drug resistance is needed to improve the clinical utility of novel agents, and to predict the success of targeted personalized therapy based on tumor-specific mutations. Little is known about the clinical significance of HER family mutations in breast cancer. Because mutations within HER1/EGFR are predictive of response to tyrosine kinase inhibitors (TKI) in lung cancer, we investigated whether mutations in HER family kinase domains are predictive of response to targeted therapy in HER2-overexpressing breast cancer. We sequenced the HER family kinase domains from 76 HER2-overexpressing invasive carcinomas and identified 12 missense variants. Patients whose tumors carried any of these mutations did not respond to HER2 directed therapy in the metastatic setting. We developed mutant cell lines and used structural analyses to determine whether changes in protein conformation could explain the lack of response to therapy. We also functionally studied all HER2 mutants and showed that they conferred an aggressive phenotype and altered effects of the TKI lapatinib. Our data demonstrate that mutations in the finely tuned HER kinase domains play a critical function in breast cancer progression and may serve as prognostic and predictive markers.

  6. Mutation in the sixth immunoglobulin domain of L1CAM is associated with migrational brain anomalies

    Science.gov (United States)

    Shieh, Christine; Moser, Franklin; Graham, John M.; Watiker, Valerie

    2015-01-01

    Objective: To describe the phenotype of a patient with classical features of X-linked L1 syndrome associated with novel brain malformations. Methods: Diagnostic analysis included physical and dysmorphology examinations, MRI of the brain, and exome sequencing of the family trio. Results: We report a 2.5-year-old boy with developmental delay, dysmorphic facies, and adducted thumbs. MRI of the brain showed a truncated corpus callosum and periventricular heterotopias associated with polymicrogyria (PMG). Variant segregation analysis with exome sequencing discovered a novel maternally derived hemizygous variant in exon 14 of the L1CAM gene (c.1759 G>C; p.G587R). Conclusions: This novel L1CAM mutation was located in the protein's sixth immunoglobin domain and involved glycine-587, a key residue in the structure of L1CAM because of its interactions with lysine-606, which indicates that any mutation at this site would likely affect the secondary structure and function of the protein. The replacement of the small nonpolar glycine residue with a large basic arginine would have an even more dramatic result. The presentation of periventricular nodular heterotopias with overlying PMG is very uncommon, and its association with L1CAM may provide insight into other similar cases. Furthermore, this presentation indicates the important role that L1CAM plays in neuronal migration and brain development and extends the phenotype associated with L1CAM-associated disorders. PMID:27066571

  7. Efficient affinity maturation of antibody variable domains requires co-selection of compensatory mutations to maintain thermodynamic stability

    Science.gov (United States)

    Julian, Mark C.; Li, Lijuan; Garde, Shekhar; Wilen, Rebecca; Tessier, Peter M.

    2017-01-01

    The ability of antibodies to accumulate affinity-enhancing mutations in their complementarity-determining regions (CDRs) without compromising thermodynamic stability is critical to their natural function. However, it is unclear if affinity mutations in the hypervariable CDRs generally impact antibody stability and to what extent additional compensatory mutations are required to maintain stability during affinity maturation. Here we have experimentally and computationally evaluated the functional contributions of mutations acquired by a human variable (VH) domain that was evolved using strong selections for enhanced stability and affinity for the Alzheimer’s Aβ42 peptide. Interestingly, half of the key affinity mutations in the CDRs were destabilizing. Moreover, the destabilizing effects of these mutations were compensated for by a subset of the affinity mutations that were also stabilizing. Our findings demonstrate that the accumulation of both affinity and stability mutations is necessary to maintain thermodynamic stability during extensive mutagenesis and affinity maturation in vitro, which is similar to findings for natural antibodies that are subjected to somatic hypermutation in vivo. These findings for diverse antibodies and antibody fragments specific for unrelated antigens suggest that the formation of the antigen-binding site is generally a destabilizing process and that co-enrichment for compensatory mutations is critical for maintaining thermodynamic stability. PMID:28349921

  8. A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog.

    Science.gov (United States)

    Katz, W S; Lesa, G M; Yannoukakos, D; Clandinin, T R; Schlessinger, J; Sternberg, P W

    1996-01-01

    The let-23 gene encodes a Caenorhabditis elegans homolog of the epidermal growth factor receptor (EGFR) necessary for vulval development. We have characterized a mutation of let-23 that activates the receptor and downstream signal transduction, leading to excess vulval differentiation. This mutation alters a conserved cysteine residue in the extracellular domain and is the first such point mutation in the EGFR subfamily of tyrosine kinases. Mutation of a different cysteine in the same subdomain causes a strong loss-of-function phenotype, suggesting that cysteines in this region are important for function and nonequivalent. Vulval precursor cells can generate either of two subsets of vulval cells (distinct fates) in response to sa62 activity. The fates produced depended on the copy number of the mutation, suggesting that quantitative differences in receptor activity influence the decision between these two fates. PMID:8552080

  9. Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro.

    Science.gov (United States)

    Li, Yan; Burclaff, Joseph; Anderson, James T

    2016-01-01

    RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.

  10. Mutation S233L in the 1B domain of keratin 1 causes epidermolytic palmoplantar keratoderma with "tonotubular" keratin.

    Science.gov (United States)

    Terron-Kwiatkowski, Ana; van Steensel, Maurice A M; van Geel, Michel; Lane, E Birgitte; McLean, W H Irwin; Steijlen, Peter M

    2006-03-01

    Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis characterized by epidermolytic hyperkeratosis restricted to the palm and sole epidermis. The disorder is normally associated with dominant-negative mutations in the keratin 9 (K9) gene; however, a small number of cases have been reported where causative mutations were identified in the K1 gene. Here, we present two unrelated Dutch EPPK families with striking ultrastructural findings: tubular keratin structures in the cytoplasm of suprabasal cells. Similar structures were reported previously in a German EPPK family and were termed "tonotubular" keratin. After excluding the involvement of the K9 gene by complete sequencing, we identified a novel mutation, S233L, at the beginning of the 1B domain of K1 in both families. Protein expression studies in cultured cells indicated pathogenicity of this mutation. This is the first report of a genetic defect in this domain of K1. The unusual gain-of-function mutation points to a subtle role of the 1B domain in mediating filament-filament interactions with regular periodicity.

  11. Fine tuning of the catalytic activity of colicin e7 nuclease domain by systematic n-terminal mutations

    DEFF Research Database (Denmark)

    Németh, Eszter; Körtvélyesi, Tamás; Thulstrup, Peter W.;

    2014-01-01

    The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446–449NColE75KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuc...

  12. N-linked glycosylation of a subunit isoforms is critical for vertebrate vacuolar H(+) -ATPase (V-ATPase) biosynthesis.

    Science.gov (United States)

    Esmail, Sally; Kartner, Norbert; Yao, Yeqi; Kim, Joo Wan; Reithmeier, Reinhart A F; Manolson, Morris F

    2017-06-29

    The a subunit of the V0 membrane-integrated sector of human V-ATPase has four isoforms, a1-a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N-glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N-glycosylated. Here we extend our investigation to the other isoforms by transiently transfecting HEK 293 cells to express cDNA constructs of epitope-tagged human a1-a3 subunits, with or without mutations that convert Asn to Gln at putative N-glycosylation sites. Expression and N-glycosylation were characterized by immunoblotting and mobility shifts after enzymatic deglycosylation, and intracellular localization was determined using immunofluorescence microscopy. All unglycosylated mutants, where predicted N-glycosylation sites had been eliminated by sequon mutagenesis, showed increased relative mobility on immunoblots, identical to what was seen for wild-type a subunits after enzymatic deglycosylation. Cycloheximide-chase experiments showed that unglycosylated subunits were turned over at a higher rate than N-glycosylated forms by degradation in the proteasomal pathway. Immunofluorescence colocalization analysis showed that unglycosylated a subunits were retained in the ER, and co-immunoprecipitation studies showed that they were unable to associate with the V-ATPase assembly chaperone, VMA21. Taken together with our previous a4 subunit studies, these observations show that N-glycosylation is crucial in all four human V-ATPase a subunit isoforms for protein stability and ultimately for functional incorporation into V-ATPase complexes. © 2017 Wiley Periodicals, Inc.

  13. Combination of genetic screening and molecular dynamics as a useful tool for identification of disease-related mutations: ZASP PDZ domain G54S mutation case.

    Science.gov (United States)

    Fratev, Filip; Mihaylova, Elina; Pajeva, Ilza

    2014-05-27

    Cypher/ZASP (LDB3 gene) is known to interact with a network of proteins. It binds to α-actinin and the calcium voltage channels (LTCC) via its PDZ domain. Here we report the identification of a highly conserved ZASP G54S mutation classified as a variant of unknown significance in a sample of an adult with hypertrophic cardiomyopathy (HCM). The initial bioinformatics calculations strongly evaluated G54S as damaging. Furthermore, we employed accelerated and classical molecular dynamics and free energy calculations to study the structural impact of this mutation on the ZASP apo form and to address the question of whether it can be linked to HCM. Seventeen independent MD runs and simulations of 2.5 μs total were performed and showed that G54S perturbs the α2 helix position via destabilization of the adjacent loop linked to the β5 sheet. This also leads to the formation of a strong H-bond between peptide target residues Leu17 and Gln66, thus restricting both the α-actinin2 and LTCC C-terminal peptides to access their natural binding site and reducing in this way their binding capacity. On the basis of these observations and the adult's clinical data, we propose that ZASP(G54S) and presumably other ZASP PDZ domain mutations can cause HCM. To the best of our knowledge, this is the first reported ZASP PDZ domain mutation that might be linked to HCM. The integrated workflow used in this study can be applied for the identification and description of other mutations that might be related to particular diseases.

  14. Mutational analysis of VAMP domains implicated in Ca2+-induced insulin exocytosis.

    Science.gov (United States)

    Regazzi, R; Sadoul, K; Meda, P; Kelly, R B; Halban, P A; Wollheim, C B

    1996-01-01

    Vesicle-associated membrane protein-2 (VAMP-2) and cellubrevin are associated with the membrane of insulin-containing secretory granules and of gamma-aminobutyric acid (GABA)-containing synaptic-like vesicles of pancreatic beta-cells. We found that a point mutation in VAMP-2 preventing targeting to synaptic vesicles also impairs the localization on insulin-containing secretory granules, suggesting a similar requirement for vesicular targeting. Tetanus toxin (TeTx) treatment of permeabilized HIT-T15 cells leads to the proteolytic cleavage of VAMP-2 and cellubrevin and causes the inhibition of Ca2+-triggered insulin exocytosis. Transient transfection of HIT-T15 cells with VAMP-1, VAMP-2 or cellubrevin made resistant to the proteolytic action of TeTx by amino acid replacements in the cleavage site restored Ca2+-stimulated secretion. Wild-type VAMP-2, wild-type cellubrevin or a mutant of VAMP-2 resistant to TeTx but not targeted to secretory granules were unable to rescue Ca2+-evoked insulin release. The transmembrane domain and the N-terminal region of VAMP-2 were not essential for the recovery of stimulated exocytosis, but deletions preventing the binding to SNAP-25 and/or to syntaxin I rendered the protein inactive in the reconstitution assay. Mutations of putative phosphorylation sites or of negatively charged amino acids in the SNARE motif recognized by clostridial toxins had no effect on the ability of VAMP-2 to mediate Ca2+-triggered secretion. We conclude that: (i) both VAMP-2 and cellubrevin can participate in the exocytosis of insulin; (ii) the interaction of VAMP-2 with syntaxin and SNAP-25 is required for docking and/or fusion of secretory granules with the plasma membrane; and (iii) the phosphorylation of VAMP-2 is not essential for Ca2+-stimulated insulin exocytosis. Images PMID:9003771

  15. Mutational Analysis and Allosteric Effects in the HIV-1 Capsid Protein Carboxyl-Terminal Dimerization Domain

    Science.gov (United States)

    2009-01-01

    The carboxyl-terminal domain (CTD, residues 146−231) of the HIV-1 capsid (CA) protein plays an important role in the CA−CA dimerization and viral assembly of the human immunodeficiency virus type 1. Disrupting the native conformation of the CA is essential for blocking viral capsid formation and viral replication. Thus, it is important to identify the exact nature of the structural changes and driving forces of the CTD dimerization that take place in mutant forms. Here, we compare the structural stability, conformational dynamics, and association force of the CTD dimers for both wild-type and mutated sequences using all-atom explicit-solvent molecular dynamics (MD). The simulations show that Q155N and E159D at the major homology region (MHR) and W184A and M185A at the helix 2 region are energetically less favorable than the wild-type, imposing profound negative effects on intermolecular CA−CA dimerization. Detailed structural analysis shows that three mutants (Q155N, E159D, and W184A) display much more flexible local structures and weaker CA−CA association than the wild-type, primarily due to the loss of interactions (hydrogen bonds, side chain hydrophobic contacts, and π-stacking) with their neighboring residues. Most interestingly, the MHR that is far from the interacting dimeric interface is more sensitive to the mutations than the helix 2 region that is located at the CA−CA dimeric interface, indicating that structural changes in the distinct motif of the CA could similarly allosterically prevent the CA capsid formation. In addition, the structural and free energy comparison of the five residues shorter CA (151−231) dimer with the CA (146−231) dimer further indicates that hydrophobic interactions, side chain packing, and hydrogen bonds are the major, dominant driving forces in stabilizing the CA interface. PMID:19199580

  16. Paget disease of bone-associated UBA domain mutations of SQSTM1 exert distinct effects on protein structure and function

    Science.gov (United States)

    Goode, Alice; Long, Jed E.; Shaw, Barry; Ralston, Stuart H.; Visconti, Micaela Rios; Gianfrancesco, Fernando; Esposito, Teresa; Gennari, Luigi; Merlotti, Daniela; Rendina, Domenico; Rea, Sarah L.; Sultana, Melanie; Searle, Mark S.; Layfield, Robert

    2014-01-01

    SQSTM1 mutations are common in patients with Paget disease of bone (PDB), with most affecting the C-terminal ubiquitin-associated (UBA) domain of the SQSTM1 protein. We performed structural and functional analyses of two UBA domain mutations, an I424S mutation relatively common in UK PDB patients, and an A427D mutation associated with a severe phenotype in Southern Italian patients. Both impaired SQSTM1's ubiquitin-binding function in pull-down assays and resulted in activation of basal NF-κB signalling, compared to wild-type, in reporter assays. We found evidence for a relationship between the ability of different UBA domain mutants to activate NF-κB signalling in vitro and number of affected sites in vivo in 1152 PDB patients from the UK and Italy, with A427D-SQSTM1 producing the greatest level of activation (relative to wild-type) of all PDB mutants tested to date. NMR and isothermal titration calorimetry studies were able to demonstrate that I424S is associated with global structural changes in the UBA domain, resulting in 10-fold weaker UBA dimer stability than wild-type and reduced ubiquitin-binding affinity of the UBA monomer. Our observations provide insights into the role of SQSTM1-mediated NF-κB signalling in PDB aetiology, and demonstrate that different mutations in close proximity within loop 2/helix 3 of the SQSTM1 UBA domain exert distinct effects on protein structure and stability, including indirect effects at the UBA/ubiquitin-binding interface. PMID:24642144

  17. Directed mutagenesis of the strongly conserved aspartate 242 in the beta-subunit of Escherichia coli proton-ATPase.

    Science.gov (United States)

    Al-Shawi, M K; Parsonage, D; Senior, A E

    1988-12-25

    Oligonucleotide-directed mutagenesis was used to substitute Asn or Val for residue Asp-242 in the beta-subunit of Escherichia coli F1-ATPase. Asp-242 is strongly conserved in beta-subunits of F1-ATPase enzymes, in a region of sequence which shows homology with numerous nucleotide-binding proteins. By analogy with adenylate kinase (Fry, D.C., Kuby, S.A., and Mildvan, A.S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 907-911), beta-Asp-242 of F1-ATPase might participate in catalysis through electrostatic effects on the substrate Mg2+ or through hydrogen bonding to the substrate(s); an acid-base catalytic role is also plausible. The substitutions Asn and Val were chosen to affect the charge, hydrogen-bonding ability, and hydrophobicity of residue beta-Asp-242. Both mutations significantly impaired oxidative phosphorylation rates in vivo and membrane ATPase and ATP-driven proton-pumping activities in vitro. Asn-242 was more detrimental than Val-242. Purified soluble mutant F1-ATPases had normal molecular size and subunit composition, and displayed 7% (beta-Asn-242) and 17% (beta-Val-242) of normal specific Mg-ATPase activity. The relative MgATPase activities of both mutant enzymes showed similar pH dependence to normal. Relative MgATPase and CaATPase activities of normal and mutant enzymes were compared at widely varied pMg and pCa. The mutations had little effect on KM MgATP, but KM CaATP was reduced. The data showed that the carboxyl side-chain of beta-Asp-242 is not involved in catalysis either as a general acid-base catalyst or through direct involvement in any protonation/deprotonation-linked mechanism, nor is it likely to be directly involved in liganding to substrate Mg2+ during the reaction. Specificity constants (kcat/KM) for MgATP and CaATP were reduced in both mutant enzymes, showing that the mutations destabilized interactions between the catalytic nucleotide-binding domain and the transition state.

  18. Thermodynamics of the ATPase cycle of GlcV, the nucleotide-binding domain of the glucose ABC transporter of Sulfolobus solfataricus

    NARCIS (Netherlands)

    Pretz, Monika G.; Albers, Sonja-Verena; Schuurman-Wolters, Gea; Tampe, Robert; Driessen, Arnold J. M.; van der Does, Chris

    2006-01-01

    ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains ( NBDs) that energize the

  19. Disease-associated mutations in the actin-binding domain of filamin B cause cytoplasmic focal accumulations correlating with disease severity

    DEFF Research Database (Denmark)

    Daniel, Philip B; Morgan, Tim; Alanay, Yasemin;

    2012-01-01

    repeats surrounding the flexible hinge 1 region of the FLNB rod domain. Despite being positioned in domains that bind actin, it is unknown if these mutations perturb cytoskeletal structure. Expression of several full-length FLNB constructs containing ABD mutations resulted in the appearance of actin...

  20. Mutational analysis of the GAP-related domain of the neurofibromatosis type 1 gene in Brazilian NF1 patients

    Directory of Open Access Journals (Sweden)

    Alessandra B. Trovó

    2004-01-01

    Full Text Available Neurofibromatosis type 1 (NF1 is a common autosomal dominant disorder caused by mutations in the NF1 gene. In the present study, a total of 55 unrelated NF1 patients were screened for mutations in the GAP-related domain/GRD (exons 20-27a by single-strand conformation polymorphism (SSCP. Four different mutations were identified and, taken together, they comprise one nonsense substitution (Q1189X, one deletion (3525-3526delAA, one missense substitution (E1356G and one mutation in the splice acceptor site (c.4111-1G>A. One novel polymorphism (c.4514+11C>G and other three putative polymorphisms were also found (c.3315-27G>A, V1146I and V1317A. Genotype-phenotype correlations were investigated, but no particular association was detected.

  1. Regulation of V-ATPase assembly and function of V-ATPases in tumor cell invasiveness.

    Science.gov (United States)

    McGuire, Christina; Cotter, Kristina; Stransky, Laura; Forgac, Michael

    2016-08-01

    V-ATPases are ATP-driven proton pumps that function within both intracellular compartments and the plasma membrane in a wide array of normal physiological and pathophysiological processes. V-ATPases are composed of a peripheral V(1) domain that hydrolyzes ATP and an integral V(0) domain that transports protons. Regulated assembly of the V-ATPase represents an important mechanism of regulating V-ATPase activity in response to a number of environmental cues. Our laboratory has demonstrated that glucose-dependent assembly of the V-ATPase complex in yeast is controlled by the Ras/cAMP/PKA pathway. By contrast, increased assembly of the V-ATPase during dendritic cell maturation involves the PI-3 kinase and mTORC1 pathways. Recently, we have shown that amino acids regulate V-ATPase assembly in mammalian cells, possibly as a means to maintain adequate levels of amino acids upon nutrient starvation. V-ATPases have also been implicated in cancer cell survival and invasion. V-ATPases are targeted to different cellular membranes by isoforms of subunit a, with a3 targeting V-ATPases to the plasma membrane of osteoclasts. We have shown that highly invasive human breast cancer cell lines express higher levels of the a3 isoform than poorly invasive lines and that knockdown of a3 reduces both expression of V-ATPases at the plasma membrane and in vitro invasion of breast tumor cells. Moreover, overexpression of a3 in a non-invasive breast epithelial line increases both plasma membrane V-ATPases and in vitro invasion. Finally, specific ablation of plasma membrane V-ATPases in highly invasive human breast cancer cells using either an antibody or small molecule approach inhibits both in vitro invasion and migration. These results suggest that plasma membrane and a3-containing V-ATPases represent a novel and important target in the development of therapeutics to limit breast cancer metastasis. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics

  2. Mutations to the cardiotonic steroid binding site of Na(+)/K(+)-ATPase are associated with high level of resistance to gamabufotalin in a natricine snake.

    Science.gov (United States)

    Mohammadi, Shabnam; Brodie, Edmund D; Neuman-Lee, Lorin A; Savitzky, Alan H

    2016-05-01

    Although toads are defended by bufadienolide toxins, some snakes have evolved resistance to bufadienolides and feed heavily on toads. We compared resistance in Nerodia rhombifer, which possesses mutations that confer target-site resistance, to Pituophis catenifer, which lacks those mutations. Even at the highest dosage tested, Nerodia showed no effects, whereas the lowest dose was lethal to Pituophis. Our results demonstrate a striking level of resistance to bufadienolides in a species possessing the mutations for resistance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. A mutation in the V1 end domain of keratin 1 in non-epidermolytic palmar-plantar keratoderma.

    Science.gov (United States)

    Kimonis, V; DiGiovanna, J J; Yang, J M; Doyle, S Z; Bale, S J; Compton, J G

    1994-12-01

    Mutations in keratin 9 have been found in families with an epidermolytic form of palmar-plantar keratoderma (PPK). In another form of PPK (Unna-Thost type), epidermolysis is not observed histologically. We studied a pedigree with this non-epidermolytic form of PPK. By gene linkage analysis, the type I keratin locus could be excluded but complete linkage with the type II keratin region was found. Sequence analysis identified a single base change in the amino-terminal V1 variable subdomain of keratin 1, which caused a lysine to isoleucine substitution. This non-conservative mutation completely cosegregated with the disease and was not observed in 50 unrelated unaffected individuals. An examination of keratin amino-terminal sequences revealed a previously unreported 22-residue window in the V1 subdomain that is conserved among most type II keratins. The altered lysine is an invariant residue in this conserved sequence. Previously described keratin mutations affect the central regions important for filament assembly and stability, and cause diseases characterized by cellular degeneration or disruption. This is the first disease mutation in a keratin chain variable end region. The observation that it is not associated with epidermolysis supports the concept that the amino-terminal domain of keratins may be involved in supramolecular interactions of keratin filaments rather than stability. Therefore, hyperkeratosis associated with this mutation may be due to perturbations in the interactions of the keratin end domain with other cellular components.

  4. Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)*

    Science.gov (United States)

    Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I.; Hill, Christopher P.

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. PMID:25833946

  5. Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs).

    Science.gov (United States)

    Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I; Hill, Christopher P

    2015-05-22

    The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations

    Science.gov (United States)

    Scott, John W.; Hawley, Simon A.; Green, Kevin A.; Anis, Miliea; Stewart, Greg; Scullion, Gillian A.; Norman, David G.; Hardie, D. Grahame

    2004-01-01

    CBS domains are defined as sequence motifs that occur in several different proteins in all kingdoms of life. Although thought to be regulatory, their exact functions have been unknown. However, their importance was underlined by findings that mutations in conserved residues within them cause a variety of human hereditary diseases, including (with the gene mutated in parentheses): Wolff-Parkinson-White syndrome (γ2 subunit of AMP-activated protein kinase); retinitis pigmentosa (IMP dehydrogenase-1); congenital myotonia, idiopathic generalized epilepsy, hypercalciuric nephrolithiasis, and classic Bartter syndrome (CLC chloride channel family members); and homocystinuria (cystathionine β-synthase). AMP-activated protein kinase is a sensor of cellular energy status that is activated by AMP and inhibited by ATP, but the location of the regulatory nucleotide-binding sites (which are prime targets for drugs to treat obesity and diabetes) was not characterized. We now show that tandem pairs of CBS domains from AMP-activated protein kinase, IMP dehydrogenase-2, the chloride channel CLC2, and cystathionine β-synthase bind AMP, ATP, or S-adenosyl methionine,while mutations that cause hereditary diseases impair this binding. This shows that tandem pairs of CBS domains act, in most cases, as sensors of cellular energy status and, as such, represent a newly identified class of binding domain for adenosine derivatives. PMID:14722619

  7. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

    Science.gov (United States)

    Zwick, Matthias; Esposito, Cinzia; Hellstern, Manuel; Seelig, Anna

    2016-07-08

    The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators.

  8. General interaction mode of CIDE:CIDE complex revealed by a mutation study of the Drep2 CIDE domain.

    Science.gov (United States)

    Lee, Seung Mi; Park, Hyun Ho

    2013-04-02

    The CIDE domain is a well known protein-protein interaction module that is initially detected at the apoptotic DNA fragmentation factor (DFF40/45). The interaction mechanism via the CIDE domain is not well understood. To elucidate CIDE domain mediated interactions in the apoptotic DNA fragmentation system, we conducted biochemical and mutational studies and found that the surface of CIDE domains can be divided into an acidic side and a basic side. In addition, a mutagenesis study revealed that the basic surface side of Drep2 CIDE is involved in the interaction with the acidic surface side of Drep1 CIDE and Drep3 CIDE. Our research supports the idea that a charge-charge interaction might be the general interaction mode of the CIDE:CIDE interaction.

  9. Deletion and deletion/insertion mutations in the juxtamembrane domain of the FLT3 gene in adult acute myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Kristin K. Deeb

    2014-01-01

    Full Text Available In contrast to FLT3 ITD mutations, in-frame deletions in the FLT3 gene have rarely been described in adult acute leukemia. We report two cases of AML with uncommon in-frame mutations in the juxtamembrane domain of the FLT3 gene: a 3-bp (c.1770_1774delCTACGinsGT; p.F590_V592delinsLF deletion/insertion and a 12-bp (c.1780_1791delTTCAGAGAATAT; p.F594_Y597del deletion. We verified by sequencing that the reading frame of the FLT3 gene was preserved and by cDNA analysis that the mRNA of the mutant allele was expressed in both cases. Given the recent development of FLT3 inhibitors, our findings may be of therapeutic value for AML patients harboring similar FLT3 mutations.

  10. Comparison of current docking tools for the simulation of inhibitor binding by the transmembrane domain of the sarco/endoplasmic reticulum calcium ATPase

    Science.gov (United States)

    Lape, Michael; Elam, Christopher; Paula, Stefan

    2010-01-01

    Inhibitors of the transmembrane protein sarco/endoplasmic reticulum calcium ATPase (SERCA) are invaluable tools for the study of the enzyme’s physiological functions and they have been recognized as a promising new class of anticancer agents. For the discovery of novel enzyme inhibitors, small molecule docking for virtual screens of large compound libraries has become increasingly important. Since the performance of various docking routines varies considerably, depending on the target and the chemical nature of the ligand, we critically evaluated the performance of four frequently used programs – GOLD, AutoDock, Surflex-Dock, and FRED – for the docking of SERCA inhibitors based on the structures of thapsigargin, di-tert-butylhydroquinone, and cyclopiazonic acid. Evaluation criteria were docking accuracy using crystal structures as references, docking reproducibility, and correlation between docking scores and known bioactivities. The best overall results were obtained by GOLD and FRED. Docking runs with conformationally flexible binding sites produced no significant improvement of the results. PMID:20167416

  11. Oncogenic CARD11 mutations induce hyperactive signaling by disrupting autoinhibition by the PKC-responsive inhibitory domain.

    Science.gov (United States)

    Lamason, Rebecca L; McCully, Ryan R; Lew, Stefanie M; Pomerantz, Joel L

    2010-09-28

    The regulated activation of NF-κB by antigen receptor signaling is required for normal B and T lymphocyte activation during the adaptive immune response. Dysregulated NF-κB activation is associated with several types of lymphoma, including diffuse large B cell lymphoma (DLBCL). During normal antigen receptor signaling, the multidomain scaffold protein CARD11 undergoes a transition from a closed, inactive state to an open, active conformation that recruits several signaling proteins into a complex, leading to IKK kinase activation. This transition is regulated by the CARD11 inhibitory domain (ID), which participates in intramolecular interactions that prevent cofactor binding to CARD11 prior to signaling, but which is neutralized after receptor engagement by phosphorylation. Several oncogenic CARD11 mutations have been identified in DLBCL that enhance activity and that are mostly found in the coiled-coil domain. However, the mechanisms by which these mutations cause CARD11 hyperactivity and spontaneous NF-κB activation are poorly understood. In this report, we provide several lines of evidence that oncogenic mutations F123I and L225LI induce CARD11 hyperactivity by disrupting autoinhibition by the CARD11 ID. These mutations disrupt ID-mediated intramolecular interactions and ID-dependent inhibition and bypass the requirement for ID phosphorylation during T cell receptor signaling. Intriguingly, these mutations selectively enhance the apparent affinity of CARD11 for Bcl10, but not for other signaling proteins that are recruited to CARD11 in an ID-dependent manner during normal antigen receptor signaling. Our results establish a mechanism that explains how DLBCL-associated mutations in CARD11 can initiate spontaneous, receptor-independent activation of NF-κB.

  12. A frequent kinase domain mutation that changes the interaction between PI3K[alpha] and the membrane

    Energy Technology Data Exchange (ETDEWEB)

    Mandelker, Diana; Gabelli, Sandra B.; Schmidt-Kittler, Oleg; Zhu, Jiuxiang; Cheong, Ian; Huang, Chuan-Hsiang; Kinzler, Kenneth W.; Vogelstein, Bert; Amzel, L. Mario; (JHU-MED); (HHMI)

    2009-12-01

    Mutations in oncogenes often promote tumorigenesis by changing the conformation of the encoded proteins, thereby altering enzymatic activity. The PIK3CA oncogene, which encodes p110{alpha}, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3K{alpha}), is one of the two most frequently mutated oncogenes in human cancers. We report the structure of the most common mutant of p110{alpha} in complex with two interacting domains of its regulatory partner (p85{alpha}), both free and bound to an inhibitor (wortmannin). The N-terminal SH2 (nSH2) domain of p85{alpha} is shown to form a scaffold for the entire enzyme complex, strategically positioned to communicate extrinsic signals from phosphopeptides to three distinct regions of p110{alpha}. Moreover, we found that Arg-1047 points toward the cell membrane, perpendicular to the orientation of His-1047 in the WT enzyme. Surprisingly, two loops of the kinase domain that contact the cell membrane shift conformation in the oncogenic mutant. Biochemical assays revealed that the enzymatic activity of the p110{alpha} His1047Arg mutant is differentially regulated by lipid membrane composition. These structural and biochemical data suggest a previously undescribed mechanism for mutational activation of a kinase that involves perturbation of its interaction with the cellular membrane.

  13. Mutational analysis of Raf-1 cysteine rich domain: requirement for a cluster of basic aminoacids for interaction with phosphatidylserine.

    Science.gov (United States)

    Improta-Brears, T; Ghosh, S; Bell, R M

    1999-08-01

    Activation of Raf-1 kinase is preceded by a translocation of Raf-1 to the plasma membrane in response to external stimuli. The membrane localization of Raf-1 is facilitated through its interaction with activated Ras and with membrane phospholipids. Previous evidence suggests that the interaction of Raf-1 with Ras is mediated by two distinct domains within the N-terminal region of Raf-1 comprising amino acid residues 51-131 and residues 139-184, the latter of which codes for a zinc containing cysteine-rich domain. The cysteine-rich domain of Raf-1 is also reported to associate with other proteins, such as 14-3-3, and for selectively binding acidic phospholipids, particularly phosphatidylserine (PS). In the present study, we have investigated the consequences of progressive deletions and point mutations within the cysteine-rich domain of Raf-1 on its ability to bind PS. A reduced interaction with PS was observed in vitro for all deletion mutants of Raf-1 expressed either as full-length proteins or as fragments containing the isolated cysteine-rich domain. In particular, the cluster of basic amino acids R143, K144, and K148 appeared to be critical for interaction with PS, since substitution of all three residues to alanine resulted in a protein that failed to interact with liposomes enriched for PS. Expression of Raf-1 in vivo, containing point mutations in the cysteine-rich domain resulted in a truncated polypeptide that lacked both the Ras and PS binding sites and could no longer translocate to the plasma membrane upon serum stimulation. These results indicate that the basic residues 143, 144 and 148 in the anterior half of Raf-1 cysteine-rich domain play a role in the association with the lipid bilayer and possibly in protein stability, therefore they might contribute to Raf-1 localization and subsequent activation.

  14. A Novel Mutation in the Pyrin Domain of the NOD-like Receptor Family Pyrin Domain Containing Protein 3 in Muckle-Wells Syndrome

    Science.gov (United States)

    Hu, Jian; Zhu, Yun; Zhang, Jian-Zhong; Zhang, Rong-Guang; Li, Hou-Min

    2017-01-01

    Background: Cryopyrin-associated periodic syndrome (CAPS) is a group of rare, heterogeneous autoinflammatory disease characterized by interleukin (IL)-1β-mediated systemic inflammation and clinical symptoms involving skin, joints, central nervous system, and eyes. It encompasses a spectrum of three clinically overlapping autoinflammatory syndromes including familial cold autoinflammatory syndrome, Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease. CAPS is associated with gain-of-function missense mutations in NOD-like receptor family pyrin domain-containing protein 3 (NLRP3), the gene encoding NLRP3. Moreover, most mutations leading to MWS occurred in exon 3 of NLRP3 gene. Here, we reported a novel mutation occurred in exon 1 of NLRP3 gene in an MWS patient and attempted to explore the pathogenic mechanism. Methods: Genetic sequence analysis of NLRP3 was performed in an MWS patient who presented with periodic fever, arthralgia, and multiform skin lesions. NLRP3 was also analyzed in this patient's parents and 50 healthy individuals. Clinical examinations including X-ray examination, skin biopsy, bone marrow aspiration smear, and blood test of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), serum levels of IL-1β, immunoglobulin E (IgE), antineutrophil cytoplasmic antibodies, antinuclear antibodies, and extractable nuclear antigen were also analyzed. The protein structure of mutant NLRP3 inflammasome was calculated by SWISS-MODEL software. Proteins of wild type and mutant components of NLRP3 inflammasome were expressed and purified, and the interaction abilities between these proteins were tested by surface plasmon resonance (SPR) assay. Results: X-ray examination showed no abnormality in the patient's knees. Laboratory tests indicated an elevation of CRP (233.24 mg/L) and ESR (67 mm/h) when the patient had fever. Serum IL-1β increased to 24.37 pg/ml, and serum IgE was higher than 2500.00 IU/ml. Other blood tests were

  15. Random domain name and address mutation (RDAM) for thwarting reconnaissance attacks

    National Research Council Canada - National Science Library

    Kai Wang; Xi Chen; Yuefei Zhu

    2017-01-01

    ... on the host’s static domain name to map to its dynamic address; therefore these methods cannot effectively defend against random scanning attacks, and cannot defend against an attacker who knows the target’s domain name...

  16. Mutations in the 1A rod domain segment of the keratin 9 gene in epidermolytic palmoplantar keratoderma.

    Science.gov (United States)

    Yang, J M; Lee, S; Kang, H J; Lee, J H; Yeo, U C; Son, I Y; Park, K B; Steinert, P M; Lee, E S

    1998-11-01

    Palmoplantar keratodermas (PPK) constitute a heterogeneous group of diseases marked by the thickening of palms and soles of affected individuals. They are divided into autosomal dominant and autosomal recessive groups by the mode of transmission. The autosomal dominantly transmitted group is further divided into epidermolytic (EPPK, Voerner) and non-epidermolytic (NEPPK, Unna-Thost) types according to the histopathologic findings. Recent development of molecular approaches has confirmed that EPPK and NEPPK are caused by the mutations in keratin 9 and 1 genes, respectively. We have studied three families of EPPK to find the mutation in the keratin 9 gene. DNA sequence analyses revealed single base changes in sequences encoding the highly conserved 1A rod domain segment of the keratin 9 gene in two of the three families. These mutations caused Arg (CGG) to Glu (CAG; R162Q) and Arg (CGG) to Try (TGG; R162W) substitutions. The same arginine position has been mutated in the keratin 10 gene in epidermolytic hyperkeratosis, the keratin 14 gene in epidermolysis bullosa simplex, and the keratin 9 gene in hereditary EPPK in Western patients. In this study we show that unrelated Korean patients have similar mutations.

  17. Hereditary angioedema in a Jordanian family with a novel missense mutation in the C1-inhibitor N-terminal domain.

    Science.gov (United States)

    Jaradat, Saied A; Caccia, Sonia; Rawashdeh, Rifaat; Melhem, Motasem; Al-Hawamdeh, Ali; Carzaniga, Thomas; Haddad, Hazem

    2016-03-01

    Hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) is an autosomal dominant disease caused by mutations in the SERPING1 gene. A Jordanian family, including 14 individuals with C1-INH-HAE clinical symptoms, was studied. In the propositus and his parents, SERPING1 had four mutations leading to amino acid substitutions. Two are known polymorphic variants (c.167T>C; p.Val34Ala and c.1438G>A; p.Val458Met), the others are newly described. One (c.203C>T; p.Thr46Ile) is located in the N-terminal domain of the C1-inhibitor protein and segregates with angioedema symptoms in the family. The other (c.800C>T; p.Ala245Val) belongs to the serpin domain, and derives from the unaffected father. DNA from additional 24 family members were screened for c.203C>T mutation in the target gene. All individuals heterozygous for the c.203C>T mutation had antigenic and functional plasma levels of C1-inhibitor below 50% of normal, confirming the diagnosis of type I C1-INH-HAE. Angioedema symptoms were present in 14 of 16 subjects carrier for the c.203T allele. Among these subjects, those carrying the c.800T variation had more severe and frequent symptoms than subjects without this mutation. This family-based study provides the first evidence that multiple amino acid substitutions in SERPING1 could influence C1-INH-HAE phenotype.

  18. Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain.

    Science.gov (United States)

    Barfoot, Tasida; Herdendorf, Timothy J; Behning, Bryanna R; Stohr, Bradley A; Gao, Yang; Kreuzer, Kenneth N; Nelson, Scott W

    2015-09-25

    Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks. Rad50 contains an anti-parallel coiled-coil with two absolutely conserved cysteine residues at its apex. These cysteine residues serve as a dimerization domain and bind a Zn(2+) cation in a tetrathiolate coordination complex known as the zinc-hook. Mutation of the zinc-hook in bacteriophage T4 is lethal, indicating the ability to bind Zn(2+) is critical for the functioning of the MR complex. In vitro, we found that complex formation between Rad50 and a peptide corresponding to the C-terminal domain of Mre11 enhances the ATPase activity of Rad50, supporting the hypothesis that the coiled-coil is a major conduit for communication between Mre11 and Rad50. We constructed mutations to perturb this domain in the bacteriophage T4 Rad50 homolog. Deletion of the Rad50 coiled-coil and zinc-hook eliminates Mre11 binding and ATPase activation but does not affect its basal activity. Mutation of the zinc-hook or disruption of the coiled-coil does not affect Mre11 or DNA binding, but their activation of Rad50 ATPase activity is abolished. Although these mutants excise a single nucleotide at a normal rate, they lack processivity and have reduced repetitive exonuclease rates. Restricting the mobility of the coiled-coil eliminates ATPase activation and repetitive exonuclease activity, but the ability to support single nucleotide excision is retained. These results suggest that the coiled-coiled domain adopts at least two conformations throughout the ATPase/nuclease cycle, with one conformation supporting enhanced ATPase activity and processivity and the other supporting nucleotide excision.

  19. Mutational analysis of the structure basis for the multimerization function of NifA central domain

    Institute of Scientific and Technical Information of China (English)

    YANG; Chengtao; (杨成涛); YU; Guanqiao; (俞冠翘); SHEN; Shanjiong; (San-Chiun; Shen,; 沈善炯); ZHU; Jiabi; (朱家璧)

    2001-01-01

    In Klebsiella pneumoniae (Kp) NifA central domain, when the conservative amino acid residue Thr-290 in C3 region was replaced by Val, the function of NifA for activating the transcription of nif genes was lost. Thus the conservative Thr-290 residue seems critical for the activation function of NifA central domain. This point mutant of NifA central domain is used to examine the putative multimerization function of NifA central domain by merodiploid experiment. The results showed that the NifA central domain bore the multimerization determinants of NifA protein. A series of truncated mutants of NifA were constructed to determine the structural elements at the central domain critical for multimerization. It demonstrates that amino acid residues 252-453 are involved in the multimerization function of NifA central domain.

  20. Mutational analysis of the structure basis for the multimerization function of NifA central domain

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In Klebsiella pneumoniae (Kp) NifA central domain, when theconservative amino acid residue Thr-290 in C3 region was replaced by Val, the function of NifA for activating the transcription of nif genes was lost. Thus the conservative Thr-290 residue seems critical for the activation function of NifA central domain. This point mutant of NifA central domain is used to examine the putative multimerization function of NifA central domain by merodiploid experiment. The results showed that the NifA central domain bore the multimerization determinants of NifA protein. A series of truncated mutants of NifA were constructed to determine the structural elements at the central domain critical for multimerization. It demonstrates that amino acid residues 252-453 are involved in the multimerization function of NifA central domain.

  1. Suramin inhibits Hsp104 ATPase and disaggregase activity.

    Directory of Open Access Journals (Sweden)

    Mariana P Torrente

    Full Text Available Hsp104 is a hexameric AAA+ protein that utilizes energy from ATP hydrolysis to dissolve disordered protein aggregates as well as amyloid fibers. Interestingly, Hsp104 orthologues are found in all kingdoms of life except animals. Thus, Hsp104 could represent an interesting drug target. Specific inhibition of Hsp104 activity might antagonize non-metazoan parasites that depend on a potent heat shock response, while producing little or no side effects to the host. However, no small molecule inhibitors of Hsp104 are known except guanidinium chloride. Here, we screen over 16,000 small molecules and identify 16 novel inhibitors of Hsp104 ATPase activity. Excluding compounds that inhibited Hsp104 activity by non-specific colloidal effects, we defined Suramin as an inhibitor of Hsp104 ATPase activity. Suramin is a polysulphonated naphthylurea and is used as an antiprotozoal drug for African Trypanosomiasis. Suramin also interfered with Hsp104 disaggregase, unfoldase, and translocase activities, and the inhibitory effect of Suramin was not rescued by Hsp70 and Hsp40. Suramin does not disrupt Hsp104 hexamers and does not effectively inhibit ClpB, the E. coli homolog of Hsp104, establishing yet another key difference between Hsp104 and ClpB behavior. Intriguingly, a potentiated Hsp104 variant, Hsp104A503V, is more sensitive to Suramin than wild-type Hsp104. By contrast, Hsp104 variants bearing inactivating sensor-1 mutations in nucleotide-binding domain (NBD 1 or 2 are more resistant to Suramin. Thus, Suramin depends upon ATPase events at both NBDs to exert its maximal effect. Suramin could develop into an important mechanistic probe to study Hsp104 structure and function.

  2. Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase.

    Science.gov (United States)

    Wang, Li Kai; Nair, Pravin A; Shuman, Stewart

    2008-08-22

    NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.

  3. Mutations in nonconserved domains of Ty3 integrase affect multiple stages of the Ty3 life cycle.

    Science.gov (United States)

    Nymark-McMahon, M H; Sandmeyer, S B

    1999-01-01

    Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3' ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3' ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle.

  4. The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination

    DEFF Research Database (Denmark)

    Burgess, Rebecca C; Sebesta, Marek; Sisakova, Alexandra

    2013-01-01

    Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54...... and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage...

  5. Variable pathogenic potentials of mutations located in the desmin alpha-helical domain

    NARCIS (Netherlands)

    B. Goudeau; F. Rodrigues-Lima; D. Fischer; M. Casteras-Simon; N. Sambuughin; M. de Visser; P. Laforet; X. Ferrer; F. Chapon; G. Sjoberg; A. Kostareva; T. Sejersen; M.C. Dalakas; L.G. Goldfarb; P. Vicart

    2006-01-01

    Mutations in the desmin gene have been recognized as a cause of desminopathy, a familial or sporadic disorder characterized by skeletal muscle weakness, often associated with cardiomyopathy or respiratory insufficiency. Distinctive histopathologic features include aberrant intracytoplasmic accumulat

  6. The Nonlinear Structure of the Desmoplakin Plakin Domain and the Effects of Cardiomyopathy-Linked Mutations

    NARCIS (Netherlands)

    C. Al-Jassar; T. Knowles; M. Jeeves; K. Kami; E. Behr; H. Bikker; M. Overduin; M. Chidgey

    2011-01-01

    Desmoplakin is a cytoplasmic desmosomal protein that plays a vital role in normal intercellular adhesion. Mutations in desmoplakin can result in devastating skin blistering diseases and arrhythmogenic right ventricular cardiomyopathy, a heart muscle disorder associated with ventricular arrhythmias,

  7. Evolution of copper transporting ATPases in eukaryotic organisms.

    Science.gov (United States)

    Gupta, Arnab; Lutsenko, Svetlana

    2012-04-01

    Copper is an essential nutrient for most life forms, however in excess it can be harmful. The ATP-driven copper pumps (Copper-ATPases) play critical role in living organisms by maintaining appropriate copper levels in cells and tissues. These evolutionary conserved polytopic membrane proteins are present in all phyla from simplest life forms (bacteria) to highly evolved eukaryotes (Homo sapiens). The presumed early function in metal detoxification remains the main function of Copper-ATPases in prokaryotic kingdom. In eukaryotes, in addition to removing excess copper from the cell, Copper-ATPases have another equally important function - to supply copper to copper dependent enzymes within the secretory pathway. This review focuses on the origin and diversification of Copper ATPases in eukaryotic organisms. From a single Copper ATPase in protozoans, a divergence into two functionally distinct ATPases is observed with the evolutionary appearance of chordates. Among the key functional domains of Copper-ATPases, the metal-binding N-terminal domain could be responsible for functional diversification of the copper ATPases during the course of evolution.

  8. Insights into the folding and unfolding processes of wild-type and mutated SH3 domain by molecular dynamics and replica exchange molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Wen-Ting Chu

    Full Text Available Src-homology regions 3 (SH3 domain is essential for the down-regulation of tyrosine kinase activity. Mutation A39V/N53P/V55L of SH3 is found to be relative to the urgent misfolding diseases. To gain insight, the human and gallus SH3 domains (PDB ID: 1NYG and 2LP5, including 58 amino acids in each protein, were selected for MD simulations (Amber11, ff99SB force field and cluster analysis to investigate the influence of mutations on the spatial structure of the SH3 domain. It is found that the large conformational change of mutations mainly exists in three areas in the vicinity of protein core: RT loop, N-src loop, distal β-hairpin to 310 helix. The C-terminus of the mutated gallus SH3 is disordered after simulation, which represents the intermediate state of aggregation. The disappeared strong Hbond net in the mutated human and gallus systems will make these mutated proteins looser than the wild-type proteins. Additionally, by performing the REMD simulations on the gallus SH3 domain, the mutated domain is found to have an obvious effect on the unfolding process. These studies will be helpful for further aggregation mechanisms investigations on SH3 family.

  9. Mutation in S6 domain of HCN4 channel in patient with suspected Brugada syndrome modifies channel function.

    Science.gov (United States)

    Biel, Stephanie; Aquila, Marco; Hertel, Brigitte; Berthold, Anne; Neumann, Thomas; DiFrancesco, Dario; Moroni, Anna; Thiel, Gerhard; Kauferstein, Silke

    2016-10-01

    Diseases such as the sick sinus and the Brugada syndrome are cardiac abnormalities, which can be caused by a number of genetic aberrances. Among them are mutations in HCN4, a gene, which encodes the hyperpolarization-activated, cyclic nucleotide-gated ion channel 4; this pacemaker channel is responsible for the spontaneous activity of the sinoatrial node. The present genetic screening of patients with suspected or diagnosed Brugada or sick sinus syndrome identified in 1 out of 62 samples the novel mutation V492F. It is located in a highly conserved site of hyperpolarization-activated cyclic nucleotide-gated (HCN)4 channel downstream of the filter at the start of the last transmembrane domain S6. Functional expression of mutant channels in HEK293 cells uncovered a profoundly reduced channel function but no appreciable impact on channel synthesis and trafficking compared to the wild type. The inward rectifying HCN4 current could be partially rescued by an expression of heteromeric channels comprising wt and mutant monomers. These heteromeric channels were responsive to cAMP but they required a more negative voltage for activation and they exhibited a lower current density than the wt channel. This suggests a dominant negative effect of the mutation in patients, which carry this heterozygous mutation. Such a modulation of HCN4 activity could be the cause of the diagnosed cardiac abnormality.

  10. The H{sub 1}–H{sub 2} domain of the α{sub 1} isoform of Na{sup +}–K{sup +}–ATPase is involved in ouabain toxicity in rat ventricular myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Chen; Li, Jun-xia; Guo, Hui-cai; Zhang, Li-nan; Guo, Wei; Meng, Jing; Wang, Yong-li, E-mail: wangyongli@gmail.com

    2012-07-01

    The composition of different isoforms of Na{sup +}-K{sup +}-ATPase (NKA, Na/K pump) in ventricular myocytes is an important factor in determining the therapeutic effect and toxicity of cardiac glycosides (CGs) on heart failure. The mechanism whereby CGs cause these effects is still not completely clear. In the present study, we prepared two site-specific antibodies (SSA78 and WJS) against the H{sub 1}–H{sub 2} domain of α{sub 1} and α{sub 2} isoforms of NKA in rat heart, respectively, and compared their influences on the effect of ouabain (OUA) in isolated rat ventricular myocytes. SSA78 or WJS, which can specifically bind with the α{sub 1} or α{sub 2} isoform, were assessed with enzyme linked immunosorbent assay (ELISA), Western blot and immunofluorescent staining methods. Preincubation of myocytes with SSA78 inhibited low OUA affinity pump current but not high OUA affinity pump current, reduced the rise in cytosolic calcium concentration ([Ca{sup 2+}]{sub i}), attenuated mitochondrial Ca{sup 2+} overload, restored mitochondrial membrane potential reduction, and delayed the decrease of the myocardial contractile force as well as the occurrence of arrhythmic contraction induced by high concentrations (1 mM) but not low concentrations (1 μM) of OUA. Similarly, preincubation of myocytes with WJS inhibited high OUA affinity pump current, reduced the increase of [Ca{sup 2+}]{sub i} and the contractility induced by 1 μM but not that induced by 1 mM OUA. These results indicate that the H{sub 1}–H{sub 2} domain of the NKA α{sub 1} isoform mediates OUA-induced cardiac toxicity in rat ventricular myocytes, and inhibitors for this binding site may be used as an adjunct to CGs treatment for cardiovascular disease. -- Highlights: ► We prepared two antibodies against the H{sub 1}-H{sub 2} domain of α{sub 1} and α{sub 2} isoforms of NKA. ► The H{sub 1}-H{sub 2} domain of the NKA α{sub 1} isoform mediates OUA-induced cardiac toxicity. ► The H{sub 1}-H{sub 2

  11. Essential Role for Pro(21) in Phospholamban for Optimal Inhibition of the Ca-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jinhui; Boschek, Curt B.; Xiong, Yijia; Sacksteder, Colette A.; Squier, Thomas C.; Bigelow, Diana J.

    2005-12-13

    We have investigated the functional role of the flexible hinge region centered near the sequence TIEMP21, which connects the N-terminal cytosolic and C-terminal membrane-spanning helical domains of phospholamban (PLB). Specifically, we ask if the conformation of this region is important to attaining optimal inhibitory interactions with the Ca-ATPase. A genetically engineered PLB mutant was constructed in which Pro21 was mutated to an alanine (P21A-PLBC); in this construct all three transmembrane cysteines were substituted with alanines to stabilize the monomeric form of PLB and a unique cysteine was introduced at position 24 near the hinge element (A24C), permitting the site-specific attachment of fluorescein-5-maleimide (FMal) to monitor structure changes. In agreement with prior measurements in cardiac SR microsomes, the calcium concentration associated with half-maximal activation (Ca1/2) of the Ca-ATPase, 290 ? 10 nM, is shifted to 580 ? 20 nM when co-reconstituted with PLBC (Pro21) as a result of a 75% reduction in the rate of formation of the second high-affinity calcium binding site associated with calcium activation. In comparison, there is a 43% reduction in ?Ca1/2 upon reconstitution of the Ca-ATPase with P21A-PLBC, which can be simulated by decreasing the rate constant associated with calcium activation by 50%. The diminished inhibitory action of P21A-PLBC is associated with alterations in the structure of the hinge element, as evidenced by the diminished solvent accessibility of FMal relative to the native structure. Likewise, increases in the ?-helical content and decreases in the mobility of the carboxyl-terminal domain of P21A-PLBC are observed using circular dichroism and fluorescence spectroscopy. Collectively, these results indicate that the overall dimensions of the carboxyl-terminal domain of PLB are increased through a stabilization of secondary structural elements upon mutation in P21A-PLBC that result in a reduction in the ability of the

  12. A secreted WNT-ligand-binding domain of FZD5 generated by a frameshift mutation causes autosomal dominant coloboma.

    Science.gov (United States)

    Liu, Chunqiao; Widen, Sonya A; Williamson, Kathleen A; Ratnapriya, Rinki; Gerth-Kahlert, Christina; Rainger, Joe; Alur, Ramakrishna P; Strachan, Erin; Manjunath, Souparnika H; Balakrishnan, Archana; Floyd, James A; Li, Tiansen; Waskiewicz, Andrew; Brooks, Brian P; Lehmann, Ordan J; FitzPatrick, David R; Swaroop, Anand

    2016-04-01

    Ocular coloboma is a common eye malformation resulting from incomplete fusion of the optic fissure during development. Coloboma is often associated with microphthalmia and/or contralateral anophthalmia. Coloboma shows extensive locus heterogeneity associated with causative mutations identified in genes encoding developmental transcription factors or components of signaling pathways. We report an ultra-rare, heterozygous frameshift mutation in FZD5 (p.Ala219Glufs*49) that was identified independently in two branches of a large family with autosomal dominant non-syndromic coloboma. FZD5 has a single-coding exon and consequently a transcript with this frameshift variant is not a canonical substrate for nonsense-mediated decay. FZD5 encodes a transmembrane receptor with a conserved extracellular cysteine rich domain for ligand binding. The frameshift mutation results in the production of a truncated protein, which retains the Wingless-type MMTV integration site family member-ligand-binding domain, but lacks the transmembrane domain. The truncated protein was secreted from cells, and behaved as a dominant-negative FZD5 receptor, antagonizing both canonical and non-canonical WNT signaling. Expression of the resultant mutant protein caused coloboma and microphthalmia in zebrafish, and disruption of the apical junction of the retinal neural epithelium in mouse, mimicking the phenotype of Fz5/Fz8 compound conditional knockout mutants. Our studies have revealed a conserved role of Wnt-Frizzled (FZD) signaling in ocular development and directly implicate WNT-FZD signaling both in normal closure of the human optic fissure and pathogenesis of coloboma.

  13. Macrolides and lincomycin susceptibility of Mycoplasma hyorhinis and variable mutation of domain II and V in 23S ribosomal RNA.

    Science.gov (United States)

    Kobayashi, Hideki; Nakajima, Hiromi; Shimizu, Yuka; Eguchi, Masashi; Hata, Eiji; Yamamoto, Koshi

    2005-08-01

    A total of 151 strains of Mycoplasma hyorhinis isolated from porcine lung lesions (weaned pigs, n=71, and finishers, n=80) were investigated for their in vitro susceptibility to 10 antimicrobial agents. Thirty-one strains (28 from weaned pigs and 3 from finishers) showed resistance to 16-membered macrolide antibiotics and lincomycin. The prevalence of the 16-membered macrolide-resistant M. hyorhinis strain in weaned pigs from Japanese herds has approximately quadrupled in the past 10 years. Several of the 31 strains were examined for mutations in the 23S ribosomal RNA (rRNA). All field strains tested showed a transition of A to G at position 2059 of 23S rRNA-rendered Escherichia coli. On the other hand, individual tylosin- and lincomycin-resistant mutants of M. hyorhinis were selected in vitro from the susceptible type strain BTS7 by 3 to 9 serial passages in subinhibitory concentrations of each antibiotic. The 23S rRNA sequences of both tylosin and lincomycin-resistant mutants were compared with that of the radical BTS7 strain. The BTS7 mutant strain selected by tylosin showed the same transition as the field-isolated strains of A2059G. However, the transition selected in lincomycin showed mutations in domains II and V of 23S rRNA, G2597U, C2611U in domain V, and the addition of an adenine at the pentameric adenine loop in domain II. The strain selected by lincomycin showed an additional point mutation of A2062G selected by tylosin.

  14. P4-ATPases

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Theorin, Lisa; Palmgren, Michael Broberg;

    2014-01-01

    Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Among these ATP-driven transporters, the P4 subfamily of P-type ATPases (P4-ATPases......) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4...... to include the regulation of membrane traffic, cytoskeletal dynamics, cell division, lipid metabolism, and lipid signaling. In this review, we will summarize the basic features of P4-ATPases and the physiological implications of their lipid transport activity in the cell. © 2013 The Author(s)....

  15. Mutational analysis of the prohead binding domain of the large subunit of terminase, the bacteriophage lambda DNA packaging enzyme.

    Science.gov (United States)

    Yeo, A; Feiss, M

    1995-01-13

    Terminase, the DNA packaging enzyme of bacteriophage lambda, is made up of two subunits, gpNul and gpA, the products of the Nu1 and A genes. The activities of terminase include DNA binding, cos cleavage and prohead binding. Specificity domains within the structure of terminase have previously been defined by genetic studies of lambda-21 hybrids. The prohead binding domain of terminase is localized to the last 32 amino acid residues of gpA. Mutations in the prohead binding domain of gpA were constructed by introducing the corresponding amino acids from gp2, the gpA analog of bacteriophage 21. The last five residues of gpA can be replaced with little effect on the burst size of lambda. A phage with a replacement of the last six residues of gpA with the corresponding residues of gp2 was unable to form plaques, indicating that the sixth-to-last residues of gpA is crucial for prohead binding. Site-specific mutagenesis of the sixth-to-last position of gpA indicated that the sixth-to-last residue of gpA must be hydrophobic, of the seven amino acids tested, only isoleucine and valine can substitute for leucine at this position. Although the last five residues of gp2 were functional when they replaced the last five residues of gpA, two results indicated that the last five residues of gpA functioned better than the corresponding residues of gp2. First, the presence of a valine residue at the sixth-to-last position of gpA allowed plaque formation, whereas replacement of the last six residues of gpA with those of gp2, which substitutes a valine residue at the sixth-to-last position, was lethal. The second set of results indicating that the last five residues of gpA function better than the gp2 residues were obtained by study of revertants of lethal substitution mutations. In constructing the replacement mutations, a short linker was inserted into the C terminus of the A gene; this insertion created a short duplication of the end of the A gene, so that the normal C

  16. Mutation of Gly717Phe in human topoisomerase 1B has an effect on enzymatic function, reactivity to the camptothecin anticancer drug and on the linker domain orientation

    DEFF Research Database (Denmark)

    Wang, Zhenxing; D'Annessa, Ilda; Tesauro, Cinzia;

    2015-01-01

    –DNA covalent adduct. In this work the role of the Gly717 residue, located in a α-helix structure bridging the active site and the linker domain, has been investigated mutating it in Phe. The mutation gives rise to drug resistance in vivo as observed through a viability assay of yeast cells. In vitro activity...... assays show that the mutant is characterized by a fast religation rate, only partially reduced by the presence of the drug. Comparative molecular dynamics simulations of the native and mutant proteins indicate that the mutation of Gly717 affects the motion orientation of the linker domain, changing its...

  17. Structures of the thermophilic F1-ATPase epsilon subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1.

    Science.gov (United States)

    Yagi, Hiromasa; Kajiwara, Nobumoto; Tanaka, Hideaki; Tsukihara, Tomitake; Kato-Yamada, Yasuyuki; Yoshida, Masasuke; Akutsu, Hideo

    2007-07-03

    The epsilon subunit of bacterial and chloroplast F(o)F(1)-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound epsilon subunit from a thermophilic Bacillus PS3 at 1.9-A resolution. The C-terminal two alpha-helices were folded into a hairpin, sitting on the beta sandwich structure, as reported for Escherichia coli. A previously undescribed ATP binding motif, I(L)DXXRA, recognizes ATP together with three arginine and one glutamate residues. The E. coli epsilon subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 epsilon subunit and relaxation parameters of the whole molecule by NMR. The two helices fold into a hairpin in the presence of ATP but extend in the absence of ATP. The latter structure has more helical regions and is much more flexible than the former. These results suggest that the epsilon C-terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of F(o)F(1)-ATP synthase.

  18. Risk Factors Of Mutation Nucleotide Oligomerazation Domain 2 Gene To Chronic Periodontitis

    OpenAIRE

    Asmawati, Dr.drg.M. Kes

    2011-01-01

    The results showed that there was 12,3% of samples that had been mutation of NOD2 gene, and in the control group only 1,2%,and ststistically there was a significant difference ( p=0,005). In conclusion,mutation NOD2 gen is a risk factor to chronic periodontitis. Chronic Periodontitis is a chronic inflammatory disease that causes a damage of tooth supporting tissue. The incidence of chronic periodontitis disease is highly reported in Indonesia.Survey in Java, Bali and Sulawesi reported the...

  19. The O-glycosylated ectodomain of FXYD5 impairs adhesion by disrupting cell-cell trans-dimerization of Na,K-ATPase β1 subunits.

    Science.gov (United States)

    Tokhtaeva, Elmira; Sun, Haying; Deiss-Yehiely, Nimrod; Wen, Yi; Soni, Pritin N; Gabrielli, Nieves M; Marcus, Elizabeth A; Ridge, Karen M; Sachs, George; Vazquez-Levin, Mónica; Sznajder, Jacob I; Vagin, Olga; Dada, Laura A

    2016-06-15

    FXYD5 (also known as dysadherin), a regulatory subunit of the Na,K-ATPase, impairs intercellular adhesion by a poorly understood mechanism. Here, we determined whether FXYD5 disrupts the trans-dimerization of Na,K-ATPase molecules located in neighboring cells. Mutagenesis of the Na,K-ATPase β1 subunit identified four conserved residues, including Y199, that are crucial for the intercellular Na,K-ATPase trans-dimerization and adhesion. Modulation of expression of FXYD5 or of the β1 subunit with intact or mutated β1-β1 binding sites demonstrated that the anti-adhesive effect of FXYD5 depends on the presence of Y199 in the β1 subunit. Immunodetection of the plasma membrane FXYD5 was prevented by the presence of O-glycans. Partial FXYD5 deglycosylation enabled antibody binding and showed that the protein level and the degree of O-glycosylation were greater in cancer than in normal cells. FXYD5-induced impairment of adhesion was abolished by both genetic and pharmacological inhibition of FXYD5 O-glycosylation. Therefore, the extracellular O-glycosylated domain of FXYD5 impairs adhesion by interfering with intercellular β1-β1 interactions, suggesting that the ratio between FXYD5 and α1-β1 heterodimer determines whether the Na,K-ATPase acts as a positive or negative regulator of intercellular adhesion.

  20. A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

    DEFF Research Database (Denmark)

    Syed, Salahuddin; Madsen, Claus Desler; Rasmussen, Lene J.;

    2016-01-01

    In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N......-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor......-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each...

  1. Nephrocystin-5, a ciliary IQ domain protein, is mutated in Senior-Loken syndrome and interacts with RPGR and calmodulin.

    Science.gov (United States)

    Otto, Edgar A; Loeys, Bart; Khanna, Hemant; Hellemans, Jan; Sudbrak, Ralf; Fan, Shuling; Muerb, Ulla; O'Toole, John F; Helou, Juliana; Attanasio, Massimo; Utsch, Boris; Sayer, John A; Lillo, Concepcion; Jimeno, David; Coucke, Paul; De Paepe, Anne; Reinhardt, Richard; Klages, Sven; Tsuda, Motoyuki; Kawakami, Isao; Kusakabe, Takehiro; Omran, Heymut; Imm, Anita; Tippens, Melissa; Raymond, Pamela A; Hill, Jo; Beales, Phil; He, Shirley; Kispert, Andreas; Margolis, Benjamin; Williams, David S; Swaroop, Anand; Hildebrandt, Friedhelm

    2005-03-01

    Nephronophthisis (NPHP) is the most frequent genetic cause of chronic renal failure in children. Identification of four genes mutated in NPHP subtypes 1-4 (refs. 4-9) has linked the pathogenesis of NPHP to ciliary functions. Ten percent of affected individuals have retinitis pigmentosa, constituting the renal-retinal Senior-Loken syndrome (SLSN). Here we identify, by positional cloning, mutations in an evolutionarily conserved gene, IQCB1 (also called NPHP5), as the most frequent cause of SLSN. IQCB1 encodes an IQ-domain protein, nephrocystin-5. All individuals with IQCB1 mutations have retinitis pigmentosa. Hence, we examined the interaction of nephrocystin-5 with RPGR (retinitis pigmentosa GTPase regulator), which is expressed in photoreceptor cilia and associated with 10-20% of retinitis pigmentosa. We show that nephrocystin-5, RPGR and calmodulin can be coimmunoprecipitated from retinal extracts, and that these proteins localize to connecting cilia of photoreceptors and to primary cilia of renal epithelial cells. Our studies emphasize the central role of ciliary dysfunction in the pathogenesis of SLSN.

  2. Mutational analysis of βCOP (Sec26p identifies an appendage domain critical for function

    Directory of Open Access Journals (Sweden)

    Cerione Richard A

    2008-01-01

    Full Text Available Abstract Background The appendage domain of the γCOP subunit of the COPI vesicle coat bears a striking structural resemblance to adaptin-family appendages despite limited primary sequence homology. Both the γCOP appendage domain and an equivalent region on βCOP contain the FxxxW motif; the conservation of this motif suggested the existence of a functional appendage domain in βCOP. Results Sequence comparisons in combination with structural prediction tools show that the fold of the COOH-terminus of Sec26p is strongly predicted to closely mimic that of adaptin-family appendages. Deletion of the appendage domain of Sec26p results in inviability in yeast, over-expression of the deletion construct is dominant negative and mutagenesis of this region identifies residues critical for function. The ArfGAP Glo3p was identified via suppression screening as a potential downstream modulator of Sec26p in a manner that is independent of the GAP activity of Glo3p but requires the presence of the COOH-terminal ISS motifs. Conclusion Together, these results indicate an essential function for the predicted βCOP appendage and suggest that both COPI appendages perform a biologically active regulatory role with a structure related to adaptin-family appendage domains.

  3. Cancer associated E17K mutation causes rapid conformational drift in AKT1 pleckstrin homology (PH domain.

    Directory of Open Access Journals (Sweden)

    Ambuj Kumar

    Full Text Available BACKGROUND: AKT1 (v-akt murine thymoma viral oncogene homologue 1 kinase is one of the most frequently activated proliferated and survival pathway of cancer. Recently it has been shown that E17K mutation in the Pleckstrin Homology (PH domain of AKT1 protein leads to cancer by amplifying the phosphorylation and membrane localization of protein. The mutant has shown resistance to AKT1/2 inhibitor VIII drug molecule. In this study we have demonstrated the detailed structural and molecular consequences associated with the activity regulation of mutant protein. METHODS: The docking score exhibited significant loss in the interaction affinity to AKT1/2 inhibitor VIII drug molecule. Furthermore, the molecular dynamics simulation studies presented an evidence of rapid conformational drift observed in mutant structure. RESULTS: There was no stability loss in mutant as compared to native structure and the major cation-π interactions were also shown to be retained. Moreover, the active residues involved in membrane localization of protein exhibited significant rise in NHbonds formation in mutant. The rise in NHbond formation in active residues accounts for the 4-fold increase in the membrane localization potential of protein. CONCLUSION: The overall result suggested that, although the mutation did not induce any stability loss in structure, the associated pathological consequences might have occurred due to the rapid conformational drifts observed in the mutant AKT1 PH domain. GENERAL SIGNIFICANCE: The methodology implemented and the results obtained in this work will facilitate in determining the core molecular mechanisms of cancer-associated mutations and in designing their potential drug inhibitors.

  4. Neonatal Marfan Syndrome: Report of a Case with an Inherited Splicing Mutation outside the Neonatal Domain.

    Science.gov (United States)

    Le Gloan, Laurianne; Hauet, Quentin; David, Albert; Hanna, Nadine; Arfeuille, Chloé; Arnaud, Pauline; Boileau, Catherine; Romefort, Bénédicte; Benbrik, Nadir; Gournay, Véronique; Joram, Nicolas; Baron, Olivier; Isidor, Bertrand

    2016-02-01

    We report a child and her mother affected by Marfan syndrome. The child presented with a phenotype of neonatal Marfan syndrome, revealed by acute and refractory heart failure, finally leading to death within the first 4 months of life. Her mother had a common clinical presentation. Genetic analysis revealed an inherited FBN1 mutation. This intronic mutation (c.6163+3_6163+6del), undescribed to date, leads to exon 49 skipping, corresponding to in-frame deletion of 42 amino acids (p.Ile2014_Asp2055del). FBN1 next-generation sequencing did not show any argument for mosaicism. Association in the same family of severe neonatal and classical Marfan syndrome illustrates the intrafamilial phenotype variability.

  5. Targeting oncoprotein stability overcomes drug resistance caused by FLT3 kinase domain mutations.

    Directory of Open Access Journals (Sweden)

    Chuanjiang Yu

    Full Text Available FLT3 is the most frequently mutated kinase in acute myeloid leukemia (AML. Internal tandem duplications (ITDs in the juxta-membrane region constitute the majority of activating FLT3 mutations. Several FLT3 kinase inhibitors were developed and tested in the clinic with significant success. However, recent studies have reported the development of secondary drug resistance in patients treated with FLT3 inhibitors. Since FLT3-ITD is an HSP90 client kinase, we here explored if targeting the stability of drug-resistant FLT3 mutant protein could be a potential therapeutic option. We observed that HSP90 inhibitor treatment resulted in the degradation of inhibitor-resistant FLT3-ITD mutants and selectively induced toxicity in cells expressing FLT3-ITD mutants. Thus, HSP90 inhibitors provide a potential therapeutic choice to overcome secondary drug resistance following TKI treatment in FLT3-ITD positive AML.

  6. Hinge-loop mutation can be used to control 3D domain swapping and amyloidogenesis of human cystatin C.

    Science.gov (United States)

    Orlikowska, Marta; Jankowska, Elżbieta; Kołodziejczyk, Robert; Jaskólski, Mariusz; Szymańska, Aneta

    2011-02-01

    Cystatins are natural inhibitors of cysteine proteases, enzymes that are widely distributed in animals, plants, and microorganisms. Human cystatin C (hCC) has been also recognized as an aggregating protein directly involved in the formation of pathological amyloid fibrils, and these amyloidogenic properties greatly increase in a naturally occurring L68Q hCC variant. For a long time only dimeric structure of wild-type hCC has been known. The dimer is created through 3D domain swapping process, in which two parts of the cystatin structure become separated from each other and next exchanged between two molecules. Important role in the domain swapping plays the L1 loop, which connects the exchanging segments and, upon dimerization, transforms from a β-turn into a part of a long β-strand. In the very recently published first monomeric structure of human cystatin C (hCC-stab1), dimerization was abrogated due to clasping of the β-strands from the swapping domains by an engineered disulfide bridge. We have designed and constructed another mutated cystatin C with the smallest possible structural intervention, that is a single-point mutation replacing hydrophobic V57 from the L1 loop by polar asparagine, known as a stabilizer of a β-turn motif. V57N hCC mutant occurred to be stable in its monomeric form and crystallized as a monomer, revealing typical cystatin fold with a five-stranded antiparallel β-sheet wrapped around an α-helix. Here we report a 2.04 Å resolution crystal structure of V57N hCC and discuss the architecture of the protein in comparison to chicken cystatin, hCC-stab1 and dimeric hCC.

  7. Mutations in domain II of 23 S rRNA facilitate translation of a 23 S rRNA-encoded pentapeptide conferring erythromycin resistance

    DEFF Research Database (Denmark)

    Dam, M; Douthwaite, S; Tenson, T

    1996-01-01

    Mutations in domain II of Escherichia coli 23 S rRNA that cause resistance to erythromycin do so in a manner fundamentally different from mutations at the drug binding site in domain V of the 23 S rRNA. The domain II mutations are located in a hairpin structure between nucleotides 1198 and 1247....... This is close to a short open reading frame in the 23 S rRNA that encodes a pentapeptide (E-peptide) whose expression in vivo renders cells resistant to erythromycin. Therefore, a possible mechanism of resistance caused by domain II mutations may be related to an increased expression of the E-peptide. To test...... this hypothesis, a range of point mutations was generated in domain II of 23 S rRNA in the vicinity of the E-peptide open reading frame. We find a correlation between erythromycin resistance of the mutant clones and increased accessibility of the ribosome binding site of the E-peptide gene. Furthermore...

  8. The Clinical Spectrum of Missense Mutations of the First Aspartic Acid of cbEGF-like Domains in Fibrillin-1 Including a Recessive Family

    Science.gov (United States)

    Hilhorst-Hofstee, Yvonne; Rijlaarsdam, Marry EB; Scholte, Arthur JHA; Swart-van den Berg, Marietta; Versteegh, Michel IM; van der Schoot-van Velzen, Iris; Schäbitz, Hans-Joachim; Bijlsma, Emilia K; Baars, Marieke J; Kerstjens-Frederikse, Wilhelmina S; Giltay, Jacques C; Hamel, Ben C; Breuning, Martijn H; Pals, Gerard

    2010-01-01

    Marfan syndrome (MFS) is a dominant disorder with a recognizable phenotype. In most patients with the classical phenotype mutations are found in the fibrillin-1 gene (FBN1) on chromosome 15q21. It is thought that most mutations act in a dominant negative way or through haploinsufficiency. In 9 index cases referred for MFS we detected heterozygous missense mutations in FBN1 predicted to substitute the first aspartic acid of different calcium-binding Epidermal Growth Factor-like (cbEGF) fibrillin-1 domains. A similar mutation was found in homozygous state in 3 cases in a large consanguineous family. Heterozygous carriers of this mutation had no major skeletal, cardiovascular or ophthalmological features of MFS. In the literature 14 other heterozygous missense mutations are described leading to the substitution of the first aspartic acid of a cbEGF domain and resulting in a Marfan phenotype. Our data show that the phenotypic effect of aspartic acid substitutions in the first position of a cbEGF domain can range from asymptomatic to a severe neonatal phenotype. The recessive nature with reduced expression of FBN1 in one of the families suggests a threshold model combined with a mild functional defect of this specific mutation. © 2010 Wiley-Liss, Inc. PMID:20886638

  9. ALK kinase domain mutations in primary anaplastic large cell lymphoma: consequences on NPM-ALK activity and sensitivity to tyrosine kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Federica Lovisa

    Full Text Available ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.

  10. ALK kinase domain mutations in primary anaplastic large cell lymphoma: consequences on NPM-ALK activity and sensitivity to tyrosine kinase inhibitors.

    Science.gov (United States)

    Lovisa, Federica; Cozza, Giorgio; Cristiani, Andrea; Cuzzolin, Alberto; Albiero, Alessandro; Mussolin, Lara; Pillon, Marta; Moro, Stefano; Basso, Giuseppe; Rosolen, Angelo; Bonvini, Paolo

    2015-01-01

    ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.

  11. A mutational analysis of the endophilin-A N-BAR domain performed in living flies

    DEFF Research Database (Denmark)

    Jung, Anita G; Mønsted, Christina Labarrera; Jansen, Anna M

    2010-01-01

    BAR domain clearly affected adult flies, larval endophilin function was surprisingly resistant to mutagenesis. Previous reports have stressed the importance of a central appendage on the convex BAR surface, which forms a hydrophobic ridge able to directly insert into the lipid bilayer. We found...

  12. Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

    Energy Technology Data Exchange (ETDEWEB)

    Mathur, Chhavi [Department of Biochemistry, Indian Institute of Science, Bangalore 560012 (India); Savithri, Handanahal S., E-mail: bchss@biochem.iisc.ernet.in [Department of Biochemistry, Indian Institute of Science, Bangalore 560012 (India)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Pepper vein banding potyvirus VPg harbors Walker motifs. Black-Right-Pointing-Pointer VPg exhibits ATPase activity in the presence of NIa-Pro. Black-Right-Pointing-Pointer Plausible structural and functional interplay between VPg and NIa-Pro. Black-Right-Pointing-Pointer Functional relevance of prolonged presence of VPg-Pro during infection. -- Abstract: Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity.

  13. Detailed characterization of the cooperative mechanism of Ca(2+) binding and catalytic activation in the Ca(2+) transport (SERCA) ATPase.

    Science.gov (United States)

    Zhang, Z; Lewis, D; Strock, C; Inesi, G; Nakasako, M; Nomura, H; Toyoshima, C

    2000-08-01

    Expression of heterologous SERCA1a ATPase in Cos-1 cells was optimized to yield levels that account for 10-15% of the microsomal protein, as revealed by protein staining on electrophoretic gels. This high level of expression significantly improved our characterization of mutants, including direct measurements of Ca(2+) binding by the ATPase in the absence of ATP, and measurements of various enzyme functions in the presence of ATP or P(i). Mutational analysis distinguished two groups of amino acids within the transmembrane domain: The first group includes Glu771 (M5), Thr799 (M6), Asp800 (M6), and Glu908 (M8), whose individual mutations totally inhibit binding of the two Ca(2+) required for activation of one ATPase molecule. The second group includes Glu309 (M4) and Asn796 (M6), whose individual or combined mutations inhibit binding of only one and the same Ca(2+). The effects of mutations of these amino acids were interpreted in the light of recent information on the ATPase high-resolution structure, explaining the mechanism of Ca(2+) binding and catalytic activation in terms of two cooperative sites. The Glu771, Thr799, and Asp800 side chains contribute prominently to site 1, together with less prominent contributions by Asn768 and Glu908. The Glu309, Asn796, and Asp800 side chains, as well as the Ala305 (and possibly Val304 and Ile307) carbonyl oxygen, contribute to site 2. Sequential binding begins with Ca(2+) occupancy of site 1, followed by transition to a conformation (E') sensitive to Ca(2+) inhibition of enzyme phosphorylation by P(i), but still unable to utilize ATP. The E' conformation accepts the second Ca(2+) on site 2, producing then a conformation (E' ') which is able to utilize ATP. Mutations of residues (Asp813 and Asp818) in the M6/M7 loop reduce Ca(2+) affinity and catalytic turnover, suggesting a strong influence of this loop on the correct positioning of the M6 helix. Mutation of Asp351 (at the catalytic site within the cytosolic domain

  14. An Activating Mutation in sos-1 Identifies Its Dbl Domain as a Critical Inhibitor of the Epidermal Growth Factor Receptor Pathway during Caenorhabditis elegans Vulval Development▿

    Science.gov (United States)

    Modzelewska, Katarzyna; Elgort, Marc G.; Huang, Jingyu; Jongeward, Gregg; Lauritzen, Amara; Yoon, Charles H.; Sternberg, Paul W.; Moghal, Nadeem

    2007-01-01

    Proper regulation of receptor tyrosine kinase (RTK)-Ras-mitogen-activated protein kinase (MAPK) signaling pathways is critical for normal development and the prevention of cancer. SOS is a dual-function guanine nucleotide exchange factor (GEF) that catalyzes exchange on Ras and Rac. Although the physiologic role of SOS and its CDC25 domain in RTK-mediated Ras activation is well established, the in vivo function of its Dbl Rac GEF domain is less clear. We have identified a novel gain-of-function missense mutation in the Dbl domain of Caenorhabditis elegans SOS-1 that promotes epidermal growth factor receptor (EGFR) signaling in vivo. Our data indicate that a major developmental function of the Dbl domain is to inhibit EGF-dependent MAPK activation. The amount of inhibition conferred by the Dbl domain is equal to that of established trans-acting inhibitors of the EGFR pathway, including c-Cbl and RasGAP, and more than that of MAPK phosphatase. In conjunction with molecular modeling, our data suggest that the C. elegans mutation, as well as an equivalent mutation in human SOS1, activates the MAPK pathway by disrupting an autoinhibitory function of the Dbl domain on Ras activation. Our work suggests that functionally similar point mutations in humans could directly contribute to disease. PMID:17339331

  15. An activating mutation in sos-1 identifies its Dbl domain as a critical inhibitor of the epidermal growth factor receptor pathway during Caenorhabditis elegans vulval development.

    Science.gov (United States)

    Modzelewska, Katarzyna; Elgort, Marc G; Huang, Jingyu; Jongeward, Gregg; Lauritzen, Amara; Yoon, Charles H; Sternberg, Paul W; Moghal, Nadeem

    2007-05-01

    Proper regulation of receptor tyrosine kinase (RTK)-Ras-mitogen-activated protein kinase (MAPK) signaling pathways is critical for normal development and the prevention of cancer. SOS is a dual-function guanine nucleotide exchange factor (GEF) that catalyzes exchange on Ras and Rac. Although the physiologic role of SOS and its CDC25 domain in RTK-mediated Ras activation is well established, the in vivo function of its Dbl Rac GEF domain is less clear. We have identified a novel gain-of-function missense mutation in the Dbl domain of Caenorhabditis elegans SOS-1 that promotes epidermal growth factor receptor (EGFR) signaling in vivo. Our data indicate that a major developmental function of the Dbl domain is to inhibit EGF-dependent MAPK activation. The amount of inhibition conferred by the Dbl domain is equal to that of established trans-acting inhibitors of the EGFR pathway, including c-Cbl and RasGAP, and more than that of MAPK phosphatase. In conjunction with molecular modeling, our data suggest that the C. elegans mutation, as well as an equivalent mutation in human SOS1, activates the MAPK pathway by disrupting an autoinhibitory function of the Dbl domain on Ras activation. Our work suggests that functionally similar point mutations in humans could directly contribute to disease.

  16. Mutations within or upstream of the basic helix-loop-helix domain of the TWIST gene are specific to Saethre-Chotzen syndrome.

    Science.gov (United States)

    El Ghouzzi, V; Lajeunie, E; Le Merrer, M; Cormier-Daire, V; Renier, D; Munnich, A; Bonaventure, J

    1999-01-01

    Saethre-Chotzen syndrome (ACS III) is an autosomal dominant craniosynostosis syndrome recently ascribed to mutations in the TWIST gene, a basic helix-loop-helix (b-HLH) transcription factor regulating head mesenchyme cell development during cranial neural tube formation in mouse. Studying a series of 22 unrelated ACS III patients, we have found TWIST mutations in 16/22 cases. Interestingly, these mutations consistently involved the b-HLH domain of the protein. Indeed, mutant genotypes included frameshift deletions/insertions, nonsense and missense mutations, either truncating or disrupting the b-HLH motif of the protein. This observation gives additional support to the view that most ACS III cases result from loss-of-function mutations at the TWIST locus. The P250R recurrent FGFR 3 mutation was found in 2/22 cases presenting mild clinical manifestations of the disease but 4/22 cases failed to harbour TWIST or FGFR 3 mutations. Clinical re-examination of patients carrying TWIST mutations failed to reveal correlations between the mutant genotype and severity of the phenotype. Finally, since no TWIST mutations were detected in 40 cases of isolated coronal craniosynostosis, the present study suggests that TWIST mutations are specific to Saethre-Chotzen syndrome.

  17. DNA Repair Domain Modeling Can Predict Cell Death and Mutation Frequency for Wide Range Spectrum of Radiation

    Science.gov (United States)

    Viger, Louise; Ponomarev, Artem L.; Plante, Ianik; Evain, Trevor; Penninckx, Sebastien; Blattnig, Steve R.; Costes, Sylvain V.

    2017-01-01

    Exploration missions to Mars and other destinations raise many questions about the health of astronauts. The continuous exposure of astronauts to galactic cosmic rays is one of the main concerns for long-term missions. Cosmic ionizing radiations are composed of different ions of various charges and energies notably, highly charged energy (HZE) particles. The HZE particles have been shown to be more carcinogenic than low-LET radiation, suggesting the severity of chromosomal aberrations induced by HZE particles is one possible explanation. However, most mathematical models predicting cell death and mutation frequency are based on directly fitting various HZE dose response and are in essence empirical approaches. In this work, we assume a simple biological mechanism to model DNA repair and use it to simultaneously explain the low- and high-LET response using the exact same fitting parameters. Our work shows that the geometrical position of DNA repair along tracks of heavy ions are sufficient to explain why high-LET particles can induce more death and mutations. Our model is based on assuming DNA double strand breaks (DSBs) are repaired within repair domain, and that any DSBs located within the same repair domain cluster into one repair unit, facilitating chromosomal rearrangements and increasing the probability of cell death. We introduced this model in 2014 using simplified microdosimetry profiles to predict cell death. In this work, we collaborated with NASA Johnson Space Center to generate more accurate microdosimetry profiles derived by Monte Carlo techniques, taking into account track structure of HZE particles and simulating DSBs in realistic cell geometry. We simulated 224 data points (D, A, Z, E) with the BDSTRACKS model, leading to a large coverage of LET from 10 to 2,400 keV/µm. This model was used to generate theoretical RBE for various particles and energies for both cell death and mutation frequencies. The RBE LET dependence is in agreement with

  18. New SMARCA2 mutation in a patient with Nicolaides–Baraitser syndrome and myoclonic astatic epilepsy

    OpenAIRE

    S. Tang; Hughes, E.(Rutgers, The State University of New Jersey, Piscataway, USA); Lascelles, K.; Simpson, M A; Pal, D. K.; Marini, C.; Guerrini, R.; Neubauer, B; Korff, C. M.; D. Craiu; Pal, D.; Caglayan, H.; Helbig, I.; De Jonghe, P; Thomas, R.

    2017-01-01

    We report a de novo SMARCA2 missense mutation discovered on exome sequencing in a patient with myoclonic astatic epilepsy, leading to reassessment and identification of Nicolaides–Baraitser syndrome. This de novo SMARCA2 missense mutation c.3721C>G, p.Gln1241Glu is the only reported mutation on exon 26 outside the ATPase domain of SMARCA2 to be associated with Nicolaides–Baraitser syndrome and adds to chromatin remodeling as a pathway for epileptogenesis. © 2016 The Authors. American Journal ...

  19. Vacuolar H+-ATPase: An Essential Multitasking Enzyme in Physiology and Pathophysiology

    Directory of Open Access Journals (Sweden)

    L. Shannon Holliday

    2014-01-01

    Full Text Available Vacuolar H+-ATPases (V-ATPases are large multisubunit proton pumps that are required for housekeeping acidification of membrane-bound compartments in eukaryotic cells. Mammalian V-ATPases are composed of 13 different subunits. Their housekeeping functions include acidifying endosomes, lysosomes, phagosomes, compartments for uncoupling receptors and ligands, autophagosomes, and elements of the Golgi apparatus. Specialized cells, including osteoclasts, intercalated cells in the kidney and pancreatic beta cells, contain both the housekeeping V-ATPases and an additional subset of V-ATPases, which plays a cell type specific role. The specialized V-ATPases are typically marked by the inclusion of cell type specific isoforms of one or more of the subunits. Three human diseases caused by mutations of isoforms of subunits have been identified. Cancer cells utilize V-ATPases in unusual ways; characterization of V-ATPases may lead to new therapeutic modalities for the treatment of cancer. Two accessory proteins to the V-ATPase have been identified that regulate the proton pump. One is the (prorenin receptor and data is emerging that indicates that V-ATPase may be intimately linked to renin/angiotensin signaling both systemically and locally. In summary, V-ATPases play vital housekeeping roles in eukaryotic cells. Specialized versions of the pump are required by specific organ systems and are involved in diseases.

  20. Structure of the vacuolar H+-ATPase rotary motor reveals new mechanistic insights.

    Science.gov (United States)

    Rawson, Shaun; Phillips, Clair; Huss, Markus; Tiburcy, Felix; Wieczorek, Helmut; Trinick, John; Harrison, Michael A; Muench, Stephen P

    2015-03-03

    Vacuolar H(+)-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼ 1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases.

  1. Transformation of the mechanism of triple-helix peptide folding in the absence of a C-terminal nucleation domain and its implications for mutations in collagen disorders.

    Science.gov (United States)

    Buevich, Alexei V; Silva, Teresita; Brodsky, Barbara; Baum, Jean

    2004-11-05

    Folding abnormalities of the triple helix have been demonstrated in collagen diseases such as osteogenesis imperfecta in which the mutation leads to the substitution of a single Gly in the (Gly-X-Y)n sequence pattern by a larger residue. Model peptides can be used to clarify the details of normal collagen folding and the consequences of the interruption of that folding by a Gly substitution. NMR and CD studies show that placement of a (GPO)4 nucleation domain at the N terminus rather than the C terminus of a native collagen sequence allows the formation of a stable triple helix but alters the folding mechanism. Although C- to N-terminal directional folding occurs when the nucleation domain is at the C terminus, there is no preferential folding direction when the nucleation domain is at the N terminus. The lack of zipper-like directional folding does not interfere with triple-helix formation, and when a Gly residue is replaced by Ser to model an osteogenesis imperfecta mutation, the peptide with the N-terminal (GPO)4 domain can still form a good triple helix N-terminal to the mutation site. These peptide studies raise the possibility that mutant collagen could fold in a C to N direction in a zipper-like manner up to the mutation site and that completion of the triple helix N-terminal to the mutation would involve an alternative mechanism.

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

    Science.gov (United States)

    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

  3. P4-ATPases

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Theorin, Lisa; Palmgren, Michael Broberg

    2014-01-01

    ) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4...

  4. Collagen VI microfibril formation is abolished by an {alpha}2(VI) von Willebrand factor type A domain mutation in a patient with Ullrich congenital muscular dystrophy.

    Science.gov (United States)

    Tooley, Leona D; Zamurs, Laura K; Beecher, Nicola; Baker, Naomi L; Peat, Rachel A; Adams, Naomi E; Bateman, John F; North, Kathryn N; Baldock, Clair; Lamandé, Shireen R

    2010-10-22

    Collagen VI is an extracellular protein that most often contains the three genetically distinct polypeptide chains, α1(VI), α2(VI), and α3(VI), although three recently identified chains, α4(VI), α5(VI), and α6(VI), may replace α3(VI) in some situations. Each chain has a triple helix flanked by N- and C-terminal globular domains that share homology with the von Willebrand factor type A (VWA) domains. During biosynthesis, the three chains come together to form triple helical monomers, which then assemble into dimers and tetramers. Tetramers are secreted from the cell and align end-to-end to form microfibrils. The precise molecular mechanisms responsible for assembly are unclear. Mutations in the three collagen VI genes can disrupt collagen VI biosynthesis and matrix organization and are the cause of the inherited disorders Bethlem myopathy and Ullrich congenital muscular dystrophy. We have identified a Ullrich congenital muscular dystrophy patient with compound heterozygous mutations in α2(VI). The first mutation causes skipping of exon 24, and the mRNA is degraded by nonsense-mediated decay. The second mutation is a two-amino acid deletion in the C1 VWA domain. Recombinant C1 domains containing the deletion are insoluble and retained intracellularly, indicating that the mutation has detrimental effects on domain folding and structure. Despite this, mutant α2(VI) chains retain the ability to associate into monomers, dimers, and tetramers. However, we show that secreted mutant tetramers containing structurally abnormal C1 VWA domains are unable to associate further into microfibrils, directly demonstrating the critical importance of a correctly folded α2(VI) C1 domain in microfibril formation.

  5. Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

    Energy Technology Data Exchange (ETDEWEB)

    Inada, Hitoshi; Procko, Erik; Sotomayor, Marcos; Gaudet, Rachelle (Harvard-Med); (Harvard)

    2012-10-23

    The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.

  6. Missense mutations in the TP53 DNA-binding domain predict outcomes in patients with advanced oral cavity squamous cell carcinoma.

    Science.gov (United States)

    Lapke, Nina; Lu, Yen-Jung; Liao, Chun-Ta; Lee, Li-Yu; Lin, Chien-Yu; Wang, Hung-Ming; Ng, Shu-Hang; Chen, Shu-Jen; Yen, Tzu-Chen

    2016-07-12

    TP53 mutations have been linked to reduced survival in patients with oral cavity squamous cell carcinoma (OSCC). However, the impact of different types of TP53 mutations remains unclear. Here, we demonstrate that the carriage of missense mutations in the TP53 DNA binding domain (DBD missense mutations) is associated with decreased disease-specific survival (DSS) compared with wild-type TP53 (P=0.002) in a cohort of 345 OSCC patients. In contrast, DSS of patients bearing all of the remaining TP53 mutations did not differ from that observed in wild-type TP53 patients (P=0.955). Our classification method for TP53 mutations was superior to previously reported approaches (disruptive, truncating, Evolutionary Action score, mutations in L2/L3/LSH) for distinguishing between low- and high-risk patients. When analyzed in combination with traditional clinicopathological factors, TP53 DBD missense mutations were an independent prognostic factor for shorter DSS (P=0.014) alongside with advanced AJCC T- and N-classifications and the presence of extracapsular spread. A scoring system that included the four independent prognostic factors allowed a reliable patient stratification into distinct risk groups (high-risk patients, 16.2%). Our results demonstrate the usefulness of TP53 DBD missense mutations combined with clinicopathological factors for improving the prognostic stratification of OSCC patients.

  7. Massively Parallel Sequencing of a Chinese Family with DFNA9 Identified a Novel Missense Mutation in the LCCL Domain of COCH

    Directory of Open Access Journals (Sweden)

    Xiaodong Gu

    2016-01-01

    Full Text Available DFNA9 is a late-onset, progressive, autosomal dominantly inherited sensorineural hearing loss with vestibular dysfunction, which is caused by mutations in the COCH (coagulation factor C homology gene. In this study, we investigated a Chinese family segregating autosomal dominant nonsyndromic sensorineural hearing loss. We identified a missense mutation c.T275A p.V92D in the LCCL domain of COCH cosegregating with the disease and absent in 100 normal hearing controls. This mutation leads to substitution of the hydrophobic valine to an acidic amino acid aspartic acid. Our data enriched the mutation spectrum of DFNA9 and implied the importance for mutation screening of COCH in age related hearing loss with vestibular dysfunctions.

  8. A novel COL4A1 frameshift mutation in familial kidney disease: the importance of the C-terminal NC1 domain of type IV collagen

    Science.gov (United States)

    Gale, Daniel P.; Oygar, D. Deren; Lin, Fujun; Oygar, P. Derin; Khan, Nadia; Connor, Thomas M.F.; Lapsley, Marta; Maxwell, Patrick H.; Neild, Guy H.

    2016-01-01

    Background Hereditary microscopic haematuria often segregates with mutations of COL4A3, COL4A4 or COL4A5 but in half of families a gene is not identified. We investigated a Cypriot family with autosomal dominant microscopic haematuria with renal failure and kidney cysts. Methods We used genome-wide linkage analysis, whole exome sequencing and cosegregation analyses. Results We identified a novel frameshift mutation, c.4611_4612insG:p.T1537fs, in exon 49 of COL4A1. This mutation predicts truncation of the protein with disruption of the C-terminal part of the NC1 domain. We confirmed its presence in 20 family members, 17 with confirmed haematuria, 5 of whom also had stage 4 or 5 chronic kidney disease. Eleven family members exhibited kidney cysts (55% of those with the mutation), but muscle cramps or cerebral aneurysms were not observed and serum creatine kinase was normal in all individuals tested. Conclusions Missense mutations of COL4A1 that encode the CB3 [IV] segment of the triple helical domain (exons 24 and 25) are associated with HANAC syndrome (hereditary angiopathy, nephropathy, aneurysms and cramps). Missense mutations of COL4A1 that disrupt the NC1 domain are associated with antenatal cerebral haemorrhage and porencephaly, but not kidney disease. Our findings extend the spectrum of COL4A1 mutations linked with renal disease and demonstrate that the highly conserved C-terminal part of the NC1 domain of the α1 chain of type IV collagen is important in the integrity of glomerular basement membrane in humans. PMID:27190376

  9. Four mutations in SH2 and SH3 domains of Bruton`s tyrosine kinase (BTK) resulting in classic X-linked agammaglobulinemia (XLA)

    Energy Technology Data Exchange (ETDEWEB)

    Chen, S.H.; Zhang, M.; Zhu, Q.; Scott, C.R.; Och, H.D. [Univ. of Washington, Seattle, WA (United States)

    1994-09-01

    XLA is an X-linked immunodeficient disease in man resulted from mutations in the BTK gene. BTK contains a unique amino-region of unknown function, SH2 and SH3 (src homology) domains, and a carboxyl-terminal kinase (SH1) domain. We have studied the normal genomic organizations of the SH2 and SH3 domains and found the regions containing 6 exons are about 3000 bp in length. We also carried out sequence analyses of cDNA and genomic DNA of XLA patients to identify mutations. Four of fourteen families with XLA were found to have mutations within the regions. (1) A point mutation G to T in codon 240 resulted in a stop codon. (2) A transition mutation (g to a) at first nucleotide of intron 8 resulted in exon 8 skipping, missing 21 codons and shorter polypeptide but with normal kinase activity and ATP binding ability. (3) An a to t transversion at one of the invariant dinucleotides (ag) of the 3{prime} end of intron 11 resulted in alternative splicing at a position 13 nucleotides downstream from the normal one. The mutation produced mRNA with 13 nucleotide deletion and presumably resulted in a frameshift at codon 372 leading to a stop codon at 398. (4) A 16 nucleotide duplication (1248 to 1263 of the cDNA sequence) consistently present in mRNA of three brothers with XLA. However, genomic sequence of patient DNA of the regions did not reveal the anormaly. The observation that mutations within SH2 and SH3 causing severe B-cell defects typical for XLA suggests that these two domains are crucial for the function of BTK, possibly by regulating the interaction of cytoplasmic proteins involved in signal transduction.

  10. Mechanistic Basis for Differential Inhibition of the F1Fo-ATPase by Aurovertin

    OpenAIRE

    2009-01-01

    The mitochondrial F1Fo-ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F1Fo-ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the β subunits in the F1 domain and inhibits F1Fo-ATPase-catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and ...

  11. Origin and evolution of metal P-type ATPases in Plantae (Archaeplastida)

    OpenAIRE

    2014-01-01

    Metal ATPases are a subfamily of P-type ATPases involved in the transport of metal cations across biological membranes. They all share an architecture featuring eight transmembrane domains in pairs of two and are found in prokaryotes as well as in a variety of Eukaryotes. In Arabidopsis thaliana, eight metal P-type ATPases have been described, four being specific to copper transport and four displaying a broader metal specificity, including zinc, cadmium and possibly copper and calcium. So fa...

  12. A Novel Heterozygous Mutation in the STAT1 SH2 Domain Causes Chronic Mucocutaneous Candidiasis, Atypically Diverse Infections, Autoimmunity, and Impaired Cytokine Regulation

    Science.gov (United States)

    Meesilpavikkai, Kornvalee; Dik, Willem A.; Schrijver, Benjamin; Nagtzaam, Nicole M. A.; van Rijswijk, Angelique; Driessen, Gertjan J.; van der Spek, Peter J.; van Hagen, P. Martin; Dalm, Virgil A. S. H.

    2017-01-01

    Chronic mucocutaneous candidiasis (CMC) is a primary immunodeficiency characterized by persistent or recurrent skin and mucosal surface infections with Candida species. Different gene mutations leading to CMC have been identified. These include various heterozygous gain-of-function (GOF) mutations in signal transducer and activator of transcription 1 (STAT1) that are not only associated with infections but also with autoimmune manifestations. Recently, two STAT1 GOF mutations involving the Src homology 2 (SH2) domain have been reported, while so far, over 50 mutations have been described mainly in the coiled coil and the DNA-binding domains. Here, we present two members of a Dutch family with a novel STAT1 mutation located in the SH2 domain. T lymphocytes of these patients revealed STAT1 hyperphosphorylation and higher expression of STAT1 target genes. The clinical picture of CMC in our patients could be explained by diminished production of interleukin (IL)-17 and IL-22, cytokines important in the protection against fungal infections.

  13. Mutation Scanning of D1705 and D1709 in the RNAse IIIb Domain of MicroRNA Processing Enzyme Dicer in Cutaneous Melanoma.

    Science.gov (United States)

    Sand, Michael; Bechara, Falk G; Skrygan, Marina; Sand, Daniel; Gambichler, Thilo; Bromba, Michael; Stockfleth, Eggert; Hessam, Schapoor

    2016-07-01

    Since the discovery of microRNAs (miRNAs) there have been performed several studies showing perturbations in the expression of miRNAs and the miRNA expression machinery in cutaneous melanoma. Dicer, a pivotal cytosolic enzyme of miRNA maturation has shown to be affected by both somatic and germline mutations in a variety of cancers. Recent studies have shown that recurrent somatic mutations of Dicer frequently affect the metal-ion-binding sites D1709 and D1705 of its RNase IIIb domain, therefore called hot spot mutations. The present study investigates metal-ion-binding sites D1709 and D1705 of the Dicer RNase IIIb domain in cutaneous melanomas and melanoma metastasis by Sanger sequencing. All investigated samples showed wildtype sequence and no single mutation was detected. The miRNA processing enzyme Dicer of melanoma and melanoma metastasis does not appear to be affected by mutation in the metal-ion-binding sites D1709 and D1705 of its RNase IIIb domain.

  14. BCR-ABL isoforms associated with intrinsic or acquired resistance to imatinib : more heterogeneous than just ABL kinase domain point mutations?

    NARCIS (Netherlands)

    Gruber, Franz X.; Lundan, Tuija; Goll, Rasmus; Silye, Aleksandra; Mikkola, Ingvild; Rekvig, Ole Petter; Knuutila, Sakari; Remes, Kari; Gedde-Dahl, Tobias; Porkka, Kimmo; Hjorth-Hansen, Henrik

    2012-01-01

    Imatinib, a small molecule inhibitor of ABL, PDGFR and C-KIT, has revolutionized treatment of chronic myeloid leukaemia (CML). However, resistance to treatment is of increasing importance and often is due to point mutations in the Abl kinase domain (Abl KD). Here, we analysed clinical outcome and mu

  15. The human epidermal growth factor receptor (EGFR gene in European patients with advanced colorectal cancer harbors infrequent mutations in its tyrosine kinase domain

    Directory of Open Access Journals (Sweden)

    Delvenne Philippe

    2011-10-01

    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR, a member of the ErbB family of receptors, is a transmembrane tyrosine kinase (TK activated by the binding of extracellular ligands of the EGF-family and involved in triggering the MAPK signaling pathway, which leads to cell proliferation. Mutations in the EGFR tyrosine kinase domain are frequent in non-small-cell lung cancer (NSCLC. However, to date, only very few, mainly non-European, studies have reported rare EGFR mutations in colorectal cancer (CRC. Methods We screened 236 clinical tumor samples from European patients with advanced CRC by direct DNA sequencing to detect potential, as yet unknown mutations, in the EGFR gene exons 18 to 21, mainly covering the EGFR TK catalytic domain. Results EGFR sequences showed somatic missense mutations in exons 18 and 20 at a frequency of 2.1% and 0.4% respectively. Somatic SNPs were also found in exons 20 and 21 at a frequency of about 3.1% and 0.4% respectively. Of these mutations, four have not yet been described elsewhere. Conclusions These mutation frequencies are higher than in a similarly sized population characterized by Barber and colleagues, but still too low to account for a major role played by the EGFR gene in CRC.

  16. Testing ERBB2 p.L755S kinase domain mutation as a druggable target in a patient with advanced colorectal cancer

    Science.gov (United States)

    Aung, Kyaw L.; Stockley, Tracy L.; Serra, Stefano; Kamel-Reid, Suzanne; Bedard, Philippe L.; Siu, Lillian L.

    2016-01-01

    Recent advances in molecular profiling technologies allow genetic driver events in individual tumors to be identified. The hypothesis behind this ongoing molecular profiling effort is that improvement in patients’ clinical outcomes will be achieved by inhibiting these discovered genetic driver events with matched targeted drugs. This hypothesis is currently being tested in oncology clinics with variable early results. Herein, we present our experience with a case of advanced colorectal cancer (CRC) with an ERBB2 p.L755S kinase domain mutation, a BRAF p.N581S mutation, and an APC p.Q1429fs mutation, together with a brief review of the literature describing the biological and clinical significance of ERRB2 kinase domain mutations in CRC. The patient was treated with trastuzumab combined with infusional 5-fluorouracil and leucovorin based on the presence of ERBB2 p.L755S kinase mutation in the tumor and based on the available evidence at the time when standard treatment options had been exhausted. However, there was no therapeutic response illustrating the challenges we face in managing patients with potentially targetable mutations where results from functional in vitro and in vivo studies lag behind those of genomic sequencing studies. Also lagging behind are clinical utility data from oncology clinics, hampering rapid therapeutic advances. Our case also highlights the logistical barriers associated with getting the most optimal therapeutic agents to the right patient in this era of personalized therapeutics based on cancer genomics. PMID:27626067

  17. Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance.

    Science.gov (United States)

    Barber-Zucker, Shiran; Uebe, René; Davidov, Geula; Navon, Yotam; Sherf, Dror; Chill, Jordan H; Kass, Itamar; Bitton, Ronit; Schüler, Dirk; Zarivach, Raz

    2016-08-23

    Cation diffusion facilitators (CDF) are highly conserved, metal ion efflux transporters that maintain divalent transition metal cation homeostasis. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). MamM is a magnetosome-associated CDF protein essential for the biomineralization of magnetic iron-oxide particles in magnetotactic bacteria. To investigate the structure-function relationship of CDF cytoplasmic domains, we characterized a MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein ZnT-10. Our results show that the M250P exchange in MamM causes severe structural changes in its CTD resulting in abnormal reduced function. Our in vivo, in vitro and in silico studies indicate that the CTD fold is critical for CDF proteins' proper function and support the previously suggested role of the CDF cytoplasmic domain as a CDF regulatory element. Based on our results, we also suggest a mechanism for the effects of the ZnT-10 L349P mutation in human.

  18. Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX.

    Directory of Open Access Journals (Sweden)

    Raphael Roduit

    Full Text Available BACKGROUND: NR2E3 (PNR is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S- cone syndrome (ESCS and, more recently, with autosomal dominant retinitis pigmentosa (adRP. NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD. The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2. NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE: These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients.

  19. Do Src Kinase and Caveolin Interact Directly with Na,K-ATPase?

    Science.gov (United States)

    Yosef, Eliyahu; Katz, Adriana; Peleg, Yoav; Mehlman, Tevie; Karlish, Steven J D

    2016-05-27

    Much evidence points to a role of Na,K-ATPase in ouabain-dependent signal transduction. Based on experiments with different cell lines and native tissue membranes, a current hypothesis postulates direct interactions between the Na,K-ATPase and Src kinase (non-receptor tyrosine kinase). Na,K-ATPase is proposed to bind Src kinase and inhibit its activity, whereas ouabain, the specific Na,K-ATPase inhibitor, binds and stabilizes the E2 conformation, thus exposing the Src kinase domain and its active site Tyr-418 for activation. Ouabain-dependent signaling is thought to be mediated within caveolae by a complex consisting of Na,K-ATPase, caveolin, and Src kinase. In the current work, we have looked for direct interactions utilizing purified recombinant Na,K-ATPase (human α1β1FXYD1 or porcine α1D369Nβ1FXYD1) and purified human Src kinase and human caveolin 1 or interactions between these proteins in native membrane vesicles isolated from rabbit kidney. By several independent criteria and techniques, no stable interactions were detected between Na,K-ATPase and purified Src kinase. Na,K-ATPase was found to be a substrate for Src kinase phosphorylation at Tyr-144. Clear evidence for a direct interaction between purified human Na,K-ATPase and human caveolin was obtained, albeit with a low molar stoichiometry (1:15-30 caveolin 1/Na,K-ATPase). In native renal membranes, a specific caveolin 14-5 oligomer (95 kDa) was found to be in direct interaction with Na,K-ATPase. We inferred that a small fraction of the renal Na,K-ATPase molecules is in a ∼1:1 complex with a caveolin 14-5 oligomer. Thus, overall, whereas a direct caveolin 1/Na,K-ATPase interaction is confirmed, the lack of direct Src kinase/Na,K-ATPase binding requires reassessment of the mechanism of ouabain-dependent signaling.

  20. Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain.

    Science.gov (United States)

    Fahsold, R; Hoffmeyer, S; Mischung, C; Gille, C; Ehlers, C; Kücükceylan, N; Abdel-Nour, M; Gewies, A; Peters, H; Kaufmann, D; Buske, A; Tinschert, S; Nürnberg, P

    2000-03-01

    More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles

  1. An activating mutation reveals a second binding mode of the integrin α2 I domain to the GFOGER motif in collagens.

    Directory of Open Access Journals (Sweden)

    Federico Carafoli

    Full Text Available The GFOGER motif in collagens (O denotes hydroxyproline represents a high-affinity binding site for all collagen-binding integrins. Other GxOGER motifs require integrin activation for maximal binding. The E318W mutant of the integrin α2β1 I domain displays a relaxed collagen specificity, typical of an active state. E318W binds more strongly than the wild-type α2 I domain to GMOGER, and forms a 2:1 complex with a homotrimeric, collagen-like, GFOGER peptide. Crystal structure analysis of this complex reveals two E318W I domains, A and B, bound to a single triple helix. The E318W I domains are virtually identical to the collagen-bound wild-type I domain, suggesting that the E318W mutation activates the I domain by destabilising the unligated conformation. E318W I domain A interacts with two collagen chains similarly to wild-type I domain (high-affinity mode. E318W I domain B makes favourable interactions with only one collagen chain (low-affinity mode. This observation suggests that single GxOGER motifs in the heterotrimeric collagens V and IX may support binding of activated integrins.

  2. Single point mutations in the helicase domain of the NS3 protein enhance dengue virus replicative capacity in human monocyte-derived dendritic cells and circumvent the type I interferon response.

    Science.gov (United States)

    Silveira, G F; Strottmann, D M; de Borba, L; Mansur, D S; Zanchin, N I T; Bordignon, J; dos Santos, C N Duarte

    2016-01-01

    Dengue is the most prevalent arboviral disease worldwide. The outcome of the infection is determined by the interplay of viral and host factors. In the present study, we evaluated the cellular response of human monocyte-derived DCs (mdDCs) infected with recombinant dengue virus type 1 (DV1) strains carrying a single point mutation in the NS3hel protein (L435S or L480S). Both mutated viruses infect and replicate more efficiently and produce more viral progeny in infected mdDCs compared with the parental, non-mutated virus (vBACDV1). Additionally, global gene expression analysis using cDNA microarrays revealed that the mutated DVs induce the up-regulation of the interferon (IFN) signalling and pattern recognition receptor (PRR) canonical pathways in mdDCs. Pronounced production of type I IFN were detected specifically in mdDCs infected with DV1-NS3hel-mutated virus compared with mdDCs infected with the parental virus. In addition, we showed that the type I IFN produced by mdDCs is able to reduce DV1 infection rates, suggesting that cytokine function is effective but not sufficient to mediate viral clearance of DV1-NS3hel-mutated strains. Our results demonstrate that single point mutations in subdomain 2 have important implications for adenosine triphosphatase (ATPase) activity of DV1-NS3hel. Although a direct functional connection between the increased ATPase activity and viral replication still requires further studies, these mutations speed up viral RNA replication and are sufficient to enhance viral replicative capacity in human primary cell infection and circumvent type I IFN activity. This information may have particular relevance for attenuated vaccine protocols designed for DV.

  3. Investigating Mutations to Reduce Huntingtin Aggregation by Increasing Htt-N-Terminal Stability and Weakening Interactions with PolyQ Domain

    Science.gov (United States)

    Mazza-Anthony, Cody; Waldispühl, Jérôme

    2016-01-01

    Huntington's disease is a fatal autosomal genetic disorder characterized by an expanded glutamine-coding CAG repeat sequence in the huntingtin (Htt) exon 1 gene. The Htt protein associated with the disease misfolds into toxic oligomers and aggregate fibril structures. Competing models for the misfolding and aggregation phenomena have suggested the role of the Htt-N-terminal region and the CAG trinucleotide repeats (polyQ domain) in affecting aggregation propensities and misfolding. In particular, one model suggests a correlation between structural stability and the emergence of toxic oligomers, whereas a second model proposes that molecular interactions with the extended polyQ domain increase aggregation propensity. In this paper, we computationally explore the potential to reduce Htt aggregation by addressing the aggregation causes outlined in both models. We investigate the mutation landscape of the Htt-N-terminal region and explore amino acid residue mutations that affect its structural stability and hydrophobic interactions with the polyQ domain. Out of the millions of 3-point mutation combinations that we explored, the (L4K E12K K15E) was the most promising mutation combination that addressed aggregation causes in both models. The mutant structure exhibited extreme alpha-helical stability, low amyloidogenicity potential, a hydrophobic residue replacement, and removal of a solvent-inaccessible intermolecular side chain that assists oligomerization. PMID:28096892

  4. Mutation in the RRM2 domain of TDP-43 in Amyotrophic Lateral Sclerosis with rapid progression associated with ubiquitin positive aggregates in cultured motor neurons.

    Science.gov (United States)

    Maurel, Cindy; Madji-Hounoum, Blandine; Thepault, Rose-Anne; Marouillat, Sylviane; Brulard, Céline; Danel-Brunaud, Véronique; Camdessanche, Jean-Philippe; Blasco, Helene; Corcia, Philippe; Andres, Christian R; Vourc'h, Patrick

    2017-07-13

    Mutations in the TAR-DNA Binding Protein-43 (TDP-43) encoding the TARDBP gene are present in amyotrophic lateral sclerosis (ALS). TDP-43 is the major component of ubiquitin-positive inclusions in motor neurons in ALS patients. We report here a novel heterozygous missense mutation in TARDBP in an ALS patient presenting a rapid form of ALS. This mutation p.N259S is located within the RNA recognition motif 2 (RRM2) in very close proximity with nucleotides in RNA. It is the first time a mutation was reported in this RRM2 domain of TDP-43. Expression of TDP-43(N259S) in neuronal cells NSC-34 and in primary cultures of motor neurons was associated with cytoplasmic TDP-43/ubiquitin positive inclusions. Our findings identified for the first time a mutation in ALS in the RRM2 domain of TDP-43, reinforcing the link between this RNA-binding protein, perturbations in RNA metabolism, disruption in protein homeostasis and ALS.

  5. Lethal mutations in the major homology region and their suppressors act by modulating the dimerization of the rous sarcoma virus capsid protein C-terminal domain.

    Science.gov (United States)

    Dalessio, Paula M; Craven, Rebecca C; Lokhandwala, Parvez M; Ropson, Ira J

    2013-02-01

    An infective retrovirus requires a mature capsid shell around the viral replication complex. This shell is formed by about 1500 capsid protein monomers, organized into hexamer and pentamer rings that are linked to each other by the dimerization of the C-terminal domain (CTD). The major homology region (MHR), the most highly conserved protein sequence across retroviral genomes, is part of the CTD. Several mutations in the MHR appear to block infectivity by preventing capsid formation. Suppressor mutations have been identified that are distant in sequence and structure from the MHR and restore capsid formation. The effects of two lethal and two suppressor mutations on the stability and function of the CTD were examined. No correlation with infectivity was found for the stability of the lethal mutations (D155Y-CTD, F167Y-CTD) and suppressor mutations (R185W-CTD, I190V-CTD). The stabilities of three double mutant proteins (D155Y/R185W-CTD, F167Y/R185W-CTD, and F167Y/I190V-CTD) were additive. However, the dimerization affinity of the mutant proteins correlated strongly with biological function. The CTD proteins with lethal mutations did not dimerize, while those with suppressor mutations had greater dimerization affinity than WT-CTD. The suppressor mutations were able to partially correct the dimerization defect caused by the lethal MHR mutations in double mutant proteins. Despite their dramatic effects on dimerization, none of these residues participate directly in the proposed dimerization interface in a mature capsid. These findings suggest that the conserved sequence of the MHR has critical roles in the conformation(s) of the CTD that are required for dimerization and correct capsid maturation. Copyright © 2012 Wiley Periodicals, Inc.

  6. The α2β2 isoform combination dominates the astrocytic Na(+) /K(+) -ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation.

    Science.gov (United States)

    Stoica, Anca; Larsen, Brian Roland; Assentoft, Mette; Holm, Rikke; Holt, Leanne Melissa; Vilhardt, Frederik; Vilsen, Bente; Lykke-Hartmann, Karin; Olsen, Michelle Lynne; MacAulay, Nanna

    2017-08-08

    Synaptic activity results in transient elevations in extracellular K(+) , clearance of which is critical for sustained function of the nervous system. The K(+) clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na(+) /K(+) -ATPase. The Na(+) /K(+) -ATPase consists of an α and a β subunit, each with several isoforms present in the central nervous system, of which the α2β2 and α2β1 isoform combinations are kinetically geared for astrocytic K(+) clearance. While transcript analysis data designate α2β2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and β isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi-quantification was carried out with polyhistidine-tagged Na(+) /K(+) -ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both β1 and β2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na(+) /K(+) -ATPase is dominated by α2, paired with β1 or β2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2(+/G301R) mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K(+) dynamics. © 2017 Wiley Periodicals, Inc.

  7. The α2β2 isoform combination dominates the astrocytic Na+/K+-ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation

    DEFF Research Database (Denmark)

    Stoica, Anca; Larsen, Brian Roland; Assentoft, Mette

    2017-01-01

    with both β1 and β2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na+/K+-ATPase is dominated by α2, paired with β1 or β2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma...

  8. A Two-amino Acid Mutation Encountered in Duchenne Muscular Dystrophy Decreases Stability of the Rod Domain 23 (R23) Spectrin-like Repeat of Dystrophin.

    Science.gov (United States)

    Legardinier, Sébastien; Legrand, Baptiste; Raguénès-Nicol, Céline; Bondon, Arnaud; Hardy, Serge; Tascon, Christophe; Le Rumeur, Elisabeth; Hubert, Jean-François

    2009-03-27

    Lack of functional dystrophin causes severe Duchenne muscular dystrophy. The subsarcolemmal location of dystrophin, as well as its association with both cytoskeleton and membrane, suggests a role in the mechanical regulation of muscular membrane stress. In particular, phenotype rescue in a Duchenne muscular dystrophy mice model has shown that some parts of the central rod domain of dystrophin, constituted by 24 spectrin-like repeats, are essential. In this study, we made use of rare missense pathogenic mutations in the dystrophin gene and analyzed the biochemical properties of the isolated repeat 23 bearing single or double mutations E2910V and N2912D found in muscle dystrophy with severity grading. No dramatic effect on secondary and tertiary structure of the repeat was found in mutants compared with wild type as revealed by circular dichroism and NMR. Thermal and chemical unfolding data from circular dichroism and tryptophan fluorescence show significant decrease of stability for the mutants, and stopped-flow spectroscopy shows decreased refolding rates. The most deleterious single mutation is the N2912D replacement, although we observe additive effects of the two mutations on repeat stability. Based on three-dimensional structures built by homology molecular modeling, we discuss the modifications of the mutation-induced repeat stability. We conclude that the main forces involved in repeat stability are electrostatic inter-helix interactions that are disrupted following mutations. This study represents the first analysis at the protein level of the consequences of missense mutations in the human dystrophin rod domain. Our results suggest that it may participate in mechanical weakening of dystrophin-deficient muscle.

  9. Probing subunit-subunit interactions in the yeast vacuolar ATPase by peptide arrays.

    Directory of Open Access Journals (Sweden)

    Lee S Parsons

    Full Text Available BACKGROUND: Vacuolar (H(+-ATPase (V-ATPase; V(1V(o-ATPase is a large multisubunit enzyme complex found in the endomembrane system of all eukaryotic cells where its proton pumping action serves to acidify subcellular organelles. In the plasma membrane of certain specialized tissues, V-ATPase functions to pump protons from the cytoplasm into the extracellular space. The activity of the V-ATPase is regulated by a reversible dissociation mechanism that involves breaking and re-forming of protein-protein interactions in the V(1-ATPase - V(o-proton channel interface. The mechanism responsible for regulated V-ATPase dissociation is poorly understood, largely due to a lack of detailed knowledge of the molecular interactions that are responsible for the structural and functional link between the soluble ATPase and membrane bound proton channel domains. METHODOLOGY/PRINCIPAL FINDINGS: To gain insight into where some of the stator subunits of the V-ATPase associate with each other, we have developed peptide arrays from the primary sequences of V-ATPase subunits. By probing the peptide arrays with individually expressed V-ATPase subunits, we have identified several key interactions involving stator subunits E, G, C, H and the N-terminal domain of the membrane bound a subunit. CONCLUSIONS: The subunit-peptide interactions identified from the peptide arrays complement low resolution structural models of the eukaryotic vacuolar ATPase obtained from transmission electron microscopy. The subunit-subunit interaction data are discussed in context of our current model of reversible enzyme dissociation.

  10. An SMC ATPase mutant disrupts chromosome segregation in Caulobacter.

    Science.gov (United States)

    Schwartz, Monica A; Shapiro, Lucy

    2011-12-01

    Accurate replication and segregation of the bacterial genome are essential for cell cycle progression. We have identified a single amino acid substitution in the Caulobacter structural maintenance of chromosomes (SMC) protein that disrupts chromosome segregation and cell division. The E1076Q point mutation in the SMC ATPase domain caused a dominant-negative phenotype in which DNA replication was able to proceed, but duplicated parS centromeres, normally found at opposite cell poles, remained at one pole. The cellular positions of other chromosomal loci were in the wild-type order relative to the parS centromere, but chromosomes remained unsegregated and appeared to be stacked upon one another. Purified SMC-E1076Q was deficient in ATP hydrolysis and exhibited abnormally stable binding to DNA. We propose that SMC spuriously links the duplicated chromosome immediately after passage of the replication fork. In wild-type cells, ATP hydrolysis opens the SMC dimer, freeing one chromosome to segregate to the opposite pole. The loss of ATP hydrolysis causes the SMC-E1076Q dimer to remain bound to both chromosomes, inhibiting segregation.

  11. Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast.

    Science.gov (United States)

    Banerjee, Subhrajit; Kane, Patricia M

    2017-07-18

    Luminal pH and phosphoinositide content are fundamental features of organelle identity. V-ATPases drive organelle acidification in all eukaryotes, and membrane-bound a-subunit isoforms of the V-ATPase are implicated in organelle-specific targeting and regulation. Earlier work demonstrated that the endo-lysosomal lipid PI(3,5)P2 activates V-ATPases containing the vacuolar a-subunit isoform in S. cerevisiae Here we demonstrate that PI4P, the predominant Golgi PI species, directly interacts with the cytosolic amino terminal (NT) domain of the yeast Golgi V-ATPase a-isoform, Stv1. Lysine 84 of Stv1NT is essential for interaction with PI4P in vitro and in vivo, and interaction with PI4P is required for efficient localization of Stv1-containing V-ATPases. The cytosolic NT domain of the human V-ATPase a2 isoform specifically interacts with PI4P in vitro, consistent with its Golgi localization and function. We propose that NT domains of Vo a-subunit isoforms interact specifically with PI lipids in their organelles of residence. These interactions can transmit organelle-specific targeting or regulation information to V-ATPases. © 2017 by The American Society for Cell Biology.

  12. Effects of naturally occurring missense mutations and G525V in the hydratase domain of human d-bifunctional protein on hydratase activity

    Directory of Open Access Journals (Sweden)

    Shirou Tsuchida

    2015-03-01

    Full Text Available d-bifunctional protein (d-BP deficiency is thought to lead to severe lipid metabolism disorders. To investigate the effect of naturally occurring missense mutations in the hydratase domain in d-BP, we constructed several d-BP hydratase variants and measured their activities. Missense mutations at sites whose conservation rates among 30 eukaryotes were < 70% did not affect hydratase activity. We predicted that missense mutations of highly conserved amino acids would markedly reduce activity. However, R562H and R562L, naturally occurring missense mutations of highly conserved amino acids, did not reduce activity. This result suggests that a missense mutation in a highly conserved amino acid does not always lead to severe lipid metabolism disorders. We also investigated the effect of G525V, which had been found in a mildly symptomatic patient with d-BP deficiency who was heterozygous for G525 and G658X. G525V markedly reduced hydratase activity. We had predicted that heterozygous G525V and G658X would lead to severely disordered lipid metabolism. However, the symptoms were inconsistent with this prediction. Characterizing mutations in the d-BP gene and the symptoms of d-BP deficiency may require pleiotropy, not only in vitro, studies.

  13. Two different point mutations in ABL gene ATP-binding domain conferring Primary Imatinib resistance in a Chronic Myeloid Leukemia (CML patient: A case report

    Directory of Open Access Journals (Sweden)

    Iqbal Zafar

    2004-01-01

    Full Text Available Imatinib (Gleevec is the effective therapy for BCR-ABL positive CML patients. Point mutations have been detected in ATP-binding domain of ABL gene which disturbs the binding of Gleevec to this target leading to resistance. Detection of mutations is helpful in clinical management of imatinib resistance. We established a very sensitive (ASO PCR to detect mutations in an imatinib-resistant CML patient. Mutations C944T and T1052C were detected which cause complete partial imatinib resistance, respectively. This is the first report of multiple point mutations conferring primary imatinib resistance in same patient at the same time. Understanding the biological reasons of primary imatinib resistance is one of the emerging issues of pharmacogenomics and will be helpful in understanding primary resistance of molecularly-targeted cancer therapies. It will also be of great utilization in clinical management of imatinib resistance. Moreover, this ASO-PCR assay is very effective in detecting mutations related to imatinib resistance.

  14. The free energy barrier for arginine gating charge translation is altered by mutations in the voltage sensor domain.

    Directory of Open Access Journals (Sweden)

    Christine S Schwaiger

    Full Text Available The gating of voltage-gated ion channels is controlled by the arginine-rich S4 helix of the voltage-sensor domain moving in response to an external potential. Recent studies have suggested that S4 moves in three to four steps to open the conducting pore, thus visiting several intermediate conformations during gating. However, the exact conformational changes are not known in detail. For instance, it has been suggested that there is a local rotation in the helix corresponding to short segments of a 3(10-helix moving along S4 during opening and closing. Here, we have explored the energetics of the transition between the fully open state (based on the X-ray structure and the first intermediate state towards channel closing (C1, modeled from experimental constraints. We show that conformations within 3 Å of the X-ray structure are obtained in simulations starting from the C1 model, and directly observe the previously suggested sliding 3(10-helix region in S4. Through systematic free energy calculations, we show that the C1 state is a stable intermediate conformation and determine free energy profiles for moving between the states without constraints. Mutations indicate several residues in a narrow hydrophobic band in the voltage sensor contribute to the barrier between the open and C1 states, with F233 in the S2 helix having the largest influence. Substitution for smaller amino acids reduces the transition cost, while introduction of a larger ring increases it, largely confirming experimental activation shift results. There is a systematic correlation between the local aromatic ring rotation, the arginine barrier crossing, and the corresponding relative free energy. In particular, it appears to be more advantageous for the F233 side chain to rotate towards the extracellular side when arginines cross the hydrophobic region.

  15. GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site.

    Science.gov (United States)

    Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

    2015-11-27

    K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4B(WT)-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4B(WT)-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding.

  16. A novel missense mutation in the C-terminal domain of lipoprotein lipase (Glu410-->Val) leads to enzyme inactivation and familial chylomicronemia.

    Science.gov (United States)

    Previato, L; Guardamagna, O; Dugi, K A; Ronan, R; Talley, G D; Santamarina-Fojo, S; Brewer, H B

    1994-09-01

    Lipoprotein lipase (LPL) is a complex enzyme consisting of multiple functional domains essential for the initial hydrolysis of triglycerides present in plasma lipoproteins. Previous studies have localized the catalytic domain of LPL, responsible for the hydrolytic function of the enzyme, to the N-terminus whereas the C-terminal end may play a role in lipid and heparin binding. To date, most described missense mutations resulting in a nonfunctional LPL have been located in the N-terminal region of the enzyme. In this manuscript we describe the defect in the LPL gene of a patient with triglycerides ranging from normal to 12,000 mg/dl, low LPL mass, and no LPL activity in post-heparin plasma. Sequencing of patient PCR-amplified DNA identified two separate mutations in the C-terminal domain of LPL: an A-->T transversion at nucleotide 1484 resulting in a Glu410-->Val substitution and a C-->G mutation at position 1595 that introduces a premature stop codon at position 447. Digestion with MaeIII and MnII established that the patient is a true homozygote for both mutations. In order to investigate the functional significance of these defects, mutant enzymes containing either the Val410 or the Ter447 mutations as well as both Val410 and Ter447, were expressed in vitro. Compared to the wild-type enzyme, LPL447 demonstrated a moderate reduction of specific activity using triolein (70% of normal) and tributyrin (74% of normal) substrates, while LPL410 had a significant (11% and 23% of normal) reduction of the normal lipase and esterase specific activities, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Structural mutations of C-domains in members of the Ig superfamily. Consequences for the interactions between the T cell antigen receptor and the zeta 2 homodimer

    DEFF Research Database (Denmark)

    Geisler, C; Rubin, B; Caspar-Bauguil, S;

    1992-01-01

    not fully understood. We locate critical amino acid residues for TCR assembly in the Ti-alpha and -beta extracellular C-domains. A point mutation (phenylalanine195----valine) in a highly conserved residue in the Ti-alpha chain of the Jurkat variant J79 was identified by DNA sequencing. This mutation did......-alpha-deficient Jurkat variant. Computer model analysis showed that the Ti-alpha phenylalanine195 directly contributed to the beta-sheet facing away from the Ti-beta chain, indicating that it could be directly involved in the interactions between one or more of the CD3 chains or the zeta 2 dimer. Site......-directed mutagenesis of the corresponding residue in the Ti-beta chain demonstrated that a phenylalanine216----valine substitution had similar effects on TCR assembly as the Ti-alpha mutation, whereas a phenylalanine216----histidine substitution allowed TCR assembly and expression. Whether the consequences for TCR...

  18. Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2012-01-01

    ATP7A and ATP7B are copper-transporting P-1B-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and C

  19. Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2012-01-01

    ATP7A and ATP7B are copper-transporting P-1B-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and C

  20. Investigating the Inhibitory Effect of Wortmannin in the Hotspot Mutation at Codon 1047 of PIK3CA Kinase Domain: A Molecular Docking and Molecular Dynamics Approach.

    Science.gov (United States)

    Kumar, D Thirumal; Doss, C George Priya

    2016-01-01

    Oncogenic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) are the most frequently reported in association with various forms of cancer. Several studies have reported the significance of hotspot mutations in a catalytic subunit of PIK3CA in association with breast cancer. Mutations are frequently observed in the highly conserved region of the kinase domain (797-1068 amino acids) of PIK3CA are activating or gain-of-function mutations. Mutation in codon 1047 occurs in the C-terminal region of the kinase domain with histidine (H) replaced by arginine (R), lysine (L), and tyrosine (Y). Pathogenicity and protein stability predictors PhD-SNP, Align GVGD, HANSA, iStable, and MUpro classified H1047R as highly deleterious when compared to H1047L and H1047Y. To explore the inhibitory activity of Wortmannin toward PIK3CA, the three-dimensional structure of the mutant protein was determined using homology modeling followed by molecular docking and molecular dynamics analysis. Docking studies were performed for the three mutants and native with Wortmannin to measure the differences in their binding pattern. Comparative docking study revealed that H1047R-Wortmannin complex has a higher number of hydrogen bonds as well as the best binding affinity next to the native protein. Furthermore, 100 ns molecular dynamics simulation was initiated with the docked complexes to understand the various changes induced by the mutation. Though Wortmannin was found to nullify the effect of H1047R over the protein, further studies are required for designing a better compound. As SNPs are major genetic variations observed in disease condition, personalized medicine would provide enhanced drug therapy.

  1. V-type ATPase proton pump expression during enamel formation.

    Science.gov (United States)

    Sarkar, Juni; Wen, Xin; Simanian, Emil J; Paine, Michael L

    2016-01-01

    Several diseases such as proximal and distal renal tubular acidosis and osteoporosis are related to intracellular pH dysregulation resulting from mutations in genes coding for ion channels, including proteins comprising the proton-pumping V-type ATPase. V-type ATPase is a multi-subunit protein complex expressed in enamel forming cells. V-type ATPase plays a key role in enamel development, specifically lysosomal acidification, yet our understanding of the relationship between the endocytotic activities and dental health and disease is limited. The objective of this study is to better understand the ameloblast-associated pH regulatory networks essential for amelogenesis. Quantitative RT-PCR was performed on tissues from secretory-stage and maturation-stage enamel organs to determine which of the V-type ATPase subunits are most highly upregulated during maturation-stage amelogenesis: a time when ameloblast endocytotic activity is highest. Western blot analyses, using specific antibodies to four of the V-type ATPase subunits (Atp6v0d2, Atp6v1b2, Atp6v1c1 and Atp6v1e1), were then applied to validate much of the qPCR data. Immunohistochemistry using these same four antibodies was also performed to identify the spatiotemporal expression profiles of individual V-type ATPase subunits. Our data show that cytoplasmic V-type ATPase is significantly upregulated in enamel organ cells during maturation-stage when compared to secretory-stage. These data likely relate to the higher endocytotic activities, and the greater need for lysosomal acidification, during maturation-stage amelogenesis. It is also apparent from our immunolocalization data, using antibodies against two of the V-type ATPase subunits (Atp6v1c1 and Atp6v1e1), that significant expression is seen at the apical membrane of maturation-stage ameloblasts. Others have also identified this V-type ATPase expression profile at the apical membrane of maturation ameloblasts. Collectively, these data better define the

  2. In and out of the cation pumps: P-type ATPase structure revisited

    DEFF Research Database (Denmark)

    Bublitz, Maike; Poulsen, Hanne; Morth, Jens Preben

    2010-01-01

    Active transport across membranes is a crucial requirement for life. P-type ATPases build up electrochemical gradients at the expense of ATP by forming and splitting a covalent phosphoenzyme intermediate, coupled to conformational changes in the transmembrane section where the ions are translocated....... The marked increment during the last three years in the number of crystal structures of P-type ATPases has greatly improved our understanding of the similarities and differences of pumps with different ion specificities, since the structures of the Ca2+-ATPase, the Na+,K+-ATPase and the H+-ATPase can now...... be compared directly. Mechanisms for ion gating, charge neutralization and backflow prevention are starting to emerge from comparative structural analysis; and in combination with functional studies of mutated pumps this provides a framework for speculating on how the ions are bound and released as well...

  3. Quantitative Gait Analysis Using a Motorized Treadmill System Sensitively Detects Motor Abnormalities in Mice Expressing ATPase Defective Spastin.

    Science.gov (United States)

    Connell, James W; Allison, Rachel; Reid, Evan

    2016-01-01

    The hereditary spastic paraplegias (HSPs) are genetic conditions in which there is progressive axonal degeneration in the corticospinal tract. Autosomal dominant mutations, including nonsense, frameshift and missense changes, in the gene encoding the microtubule severing ATPase spastin are the most common cause of HSP in North America and northern Europe. In this study we report quantitative gait analysis using a motorized treadmill system, carried out on mice knocked-in for a disease-associated mutation affecting a critical residue in the Walker A motif of the spastin ATPase domain. At 4 months and at one year of age homozygous mutant mice had a number of abnormal gait parameters, including in stride length and stride duration, compared to heterozygous and wild-type littermates. Gait parameters in heterozygous animals did not differ from wild-type littermates. We conclude that quantitative gait analysis using the DigiGait system sensitively detects motor abnormalities in a hereditary spastic paraplegia model, and would be a useful method for analyzing the effects of pharmacological treatments for HSP.

  4. Mutations in the basic domain and the loop-helix II junction of TWIST abolish DNA binding in Saethre-Chotzen syndrome.

    Science.gov (United States)

    El Ghouzzi, V; Legeai-Mallet, L; Benoist-Lasselin, C; Lajeunie, E; Renier, D; Munnich, A; Bonaventure, J

    2001-03-09

    Saethre-Chotzen syndrome is an autosomal dominant skull disorder resulting from premature fusion of coronal sutures (craniosynostosis). It is caused by mutations in the TWIST gene encoding a basic Helix-Loop-Helix transcription factor. Here we report on the identification of a novel mutation affecting a highly conserved residue of the basic domain. Unlike nonsense and missense mutations lying within helices, this mutation does not affect protein stability or heterodimerisation of TWIST with its partner E12. However, it does abolish TWIST binding capacity to a target E-box as efficiently as two missense mutations in the loop-helix II junction. By contrast, elongation of the loop through a 7 amino acid insertion appears not to hamper binding to the DNA target. We conclude that loss of TWIST protein function in Saethre-Chotzen patients can occur at three different levels, namely protein stability, dimerisation, and DNA binding and that the loop-helix II junction is essential for effective protein-DNA interaction.

  5. Mutations in NOTCH1 PEST-domain orchestrate CCL19-driven homing of Chronic Lymphocytic Leukemia cells by modulating the tumor suppressor gene DUSP22.

    Science.gov (United States)

    Arruga, F; Gizdic, B; Bologna, C; Cignetto, S; Buonincontri, R; Serra, S; Vaisitti, T; Gizzi, K; Vitale, N; Garaffo, G; Mereu, E; Diop, F; Neri, F; Incarnato, D; Coscia, M; Allan, J; Piva, R; Oliviero, S; Furman, R R; Rossi, D; Gaidano, G; Deaglio, S

    2016-12-26

    Even if NOTCH1 is commonly mutated in Chronic Lymphocytic Leukemia (CLL), its functional impact in the disease remains unclear. Using CRISPR/Cas9-generated Mec-1 cell line models, we show that NOTCH1 regulates growth and homing of CLL cells by dictating expression levels of the tumor suppressor gene DUSP22. Specifically, NOTCH1 affects the methylation of DUSP22 promoter by modulating a nuclear complex, which tunes the activity of DNA methyltransferase 3A (DNMT3A). These effects are enhanced by PEST-domain mutations, which stabilize the molecule and prolong signaling. CLL patients with a NOTCH1-mutated clone showed low levels of DUSP22 and active chemotaxis to CCL19. Lastly, in xenograft models, NOTCH1-mutated cells displayed a unique homing behavior, localizing preferentially to the spleen and brain. These findings connect NOTCH1, DUSP22, and CCL19-driven chemotaxis within a single functional network, suggesting that modulation of the homing process may provide a relevant contribution to the unfavorable prognosis associated with NOTCH1 mutations in CLL.Leukemia accepted article preview online, 26 December 2016. doi:10.1038/leu.2016.383.

  6. Novel human mutation and CRISPR/Cas genome-edited mice reveal the importance of C-terminal domain of MSX1 in tooth and palate development.

    Science.gov (United States)

    Mitsui, Silvia Naomi; Yasue, Akihiro; Masuda, Kiyoshi; Naruto, Takuya; Minegishi, Yoshiyuki; Oyadomari, Seiichi; Noji, Sumihare; Imoto, Issei; Tanaka, Eiji

    2016-12-05

    Several mutations, located mainly in the MSX1 homeodomain, have been identified in non-syndromic tooth agenesis predominantly affecting premolars and third molars. We identified a novel frameshift mutation of the highly conserved C-terminal domain of MSX1, known as Msx homology domain 6 (MH6), in a Japanese family with non-syndromic tooth agenesis. To investigate the importance of MH6 in tooth development, Msx1 was targeted in mice with CRISPR/Cas system. Although heterozygous MH6 disruption did not alter craniofacial development, homozygous mice exhibited agenesis of lower incisors with or without cleft palate at E16.5. In addition, agenesis of the upper third molars and the lower second and third molars were observed in 4-week-old mutant mice. Although the upper second molars were present, they were abnormally small. These results suggest that the C-terminal domain of MSX1 is important for tooth and palate development, and demonstrate that that CRISPR/Cas system can be used as a tool to assess causality of human disorders in vivo and to study the importance of conserved domains in genes.

  7. Combinations of mutations in the connection domain of human immunodeficiency virus type 1 reverse transcriptase: assessing the impact on nucleoside and nonnucleoside reverse transcriptase inhibitor resistance.

    Science.gov (United States)

    Gupta, Soumi; Fransen, Signe; Paxinos, Ellen E; Stawiski, Eric; Huang, Wei; Petropoulos, Christos J

    2010-05-01

    Recent reports have described the effect of mutations in the connection and RNase H domains of reverse transcriptase (RT) on nucleoside and nonnucleoside reverse transcriptase inhibitor (NRTI and NNRTI, respectively) resistance in the presence of thymidine analog resistance mutations (TAMs) and NNRTI mutations (J. H. Brehm, D. Koontz, J. D. Meteer, V. Pathak, N. Sluis-Cremer, and J. W. Mellors, J. Virol. 81:7852-7859, 2007; K. A. Delviks-Frankenberry, G. N. Nikolenko, R. Barr, and V. K. Pathak, J. Virol. 81:6837-6845, 2007; G. N. Nikolenko, K. A. Delviks-Frankenberry, S. Palmer, F. Maldarelli, M. J. Fivash, Jr., J. M. Coffin, and V. K. Pathak, Proc. Natl. Acad. Sci. U. S. A. 104:317-322, 2007; G. N. Nikolenko, S. Palmer, F. Maldarelli, J. W. Mellors, J. M. Coffin, and V. K. Pathak, Proc. Natl. Acad. Sci. U. S. A. 102:2093-2098, 2005; and S. H. Yap, C. W. Sheen, J. Fahey, M. Zanin, D. Tyssen, V. D. Lima, B. Wynhoven, M. Kuiper, N. Sluis-Cremer, P. R. Harrigan, and G. Tachedjian, PLoS Med. 4:e335, 2007). In the present study, novel mutations in the connection domain of RT (T369I/V), first identified in patient-derived viruses, were characterized, and their effects on NNRTI and NNRTI susceptibility were determined. Furthermore, the effect of N348I on NRTI and NNRTI resistance was confirmed. HIV-1 with either N348I or T369I/V demonstrated reduced susceptibility to nevirapine (NVP), efavirenz (EFV), delaviridine (DLV), and zidovudine (ZDV) compared to wild-type HIV-1. However, HIV-1 with T369I and N348I demonstrated 10- to 60-fold resistance to these same drugs. In clinical samples, these two connection domain RT mutations were predominantly observed in viruses containing TAMs and NNRTI mutations and did not alter the susceptible-resistant classifications of these samples. Introduction of T369I, N348I, or T369I/N348I also reduced replication capacity (RC). These observations suggest that it may be of scientific interest to test these mutations against new NNRTI

  8. Functional coupling of V-ATPase and CLC-5

    Science.gov (United States)

    Satoh, Nobuhiko; Suzuki, Masashi; Nakamura, Motonobu; Suzuki, Atsushi; Horita, Shoko; Seki, George; Moriya, Kyoji

    2017-01-01

    Dent’s disease is an X-linked renal tubulopathy characterized by low molecular weight proteinuria, hypercalciuria and progressive renal failure. Disease aetiology is associated with mutations in the CLCN5 gene coding for the electrogenic 2Cl-/H+ antiporter chloride channel 5 (CLC-5), which is expressed in the apical endosomes of renal proximal tubules with the vacuolar type H+-ATPase (V-ATPase). Initially identified as a member of the CLC family of Cl- channels, CLC-5 was presumed to provide Cl- shunt into the endosomal lumen to dissipate H+ accumulation by V-ATPase, thereby facilitating efficient endosomal acidification. However, recent findings showing that CLC-5 is in fact not a Cl- channel but a 2Cl-/H+ antiporter challenged this classical shunt model, leading to a renewed and intense debate on its physiological roles. Cl- accumulation via CLC-5 is predicted to play a critical role in endocytosis, as illustrated in mice carrying an artificial Cl- channel mutation E211A that developed defective endocytosis but normal endosomal acidification. Conversely, a recent functional analysis of a newly identified disease-causing Cl- channel mutation E211Q in a patient with typical Dent’s disease confirmed the functional coupling between V-ATPase and CLC-5 in endosomal acidification, lending support to the classical shunt model. In this editorial, we will address the current recognition of the physiological role of CLC-5 with a specific focus on the functional coupling of V-ATPase and CLC-5. PMID:28101447

  9. Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

    Science.gov (United States)

    Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Razafsky, David S.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

    2012-01-01

    Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies. PMID:22649085

  10. A novel LQT3 mutation implicates the human cardiac sodium channel domain IVS6 in inactivation kinetics

    NARCIS (Netherlands)

    Groenewegen, WA; Bezzina, CR; van Tintelen, JP; Hoorntje, TM; Mannens, MMAM; Wilde, AAM; Jongsma, HJ; Rook, MB

    2003-01-01

    The Long QT3 syndrome is associated with mutations in the cardiac sodium channel gene SCN5A. Objective: The aim of the present study was the identification and functional characterization of a mutation in a family with the long QT3 syndrome. Methods: The human cardiac sodium channel gene SCN5A was s

  11. Genetic Mutation of Vitamin K-dependent Gamma-glutamyl Car-boxylase Domain in Patients with Calcium Oxalate Urolithiasis

    Institute of Scientific and Technical Information of China (English)

    Jiankun QIAO; Tao WANG; Jun YANG; Jihong LIU; Xiaoxin GONG; Xiaolin GUO; Shaogang WANG; Zhangqun YE

    2009-01-01

    To investigate the exon mutation of vitamin K-dependent gamma-glutamyl carboxylase (GGCX or VKDC) in patients with calcium oxalate urolithasis, renal cortex and peripheral blood sam-ples were obtained from severe hydronephrosis patients (with or without calculi), and renal tumor pa-tients undergoing nephrectomy. GGCX mutations in all 15 exons were examined in 44 patients with calcium oxalate urolithiasis (COU) by polymerase chain reaction (PCR) and denatured high pressure liquid chromatography (DHPLC), and confirmed by sequencing. Mutation was not found in all COU samples compared to the controls. These data demonstrated that functional GGCX mutations in all 15 exons do not occur in most COU patients. It was suggested that there may be no significant association between the low activity and mutation of GGCX in COU.

  12. Helical stalk segments S4 and S5 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae are optimized to impact catalytic site environment.

    Science.gov (United States)

    Soteropoulos, P; Valiakhmetov, A; Kashiwazaki, R; Perlin, D S

    2001-05-11

    The stalk segments of P-type ion-translocating enzymes are presumed to play important roles in energy coupling. In this work, stalk segments S4 and S5 of the yeast H(+)-ATPase were examined for helical character, optimal length, and segment orientation by a combination of proline substitution, insertion/deletion mutagenesis, and second-site suppressor analyses. The substitution of various residues for helix-disrupting proline in both S4 (L353P,L353G; A354P; and G371P) and S5 (D676P and I684P) resulted in highly defective or inactive enzymes supporting the importance of helical character and/or the maintenance of essential interactions. The contiguous helical nature of transmembrane segment M5 and stalk element S5 was explored and found to be favorable, although not essential. The deletion or addition of one or more amino acids at positions Ala(354) in S4 and Asp(676) in S5, which were intended to either rotate helical faces or extend/reduce the length of helical segments, resulted in enzyme destabilization that abolished most enzyme assembly. Second-site suppressor mutations were obtained to primary site mutations G371A (S4) and D676G (S5) and were analyzed with a molecular structure model of the H(+)-ATPase. Primary site mutations were predicted to alter the site of phosphorylation either directly or indirectly. The suppressor mutations either directly changed packing around the primary site or altered the environment of the site of phosphorylation. Overall, these data support the view that stalk segments S4 and S5 of the H(+)-ATPase are helical elements that are optimized for length and interactions with other stalk elements and can influence the phosphorylation domain.

  13. A novel angiotensin I-converting enzyme mutation (S333W impairs N-domain enzymatic cleavage of the anti-fibrotic peptide, AcSDKP.

    Directory of Open Access Journals (Sweden)

    Sergei M Danilov

    Full Text Available BACKGROUND: Angiotensin I-converting enzyme (ACE has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis. RESULTS: We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold to cleave the physiological substrate AcSDKP compared to wild-type ACE. CONCLUSIONS AND SIGNIFICANCE: A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues, may confer protection against fibrosis.

  14. Analysis of evolution and virological characteristics of rtI233V mutations in the hepatitis B virus reverse transcriptase domain

    Directory of Open Access Journals (Sweden)

    Xiao-ling YE

    2015-04-01

    Full Text Available Objective To analyze the evolution of rtI233V mutation in the reverse transcriptase domain of hepatitis B virus (HBV and its association with adefovir dipivoxil (ADV resistance. Methods The rate of detection of rtI233V mutation in 9830 patients with chronic HBV infection was analyzed. HBV reverse transcriptase genes isolated from serial serum samples of two patients were amplified by nested PCR, and clonal sequencing (>20 clones/sample was performed to analyze the evolution of rtI233V mutations. The replica of pTriEx-HBV1.1 vectors harboring wild-type and mutant strains (rtI233V, rtN236T, rtI233V+rtN236T were respectively constructed and transiently transfected into HepG2 cells. Media containing serial concentrations of lamivudine (LAM, ADV, entecavir (ETV, or tenofovir (TDF were used to treat the cells. Then HBV DNA in the supernatants was quantitatively determined by real-time PCR in order to analyze HBV mutants' replication competence and phenotypic characteristics under the drug pressures. Results The detection rate of rtI233V mutation in 9830 nucleos(tide analogues-treated patients was 0.28% (28/9830, including 0.19% (19 patients with rtI233V individual mutation and 0.09% (9 patients with rtI233V mutation combining with rtN236T or other mutations. All of the patients had rtI233V had ADV exposure history: 16 (57.1% of them received ADV monotherapy for over six months, and 12(42.9% of them received ADV combined sequential therapy for over 12 months. Replication competence and phenotypic resistance analysis showed rtI233V and wild-type strains had similar viral replication competence, while rtN236T exhibited significantly lower replication competence compared with wild-type strains. rtI233V strains remained sensitive to LAM, ADV, ETV, and TDF and showed little influence on drug resistance when combined with rtN236T, but it showed ability to restore the defected replication capacity of rtN236T strains. Conclusions  The rtI233V mutation

  15. Regulatory Mechanisms in the P4-ATPase Complex

    DEFF Research Database (Denmark)

    Costa, Sara

    Eukaryotic cell membranes are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Several relevant physiological processes depend on trans-bilayer phospholipid asymmetry, including vesiculation...... of their activity and regulation remain to be elucidated. Therefore, these studies focus on the role of the catalytic and CDC50 β-subunit in the phospholipid translocation and the regulation processes behind it. Recent studies suggested that P4-ATPase complex functionality is highly dependent on the conformation...... autoinhibitory domains in both N- and C-terminus of the P4-ATPase catalytic subunit from yeast. They regulate flippase activity in a coordinated manner which suggests the presence of a cross-talk between both protein termini. Furthermore, characterizing P4-ATPase activity is not a trivial task...

  16. Complete kinetic and thermodynamic characterization of the unisite catalytic pathway of Escherichia coli F1-ATPase. Comparison with mitochondrial F1-ATPase and application to the study of mutant enzymes.

    Science.gov (United States)

    Al-Shawi, M K; Senior, A E

    1988-12-25

    A complete analysis is presented of the component rate constants of the "unisite" reaction pathway in normal Escherichia coli F1-ATPase. Gibbs free energy profiles of the unisite reaction pathway were constructed for both normal E. coli F1 and bovine-heart mitochondrial F1, and comparison indicated that E. coli F1 is an ancestral form of the mitochondrial enzyme. Similar kinetic and thermodynamic analyses of the unisite reaction pathway were done for mutant beta-Asn-242 and beta-Val-242 E. coli F1-ATPases. Both mutations affected unisite binding and hydrolysis of MgATP but had little effect on release of products or binding of MgADP. It was apparent that a primary effect of the mutations was on the interaction between the catalytic nucleotide-binding domain and the substrate MgATP. The catalytic transition state [F1-ATP]++ was the most destabilized step in the reaction sequence. Measurements of delta delta G[F1.ATP]++ and linear free energy plots for the catalytic step were consistent with the view that, in normal enzyme, residue beta-Asp-242 accepts an H-bond from the transition-state substrate in order to facilitate catalysis. Both mutations impaired positive catalytic cooperativity. This was caused by energetic destabilization of the catalytic transition state and was an indirect effect, not a direct effect on signal transmission per se between catalytic nucleotide-binding domains on beta-subunits. Therefore, impairment of unisite catalysis and of positive catalytic cooperativity appeared to be linked. This may provide a unifying explanation as to why a series of other, widely separated mis-sense mutations within the catalytic nucleotide-binding domain on F1-beta-subunit, which have been reported to affect unisite catalysis, also impair positive catalytic cooperativity. Linear free energy plots for the ATP-binding step of unisite catalysis demonstrated that beta-Asn-242 and beta-Val-242 mutant enzymes did not suffer any gross disruptive change in structure of

  17. The Domain II S4-S5 Linker in Nav1.9: A Missense Mutation Enhances Activation, Impairs Fast Inactivation, and Produces Human Painful Neuropathy.

    Science.gov (United States)

    Han, Chongyang; Yang, Yang; de Greef, Bianca T A; Hoeijmakers, Janneke G J; Gerrits, Monique M; Verhamme, Camiel; Qu, Jian; Lauria, Giuseppe; Merkies, Ingemar S J; Faber, Catharina G; Dib-Hajj, Sulayman D; Waxman, Stephen G

    2015-06-01

    Painful small fiber neuropathy is a challenging medical condition with no effective treatment. Non-genetic causes can be identified in one half of the subjects. Gain-of-function variants of sodium channels Nav1.7 and Nav1.8 have recently been associated with painful small fiber neuropathy. More recently, mutations of sodium channel Nav1.9 have been linked to human pain disorders, with two gain-of-function mutations found in patients with painful small fiber neuropathy. Here we report a novel Nav1.9 mutation, a glycine 699 substitution by arginine (G699R) in the domain II S4-S5 linker, identified in a patient with painful small fiber neuropathy. In this study, we assayed the mutant channels by voltage-clamp in superior cervical ganglion neurons, which do not produce endogenous Nav1.8 or Nav1.9 currents, and provide a novel platform where Nav1.9 is expressed at relatively high levels. Voltage-clamp analysis showed that the mutation hyperpolarizes (-10.1 mV) channel activation, depolarizes (+6.3 mV) steady-state fast inactivation, slows deactivation, and enhances ramp responses compared with wild-type Nav1.9 channels. Current-clamp analysis showed that the G699R mutant channels render dorsal root ganglion neurons hyperexcitable, via depolarized resting membrane potential, reduced current threshold and increased evoked firing. These observations show that the domain II S4-S5 linker plays an important role in the gating of Nav1.9 and demonstrates that a mutation in this linker is linked to a common pain disorder.

  18. A novel mutation causing nephronophthisis in the Lewis polycystic kidney rat localises to a conserved RCC1 domain in Nek8

    Directory of Open Access Journals (Sweden)

    McCooke John K

    2012-08-01

    Full Text Available Abstract Background Nephronophthisis (NPHP as a cause of cystic kidney disease is the most common genetic cause of progressive renal failure in children and young adults. NPHP is characterized by abnormal and/or loss of function of proteins associated with primary cilia. Previously, we characterized an autosomal recessive phenotype of cystic kidney disease in the Lewis Polycystic Kidney (LPK rat. Results In this study, quantitative trait locus analysis was used to define a ~1.6Mbp region on rat chromosome 10q25 harbouring the lpk mutation. Targeted genome capture and next-generation sequencing of this region identified a non-synonymous mutation R650C in the NIMA (never in mitosis gene a- related kinase 8 ( Nek8 gene. This is a novel Nek8 mutation that occurs within the regulator of chromosome condensation 1 (RCC1-like region of the protein. Specifically, the R650C substitution is located within a G[QRC]LG repeat motif of the predicted seven bladed beta-propeller structure of the RCC1 domain. The rat Nek8 gene is located in a region syntenic to portions of human chromosome 17 and mouse 11. Scanning electron microscopy confirmed abnormally long cilia on LPK kidney epithelial cells, and fluorescence immunohistochemistry for Nek8 protein revealed altered cilia localisation. Conclusions When assessed relative to other Nek8 NPHP mutations, our results indicate the whole propeller structure of the RCC1 domain is important, as the different mutations cause comparable phenotypes. This study establishes the LPK rat as a novel model system for NPHP and further consolidates the link between cystic kidney disease and cilia proteins.

  19. Episodic ataxia type 1 mutations affect fast inactivation of K+ channels by a reduction in either subunit surface expression or affinity for inactivation domain.

    Science.gov (United States)

    Imbrici, Paola; D'Adamo, Maria Cristina; Grottesi, Alessandro; Biscarini, Andrea; Pessia, Mauro

    2011-06-01

    Episodic ataxia type 1 (EA1) is an autosomal dominant disorder characterized by continuous myokymia and episodic attacks of ataxia. Mutations in the gene KCNA1 that encodes the voltage-gated potassium channel Kv1.1 are responsible for EA1. In several brain areas, Kv1.1 coassembles with Kv1.4, which confers N-type inactivating properties to heteromeric channels. It is therefore likely that the rate of inactivation will be determined by the number of Kv1.4 inactivation particles, as set by the precise subunit stoichiometry. We propose that EA1 mutations affect the rate of N-type inactivation either by reduced subunit surface expression, giving rise to a reduced number of Kv1.1 subunits in heterotetramer Kv1.1-Kv1.4 channels, or by reduced affinity for the Kv1.4 inactivation domain. To test this hypothesis, quantified amounts of mRNA for Kv1.4 or Kv1.1 containing selected EA1 mutations either in the inner vestibule of Kv1.1 on S6 or in the transmembrane regions were injected into Xenopus laevis oocytes and the relative rates of inactivation and stoichiometry were determined. The S6 mutations, V404I and V408A, which had normal surface expression, reduced the rate of inactivation by a decreased affinity for the inactivation domain while the mutations I177N in S1 and E325D in S5, which had reduced subunit surface expression, increased the rate of N-type inactivation due to a stoichiometric increase in the number of Kv1.4 subunits.

  20. The molecular structure of the Na(+)-translocating F1F0-ATPase of Acetobacterium woodii, as revealed by electron microscopy, resembles that of H(+)-translocating ATPases.

    Science.gov (United States)

    Reidlinger, J; Mayer, F; Müller, V

    1994-12-12

    The Na(+)-translocating F1F0-ATPase of Acetobacterium woodii was examined by electron microscopy. After reconstitution into proteoliposomes, knobs typical for the F1 domain were visible on the outside of the membrane. The F1-part of the isolated enzyme showed a hexagonal symmetry suggesting an alpha 3 beta 3 structure, and the F1F0 complex had molecular dimensions very similar to those of H(+)-translocating ATPases of E. coli, chloroplasts, and mitochondria.

  1. Intronic mutations outside of Alu-repeat-rich domains of the LDL receptor gene are a cause of familial hypercholesterolemia.

    Science.gov (United States)

    Amsellem, Sabine; Briffaut, Dorothée; Carrié, Alain; Rabès, Jean Pierre; Girardet, Jean Philippe; Fredenrich, Alexandre; Moulin, Philippe; Krempf, Michel; Reznik, Yves; Vialettes, Bernard; de Gennes, Jean Luc; Brukert, Eric; Benlian, Pascale

    2002-12-01

    Familial hypercholesterolemia (FH), a frequent monogenic condition complicated by premature cardiovascular disease, is characterized by high allelic heterogeneity at the low-density lipoprotein receptor ( LDLR) locus. Despite more than a decade of genetic testing, knowledge about intronic disease-causing mutations has remained limited because of lack of available genomic sequences. Based on the finding from bioinformatic analysis that Alu repeats represent 85% of LDLR intronic sequences outside exon-intron junctions, we designed a strategy to improve the exploration of genomic regions in the vicinity of exons in 110 FH subjects from an admixed population. In the first group of 42 patients of negative mutation carriers, as previously established by former screening strategies (denaturing gradient gel electrophoresis, DNA sequencing with former primers overlapping splice-sites, Southern Blotting), about half ( n=22) were found to be carriers of at least one heterozygous mutation. Among a second group of 68 newly recruited patients, 27% of mutation carriers ( n=37) had a splicing regulatory mutation. Overall, out of the 54 mutations identified, 13 were intronic, and 18 were novel, out of which nearly half were intronic. Two novel intronic mutations (IVS8-10G-->A within the polypyrimidine tract and IVS7+10G-->A downstream of donor site) might create potential aberrant splice sites according to neural-network computed estimation, contrary to 31 common single nucleotide variations also identified at exon-intron junctions. This new strategy of detecting the most likely disease-causing LDLR mutations outside of Alu-rich genomic regions reveals that intronic mutations may have a greater impact than previously reported on the molecular basis of FH.

  2. Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Baron, J. Allen; Chen, Janice S.; Culotta, Valeria C., E-mail: vculott1@jhu.edu

    2015-07-03

    In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain. This “synthetic lethality” was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection. - Highlights: • In yeast, the anti-oxidant enzyme SOD1 promotes activity of the proton ATPase Pma1p. • Cells expressing a T912D variant of Pma1p are not viable without SOD1. • SOD1 is needed to protect Pma1-T912D expressing cells from severe oxidative damage. • SOD1 activates Pma1p through casein kinase signaling and oxidative stress protection.

  3. Elucidating Functional Aspects of P-type ATPases

    DEFF Research Database (Denmark)

    Autzen, Henriette Elisabeth

    2015-01-01

    similar to that of the wild type (WT) protein. The discrepancy between the newly determined crystal structure of LpCopA and the functional manifestations of the missense mutation in human CopA, could indicate that LpCopA is insufficient in structurally elucidating the effect of disease-causing mutations...... cancer and pathogenic microbes. The goal of this Ph.D. dissertation was to functionally characterize SERCA1a and CopA from Legionella pneumophila (LpCopA) through a range of different methods within structural biology. Crystallographic studies of SERCA1a led to a newly determined crystal structure......P-type ATPases are proteins that act to maintain ion homeostasis and electrochemical gradients through the translocation of cations across cell membranes. Underscoring their significance in humans, dysfunction of the ATPases can lead to crucial diseases. Dysfunction of the sarco...

  4. Crystal structure of a copper-transporting PIB-type ATPase

    DEFF Research Database (Denmark)

    Gourdon, Pontus; Liu, Xiang-Yu; Skjørringe, Tina

    2011-01-01

    Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu(+)-ATPase, ......Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu......(+)-ATPase, in a copper-free form, as determined by X-ray crystallography at 3.2 Å resolution. The structure indicates a three-stage copper transport pathway involving several conserved residues. A PIB-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a putative...... copper entry point at the intracellular interface. Comparisons to Ca(2+)-ATPase suggest an ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site. The structure also provides a framework to analyse missense mutations in the human ATP7A and ATP7B...

  5. Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain.

    Science.gov (United States)

    Laitinen, O H; Airenne, K J; Marttila, A T; Kulik, T; Porkka, E; Bayer, E A; Wilchek, M; Kulomaa, M S

    1999-11-12

    Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W-->K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K(d) approximately 10(-15) M of the wild-type tetramer down to K(d) approximately 10(-8) M for both W-->K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both W-->K mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization.

  6. Sensitive detection of pre-existing BCR-ABL kinase domain mutations in CD34+ cells of newly diagnosed chronic-phase chronic myeloid leukemia patients is associated with imatinib resistance: implications in the post-imatinib era.

    Directory of Open Access Journals (Sweden)

    Zafar Iqbal

    Full Text Available BACKGROUND: BCR-ABL kinase domain mutations are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CML patients. Recent studies indicate the presence of pre-existing BCR-ABL mutations in a higher percentage of CML patients when CD34+ stem/progenitor cells are investigated using sensitive techniques, and these mutations are associated with imatinib resistance and disease progression. However, such studies were limited to smaller number of patients. METHODS: We investigated BCR-ABL kinase domain mutations in CD34+ cells from 100 chronic-phase CML patients by multiplex allele-specific PCR and sequencing at diagnosis. Mutations were re-investigated upon manifestation of imatinib resistance using allele-specific PCR and direct sequencing of BCR-ABL kinase domain. RESULTS: Pre-existing BCR-ABL mutations were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8-48, all patients with pre-existing BCR-ABL mutations exhibited imatinib resistance. Of the 68 patients without pre-existing BCR-ABL mutations, 24 developed imatinib resistance; allele-specific PCR and BCR-ABL kinase domain sequencing detected mutations in 22 of these patients. All 32 patients with pre-existing BCR-ABL mutations had the same mutations after manifestation of imatinib-resistance. In imatinib-resistant patients without pre-existing BCR-ABL mutations, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients except T315I and Y253F mutations responded to imatinib dose escalation. CONCLUSION: Pre-existing BCR-ABL mutations can be detected in a substantial number of chronic-phase CML patients by sensitive allele-specific PCR technique using CD34+ cells. These mutations are associated with imatinib resistance if affecting drug binding directly or indirectly. After the recent approval of nilotinib, dasatinib, bosutinib and ponatinib for treatment of chronic myeloid

  7. Pathogenic Cysteine Removal Mutations in FGFR Extracellular Domains Stabilize Receptor Dimers and Perturb the TM Dimer Structure.

    Science.gov (United States)

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-10-09

    Missense mutations that introduce or remove cysteine residues in receptor tyrosine kinases are believed to cause pathologies by stabilizing the active receptor tyrosine kinase dimers. However, the magnitude of this stabilizing effect has not been measured for full-length receptors. Here, we characterize the dimer stabilities of three full-length fibroblast growth factor receptor (FGFR) mutants harboring pathogenic cysteine substitutions: the C178S FGFR1 mutant, the C342R FGFR2 mutant, and the C228R FGFR3 mutant. We find that the three mutations stabilize the FGFR dimers. We further see that the mutations alter the configuration of the FGFR transmembrane dimers. Thus, both aberrant dimerization and perturbed dimer structure likely contribute to the pathological phenotypes arising due to these mutations.

  8. A novel mutation in the L12 domain of keratin 1 is associated with mild epidermolytic ichthyosis

    NARCIS (Netherlands)

    Bolling, M. C.; Bladergroen, R. S.; van Steensel, M. A. M.; Willemsen, M.; Jonkman, M. F.; van Geel, M.

    P>Background Epidermolytic ichthyosis (EI), previously termed bullous congenital ichthyosiform erythroderma or epidermolytic hyperkeratosis, is a clinically heterogeneous genodermatosis caused by mutations in the genes encoding the suprabasal keratins 1 and 10. Classical EI is clinically

  9. Disulfide bond formation between the COOH-terminal domain of the beta subunits and the gamma and epsilon subunits of the Escherichia coli F1-ATPase. Structural implications and functional consequences.

    Science.gov (United States)

    Aggeler, R; Haughton, M A; Capaldi, R A

    1995-04-21

    A set of mutants of the Escherichia coli F1F0-type ATPase has been generated by site-directed mutagenesis as follows: beta E381C, beta S383C, beta E381C/epsilon S108C, and beta S383C/epsilon S108C. Treatment of ECF1 isolated from any of these mutants with CuCl2 induces disulfide bond formation. For the single mutants, beta E381C and beta S383C, a disulfide bond is formed in essentially 100% yield between a beta subunit and the gamma subunit, probably at Cys87 based on the recent structure determination of F1 (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). In the double mutants, two disulfide bonds are formed, again in essentially full yield, one between beta and gamma, the other between a beta and the epsilon subunit via Cys108. The same two cross-links are produced with CuCl2 treatment of ECF1F0 isolated from either of the double mutants. These results show that the parts of gamma around residue 87 (a short alpha-helix) and the epsilon subunit interact with different beta subunits. The yield of covalent linkage of beta to gamma is nucleotide dependent and highest in ATP and much lower with ADP in catalytic sites. The yield of covalent linkage of beta to epsilon is also nucleotide dependent but in this case is highest in ADP and much lower in ATP. Disulfide bond formation between either beta and gamma, or beta and epsilon inhibits the ATPase activity of the enzyme in proportion to the yield of the cross-linked product. Chemical modification of the Cys at either position 381 or 383 of the beta subunit inhibits ATPase activity in a manner that appears to be dependent on the size of the modifying reagent. These results are as expected if movements of the catalytic site-containing beta subunits relative to the gamma and epsilon subunits are an essential part of the cooperativity of the enzyme.

  10. Identification of EGFR kinase domain mutations among lung cancer patients in China: implication for targeted cancer therapy

    Institute of Scientific and Technical Information of China (English)

    Bao Ming QIN; Xiao CHEN; Jing De ZHU; Duan Qing PEI

    2005-01-01

    Lung cancer is one of the leading causes of death with one of the lowest survival rates. However, a subset of lung cancer patients who are of Asian origin and carry somatic mutations in epidermal growth factor receptor or EGFR have responded remarkable well to two tyrosine kinase inhibitors, gefitinib and erlotinib. While EGFR mutation profiles have been reported from Japan, South Korea, and Taiwan, there is no such report from mainland of China where the largest pool of patients reside. In this report, we identified ten somatic mutations from a total of 41 lung cancer patients in China. Among them, seven mutations were found in 17 adenocarcinomas. In contrast to previous reports, eight of these mutations are deletions in exon 19 and two of these deletions are homozygous. These results suggest that a large portion of Chinese adenocarcinoma patients could benefit from gefitinib or erlotinib. This unique mutation profile provides a rationale to develop the next generation of EGFR inhibitors more suitable for the Chinese population.

  11. Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S.cerevisiae

    DEFF Research Database (Denmark)

    L. Rudashevskaya, Elena; Ye, Juanying; Jensen, Ole Nørregaard

    +-ATPases are app. 60 amino acid residues longer than their yeast homologous. Yeast is found to phosphorylate at least one residue within the plant C-terminus. At the same time a wide range of investigations on structure, function, regulation and interaction of H+-ATPase is carried out with implication...... It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H...... of heterologous system of yeast cells, expressing plant proton pump. Therefore identification of possible regulatory effects by phosphorylation events in plant H+-ATPase in the system is significant. A number of putative phosphorylation sites at regulatory C-domain of H+-ATPase (AHA2) have been point...

  12. The de novo cytosine methyltransferase DRM2 requires intact UBA domains and a catalytically mutated paralog DRM3 during RNA-directed DNA methylation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Ian R Henderson

    2010-10-01

    Full Text Available Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS rearranged methyltransferase2 (DRM2 is required for establishment of small interfering RNA (siRNA directed DNA methylation. In mammals PIWI proteins and piRNA act in a convergently evolved RNA-directed DNA methylation system that is required to repress transposon expression in the germ line. De novo methylation may also be independent of RNA interference and small RNAs, as in Neurospora crassa. Here we identify a clade of catalytically mutated DRM2 paralogs in flowering plant genomes, which in A.thaliana we term domains rearranged methyltransferase3 (DRM3. Despite being catalytically mutated, DRM3 is required for normal maintenance of non-CG DNA methylation, establishment of RNA-directed DNA methylation triggered by repeat sequences and accumulation of repeat-associated small RNAs. Although the mammalian catalytically inactive Dnmt3L paralogs act in an analogous manner, phylogenetic analysis indicates that the DRM and Dnmt3 protein families diverged independently in plants and animals. We also show by site-directed mutagenesis that both the DRM2 N-terminal UBA domains and C-terminal methyltransferase domain are required for normal RNA-directed DNA methylation, supporting an essential targeting function for the UBA domains. These results suggest that plant and mammalian RNA-directed DNA methylation systems consist of a combination of ancestral and convergent features.

  13. Cysteine residues 244 and 458-459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions.

    Science.gov (United States)

    Petrushanko, Irina Yu; Mitkevich, Vladimir A; Lakunina, Valentina A; Anashkina, Anastasia A; Spirin, Pavel V; Rubtsov, Peter M; Prassolov, Vladimir S; Bogdanov, Nikolay B; Hänggi, Pascal; Fuller, William; Makarov, Alexander A; Bogdanova, Anna

    2017-10-01

    Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines' thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na,K-ATPase

  14. Cysteine residues 244 and 458–459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions

    Directory of Open Access Journals (Sweden)

    Irina Yu. Petrushanko

    2017-10-01

    Full Text Available Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD and nucleotide binding domain (NBD of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines’ thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG. Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459 were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor

  15. Mutational library analysis of selected amino acids in the receptor binding domain of envelope of Akv murine leukemia virus by conditionally replication competent bicistronic vectors

    DEFF Research Database (Denmark)

    Bahrami, Shervin; Pedersen, Finn Skou; Duch, Mogens R.

    2003-01-01

    envelope expression. This vector functions as a replication competent mini-virus in a culture of NIH 3T3 derived semi-packaging cells that express the viral Gag and Pol proteins. Titers comparable to those of wild type virus were achieved by this system. To test this vector system, we created a random......The envelope protein of retroviruses is responsible for viral entry into host cells. Here, we describe a mutational library approach to dissect functional domains of the envelope protein involving a retroviral vector, which expresses both the envelope protein of Akv murine leukemia virus (MLV...... mutational library of Arg 85 and Asp 86 in the first variable region of Akv envelope protein. Homologous amino acids to Asp 86 in Moloney and Friend murine leukemia viruses are thought to be directly involved in receptor binding. Subsequent selection of mutants capable of infecting murine NIH 3T3 cells...

  16. Geographic structuring of the Plasmodium falciparum sarco(endoplasmic reticulum Ca2+ ATPase (PfSERCA gene diversity.

    Directory of Open Access Journals (Sweden)

    Ronan Jambou

    Full Text Available Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp, of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC(50 for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates. This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte.

  17. Conformational heterogeneity of the Roc domains in C. tepidum Roc-COR and implications for human LRRK2 Parkinson mutations

    NARCIS (Netherlands)

    Rudi, Katharina; Ho, Franz Y; Gilsbach, Bernd Karl; Pots, Henderikus; Wittinghofer, Alfred; Kortholt, Arjan; Klare, Johann P

    2015-01-01

    Ras of complex proteins (Roc) is a Ras-like GTP binding domain that always occurs in tandem with the C-terminal of Roc (COR) domain, and is found in bacteria, plants and animals. Recently, it has been shown that Roco proteins belong to the family of G-proteins activated by nucleotide-dependent dimer

  18. Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Thea Bismo Strøm

    2014-01-01

    Full Text Available More than 1700 mutations in the low density lipoprotein receptor (LDLR gene have been found to cause familial hypercholesterolemia (FH. These are commonly divided into five classes based upon their effects on the structure and function of the LDLR. However, little is known about the mechanism by which mutations in the transmembrane domain of the LDLR gene cause FH. We have studied how the transmembrane mutation G805R affects the function of the LDLR. Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum. This led to reduced amounts of the mature 160 kDa LDLR at the cell surface. However, significant amounts of a secreted 140 kDa G805R-LDLR ectodomain fragment was observed in the culture media. Treatment of the cells with the metalloproteinase inhibitor batimastat largely restored the amounts of the 120 and 160 kDa forms in cell lysates, and prevented secretion of the 140 kDa ectodomain fragment. Together, these data indicate that a metalloproteinase cleaved the ectodomain of the 120 kDa precursor G805R-LDLR in the endoplasmic reticulum. It was the presence of the polar Arg805 and not the lack of Gly805 which led to ectodomain cleavage. Arg805 also prevented γ-secretase cleavage within the transmembrane domain. It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

  19. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase

    DEFF Research Database (Denmark)

    Jahn, T.; Fuglsang, A.T.; Olsson, A.

    1997-01-01

    Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H...... plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase....

  20. Dominant Rio1 kinase/ATPase catalytic mutant induces trapping of late pre-40S biogenesis factors in 80S-like ribosomes.

    Science.gov (United States)

    Ferreira-Cerca, Sébastien; Kiburu, Irene; Thomson, Emma; LaRonde, Nicole; Hurt, Ed

    2014-07-01

    During eukaryotic ribosome biogenesis, members of the conserved atypical serine/threonine protein kinase family, the RIO kinases (Rio1, Rio2 and Rio3) function in small ribosomal subunit biogenesis. Structural analysis of Rio2 indicated a role as a conformation-sensing ATPase rather than a kinase to regulate its dynamic association with the pre-40S subunit. However, it remained elusive at which step and by which mechanism the other RIO kinase members act. Here, we have determined the crystal structure of the human Rio1-ATP-Mg(2+) complex carrying a phosphoaspartate in the active site indicative of ATPase activity. Structure-based mutations in yeast showed that Rio1's catalytic activity regulates its pre-40S association. Furthermore, we provide evidence that Rio1 associates with a very late pre-40S via its conserved C-terminal domain. Moreover, a rio1 dominant-negative mutant defective in ATP hydrolysis induced trapping of late biogenesis factors in pre-ribosomal particles, which turned out not to be pre-40S but 80S-like ribosomes. Thus, the RIO kinase fold generates a versatile ATPase enzyme, which in the case of Rio1 is activated following the Rio2 step to regulate one of the final 40S maturation events, at which time the 60S subunit is recruited for final quality control check.

  1. Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S.cerevisiae

    DEFF Research Database (Denmark)

    L. Rudashevskaya, Elena; Ye, Juanying; Jensen, Ole Nørregaard

     It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H......+-ATPases are app. 60 amino acid residues longer than their yeast homologous. Yeast is found to phosphorylate at least one residue within the plant C-terminus. At the same time a wide range of investigations on structure, function, regulation and interaction of H+-ATPase is carried out with implication......-mutated to alanine residues (to prevent possible phosphorylation) or aspartate residues (to mimic phosphorylation of residue) and the mutated aha2 enzyme expressed in the yeast strain RS-72. Most of the mutations show positive or negative effect on yeast growth in functional complementation assays. It shows in vivo...

  2. The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis.

    Science.gov (United States)

    Maheshwar, M M; Cheadle, J P; Jones, A C; Myring, J; Fryer, A E; Harris, P C; Sampson, J R

    1997-10-01

    Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.

  3. 肺癌患者KDR基因酪氨酸激酶结构域的突变和多态性研究%Mutation and polymorphism in the tyrosine kinase domain of KDR in Chinese human lung cancer patients

    Institute of Scientific and Technical Information of China (English)

    Shejuan An; Zhihong Chen; Jian Su; Jiaying Lin; Ying Huang; Hongyan Tang; Yilong Wu

    2009-01-01

    Objective: Although the kinase insert domain-containing receptor (KDR) gene play an very important role in the metastasis of cancer and is also as one of the molecular targets used in cancer therapy, mutation in the tyrosine kinase (TK) domain of the KDR gene has not been reported. Here we detected the mutations and polymorphisms in the TK domain of KDR gene in human lung cancer patients and to give the basic evidence and clue for cancer prevention and target therapy. Methods: The entire sequence of exons 21, 22, 23 and 27 (which contain the coding sequence of tyrosine phosphorylation)in the TK domain of KDR gene in the patients with lung cancer and control healthy individuals were assayed by PCR and DNA sequencing. We also analyzed one non-coding single nucteotide polymorphisms (SNPs) in the KDR gene. Results: No mutations were found in exon 22, 23 and 27. One heterozygous mutation of c.+2837 in exon 21 was found at a frequency of 2.08% (2/96) in the patients with lung cancer and none were detected in the healthy control individuals. The mutation was from a G to a A resulting in substitution of arginine with histidine residue. Conclusion: Our data suggested that we should focus on the mutation or SNP in the other regions or the expression levels of KDR gene, and the function of c.+2837 mutation of KDR gene may be needed further study in the future.

  4. Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation

    DEFF Research Database (Denmark)

    Cornelius, Flemming; Mahmmoud, Yasser Ahmed

    2007-01-01

    with anionic phospholipids. Specifically, the effects of the cytoplasmic domain of FXYD10, which contains the phosphorylation sites for protein kinases, on the kinetics of the Na,K-ATPase reaction were investigated by a comparison of the reconstituted native enzyme and the enzyme where 23 C-terminal amino...... acids of FXYD10 had been cleaved by mild, controlled trypsin treatment. Several kinetic properties of the Na,K-ATPase reaction cycle as well as the FXYD-regulation of Na,K-ATPase activity were found to be affected by acidic phospholipids like PI, PS, and PG. This takes into consideration the Na+ and K......+ activation, the K+-deocclusion reaction, and the poise of the E1/E2 conformational equilibrium, whereas the ATP activation was unchanged. Anionic phospholipids increased the intermolecular cross-linking between the FXYD10 C-terminus (Cys74) and the Cys254 in the Na,K-ATPase A-domain. However, neither...

  5. Amyloidogenic Properties of a D/N Mutated 12 Amino Acid Fragment of the C-Terminal Domain of the Cholesteryl-Ester Transfer Protein (CETP

    Directory of Open Access Journals (Sweden)

    Victor García-González

    2011-03-01

    Full Text Available The cholesteryl-ester transfer protein (CETP facilitates the transfer of cholesterol esters and triglycerides between lipoproteins in plasma where the critical site for its function is situated in the C-terminal domain. Our group has previously shown that this domain presents conformational changes in a non-lipid environment when the mutation D470N is introduced. Using a series of peptides derived from this C-terminal domain, the present study shows that these changes favor the induction of a secondary β-structure as characterized by spectroscopic analysis and fluorescence techniques. From this type of secondary structure, the formation of peptide aggregates and fibrillar structures with amyloid characteristics induced cytotoxicity in microglial cells in culture. These supramolecular structures promote cell cytotoxicity through the formation of reactive oxygen species (ROS and change the balance of a series of proteins that control the process of endocytosis, similar to that observed when β-amyloid fibrils are employed. Therefore, a fine balance between the highly dynamic secondary structure of the C-terminal domain of CETP, the net charge, and the physicochemical characteristics of the surrounding microenvironment define the type of secondary structure acquired. Changes in this balance might favor misfolding in this region, which would alter the lipid transfer capacity conducted by CETP, favoring its propensity to substitute its physiological function.

  6. Mutational analysis of dimeric linkers in peri- and cytoplasmic domains of histidine kinase DctB reveals their functional roles in signal transduction.

    Science.gov (United States)

    Liu, Jiwei; Yang, Jianguo; Wen, Jin; Yang, Yun; Wei, Xiaolu; Zhang, Xiaodong; Wang, Yi-Ping

    2014-06-01

    Membrane-associated histidine kinases (HKs) in two-component systems respond to environmental stimuli by autophosphorylation and phospho-transfer. HK typically contains a periplasmic sensor domain that regulates the cytoplasmic kinase domain through a conserved cytoplasmic linker. How signal is transduced from the ligand-binding site across the membrane barrier remains unclear. Here, we analyse two linker regions of a typical HK, DctB. One region connects the first transmembrane helix with the periplasmic Per-ARNT-Sim domains, while the other one connects the second transmembrane helix with the cytoplasmic kinase domains. We identify a leucine residue in the first linker region to be essential for the signal transduction and for maintaining the delicate balance of the dimeric interface, which is key to its activities. We also show that the other linker, belonging to the S-helix coiled-coil family, plays essential roles in signal transduction inside the cell. Furthermore, by combining mutations with opposing activities in the two regions, we show that these two signalling transduction elements are integrated to produce a combined effect on the final activity of DctB.

  7. Mutation and crystallization of the first KH domain of human polycytosine-binding protein 1 (PCBP1) in complex with DNA.

    Science.gov (United States)

    Yoga, Yano M K; Traore, Daouda A K; Wilce, Jacqueline A; Wilce, Matthew C J

    2011-10-01

    Polycytosine-binding proteins (PCBPs) are triple KH-domain proteins that play an important role in the regulation of translation of eukaryotic mRNA. They are also utilized by viral RNA and have been shown to interact with ssDNA. Underlying their function is the specific recognition of C-rich nucleotides by their KH domains. However, the structural basis of this recognition is only partially understood. Here, the preparation of a His-tagged KH domain is described, representing the first domain of PCBP1 that incorporates a C54S mutation as well as the addition of a C-terminal tryptophan. This construct has facilitated the preparation of highly diffracting crystals in complex with C-rich DNA (sequence ACCCCA). Crystals of the KH1-DNA complex were grown using the hanging-drop vapour-diffusion method in 0.1 M phosphate-citrate pH 4.2, 40%(v/v) PEG 300. X-ray diffraction data were collected to 1.77 Å resolution and the diffraction was consistent with space group P2(1), with unit-cell parameters a = 38.59, b = 111.88, c = 43.42 Å, α = γ = 90.0, β = 93.37°. The structure of the KH1-DNA complex will further our insight into the basis of cytosine specificity by PCBPs.

  8. Mutational analysis of residues in the regulatory CBS domains of Moorella thermoacetica pyrophosphatase corresponding to disease-related residues of human proteins.

    Science.gov (United States)

    Jämsen, Joonas; Tuominen, Heidi; Baykov, Alexander A; Lahti, Reijo

    2011-02-01

    mtCBS-PPase [CBS (cystathionine β-synthase) domain-containing pyrophosphatase from Moorella thermoacetica] contains a pair of CBS domains that strongly bind adenine nucleotides, thereby regulating enzyme activity. Eight residues associated with the CBS domains of mtCBS-PPase were screened to explore possible associations with regulation of enzyme activity. The majority of the substitutions (V99A, R168A, Y169A, Y169F, Y188A and H189A) enhanced the catalytic activity of mtCBS-PPase, two substitutions (R170A and R187G) decreased activity, and one substitution (K100G) had no effect. AMP-binding affinity was markedly decreased in the V99A, R168A and Y169A mutant proteins, and elevated in the R187G and H189A mutant proteins. Remarkably, the R168A and Y169A substitutions changed the effect of AMP from inhibition to activation. The stoichiometry of AMP binding increased from one to two AMP molecules per CBS domain pair in the Y169F, R170A, R187G and Y188A variants. The ADP-binding affinity decreased in three and increased in four mutant proteins. These findings identify residues determining the strength and selectivity of nucleotide binding, as well as the direction (inhibition or activation) of the subsequent effect. The data suggest that mutations in human CBS domain-containing proteins can be translated into a bacterial context. Furthermore, our data support the hypothesis that the CBS domains act as an 'internal inhibitor' of mtCBS-PPase.

  9. Symptomatic type 1 protein C deficiency caused by a de novo Ser270Leu mutation in the catalytic domain

    DEFF Research Database (Denmark)

    Lind, B; Koefoed, P; Thorsen, S

    2001-01-01

    the intracellular content of mutant and wild-type protein was similar. Northern blot analysis of total mRNA from transfected cells showed no reduction of the mutant protein C mRNA compared with wild-type protein C mRNA. Collectively, these results indicate that the Ser270Leu mutation in the affected family caused...

  10. Roles and mechanisms of copper transporting ATPases in cancer pathogenesis.

    Science.gov (United States)

    Zhang, Yuqing; Li, Min; Yao, Qizhi; Chen, Changyi

    2009-01-01

    Copper (Cu) is an essential trace element for cell metabolism as a cofactor to many key metabolic enzymes. Numerous physiological processes rely on the adequate and timely transport of copper ions mediated by copper-transporting ATPases (Cu-ATPases), which are essential for human cell growth and development. Inherited gene mutations of ATP7A and ATP7B result in clinical diseases related to damage in the multiple organ systems. Increased expression of these genes has been recently observed in some human cancer specimens, and may be associated with tumorigenesis and chemotherapy resistance. However, underlying mechanisms of Cu-ATPases in human cancer progression and treatment are largely unknown. In this review, we summarize current progress on the copper transport system, the structural and functional properties of the Cu-ATPases, ATP7A and ATP7B, in copper homeostasis, and their roles in anti-tumor drug resistance and cancer metastasis. This review provides valuable information for clinicians and researchers who want to recognize the newest advances in this new field and identify possible lines of investigation in copper transport as important mediators in human physiology and cancer.

  11. Structural Insight for Roles of DR5 Death Domain Mutations on Oligomerization of DR5 Death Domain-FADD Complex in the Death-Inducing Signaling Complex Formation: A Computational Study.

    Science.gov (United States)

    Yang, Hongyi; Song, Yuhua

    2016-04-01

    Death receptor 5 (DR5)-induced apoptosis that prioritizes the death of tumor cells has been proposed as one of the promising cancer therapies. In this process, oligomerized DR5 death domain (DD) binding to Fas-associated death domain (FADD) leads to FADD activating caspase-8, which marks the formation of the death-inducing signaling complex (DISC) that initiates apoptosis. DR5 DD mutations found in cancer cells have been suggested to play an important pathological role, the mechanism through which those mutants prevent the DR5-activated DISC formation is not clear yet. This study sought to provide structural and molecular insight for the roles of four selected DR5 DD mutations (E355K, E367K, K415N, and L363F) in the oligomerization of DR5 DD-FADD complex during the DISC formation. Results from the molecular dynamics simulations show that the simulated mutants induce conformational, dynamical motions and interactions changes in the DR5 DD-FADD tetramer complex, including changes in a protein's backbone flexibility, less exposure of FADD DED's caspase-8 binding site, reduced H-bonding and hydrophobic contacts at the DR5 DD-FADD DD binding, altered distribution of the electrostatic potentials and correlated motions of residues, and reduced binding affinity of DR5 DD binding to FADD. This study provides structural and molecular insight for the influence of DR5 DD mutations on oligomerization of DR5 DD-FADD complex, which is expected to foster understanding of the DR5 DD mutants' resistance mechanism against DR5-activated DISC formation.

  12. Penicillin-binding protein 2x of Streptococcus pneumoniae: the mutation Ala707Asp within the C-terminal PASTA2 domain leads to destabilization.

    Science.gov (United States)

    Schweizer, Inga; Peters, Katharina; Stahlmann, Christoph; Hakenbeck, Regine; Denapaite, Dalia

    2014-06-01

    Streptococcus pneumoniae penicillin-binding protein 2x (PBP2x) is an enzyme involved in the last stages of peptidoglycan assembly and essential for bacterial growth and survival. PBP2x localizes to the division site, a process that depends on its Penicillin-Binding Protein And Serine-Threonine-kinase Associated (PASTA) domains, which was previously demonstrated via GFP-PBP2x in living cells. During this study a mutant strain was isolated in which the GFP-PBP2x fusion protein did not localize at division sites and it contained reduced amounts of the full-length GFP-PBP2x. We now show that this defect is due to a point mutation within the C-terminal PASTA2 domain of PBP2x. The mutant protein was analyzed in detail in terms of beta-lactam binding, functionality, and localization in live cells. We demonstrate that the mutation affects the GFP-tagged PBP2x variant severely and renders it susceptible to the protease/chaperone HtrA.

  13. Field-collected permethrin-resistant Aedes aegypti from central Thailand contain point mutations in the domain IIS6 of the sodium channel gene (KDR).

    Science.gov (United States)

    Srisawat, Raweewan; Komalamisra, Narumon; Apiwathnasorn, Chamnarn; Paeporn, Pungasem; Roytrakul, Sittiruk; Rongsriyam, Yupha; Eshita, Yuki

    2012-11-01

    One of the mechanisms responsible for pyrethroid resistance in mosquitoes is mutations in domain IIS6 of voltage-gated sodium channel gene (kdr). Aedes aegypti larvae were collected from the central provinces of Thailand (Bangkok, Prachin Buri and Ratchaburi) and colonized until they became adults. Partial fragment of kdr of permethrin-resistant mosquitoes were amplified by RT-PCR and sequenced. Among the four nucleotide mutations detected, two mutations resulted in two amino acid substitutions, S(TCC) 989 P(CCC) and V(GTA)1016 G(GGA). Among 94 permethrin-resistant mosquitoes, the SS genotype (SS/VV) was found to predominate (n = 74), followed by SR (SP/VG) (n = 15) and RR (PP/ GG) genotypes (n = 5), with the resistant allele frequency ranging from 0.03 to 0.17. As pyrethroid insecticides are currently being advocated for use in Thailand, investigations of pyrethroid resistance in other regions of the country are needed to prevent potential cross-resistance among different types of insecticides.

  14. New ATPase regulators-p97 goes to the PUB

    DEFF Research Database (Denmark)

    Madsen, Louise; Seeger, Michael; Semple, Colin A;

    2009-01-01

    The conserved eukaryotic AAA-type ATPase complex, known as p97 or VCP in mammals and Cdc48 in yeast, is involved in a number of cellular pathways, including fusion of homotypic membranes, protein degradation, and activation of membrane-bound transcription factors. Most likely, p97 is directed to ...... of the currently known PUB-domain proteins and other p97-interacting proteins....

  15. Mutations of Asp540 and the domain-connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity.

    Science.gov (United States)

    Tanabe, Maiko; Ishino, Sonoko; Ishino, Yoshizumi; Nishida, Hirokazu

    2014-01-21

    The structure of Pyrococcus furiosus DNA ligase (PfuLig), which architecturally resembles human DNA ligase I (hLigI), revealed that the C-terminal helix stabilizes the closed conformation through several ionic interactions between two domains (adenylylation domain (AdD) and C-terminal OB-fold domain (OBD)). This helix is oriented differently in DNA-bound hLigI, suggesting that the disruption of its interactions with AdD facilitates DNA binding. Previously, we demonstrated that the replacement of Asp540 with arginine improves the ligation activity. Here we report that the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhanced the activity.

  16. The SMAD-binding domain of SKI: a hotspot for de novo mutations causing Shprintzen–Goldberg syndrome

    OpenAIRE

    Schepers, Dorien; Doyle, Alexander J; Oswald, Gretchen; Sparks, Elizabeth; Myers, Loretha; Patrick J Willems; Mansour, Sahar; Simpson, Michael A; Frysira, Helena; Maat-Kievit, Anneke; Van Minkelen, Rick; Hoogeboom, Jeanette M; Mortier, Geert R.; Titheradge, Hannah; Brueton, Louise

    2014-01-01

    Shprintzen–Goldberg syndrome (SGS) is a rare, systemic connective tissue disorder characterized by craniofacial, skeletal, and cardiovascular manifestations that show a significant overlap with the features observed in the Marfan (MFS) and Loeys–Dietz syndrome (LDS). A distinguishing observation in SGS patients is the presence of intellectual disability, although not all patients in this series present this finding. Recently, SGS was shown to be due to mutations in the SKI gene, encoding the ...

  17. Angiotensin I-converting enzyme Gln1069Arg mutation impairs trafficking to the cell surface resulting in selective denaturation of the C-domain.

    Directory of Open Access Journals (Sweden)

    Sergei M Danilov

    Full Text Available BACKGROUND: Angiotensin-converting enzyme (ACE; Kininase II; CD143 hydrolyzes small peptides such as angiotensin I, bradykinin, substance P, LH-RH and several others and thus plays a key role in blood pressure regulation and vascular remodeling. Complete absence of ACE in humans leads to renal tubular dysgenesis (RTD, a severe disorder of renal tubule development characterized by persistent fetal anuria and perinatal death. METHODOLOGY/PRINCIPAL FINDINGS: Patient with RTD in Lisbon, Portugal, maintained by peritoneal dialysis since birth, was found to have a homozygous substitution of Arg for Glu at position 1069 in the C-terminal domain of ACE (Q1069R resulting in absence of plasma ACE activity; both parents and a brother who are heterozygous carriers of this mutation had exactly half-normal plasma ACE activity compared to healthy individuals. We hypothesized that the Q1069R substitution impaired ACE trafficking to the cell surface and led to accumulation of catalytically inactive ACE in the cell cytoplasm. CHO cells expressing wild-type (WT vs. Q1069R-ACE demonstrated the mutant accumulates intracellularly and also that it is significantly degraded by intracellular proteases. Q1069R-ACE retained catalytic and immunological characteristics of WT-ACE N domain whereas it had 10-20% of the nativity of the WT-ACE C domain. A combination of chemical (sodium butyrate or pharmacological (ACE inhibitor chaperones with proteasome inhibitors (MG 132 or bortezomib significantly restored trafficking of Q1069R-ACE to the cell surface and increased ACE activity in the cell culture media 4-fold. CONCLUSIONS/SIGNIFICANCE: Homozygous Q1069R substitution results in an ACE trafficking and processing defect which can be rescued, at least in cell culture, by a combination of chaperones and proteasome inhibitors. Further studies are required to determine whether similar treatment of individuals with this ACE mutation would provide therapeutic benefits such as

  18. A frameshift mutation in the HuP2 paired domain of the probable human homolog of murine Pax-3 is responsible for Waardenburg syndrome type 1 in an Indonesian family.

    Science.gov (United States)

    Morell, R; Friedman, T B; Moeljopawiro, S; Hartono; Soewito; Asher, J H

    1992-07-01

    Waardenburg syndrome type 1 (WS1) is an autosomal dominant disorder characterized by deafness, dystopia canthorum, heterochromia iridis, white forelock, and premature greying. A similar phenotype is caused in the mouse by mutations in the Pax-3 gene. This observation, together with comparisons of conserved syntenies in the murine and human genetic maps, suggested that at least some WS1 mutations should occur in HuP2, the probable human homolog of Pax-3. Two mutations in the HuP2 sequence of individuals with WS1 have been reported recently. Both of them occur in the highly conserved paired box region of the gene, which encodes a DNA binding domain. The functional consequences of these mutations are at present speculative. We report here a 14 bp deletion in the paired domain encoded by exon 2 of HuP2 in an Indonesian family segregating for WS1. This frameshift mutation results in a premature termination codon in exon 3. The HuP2 product is a truncated protein lacking most of the paired domain and all of the predicted homeo domain. We propose that the WS1 phenotype in this family is due to loss of function of HuP2 and discuss two mechanisms for the dominant effect of this mutation.

  19. A single mutation in framework 2 of the heavy variable domain improves the properties of a diabody and a related single-chain antibody.

    Science.gov (United States)

    Rodríguez-Rodríguez, Everardo Remi; Ledezma-Candanoza, Luis M; Contreras-Ferrat, Luis Gabriel; Olamendi-Portugal, Timoteo; Possani, Lourival D; Becerril, Baltazar; Riaño-Umbarila, Lidia

    2012-10-26

    Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single-chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy-chain variable domain. Diabody D4 displayed an increase in T(m) (thermal transition midpoint temperature) of 6.3°C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem.286, 6143-6151]. The new variant, scFv LER, displayed an increase in T(m) of 3.4°C and was capable of neutralizing 2 LD(50) of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.

  20. Regulation of vacuolar proton pumping ATPase-dependent luminal acidification in the epididymis

    Institute of Scientific and Technical Information of China (English)

    Nicolas Da Silva; Winnie W. C. Shum; Sylvie Breton

    2007-01-01

    Luminal acidification in the epididymis is an important process for the regulation of male fertility. Low pH and low bicarbonate concentration are among key factors that keep spermatozoa in a dormant state while they mature and are stored in this organ. Although significant bicarbonate reabsorption is achieved by principal cells in the proximal regions of the epididymis, clear and narrow cells are specialized for net proton secretion. Clear cells express very high levels of the vacuolar proton pumping ATPase (V-ATPase) in their apical membrane and are responsible for the bulk of proton secretion. In the present paper, selected aspects of V-ATPase regulation in clear cells are described and potential pathologies associated with mutations of some of the V-ATPase subunits are discussed.

  1. A Yersinia pestis YscN ATPase mutant functions as a live attenuated vaccine against bubonic plague in mice.

    Science.gov (United States)

    Bozue, Joel; Cote, Christopher K; Webster, Wendy; Bassett, Anthony; Tobery, Steven; Little, Stephen; Swietnicki, Wieslaw

    2012-07-01

    Yersinia pestis is the causative agent responsible for bubonic and pneumonic plague. The bacterium uses the pLcr plasmid-encoded type III secretion system to deliver virulence factors into host cells. Delivery requires ATP hydrolysis by the YscN ATPase encoded by the yscN gene also on pLcr. A yscN mutant was constructed in the fully virulent CO92 strain containing a nonpolar, in-frame internal deletion within the gene. We demonstrate that CO92 with a yscN mutation was not able to secrete the LcrV protein (V-Antigen) and attenuated in a subcutaneous model of plague demonstrating that the YscN ATPase was essential for virulence. However, if the yscN mutant was complemented with a functional yscN gene in trans, virulence was restored. To evaluate the mutant as a live vaccine, Swiss-Webster mice were vaccinated twice with the ΔyscN mutant at varying doses and were protected against bubonic plague in a dose-dependent manner. Antibodies to F1 capsule but not to LcrV were detected in sera from the vaccinated mice. These preliminary results suggest a proof-of-concept for an attenuated, genetically engineered, live vaccine effective against bubonic plague. Published 2012. This article is a US Government work and is in the public domain in the USA.

  2. Enhancement of Cry19Aa Mosquitocidal Activity against Aedes aegypti by Mutations in the Putative Loop Regions of Domain II

    OpenAIRE

    Abdullah, Mohd Amir F.; Donald H Dean

    2004-01-01

    Improvements in the mosquitocidal activity of Bacillus thuringiensis Cry19Aa were achieved by protein engineering of putative surface loop residues in domain II through rational design. The improvement of Aedes toxicity in Cry19Aa was 42,000-fold and did not affect its toxicity against Anopheles or Culex.

  3. Structure of the active form of human origin recognition complex and its ATPase motor module

    Energy Technology Data Exchange (ETDEWEB)

    Tocilj, Ante; On, Kin Fan; Yuan, Zuanning; Sun, Jingchuan; Elkayam, Elad; Li, Huilin; Stillman, Bruce; Joshua-Tor, Leemor

    2017-01-23

    Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations.

  4. Structure of the active form of human origin recognition complex and its ATPase motor module

    Science.gov (United States)

    Tocilj, Ante; On, Kin Fan; Yuan, Zuanning; Sun, Jingchuan; Elkayam, Elad; Li, Huilin; Stillman, Bruce; Joshua-Tor, Leemor

    2017-01-01

    Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations. DOI: http://dx.doi.org/10.7554/eLife.20818.001 PMID:28112645

  5. Ser217Cys mutation in the Ig Ⅱ domain of FGFR3 in a Chinese family with autosomal dominant achondroplasia

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shi-rong; ZHOU Xiao-qing; REN Xiang; WANG Tian-tian; YUAN Ming-xiong; WANG Qing; LIU Jing-yu; LIU Mu-gen

    2007-01-01

    @@ Achondroplasia (ACH) is the most common form of skeletal dysplasia characterized by disproportionately short stature, lumbar lordosis, relative macrocephaly and other skeletal anomalies resulting from a defect in the maturation of the chondrocytes in the growth plate of the cartilage. The combined frequency of the disease has been estimated to be 1 in 15 000 live births.1 ACH is inherited in autosomal dominant fashion with a complete penetrance, more than 80% of affected individuals have de novo mutations associated with increased paternal age.

  6. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis

    Directory of Open Access Journals (Sweden)

    Jonathon eTelianidis

    2013-08-01

    Full Text Available Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer’s, Parkinson’s and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-Type ATPases (copper-ATPases, ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.

  7. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis.

    Science.gov (United States)

    Telianidis, Jonathon; Hung, Ya Hui; Materia, Stephanie; Fontaine, Sharon La

    2013-01-01

    Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-type ATPases (copper-ATPases), ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains, and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis, and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.

  8. Effects of mutations in the Arabidopsis Cold Shock Domain Protein 3 (AtCSP3) gene on leaf cell expansion.

    Science.gov (United States)

    Yang, Yongil; Karlson, Dale

    2012-08-01

    The cold shock domain is among the most evolutionarily conserved nucleic acid binding domains from prokaryotes to higher eukaryotes, including plants. Although eukaryotic cold shock domain proteins have been extensively studied as transcriptional and post-transcriptional regulators during various developmental processes, their functional roles in plants remains poorly understood. In this study, AtCSP3 (At2g17870), which is one of four Arabidopsis thaliana c old s hock domain proteins (AtCSPs), was functionally characterized. Quantitative RT-PCR analysis confirmed high expression of AtCSP3 in reproductive and meristematic tissues. A homozygous atcsp3 loss-of-function mutant exhibits an overall reduced seedling size, stunted and orbicular rosette leaves, reduced petiole length, and curled leaf blades. Palisade mesophyll cells are smaller and more circular in atcsp3 leaves. Cell size analysis indicated that the reduced size of the circular mesophyll cells appears to be generated by a reduction of cell length along the leaf-length axis, resulting in an orbicular leaf shape. It was also determined that leaf cell expansion is impaired for lateral leaf development in the atcsp3 loss-of-function mutant, but leaf cell proliferation is not affected. AtCSP3 loss-of-function resulted in a dramatic reduction of LNG1 transcript, a gene that is involved in two-dimensional leaf polarity regulation. Transient subcellular localization of AtCSP3 in onion epidermal cells confirmed a nucleocytoplasmic localization pattern. Collectively, these data suggest that AtCSP3 is functionally linked to the regulation of leaf length by affecting LNG1 transcript accumulation during leaf development. A putative function of AtCSP3 as an RNA binding protein is also discussed in relation to leaf development.

  9. High-efficiency localization of Na+-K+ ATPases on the cytoplasmic side by direct stochastic optical reconstruction microscopy

    Science.gov (United States)

    Wu, Jiazhen; Gao, Jing; Qi, Miao; Wang, Jianzhong; Cai, Mingjun; Liu, Shuheng; Hao, Xian; Jiang, Junguang; Wang, Hongda

    2013-11-01

    We describe a concise and effective strategy towards precisely mapping Na+-K+ ATPases on the cytoplasmic side of cell membranes by direct stochastic optical reconstruction microscopy (dSTORM). We found that most Na+-K+ ATPases are localized in different sizes of clusters on human red blood cell (hRBC) membranes, revealed by Ripley's K-function analysis. Further evidence that cholesterol depletion causes the dispersion of Na+-K+ ATPase clusters indicates that such clusters could be localized in cholesterol-enriched domains. Our results suggest that Na+-K+ ATPases might aggregate within the lipid rafts to fulfill their functions.We describe a concise and effective strategy towards precisely mapping Na+-K+ ATPases on the cytoplasmic side of cell membranes by direct stochastic optical reconstruction microscopy (dSTORM). We found that most Na+-K+ ATPases are localized in different sizes of clusters on human red blood cell (hRBC) membranes, revealed by Ripley's K-function analysis. Further evidence that cholesterol depletion causes the dispersion of Na+-K+ ATPase clusters indicates that such clusters could be localized in cholesterol-enriched domains. Our results suggest that Na+-K+ ATPases might aggregate within the lipid rafts to fulfill their functions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr03665k

  10. The oncogenic 70Z Cbl mutation blocks the phosphotyrosine binding domain-dependent negative regulation of ZAP-70 by c-Cbl in Jurkat T cells.

    Science.gov (United States)

    van Leeuwen, J E; Paik, P K; Samelson, L E

    1999-10-01

    T-cell receptor (TCR) engagement results in the activation of Src family (Lck and Fyn) and ZAP-70 protein tyrosine kinases, leading to tyrosine phosphorylation of multiple cellular substrates including the complex adapter protein c-Cbl. Moreover, Cbl is tyrosine phosphorylated upon engagement of growth factor receptors, cytokine receptors, and immunoreceptors and functions as a negative regulator of tyrosine kinase signalling pathways. Cbl associates via its phosphotyrosine binding (PTB) domain to the ZAP-70 pY292 negative regulatory phosphotyrosine. We recently demonstrated that the oncogenic Cbl mutant, 70Z Cbl, requires its PTB domain to upregulate NFAT in unstimulated Jurkat T cells. Here, we demonstrate that kinase-dead but not wild-type forms of Fyn, Lck, and ZAP-70 block 70Z Cbl-mediated NFAT activation. Moreover, 70Z Cbl does not upregulate NFAT in the ZAP-70-deficient P116 Jurkat T-cell line. The requirement for Fyn, Lck, and ZAP-70 is not due to tyrosine phosphorylation of 70Z Cbl, as mutation of all tyrosines in, or deletion of, the C-terminal region of 70Z Cbl (amino acids 655 to 906) blocks 70Z Cbl tyrosine phosphorylation but enhances 70Z Cbl-mediated NFAT activation. Further, 70Z Cbl does not cooperate with ZAP-70 Y292F to upregulate NFAT, indicating that 70Z Cbl and ZAP-70 do not activate parallel signalling pathways. Finally, the upregulation of NFAT observed upon ZAP-70 overexpression is blocked by Cbl in a PTB domain-dependent manner. We conclude that oncogenic 70Z Cbl acts as a dominant negative to block the PTB domain-dependent negative regulatory role of endogenous Cbl on ZAP-70, leading to constitutive ZAP-70 signalling and activation of transcription factors.

  11. ATPase-dependent auto-phosphorylation of the open condensin hinge diminishes DNA binding.

    Science.gov (United States)

    Akai, Yuko; Kanai, Ryuta; Nakazawa, Norihiko; Ebe, Masahiro; Toyoshima, Chikashi; Yanagida, Mitsuhiro

    2014-12-01

    Condensin, which contains two structural maintenance of chromosome (SMC) subunits and three regulatory non-SMC subunits, is essential for many chromosomal functions, including mitotic chromosome condensation and segregation. The ATPase domain of the SMC subunit comprises two termini connected by a long helical domain that is interrupted by a central hinge. The role of the ATPase domain has remained elusive. Here we report that the condensin SMC subunit of the fission yeast Schizosaccharomyces pombe is phosphorylated in a manner that requires the presence of the intact SMC ATPase Walker motif. Principal phosphorylation sites reside in the conserved, glycine-rich stretch at the hinge interface surrounded by the highly basic DNA-binding patch. Phosphorylation reduces affinity for DNA. Consistently, phosphomimetic mutants produce severe mitotic phenotypes. Structural evidence suggests that prior opening (though slight) of the hinge is necessary for phosphorylation, which is implicated in condensin's dissociation from and its progression along DNA.

  12. ATP-dependent inactivation of the beta-Ser339Cys mutant F1-ATPase from Escherichia coli by N-ethylmaleimide.

    Science.gov (United States)

    Schmidt, G; Senior, A E

    1995-08-01

    We introduced mutations at the highly-conserved residue Ser-339 in subunit beta of Escherichia coli F1-ATPase. The mutations beta S339Y and beta S339F abolished ATPase activity and impaired enzyme assembly. In contrast beta S339C F1 retained function to a substantial degree. N-Ethylmaleimide (NEM) at 0.2-0.3 mM inactivated beta S339C F1-ATPase by 80-95% in the presence of MgATP or MgADP but did not inactivate appreciably in absence of nucleotide or presence of EDTA. In absence of nucleotide, 0.7 mol of [14C-NEM] was incorporated into beta-subunits of 1.0 mol F1: in presence of MgATP the amount was 1.7 mol/mol, i.e. the introduced Cys residue became more accessible to reaction in the presence of MgATP. In the X-ray structure of F1 (Abrahams et al. (1994) Nature 370, 621-628) one of the catalytic nucleotide-binding domains is empty (on the "beta E subunit") and contains a cleft. Residue beta-339 lies within this cleft; the cleft does not occur in the other two beta-subunits. Our data are consistent with the conclusion that in wild-type enzyme under physiological conditions, MgATP or MgADP induce an enzyme conformation in which residue beta-Ser-339 becomes more exposed, possibly similar to the situation seen in the "beta E-subunit" in the X-ray structure.

  13. Caution Is Required in Interpretation of Mutations in the Voltage Sensing Domain of Voltage Gated Channels as Evidence for Gating Mechanisms

    Directory of Open Access Journals (Sweden)

    Alisher M. Kariev

    2015-01-01

    Full Text Available The gating mechanism of voltage sensitive ion channels is generally considered to be the motion of the S4 transmembrane segment of the voltage sensing domains (VSD. The primary supporting evidence came from R→C mutations on the S4 transmembrane segment of the VSD, followed by reaction with a methanethiosulfonate (MTS reagent. The cys side chain is –SH (reactive form –S−; the arginine side chain is much larger, leaving space big enough to accommodate the MTS sulfonate head group. The cavity created by the mutation has space for up to seven more water molecules than were present in wild type, which could be displaced irreversibly by the MTS reagent. Our quantum calculations show there is major reorientation of three aromatic residues that face into the cavity in response to proton displacement within the VSD. Two phenylalanines reorient sufficiently to shield/unshield the cysteine from the intracellular and extracellular ends, depending on the proton positions, and a tyrosine forms a hydrogen bond to the cysteine sulfur with its side chain –OH. These could produce the results of the experiments that have been interpreted as evidence for physical motion of the S4 segment, without physical motion of the S4 backbone. The computations strongly suggest that the interpretation of cysteine substitution reaction experiments be re-examined in the light of these considerations.

  14. Point mutations in the N-terminal domain of transactive response DNA-binding protein 43 kDa (TDP-43) compromise its stability, dimerization, and functions.

    Science.gov (United States)

    Mompeán, Miguel; Romano, Valentina; Pantoja-Uceda, David; Stuani, Cristiana; Baralle, Francisco E; Buratti, Emanuele; Laurents, Douglas V

    2017-07-14

    Transactive response DNA-binding protein 43 (TDP-43) performs multiple tasks in mRNA processing, transport, and translational regulation, but it also forms aggregates implicated in amyotrophic lateral sclerosis. TDP-43's N-terminal domain (NTD) is important for these activities and dysfunctions; however, there is an open debate about whether or not it adopts a specifically folded, stable structure. Here, we studied NTD mutations designed to destabilize its structure utilizing NMR and fluorescence spectroscopies, analytical ultracentrifugation, splicing assays, and cell microscopy. The substitutions V31R and T32R abolished TDP-43 activity in splicing and aggregation processes, and even the rather mild L28A mutation severely destabilized the NTD, drastically reducing TDP-43's in vitro splicing activity and inducing aberrant localization and aggregation in cells. These findings strongly support the idea that a stably folded NTD is essential for correct TDP-43 function. The stably folded NTD also promotes dimerization, which is pertinent to the protein's activities and pathological aggregation, and we present an atomic-level structural model for the TDP-43 dimer based on NMR data. Leu-27 is evolutionarily well conserved even though it is exposed in the monomeric NTD. We found here that Leu-27 is buried in the dimer and that the L27A mutation promotes monomerization. In conclusion, our study sheds light on the structural and biological properties of the TDP-43 NTD, indicating that the NTD must be stably folded for TDP-43's physiological functions, and has implications for understanding the mechanisms promoting the pathological aggregation of this protein. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Fitness conferred by BCR-ABL kinase domain mutations determines the risk of pre-existing resistance in chronic myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Kevin Leder

    Full Text Available Chronic myeloid leukemia (CML is the first human malignancy to be successfully treated with a small molecule inhibitor, imatinib, targeting a mutant oncoprotein (BCR-ABL. Despite its successes, acquired resistance to imatinib leads to reduced drug efficacy and frequent progression of disease. Understanding the characteristics of pre-existing resistant cells is important for evaluating the benefits of first-line combination therapy with second generation inhibitors. However, due to limitations of assay sensitivity, determining the existence and characteristics of resistant cell clones at the start of therapy is difficult. Here we combined a mathematical modeling approach using branching processes with experimental data on the fitness changes (i.e., changes in net reproductive rate conferred by BCR-ABL kinase domain mutations to investigate the likelihood, composition, and diversity of pre-existing resistance. Furthermore, we studied the impact of these factors on the response to tyrosine kinase inhibitors. Our approach predicts that in most patients, there is at most one resistant clone present at the time of diagnosis of their disease. Interestingly, patients are no more likely to harbor the most aggressive, pan-resistant T315I mutation than any other resistance mutation; however, T315I cells on average establish larger-sized clones at the time of diagnosis. We established that for patients diagnosed late, the relative benefit of combination therapy over monotherapy with imatinib is significant, while this benefit is modest for patients with a typically early diagnosis time. These findings, after pre-clinical validation, will have implications for the clinical management of CML: we recommend that patients with advanced-phase disease be treated with combination therapy with at least two tyrosine kinase inhibitors.

  16. Mutational analysis of the C-terminal domain of the Rhodobacter sphaeroides response regulator PrrA

    OpenAIRE

    Jones, Denise F.; Stenzel, Rachelle A.; Donohue, Timothy J.

    2005-01-01

    The Rhodobacter sphaeroides response regulator PrrA directly activates transcription of genes necessary for energy conservation at low O2 tensions and under anaerobic conditions. It is proposed that PrrA homologues contain a C-terminal DNA-binding domain (PrrA-CTD) that lacks significant amino acid sequence similarity to those found in other response regulators. To test this hypothesis, single amino acid substitutions were created at 12 residues in the PrrA-CTD. These mutant PrrA proteins wer...

  17. Mutational analysis of a predicted double β-propeller domain of the DspA/E effector of Erwinia amylovora.

    Science.gov (United States)

    Siamer, Sabrina; Gaubert, Stéphane; Boureau, Tristan; Brisset, Marie-Noëlle; Barny, Marie-Anne

    2013-05-01

    The bacterium Erwinia amylovora causes fire blight, an invasive disease that threatens apple trees, pear trees and other plants of the Rosaceae family. Erwinia amylovora pathogenicity relies on a type III secretion system and on a single effector DspA/E. This effector belongs to the widespread AvrE family of effectors whose biological function is unknown. In this manuscript, we performed a bioinformatic analysis of DspA/E- and AvrE-related effectors. Motif search identified nuclear localization signals, peroxisome targeting signals, endoplasmic reticulum membrane retention signals and leucine zipper motifs, but none of these motifs were present in all the AvrE-related effectors analysed. Protein threading analysis, however, predicted a conserved double β-propeller domain in the N-terminal part of all the analysed effector sequences. We then performed a random pentapeptide mutagenesis of DspA/E, which led to the characterization of 13 new altered proteins with a five amino acids insertion. Eight harboured the insertion inside the predicted β-propeller domain and six of these eight insertions impaired DspA/E stability or function. Conversely, the two remaining insertions generated proteins that were functional and abundantly secreted in the supernatant suggesting that these two insertions stabilized the protein.

  18. Effects of C-terminal truncations on trafficking of the yeast plasma membrane H+-ATPase.

    Science.gov (United States)

    Mason, A Brett; Allen, Kenneth E; Slayman, Carolyn W

    2006-08-18

    Within the large family of P-type cation-transporting ATPases, members differ in the number of C-terminal transmembrane helices, ranging from two in Cu2+-ATPases to six in H+-, Na+,K+-, Mg2+-, and Ca2+-ATPases. In this study, yeast Pma1 H+-ATPase has served as a model to examine the role of the C-terminal membrane domain in ATPase stability and targeting to the plasma membrane. Successive truncations were constructed from the middle of the major cytoplasmic loop to the middle of the extended cytoplasmic tail, adding back the C-terminal membrane-spanning helices one at a time. When the resulting constructs were expressed transiently in yeast, there was a steady increase in half-life from 70 min in Pma1 delta452 to 348 min in Pma1 delta901, but even the longest construct was considerably less stable than wild-type ATPase (t(1/2) = 11 h). Confocal immunofluorescence microscopy showed that 11 of 12 constructs were arrested in the endoplasmic reticulum and degraded in the proteasome. The only truncated ATPase that escaped the ER, Pma1 delta901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1 delta901 to be misfolded, however, resulting in premature delivery to the vacuole for degradation. As model substrates, this series of truncations affirms the importance of the entire C-terminal domain to yeast H+-ATPase biogenesis and defines a sequence element of 20 amino acids in the carboxyl tail that is critical to ER escape and trafficking to the plasma membrane.

  19. Mutations Phe785Leu and Thr618Met in Na+, K+-ATPase, Associated with Familial Rapid-Onset Dystonia Parkinsonism, Interfere with Na+ Interaction by Distinct Mechanisms

    DEFF Research Database (Denmark)

    Schack, Vivien Rodacker; Toustrup-Jensen, Mads Schak; Vilsen, Bente

    lead to functionally altered, but active, Na+, K+-pumps that display reduced apparent affinity for cytoplasmic Na+, but the underlying mechanism differs between the mutants. In Phe785Leu, the interaction of the E1 form with Na+ is defective, and the E1-E2 equilibrium is not displaced. In Thr618Met......, the Na+ affinity is reduced because of displacement of the conformational equilibrium in favor of the K+-occluded E2(K2) form. In both mutants, K+ interaction at the external activating sites of the E2P phosphoenzyme is normal. The change of cellular Na+ homeostasis is likely a major factor contributing...... that the aromatic function of the side chain, as well as its exact position, is critical for Na+ and ouabain binding. Structural modeling demonstrates that substitution of Phe785 disturbs its participation in a hydrophobic network between three transmembrane segments. It also indicates that the Thr618Met mutation...

  20. A mutation correcting the DNA interaction defects of a mutant phage lambda terminase, gpNu1 K35A terminase.

    Science.gov (United States)

    Hwang, Y; Feiss, M

    1999-12-20

    Terminase, the DNA packaging enzyme of bacteriophage lambda, is a heteromultimer composed of gpNu1 (181 aa) and gpA (641 aa) subunits, encoded by the lambda Nu1 and A genes, respectively. Similarity between the deduced amino acid sequences of gpNu1 and gpA and the nucleotide binding site consensus sequence suggests that each terminase subunit has an ATP reactive center. Terminase has been shown to have two distinct ATPase activities. The gpNu1 subunit has a low-affinity ATPase stimulated by nonspecific DNA and gpA has a high-affinity ATPase. In previous work, a mutant terminase, gpNu1 K35A holoterminase, had a mild defect in interactions with DNA, such that twofold increased DNA concentrations were required both for full stimulation of the low-affinity ATPase and for saturation of the cos cleavage reaction. In addition, the gpNu1 K35A terminase exhibited a post-cleavage defect in DNA packaging that accounted for the lethality of the Nu1 K35A mutation [Y. Hwang and M. Feiss (1997) Virology 231, 218-230]. In the work reported here, a mutation in the turn of the putative helix-turn-helix DNA binding domain has been isolated as a suppressor of the gpNu1 K35A change. This suppressor mutation causes the change A14V in gpNu1. A14V reverses the DNA-binding defects of gpNu1 K35A terminase, both for stimulation of the low-affinity ATPase and for saturation of the cos cleavage defect. A14V suppresses the post-cleavage DNA packaging defect caused by the gpNu1 K35A change.

  1. Analysis of mucolipidosis II/III GNPTAB missense mutations identifies domains of UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase involved in catalytic function and lysosomal enzyme recognition.

    Science.gov (United States)

    Qian, Yi; van Meel, Eline; Flanagan-Steet, Heather; Yox, Alex; Steet, Richard; Kornfeld, Stuart

    2015-01-30

    UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase tags newly synthesized lysosomal enzymes with mannose 6-phosphate recognition markers, which are required for their targeting to the endolysosomal system. GNPTAB encodes the α and β subunits of GlcNAc-1-phosphotransferase, and mutations in this gene cause the lysosomal storage disorders mucolipidosis II and III αβ. Prior investigation of missense mutations in GNPTAB uncovered amino acids in the N-terminal region and within the DMAP domain involved in Golgi retention of GlcNAc-1-phosphotransferase and its ability to specifically recognize lysosomal hydrolases, respectively. Here, we undertook a comprehensive analysis of the remaining missense mutations in GNPTAB reported in mucolipidosis II and III αβ patients using cell- and zebrafish-based approaches. We show that the Stealth domain harbors the catalytic site, as some mutations in these regions greatly impaired the activity of the enzyme without affecting its Golgi localization and proteolytic processing. We also demonstrate a role for the Notch repeat 1 in lysosomal hydrolase recognition, as missense mutations in conserved cysteine residues in this domain do not affect the catalytic activity but impair mannose phosphorylation of certain lysosomal hydrolases. Rescue experiments using mRNA bearing Notch repeat 1 mutations in GNPTAB-deficient zebrafish revealed selective effects on hydrolase recognition that differ from the DMAP mutation. Finally, the mutant R587P, located in the spacer between Notch 2 and DMAP, was partially rescued by overexpression of the γ subunit, suggesting a role for this region in γ subunit binding. These studies provide new insight into the functions of the different domains of the α and β subunits.

  2. Mutations in the G-domain of Ski7 cause specific dysfunction in non-stop decay

    Science.gov (United States)

    Horikawa, Wataru; Endo, Kei; Wada, Miki; Ito, Koichi

    2016-01-01

    Ski7 functions as a cofactor in both normal mRNA turnover and non-stop mRNA decay (NSD) mRNA surveillance in budding yeast. The N-terminal region of Ski7 (Ski7N) interacts with the ski-complex and the exosome. The C-terminal region of Ski7 (Ski7C) binds guanine nucleotides and shares overall sequence and structural homology with the proteins of the translational GTPase superfamily, especially the tRNA/tRNA-mimic carrier protein subfamilies such as EF1α, eRF3, and Hbs1. Previous reports showed that Ski7N polypeptide functions adequately in vivo, while Ski7C, if any, only slightly. Furthermore, Ski7C does not exhibit GTP-hydrolysing activities under normal conditions. Therefore, the physiological and functional significance of the conserved Ski7C is unclear. Here, we report strong genetic evidence suggesting differential roles for Ski7N and Ski7C in normal and specific mRNA turnover pathways by creating/isolating mutations in both Ski7N and Ski7C conserved motifs using indicator yeast strains. We concluded that Ski7C participates in mRNA surveillance as a regulatory module competitively with the Hbs1/Dom34 complex. Our results provide insights into the molecular regulatory mechanisms underlying mRNA surveillance. PMID:27381255

  3. Molecular dynamics simulation reveals insights into the mechanism of unfolding by the A130T/V mutations within the MID1 zinc-binding Bbox1 domain.

    Directory of Open Access Journals (Sweden)

    Yunjie Zhao

    Full Text Available The zinc-binding Bbox1 domain in protein MID1, a member of the TRIM family of proteins, facilitates the ubiquitination of the catalytic subunit of protein phosphatase 2A and alpha4, a protein regulator of PP2A. The natural mutation of residue A130 to a valine or threonine disrupts substrate recognition and catalysis. While NMR data revealed the A130T mutant Bbox1 domain failed to coordinate both structurally essential zinc ions and resulted in an unfolded structure, the unfolding mechanism is unknown. Principle component analysis revealed that residue A130 served as a hinge point between the structured β-strand-turn-β-strand (β-turn-β and the lasso-like loop sub-structures that constitute loop1 of the ββα-RING fold that the Bbox1 domain adopts. Backbone RMSD data indicate significant flexibility and departure from the native structure within the first 5 ns of the molecular dynamics (MD simulation for the A130V mutant (>6 Å and after 30 ns for A130T mutant (>6 Å. Overall RMSF values were higher for the mutant structures and showed increased flexibility around residues 125 and 155, regions with zinc-coordinating residues. Simulated pKa values of the sulfhydryl group of C142 located near A130 suggested an increased in value to ~9.0, paralleling the increase in the apparent dielectric constants for the small cavity near residue A130. Protonation of the sulfhydryl group would disrupt zinc-coordination, directly contributing to unfolding of the Bbox1. Together, the increased motion of residues of loop 1, which contains four of the six zinc-binding cysteine residues, and the increased pKa of C142 could destabilize the structure of the zinc-coordinating residues and contribute to the unfolding.

  4. Dynamically-driven inactivation of the catalytic machinery of the SARS 3C-like protease by the N214A mutation on the extra domain.

    Science.gov (United States)

    Shi, Jiahai; Han, Nanyu; Lim, Liangzhong; Lua, Shixiong; Sivaraman, J; Wang, Lushan; Mu, Yuguang; Song, Jianxing

    2011-02-01

    Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro.

  5. Effect of propranolol on rat brain synaptosomal Na(+)-K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase.

    Science.gov (United States)

    Gopalaswamy, U V; Satav, J G; Katyare, S S; Bhattacharya, R K

    1997-01-24

    The beta blocker drug propranolol (PPL) significantly inhibited Na(+)-K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase activities in a concentration dependent manner in rat brain synaptosomes. The concentrations required for 50% inhibition (IC50) in the activity of these enzymes were 1.5-1.8 mM. The double-reciprocal plot of ATP-stimulated Na(+)-K(+)-ATPase activity in the presence of PPL showed apparent decrease in K(m) and Vmax and the inhibition was of an uncompetitive type with respect to ATP. The nature of inhibition by PPL of Na(+)-activated Na(+)-K(+)-ATPase activity was of a mixed type showing an increase in Km and decrease in Vmax. Potassium activation kinetics of Na(+)-K(+)-ATPase displayed uncompetitive type of inhibition with PPL since Km and Vmax were decreased. Magnesium activation of Mg(2+)-ATPase showed decrease in Vmax with no apparent change in Km in the presence of PPL. The drug inhibited synaptosomal Ca(2+)-ATPase in an uncompetitive manner. The observed inhibition of synaptosomal ATPases indicates possible alterations in the synaptic transmission by the beta blocker drug PPL.

  6. Structural and functional studies of heavy metal ATPases

    DEFF Research Database (Denmark)

    Sitsel, Oleg

    2015-01-01

    to handle heavy metal ions. LpCopA is then compared to its two human homologues ATP7A and ATP7B, which cause the severe Menkes and Wilson diseases when malfunctioning. The differences between the three proteins are described and disease-causing mutations in the human proteins are analyzed. The crystal......Copper and zinc are trace elements that are crucial for the well-being of all cells and are an indispensable part of many proteins. At the same time, the intracellular levels of these metals require careful regulation, as an excess or deficiency may be lethal. P1B-ATPases are key players in Cu...

  7. Oxidative phosphorylation in Escherichia coli. Characterization of mutant strains in which F1-ATPase contains abnormal beta-subunits.

    Science.gov (United States)

    Senior, A E; Langman, L; Cox, G B; Gibson, F

    1983-02-15

    To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified. Seventeen strains of E. coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied. The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength. A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength. Presumably in these two strains F1-ATPase is not released in soluble form by this procedure. F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484). F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity. The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity. The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain. The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations. It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism.

  8. Functional Analysis of P4-ATPases

    DEFF Research Database (Denmark)

    Theorin, Lisa

    studies have identified P4-ATPases as phospholipid transporters that interact and cooperate with cytosolic coat proteins to drive vesicle formation at the trans-Golgi network, endosome compartment and plasma membrane. Our hypothesis is that by flipping a lipid the P4-ATPases either provide essential......Across membranes of the late secretory pathway in eukaryotic cells an asymmetric lipid distribution is maintained, with the lipids phosphatidylserine and phosphatidylethanolamine restricted to the cytoplasmic leaflet of the membrane. In recent years a subgroup of P-type ATPases, P4-ATPases, has...... and mammalian P4-ATPases have been studied extensively and the physiological function is mostly known, while the exact biochemistry and specific activity is mostly unknown. Even though the plant Arabidopsis thaliana has 12 P4-ATPases, not much is known about their function. In this study, the biochemical...

  9. Ion pathways in the sarcoplasmic reticulum Ca2+-ATPase.

    Science.gov (United States)

    Bublitz, Maike; Musgaard, Maria; Poulsen, Hanne; Thøgersen, Lea; Olesen, Claus; Schiøtt, Birgit; Morth, J Preben; Møller, Jesper Vuust; Nissen, Poul

    2013-04-12

    The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is a transmembrane ion transporter belonging to the P(II)-type ATPase family. It performs the vital task of re-sequestering cytoplasmic Ca(2+) to the sarco/endoplasmic reticulum store, thereby also terminating Ca(2+)-induced signaling such as in muscle contraction. This minireview focuses on the transport pathways of Ca(2+) and H(+) ions across the lipid bilayer through SERCA. The ion-binding sites of SERCA are accessible from either the cytoplasm or the sarco/endoplasmic reticulum lumen, and the Ca(2+) entry and exit channels are both formed mainly by rearrangements of four N-terminal transmembrane α-helices. Recent improvements in the resolution of the crystal structures of rabbit SERCA1a have revealed a hydrated pathway in the C-terminal transmembrane region leading from the ion-binding sites to the cytosol. A comparison of different SERCA conformations reveals that this C-terminal pathway is exclusive to Ca(2+)-free E2 states, suggesting that it may play a functional role in proton release from the ion-binding sites. This is in agreement with molecular dynamics simulations and mutational studies and is in striking analogy to a similar pathway recently described for the related sodium pump. We therefore suggest a model for the ion exchange mechanism in P(II)-ATPases including not one, but two cytoplasmic pathways working in concert.

  10. Familial hemiplegic migraine type 2 (FHM2) associated mutation in the 2 isoform of Na,KATPase leads to elevated contractility and vasodilatation

    DEFF Research Database (Denmark)

    Hangaard, Lise; Lykke-Hartmann, Karin; Xie, Zijian;

    is associated with few point mutations in the 2 isoform Na,K-ATPase. Mice bearing a mutation corresponding to the inherited mutation in FHM2 patients (G301R) were used in functional studies of middle cerebral arteries. Arteries from heterozygote G301R mice were not different in total 2 Na,K-ATPase m...

  11. Structural studies of conformational changes of proteins upon phosphorylation: Structures of activated CheY, CheY-N16-FliM complex, and AAA {sup +} ATPase domain of NtrC1 in both inactive and active states

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seok-Yong

    2003-04-10

    Protein phosphorylation is a general mechanism for signal transduction as well as regulation of cellular function. Unlike phosphorylation in eukaryotic systems that uses Ser/Thr for the sites of modification, two-component signal transduction systems, which are prevalent in bacteria, archea, and lower eukaryotes, use an aspartate as the site of phosphorylation. Two-component systems comprise a histidine kinase and a receiver domain. The conformational change of the receiver domain upon phosphorylation leads to signal transfer to the downstream target, a process that had not been understood well at the molecular level. The transient nature of the phospho-Asp bond had made structural studies difficult. The discovery of an excellent analogue for acylphosphate, BeF{sub 3}{sup -}, enabled structural study of activated receiver domains. The structure of activated Chemotaxis protein Y (CheY) was determined both by NMR spectroscopy and X-ray crystallography. These structures revealed the molecular basis of the conformational change that is coupled to phosphorylation. Phosphorylation of the conserved Asp residue in the active site allows hydrogen bonding of the T87 O{gamma} to phospho-aspartate, which in turn leads to the rotation of Y106 into the ''in'' position (termed Y-T coupling). The structure of activated CheY complexed with the 16 N-terminal residues of FliM (N16-FliM), its target, was also determined by X-ray crystallography and confirmed the proposed mechanism of activation (Y-T coupling). First, N16-FliM binds to the region on CheY that undergoes a significant conformational change. Second, the ''in'' position of Y106 presents a better binding surface for FliM because the sidechain of Y106 in the inactive form of CheY (''out'' position) sterically interferes with binding of N16-FliM. In addition to confirmation of Y-T coupling, the structure of the activated CheY-N16-FliM complex suggested that the N16

  12. Kinase domain mutations of BCR-ABL frequently precede imatinib-based therapy and give rise to relapse in patients with de novo Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL).

    Science.gov (United States)

    Pfeifer, Heike; Wassmann, Barbara; Pavlova, Anna; Wunderle, Lydia; Oldenburg, Johannes; Binckebanck, Anja; Lange, Thoralf; Hochhaus, Andreas; Wystub, Silvia; Brück, Patrick; Hoelzer, Dieter; Ottmann, Oliver G

    2007-07-15

    Acquired imatinib resistance in advanced Philadelphia-positive acute lymphoblastic leukemia (Ph(+) ALL) has been associated with mutations in the kinase domain (KD) of BCR-ABL. We examined the prevalence of KD mutations in newly diagnosed and imatinib-naive Ph(+) ALL patients and assessed their clinical relevance in the setting of uniform frontline therapy with imatinib in combination with chemotherapy. Patients enrolled in the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL) trial ADE10 for newly diagnosed elderly Ph(+) ALL were retrospectively examined for the presence of BCR-ABL KD mutations by denaturing high-performance liquid chromatography (D-HPLC), cDNA sequencing, and allele-specific polymerase chain reaction (PCR). A KD mutation was detected in a minor subpopulation of leukemic cells in 40% of newly diagnosed and imatinib-naive patients. At relapse, the dominant cell clone harbored an identical mutation in 90% of cases, the overall prevalence of mutations at relapse was 80%. P-loop mutations predominated and were not associated with an inferior hematologic or molecular remission rate or shorter remission duration compared with unmutated BCR-ABL. BCR-ABL mutations conferring high-level imatinib resistance are present in a substantial proportion of patients with de novo Ph(+) ALL and eventually give rise to relapse. This provides a rationale for the frontline use of kinase inhibitors active against these BCR-ABL mutants.

  13. Structure-function analysis of the NB-ARC domain of plant disease resistance proteins.

    Science.gov (United States)

    van Ooijen, Gerben; Mayr, Gabriele; Kasiem, Mobien M A; Albrecht, Mario; Cornelissen, Ben J C; Takken, Frank L W

    2008-01-01

    Resistance (R) proteins in plants are involved in pathogen recognition and subsequent activation of innate immune responses. Most resistance proteins contain a central nucleotide-binding domain. This so-called NB-ARC domain consists of three subdomains: NB, ARC1, and ARC2. The NB-ARC domain is a functional ATPase domain, and its nucleotide-binding state is proposed to regulate activity of the R protein. A highly conserved methionine-histidine-aspartate (MHD) motif is present at the carboxy-terminus of ARC2. An extensive mutational analysis of the MHD motif in the R proteins I-2 and Mi-1 is reported. Several novel autoactivating mutations of the MHD invariant histidine and conserved aspartate were identified. The combination of MHD mutants with autoactivating hydrolysis mutants in the NB subdomain showed that the autoactivation phenotypes are not additive. This finding indicates an important regulatory role for the MHD motif in the control of R protein activity. To explain these observations, a three-dimensional model of the NB-ARC domain of I-2 was built, based on the APAF-1 template structure. The model was used to identify residues important for I-2 function. Substitution of the selected residues resulted in the expected distinct phenotypes. Based on the model, it is proposed that the MHD motif fulfils the same function as the sensor II motif found in AAA+ proteins (ATPases associated with diverse cellular activities)-co-ordination of the nucleotide and control of subdomain interactions. The presented 3D model provides a framework for the formulation of hypotheses on how mutations in the NB-ARC exert their effects.

  14. Mutations in TBCK, Encoding TBC1-Domain-Containing Kinase, Lead to a Recognizable Syndrome of Intellectual Disability and Hypotonia

    Science.gov (United States)

    Bhoj, Elizabeth J.; Li, Dong; Harr, Margaret; Edvardson, Shimon; Elpeleg, Orly; Chisholm, Elizabeth; Juusola, Jane; Douglas, Ganka; Guillen Sacoto, Maria J.; Siquier-Pernet, Karine; Saadi, Abdelkrim; Bole-Feysot, Christine; Nitschke, Patrick; Narravula, Alekhya; Walke, Maria; Horner, Michele B.; Day-Salvatore, Debra-Lynn; Jayakar, Parul; Vergano, Samantha A. Schrier; Tarnopolsky, Mark A.; Hegde, Madhuri; Colleaux, Laurence; Crino, Peter; Hakonarson, Hakon

    2016-01-01

    Through an international multi-center collaboration, 13 individuals from nine unrelated families and affected by likely pathogenic biallelic variants in TBC1-domain-containing kinase (TBCK) were identified through whole-exome sequencing. All affected individuals were found to share a core phenotype of intellectual disability and hypotonia, and many had seizures and showed brain atrophy and white-matter changes on neuroimaging. Minor non-specific facial dysmorphism was also noted in some individuals, including multiple older children who developed coarse features similar to those of storage disorders. TBCK has been shown to regulate the mammalian target of rapamycin (mTOR) signaling pathway, which is also stimulated by exogenous leucine supplementation. TBCK was absent in cells from affected individuals, and decreased phosphorylation of phospho-ribosomal protein S6 was also observed, a finding suggestive of downregulation of mTOR signaling. Lastly, we demonstrated that activation of the mTOR pathway in response to L-leucine supplementation was retained, suggesting a possible avenue for directed therapies for this condition. PMID:27040691

  15. A dominant mutation in the light-oxygen and voltage2 domain vicinity impairs phototropin1 signaling in tomato.

    Science.gov (United States)

    Sharma, Sulabha; Kharshiing, Eros; Srinivas, Ankanagari; Zikihara, Kazunori; Tokutomi, Satoru; Nagatani, Akira; Fukayama, Hiroshi; Bodanapu, Reddaiah; Behera, Rajendra K; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2014-04-01

    In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A'α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A'α helix region in phototropic signaling of tomato.

  16. Interdependence of the Rad50 hook and globular domain functions

    Science.gov (United States)

    Hohl, Marcel; Kochańczyk, Tomasz; Tous, Cristina; Aguilera, Andrés; Krężel, Artur; Petrini, John H J

    2015-01-01

    SUMMARY Rad50 contains a conserved Zn2+ coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here we focused on rad50 mutations flanking the Zn2+-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end-joining, and DNA double strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled coil and globular ATPase domain, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions. PMID:25601756

  17. Mutations in the paired domain of the human PAX3 gene cause Klein-Waardenburg syndrome (WS-III) as well as Waardenburg syndrome type I (WS-I)

    Energy Technology Data Exchange (ETDEWEB)

    Hoth, C.F.; Milunsky, A.; Lipsky, N.; Baldwin, C.T. (Boston Univ. School of Medicine, MA (United States)); Sheffer, R. (Hadassah-Hebrew Univ. Medical Center, Jerusalem (Israel)); Clarren, S.K. (Univ. of Washington School of Medicine, Seattle (United States))

    1993-03-01

    Waardenburg syndrome type I (WS-I) is an autosomal dominant disorder characterized by sensorineural hearing loss, dystopia canthorum, pigmentary disturbances, and other developmental defects. Klein-Waardenburg syndrome (WS-III) is a disorder with many of the same characteristics as WS-I and includes musculoskeletal abnormalities. The authors have recently reported the identification and characterization of one of the first gene defects, in the human PAX3 gene, which causes WS-I. PAX3 is a DNA-binding protein that contains a structural motif known as the paired domain and is believed to regulate the expression of other genes. In this report they describe two new mutations, in the human PAX3 gene, that are associated with WS. One mutation was found in a family with WS-I, while the other mutation was found in a family with WS-III. Both mutations were in the highly conserved paired domain of the human PAX3 gene and are similar to other mutations that cause WS. The results indicate that mutations in the PAX3 gene can cause both WS-I and WS-III. 36 refs., 4 figs.

  18. Insight into the flagella type III export revealed by the complex structure of the type III ATPase and its regulator.

    Science.gov (United States)

    Imada, Katsumi; Minamino, Tohru; Uchida, Yumiko; Kinoshita, Miki; Namba, Keiichi

    2016-03-29

    FliI and FliJ form the FliI6FliJ ATPase complex of the bacterial flagellar export apparatus, a member of the type III secretion system. The FliI6FliJ complex is structurally similar to the α3β3γ complex of F1-ATPase. The FliH homodimer binds to FliI to connect the ATPase complex to the flagellar base, but the details are unknown. Here we report the structure of the homodimer of a C-terminal fragment of FliH (FliHC2) in complex with FliI. FliHC2 shows an unusually asymmetric homodimeric structure that markedly resembles the peripheral stalk of the A/V-type ATPases. The FliHC2-FliI hexamer model reveals that the C-terminal domains of the FliI ATPase face the cell membrane in a way similar to the F/A/V-type ATPases. We discuss the mechanism of flagellar ATPase complex formation and a common origin shared by the type III secretion system and the F/A/V-type ATPases.

  19. Identification and functional analysis of a novel PRKAG2 mutation responsible for Chinese PRKAG2 cardiac syndrome reveal an important role of non-CBS domains in regulating the AMPK pathway.

    Science.gov (United States)

    Zhang, Bi-li; Xu, Rong-liang; Zhang, Jing; Zhao, Xian-xian; Wu, Hong; Ma, Li-ping; Hu, Jian-qiang; Zhang, Jian-liang; Ye, Zhong; Zheng, Xing; Qin, Yong-wen

    2013-10-01

    PRKAG2 gene encodes the γ2 regulatory subunit of AMP-activated protein kinase (AMPK) that acts as a sensor of cellular energy status, and its germline mutations are responsible for PRKAG2 cardiac syndrome (PCS). The majority of missense mutations of cystathionine beta-synthase (CBS) domains found in PCS impair the binding activity of PRKAG2 to adenosine derivatives, and therefore lead to PRKAG2 function impairment and AMPK activity alteration, resulting in a familial syndrome of ventricular preexcitation, conduction defects, and cardiac hypertrophy. However, it is unclear about the PRKAG2 mutation in the non-CBS domain. Here, a Chinese family exhibiting the cardiac syndrome associated with a novel heterozygous PRKAG2 mutation (Gly100Ser) mapped to exon 3 encoding a non-CBS domain is described and the function of this novel mutation was investigated in vitro. The PRKAG2 G100S and R302Q mutations were constructed by a two-step polymerase chain reaction and then transfected into CCL13 cells by lentivirus vectors. Wild-type PRKAG2 gene transfection was used as a negative control. PRKAG2 expression was determined by Western blot. Immunofluorescence was used to localize the intracellular PRKAG2 proteins. MTT assay was performed to explore the effect of mutations on cell proliferation. Periodic acid-Schiff staining was used for detecting glycogen accumulation. AMPK concentration was measured with enzyme-linked immunosorbent assay. Our results showed neither intracellular localization of PRKAG2 nor cell growth was altered. In contrast, PRKAG2 protein expression levels were significantly reduced by this mutation. Furthermore, PRKAG2-mediated activity of AMPK was attenuated, resulting in glycogen metabolism dysregulation. These findings revealed that non-CBS domains of PRKAG2 were essential to the regulation of AMPK activity, similar to CBS. Our study ascribes a crucial regulatory role to the novel PRKAG2 G100S mutation, and reiterates that PCS occurs as a consequence of

  20. Directed mutagenesis of the dicyclohexylcarbodiimide-reactive carboxyl residues in beta-subunit of F1-ATPase of Escherichia coli.

    Science.gov (United States)

    Parsonage, D; Wilke-Mounts, S; Senior, A E

    1988-02-15

    Previous studies in which dicyclohexylcarbodiimide (DCCD) was used to inactivate F1-ATPase enzymes have suggested that two glutamate residues in the beta-subunit are essential for catalysis. In the Escherichia coli F1-ATPase, these are residues beta-Glu-181 and beta-Glu-192. Oligonucleotide-directed mutagenesis was used to change these residues to beta-Gln-181 and beta-Gln-192. The beta-Gln-181 mutation produced strong impairment of oxidative phosphorylation in vivo and also of ATPase and ATP-driven proton-pumping activities in membranes assayed in vitro. A low level of each activity was detected and an F1-ATPase appeared to be assembled normally on the membranes. Therefore, it is suggested that the carboxyl side chain at residue beta-181 is important, although not absolutely required, for catalysis in both directions on E. coli F1-ATPase. The beta-Gln-192 mutation produced partial inhibition of oxidative phosphorylation in vivo and membrane ATPase activity was reduced by 78%. These results contrast with the complete or near-complete inactivation seen when E. coli F1-ATPase is reacted with DCCD and imply that DCCD-inactivation is attributable more to the attachment of the bulky DCCD molecule than to the derivatization of the carboxyl side chain of residue beta-Glu-192. M. Ohtsubo and colleagues (Biochem. Biophys. Res. Commun. (1987) 146, 705-710) described mutagenesis of the F1-beta-subunit of thermophilic bacterium PS3. Mutations (Glu----Gln) of the residues homologous to Glu-181 and Glu-192 of E. coli F1-beta-subunit both caused total inhibition of ATPase activity. Therefore, there was a marked difference in results obtained when the same residues were modified in the PS3 and E. coli F1-beta-subunits.

  1. Mutational analysis of the cytoplasmic tail of the human transferrin receptor. Identification of a sub-domain that is required for rapid endocytosis.

    Science.gov (United States)

    Gironès, N; Alverez, E; Seth, A; Lin, I M; Latour, D A; Davis, R J

    1991-10-05

    It has been reported that the sequence Tyr20-X-Arg-Phe23 present within the cytoplasmic tail of the transferrin receptor may represent a tyrosine internalization signal (Collawn, J.F., Stangel, M., Kuhn, L.A., Esekogwu, V., Jing, S., Trowbridge, I.S., and Tainer, J. A. (1990) Cell 63, 1061-1072). However, as Tyr20 is not conserved between species (Alvarez, E., Gironès, N., and Davis, R. J. (1990) Biochem. J. 267, 31-35), the functional role of the putative tyrosine internalization signal is not clear. To address this question, we constructed a series of 32 deletions and point mutations within the cytoplasmic tail of the human transferrin receptor. The effect of these mutations on the apparent first order rate constant for receptor endocytosis was examined. It was found that the region of the cytoplasmic tail that is proximal to the transmembrane domain (residues 28-58) is dispensable for rapid endocytosis. In contrast, the distal region of the cytoplasmic tail (residues 1-27) was found to be both necessary and sufficient for the rapid internalization of the transferrin receptor. The region identified includes Tyr20-X-Arg-Phe23, but is significantly larger than this tetrapeptide. It is therefore likely that structural information in addition to the proposed tyrosine internalization signal is required for endocytosis. To test this hypothesis, we investigated whether a heterologous tyrosine internalization signal (from the low density lipoprotein receptor) could function to cause the rapid endocytosis of the transferrin receptor. It was observed that this heterologous tyrosine internalization signal did not allow rapid endocytosis. We conclude that the putative tyrosine internalization signal (Tyr20-Thr-Arg-Phe23) is not sufficient to determine rapid endocytosis of the transferrin receptor. The data reported here indicate that the transferrin receptor internalization signal is formed by a larger cytoplasmic tail structure located at the amino terminus of the receptor.

  2. Alternating hemiplegia of childhood-related neural and behavioural phenotypes in Na+,K+-ATPase α3 missense mutant mice.

    Directory of Open Access Journals (Sweden)

    Greer S Kirshenbaum

    Full Text Available Missense mutations in ATP1A3 encoding Na(+,K(+-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC, a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na(+,K(+-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N that affects the same position in Na(+,K(+-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na(+,K(+-ATPase α3, including upon the K(+ pore and predicted K(+ binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality, directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na(+,K(+-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC.

  3. P(1B)-ATPases--an ancient family of transition metal pumps with diverse functions in plants.

    Science.gov (United States)

    Williams, Lorraine E; Mills, Rebecca F

    2005-10-01

    P(1B)-ATPases form a distinct evolutionary sub-family of P-type ATPases, transporting transition metals such as Cu, Zn, Cd, Pb and Co across membranes in a wide range of organisms, including plants. Structurally they are distinct from other P-types, possessing eight transmembrane helices, a CPx/SPC motif in transmembrane domain six, and putative transition metal-binding domains at the N- and/or C-termini. Arabidopsis has eight P(1B)-ATPases (AtHMA1-AtHMA8), which differ in their structure, function and regulation. They perform a variety of important physiological tasks relating to transition metal transport and homeostasis. The crucial roles of plant P(1B)-ATPases in micronutrient nutrition, delivery of essential metals to target proteins, and toxic metal detoxification are discussed.

  4. Quaternary structure of V1 and F1 ATPase: significance of structural homologies and diversities.

    Science.gov (United States)

    Svergun, D I; Konrad, S; Huss, M; Koch, M H; Wieczorek, H; Altendorf, K; Volkov, V V; Grüber, G

    1998-12-22

    The V1 ATPase from the tobacco hornworm Manduca sexta and the Escherichia coli F1 ATPase were characterized by small-angle X-ray scattering (SAXS). The radii of gyration (Rg) of the complexes were 6.2 +/- 0.1 and 4.7 +/- 0.02 nm, respectively. The shape of the M. sexta V1 ATPase was determined ab initio from the scattering data showing six masses, presumed to be the A and B subunits, arranged in an alternating manner about a 3-fold axis. A seventh mass with a length of about 11.0 nm extends perpendicularly to the center of the hexameric unit. This central mass is presumed to be the stalk that connects V1 with the membrane domain (V(O)) in the intact V1V(O)-ATPase. In comparison, the shape of the F1 ATPase from E. coli possesses a quasi-3-fold symmetry over the major part of the enzyme. The overall asymmetry of the structure is given by a stem, assumed to include the central stalk subunits. The features of the V1 and F1 ATPase reveal structural homologies and diversities of the key components of the complexes.

  5. Origin and evolution of metal p-Type ATPases in Plantae (Archaeplastida

    Directory of Open Access Journals (Sweden)

    Marc eHanikenne

    2014-01-01

    Full Text Available Metal ATPases are a subfamily of P-type ATPases involved in the transport of metal cations across biological membranes. They all share an architecture featuring eight transmembrane domains in pairs of two and are found in prokaryotes as well as in a variety of Eukaryotes. In Arabidopsis thaliana, eight metal P-type ATPases have been described, four being specific to copper transport and four displaying a broader metal specificity, including zinc, cadmium and possibly copper and calcium. So far, few efforts have been devoted to elucidating the origin and evolution of these proteins in Eukaryotes. In this work, we use large-scale phylogenetics to show that metal P-type ATPases form a homogenous group among P-type ATPases and that their specialisation into either monovalent (Cu or divalent (Zn, Cd… metal transport stems from a gene duplication that took place early in the evolution of Life. Then, we demonstrate that the four subgroups of plant metal ATPases all have a different evolutionary origin and a specific taxonomic distribution, only one tracing back to the cyanobacterial progenitor of the chloroplast. Finally, we examine the subsequent evolution of these proteins in green plants and conclude that the genes thoroughly characterised in model organisms are often the result of lineage-specific gene duplications, which calls for caution when attempting to infer function from sequence similarity alone in non-model organisms.

  6. Optimization of pyrrolamides as mycobacterial GyrB ATPase inhibitors: structure-activity relationship and in vivo efficacy in a mouse model of tuberculosis.

    Science.gov (United States)

    P, Shahul Hameed; Solapure, Suresh; Mukherjee, Kakoli; Nandi, Vrinda; Waterson, David; Shandil, Radha; Balganesh, Meenakshi; Sambandamurthy, Vasan K; Raichurkar, Anand Kumar; Deshpande, Abhijeet; Ghosh, Anirban; Awasthy, Disha; Shanbhag, Gajanan; Sheikh, Gulebahar; McMiken, Helen; Puttur, Jayashree; Reddy, Jitendar; Werngren, Jim; Read, Jon; Kumar, Mahesh; R, Manjunatha; Chinnapattu, Murugan; Madhavapeddi, Prashanti; Manjrekar, Praveena; Basu, Reetobrata; Gaonkar, Sheshagiri; Sharma, Sreevalli; Hoffner, Sven; Humnabadkar, Vaishali; Subbulakshmi, Venkita; Panduga, Vijender

    2014-01-01

    Moxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosis through inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosis H37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatis GyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of <5 nM, an MIC of 0.03 μg/ml against M. tuberculosis H37Rv, and an MIC90 of <0.25 μg/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was ∼10(-6) to 10(-8), and the point mutation mapped to the M. tuberculosis GyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivo efficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents.

  7. Mutations in the ExbB cytoplasmic carboxy terminus prevent energy-dependent interaction between the TonB and ExbD periplasmic domains.

    Science.gov (United States)

    Jana, Bimal; Manning, Marta; Postle, Kathleen

    2011-10-01

    The TonB system of Gram-negative bacteria provides passage across the outer membrane (OM) diffusion barrier that otherwise limits access to large, scarce, or important nutrients. In Escherichia coli, the integral cytoplasmic membrane (CM) proteins TonB, ExbB, and ExbD couple the CM proton motive force (PMF) to active transport of iron-siderophore complexes and vitamin B(12) across the OM through high-affinity transporters. ExbB is an integral CM protein with three transmembrane domains. The majority of ExbB occupies the cytoplasm. Here, the importance of the cytoplasmic ExbB carboxy terminus (residues 195 to 244) was evaluated by cysteine scanning mutagenesis. D211C and some of the substitutions nearest the carboxy terminus spontaneously formed disulfide cross-links, even though the cytoplasm is a reducing environment. ExbB N196C and D211C substitutions were converted to Ala substitutions to stabilize them. Only N196A, D211A, A228C, and G244C substitutions significantly decreased ExbB activity. With the exception of ExbB(G244C), all of the substituted forms were dominant. Like wild-type ExbB, they all formed a formaldehyde cross-linked tetramer, as well as a tetramer cross-linked to an unidentified protein(s). In addition, they could be formaldehyde cross-linked to ExbD and TonB. Taken together, the data suggested that they assembled normally. Three of four ExbB mutants were defective in supporting both the PMF-dependent formaldehyde cross-link between the periplasmic domains of TonB and ExbD and the proteinase K-resistant conformation of TonB. Thus, mutations in a cytoplasmic region of ExbB prevented a periplasmic event and constituted evidence for signal transduction from cytoplasm to periplasm in the TonB system.

  8. Coil-to-Helix Transition within Phospholamban Underlies Release of Ca-ATPase Inhibition in Response to β-Adrenergic Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Bigelow, Diana J.; Squier, Thomas C.

    2006-02-01

    Phospholamban (PLB) is a major target of beta-adrenergic signaling, whose phosphorylation results in enhanced rates of relaxation in the heart. Prior to phosphorylation, PLB functions to reduce the calcium sensitivity of the Ca-ATPase, resulting in slower rates of calcium resequestration into the sarcoplasmic reticulum after each contractile event. Recent structures indicate that the inhibitory interaction between PLB and the Ca-ATPase requires PLB to assume an extended structure, where the transmembrane and cytosolic portions of PLB undergo specific binding interactions with distant sites on the Ca-ATPase. In the extended conformation, PLB binding to the Ca-ATPase functions to inhibit the Ca-ATPase through a reduction in the rates of catalytically important motions involving the nucleotide binding domain. Phosphorylation of PLB at either Ser16 or Thr17 releases the inhibitory interaction between PLB and the Ca-ATPase. These sites of phosphorylation are within a hinge region in PLB that separates the highly structured transmembrane and cytosolic portions that associate with the Ca-ATPase. The helical content of the hinge region increases following the phosphorylation of PLB, which induces a shortening of the maximal dimensions of PLB and a release of the inhibitory interaction with the Ca-ATPase. Following phosphorylation, PLB remains associated with the Ca-ATPase in a more compact form that has no inhibitory capability. Thus, the conformational switch involving PLB regulation of the Ca-ATPase relies upon a physical mechanism, whereby the phosphorylation-dependent stabilization of the structure of PLB functions to destabilize the inhibitory interaction between PLB and the Ca-ATPase. Upon hydrolysis of the phosphoester linkages by endogenous phosphatases PLB is poised to reassume the inhibited state through re-association with inhibitory sites on the nucleotide binding domain of the Ca-ATPase.

  9. Membrane Structure of CtrA3, a Copper-transporting P-type-ATPase from Aquifex aeolicus

    NARCIS (Netherlands)

    Chintalapati, Sivaram; Kurdi, Rana Al; Terwisscha van Scheltinga, Anke C.; Kühlbrandt, Werner

    2008-01-01

    We have produced and characterized two new copper-transporting ATPases, CtrA2 and CtrA3 from Aquifex aeolicus, that belong to the family of heavy metal ion-transporting PIB-type ATPases. CtrA2 has a CPC metal-binding sequence in TM6 and a CxxC metal-binding N-terminal domain, while CtrA3 has a CPH m

  10. Membrane Structure of CtrA3, a Copper-transporting P-type-ATPase from Aquifex aeolicus

    NARCIS (Netherlands)

    Chintalapati, Sivaram; Kurdi, Rana Al; Terwisscha van Scheltinga, Anke C.; Kühlbrandt, Werner

    2008-01-01

    We have produced and characterized two new copper-transporting ATPases, CtrA2 and CtrA3 from Aquifex aeolicus, that belong to the family of heavy metal ion-transporting PIB-type ATPases. CtrA2 has a CPC metal-binding sequence in TM6 and a CxxC metal-binding N-terminal domain, while CtrA3 has a CPH m

  11. ATPase Activity Measurements Using Radiolabeled ATP

    NARCIS (Netherlands)

    Swarts, H.G.; Koenderink, J.B.

    2016-01-01

    ATP provides the energy that is essential for all P-type ATPases to actively transport their substrates against an existing gradient. This ATP hydrolysis can be measured using different methods. Here, we describe a method that uses radiolabeled [gamma-(32)P]ATP, which is hydrolyzed by P-type ATPases

  12. ATPase activity of the cystic fibrosis transmembrane conductance regulator.

    Science.gov (United States)

    Li, C; Ramjeesingh, M; Wang, W; Garami, E; Hewryk, M; Lee, D; Rommens, J M; Galley, K; Bear, C E

    1996-11-08

    The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-activated chloride channel thought to be critical for salt and water transport by epithelial cells. Plausible models exist to describe a role for ATP hydrolysis in CFTR channel activity; however, biochemical evidence that CFTR possesses intrinsic ATPase activity is lacking. In this study, we report the first measurements of the rate of ATP hydrolysis by purified, reconstituted CFTR. The mutation CFTRG551D resides within a motif conserved in many nucleotidases and is known to cause severe human disease. Following reconstitution the mutant protein exhibited both defective ATP hydrolysis and channel gating, providing direct evidence that CFTR utilizes ATP to gate its channel activity.

  13. Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain.

    Science.gov (United States)

    Ito, H; Fujitani, K; Usui, K; Shimizu-Nishikawa, K; Tanaka, S; Yamamoto, D

    1996-09-03

    We have isolated a new Drosophila mutant, satori (sat), the males of which do not court or copulate with female flies. The sat mutation comaps with fruitless (fru) at 91B and does not rescue the bisexual phenotype of fru, indicating that sat is allelic to fru (fru(sat)). The fru(sat) adult males lack a male-specific muscle, the muscle of Lawrence, as do adult males with other fru alleles. Molecular cloning and analyses of the genomic and complementary DNAs indicated that transcription of the fru locus yields several different transcripts. The sequence of fru cDNA clones revealed a long open reading frame that potentially encodes a putative transcription regulator with a BTB domain and two zinc finger motifs. In the 5' noncoding region, three putative transformer binding sites were identified in the female transcript but not in male transcripts. The fru gene is expressed in a population of brain cells, including those in the antennal lobe, that have been suggested to be involved in determination of male sexual orientation. We suggest that fru functions downstream of tra in the sex-determination cascade in some neural cells and that inappropriate sexual development of these cells in the fru mutants results in altered sexual orientation of the fly.

  14. Rapid Evolution to Blast Crisis Associated with a Q252H ABL1 Kinase Domain Mutation in e19a2 BCR-ABL1 Chronic Myeloid Leukaemia

    Directory of Open Access Journals (Sweden)

    Sarah L. McCarron

    2013-01-01

    Full Text Available A minority of chronic myeloid leukaemia (CML patients express variant transcripts of which the e19a2 BCR-ABL1 fusion is the most common. Instances of tyrosine kinase inhibitor (TKI resistance in e19a2 BCR-ABL1 CML patients have rarely been reported. A case of e19a2 BCR-ABL1 CML is described in whom imatinib resistance, associated with a Q252H ABL1 kinase domain mutation, became apparent soon after initiation of TKI therapy. The patient rapidly transformed to myeloid blast crisis (BC with considerable bone marrow fibrosis and no significant molecular response to a second generation TKI. The clinical course was complicated by comorbidities with the patient rapidly succumbing to advanced disease. This scenario of Q252H-associated TKI resistance with rapid BC transformation has not been previously documented in e19a2 BCR-ABL1 CML. This case highlights the considerable challenges remaining in the management of TKI-resistant BC CML, particularly in the elderly patient.

  15. Mutations in a Conserved Domain of E. coli MscS to the Most Conserved Superfamily Residue Leads to Kinetic Changes.

    Directory of Open Access Journals (Sweden)

    Hannah R Malcolm

    Full Text Available In Escherichia coli (E. coli the mechanosensitive channel of small conductance, MscS, gates in response to membrane tension created from acute external hypoosmotic shock, thus rescuing the bacterium from cell lysis. E. coli MscS is the most well studied member of the MscS superfamily of channels, whose members are found throughout the bacterial and plant kingdoms. Homology to the pore lining helix and upper vestibule domain of E. coli MscS is required for inclusion into the superfamily. Although highly conserved, in the second half of the pore lining helix (TM3B, E. coli MscS has five residues significantly different from other members of the superfamily. In superfamilies such as this, it remains unclear why variations within such a homologous region occur: is it tolerance of alternate residues, or does it define functional variance within the superfamily? Point mutations (S114I/T, L118F, A120S, L123F, F127E/K/T and patch clamp electrophysiology were used to study the effect of changing these residues in E. coli MscS on sensitivity and gating. The data indicate that variation at these locations do not consistently lead to wildtype channel phenotypes, nor do they define large changes in mechanosensation, but often appear to effect changes in the E. coli MscS channel gating kinetics.

  16. Mutations in a Conserved Domain of E. coli MscS to the Most Conserved Superfamily Residue Leads to Kinetic Changes.

    Science.gov (United States)

    Malcolm, Hannah R; Blount, Paul

    2015-01-01

    In Escherichia coli (E. coli) the mechanosensitive channel of small conductance, MscS, gates in response to membrane tension created from acute external hypoosmotic shock, thus rescuing the bacterium from cell lysis. E. coli MscS is the most well studied member of the MscS superfamily of channels, whose members are found throughout the bacterial and plant kingdoms. Homology to the pore lining helix and upper vestibule domain of E. coli MscS is required for inclusion into the superfamily. Although highly conserved, in the second half of the pore lining helix (TM3B), E. coli MscS has five residues significantly different from other members of the superfamily. In superfamilies such as this, it remains unclear why variations within such a homologous region occur: is it tolerance of alternate residues, or does it define functional variance within the superfamily? Point mutations (S114I/T, L118F, A120S, L123F, F127E/K/T) and patch clamp electrophysiology were used to study the effect of changing these residues in E. coli MscS on sensitivity and gating. The data indicate that variation at these locations do not consistently lead to wildtype channel phenotypes, nor do they define large changes in mechanosensation, but often appear to effect changes in the E. coli MscS channel gating kinetics.

  17. Mutation of FVS1, encoding a protein with a sterile alpha motif domain, affects asexual reproduction in the fungal plant pathogen Fusarium oxysporum.

    Science.gov (United States)

    Iida, Yuichiro; Fujiwara, Kazuki; Yoshioka, Yosuke; Tsuge, Takashi

    2014-02-01

    Fusarium oxysporum produces three kinds of asexual spores: microconidia, macroconidia and chlamydospores. We previously analysed expressed sequence tags during vegetative growth and conidiation in F. oxysporum and found 42 genes that were markedly upregulated during conidiation compared to vegetative growth. One of the genes, FVS1, encodes a protein with a sterile alpha motif (SAM) domain, which functions in protein-protein interactions that are involved in transcriptional or post-transcriptional regulation and signal transduction. Here, we made FVS1-disrupted mutants from the melon wilt pathogen F. oxysporum f. sp. melonis. Although the mutants produced all three kinds of asexual spores with normal morphology, they formed markedly fewer microconidia and macroconidia than the wild type. The mutants appeared to have a defect in the development of the conidiogenesis cells, conidiophores and phialides, required for the formation of microconidia and macroconidia. In contrast, chlamydospore formation was dramatically promoted in the mutants. The growth rates of the mutants on media were slightly reduced, indicating that FVS1 is also involved in, but not essential for, vegetative growth. We also observed that mutation of FVS1 caused defects in conidial germination and virulence, suggesting that the Fvs1 has pleiotropic functions in F. oxysporum.

  18. Domains of laminin

    DEFF Research Database (Denmark)

    Engvall, E; Wewer, U M

    1996-01-01

    Extracellular matrix molecules are often very large and made up of several independent domains, frequently with autonomous activities. Laminin is no exception. A number of globular and rod-like domains can be identified in laminin and its isoforms by sequence analysis as well as by electron...... microscopy. Here we present the structure-function relations in laminins by examination of their individual domains. This approach to viewing laminin is based on recent results from several laboratories. First, some mutations in laminin genes that cause disease have affected single laminin domains, and some...... laminin isoforms lack particular domains. These mutants and isoforms are informative with regard to the activities of the mutated and missing domains. These mutants and isoforms are informative with regard to the activities of the mutated and missing domains. Second, laminin-like domains have now been...

  19. The subunit b dimer of the FOF1-ATP synthase: interaction with F1-ATPase as deduced by site-specific spin-labeling.

    Science.gov (United States)

    Motz, Christian; Hornung, Tassilo; Kersten, Michael; McLachlin, Derek T; Dunn, Stanley D; Wise, John G; Vogel, Pia D

    2004-11-19

    We have used site-specific spin-labeling of single cysteine mutations within a water-soluble mutant of subunit b of the ATP synthase and employed electron spin resonance (ESR) spectroscopy to obtain information about the binding interactions of the b dimer with F1-ATPase. Interaction of b2 with a delta-depleted F1 (F1-delta) was also studied. The cysteine mutations used for spin-labeling were distributed throughout the cytosolic domain of the b subunit. In addition, each position between residues 101 and 114 of b was individually mutated to cysteine. All mutants were modified with a cysteine-reactive spin label. The room temperature ESR spectra of spin-labeled b2 in the presence of F1 or F1-delta when compared with the spectra of free b2 indicate a tight binding interaction between b2 and F1. The data suggest that b2 packs tightly to F1 between residues 80 and the C terminus but that there are segments of b2 within that region where packing interactions are quite loose. Two-dimensional gel electrophoresis confirmed binding of the modified b mutants to F1-ATPase as well as to F1-delta. Subsequent addition of delta to F1-delta.b2 complex resulted in changes in the ESR spectra, indicating different binding interactions of b to F1 in the presence or absence of delta. The data also suggest that the reconstitution of the ATP synthase is not ordered with respect to these subunits. Additional spectral components observed in b preparations that were spin-labeled between amino acid position 101 and 114 are indicative of either two populations of b subunits with different packing interactions or to helical bending within this region.

  20. Mutations in Streptococcus pneumoniae penicillin-binding protein 2x: importance of the C-terminal penicillin-binding protein and serine/threonine kinase-associated domains for beta-lactam binding.

    Science.gov (United States)

    Maurer, Patrick; Todorova, Katya; Sauerbier, Julia; Hakenbeck, Regine

    2012-06-01

    Penicillin-binding protein 2x (PBP2x) mutations that occur during the selection with beta-lactams are located within the central penicillin-binding/transpeptidase (TP) domain, and are believed to mediate resistance by interfering with the formation of a covalent complex of the active site serine with the antibiotic. We now investigated the effect of two point mutations found in two independently obtained laboratory mutants that are located at the surface of the TP domain with their side chains facing outside (G422D respectively R426C). They have no significant effect on resistance to cefotaxime in vivo or on binding to Bocillin™FL to the active site in vitro using purified PBP2x derivatives, thus apparently do not affect the active site directly. In contrast, in silico modeling revealed that they affect van der Waal's interactions with the PASTA1 (PBP and serine/threonine kinase associated) domain of the C-terminal extension and a noncovalent cefuroxime molecule found in the X-ray structure of an acylated PBP2x, suggesting some effect of the mutations on the interaction of the TP domain with PASTA1 and/or with the antibiotic associated with PASTA1. The effect of the PASTA domains on covalent binding of PBP2x to Bocillin FL was then investigated using a series of soluble truncated PBP2x derivatives. Deletion of 127 C-terminal residues, that is, of both PASTA domains, decreased binding dramatically by ∼90%. Surprisingly, deletion of only 40 amino acids resulted in the same phenotype, whereas the absence of 30 amino acids affected binding marginally by 10%, documenting a crucial role of the C-terminal domain for beta-lactam binding.

  1. Curcumin is a lipid dependent inhibitor of the Na,K-ATPase that likely interacts at the protein-lipid interface.

    Science.gov (United States)

    Mahmmoud, Yasser A

    2011-01-01

    Curcumin is an important nutraceutical widely used in disease treatment and prevention. We have previously suggested that curcumin interferes with K(+) binding to pig kidney Na,K-ATPase by interaction with its extracellular domains. The aim of this study was to further characterize the site of curcumin interaction with the ATPase. We have performed pair inhibitor studies and investigated the sided action of curcumin on pig kidney Na,K-ATPase reconstituted into lipid vesicles of defined composition. An addition of curcumin to either the intracellular or extracellular domains of the Na,K-ATPase produced similar inhibition. The lipid environment and temperature strongly influenced the potency of the drug. Curcumin inhibition decreased following insertion of the ATPase in sphingomyelin-cholesterol 'raft' domains and fully abolished following treatment with non-ionic detergents. The drug induced cross-linking of membrane embedded domains of the Na,K-ATPase. We conclude that curcumin interacts with Na,K-ATPase at the protein-lipid interface. Non-annulus lipids likely participate in this interaction. These results provide new information on the molecular mechanism of curcumin action and explain (at least partly) the ambiguous effectiveness of this polyphenol in the different systems.

  2. NVL2, a nucleolar AAA-ATPase, is associated with the nuclear exosome and is involved in pre-rRNA processing

    Energy Technology Data Exchange (ETDEWEB)

    Yoshikatsu, Yuki [Department of Life Systems, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima 770-8506 (Japan); Ishida, Yo-ichi; Sudo, Haruka [Department of Molecular and Cellular Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan); Yuasa, Keizo; Tsuji, Akihiko [Department of Life Systems, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima 770-8506 (Japan); Nagahama, Masami, E-mail: nagahama@my-pharm.ac.jp [Department of Molecular and Cellular Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan)

    2015-08-28

    Nuclear VCP-like 2 (NVL2) is a member of the chaperone-like AAA-ATPase family and is involved in the biosynthesis of 60S ribosomal subunits in mammalian cells. We previously showed the interaction of NVL2 with a DExD/H-box RNA helicase MTR4/DOB1, which is a known cofactor for an exoribonuclease complex, the exosome. This finding implicated NVL2 in RNA metabolic processes during ribosome biogenesis. In the present study, we found that a series of mutations within the ATPase domain of NVL2 causes a defect in pre-rRNA processing into mature 28S and 5.8S rRNAs. Co-immunoprecipitation analysis showed that NVL2 was associated with the nuclear exosome complex, which includes RRP6 as a nucleus-specific catalytic subunit. This interaction was prevented by depleting either MTR4 or RRP6, indicating their essential role in mediating this interaction with NVL2. Additionally, knockdown of MPP6, another cofactor for the nuclear exosome, also prevented the interaction by causing MTR4 to dissociate from the nuclear exosome. These results suggest that NVL2 is involved in pre-rRNA processing by associating with the nuclear exosome complex and that MPP6 is required for maintaining the integrity of this rRNA processing complex. - Highlights: • ATPase-deficient mutants of NVL2 have decreased pre-rRNA processing. • NVL2 associates with the nuclear exosome through interactions with MTR4 and RRP6. • MPP6 stabilizes MTR4-RRP6 interaction and allows NVL2 to interact with the complex.

  3. Functional diversity of five homologous Cu+-ATPases present in Sinorhizobium meliloti.

    Science.gov (United States)

    Patel, Sarju J; Padilla-Benavides, Teresita; Collins, Jessica M; Argüello, José M

    2014-06-01

    Copper is an important element in host-microbe interactions, acting both as a catalyst in enzymes and as a potential toxin. Cu(+)-ATPases drive cytoplasmic Cu(+) efflux and protect bacteria against metal overload. Many pathogenic and symbiotic bacteria contain multiple Cu(+)-ATPase genes within particular genetic environments, suggesting alternative roles for each resulting protein. This hypothesis was tested by characterizing five homologous Cu(+)-ATPases present in the symbiotic organism Sinorhizobium meliloti. Mutation of each gene led to different phenotypes and abnormal nodule development in the alfalfa host. Distinct responses were detected in free-living S. meliloti mutant strains exposed to metal and redox stresses. Differential gene expression was detected under Cu(+), oxygen or nitrosative stress. These observations suggest that CopA1a maintains the cytoplasmic Cu(+) quota and its expression is controlled by Cu(+) levels. CopA1b is also regulated by Cu(+) concentrations and is required during symbiosis for bacteroid maturation. CopA2-like proteins, FixI1 and FixI2, are necessary for the assembly of two different cytochrome c oxidases at different stages of bacterial life. CopA3 is a phylogenetically distinct Cu(+)-ATPase that does not contribute to Cu(+) tolerance. It is regulated by redox stress and required during symbiosis. We postulated a model where non-redundant homologous Cu(+)-ATPases, operating under distinct regulation, transport Cu(+) to different target proteins.

  4. Presenilin 1 maintains lysosomal Ca2+ homeostasis by regulating vATPase-mediated lysosome acidification

    Science.gov (United States)

    Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M.; Haslett, Luke J.; Kumar, Asok; Sato, Yutaka; Lie, Pearl P. Y.; Mohan, Panaiyur; Coffey, Erin E.; Kompella, Uday; Mitchell, Claire H.; Lloyd-Evans, Emyr; Nixon, Ralph A.

    2015-01-01

    Summary Presenilin-1 (PS1) deletion or Alzheimer’s Disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss of function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism. PMID:26299959

  5. Crystal structure of a copper-transporting PIB-type ATPase.

    Science.gov (United States)

    Gourdon, Pontus; Liu, Xiang-Yu; Skjørringe, Tina; Morth, J Preben; Møller, Lisbeth Birk; Pedersen, Bjørn Panyella; Nissen, Poul

    2011-07-01

    Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu(+)-ATPase, in a copper-free form, as determined by X-ray crystallography at 3.2 Å resolution. The structure indicates a three-stage copper transport pathway involving several conserved residues. A PIB-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a putative copper entry point at the intracellular interface. Comparisons to Ca(2+)-ATPase suggest an ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site. The structure also provides a framework to analyse missense mutations in the human ATP7A and ATP7B proteins associated with Menkes' and Wilson's diseases.

  6. Aspects of gene structure and functional regulation of the isozymes of Na,K-ATPase

    DEFF Research Database (Denmark)

    Jorgensen, P.L.

    2001-01-01

    genomes, the genes of four alpha-subunit and at least three beta-subunit isoforms of Na,K-ATPase are identified and two gamma-subunits are expressed in kidney. The isoforms combine in a number of Na,K-ATPase isozymes that are expressed in a tissue and cell specific manner. Models of the molecular...... mechanism of regulation of these isozymes have become more reliable due to progress in understanding the three-dimensional protein structure and conformational transitions mediating transfer of energy from the P-domain to intramembrane Na+ and K+ binding sites....

  7. Structural studies of Ca2+-ATPase ligand and regulatory complexes

    DEFF Research Database (Denmark)

    Drachmann, Nikolaj Düring

    2015-01-01

    The Ca2+-ATPase (sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA is a part of the vital P-type ATPase family, which was first discovered in 1957 by Professor Jens Christian Skou. He was the first to describe the Na+,K+-ATPase and its role in generating the membrane potential across the axonal...

  8. Novel regulation of cell [Na(+)] in macula densa cells: apical Na(+) recycling by H-K-ATPase.

    Science.gov (United States)

    Peti-Peterdi, János; Bebok, Zsuzsa; Lapointe, Jean-Yves; Bell, P Darwin

    2002-02-01

    Na-K-ATPase is the nearly ubiquitous enzyme that maintains low-Na(+), high-K(+) concentrations in cells by actively extruding Na(+) in exchange for K(+). The prevailing paradigm in polarized absorbing epithelial cells, including renal nephron segments and intestine, has been that Na-K-ATPase is restricted to the basolateral membrane domain, where it plays a prominent role in Na(+) absorption. We have found, however, that macula densa (MD) cells lack functionally and immunologically detectable amounts of Na-K-ATPase protein. In fact, these cells appear to regulate their cytosolic [Na(+)] via another member of the P-type ATPase family, the colonic form of H-K-ATPase, which is located at the apical membrane in these cells. We now report that this constitutively expressed apical MD colonic H-K-ATPase can function as a Na(H)-K-ATPase and regulate cytosolic [Na(+)] in a novel manner. This apical Na(+)-recycling mechanism may be important as part of the sensor function of MD cells and represents a new paradigm in cell [Na(+)] regulation.

  9. Cellular multitasking: the dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance.

    Science.gov (United States)

    Lutsenko, Svetlana; Gupta, Arnab; Burkhead, Jason L; Zuzel, Vesna

    2008-08-01

    The human copper-transporting ATPases (Cu-ATPases) are essential for dietary copper uptake, normal development and function of the CNS, and regulation of copper homeostasis in the body. In a cell, Cu-ATPases maintain the intracellular concentration of copper by transporting copper into intracellular exocytic vesicles. In addition, these P-type ATPases mediate delivery of copper to copper-dependent enzymes in the secretory pathway and in specialized cell compartments such as secretory granules or melanosomes. The multiple functions of human Cu-ATPase necessitate complex regulation of these transporters that is mediated through the presence of regulatory domains in their structure, posttranslational modification and intracellular trafficking, as well as interactions with the copper chaperone Atox1 and other regulatory molecules. In this review, we summarize the current information on the function and regulatory mechanisms acting on human Cu-ATPases ATP7A and ATP7B. Brief comparison with the Cu-ATPase orthologs from other species is included.

  10. Molecular basis of calcium-sensitizing and desensitizing mutations of the human cardiac troponin C regulatory domain: a multi-scale simulation study.

    Directory of Open Access Journals (Sweden)

    Peter Michael Kekenes-Huskey

    Full Text Available Troponin C (TnC is implicated in the initiation of myocyte contraction via binding of cytosolic Ca²⁺ and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease Ca²⁺ sensitivity, in order to understand the structural basis of their impact on TnC function. We will use the distinction for mutants that are associated with increased Ca²⁺ affinity and for those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the physiologically-active site II Ca²⁺ binding site in the Ca²⁺-free (apo state closely resembled the Ca²⁺-bound (holo state. In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo conformation. We hypothesize that these phenomena contribute to the increased association rate, k(on, for the GOF mutants relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to Ca²⁺ affinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs that target TnC.

  11. The voltage-sensing domain of kv7.2 channels as a molecular target for epilepsy-causing mutations and anticonvulsants

    Directory of Open Access Journals (Sweden)

    Francesco eMiceli

    2011-02-01

    Full Text Available Understanding the molecular mechanisms underlying voltage-dependent gating in voltage-gated ion channels (VGICs has been a major effort over the last decades. In recent years, changes in the gating process have emerged as common denominators for several genetically-determined channelopathies affecting heart rhythm (arrhythmias, neuronal excitability (epilepsy, pain or skeletal muscle contraction (periodic paralysis. Moreover, gating changes appear as the main molecular mechanism by which several natural toxins from a variety of species affect ion channel function.In this work, we describe the pathophysiological and pharmacological relevance of the gating process in voltage-gated K+ channels encoded by the Kv7 gene family. After reviewing the current knowledge on the molecular mechanisms and on the structural models of voltage-dependent gating in VGICs, we describe the physiological relevance of these channels, with particular emphasis on those formed by Kv7.2-5 subunits having a well-established role in controlling neuronal excitability in humans. In fact, genetically-determined alterations in Kv7.2 and Kv7.3 genes are responsible for benign familial neonatal convulsions, a rare seizure disorder affecting newborns, and the pharmacological activation of Kv7.2/3 channels can exert antiepileptic activity in humans. Both mutation-triggered channel dysfunction and drug-induced channel activation can occur by impeding or facilitating, respectively, channel sensitivity to membrane voltage and can affect overlapping molecular sites within the voltage-sensing domain of these channels. Thus, understanding the molecular steps involved in voltage-sensing in Kv7 channels will allow to better define the pathogenesis of rare human epilepsy, and to design innovative pharmacological strategies for the treatment of epilepsies and, possibly, other human diseases characterized by neuronal hyperexcitability.

  12. Sodium, potassium-atpases in algae and oomycetes.

    Science.gov (United States)

    Barrero-Gil, Javier; Garciadeblás, Blanca; Benito, Begoña

    2005-08-01

    We have investigated the presence of K(+)-transporting ATPases that belong to the phylogenetic group of animal Na(+),K(+)-ATPases in the Pythium aphanidermatum Stramenopile oomycete, the Porphyra yezoensis red alga, and the Udotea petiolata green alga, by molecular cloning and expression in heterologous systems. PCR amplification and search in EST databases allowed one gene to be identified in each species that could encode ATPases of this type. Phylogenetic analysis of the sequences of these ATPases revealed that they cluster with ATPases of animal origin, and that the algal ATPases are closer to animal ATPases than the oomycete ATPase is. The P. yezoensis and P. aphanidermatum ATPases were functionally expressed in Saccharomyces cerevisiae and Escherichia coli alkali cation transport mutants. The aforementioned cloning and complementary searches in silicio for H(+)- and Na(+),K(+)-ATPases revealed a great diversity of strategies for plasma membrane energization in eukaryotic cells different from typical animal, plant, and fungal cells.

  13. Darier disease in Slovenia: spectrum of ATP2A2 mutations and relation to patients' phenotypes.

    Science.gov (United States)

    Godic, Aleksandar; Strazisar, Mojca; Zupan, Andrej; Korosec, Branka; Kansky, Aleksej; Glavac, Damjan

    2010-01-01

    ATP2A2 encodes the sarco/endoplasmic reticulum Ca2+- ATPase (SERCA2) and has been identified as a defective gene in Darier disease (DD). It is an autosomal dominant genodermatosis, which is characterized by loss of adhesion between suprabasal epidermal keratinocytes (acantholysis) and abnormal keratinization (dyskeratosis). We examined 28 Slovenian patients with DD (the cohort of patients represents over 50% of all DD patients in Slovenia) and screened genomic DNA for ATP2A2 mutations and RNA for splice site mutations. We identified 7 different ATP2A2 mutations, 4 of which are novel: A516P, R559G, 544+1del6, and 1762-6del18. We also found two previously described polymorphisms 2741+54 G>A in intron XVIII and 2172 G>A (A724A) in exon 15, with allele frequencies of 64.2% and 11.3%, respectively. The mutations are scattered throughout the gene and affect the actuator, phosphorylation, stalk and transmembrane domains of SERCA2. A P160L mutation in a Slovene patient with severe DD and a history of deafness is another consistent genotype-phenotype correlation. It seems that mutations of the ATP2A2 gene may also play a role in the pathogenesis of deafness, which seems to be a new phenotypic characteristic of DD patients.

  14. An update in the structure, function, and regulation of V-ATPases: the role of the C subunit

    Directory of Open Access Journals (Sweden)

    M Pérez-Sayáns

    Full Text Available Vacuolar ATPases (V-ATPases are present in specialized proton secretory cells in which they pump protons across the membranes of various intracellular organelles and across the plasma membrane. The proton transport mechanism is electrogenic and establishes an acidic pH and a positive transmembrane potential in these intracellular and extracellular compartments. V-ATPases have been found to be practically identical in terms of the composition of their subunits in all eukaryotic cells. They have two distinct structures: a peripheral catalytic sector (V1 and a hydrophobic membrane sector (V0 responsible for driving protons. V-ATPase activity is regulated by three different mechanisms, which control pump density, association/dissociation of the V1 and V0 domains, and secretory activity. The C subunit is a 40-kDa protein located in the V1 domain of V-ATPase. The protein is encoded by the ATP6V1C gene and is located at position 22 of the long arm of chromosome 8 (8q22.3. The C subunit has very important functions in terms of controlling the regulation of the reversible dissociation of V-ATPases.

  15. A single missense mutation in a coiled-coil domain of Escherichia coli ribosomal protein S2 confers a thermosensitive phenotype that can be suppressed by ribosomal protein S1.

    Science.gov (United States)

    Aseev, Leonid V; Chugunov, Anton O; Efremov, Roman G; Boni, Irina V

    2013-01-01

    Ribosomal protein S2 is an essential component of translation machinery, and its viable mutated variants conferring distinct phenotypes serve as a valuable tool in studying the role of S2 in translation regulation. One of a few available rpsB mutants, rpsB1, shows thermosensitivity and ensures enhanced expression of leaderless mRNAs. In this study, we identified the nature of the rpsB1 mutation. Sequencing of the rpsB1 allele revealed a G-to-A transition in the part of the rpsB gene which encodes a coiled-coil domain of S2. The resulting E132K substitution resides in a highly conserved site, TKKE, a so-called N-terminal capping box, at the beginning of the second alpha helix. The protruding coiled-coil domain of S2 is known to provide binding with 16S rRNA in the head of the 30S subunit and, in addition, to interact with a key mRNA binding protein, S1. Molecular dynamics simulations revealed a detrimental impact of the E132K mutation on the coiled-coil structure and thereby on the interactions between S2 and 16S rRNA, providing a clue for the thermosensitivity of the rpsB1 mutant. Using a strain producing a leaderless lacZ transcript from the chromosomal lac promoter, we demonstrated that not only the rpsB1 mutation generating S2/S1-deficient ribosomes but also the rpsA::IS10 mutation leading to partial deficiency in S1 alone increased translation efficiency of the leaderless mRNA by about 10-fold. Moderate overexpression of S1 relieved all these effects and, moreover, suppressed the thermosensitive phenotype of rpsB1, indicating the role of S1 as an extragenic suppressor of the E132K mutation.

  16. Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state

    Science.gov (United States)

    Morito, Daisuke; Nishikawa, Kouki; Hoseki, Jun; Kitamura, Akira; Kotani, Yuri; Kiso, Kazumi; Kinjo, Masataka; Fujiyoshi, Yoshinori; Nagata, Kazuhiro

    2014-03-01

    Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell.

  17. [ATPase and phosphatase activity of drone brood].

    Science.gov (United States)

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae.

  18. Functional Analysis of P4-ATPases

    DEFF Research Database (Denmark)

    Theorin, Lisa

    and mammalian P4-ATPases have been studied extensively and the physiological function is mostly known, while the exact biochemistry and specific activity is mostly unknown. Even though the plant Arabidopsis thaliana has 12 P4-ATPases, not much is known about their function. In this study, the biochemical...... properties, with a focus on the lipid requirements, of the Aminophospholipid ATPase 2 (ALA2), a P4-ATPase from A. thaliana, were characterized. Heterologous expression of ALA2 together with its subunit, the Cdc50 homolog ALA Interacting Subunit 5 (ALIS5), in the yeast Saccharomyces cerevisiae allowed...... is specific for phosphatidylserine and that binding of the lipid to the substrate binding site requires a unique spatial configuration of the lipid head group. Detailed information on the substrate requirements lead the way towards the full function and transport pathway of lipid flippases in plants. Recent...

  19. A Systematic Study on Structure and Function of ATPase of Wuchereria bancrofti

    Science.gov (United States)

    Islam, Md. Saiful; Patwary, Noman Ibna Amin; Muzahid, Nazmul Hasan; Shahik, Shah Md.; Sohel, Md.; Hasan, Md. Anayet

    2014-01-01

    Background: Analyzing the structures and functions of different proteins of Wuchereria bancrofti is very important because till date no effective drug or vaccine has been discovered to treat lymphatic filariasis (LF). ATPase is one of the most important proteins of Wuchereria bancrofti. Adenosine triphosphate (ATP) converts into adenosine diphosphate (ADP) and a free phosphate ion by the action of these ATPase enzymes. Energy releases from these dephosphorylation reactions drive the other chemical reactions in the cell. Materials and Methods: In this study we worked on the protein ATPase of Wuchereria bancrofti which has been annotated from National Center for Biotechnology Information (NCBI). Various computational tools and databases have been used to determine the various characteristics of that enzyme such as physiochemical properties, secondary structure, three-dimensional (3D) structure, conserved domain, epitope, and their molecular evolutionary relationship. Result: Subcellular localization of ATPase was identified and we have found that 55.5% are localized in the cytoplasm. Secondary and 3D structure of this protein was also predicted. Both structure and function analysis of ATPase of Wuchereria bancrofti showed unique nonhomologous epitope sites and nonhomologous antigenicity sites. Moreover, it resulted in 15 ligand drug-binding sites in its tertiary structure. Conclusion: Structure prediction of these proteins and detection of binding sites and antigenicity sites from this study would indicate a potential target aiding docking studies for therapeutic designing against filariasis. PMID:25948965

  20. Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

    Science.gov (United States)

    Goldgof, Gregory M.; Durrant, Jacob D.; Ottilie, Sabine; Vigil, Edgar; Allen, Kenneth E.; Gunawan, Felicia; Kostylev, Maxim; Henderson, Kiersten A.; Yang, Jennifer; Schenken, Jake; LaMonte, Gregory M.; Manary, Micah J.; Murao, Ayako; Nachon, Marie; Stanhope, Rebecca; Prescott, Maximo; McNamara, Case W.; Slayman, Carolyn W.; Amaro, Rommie E.; Suzuki, Yo; Winzeler, Elizabeth A.

    2016-01-01

    The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity. PMID:27291296

  1. The coupling ATPase complex: an evolutionary view.

    Science.gov (United States)

    Harris, D A

    1981-01-01

    Phospholipid micelles and vesicles, present in the primordial soup, formed both primitive (surface) catalyst and primitive replicative life forms. With the adoption of a common energy source, ATP, integrated biochemical systems within these vesicles became possible - cells. Fermentation within these primitive cells was favoured by the evolution, first of ion channels allowing protons to leak out, and then of an active ATP-driven pump. In the prokaryotic/mitochondria/chloroplast line, the proton channel was such as to be blocked by dicyclohexylcarbodiimide and the adenosine 5' triphosphate phosphohydrolase (ATPase) by 4-chloro 7-nitrobenzofurazan (Nbf-C1). The ATPase was initially simple (4 subunits) but later, possibly concomitant with its evolution to an ATP synthetase, became more complex (8 subunits). One of the steps in evolution probably involved gene duplication and divergence of 2 subunits (alpha and beta) from the largest of the ATPase subunits. From this stage, the general form of the ATPase was fixed, although sensitivity to, for example, oligomycin involved later, after divergence of the mitochondrial and chloroplast lines. A regulatory protein, the ATPase inhibitor, is found associated with a wide spectrum of coupling ATPases.

  2. Lack of somatic mutations in the catalytic domains of CREBBP and EP300 genes implies a role for histone deacetylase inhibition in myeloproliferative neoplasms

    DEFF Research Database (Denmark)

    Andersen, C.L.; Grønbæk, K.; Hasselbalch, H.

    2012-01-01

    with myeloproliferative neoplasms (MPNs) has not previously been performed. DNA was purified from diagnostic samples of 56 MPN patients. We designed a mutation screening assay based on denaturing gradient gel electrophoresis and direct sequencing. Our results suggest that CREBBP and EP300 mutations are not major...... pathogenetic mechanisms of MPNs. The rationale for using HDACi in these patients seems reasonable. © 2011 Elsevier Ltd....

  3. The Clinical Spectrum of Missense Mutations of the First Aspartic Acid of cbEGF-like Domains in Fibrillin-1 Including a Recessive Family

    NARCIS (Netherlands)

    Hilhorst-Hofstee, Yvonne; Rijlaarsdam, Marry E. B.; Scholte, Arthur J. H. A.; Swart-van den Berg, Marietta; Versteegh, Michel I. M.; van der Schoot-van Velzen, Iris; Schaebitz, Hans-Joachim; Bijlsma, Emilia K.; Baars, Marieke J.; Kerstjens-Frederikse, Wilhelmina S.; Giltay, Jacques C.; Hamel, Ben C.; Breuning, Martijn H.; Pals, Gerard

    2010-01-01

    Marfan syndrome (MFS) is a dominant disorder with a recognizable phenotype. In most patients with the classical phenotype mutations are found in the fibrillin-1 gene (FBN1) on chromosome 15q21. It is thought that most mutations act in a dominant negative way or through haploinsufficiency. In 9 index

  4. De novo mutations in the motor domain of KIF1A cause cognitive impairment, spastic paraparesis, axonal neuropathy, and cerebellar atrophy

    NARCIS (Netherlands)

    Lee, Jae Ran; Srour, Myriam; Kim, Doyoun; Hamdan, Fadi F.; Lim, So Hee; Brunel-Guitton, Catherine; Décarie, Jean Claude; Rossignol, Elsa; Mitchell, Grant A.; Schreiber, Allison; Moran, Rocio; Van Haren, Keith; Richardson, Randal; Nicolai, Joost; Oberndorff, Karin M E J; Wagner, Justin D.; Boycott, Kym M.; Rahikkala, Elisa; Junna, Nella; Tyynismaa, Henna; Cuppen, Inge; Verbeek, Nienke E.; Stumpel, Connie T R M; Willemsen, Michel A.; de Munnik, Sonja A.; Rouleau, Guy A.; Kim, Eunjoon; Kamsteeg, Erik Jan; Kleefstra, Tjitske; Michaud, Jacques L.

    2015-01-01

    KIF1A is a neuron-specific motor protein that plays important roles in cargo transport along neurites. Recessive mutations in KIF1A were previously described in families with spastic paraparesis or sensory and autonomic neuropathy type-2. Here, we report 11 heterozygous de novo missense mutations (p

  5. Loss or gain of function in NIH3T3 and PC12 cells produced by different mutations in the RET tyrosine kinase domain may explain phenotypic diversity between Hirchsprung disease and MEN 2B

    Energy Technology Data Exchange (ETDEWEB)

    Pasini, B.; Seri, M.; Yin, L. [Laboratorio di Genetica Molecolare, Genova (Italy)] [and others

    1994-09-01

    The RET protooncogene encodes a receptor tyrosine kinase involved in the control differentiation of neural crest derived cells. Point mutations of the RET tyrosine kinase domain were identified among others in 2 distinct genetic disorders, Hirchsprung disease (HSCR) and Multiple Endocrine Neoplasia 2B (MEN 2B). In order to test the biological effect of HSCR and MEN 2B mutations we used a system based on RET-PTC2, a chimeric activated form of the RET protoocogene isolated from a papillary thyroid carcinoma, which shows a detectable transforming activity in NIH3T3 cells and induction of differentiation in PC12 cells. By site-direct mutagenesis we introduced into RET-PTC2 cDNA the mutations at codon 918 (Met{yields}thr, typical of MEN 2B), at codon 765 (Ser{yields}Pro, observed in HSCR) and at codon 897 (Arg{yields}Gln, also observed in HSCR). The former mutation appears to increase the transforming activity of RET-PTC2 in NIH3T3 cells. The latter two mutations abolish the oncogenic activity in NIH3T3 cells as well as its differentiating effect in PC12 cells. These results suggest that RET mutations may cause MEN 2B and HSCR phenotypes through a mechanism of gain or loss of function respectively. Finally, co-transfection experiments of wild-type RET-PTC2 with either HSCR mutation are in progress in order to test the hypothesis of a dominant negative effect in heterozygous state.

  6. VHL missense mutations in the p53 binding domain show different effects on p53 signaling and HIFα degradation in clear cell renal cell carcinoma.

    Science.gov (United States)

    Razafinjatovo, Caroline Fanja; Stiehl, Daniel; Deininger, Eva; Rechsteiner, Markus; Moch, Holger; Schraml, Peter

    2017-02-07

    Clear cell Renal Cell Carcinoma (ccRCC) formation is connected to functional loss of the von Hippel-Lindau (VHL) gene. Recent data identified its gene product, pVHL, as a multifunctional adaptor protein which interacts with HIFα subunits but also with the tumor suppressor p53. p53 is hardly expressed and rarely mutated in most ccRCC. We showed that low and absent p53 expression correlated with the severity of VHL mutations in 262 analyzed ccRCC tissues. In contrast to nonsense and frameshift mutations which abrogate virtually all pVHL functions, missense mutations may rather influence one or few functions. Therefore, we focused on four VHL missense mutations, which affect the overlapping pVHL binding sites of p53 and Elongin C, by investigating their impact on HIFα degradation, p53 expression and signaling, as well as on cellular behavior using ccRCC cell lines and tissues. TP53 mRNA and its effector targets p21, Bax and Noxa, were altered both in engineered cell lines and in tumor tissues which carried the same missense mutations. Two of these mutations were not able to degrade HIFα whereas the remaining two mutations led to HIFα downregulation, suggesting the latter are p53 binding site-specific. The selected VHL missense mutations further enhanced tumor cell survival, but had no effects on cell proliferation. Whereas Sunitinib was able to efficiently reduce cell proliferation, Camptothecin was additionally able to increase apoptotic activity of the tumor cells. It is concluded that systematic characterization of the VHL mutation status may help optimizing targeted therapy for patients with metastatic ccRCC.

  7. Spectrum of mutations in the ATP binding domain of ATP7B gene of Wilson Disease in a regional Indian cohort.

    Science.gov (United States)

    Guggilla, Sreenivasa Rao; Senagari, Jalandhar Reddy; Rao, P N; Madireddi, Sujatha

    2015-09-10

    Wilson disease is an autosomal recessive disorder of abnormal copper accumulation in the liver, brain, kidney and cornea, resulting in hepatic and neurological abnormalities, which results from impaired ATP7B protein function due to mutations in candidate ATP7B gene, till date more than 500 disease causing mutations were found. In India most disease causing mutations were identified in ATP-BD. DNA samples of the 101 WD cases and 100 control population were analyzed for mutations. 11 mutations were identified in 57 chromosomes. Three novel mutations, c.3310T>A (p.Cys1104Ser), c.3337C>A (p.Leu1113Met) on exon 15 and c.3877G>A (p.Glu1293Lys) on exon 18 were identified for the first time in the ATP7B gene. Two mutations, c.3121C>T (p.Arg1041Trp) and c.3128T>C (p.Leu1043Pro) on exon 14 were discovered for the first time in Indian Wilson disease patients. Four previously reported mutations c.3008C>T, c.3029A>G on exon 13, c.3182G>A on exon 14 and c.3809A>G on exon 18 from South India were also found in this study. Our research has enriched the spectrum of mutations of the ATP7B gene in the south Indian population. The detection of new mutations in the ATP7B gene can aid in genetic counseling and clinical or/prenatal diagnosis.

  8. Transcomplementation, but not Physical Association of the CC-NB-ARC and LRR Domains of Tomato R Protein Mi-1.2 is Altered by Mutations in the ARC2 Subdomain

    Institute of Scientific and Technical Information of China (English)

    Gerben van Ooijen; Gabriele Mayr; Mario Albrecht; Ben J. C. Cornelissena; Frank L.W. Takken

    2008-01-01

    Race-specific disease resistance in plants is mediated by Resistance (R) proteins that recognize pathogen attack and initiate defence responses. Most R proteins contain a central NB-ARC domain and a C-terminal leucine-rich repeat (LRR) domain. We analyzed the intramolecular interaction of the LRR domain of tomato R protein Mi-1.2 with its Nterminus. We expressed the CC-NB-ARC and LRR parts in trans and analyzed functional transcomplementation and physical interactions. We show that these domains functionally transcomplement when expressed in trans. Known autoactivating LRR domain swaps were found to induce a hypersensitive response (HR) upon co-expression. Likewise, autoactivating mutants in the NB subdomain transcomplemented to induce HR. Point mutations in the ARC2 subdomain that induce strong autoactivation in the full-length Mi-1.2 protein, however, fail to induce HR in the transcomplementation assay. These data indicate distinct functions for the NB-ARC subdomains in induction of HR signalling. Furthermore, dissociation of the LRR is not required to release its negative regulation, as in all combinations of CC-NB-ARC and LRR domains tested, a physical interaction was observed.

  9. Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

    Science.gov (United States)

    Schulz, Sebastian; Doller, Anke; Pendini, Nicole R; Wilce, Jacqueline A; Pfeilschifter, Josef; Eberhardt, Wolfgang

    2013-12-01

    The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA.

  10. Spliceosome discards intermediates via the DEAH box ATPase Prp43p.

    Science.gov (United States)

    Mayas, Rabiah M; Maita, Hiroshi; Semlow, Daniel R; Staley, Jonathan P

    2010-06-01

    To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5' exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation, this work establishes a parallel between the discard of suboptimal intermediates and the dissociation of a genuine excised intron product.

  11. E. coli F1-ATPase: site-directed mutagenesis of the beta-subunit.

    Science.gov (United States)

    Parsonage, D; Wilke-Mounts, S; Senior, A E

    1988-05-09

    Residues beta Glu-181 and beta Glu-192 of E. coli F1-ATPase (the DCCD-reactive residues) were mutated to Gln. Purified beta Gln-181 F1 showed 7-fold impairment of 'unisite' Pi formation from ATP and a large decrease in affinity for ATP. Thus the beta-181 carboxyl group in normal F1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 X 10(4)- to 548-fold in beta Gln-181 F1. In contrast, purified beta Gln-192 F1 showed only 6-fold reduction in 'multisite' ATPase activity. Residues beta Gly-149 and beta Gly-154 were mutated to Ile singly and in combination. These mutations, affecting residues which are strongly conserved in nucleotide-binding proteins, were chosen to hinder conformational motion in a putative 'flexible loop' in beta-subunit. Impairment of purified F1-ATPase ranged from 5 to 61%, with the double mutant F1 less impaired than either single mutant. F1 preparations containing beta Ile-154 showed 2-fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants.

  12. Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation.

    Science.gov (United States)

    Hacker, Kathryn E; Fahey, Catherine C; Shinsky, Stephen A; Chiang, Yun-Chen J; DiFiore, Julia V; Jha, Deepak Kumar; Vo, Andy H; Shavit, Jordan A; Davis, Ian J; Strahl, Brian D; Rathmell, W Kimryn

    2016-09-30

    The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.

  13. Biochemical characterization of sporadic/familial hemiplegic migraine mutations

    NARCIS (Netherlands)

    Weigand, K.M.; Swarts, H.G.P.; Russel, F.G.; Koenderink, J.B.

    2014-01-01

    Sporadic hemiplegic migraine type 2 (SHM2) and familial hemiplegic migraine type 2 (FHM2) are rare forms of hemiplegic migraine caused by mutations in the Na(+),K(+)-ATPase alpha2 gene. Today, more than 70 different mutations have been linked to SHM2/FHM2, randomly dispersed over the gene. For many

  14. The F-ATPase operon from the oral streptococci S. mutans and S. sanguis: How structure relates to function

    Science.gov (United States)

    Kuhnert, Wendi Lee

    1999-10-01

    subunit do not result in structural alterations. Therefore, the contribution that the F-ATPase makes towards the aciduricity of the oral streptococci is linked to its increased expression at low pH or perhaps to structural differences in the other, less-conserved, domains of the enzyme.

  15. Hyperactivation of HUSH complex function by Charcot-Marie-Tooth disease mutation in MORC2.

    Science.gov (United States)

    Tchasovnikarova, Iva A; Timms, Richard T; Douse, Christopher H; Roberts, Rhys C; Dougan, Gordon; Kingston, Robert E; Modis, Yorgo; Lehner, Paul J

    2017-07-01

    Dominant mutations in the MORC2 gene have recently been shown to cause axonal Charcot-Marie-Tooth (CMT) disease, but the cellular function of MORC2 is poorly understood. Here, through a genome-wide CRISPR-Cas9-mediated forward genetic screen, we identified MORC2 as an essential gene required for epigenetic silencing by the HUSH complex. HUSH recruits MORC2 to target sites in heterochromatin. We exploited a new method, differential viral accessibility (DIVA), to show that loss of MORC2 results in chromatin decompaction at these target loci, which is concomitant with a loss of H3K9me3 deposition and transcriptional derepression. The ATPase activity of MORC2 is critical for HUSH-mediated silencing, and the most common alteration affecting the ATPase domain in CMT patients (p.Arg252Trp) hyperactivates HUSH-mediated repression in neuronal cells. These data define a critical role for MORC2 in epigenetic silencing by the HUSH complex and provide a mechanistic basis underpinning the role of MORC2 mutations in CMT disease.

  16. ATPase8-6基因研究杂交多倍体鱼线粒体母性遗传%Evidence for maternal inheritance of mitochondrial DNA in polyploid fish of crosses by ATPase8 and ATPase6 genes

    Institute of Scientific and Technical Information of China (English)

    郭新红; 刘少军; 刘筠

    2004-01-01

    were silent mutations. Phylogenetic trees showing three clusters were constructed with the ATPase8 and ATPase6 gene sequence data for all samples. The first cluster contained triploid common carp and diploid common carp; the second cluster contained triploid crucian carp and diploid Japanese crucian carp;and the third cluster contained allotetraploid fish and diploid red crucian carp. This finding indicated that mitochondrial ATPase8 and ATPase6 genes of the artificial polyploid fish including the allotetraploid fish, triploid crucian carp, and triploid common carp were inherited maternally. It was noticeable that after eleven generations from F1 to F1 hybrids, the F11 allotetraploid fish still kept the high homology to the female parent red crucian carp in the ATPase8 and ATPase6genes. The high homology of F11 tetraploid to the female parent red crucian carp also indicated that the F11 allotetraploid fish possessed the stable inheritable characteristic. The analysis and elucidation of the variation of the ATPase8 and ATPase6 DNA in different cyprinids proved that ATPase8 and ATPase6 genes were the useful genetic markers to monitor the variations in the progeny of the crosses [Acta Zoologica Sinica 50 (3): 408 - 413, 2004].

  17. Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit ε.

    Science.gov (United States)

    Roy, Ankoor; Hutcheon, Marcus L; Duncan, Thomas M; Cingolani, Gino

    2012-10-01

    The bacterial ATP synthase (F(O)F(1)) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ∼529 kDa), E. coli F(O)F(1) represents nature's smallest rotary motor, composed of a membrane-embedded proton transporter (F(O)) and a peripheral catalytic complex (F(1)). The ATPase activity of isolated F(1) is fully expressed by the α(3)β(3)γ 'core', whereas single δ and ε subunits are required for structural and functional coupling of E. coli F(1) to F(O). In contrast to mitochondrial F(1)-ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F(1)-ATPase catalytic core. Destabilizing the compact conformation of ε's C-terminal domain with a phosphomimetic mutation (εS65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F(1) that diffract to ∼3.15 Å resolution.

  18. Glu-857 moderates K+-dependent stimulation and SCH 28080-dependent inhibition of the gastric H,K-ATPase.

    Science.gov (United States)

    Rulli, S J; Horiba, M N; Skripnikova, E; Rabon, E C

    1999-05-21

    The rabbit H,K-ATPase alpha- and beta-subunits were transiently expressed in HEK293 T cells. The co-expression of the H,K-ATPase alpha- and beta-subunits was essential for the functional H,K-ATPase. The K+-stimulated H,K-ATPase activity of 0.82 +/- 0.2 micromol/mg/h saturated with a K0.5 (KCl) of 0.6 +/- 0.1 mM, whereas the 2-methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080)-inhibited ATPase of 0.62 +/- 0.07 micromol/mg/h saturated with a Ki (SCH 28080) of 1.0 +/- 0.3 microM. Site mutations were introduced at the N,N-dicyclohexylcarbodiimide-reactive residue, Glu-857, to evaluate the role of this residue in ATPase function. Variations in the side chain size and charge of this residue did not inhibit the specific activity of the H,K-ATPase, but reversal of the side chain charge by substitution of Lys or Arg for Glu produced a reciprocal change in the sensitivity of the H,K-ATPase to K+ and SCH 28080. The K0.5 for K+stimulated ATPase was decreased to 0.2 +/-.05 and 0.2 +/-.03 mM, respectively, in Lys-857 and Arg-857 site mutants, whereas the Ki for SCH 28080-dependent inhibition was increased to 6.5 +/- 1.4 and 5.9 +/- 1.5 microM, respectively. The H,K-ATPase kinetics were unaffected by the introduction of Ala at this site, but Leu produced a modest reciprocal effect. These data indicate that Glu-857 is not an essential residue for cation-dependent activity but that the residue influences the kinetics of both K+ and SCH 28080-mediated functions. This finding suggests a possible role of this residue in the conformational equilibrium of the H,K-ATPase.

  19. Structures of PHR Domains from Mus musculus Phr1 (Mycbp2) Explain the Loss-of-Function Mutation (Gly1092 → Glu) of the C. elegans Ortholog RPM-1

    Energy Technology Data Exchange (ETDEWEB)

    Sampathkumar, Parthasarathy; Ozyurt, Sinem A.; Miller, Stacy A.; Bain, Kevin T.; Rutter, Marc E.; Gheyi, Tarun; Abrams, Benjamin; Wang, Yingchun; Atwell, Shane; Luz, John G.; Thompson, Devon A.; Wasserman, Stephen R.; Emtage, J. Spencer; Park, Eun Chan; Rongo, Christopher; Jin, Yishi; Klemke, Richard L.; Sauder, J. Michael; Burley, Stephen K. (Rutgers); (UCSC); (Lilly); (UCSD)

    2010-11-15

    PHR [PAM (protein associated with Myc)-HIW (Highwire)-RPM-1 (regulator of presynaptic morphology 1)] proteins are conserved, large multi-domain E3 ubiquitin ligases with modular architecture. PHR proteins presynaptically control synaptic growth and axon guidance and postsynaptically regulate endocytosis of glutamate receptors. Dysfunction of neuronal ubiquitin-mediated proteasomal degradation is implicated in various neurodegenerative diseases. PHR proteins are characterized by the presence of two PHR domains near the N-terminus, which are essential for proper localization and function. Structures of both the first and second PHR domains of Mus musculus (mouse) Phr1 (MYC binding protein 2, Mycbp2) have been determined, revealing a novel {beta} sandwich fold composed of 11 antiparallel {beta}-strands. Conserved loops decorate the apical side of the first PHR domain (MmPHR1), yielding a distinct conserved surface feature. The surface of the second PHR domain (MmPHR2), in contrast, lacks significant conservation. Importantly, the structure of MmPHR1 provides insights into a loss-of-function mutation, Gly1092 {yields} Glu, observed in the Caenorhabditis elegans ortholog RPM-1.

  20. Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domain II and III in the specificity towards Spodoptera exigua larvae

    NARCIS (Netherlands)

    Herrero Sendra, S.; González-Cabrera, J.; Ferré, J.; Bakker, P.L.; Maagd, de R.A.

    2004-01-01

    Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541-544 in domain III (mutant pPB20). Addition

  1. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

      The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded H+-ATPases extrude protons from cells...... of plants and fungi to generate electrochemical proton gradients. A recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Together with biochemical and structural data presented in this thesis we are now able...... to describe the basic molecular components that allow the plasma membrane proton H+-ATPase to carry out proton transport against large membrane potentials. Moreover, a completely new paradigm for post-translational activation of these proteins is presented. The talk will focus on the following themes...

  2. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

      The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded H+-ATPases extrude protons from cells...... of plants and fungi to generate electrochemical proton gradients. A recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Together with biochemical and structural data presented in this thesis we are now able...... to describe the basic molecular components that allow the plasma membrane proton H+-ATPase to carry out proton transport against large membrane potentials. Moreover, a completely new paradigm for post-translational activation of these proteins is presented. The talk will focus on the following themes...

  3. Relationship between intracellular Na+ concentration and reduced Na+ affinity in Na+,K+-ATPase mutants causing neurological disease

    DEFF Research Database (Denmark)

    Toustrup-Jensen, Mads Schak; Einholm, Anja P.; Schack, Vivien;

    2014-01-01

    The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na+,K+-ATPase α2- and α3-isoforms, expressed in glial and neuronal cells, respectively. Although these disorders...... are distinct, they overlap in phenotypical presentation. Two Na+,K+-ATPase mutations, extending the C-terminus by either 28 residues ("+28" mutation) or an extra tyrosine ("+Y"), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease......, addressing the question to what extent they cause a change of the intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na+ affinity without disturbance of K+ binding, as did other RDP mutants. No phosphorylation from ATP...

  4. New founding mutation in MSH2 associated with hereditary nonpolyposis colorectal cancer syndrome on the Island of Tenerife.

    Science.gov (United States)

    Medina-Arana, Vicente; Barrios, Ysamar; Fernández-Peralta, Antonia; Herrera, Mercedes; Chinea, Nancy; Lorenzo, Nieves; Jiménez, Alejandro; Martín-López, Juana Victoria; González-Hermoso, Fernando; Salido, Eduardo; González-Aguilera, Juan J

    2006-12-01

    Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC) is a hereditary syndrome with genetic heterogeneity. The disease is caused by mutations or epigenetic silencing in DNA mismatch repair genes, MLH1, MSH2, MSH6, PMS2 and MLH3, although the vast majority of cases correspond to mutations of MLH1 and MSH2. We herein describe a nucleotide change, c.2063T>G in exon 13 of the MSH2 gene, present in families that fulfill the Amsterdam criteria for Lynch syndrome and originate from northern Tenerife (Canary Islands-Spain). This mutation is expected to result in a nonconservative amino acid change, M688R, at the ATPase domain of the MSH2 protein. We found five large families with this mutation, and about half the individuals heterozygous for M688R developed malignancies by the sixth decade of life. In many cases analyzed, their tumors revealed loss of the normal allele, being homozygous for M688R. There is an evidence of historical isolation for the population studied, which could have favored a considerable genetic drift. The presence of the same mutation and the disease associated-haplotype conservation in families not directly related can be probably the consequence of a bottleneck in the founding of this population (rather than a relatively recent founding of the mutation).

  5. Chaperone ligand-discrimination by the TPR-domain protein Tah1.

    Science.gov (United States)

    Millson, Stefan H; Vaughan, Cara K; Zhai, Chao; Ali, Maruf M U; Panaretou, Barry; Piper, Peter W; Pearl, Laurence H; Prodromou, Chrisostomos

    2008-07-15

    Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90alpha and Hsp90beta proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

  6. Non-syndromic tooth agenesis in two Chinese families associated with novel missense mutations in the TNF domain of EDA (ectodysplasin A.

    Directory of Open Access Journals (Sweden)

    Shufeng Li

    Full Text Available Here we report two unrelated Chinese families with congenital missing teeth inherited in an X-linked manner. We mapped the affected locus to chromosome Xp11-Xq21 in one family. In the defined region, both families were found to have novel missense mutations in the ectodysplasin-A (EDA gene. The mutation of c.947A>G caused the D316G substitution of the EDA protein. The mutation of c.1013C>T found in the other family resulted in the Thr to Met mutation at position 338 of EDA. The EDA gene has been reported responsible for X-linked hypohidrotic ectodermal dysplasia (XLHED in humans characterized by impaired development of hair, eccrine sweat glands, and teeth. In contrast, all the affected individuals in the two families that we studied here had normal hair and skin. Structural analysis suggests that these two novel mutants may account for the milder phenotype by affecting the stability of EDA trimers. Our results indicate that these novel missense mutations in EDA are associated with the isolated tooth agenesis and provide preliminary explanation for the abnormal clinical phenotype at a molecular structural level.

  7. Multi-site TBT binding skews the inhibition of oligomycin on the mitochondrial Mg-ATPase in Mytilus galloprovincialis.

    Science.gov (United States)

    Nesci, Salvatore; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Pagliarani, Alessandra

    2011-07-01

    Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F(1)F(0)-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 μM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F(0), namely oligomycin and N,N'-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F(1), maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 μM TBT concentrations, a structural detachment of the two F(1) and F(0) domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 μM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F(0) proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F(1)F(0)-ATPase oligomycin sensitivity loss.

  8. The intein of the Thermoplasma A-ATPase A subunit: Structure, evolution and expression in E. coli

    Directory of Open Access Journals (Sweden)

    Gogarten J Peter

    2001-11-01

    Full Text Available Abstract Background Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing, and religate the host protein with a peptide bond. In addition to this splicing activity, most reported inteins also contain an endonuclease domain that is important in intein propagation. Results The gene encoding the Thermoplasma acidophilum A-ATPase catalytic subunit A is the only one in the entire T. acidophilum genome that has been identified to contain an intein. This intein is inserted in the same position as the inteins found in the ATPase A-subunits encoding gene in Pyrococcus abyssi, P. furiosus and P. horikoshii and is found 20 amino acids upstream of the intein in the homologous vma-1 gene in Saccharomyces cerevisiae. In contrast to the other inteins in catalytic ATPase subunits, the T. acidophilum intein does not contain an endonuclease domain. T. acidophilum has different codon usage frequencies as compared to Escherichia coli. Initially, the low abundance of rare tRNAs prevented expression of the T. acidophilum A-ATPase A subunit in E. coli. Using a strain of E. coli that expresses additional tRNAs for rare codons, the T. acidophilum A-ATPase A subunit was successfully expressed in E. coli. Conclusions Despite differences in pH and temperature between the E. coli and the T. acidophilum cytoplasms, the T. acidophilum intein retains efficient self-splicing activity when expressed in E. coli. The small intein in the Thermoplasma A-ATPase is closely related to the endonuclease containing intein in the Pyrococcus A-ATPase. Phylogenetic analyses suggest that this intein was horizontally transferred between Pyrococcus and Thermoplasma, and that the small intein has persisted in Thermoplasma apparently without homing.

  9. Regulation of the V-ATPase along the endocytic pathway occurs through reversible subunit association and membrane localization.

    Directory of Open Access Journals (Sweden)

    Céline Lafourcade

    Full Text Available The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase, a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V(0 and the cytoplasmic V(1. Here we found that the ratio of membrane associated V(1/Vo varies along the endocytic pathway, the relative abundance of V(1 being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments.

  10. Expression of genes encoding F-1-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis

    DEFF Research Database (Denmark)

    Købmann, Brian Jensen; Solem, Christian; Pedersen, M.B.

    2002-01-01

    We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F-1 domain of the membrane-bound (F1F0) H+-ATPase were expressed from a range of synthetic constitutive promoters. Expression...

  11. The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+-binding but not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor

    DEFF Research Database (Denmark)

    Søndergaard, Mads Toft; Liu, Yingjie; Larsen, Kamilla Taunsig

    2017-01-01

    (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+-binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start methionine), which markedly reduces CaM Ca2+-binding...... to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+-release by manipulating the CaM-RyR2 interaction....

  12. Protein tyrosine phosphatase SHP2/PTPN11 mistargeting as a consequence of SH2-domain point mutations associated with Noonan Syndrome and leukemia

    DEFF Research Database (Denmark)

    Müller, Pia J; Rigbolt, Kristoffer T G; Paterok, Dirk

    2013-01-01

    SHP2/PTPN11 is a key regulator of cytokine, growth factor and integrin signaling. SHP2 influences cell survival, proliferation and differentiation by regulating major signaling pathways. Mutations in PTPN11 cause severe diseases like Noonan, LEOPARD syndrome or leukemia. Whereas several of these ......SHP2/PTPN11 is a key regulator of cytokine, growth factor and integrin signaling. SHP2 influences cell survival, proliferation and differentiation by regulating major signaling pathways. Mutations in PTPN11 cause severe diseases like Noonan, LEOPARD syndrome or leukemia. Whereas several...

  13. Roles of transmembrane segment M1 of Na(+),K (+)-ATPase and Ca (2+)-ATPase, the gatekeeper and the pivot

    DEFF Research Database (Denmark)

    Einholm, Anja P.; Andersen, Jens Peter; Vilsen, Bente

    2007-01-01

    In this review we summarize mutagenesis work on the structure-function relationship of transmembrane segment M1 in the Na(+),K(+)-ATPase and the sarco(endo)plasmic reticulum Ca(2+)-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported...... cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca(2+) interaction/occlusion in Ca(2+)-ATPase and K(+) interaction/occlusion in Na(+),K(+)-ATPase. Leu(65) of the Ca(2+)-ATPase and Leu(99) of the Na(+),K(+)-ATPase, located...... of the extracytoplasmic gate in both the Ca(2+)-ATPase and the Na(+),K(+)-ATPase. Udgivelsesdato: 2007-Dec...

  14. Transmission of stability information through the N-domain of tropomyosin is interrupted by a stabilizing mutation (A109L) in the hydrophobic core of the stability control region (residues 97-118).

    Science.gov (United States)

    Kirwan, J Paul; Hodges, Robert S

    2014-02-14

    Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97-118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1-131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1-131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.

  15. Transmission of Stability Information through the N-domain of Tropomyosin Is Interrupted by a Stabilizing Mutation (A109L) in the Hydrophobic Core of the Stability Control Region (Residues 97–118)

    Science.gov (United States)

    Kirwan, J. Paul; Hodges, Robert S.

    2014-01-01

    Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97–118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1–131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1–131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule. PMID:24362038

  16. Dynamically-driven enhancement of the catalytic machinery of the SARS 3C-like protease by the S284-T285-I286/A mutations on the extra domain.

    Directory of Open Access Journals (Sweden)

    Liangzhong Lim

    Full Text Available Previously we revealed that the extra domain of SARS 3CLpro mediated the catalysis via different mechanisms. While the R298A mutation completely abolished the dimerization, thus resulting in the inactive catalytic machinery, N214A inactivated the enzyme by altering its dynamics without significantly perturbing its structure. Here we studied another mutant with S284-T285-I286 replaced by Ala (STI/A with a 3.6-fold activity increase and slightly enhanced dimerization. We determined its crystal structure, which still adopts the dimeric structure almost identical to that of the wild-type (WT, except for slightly tighter packing between two extra-domains. We then conducted 100-ns molecular dynamics (MD simulations for both STI/A and WT, the longest reported so far for 3CLpro. In the simulations, two STI/A extra domains become further tightly packed, leading to a significant volume reduction of the nano-channel formed by residues from both catalytic and extra domains. The enhanced packing appears to slightly increase the dynamic stability of the N-finger and the first helix residues, which subsequently triggers the redistribution of dynamics over residues directly contacting them. This ultimately enhances the dynamical stability of the residues constituting the catalytic dyad and substrate-binding pockets. Further correlation analysis reveals that a global network of the correlated motions exists in the protease, whose components include all residues identified so far to be critical for the dimerization and catalysis. Most strikingly, the N214A mutation globally decouples this network while the STI/A mutation alters the correlation pattern. Together with previous results, the present study establishes that besides the classic structural allostery, the dynamic allostery also operates in the SARS 3CLpro, which is surprisingly able to relay the perturbations on the extra domain onto the catalytic machinery to manifest opposite catalytic effects. Our

  17. Dynamically-driven enhancement of the catalytic machinery of the SARS 3C-like protease by the S284-T285-I286/A mutations on the extra domain.

    Science.gov (United States)

    Lim, Liangzhong; Shi, Jiahai; Mu, Yuguang; Song, Jianxing

    2014-01-01

    Previously we revealed that the extra domain of SARS 3CLpro mediated the catalysis via different mechanisms. While the R298A mutation completely abolished the dimerization, thus resulting in the inactive catalytic machinery, N214A inactivated the enzyme by altering its dynamics without significantly perturbing its structure. Here we studied another mutant with S284-T285-I286 replaced by Ala (STI/A) with a 3.6-fold activity increase and slightly enhanced dimerization. We determined its crystal structure, which still adopts the dimeric structure almost identical to that of the wild-type (WT), except for slightly tighter packing between two extra-domains. We then conducted 100-ns molecular dynamics (MD) simulations for both STI/A and WT, the longest reported so far for 3CLpro. In the simulations, two STI/A extra domains become further tightly packed, leading to a significant volume reduction of the nano-channel formed by residues from both catalytic and extra domains. The enhanced packing appears to slightly increase the dynamic stability of the N-finger and the first helix residues, which subsequently triggers the redistribution of dynamics over residues directly contacting them. This ultimately enhances the dynamical stability of the residues constituting the catalytic dyad and substrate-binding pockets. Further correlation analysis reveals that a global network of the correlated motions exists in the protease, whose components include all residues identified so far to be critical for the dimerization and catalysis. Most strikingly, the N214A mutation globally decouples this network while the STI/A mutation alters the correlation pattern. Together with previous results, the present study establishes that besides the classic structural allostery, the dynamic allostery also operates in the SARS 3CLpro, which is surprisingly able to relay the perturbations on the extra domain onto the catalytic machinery to manifest opposite catalytic effects. Our results thus imply a

  18. Zinc Finger Domain of the PRDM9 Gene on Chromosome 1 Exhibits High Diversity in Ruminants but Its Paralog PRDM7 Contains Multiple Disruptive Mutations

    National Research Council Canada - National Science Library

    Ahlawat, Sonika; Sharma, Priyanka; Sharma, Rekha; Arora, Reena; De, Sachinandan

    2016-01-01

    ...) and found 43 different ZF domain sequences. Ruminant zinc fingers of PRDM9 were found to be diversifying under positive selection and concerted evolution, specifically at positions involved in defining their DNA-binding specificity, consistent...

  19. Functional analysis of the GAF domain of NifA in Azospirillum brasilense: effects of Tyr-->Phe mutations on NifA and its interaction with GlnB.

    Science.gov (United States)

    Chen, Sanfeng; Liu, Li; Zhou, Xiaoyu; Elmerich, Claudine; Li, Ji-Lun

    2005-06-01

    Regulation of NifA activity in Azospirillum brasilense depends on GlnB (a PII protein), and it was previously reported that the target of GlnB activity is the N-terminal domain of NifA. Furthermore, mutation of the Tyr residue at position 18 in the N-terminal domain resulted in a NifA protein that did not require GlnB for activity under nitrogen fixation conditions. We report here that a NifA double mutant in which the Tyr residues at positions 18 and 53 of NifA N-were simultaneously replaced by Phe (NifA-Y1853F) displays high nitrogenase activity, which is still regulatable by ammonia, but not by GlnB. The yeast two-hybrid technique was used to investigate whether GlnB can physically interact with wild-type and mutant NifA proteins. GlnB was found to interact directly with the N-terminal GAF domain of wild-type NifA, but not with its central or C-terminal domain. GlnB could still bind to the single NifA mutants Y18F and Y53F. In contrast, no interaction was detected between GlnB and the double mutant NifA-Y18/53F or between GlnB and NifA-Y43.

  20. Mutations in different functional domains of the human muscle acetylcholine receptor alpha subunit in patients with the slow-channel congenital myasthenic syndrome

    NARCIS (Netherlands)

    Croxen, R; Newland, C; Beeson, D; Oosterhuis, H; Chauplannaz, G; Vincent, A; NewsomDavis, J

    1997-01-01

    Congenital myasthenic syndromes are a group of rare genetic disorders that compromise neuromuscular transmission. A subset of these disorders, the slow-channel congenital myasthenic syndrome (SCCMS), is dominantly inherited and has been shown to involve mutations within the muscle acetylcholine rece

  1. Mutations in the TGF beta Binding-Protein-Like Domain 5 of FBN1 Are Responsible for Acromicric and Geleophysic Dysplasias

    NARCIS (Netherlands)

    Le Goff, Carine; Mahaut, Clementine; Wang, Lauren W.; Allali, Slimane; Abhyankar, Avinash; Jensen, Sacha; Zylberberg, Louise; Collod-Beroud, Gwenaelle; Bonnet, Damien; Alanay, Yasemin; Brady, Angela. F.; Cordier, Marie-Pierre; Devriendt, Koen; Genevieve, David; Kiper, Pelin Ozlem Simsek; Kitoh, Hiroshi; Krakow, Deborah; Lynch, Sally Ann; Le Merrer, Martine; Megarbane, Andre; Mortier, Geert; Odent, Sylvie; Polak, Michel; Rohrbach, Marianne; Sillence, David; Stolte-Dijkstra, Irene; Superti-Furga, Andrea; Rimoin, David L.; Topouchian, Vicken; Unger, Sheila; Zabel, Bernhard; Bole-Feysot, Christine; Nitschke, Patrick; Handford, Penny; Casanova, Jean-Laurent; Boileau, Catherine; Apte, Suneel S.; Munnich, Arnold; Cormier-Dairel, Valerie

    2011-01-01

    Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although All has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. Af

  2. Drosophila model of Meier-Gorlin syndrome based on the mutation in a conserved C-Terminal domain of Orc6.

    Science.gov (United States)

    Balasov, Maxim; Akhmetova, Katarina; Chesnokov, Igor

    2015-11-01

    Meier-Gorlin syndrome (MGS) is an autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears, and skeletal abnormalities. Patients with MGS often carry mutations in the genes encoding the components of the pre-replicative complex such as Origin Recognition Complex (ORC) subunits Orc1, Orc4, Orc6, and helicase loaders Cdt1 and Cdc6. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. Mutation in conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. In order to study the effects of MGS mutation in an animal model system we introduced MGS mutation in Orc6 and established Drosophila model of MGS. Mutant flies die at third instar larval stage with abnormal chromosomes and DNA replication defects. The lethality can be rescued by elevated expression of mutant Orc6 protein. Rescued MGS flies are unable to fly and display multiple planar cell polarity defects. © 2015 Wiley Periodicals, Inc.

  3. Structure-function relationships in the Na,K-ATPase. cap alpha. subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Price, E.M.; Lingrel, J.B.

    1988-11-01

    Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the ..cap alpha..1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat ..cap alpha..1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase ..cap alpha.. subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep ..cap alpha..1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep ..cap alpha..1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep ..cap alpha..1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat ..cap alpha..1 cDNA, the rat/sheep chimera, or the mutant sheep ..cap alpha..1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, /sup 86/Rb/sup +/ uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase ..cap alpha.. subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep ..cap alpha..1 subunit (glutamine and asparagine) are somehow involved in ouabain binding.

  4. Properties of F1-ATPase from the uncD412 mutant of Escherichia coli.

    Science.gov (United States)

    Wise, J G; Duncan, T M; Latchney, L R; Cox, D N; Senior, A E

    1983-11-01

    Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5'-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.

  5. Bacteriophage lambda terminase: alterations of the high-affinity ATPase affect viral DNA packaging.

    Science.gov (United States)

    Dhar, Alok; Feiss, Michael

    2005-03-18

    DNA packaging by large DNA viruses such as the tailed bacteriophages and the herpesviruses involves DNA translocation into a preformed protein shell, called the prohead. Translocation is driven by an ATP hydrolysis-powered DNA packaging motor. The bacteriophages encode a heterodimeric viral DNA packaging protein, called terminase. The terminases have an ATPase center located in the N terminus of the large subunit implicated in DNA translocation. In previous work with phage lambda, lethal mutations that changed ATP-reactive residues 46 and 84 of gpA, the large terminase subunit, were studied. These mutant enzymes retained the terminase endonuclease and helicase activities, but had severe defects in virion assembly, and lacked the terminase high-affinity ATPase activity. Surprisingly, in the work described here, we found that enzymes with the conservative gpA changes Y46F and Y46A had only mild packaging defects. These mild defects contrast with their profound virion assembly defects. Thus, these mutant enzymes have, in addition to the mild DNA packaging defects, a severe post-DNA packaging defect. In contrast, the gpA K84A enzyme had similar virion assembly and DNA packaging defects. The DNA packaging energy budget, i.e. DNA packaged/ATP hydrolyzed, was unchanged for the mutant enzymes, indicating that DNA translocation is tightly coupled to ATP hydrolysis. A model is proposed in which gpA residues 46 and 84 are important for terminase's high-affinity ATPase activity. Assembly of the translocation complex remodels this ATPase so that residues 46 and 84 are not crucial for the activated translocation ATPase. Changing gpA residues 46 and 84 primarily affects assembly, rather than the activity, of the translocation complex.

  6. Structure of a catalytic dimer of the α- and β-subunits of the F-ATPase from Paracoccus denitrificans at 2.3 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Morales-Ríos, Edgar; Montgomery, Martin G. [The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY (United Kingdom); Leslie, Andrew G. W. [The Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH (United Kingdom); García-Trejo, José J. [Universidad Nacional Autónoma de México, Mexico City (Mexico); Walker, John E., E-mail: walker@mrc-mbu.cam.ac.uk [The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY (United Kingdom)

    2015-09-23

    The structure of the αβ heterodimer of the F-ATPase from the α-proteobacterium P. denitrificans has been determined at 2.3 Å resolution. It corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases. The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F{sub 1} domain of the enzyme. This domain is a complex of three α-subunits and three β-subunits, and one copy of each of the γ-, δ- and ∊-subunits. Attempts to crystallize the F{sub 1}–ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and β-subunits only. Its structure was determined to 2.3 Å resolution and consists of a heterodimer of one α-subunit and one β-subunit. It has no bound nucleotides, and it corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized.

  7. Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Lee

    2015-09-01

    Full Text Available Presenilin 1 (PS1 deletion or Alzheimer’s disease (AD-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

  8. Presenilin 1 Maintains Lysosomal Ca(2+) Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification.

    Science.gov (United States)

    Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M; Haslett, Luke J; Kumar, Asok; Sato, Yutaka; Lie, Pearl P Y; Mohan, Panaiyur; Coffey, Erin E; Kompella, Uday; Mitchell, Claire H; Lloyd-Evans, Emyr; Nixon, Ralph A

    2015-09-01

    Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

  9. Effects of calcium binding and the hypertrophic cardiomyopathy A8V mutation on the dynamic equilibrium between closed and open conformations of the regulatory N-domain of isolated cardiac troponin C.

    Science.gov (United States)

    Cordina, Nicole M; Liew, Chu K; Gell, David A; Fajer, Piotr G; Mackay, Joel P; Brown, Louise J

    2013-03-19

    Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca(2+) binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca(2+) switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states.

  10. Obstacle Effects on One-Dimensional Translocation of ATPase

    Institute of Scientific and Technical Information of China (English)

    WANG Xian-Ju; AI Bao-Quan; LIU Liang-Gang

    2002-01-01

    We apply a general random walk model to the study of the ATPase's one-dimensional translocation along obstacle biological environment, and show the effects of random obstacles on the ATPase translocation along single stranded DNA. We find that the obstacle environment can reduce the lifetime of ATPase lattice-bound state which results in the inhibition of ATPase activity. We also carry out the ranges of rate constant of ATPase unidirectonal translocation and bidirectional translocation. Our results are consistent with the experiments and relevant theoretical consideration, and can be used to explain some physiological phenomena.

  11. Mutations at positions 547-553 of rat glucocorticoid receptors reveal that hsp90 binding requires the presence, but not defined composition, of a seven-amino acid sequence at the amino terminus of the ligand binding domain.

    Science.gov (United States)

    Kaul, Sunil; Murphy, Patrick J M; Chen, Jun; Brown, Lloyd; Pratt, William B; Simons, S Stoney

    2002-09-27

    Glucocorticoid receptors (GRs) must heterocomplex with hsp90 to have an open steroid binding cleft that can be accessed by steroid. We reported that a seven-amino acid sequence (547-553 of rat GR) overlapping the amino-terminal end of the ligand binding domain is required for hsp90 binding to GR. We have now conducted saturation mutagenesis of this sequence, which appears to be part of the surface where the ligand binding cleft merges with the surface of the ligand binding domain. No single point mutation causes significant changes in any of a variety of biochemical and biological properties in addition to hsp90 binding. A triple mutation (P548A/T549A/V551A) increases by >100-fold the steroid concentration required for half-maximal induction without affecting the level of maximal induction or coactivator response. Interestingly, this triple mutant displays reduced binding of steroid and hsp90 in whole cells, but it possesses wild type affinity for steroid and normal hsp90 binding capacity under cell-free conditions. This phenotype of a dramatic shift in the dose response for transactivation would be expected from an increase in the rate of disassembly of the triple mutant GR.hsp90 heterocomplex in the cell. Mutation of the entire seven-amino acid region to CAAAAAC maintains the presence of a critical alpha-helical structure and heterocomplex formation with hsp90 but eliminates steroid binding and transcriptional activation, thus disconnecting hsp90 binding from opening of the ligand binding cleft and steroid binding.

  12. Toxin-resistant isoforms of Na+/K+-ATPase in snakes do not closely track dietary specialization on toads.

    Science.gov (United States)

    Mohammadi, Shabnam; Gompert, Zachariah; Gonzalez, Jonathan; Takeuchi, Hirohiko; Mori, Akira; Savitzky, Alan H

    2016-11-16

    Toads are chemically defended by bufadienolides, a class of cardiotonic steroids that exert toxic effects by binding to and disabling the Na(+)/K(+)-ATPases of cell membranes. Some predators, including a number of snakes, have evolved resistance to the toxic effects of bufadienolides and prey regularly on toads. Resistance in snakes to the acute effects of these toxins is conferred by at least two amino acid substitutions in the cardiotonic steroid binding pocket of the Na(+)/K(+)-ATPase. We surveyed 100 species of snakes from a broad phylogenetic range for the presence or absence of resistance-conferring mutations. We found that such mutations occur in a much wider range of taxa than previously believed. Although all sequenced species known to consume toads exhibited the resistance mutations, many of the species possessing the mutations do not feed on toads, much less specialize on that food source. This suggests that either there is little performance cost associated with these mutations or they provide an unknown benefit. Furthermore, the distribution of the mutation among major clades of advanced snakes suggests that the origin of the mutation reflects evolutionary retention more than dietary constraint.

  13. BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia.

    Science.gov (United States)

    Zabriskie, Matthew S; Eide, Christopher A; Tantravahi, Srinivas K; Vellore, Nadeem A; Estrada, Johanna; Nicolini, Franck E; Khoury, Hanna J; Larson, Richard A; Konopleva, Marina; Cortes, Jorge E; Kantarjian, Hagop; Jabbour, Elias J; Kornblau, Steven M; Lipton, Jeffrey H; Rea, Delphine; Stenke, Leif; Barbany, Gisela; Lange, Thoralf; Hernández-Boluda, Juan-Carlos; Ossenkoppele, Gert J; Press, Richard D; Chuah, Charles; Goldberg, Stuart L; Wetzler, Meir; Mahon, Francois-Xavier; Etienne, Gabriel; Baccarani, Michele; Soverini, Simona; Rosti, Gianantonio; Rousselot, Philippe; Friedman, Ran; Deininger, Marie; Reynolds, Kimberly R; Heaton, William L; Eiring, Anna M; Pomicter, Anthony D; Khorashad, Jamshid S; Kelley, Todd W; Baron, Riccardo; Druker, Brian J; Deininger, Michael W; O'Hare, Thomas

    2014-09-08

    Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph(+) leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome.

  14. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Roehrig, John T., E-mail: jtr1@cdc.gov [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Butrapet, Siritorn; Liss, Nathan M. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Bennett, Susan L. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  15. [Effect of mutations and modifications of amino acid residues on zinc-induced interaction of the metal-binding domain of β-amyloid with DNA].

    Science.gov (United States)

    Khmeleva, S A; Mezentsev, Y V; Kozin, S A; Mitkevich, V A; Medvedev, A E; Ivanov, A S; Bodoev, N V; Makarov, A A; Radko, S P

    2015-01-01

    Interaction of intranuclear β-amyloid with DNA is considered to be a plausible mechanism of Alzheimer's disease pathogenesis. The interaction of single- and double-stranded DNA with synthetic peptides was analyzed using surface plasmon resonance. The peptides represent the metal-binding domain of β-amyloid (amino acids 1-16) and its variants with chemical modifications and point substitutions of amino acid residues which are associated with enhanced neurotoxicity of β-amyloid in cell tests. It has been shown that the presence of zinc ions is necessary for the interaction of the peptides with DNA in solution. H6R substitution has remarkably reduced the ability of domain 1-16 to bind DNA. This is in accordance with the supposition that the coordination of a zinc ion by amino acid residues His6, Glu11, His13, and His14 of the β-amyloid metal-binding domain results in the occurrence of an anion-binding site responsible for the interaction of the domain with DNA. Zinc-induced dimerization and oligomerization of domain 1-16 associated with phosphorylation of Ser8 and the presence of unblocked amino- and carboxy-terminal groups have resulted in a decrease of peptide concentrations required for detection of the peptide-DNA interaction. The presence of multiple anion-binding sites on the dimers and oligomers is responsible for the enhancement of the peptide-DNA interaction. A substitution of the negatively charged residue Asp7 for the neutral residue Asn in close proximity to the anion-binding site of the domain 1-16 of Aβ facilitates the electrostatic interaction between this site and phosphates of a polynucleotide chain, which enhances zinc-induced binding to DNA.

  16. ENU-induced mutation in the DNA-binding domain of KLF3 reveals important roles for KLF3 in cardiovascular development and function in mice.

    Directory of Open Access Journals (Sweden)

    Lois Kelsey

    Full Text Available KLF3 is a Krüppel family zinc finger transcription factor with widespread tissue expression and no previously known role in heart development. In a screen for dominant mutations affecting cardiovascular function in N-ethyl-N-nitrosourea (ENU mutagenized mice, we identified a missense mutation in the Klf3 gene that caused aortic valvular stenosis and partially penetrant perinatal lethality in heterozygotes. All homozygotes died as embryos. In the first of three zinc fingers, a point mutation changed a highly conserved histidine at amino acid 275 to arginine (Klf3(H275R . This change impaired binding of the mutant protein to KLF3's canonical DNA binding sequence. Heterozygous Klf3(H275R mutants that died as neonates had marked biventricular cardiac hypertrophy with diminished cardiac chambers. Adult survivors exhibited hypotension, cardiac hypertrophy with enlarged cardiac chambers, and aortic valvular stenosis. A dominant negative effect on protein function was inferred by the similarity in phenotype between heterozygous Klf3(H275R mutants and homozygous Klf3 null mice. However, the existence of divergent traits suggested the involvement of additional interactions. We conclude that KLF3 plays diverse and important roles in cardiovascular development and function in mice, and that amino acid 275 is critical for normal KLF3 protein function. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans.

  17. A novel mutation within the 2B rod domain of keratin 9 in a Chinese pedigree with epidermolytic palmoplantar keratoderma combined with knuckle pads and camptodactyly.

    Science.gov (United States)

    Du, Zhen-Fang; Wei, Wei; Wang, Yi-Fan; Chen, Xiao-Ling; Chen, Chun-Yue; Liu, Wen-Ting; Lu, Jia-Jun; Mao, Lian-Gen; Xu, Chen-Ming; Fang, Hong; Zhang, Xian-Ning

    2011-01-01

    Knuckle pads and camptodactyly are overlapping symptoms associated with many genetic and environmental factors. To the best of our knowledge, all reported cases of epidermolytic palmoplantar keratoderma (EPPK) with knuckle pads have been without accompanying camptodactyly. We here report a novel KRT9 mutation-EPPK family with combined knuckle pads and camptodactyly. All the EPPK-affected individuals in this southern Chinese pedigree suffered severe diffuse palmar and plantar hyperkeratosis including hyperhidrosis and cuticle splitting: 3 females presented EPPK only, 8 adult males had notably severe knuckle pads and camptodactyly as well as EPPK, and one 6-year-old boy manifested EPPK with knuckle pads. Haplotype analysis excluded the known candidate loci for camptodactyly and/or knuckle pad-like phenotypes on chromosomes 13q12, 3q11.2-q13.12, 1q24-q25, 4p16.3 and 16q11.1-q22, while only the markers D17S1787 and D17S579 flanking KRT9 showed co-segregation with EPPK. Then a novel c.T1373C (p.L458P) mutation within the sixth exon of KRT9 was validated, and this mutation presented a more severe pathogenicity than the previously reported p.L458F. We speculated that KRT9 plays a complicated role in the genesis of EPPK with knuckle pads and camptodactyly, which needs to be further investigated.

  18. Regulated assembly of vacuolar ATPase is increased during cluster disruption-induced maturation of dendritic cells through a phosphatidylinositol 3-kinase/mTOR-dependent pathway.

    Science.gov (United States)

    Liberman, Rachel; Bond, Sarah; Shainheit, Mara G; Stadecker, Miguel J; Forgac, Michael

    2014-01-17

    The vacuolar (H(+))-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation.

  19. Mutation of the Kunitz-type proteinase inhibitor domain in the amyloid β-protein precursor abolishes its anti-thrombotic properties in vivo.

    Science.gov (United States)

    Xu, Feng; Davis, Judianne; Hoos, Michael; Van Nostrand, William E

    2017-07-01

    Kunitz proteinase inhibitor (KPI) domain-containin