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Sample records for ku80 carboxy terminus

  1. The carboxy-terminus of protein 4.1r resembles Beta-tubulin.

    PubMed

    Fant, Xavier; Merdes, Andreas

    2002-01-01

    The protein 4.1R is an isoform of a larger family of 4.1 proteins. It is known as a component of the plasma membrane skeleton, but it is also found at the centrosomes in interphase and mitosis. To investigate the properties of the carboxy terminal region of protein 4.1R, we raised antibodies against a peptide representing the last 14 amino acids of 4.1R. These antibodies crossreact with an epitope in beta-tubulin and stain the microtubule network by immunofluorescence. Furthermore, sequence comparison of the carboxy terminal 4.1R peptide sequence with tubulin reveals homology with a region at the end of helix 5 in beta-tubulin, but not alpha-tubulin. A potential function of the 4.1R carboxy terminus in regulating the formation of microtubule networks is discussed. PMID:11991667

  2. Site-Specific Orthogonal Labeling of the Carboxy Terminus of α-Tubulin

    PubMed Central

    Banerjee, Abhijit; Panosian, Timothy D.; Mukherjee, Kamalika; Ravindra, Rudravajhala; Gal, Susannah; Sackett, Dan L.; Bane, Susan

    2010-01-01

    A fluorescent probe has been attached to the carboxy terminus of the α-subunit of α,β-tubulin by an enzymatic reaction followed by a chemical reaction. The unnatural amino acid 3-formyltyrosine is attached to the carboxy terminus of α-tubulin through the use of the enzyme tubulin tyrosine ligase. The aromatic aldehyde of the unnatural amino acid serves as an orthogonal electrophile that specifically reacts with a fluorophore containing an aromatic hydrazine functional group, which in this case is 7-hydrazino-4-methyl coumarin. Conditions for covalent bond formation between the unnatural amino acid and the fluorophore are mild, allowing fluorescently labeled tubulin to retain its ability to assemble into microtubules. A key feature of the labeling reaction is that it produces a red shift in the fluorophore’s absorption and emission maxima, accompanied by an increase in its quantum yield; thus, fluorescently labeled protein can be observed in the presence of unreacted fluorophore. Both the enzymatic and coupling reaction can occur in living cells. The approach presented here should be applicable to a wide variety of in vitro systems. PMID:20545322

  3. A tag at the carboxy terminus prevents membrane integration of VDAC1 in mammalian mitochondria.

    PubMed

    Kozjak-Pavlovic, Vera; Ross, Katharina; Götz, Monika; Goosmann, Christian; Rudel, Thomas

    2010-03-19

    beta-Barrel proteins are found in the outer membranes of bacteria, chloroplasts and mitochondria. The evolutionary conserved sorting and assembly machinery (SAM complex) assembles mitochondrial beta-barrel proteins, such as voltage-dependent anion-selective channel 1 (VDAC1), into complexes in the outer membrane by recognizing a sorting beta-signal in the carboxy-terminal part of the protein. Here we show that in mammalian mitochondria, masking of the C-terminus of beta-barrel proteins by a tag leads to accumulation of soluble misassembled protein in the intermembrane space, which causes mitochondrial fragmentation and loss of membrane potential. A similar phenotype is observed if the beta-signal is shortened, removed or when the conserved hydrophobic residues in the beta-signal are mutated. The length of the tag at the C-terminus is critical for the assembly of VDAC1, as well as the amino acid residues at positions 130, 222, 225 and 251 of the protein. We propose that if the recognition of the beta-signal or the folding of the beta-barrel proteins is inhibited, the nonassembled protein will accumulate in the intermembrane space, aggregate and damage mitochondria. This effect offers easy tools for studying the requirements for the membrane assembly of beta-barrel proteins, but also advises caution when interpreting the outcome of the beta-barrel protein overexpression experiments. PMID:20117113

  4. Carboxy terminus heat shock protein 70 interacting protein reduces tau-associated degenerative changes.

    PubMed

    Saidi, Laiq-Jan; Polydoro, Manuela; Kay, Kevin R; Sanchez, Laura; Mandelkow, Eva-Maria; Hyman, Bradley T; Spires-Jones, Tara L

    2015-01-01

    One of the hallmarks of Alzheimer's disease is the formation of neurofibrillary tangles, intracellular aggregates of hyperphosphorylated, mislocalized tau protein, which are associated with neuronal loss. Changes in tau are known to impair cellular transport (including that of mitochondria) and are associated with cell death in cell culture and mouse models of tauopathy. Thus clearing pathological forms of tau from cells is a key therapeutic strategy. One critical modulator in the degradation and clearance of misfolded proteins is the co-chaperone CHIP (Carboxy terminus Hsp70 interacting Protein), which is known to play a role in refolding and clearance of hyperphosphorylated tau. Here, we tested the hypothesis that CHIP could ameliorate pathological changes associated with tau. We find that co-expressing CHIP with full-length tau, tau truncated at D421 mimicking caspase cleavage, or the short tauRDΔK280 tau construct containing only the tau repeat domain with a tauopathy mutation, decreases tau protein levels in human H4 neuroglioma cells in a manner dependent on the Hsp70-binding TPR domain of CHIP. The observed reduction in tau levels by CHIP is associated with a decrease of tau phosphorylation and reduced levels of cleaved Caspase 3 indicating that CHIP plays an important role in preventing tau-induced pathological changes. Furthermore, tau-associated mitochondrial transport deficits are rescued by CHIP co-expression in H4 cells. Together, these data suggest that the co-chaperone CHIP can rescue the pathological effects of tau, and indicate that other diseases of protein misfolding and accumulation may also benefit from CHIP upregulation. PMID:25374103

  5. The HTLV-1 Tax oncoprotein represses Ku80 gene expression.

    PubMed

    Ducu, Razvan I; Dayaram, Tajhal; Marriott, Susan J

    2011-07-20

    The HTLV-I oncoprotein Tax interferes with DNA double strand break repair. Since non-homologous end joining (NHEJ) is a major pathway used to repair DNA double strand breaks we examined the effect of Tax on this pathway, with particular interest in the expression and function of Ku80, a critical component of the NHEJ pathway. Tax expression decreased Ku80 mRNA and protein levels, and repressed transcription from the Ku80 promoter. Conversely, Ku80 mRNA increased following siRNA knockdown of Tax in HTLV-I infected cells. Tax expression was associated with an elevated number of micronuclei and nucleoplasmic bridges, hallmarks of improper DNA double strand break repair. Our studies identified Tax as a transcriptional repressor of Ku80 that correlates with decreased DNA repair function. The reduction of Ku80 transcription by Tax may deplete the cell of an essential DNA break binding protein, resulting in reduced repair of DNA double strand breaks and accumulation genomic mutations. PMID:21571351

  6. The carboxy-terminus of p63 links cell cycle control and the proliferative potential of epidermal progenitor cells

    PubMed Central

    Suzuki, Daisuke; Sahu, Raju; Leu, N. Adrian; Senoo, Makoto

    2015-01-01

    The transcription factor p63 (Trp63) plays a key role in homeostasis and regeneration of the skin. The p63 gene is transcribed from dual promoters, generating TAp63 isoforms with growth suppressive functions and dominant-negative ΔNp63 isoforms with opposing properties. p63 also encodes multiple carboxy (C)-terminal variants. Although mutations of C-terminal variants have been linked to the pathogenesis of p63-associated ectodermal disorders, the physiological role of the p63 C-terminus is poorly understood. We report here that deletion of the p63 C-terminus in mice leads to ectodermal malformation and hypoplasia, accompanied by a reduced proliferative capacity of epidermal progenitor cells. Notably, unlike the p63-null condition, we find that p63 C-terminus deficiency promotes expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 (Cdkn1a), a factor associated with reduced proliferative capacity of both hematopoietic and neuronal stem cells. These data suggest that the p63 C-terminus plays a key role in the cell cycle progression required to maintain the proliferative potential of stem cells of many different lineages. Mechanistically, we show that loss of Cα, the predominant C-terminal p63 variant in epithelia, promotes the transcriptional activity of TAp63 and also impairs the dominant-negative activity of ΔNp63, thereby controlling p21Waf1/Cip1 expression. We propose that the p63 C-terminus links cell cycle control and the proliferative potential of epidermal progenitor cells via mechanisms that equilibrate TAp63 and ΔNp63 isoform function. PMID:25503409

  7. Phosphorylation at the carboxy terminus of the 55-kilodalton adenovirus type 5 E1B protein regulates transforming activity.

    PubMed Central

    Teodoro, J G; Halliday, T; Whalen, S G; Takayesu, D; Graham, F L; Branton, P E

    1994-01-01

    The 55-kDa product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation through complex formation with and inactivation of the cellular tumor suppressor p53. We have used both biochemical and genetic approaches to show that this 496-residue (496R) protein of adenovirus type 5 is phosphorylated at serine and threonine residues near the carboxy terminus within sequences characteristic of substrates of casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased viral replication and greatly reduced the efficiency of transformation of primary baby rat kidney cells. Such mutant 496R proteins interacted with p53 at efficiencies similar to those of wild-type 496R but only partially inhibited p53 transactivation activity. These results indicated that phosphorylation at these carboxy-terminal sites either regulates the inhibition of p53 or regulates some other 496R function required for cell transformation. Images PMID:8289381

  8. Carboxy-Terminus Recruitment Induced by Substrate Binding in Eukaryotic Fructose Bis-phosphate Aldolases

    SciTech Connect

    Lafrance-Vanasse,J.; Sygusch, J.

    2007-01-01

    The crystal structures of Leishmania mexicana fructose-1,6-bis(phosphate) aldolase in complex with substrate and competitive inhibitor, mannitol-1,6-bis(phosphate), were solved to 2.2 {angstrom} resolution. Crystallographic analysis revealed a Schiff base intermediate trapped in the native structure complexed with substrate while the inhibitor was trapped in a conformation mimicking the carbinolamine intermediate. Binding modes corroborated previous structures reported for rabbit muscle aldolase. Amino acid substitution of Gly-312 to Ala, adjacent to the P{sub 1}-phosphate binding site and unique to trypanosomatids, did not perturb ligand binding in the active site. Ligand attachment ordered amino acid residues 359-367 of the C-terminal region (353-373) that was disordered beyond Asp-358 in the unbound structure, revealing a novel recruitment mechanism of this region by aldolases. C-Terminal peptide ordering is triggered by P{sub 1}-phosphate binding that induces conformational changes whereby C-terminal Leu-364 contacts P{sub 1}-phosphate binding residue Arg-313. C-Terminal region capture synergizes additional interactions with subunit surface residues, not perturbed by P1-phosphate binding, and stabilizes C-terminal attachment. Amino acid residues that participate in the capturing interaction are conserved among class I aldolases, indicating a general recruitment mechanism whereby C-terminal capture facilitates active site interactions in subsequent catalytic steps. Recruitment accelerates the enzymatic reaction by using binding energy to reduce configurational entropy during catalysis thereby localizing the conserved C-terminus tyrosine, which mediates proton transfer, proximal to the active site enamine.

  9. Carboxy-terminus recruitment induced by substrate binding in eukaryotic fructose bis-phosphate aldolases.

    PubMed

    Lafrance-Vanasse, Julien; Sygusch, Jurgen

    2007-08-21

    The crystal structures of Leishmania mexicana fructose-1,6-bis(phosphate) aldolase in complex with substrate and competitive inhibitor, mannitol-1,6-bis(phosphate), were solved to 2.2 A resolution. Crystallographic analysis revealed a Schiff base intermediate trapped in the native structure complexed with substrate while the inhibitor was trapped in a conformation mimicking the carbinolamine intermediate. Binding modes corroborated previous structures reported for rabbit muscle aldolase. Amino acid substitution of Gly-312 to Ala, adjacent to the P1-phosphate binding site and unique to trypanosomatids, did not perturb ligand binding in the active site. Ligand attachment ordered amino acid residues 359-367 of the C-terminal region (353-373) that was disordered beyond Asp-358 in the unbound structure, revealing a novel recruitment mechanism of this region by aldolases. C-Terminal peptide ordering is triggered by P1-phosphate binding that induces conformational changes whereby C-terminal Leu-364 contacts P1-phosphate binding residue Arg-313. C-Terminal region capture synergizes additional interactions with subunit surface residues, not perturbed by P1-phosphate binding, and stabilizes C-terminal attachment. Amino acid residues that participate in the capturing interaction are conserved among class I aldolases, indicating a general recruitment mechanism whereby C-terminal capture facilitates active site interactions in subsequent catalytic steps. Recruitment accelerates the enzymatic reaction by using binding energy to reduce configurational entropy during catalysis thereby localizing the conserved C-terminus tyrosine, which mediates proton transfer, proximal to the active site enamine. PMID:17661446

  10. Ku80 cooperates with CBP to promote COX-2 expression and tumor growth

    PubMed Central

    Qin, Yu; Xuan, Yang; Jia, Yunlu; Hu, Wenxian; Yu, Wendan; Dai, Meng; Li, Zhenglin; Yi, Canhui; Zhao, Shilei; Li, Mei; Du, Sha; Cheng, Wei; Xiao, Xiangsheng; Chen, Yiming; Wu, Taihua; Meng, Songshu; Yuan, Yuhui; Liu, Quentin; Huang, Wenlin; Guo, Wei; Wang, Shusen; Deng, Wuguo

    2015-01-01

    Cyclooxygenase-2 (COX-2) plays an important role in lung cancer development and progression. Using streptavidin-agarose pulldown and proteomics assay, we identified and validated Ku80, a dimer of Ku participating in the repair of broken DNA double strands, as a new binding protein of the COX-2 gene promoter. Overexpression of Ku80 up-regulated COX-2 promoter activation and COX-2 expression in lung cancer cells. Silencing of Ku80 by siRNA down-regulated COX-2 expression and inhibited tumor cell growth in vitro and in a xenograft mouse model. Ku80 knockdown suppressed phosphorylation of ERK, resulting in an inactivation of the MAPK pathway. Moreover, CBP, a transcription co-activator, interacted with and acetylated Ku80 to co-regulate the activation of COX-2 promoter. Overexpression of CBP increased Ku80 acetylation, thereby promoting COX-2 expression and cell growth. Suppression of CBP by a CBP-specific inhibitor or siRNA inhibited COX-2 expression as well as tumor cell growth. Tissue microarray immunohistochemical analysis of lung adenocarcinomas revealed a strong positive correlation between levels of Ku80 and COX-2 and clinicopathologic variables. Overexpression of Ku80 was associated with poor prognosis in patients with lung cancers. We conclude that Ku80 promotes COX-2 expression and tumor growth and is a potential therapeutic target in lung cancer. PMID:25797267

  11. CD63 Interacts with the Carboxy-Terminus of the Colonic H+,K+-ATPase to Increase Plasma Membrane Localization and Rb+-Uptake

    PubMed Central

    Codina, Juan; Li, Jian; DuBose, Thomas D.

    2005-01-01

    The carboxy-terminus of the colonic H+,K+-ATPase is required for stable assembly with the β-subunit, translocation to the plasma membrane and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HKα2, we selected 84 amino acids in the carboxy-terminus of the α-subunit of mouse colonic H+,K+-ATPase (CT-HKα2) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize interaction of CT-HKα2 with CD63, recombinant CT-HKα2 and CD63 were synthesized in vitro, incubated, and complexes immunoprecipitated. CT-HKα 2 protein (but not CT-HKα1) co-precipitated with CD63, confirming stable assembly of HKα2 with CD63. In HEK-293 transfected with HKα2 plus β1-Na+,K+-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKα2/NKβ1 and 86Rb+-uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKα2/NKβ1 complex in the cell membrane. PMID:15647390

  12. Hyperthermia Induces Apoptosis of 786-O Cells through Suppressing Ku80 Expression

    PubMed Central

    Qi, Defeng; Hu, Yuan; Li, Jinhui; Peng, Tao; Su, Jialin; He, Yun; Ji, Weidong

    2015-01-01

    Hyperthermia as an anticancer method has been paid increasing attention in recent years. Several studies have shown that hyperthermia can kill tumor cells by inducing apoptosis. However, the underlying molecular mechanisms of hyperthermia-induced apoptosis are largely unknown. To investigate the effects and molecular mechanism of hyperthermia on the apoptosis in renal carcinoma 786-O cells, we firstly examined apoptosis and Ku expression in 786-O cell line treated with heat exposure (42°C for 0-4 h). The results showed that hyperthermia induced apoptosis of 786-O cells, and suppressed significantly Ku80 expression, but not Ku70 expression. Next, we knock-down Ku80 in 786-O cells, generating stable cell line 786-O-shKu80, and detected apoptosis, cell survival and cell cycle distribution. Our data showed higher apoptotic rate and lower surviving fraction in the stable cell line 786-O-shKu80 compared with those in control cells, exposed to the same heat stress (42°C for 0-4 h). Moreover, the results also showed suppression of Ku80 led to G2/M phase arrest in the stable cell line 786-O-shKu80 following heat treatment. Together, these findings indicate that Ku80 may play an important role in hyperthermia-induced apoptosis and heat-sensitivity of renal carcinoma cells through influencing the cell cycle distribution. PMID:25902193

  13. Decreased origin usage and initiation of DNA replication in haploinsufficient HCT116 Ku80+/- cells.

    PubMed

    Sibani, Sahar; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2005-08-01

    One of the functions of the abundant heterodimeric nuclear protein, Ku (Ku70/Ku80), is its involvement in the initiation of DNA replication through its ability to bind to chromosomal replication origins in a sequence-specific and cell cycle dependent manner. Here, using HCT116 Ku80+/- cells, the effect of Ku80 deficiency on cell cycle progression and origin activation was examined. Western blot analyses revealed a 75% and 36% decrease in the nuclear expression of Ku80 and Ku70, respectively. This was concomitant with a 33% and 40% decrease in chromatin binding of both proteins, respectively. Cell cycle analysis of asynchronous and late G1 synchronized Ku80+/- cells revealed a prolonged G1 phase. Furthermore, these Ku-deficient cells had a 4.5-, 3.4- and 4.3-fold decrease in nascent strand DNA abundance at the lamin B2, beta-globin and c-myc replication origins, respectively. Chromatin immunoprecipitation (ChIP) assays showed that the association of Ku80 with the lamin B2, beta-globin and c-myc origins was decreased by 1.5-, 2.3- and 2.5-fold, respectively, whereas that of Ku70 was similarly decreased (by 2.1-, 1.5- and 1.7-fold, respectively) in Ku80+/- cells. The results indicate that a deficiency of Ku80 resulted in a prolonged G1 phase, as well as decreased Ku binding to and activation of origins of DNA replication. PMID:16014376

  14. An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation

    PubMed Central

    Postow, Lisa; Funabiki, Hironori

    2013-01-01

    The Ku heterodimer, composed of Ku70 and Ku80, is the initiating factor of the nonhomologous end joining (NHEJ) double-strand break (DSB) repair pathway. Ku is also thought to impede the homologous recombination (HR) repair pathway via inhibition of DNA end resection. Using the cell-free Xenopus laevis egg extract system, we had previously discovered that Ku80 becomes polyubiquitylated upon binding to DSBs, leading to its removal from DNA and subsequent proteasomal degradation. Here we show that the Skp1-Cul1-F box (SCF) E3 ubiquitin ligase complex is required for Ku80 ubiquitylation and removal from DNA. A screen for DSB-binding F box proteins revealed that the F box protein Fbxl12 was recruited to DNA in a DSB- and Ku-sensitive manner. Immunodepletion of Fbxl12 prevented Cul1 and Skp1 binding to DSBs and Ku80 ubiquitylation, indicating that Fbxl12 is the F box protein responsible for Ku80 substrate recognition. Unlike typical F box proteins, the F box of Fbxl12 was essential for binding to both Skp1 and its substrate Ku80. Besides Fbxl12, six other chromatin-binding F box proteins were identified in our screen of a subset of Xenopus F box proteins: β-TrCP, Fbh1, Fbxl19, Fbxo24, Fbxo28 and Kdm2b. Our study unveils a novel function for the SCF ubiquitin ligase in regulating the dynamic interaction between DNA repair machineries and DSBs. PMID:23324393

  15. Enhancement of Zta-activated lytic transcription of Epstein-Barr virus by Ku80.

    PubMed

    Chen, Chien-Chang; Yang, Ya-Chun; Wang, Wen-Hung; Chen, Chien-Sin; Chang, Li-Kwan

    2011-03-01

    Zta, encoded by the BZLF1 gene of Epstein-Barr virus (EBV), is a transcription factor that is expressed during the immediate-early stage of the lytic cycle. The expression of Zta is crucial to viral lytic development. Earlier studies showed that Ku80 is a binding partner of Zta in ZKO-293 cells and is co-purified with Zta. This study verifies the interaction between Ku80 and Zta by using glutathione S-transferase-pull-down and co-immunoprecipitation assays, and also by indirect immunofluorescence analysis. This investigation also reveals that Ku80 binds to Zta on Zta-response elements in the BHLF1 promoter, enhancing the promoter activity. This study also reveals that the interaction between Zta and Ku80 involves the C-terminal region of Zta and the 425 aa N-terminal region of Ku80. The interaction between these two proteins and the enhancement of transcription that is activated by Zta suggest that Ku80 is important to EBV lytic development. PMID:21123545

  16. A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function

    PubMed Central

    Okray, Zeynep; de Esch, Celine EF; Van Esch, Hilde; Devriendt, Koen; Claeys, Annelies; Yan, Jiekun; Verbeeck, Jelle; Froyen, Guy; Willemsen, Rob; de Vrij, Femke MS; Hassan, Bassem A

    2015-01-01

    Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5′ untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes. PMID:25693964

  17. Activation of the oncogenic potential of the avian cellular src protein by specific structural alteration of the carboxy terminus.

    PubMed Central

    Reynolds, A B; Vila, J; Lansing, T J; Potts, W M; Weber, M J; Parsons, J T

    1987-01-01

    The role of tyrosine phosphorylation in the regulation of tyrosine protein kinase activity was investigated using site-directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp60c-src. Mutations that change Tyr 527 to Phe or Ser activate in vivo tyrosine protein kinase activity and induce cellular transformation of chicken cells in culture. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c-src do not induce transformation or in vivo tyrosine protein kinase activity. Amber mutations, which alter the structure of the pp60c-src C terminus by inducing premature termination of the c-src protein at either residue 518 or 523 also induce morphological transformation and increase in vivo tyrosine phosphorylation, whereas removal of the last four residues of c-src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c-src protein. We conclude from these studies that C-terminal alterations which either remove or replace Tyr 527 serve to activate the c-src protein resulting in cellular transformation and increased in vivo tyrosine protein kinase activity. Images Fig. 2. Fig. 3. Fig. 4. PMID:2822389

  18. Lethality in PARP-1/Ku80 double mutant mice reveals physiologicalsynergy during early embryogenesis

    SciTech Connect

    Henrie, Melinda S.; Kurimasa, Akihiro; Burma, Sandeep; Menissier-de Murcia, Josiane; de Murcia, Gilbert; Li, Gloria C.; Chen,David J.

    2002-09-24

    Ku is an abundant heterodimeric nuclear protein, consisting of 70-kDa and 86-kDa tightly associated subunits that comprise the DNA binding component of DNA-dependent protein kinase. Poly(ADP)ribose polymerase-1 (PARP-1) is a 113-kDa protein that catalyzes the synthesis of poly(ADP-ribose) on target proteins. Both Ku and PARP-1 recognize and bind to DNA ends. Ku functions in the non-homologous end joining (NHEJ) repair pathway whereas PARP-1 functions in the single strand break repair and base excision repair (BER) pathways. Recent studies have revealed that PARP-1 and Ku80 interact in vitro. To determine whether the association of PARP-1 and Ku80 has any physiological significance or synergistic function in vivo, mice lacking both PARP-1 and Ku80 were generated. The resulting offspring died during embryonic development displaying abnormalities around the gastrulation stage. In addition, PARP-1-/-Ku80-/- cultured blastocysts had an increased level of apoptosis. These data suggest that the functions of both Ku80 and PARP-1 are essential for normal embryogenesis and that a loss of genomic integrity leading to cell death through apoptosis is likely the cause of the embryonic lethality observed in these mice.

  19. The DNA-binding domain of the transcriptional activator protein NifA resides in its carboxy terminus, recognises the upstream activator sequences of nif promoters and can be separated from the positive control function of NifA.

    PubMed Central

    Morett, E; Cannon, W; Buck, M

    1988-01-01

    The positive control protein NifA activates transcription of nitrogen fixation promoters in Klebsiella pneumoniae. NifA is believed to bind to specific sites, the upstream activator sequences (UAS's), of the nif promoters which it activates. We have now shown by mutation of the carboxy terminus of NifA that this is the DNA-binding domain and that the DNA-binding and positive activator functions of NifA can be separated. Mutational analysis of the nifH UAS and in vivo methylation protection analysis of the interaction of NifA with the nifH promoter demonstrates that the UAS is recognised by the carboxy terminus of NifA. The UAS's of K. pneumoniae nif promoters are also required for activation by the Rhizobium meliloti NifA indicating that this activator also possesses DNA-binding activity. Images PMID:3062575

  20. Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

    PubMed Central

    Rommereim, Leah M.; Hortua Triana, Miryam A.; Falla, Alejandra; Sanders, Kiah L.; Guevara, Rebekah B.; Bzik, David J.; Fox, Barbara A.

    2013-01-01

    Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination. Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4. Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human

  1. Genetic manipulation in Δku80 strains for functional genomic analysis of Toxoplasma gondii.

    PubMed

    Rommereim, Leah M; Hortua Triana, Miryam A; Falla, Alejandra; Sanders, Kiah L; Guevara, Rebekah B; Bzik, David J; Fox, Barbara A

    2013-01-01

    Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination. Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein(1,2). The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale(1-4). Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human

  2. Overexpression of Ku80 correlates with aggressive clinicopathological features and adverse prognosis in esophageal squamous cell carcinoma

    PubMed Central

    WANG, SHUAI; WANG, ZHOU; YANG, YU; SHI, MO; SUN, ZHENGUO

    2015-01-01

    Ku80, a subunit of the heterodymeric Ku protein, is clearly implicated in nonhomologous end joining DNA repair, chemoresistance and radioresistance in malignant tumors. In the present study, the clinicopathological significance of Ku80 in esophageal squamous cell carcinoma (ESCC) was investigated. The expression levels of Ku80 were determined by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry in ESCC specimens and normal esophageal mucosa. The mRNA and protein levels of Ku80 were significantly higher in ESCC tissues than in normal esophageal mucosa, and were significantly associated with tumor differentiation, local invasion, lymph node metastasis and tumor-node-metastasis (TNM) stage. However, overexpression of Ku80 mRNA and protein levels were not significantly correlated with age, gender, tumor site or tumor size. Cox proportional hazards regression model demonstrated that tumor local invasion, lymph node metastasis, TNM stage and Ku80 mRNA and protein levels were independent risk factors indicating the overall survival of patients with ESCC. The present study demonstrated that aberrant Ku80 overexpression is observed in ESCC. In addition, high expression levels of Ku80 are associated with adverse clinicopathological features and unfavorable prognosis in ESCC patients. PMID:26722230

  3. Brain distribution of carboxy terminus of Hsc70-interacting protein (CHIP) and its nuclear translocation in cultured cortical neurons following heat stress or oxygen-glucose deprivation.

    PubMed

    Anderson, Lauren G; Meeker, Rick B; Poulton, Winona E; Huang, David Y

    2010-09-01

    Carboxy terminus of Hsc70-interacting protein (CHIP) is thought to be a cytoprotective protein with protein quality control roles in neurodegenerative diseases and myocardial ischemia. This study describes the localization of CHIP expression in normal rodent brain and the early CHIP response in primary cultures of cortical neurons following ischemic stress models: heat stress (HS) and oxygen-glucose deprivation (OGD). CHIP was highly expressed throughout the brain, predominantly in neurons. The staining pattern was primarily cytoplasmic, although small amounts were seen in the nucleus. More intense nuclear staining was observed in primary cultured neurons which increased with stress. Nuclear accumulation of CHIP occurred within 5-10 min of HS and decreased to baseline levels or lower by 30-60 min. Decrease in nuclear CHIP at 30-60 min of HS was associated with a sharp increase in delayed cell death. While no changes in cytoplasmic CHIP were observed immediately following OGD, nuclear levels of CHIP increased slightly in response to OGD durations of 30 to 240 min. OGD-induced increases in nuclear CHIP decreased slowly during post-ischemic recovery. Nuclear CHIP decreased earlier in recovery following 120 min of OGD (4 h) than 30 min of OGD (12 h). Significant cell death first appeared between 12 and 24 h after OGD, again suggesting that delayed cell death follows closely behind the disappearance of nuclear CHIP. The ability of CHIP to translocate to and accumulate in the nucleus may be a limiting variable that determines how effectively cells respond to external stressors to facilitate cell survival. Using primary neuronal cell cultures, we were able to demonstrate rapid translocation of CHIP to the nucleus within minutes of heat stress and oxygen-glucose deprivation. An inverse relationship between nuclear CHIP and delayed cell death at 24 h suggests that the decrease in nuclear CHIP following extreme stress is linked to delayed cell death. Our findings of acute

  4. Tyrosines 1021 and 1009 are phosphorylation sites in the carboxy terminus of the platelet-derived growth factor receptor beta subunit and are required for binding of phospholipase C gamma and a 64-kilodalton protein, respectively.

    PubMed Central

    Valius, M; Bazenet, C; Kazlauskas, A

    1993-01-01

    Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the

  5. The carboxy terminus of pp60c-src is a regulatory domain and is involved in complex formation with the middle-T antigen of polyomavirus.

    PubMed Central

    Cheng, S H; Piwnica-Worms, H; Harvey, R W; Roberts, T M; Smith, A E

    1988-01-01

    A large number of mutations were introduced into the carboxy-terminal domain of pp60c-src. The level of phosphorylation on Tyr-416 and Tyr-527, the transforming activity (as measured by focus formation on NIH 3T3 cells), kinase activity, and the ability of the mutant pp60c-src to associate with the middle-T antigen of polyomavirus were examined. The results indicate that Tyr-527 is a major carboxy-terminal element responsible for regulating pp60c-src in vivo. A good but not perfect correlation exists between lack of phosphorylation at Tyr-527 and increased phosphorylation at Tyr-416, between elevated phosphorylation on Tyr-416 and activated kinase activity, and between activated kinase activity and transforming activity. Phosphorylation of Tyr-527 was insensitive to the mutation of adjacent residues, indicating that the primary sequence only has a minor role in recognition by kinases or phosphatases which regulate it in vivo. Three mutants which have in common a modified Glu-524 residue were phosphorylated on Tyr-416 and Tyr-527 and were weakly transforming. This suggests that other mechanisms besides complete dephosphorylation of Tyr-527 can lead to increased phosphorylation of Tyr-416 and activation of the transforming activity of pp60c-src. Furthermore, the residues between Asp-518 and Pro-525 were required to form a stable complex with middle-T antigen. The proximity of these sequences to Tyr-527 suggests a model in which middle-T activates pp60c-src by binding directly to this region of the molecular and thereby preventing phosphorylation of Tyr-527. Alternatively, middle-T binding may mediate a conformational change in this region, which in turn induces an alteration in the level of phosphorylation at Tyr-527 and Tyr-416. Images PMID:2454396

  6. Carboxy terminus and pore-forming domain properties specific to Cx37 are necessary for Cx37-mediated suppression of insulinoma cell proliferation

    PubMed Central

    Nelson, Tasha K.; Sorgen, Paul L.

    2013-01-01

    Connexin 37 (Cx37) suppresses cell proliferation when expressed in rat insulinoma (Rin) cells, an effect also manifest in vivo during vascular development and in response to tissue injury. Mutant forms of Cx37 with nonfunctional channels but normally localized, wild-type carboxy termini are not growth suppressive. Here we determined whether the carboxy-terminal (CT) domain is required for Cx37-mediated growth suppression and whether the Cx37 pore-forming domain can be replaced with the Cx43 pore-forming domain and still retain growth-suppressive properties. We show that despite forming functional gap junction channels and hemichannels, Cx37 with residues subsequent to 273 replaced with a V5-epitope tag (Cx37–273tr*V5) had no effect on the proliferation of Rin cells, did not facilitate G1-cell cycle arrest with serum deprivation, and did not prolong cell cycle time comparably to the wild-type protein. The chimera Cx43*CT37, comprising the pore-forming domain of Cx43 and CT of Cx37, also did not suppress proliferation, despite forming functional gap junctions with a permselective profile similar to wild-type Cx37. Differences in channel behavior of both Cx37–273tr*V5 and Cx43*CT37 relative to their wild-type counterparts and failure of the Cx37-CT to interact as the Cx43-CT does with the Cx43 cytoplasmic loop suggest that the Cx37-CT and pore-forming domains are both essential to growth suppression by Cx37. PMID:24133065

  7. Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: The effects of ionizing radiation on growth, survival, and development

    PubMed Central

    Nussenzweig, André; Sokol, Karen; Burgman, Paul; Li, Ligeng; Li, Gloria C.

    1997-01-01

    We recently have shown that mice deficient for the 86-kDa component (Ku80) of the DNA-dependent protein kinase exhibit growth retardation and a profound deficiency in V(D)J (variable, diversity, and joining) recombination. These defects may be related to abnormalities in DNA metabolism that arise from the inability of Ku80 mutant cells to process DNA double-strand breaks. To further characterize the role of Ku80 in DNA double-strand break repair, we have generated embryonic stem cells and pre-B cells and examined their response to ionizing radiation. Ku80−/− embryonic stem cells are more sensitive than controls to γ-irradiation, and pre-B cells derived from Ku80 mutant mice display enhanced spontaneous and γ-ray-induced apoptosis. We then determined the effects of ionizing radiation on the survival, growth, and lymphocyte development in Ku80-deficient mice. Ku80−/− mice display a hypersensitivity to γ-irradiation, characterized by loss of hair pigmentation, severe injury to the gastrointestinal tract, and enhanced mortality. Exposure of newborn Ku80−/− mice to sublethal doses of ionizing radiation enhances their growth retardation and results in the induction of T cell-specific differentiation. However, unlike severe combined immunodeficient mice, radiation-induced T cell development in Ku80−/− mice is not accompanied by extensive thymocyte proliferation. The response of Ku80-deficient cell lines and mice to DNA-damaging agents provides important insights into the role of Ku80 in growth regulation, lymphocyte development, and DNA repair. PMID:9391070

  8. Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: the effects of ionizing radiation on growth, survival, and development.

    PubMed

    Nussenzweig, A; Sokol, K; Burgman, P; Li, L; Li, G C

    1997-12-01

    We recently have shown that mice deficient for the 86-kDa component (Ku80) of the DNA-dependent protein kinase exhibit growth retardation and a profound deficiency in V(D)J (variable, diversity, and joining) recombination. These defects may be related to abnormalities in DNA metabolism that arise from the inability of Ku80 mutant cells to process DNA double-strand breaks. To further characterize the role of Ku80 in DNA double-strand break repair, we have generated embryonic stem cells and pre-B cells and examined their response to ionizing radiation. Ku80(-/-) embryonic stem cells are more sensitive than controls to gamma-irradiation, and pre-B cells derived from Ku80 mutant mice display enhanced spontaneous and gamma-ray-induced apoptosis. We then determined the effects of ionizing radiation on the survival, growth, and lymphocyte development in Ku80-deficient mice. Ku80(-/-) mice display a hypersensitivity to gamma-irradiation, characterized by loss of hair pigmentation, severe injury to the gastrointestinal tract, and enhanced mortality. Exposure of newborn Ku80(-/-) mice to sublethal doses of ionizing radiation enhances their growth retardation and results in the induction of T cell-specific differentiation. However, unlike severe combined immunodeficient mice, radiation-induced T cell development in Ku80(-/-) mice is not accompanied by extensive thymocyte proliferation. The response of Ku80-deficient cell lines and mice to DNA-damaging agents provides important insights into the role of Ku80 in growth regulation, lymphocyte development, and DNA repair. PMID:9391070

  9. Determinants of affinity and mode of DNA binding at the carboxy terminus of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1.

    PubMed Central

    Andera, L; Geiduschek, E P

    1994-01-01

    The role of the carboxy-terminal amino acids of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1, in DNA binding was analyzed. Chain-terminating mutations truncating the normally 99-amino-acid TF1 at amino acids 96, 97, and 98 were constructed, as were missense mutations substituting cysteine, arginine, and serine for phenylalanine at amino acid 97 and tryptophan for lysine at amino acid 99. The binding of the resulting proteins to a synthetic 44-bp binding site in 5-(hydroxymethyl)uracil DNA, to binding sites in larger SPO1 [5-(hydroxymethyl)uracil-containing] DNA fragments, and to thymine-containing homologous DNA was analyzed by gel retardation and also by DNase I and hydroxy radical footprinting. We conclude that the C tail up to and including phenylalanine at amino acid 97 is essential for DNA binding and that the two C-terminal amino acids, 98 and 99, are involved in protein-protein interactions between TF1 dimers bound to DNA. Images PMID:8113176

  10. The Carboxy Terminus of the Ligand Peptide Determines the Stability of the MHC Class I Molecule H-2Kb: A Combined Molecular Dynamics and Experimental Study

    PubMed Central

    Abualrous, Esam Tolba; Saini, Sunil Kumar; Ramnarayan, Venkat Raman; Ilca, Florin Tudor; Zacharias, Martin; Springer, Sebastian

    2015-01-01

    Major histocompatibility complex (MHC) class I molecules (proteins) bind peptides of eight to ten amino acids to present them at the cell surface to cytotoxic T cells. The class I binding groove binds the peptide via hydrogen bonds with the peptide termini and via diverse interactions with the anchor residue side chains of the peptide. To elucidate which of these interactions is most important for the thermodynamic and kinetic stability of the peptide-bound state, we have combined molecular dynamics simulations and experimental approaches in an investigation of the conformational dynamics and binding parameters of a murine class I molecule (H-2Kb) with optimal and truncated natural peptide epitopes. We show that the F pocket region dominates the conformational and thermodynamic properties of the binding groove, and that therefore the binding of the C terminus of the peptide to the F pocket region plays a crucial role in bringing about the peptide-bound state of MHC class I. PMID:26270965

  11. Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection.

    PubMed

    Fox, Barbara A; Falla, Alejandra; Rommereim, Leah M; Tomita, Tadakimi; Gigley, Jason P; Mercier, Corinne; Cesbron-Delauw, Marie-France; Weiss, Louis M; Bzik, David J

    2011-09-01

    Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8(+) T cell epitopes that elicit corresponding antigen-specific CD8(+) T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8(+) T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission. PMID:21531875

  12. CHIP, a carboxy terminus HSP-70 interacting protein, prevents cell death induced by endoplasmic reticulum stress in the central nervous system

    PubMed Central

    Cabral Miranda, Felipe; Adão-Novaes, Juliana; Hauswirth, William W.; Linden, Rafael; Petrs-Silva, Hilda; Chiarini, Luciana B.

    2015-01-01

    Endoplasmic reticulum (ER) stress and protein misfolding are associated with various neurodegenerative diseases. ER stress activates unfolded protein response (UPR), an adaptative response. However, severe ER stress can induce cell death. Here we show that the E3 ubiquitin ligase and co-chaperone Carboxyl Terminus HSP70/90 Interacting Protein (CHIP) prevents neuron death in the hippocampus induced by severe ER stress. Organotypic hippocampal slice cultures (OHSCs) were exposed to Tunicamycin, a pharmacological ER stress inducer, to trigger cell death. Overexpression of CHIP was achieved with a recombinant adeno-associated viral vector (rAAV) and significantly diminished ER stress-induced cell death, as shown by analysis of propidium iodide (PI) uptake, condensed chromatin, TUNEL and cleaved caspase 3 in the CA1 region of OHSCs. In addition, overexpression of CHIP prevented upregulation of both CHOP and p53 both pro-apoptotic pathways induced by ER stress. We also detected an attenuation of eIF2a phosphorylation promoted by ER stress. However, CHIP did not prevent upregulation of BiP/GRP78 induced by UPR. These data indicate that overexpression of CHIP attenuates ER-stress death response while maintain ER stress adaptative response in the central nervous system. These results indicate a neuroprotective role for CHIP upon UPR signaling. CHIP emerge as a candidate for clinical intervention in neurodegenerative diseases associated with ER stress. PMID:25620910

  13. CHIP, a carboxy terminus HSP-70 interacting protein, prevents cell death induced by endoplasmic reticulum stress in the central nervous system.

    PubMed

    Cabral Miranda, Felipe; Adão-Novaes, Juliana; Hauswirth, William W; Linden, Rafael; Petrs-Silva, Hilda; Chiarini, Luciana B

    2014-01-01

    Endoplasmic reticulum (ER) stress and protein misfolding are associated with various neurodegenerative diseases. ER stress activates unfolded protein response (UPR), an adaptative response. However, severe ER stress can induce cell death. Here we show that the E3 ubiquitin ligase and co-chaperone Carboxyl Terminus HSP70/90 Interacting Protein (CHIP) prevents neuron death in the hippocampus induced by severe ER stress. Organotypic hippocampal slice cultures (OHSCs) were exposed to Tunicamycin, a pharmacological ER stress inducer, to trigger cell death. Overexpression of CHIP was achieved with a recombinant adeno-associated viral vector (rAAV) and significantly diminished ER stress-induced cell death, as shown by analysis of propidium iodide (PI) uptake, condensed chromatin, TUNEL and cleaved caspase 3 in the CA1 region of OHSCs. In addition, overexpression of CHIP prevented upregulation of both CHOP and p53 both pro-apoptotic pathways induced by ER stress. We also detected an attenuation of eIF2a phosphorylation promoted by ER stress. However, CHIP did not prevent upregulation of BiP/GRP78 induced by UPR. These data indicate that overexpression of CHIP attenuates ER-stress death response while maintain ER stress adaptative response in the central nervous system. These results indicate a neuroprotective role for CHIP upon UPR signaling. CHIP emerge as a candidate for clinical intervention in neurodegenerative diseases associated with ER stress. PMID:25620910

  14. Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

    PubMed Central

    Fiester, Steven E.; Nwugo, Chika C.; Penwell, William F.; Neary, John M.; Beckett, Amber C.; Arivett, Brock A.; Schmidt, Robert E.; Geiger, Sarah C.; Connerly, Pamela L.; Menke, Sharon M.; Tomaras, Andrew P.

    2015-01-01

    Acinetobacter baumannii is a Gram-negative opportunistic nosocomial pathogen that causes pneumonia and soft tissue and systemic infections. Screening of a transposon insertion library of A. baumannii ATCC 19606T resulted in the identification of the 2010 derivative, which, although capable of growing well in iron-rich media, failed to prosper under iron chelation. Genetic, molecular, and functional assays showed that 2010's iron utilization-deficient phenotype is due to an insertion within the 3′ end of secA, which results in the production of a C-terminally truncated derivative of SecA. SecA plays a critical role in protein translocation through the SecYEG membrane channel. Accordingly, the secA mutation resulted in undetectable amounts of the ferric acinetobactin outer membrane receptor protein BauA while not affecting the production of other acinetobactin membrane protein transport components, such as BauB and BauE, or the secretion of acinetobactin by 2010 cells cultured in the presence of subinhibitory concentrations of the synthetic iron chelator 2,2′-dipyridyl. Outer membrane proteins involved in nutrient transport, adherence, and biofilm formation were also reduced in 2010. The SecA truncation also increased production of 30 different proteins, including proteins involved in adaptation/tolerance responses. Although some of these protein changes could negatively affect the pathobiology of the 2010 derivative, its virulence defect is mainly due to its inability to acquire iron via the acinetobactin-mediated system. These results together indicate that although the C terminus of the A. baumannii ATCC 19606T SecA is not essential for viability, it plays a critical role in the production and translocation of different proteins and virulence. PMID:25605767

  15. KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair

    SciTech Connect

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2011-10-01

    Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear {gamma}-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response.

  16. Preclinical evaluation of 111In-DTPA-INCA-X anti-Ku70/Ku80 monoclonal antibody in prostate cancer

    PubMed Central

    Evans-Axelsson, Susan; Vilhelmsson Timmermand, Oskar; Welinder, Charlotte; Borrebaeck, Carl AK; Strand, Sven-Erik; Tran, Thuy A; Jansson, Bo; Bjartell, Anders

    2014-01-01

    The aim of this investigation was to assess the Ku70/Ku80 complex as a potential target for antibody imaging of prostate cancer. We evaluated the in vivo and ex vivo tumor targeting and biodistribution of the 111In-labeled human internalizing antibody, INCA-X (111In-DTPA-INCA-X antibody), in NMRI-nude mice bearing human PC-3, PC-3M-Lu2 or DU145 xenografts. DTPA-conjugated, non-labeled antibody was pre-administered at different time-points followed by a single intravenous injection of 111In-DTPA-INCA-X. At 48, 72 and 96 h post-injection, tissues were harvested, and the antibody distribution was determined by measuring radioactivity. Preclinical SPECT/CT imaging of mice with and without the predose was performed at 48 hours post-injection of labeled DTPA-INCA-X. Biodistribution of the labeled antibody showed enriched activity in tumor, spleen and liver. Animals pre-administered with DTPA-INCA-X showed increased tumor uptake and blood content of 111In-DTPA-INCA-X with reduced splenic and liver uptake. The in vitro and in vivo data presented show that the 111In-labeled INCA-X antibody is internalized into prostate cancer cells and by pre-administering non-labeled DTPA-INCA-X, we were able to significantly reduce the off target binding and increase the 111In-DTPA-INCA-X mAb uptake in PC-3, PC-3M-Lu2 and DU145 xenografts. The results are encouraging and identifying the Ku70/Ku80 antigen as a target is worth further investigation for functional imaging of prostate cancer. PMID:24982817

  17. Type II Toxoplasma gondii KU80 Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection ▿ †

    PubMed Central

    Fox, Barbara A.; Falla, Alejandra; Rommereim, Leah M.; Tomita, Tadakimi; Gigley, Jason P.; Mercier, Corinne; Cesbron-Delauw, Marie-France; Weiss, Louis M.; Bzik, David J.

    2011-01-01

    Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8+ T cell epitopes that elicit corresponding antigen-specific CD8+ T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8+ T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission. PMID:21531875

  18. Role of the carboxy terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: assembly requires a free carboxyl group at C-terminal position 344.

    PubMed

    Nixon, P J; Trost, J T; Diner, B A

    1992-11-10

    The D1 polypeptide of the photosystem II (PSII) reaction center is synthesized as a precursor polypeptide which is posttranslationally processed at the carboxy terminus. It has been shown in spinach that such processing removes nine amino acids, leaving Ala344 as the C-terminal residue [Takahashi, M., Shiraishi, T., & Asada, K. (1988) FEBS Lett. 240, 6-8; Takahashi, Y., Nakane, H., Kojima, H., & Satoh, K. (1990) Plant Cell Physiol. 31, 273-280]. We show here that processing on the carboxy side of Ala344 also occurs in the cyanobacterium Synechocystis 6803, resulting in the removal of 16 amino acids. By constructing a deletion strain of Synechocystis 6803 that lacks the three copies of the psbA gene encoding D1, we have developed a system for generating psbA mutants. Using this system, we have constructed mutants of Synechocystis 6803 that are modified in the region of the C-terminus of the D1 polypeptide. Characterization of these mutants has revealed that (1) processing of the D1 polypeptide is blocked when the residue after the cleavage site is changed from serine to proline (mutant Ser345Pro) with the result that the manganese cluster is unable to assemble correctly; (2) the C-terminal extension of 16 amino acid residues can be deleted with little consequence either for insertion of D1 into the thylakoid membrane or for assembly of D1 into a fully active PSII complex; (3) removal of only one more residue (mutant Ala344stop) results in a loss of assembly of the manganese cluster; and (4) the ability of detergent-solubilized PSII core complexes (lacking the manganese cluster) to bind and oxidize exogenous Mn2+ by the secondary donor, Z+, is largely unaffected in the processing mutants (the Ser345Pro mutant of Synechocystis 6803 and the LF-1 mutant of Scenedesmus obliquus) and the truncation mutant Ala344stop. Our results are consistent with a role for processing in regulating the assembly of the photosynthetic manganese cluster and a role for the free carboxy

  19. A YAC contig encompassing the XRCC5 (Ku80) DNA repair gene and complementation of defective cells by YAC protoplast fusion

    SciTech Connect

    Blunt, T.; Priestley, A.; Hafezparast, M.; McMillan, T.

    1995-11-20

    The Chinese hamster ovary xrs mutants are sensitive to ionizing radiation, defective in DNA double-strand break rejoining, and unable to carry out V(D)J recombination effectively. Recently, the gene defective in these mutants, XRCC5, has been shown to encode Ku80, a component of the Ku protein and DNA-dependent protein kinase. We present here a YAC contig involving 25 YACs mapping to the region 2q33-q34, which encompasses the XRCC5 gene. Eight new markers for this region of chromosome 2 are identified. YACs encoding the Ku80 gene were transferred to xrs cells by protoplast fusion, and complementation of all the defective phenotypes has been obtained with two YACs. We discuss the advantages and disadvantages of this approach as a strategy for cloning human genes complementing defective rodent cell lines. 44 refs., 2 figs., 4 tabs.

  20. Recombinant thyrotropin containing a beta-subunit chimera with the human chorionic gonadotropin-beta carboxy-terminus is biologically active, with a prolonged plasma half-life: role of carbohydrate in bioactivity and metabolic clearance.

    PubMed

    Joshi, L; Murata, Y; Wondisford, F E; Szkudlinski, M W; Desai, R; Weintraub, B D

    1995-09-01

    Recombinant TSH is now successfully being used in clinical studies of thyroid cancer. Because of its therapeutic potential, we have constructed a longer acting analog of TSH by fusing the carboxy-terminal extension peptide (CTEP) of hCG beta onto TSH beta. When coexpressed either with alpha-subunit complementary DNA or alpha minigene in African green monkey (COS-7) and human embryonic kidney (293) cells, the chimera was fully bioactive in vitro and exhibited enhanced in vivo potency associated with a prolonged plasma half-life. The addition of 25 amino acids with 4 O-linked oligosaccharide chains did not affect the assembly and secretion of chimeric TSH. Wild-type (WT) and chimeric TSH secreted by COS-7 and 293 cells displayed wide differences in their plasma half-lives, presumably due to the presence of terminal sialic acid and SO4 on their oligosaccharide chains, respectively. Chimeric and WT TSH secreted by both cell lines demonstrated similar bioactivity in cAMP production, with some differences in [3H]thymidine incorporation. Chimeric TSH appears to be more effective in COS-7 cells than in 293 cells, as judged by growth assay. COS-7-produced chimeric TSH showed the maximum increase in half-life, indicating the importance of sialic acid in prolonging half-life and in vivo potency. Sulfation of both subunits, predominantly beta and to a lesser extent alpha, appears to be responsible at least in part for the increased metabolic clearance of WT and chimeric TSH secreted by 293 cells. Apart from its therapeutic potential, chimeric TSH produced in various cell lines can be used as a tool to delineate the roles of sulfate and sialic acid in the in vivo clearance and, thereby, the in vivo bioactivity. PMID:7544273

  1. Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides

    PubMed Central

    2014-01-01

    Background Rhodosporidium toruloides is a β-carotenoid accumulating, oleaginous yeast that has great biotechnological potential. The lack of reliable and efficient genetic manipulation tools have been a major hurdle blocking its adoption as a biotechnology platform. Results We report for the first time the development of a highly efficient targeted gene deletion method in R. toruloides ATCC 10657 via Agrobacterium tumefaciens-mediated transformation. To further improve targeting frequency, the KU70 and KU80 homologs in R. toruloides were isolated and characterized in detail. A KU70-deficient mutant (∆ku70e) generated with the hygromycin selection cassette removed by the Cre-loxP recombination system showed a dramatically improved targeted gene deletion frequency, with over 90% of the transformants being true knockouts when homology sequence length of at least 1 kb was used. Successful gene targeting could be made with homologous flanking sequences as short as 100 bp in the ∆ku70e strain. KU70 deficiency did not perturb cell growth although an elevated sensitivity to DNA mutagenic agents was observed. Compared to the other well-known oleaginous yeast, Yarrowia lipolytica, R. toruloides KU70/KU80 genes contain much higher density of introns and are the most GC-rich KU70/KU80 genes reported. Conclusions The KU70-deficient mutant generated herein was effective in improving gene deletion frequency and allowed shorter homology sequences to be used for gene targeting. It retained the key oleaginous and fast growing features of R. toruloides. The strain should facilitate both fundamental and applied studies in this important yeast, with the approaches taken here likely to be applicable in other species in subphylum Pucciniomycotina. PMID:25188820

  2. 4. AERIAL VIEW OF MT. VERNON TERMINUS, SOUTHERN TERMINUS OF ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. AERIAL VIEW OF MT. VERNON TERMINUS, SOUTHERN TERMINUS OF GEORGE WASHINGTON MEMORIAL PARKWAY (GWMP), LOOKING SOUTHEAST. - George Washington Memorial Parkway, Along Potomac River from McLean to Mount Vernon, VA, Mount Vernon, Fairfax County, VA

  3. Role of the Cro repressor carboxy-terminal domain and flexible dimer linkage in operator and nonspecific DNA binding.

    PubMed

    Hubbard, A J; Bracco, L P; Eisenbeis, S J; Gayle, R B; Beaton, G; Caruthers, M H

    1990-10-01

    A series of mutations comprising single and multiple substitutions, deletions, and extensions within the carboxy-terminal domain of the bacteriophage lambda Cro repressor have been constructed. These mutations generally affect the affinity of repressor for specific and nonspecific DNA. Additionally, substitution of the carboxy-terminal alanine with several amino acids capable of hydrogen-bonding interactions leads to improved specific binding affinities. A mutation is also described whereby cysteine links the two Cro monomers by a disulfide bond. As a consequence, a significant improvement in nonspecific binding and a concomitant reduction in specific binding are observed with this mutant. These results provide evidence that the carboxy terminus of Cro repressor is an important DNA binding domain and that a flexible connection between the two repressor monomers is a critical factor in modulating the affinity of wild-type repressor for DNA. PMID:2271592

  4. Efficient Synthesis of 5-Carboxy-2'-Deoxypyrimidine Nucleoside 5'-Triphosphates.

    PubMed

    Gong, Shan-Shan; Sun, Jian; You, Yue-Hai; Chen, Ji-Zong; Liu, Guo-Dong; Sun, Qi

    2016-06-01

    An efficient P(V)-N activation method for the synthesis of 5-carboxy-2'-deoxyuridine and 5-carboxy-2'-deoxycytidine triphosphates directly from the corresponding phosphoropiperidate precursors has been developed. PMID:27104859

  5. Novel carboxy functionalized sol-gel precursors

    SciTech Connect

    Wolter, H.; Storch, W.; Gellermann, C.

    1996-12-31

    A novel family of inorganic-organic copolymers (ORMOCER`s) derived from urethane- and thioether(meth)acrylate alkoxysilanes has been successfully exploited for a variety of diverse applications. In order to widen the range of applications an additional functionality (carboxy group) has been incorporated int his silane type. Conventional sol-gel processing facilitates the formation of an inorganic Si-O-Si-network via hydrolysis and polycondensation reactions of alkoxysilyl moieties and in addition, the (meth)acrylate groups are available for radically induced polymerization to obtain a complementary organic polymer structure. The presence of a carboxy group would appear to have great potential for a range of diverse areas of application, such as an internal catalyst for the sol-gel process, complexation of elements such as Zr and Ti, increasing the adhesion to various substrates and modification of solubility. A number of novel silanes and their syntheses will be described in this paper.

  6. Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity.

    PubMed

    Styles, Nathan A; Shonsey, Erin M; Falany, Josie L; Guidry, Amber L; Barnes, Stephen; Falany, Charles N

    2016-07-01

    Bile acid CoA:amino acid N-acyltransferase (BAAT) is the terminal enzyme in the synthesis of bile salts from cholesterol and catalyzes the conjugation of taurine or glycine to bile acid CoA thioesters to form bile acid N-acylamidates. BAAT has a dual localization to the cytosol and peroxisomes, possibly due to an inefficient carboxy-terminal peroxisomal targeting signal (PTS), -serine-glutamine-leucine (-SQL). Mutational analysis was used to define the role of the carboxy terminus in peroxisomal localization and kinetic activity. Amidation activity of BAAT and BAAT lacking the final two amino acids (AAs) (BAAT-S) were similar, whereas the activity of BAAT with a canonical PTS sequence (BAAT-SKL) was increased >2.5-fold. Kinetic analysis of BAAT and BAAT-SKL showed that BAAT-SKL had a lower Km for taurine and glycine as well as a greater Vmax There was no difference in the affinity for cholyl-CoA. In contrast to BAAT, BAAT-SKL forms bile acid N-acylamidates with β-alanine. BAAT-S immunoprecipitated when incubated with peroxisomal biogenesis factor 5 (Pex5) and rabbit anti-Pex5 antibodies; however, deleting the final 12 AAs prevented coimmunoprecipitation with Pex5, indicating the Pex5 interaction involves more than the -SQL sequence. These results indicate that even small changes in the carboxy terminus of BAAT can have significant effects on activity and substrate specificity. PMID:27230263

  7. Automated carboxy-terminal sequence analysis of peptides.

    PubMed Central

    Bailey, J. M.; Shenoy, N. R.; Ronk, M.; Shively, J. E.

    1992-01-01

    Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these

  8. Frizzled-4 C-terminus Distal to KTXXXW Motif is Essential for Normal Dishevelled Recruitment and Norrin-stimulated Activation of Lef/Tcf-dependent Transcriptional Activation.

    PubMed

    Bertalovitz, Alexander C; Pau, Milly S; Gao, Shujuan; Malbon, Craig C; Wang, Hsien-Yu

    2016-01-01

    The carboxy (C)-termini of G protein coupled receptors (GPCR) dictate essential functions. The KTXXXW motif C-terminus of Frizzleds (FZD) has been implicated in recruitment of Dishevelled (DVL). Through study of FZD4 and its associated ligand Norrin, we report that a minimum of three residues distal to the KTXXXW motif in the C-terminal tail of Frizzled-4 are essential for DVL recruitment and robust Lef/Tcf-dependent transcriptional activation in response to Norrin. PMID:27096005

  9. Frizzled-4 C-terminus Distal to KTXXXW Motif is Essential for Normal Dishevelled Recruitment and Norrin-stimulated Activation of Lef/Tcf-dependent Transcriptional Activation

    PubMed Central

    Pau, Milly S.; Gao, Shujuan; Malbon, Craig C.; Wang, Hsien-yu

    2016-01-01

    The carboxy (C)-termini of G protein coupled receptors (GPCR) dictate essential functions. The KTXXXW motif C-terminus of Frizzleds (FZD) has been implicated in recruitment of Dishevelled (DVL). Through study of FZD4 and its associated ligand Norrin, we report that a minimum of three residues distal to the KTXXXW motif in the C-terminal tail of Frizzled-4 are essential for DVL recruitment and robust Lef/Tcf-dependent transcriptional activation in response to Norrin. PMID:27096005

  10. The Carboxy-terminus of BAK1 regulates kinase activity and is required for normal growth of Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In brassinosteroid (BR) signaling, binding of brassinolide to the BRI1 receptor kinase promotes interaction with its co-receptor, BAK1. Juxtaposition of the kinase domains that occurs then allows reciprocal transphosphorylation and activation of both kinases, but details of that process are not enti...

  11. Immunoreactivity of polyclonal antibodies generated against the carboxy terminus of the predicted amino acid sequence of the Huntington disease gene

    SciTech Connect

    Alkatib, G.; Graham, R.; Pelmear-Telenius, A.

    1994-09-01

    A cDNA fragment spanning the 3{prime}-end of the Huntington disease gene (from 8052 to 9252) was cloned into a prokaryotic expression vector containing the E. Coli lac promoter and a portion of the coding sequence for {beta}-galactosidase. The truncated {beta}-galactosidase gene was cleaved with BamHl and fused in frame to the BamHl fragment of the Huntington disease gene 3{prime}-end. Expression analysis of proteins made in E. Coli revealed that 20-30% of the total cellular proteins was represented by the {beta}-galactosidase-huntingtin fusion protein. The identity of the Huntington disease protein amino acid sequences was confirmed by protein sequence analysis. Affinity chromatography was used to purify large quantities of the fusion protein from bacterial cell lysates. Affinity-purified proteins were used to immunize New Zealand white rabbits for antibody production. The generated polyclonal antibodies were used to immunoprecipitate the Huntington disease gene product expressed in a neuroblastoma cell line. In this cell line the antibodies precipitated two protein bands of apparent gel migrations of 200 and 150 kd which together, correspond to the calculated molecular weight of the Huntington disease gene product (350 kd). Immunoblotting experiments revealed the presence of a large precursor protein in the range of 350-750 kd which is in agreement with the predicted molecular weight of the protein without post-translational modifications. These results indicate that the huntingtin protein is cleaved into two subunits in this neuroblastoma cell line and implicate that cleavage of a large precursor protein may contribute to its biological activity. Experiments are ongoing to determine the precursor-product relationship and to examine the synthesis of the huntingtin protein in freshly isolated rat brains, and to determine cellular and subcellular distribution of the gene product.

  12. A motif shared by TFIIF and TFIIB mediates their interaction with the RNA polymerase II carboxy-terminal domain phosphatase Fcp1p in Saccharomyces cerevisiae.

    PubMed

    Kobor, M S; Simon, L D; Omichinski, J; Zhong, G; Archambault, J; Greenblatt, J

    2000-10-01

    Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo. PMID:11003641

  13. A Motif Shared by TFIIF and TFIIB Mediates Their Interaction with the RNA Polymerase II Carboxy-Terminal Domain Phosphatase Fcp1p in Saccharomyces cerevisiae

    PubMed Central

    Kobor, Michael S.; Simon, Lisa D.; Omichinski, Jim; Zhong, Guoqing; Archambault, Jacques; Greenblatt, Jack

    2000-01-01

    Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo. PMID:11003641

  14. The genomic 5' terminus of Manchester calicivirus.

    PubMed

    Liu, B; Clarke, I N; Caul, E O; Lambden, P R

    1997-01-01

    An enteric calicivirus showing the classic cup-shaped surface morphology was identified in a stool sample obtained from a child with symptoms of acute gastroenteritis (Portishead virus, PHV). Genomic RNA was extracted directly from the PHV stool sample and amplified by RT-PCR using primers based on the Manchester isolate of HuCV. The 3' terminus of the cDNA was defined by homopolymer tailing with dATP and revealed an additional 165 nucleotides suggesting that the previously determined Manchester HuCV (MV) genome sequence was incomplete. Homopolymer tailing of MV cDNA primed using sequence data from the 5' terminus of PHV allowed extension of the MV genome by a further 165 nucleotides thereby increasing the overall genome length to 7431 nucleotides and resulting in an additional 72 amino acids at the N-terminus of the polyprotein. A conserved sequence motif typical of other caliciviruses was also identified at the extreme 5'-terminus of the genome. PMID:9354265

  15. Automated carboxy-terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate.

    PubMed Central

    Bailey, J. M.; Nikfarjam, F.; Shenoy, N. R.; Shively, J. E.

    1992-01-01

    Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on the automation of the thiocyanate chemistry using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80). A major limitation of this approach was the need to activate the C-terminus with acetic anhydride. We now describe the use of a new reagent, diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine, which combines the activation and derivatization steps to produce peptidylthiohydantoins. Previous work by Kenner et al. (Kenner, G.W., Khorana, H.G., & Stedman, R.J., 1953, Chem. Soc. J., 673-678) with this reagent demonstrated slow kinetics. Several days were required for complete reaction. We show here that the inclusion of pyridine was found to promote the formation of C-terminal thiohydantoins by DPP-ITC resulting in complete conversion of the C-terminal amino acid to a thiohydantoin in less than 1 h. Reagents such as imidazole, triazine, and tetrazole were also found to promote the reaction with DPP-ITC as effectively as pyridine. General base catalysts, such as triethylamine, do not promote the reaction, but are required to convert the C-terminal carboxylic acid to a salt prior to the reaction with DPP-ITC and pyridine. By introducing the DPP-ITC reagent and pyridine in separate steps in an automated sequencer, we observed improved sequencing yields for amino acids normally found difficult to derivatize with acetic anhydride/TMS-ITC. This was particularly true for aspartic acid, which now can be sequenced in yields comparable to most of the other amino acids. Automated programs are described for the C-terminal sequencing of

  16. Anthropomorphic Telemanipulation System in Terminus Control Mode

    NASA Technical Reports Server (NTRS)

    Jau, Bruno M.; Lewis, M. Anthony; Bejczy, Antal K.

    1994-01-01

    This paper describes a prototype anthropomorphic kinesthetic telepresence system that is being developed at JPL. It utilizes dexterous terminus devices in the form of an exoskeleton force-sensing master glove worn by the operator and a replica four finger anthropomorphic slave hand. The newly developed master glove is integrated with our previously developed non-anthropomorphic six degree of freedom (DOF) universal force-reflecting hand controller (FRHC). The mechanical hand and forearm are mounted to an industrial robot (PUMA 560), replacing its standard forearm. The notion of 'terminus control mode' refers to the fact that only the terminus devices (glove and robot hand) are of anthropomorphic nature, and the master and slave arms are non-anthropomorphic. The system is currently being evaluated, focusing on tool handling and astronaut equivalent task executions. The evaluation revealed the system's potential for tool handling but it also became evident that hand tool manipulations and space operations require a dual arm robot. This paper describes the system's principal components, its control and computing architecture, discusses findings of the tool handling evaluation, and explains why common tool handling and EVA space tasks require dual arm robots.

  17. Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase.

    PubMed

    Hüssy, P; Langen, H; Mous, J; Jacobsen, H

    1996-10-01

    The expression and processing of hepatitis C virus core protein was analyzed. Two protein bands, 21 kDa (P21), corresponding to the full-length core, and 19 kDa (P19), were detected as major products when core protein was expressed in the standard rabbit reticulocyte lysate system or in Sf9 insect cells. Core proteins with amino-terminal hexa-histidine tags were expressed which allowed the purification of the hexa-histidine P19 core with NI(2+)-NTA columns. With the help of mass spectrometry, the molecular weight of hexa-histidine-P19 was analyzed and its carboxy-terminus could be calculated. Fusion proteins of truncated core/core-E1 species fused to mouse dihydrofolate reductase (mDHFR) showed cleavage in the expected region. Cleavage sites could be determined by amino-terminal protein sequencing of the DHFR-fusion partner. Our data show that there are not one but two core products with an apparent molecular weight of about 19 kDa, ending either at amino acid leucine 179 or leucine 182, respectively. These cleavages in the hydrophobic, carboxy-terminal region of HCV core suggest processing by (a) recently proposed eucaryotic signal peptide peptidase(s) (F. Lyko et al. (1995) J. Biol. Chem. 270, 19873-19878). Furthermore, our results demonstrate that cleavage at these sites and the formation of the P19 species does not require previous processing at the signalase site (position 191/192) of the HCV-polyprotein. PMID:8862403

  18. Identification of human adenovirus early region 1 products by using antisera against synthetic peptides corresponding to the predicted carboxy termini.

    PubMed Central

    Yee, S P; Rowe, D T; Tremblay, M L; McDermott, M; Branton, P E

    1983-01-01

    Synthetic peptides were prepared which corresponded to the carboxy termini of the human adenovirus type 5 early region 1B (E1B) 58,000-molecular-weight (58K) protein (Tyr-Ser-Asp-Glu-Asp-Thr-Asp) and of the E1A gene products (Tyr-Gly-Lys-Arg-Pro-Arg-Pro). Antisera raised against these peptides precipitated polypeptides from adenovirus type 5-infected KB cells; serum raised against the 58K carboxy terminus was active against the E1B 58K phosphoprotein, whereas serum raised against the E1A peptide immunoprecipitated four major and at least two minor polypeptides. These latter proteins migrated with apparent molecular weights of 52K, 50K, 48.5K, 45K, 37.5K, and 35K, and all were phosphoproteins. By using tryptic phosphopeptide analysis, the four major species (52K, 50K, 48.5K, and 45K) were found to be related, as would be expected if all were products of the E1A region. The ability of the antipeptide sera to precipitate these viral proteins thus confirmed that the previously proposed sequence of E1 DNA and mRNA and the reading frame of the mRNA are correct. Immunofluorescent-antibody staining with the antipeptide sera indicated that the 58K E1B protein was localized both in the nucleus and in the cytoplasm, especially in the perinuclear region. The E1A-specific serum also stained both discrete patches in the nucleus and diffuse areas of the cytoplasm. These data suggest that both the 58K protein and the E1A proteins may function in or around the nucleus. These highly specific antipeptide sera should allow for a more complete identification and characterization of these important viral proteins. Images PMID:6343626

  19. Modification of the carboxy-terminal flanking region of a universal influenza epitope alters CD4+ T-cell repertoire selection

    PubMed Central

    Cole, David K.; Gallagher, Kathleen; Lemercier, Brigitte; Holland, Christopher J.; Junaid, Sayed; Hindley, James P.; Wynn, Katherine K.; Gostick, Emma; Sewell, Andrew K.; Gallimore, Awen M.; Ladell, Kristin; Price, David A.; Gougeon, Marie-Lise; Godkin, Andrew

    2012-01-01

    Human CD4+ αβ T cells are activated via T-cell receptor recognition of peptide epitopes presented by major histocompatibility complex (MHC) class II (MHC-II). The open ends of the MHC-II binding groove allow peptide epitopes to extend beyond a central nonamer core region at both the amino- and carboxy-terminus. We have previously found that these non-bound C-terminal residues can alter T cell activation in an MHC allele-transcending fashion, although the mechanism for this effect remained unclear. Here we show that modification of the C-terminal peptide-flanking region of an influenza hemagglutinin (HA305−320) epitope can alter T-cell receptor binding affinity, T-cell activation and repertoire selection of influenza-specific CD4+ T cells expanded from peripheral blood. These data provide the first demonstration that changes in the C-terminus of the peptide-flanking region can substantially alter T-cell receptor binding affinity, and indicate a mechanism through which peptide flanking residues could influence repertoire selection. PMID:22314361

  20. Alpha-carboxy nucleoside phosphonates as universal nucleoside triphosphate mimics

    PubMed Central

    Balzarini, Jan; Das, Kalyan; Bernatchez, Jean A.; Martinez, Sergio E.; Ngure, Marianne; Keane, Sarah; Ford, Alan; Maguire, Nuala; Mullins, Niki; John, Jubi; Kim, Youngju; Dehaen, Wim; Vande Voorde, Johan; Liekens, Sandra; Naesens, Lieve; Götte, Matthias; Maguire, Anita R.; Arnold, Eddy

    2015-01-01

    Polymerases have a structurally highly conserved negatively charged amino acid motif that is strictly required for Mg2+ cation-dependent catalytic incorporation of (d)NTP nucleotides into nucleic acids. Based on these characteristics, a nucleoside monophosphonate scaffold, α-carboxy nucleoside phosphonate (α-CNP), was designed that is recognized by a variety of polymerases. Kinetic, biochemical, and crystallographic studies with HIV-1 reverse transcriptase revealed that α-CNPs mimic the dNTP binding through a carboxylate oxygen, two phosphonate oxygens, and base-pairing with the template. In particular, the carboxyl oxygen of the α-CNP acts as the potential equivalent of the α-phosphate oxygen of dNTPs and two oxygens of the phosphonate group of the α-CNP chelate Mg2+, mimicking the chelation by the β- and γ-phosphate oxygens of dNTPs. α-CNPs (i) do not require metabolic activation (phosphorylation), (ii) bind directly to the substrate-binding site, (iii) chelate one of the two active site Mg2+ ions, and (iv) reversibly inhibit the polymerase catalytic activity without being incorporated into nucleic acids. In addition, α-CNPs were also found to selectively interact with regulatory (i.e., allosteric) Mg2+-dNTP-binding sites of nucleos(t)ide-metabolizing enzymes susceptible to metabolic regulation. α-CNPs represent an entirely novel and broad technological platform for the development of specific substrate active- or regulatory-site inhibitors with therapeutic potential. PMID:25733891

  1. Rapid elimination of Carboxy-THC in a cohort of chronic cannabis users.

    PubMed

    Lewis, John; Molnar, Anna; Allsop, David; Copeland, Jan; Fu, Shanlin

    2016-01-01

    Urinary 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (Carboxy-THC) concentrations, normalised to creatinine output, have been demonstrated to be a useful tool in the interpretation of the results of a series of urine tests for cannabis. These tests, often termed historical data, can be used to identify potential chronic cannabis users who may present occupational health and safety risks within the workplace. Conversely, the data can also be used to support employee claims of previous regular, rather than recent, cannabis use. This study aimed at examining the mean elimination of Carboxy-THC in 37 chronic users undergoing voluntary abstinence over a 2-week period. Urine specimens were collected prior to the study and after 1 and 2 weeks of abstinence. Carboxy-THC levels in urine were measured by gas chromatography-mass spectrometry (GC-MS) following alkaline hydrolysis, organic solvent extraction and derivatisation to form its pentafluoropropionic derivative. The creatinine-normalised Carboxy-THC concentrations declined rapidly over the 2 weeks of abstinence period and the majority of chronic cannabis users (73%) reduced their urinary Carboxy-THC levels to below the 15-μg/L confirmatory cutoff within that time. The study further highlights the value of historical urinary Carboxy-THC data as a means of identifying potential occupational health and safety risks among chronic cannabis users. PMID:26233612

  2. Protein-Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP).

    PubMed

    Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D; Blackburn, Elizabeth A; Ball, Kathryn L

    2015-11-01

    CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

  3. The penultimate arginine of the carboxy terminus determines slow desensitisation in a P2X receptor from the cattle tick Boophilus microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    P2X ion channels have been functionally characterised from a range of eukaryotes. Whilst these receptors can be broadly classified into fast and slow desensitising, the molecular mechanisms underlying current desensitisation are not fully understood. Here we describe the characterisation of a P2X ch...

  4. The N Terminus and C Terminus of Herpes Simplex Virus 1 ICP4 Cooperate To Activate Viral Gene Expression

    PubMed Central

    Wagner, Lauren M.; Lester, Jonathan T.; Sivrich, Frances L.

    2012-01-01

    Infected cell polypeptide 4 (ICP4) activates transcription from most viral promoters. Two transactivation domains, one N-terminal and one C terminal, are largely responsible for the activation functions of ICP4. A mutant ICP4 molecule lacking the C-terminal activation domain (n208) efficiently activates many early genes, whereas late genes are poorly activated, and virus growth is severely impaired. The regions within the N terminus of ICP4 (amino acids 1 to 210) that contribute to activation were investigated by analysis of deletion mutants in the presence or absence of the C-terminal activation domain. The mutants were assessed for their abilities to support viral replication and to regulate gene expression. Several deletions in regions conserved in other alphaherpesviruses resulted in impaired activation and viral growth, without affecting DNA binding. The single small deletion that had the greatest effect on activation in the absence of the C terminus corresponded to a highly conserved stretch of amino acids between 81 and 96, rendering the molecule nonfunctional. However, when the C terminus was present, the same deletion had a minimal effect on activity. The amino terminus of ICP4 was predicted to be relatively disordered compared to the DNA-binding domain and the C-terminal 500 amino acids. Moreover, the amino terminus appears to be in a relatively extended conformation as determined by the hydrodynamic properties of several mutants. The data support a model where the amino terminus is an extended and possibly flexible region of the protein, allowing it to efficiently interact with multiple transcription factors at a distance from where it is bound to DNA, thereby enabling ICP4 to function as a general activator of polymerase II transcription. The C terminus of ICP4 can compensate for some of the mutations in the N terminus, suggesting that it either specifies redundant interactions or enables the amino terminus to function more efficiently. PMID:22496239

  5. 32. VIEW OF TERMINUS OF GRAND CANAL, SHOWING TURNOUT GATES, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    32. VIEW OF TERMINUS OF GRAND CANAL, SHOWING TURNOUT GATES, LOOKING SOUTHWEST. WASTE WATER IS TURNED INTO THE BED OF NEW RIVER. Photographer: Mark Durben, April 1989 - Grand Canal, North side of Salt River, Tempe, Maricopa County, AZ

  6. 2. View of hydraulic gates at terminus of pipes to ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. View of hydraulic gates at terminus of pipes to feeder canal. The Siphon-Breaker Building is to the right, looking south. - Columbia Basin Project, Grand Coulee Siphon Breaker Building, Grand Coulee, Grant County, WA

  7. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility.

    PubMed

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K; Kline, Douglas W; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

  8. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility

    PubMed Central

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K.; Kline, Douglas W.; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

  9. Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus

    PubMed Central

    Liu, Pengda; Begley, Michael; Michowski, Wojciech; Inuzuka, Hiroyuki; Ginzberg, Miriam; Gao, Daming; Tsou, Peiling; Gan, Wenjian; Papa, Antonella; Kim, Byeong Mo; Wan, Lixin; Singh, Amrik; Zhai, Bo; Yuan, Min; Wang, Zhiwei; Gygi, Steven P.; Lee, Tae Ho; Lu, Kun-Ping; Toker, Alex; Pandolfi, Pier Paolo; Asara, John M.; Kirschner, Marc W.; Sicinski, Piotr; Cantley, Lewis; Wei, Wenyi

    2014-01-01

    Akt, also known as protein kinase B, plays key roles in cell proliferation, survival and metabolism. Akt hyperactivation contributes to many pathophysiological conditions, including human cancers1–3, and is closely associated with poor prognosis and chemo- or radio-therapeutic resistance4. Phosphorylation of Akt at S473 (ref. 5) and T308 (ref. 6) activates Akt. However, it remains unclear whether further mechanisms account for full Akt activation, and whether Akt hyperactivation is linked to misregulated cell cycle progression, another cancer hallmark7. Here we report that Akt activity fluctuates across the cell cycle, mirroring cyclin A expression. Mechanistically, phosphorylation of S477 and T479 at the Akt extreme carboxy terminus by cyclin-dependent kinase 2 (Cdk2)/cyclin A or mTORC2, under distinct physiological conditions, promotes Akt activation through facilitating, or functionally compensating for, S473 phosphorylation. Furthermore, deletion of the cyclin A2 allele in the mouse olfactory bulb leads to reduced S477/T479 phosphorylation and elevated cellular apoptosis. Notably, cyclin A2-deletion-induced cellular apoptosis in mouse embryonic stem cells is partly rescued by S477D/T479E-Akt1, supporting a physiological role for cyclin A2 in governing Akt activation. Together, the results of our study show Akt S477/T479 phosphorylation to be an essential layer of the Akt activation mechanism to regulate its physiological functions, thereby providing a new mechanistic link between aberrant cell cycle progression and Akt hyperactivation in cancer. PMID:24670654

  10. Ultrafast Dynamics of Carboxy-Hemoglobin: Two-Dimensional Infrared Spectroscopy Experiments and Simulations.

    PubMed

    Falvo, Cyril; Daniault, Louis; Vieille, Thibault; Kemlin, Vincent; Lambry, Jean-Christophe; Meier, Christoph; Vos, Marten H; Bonvalet, Adeline; Joffre, Manuel

    2015-06-18

    This Letter presents a comparison between experimental and simulated 2D mid-infrared spectra of carboxy-hemoglobin in the spectral region of the carbon monoxide stretching mode. The simulations rely on a fluctuating potential energy surface that includes both the effect of heme and the protein surroundings computed from molecular dynamics simulations. A very good agreement between theory and experiment is obtained with no adjustable parameters. The simulations show that the effect of the distal histidine through the hydrogen bond is strong and is directly responsible for the slow decay of the frequency-frequency correlation function on a 10 ps time scale. This study confirms that fluctuations in carboxy-hemoglobin are more inhomogeneous than those in the more frequently studied carboxy-myoglobin. The comparison between simulations and experiments brings valuable information on the complex relation between protein structure and spectral diffusion. PMID:26266594

  11. Channel Catfish, Ictalurus punctatus, ubiquitin carboxy-terminal hydrolase L5 cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome cycle is a complex, non-lysosomal biochemical process for intracellular protein degradation. This process involves many enzymes. One of them is ubiquitin carboxy-terminal hydrolase (UCT). In this report, we cloned, sequenced and characterized the channel catfish UCT L5 cDNA....

  12. Molecular dissection of radixin: distinct and interdependent functions of the amino- and carboxy-terminal domains

    PubMed Central

    1995-01-01

    The ERM proteins--ezrin, radixin, and moesin--occur in particular cortical cytoskeletal structures. Several lines of evidence suggest that they interact with both cytoskeletal elements and plasma membrane components. Here we described the properties of full-length and truncated radixin polypeptides expressed in transfected cells. In stable transfectants, exogenous full-length radixin behaves much like endogenous ERM proteins, localizing to the same cortical structures. However, the presence of full-length radixin or its carboxy-terminal domain in cortical structures correlates with greatly diminished staining of endogenous moesin in those structures, suggesting that radixin and moesin compete for a limiting factor required for normal associations in the cell. The results also reveal distinct roles for the amino- and carboxy-terminal domains. At low levels relative to endogenous radixin, the carboxy-terminal polypeptide is associated with most of the correct cortical targets except cleavage furrows. In contrast, the amino-terminal polypeptide is diffusely localized throughout the cell. Low level expression of full-length radixin or either of the truncated polypeptides has no detectable effect on cell physiology. However, high level expression of the carboxy-terminal domain dramatically disrupts normal cytoskeletal structures and functions. At these high levels, the amino-terminal polypeptide does localize to cortical structures, but does not affect the cells. We conclude that the behavior of radixin in cells depends upon activities contributed by separate domains of the protein, but also requires modulating interactions between those domains. PMID:7744951

  13. Proliferation-associated nuclear antigen Ki-S1 is identical with topoisomerase II alpha. Delineation of a carboxy-terminal epitope with peptide antibodies.

    PubMed Central

    Boege, F.; Andersen, A.; Jensen, S.; Zeidler, R.; Kreipe, H.

    1995-01-01

    Proliferation-linked expression of the nuclear Ki-S1 antigen is a significant prognostic indicator in mammary carcinomas. Here, we show staining of a protein of 170 kd by Ki-S1 antibody in immunoblots of Saccharomyces cerevisiae expressing human topoisomerase II alpha but not in the parental strain. In HL-60 cells containing both isoforms of human topoisomerase II, Ki-S1 antibody binds selectively to the 170-kd isoenzyme in a similar fashion as peptide-antibodies directed against amino acid residues 1 to 15 or 1512 to 1530 of human topoisomerase II alpha. Conversely, antibodies directed against carboxyl-terminal sequences of human topoisomerase II beta selectively stain a 180-kd protein. The immunoreactive pattern of V8 endoproteinase restriction digests of human topoisomerase II alpha was identical for Ki-S1-antibody and peptide-antibodies directed against residues 1512 to 1530 but different for peptide-antibodies directed against residues 1 to 15. The Rf values of the smallest fragment commonly recognized by Ki-S1 antibody and the carboxy terminus-specific peptide-antibody place the Ki-S1 epitope within the last 495 carboxyl-terminal amino acid residues of topoisomerase II alpha. Images Figure 1 Figure 2 Figure 3 PMID:7539979

  14. Characterization of pseudorabies virus mutants expressing carboxy-terminal truncations of gE: evidence for envelope incorporation, virulence, and neurotropism domains.

    PubMed Central

    Tirabassi, R S; Townley, R A; Eldridge, M G; Enquist, L W

    1997-01-01

    Glycoprotein E (gE) gene of pseudorabies virus (PRV) is conserved among diverse alphaherpesviruses and therefore is predicted to be important for virus survival. gE contributes to viral spread from cell to cell in a variety of hosts and is responsible, in part, for increased virulence or pathogenesis of the virus. Virulence and spread mediated by gE are thought to be highly correlated. We initiated this study to explore the hypothesis that these two phenotypes might reflect separate functions of the gE protein. We did so by focusing on the role of the gE carboxy terminus in neuronal spread. Viruses harboring nonsense mutations affecting the expression of the gE cytoplasmic domain had several notable phenotypes. First, the truncated gE proteins expressed from these mutants are not found in virion envelopes. Second, the mutants retain the ability to spread to all retinorecipient regions of the rodent brain after retinal infection of rats. Third, the mutants have the reduced virulence phenotype of a gE deletion mutant in rats. Finally, the mutants have distinct plaque-size phenotypes on MDBK cells but not PK15 cells. Based on these observations, we suggest that gE-mediated virulence and spread may reflect separate functions that are not mediated by gE on virus particles. PMID:9261363

  15. 23. TERMINUS, NORTH BRANCH PRAIRIE CITY DITCH. DITCH COMES FROM ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    23. TERMINUS, NORTH BRANCH PRAIRIE CITY DITCH. DITCH COMES FROM ISOLATED GROUP OF TREES IN MIDDLE DISTANCE, AND ENDS AT CENTER RIGHT. WATER THEN PROCEEDED DOWN SWALE, INTO TREES AT LEFT. VIEW TO NORTH. - Natomas Ditch System, Rhodes Ditch, West of Bidwell Street, north of U.S. Highway 50, Folsom, Sacramento County, CA

  16. 51. FRED TEICHMAN'S ROTATING SCREEN AT TERMINUS OF THE POWER ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    51. FRED TEICHMAN'S ROTATING SCREEN AT TERMINUS OF THE POWER CANAL. SETTLING BASIN IS LOCATED IN BOTTOM LEFT OF PHOTO. UPSTREAM SIDE OF ROOSEVELT DAM IS VISIBLE Photographer: Mark Durben, 1984 - Roosevelt Power Canal & Diversion Dam, Parallels Salt River, Roosevelt, Gila County, AZ

  17. 207. Oconaluffee River Bridge is the southern terminus of the ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    207. Oconaluffee River Bridge is the southern terminus of the Blue Ride Parkway. It is a concrete girder bridge completed in 1957. It is the only concrete girder bridge with stone-faced piers. Looking east-southeast. - Blue Ridge Parkway, Between Shenandoah National Park & Great Smoky Mountains, Asheville, Buncombe County, NC

  18. Four+ Years of Measurements from the Mendenhall Glacier Terminus

    NASA Astrophysics Data System (ADS)

    Heavner, M.; Fatland, D. R.

    2012-12-01

    We describe the instrumentation, power, communications, and lessons learned from ongoing four+ years of measurements at the terminus of Mendenhall Glacier. In this presentation we focus on the most successful microserver deployment. The microserver is a simple rugged computer with a radio modem that can survive and operate outdoors in harsh environments like Antarctica. The system is called a microserver because of the networking capabilities, particularly as it may act as anchor points for localized lightweight sensor networks. SEAMONSTER, the SouthEast Alaska MOnitoring Network for Science, Technology, Education and Research, is a demonstration sensor web effort. The microserver design for SEAMONSTER is intended to provide general capabilities that could be used in harsh environments specifically for cryospheric observations. At the Mendenhall terminus the observations included meteorologic data and repeat digital photography. Other SEAMONSTER stations included snow accumulation and density, precision GPS, seismic, water pressure, and other measurements. Power generation at the Mendenhall deployment is both solar and wind.

  19. Role of the synaptobrevin C terminus in fusion pore formation

    PubMed Central

    Ngatchou, Annita N.; Kisler, Kassandra; Fang, Qinghua; Walter, Alexander M.; Zhao, Ying; Bruns, Dieter; Sørensen, Jakob B.; Lindau, Manfred

    2010-01-01

    Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets. PMID:20937897

  20. Chromosomal double-strand break repair in Ku80-deficient cells.

    PubMed Central

    Liang, F; Romanienko, P J; Weaver, D T; Jeggo, P A; Jasin, M

    1996-01-01

    The x-ray sensitive hamster cell line xrs-6 is deficient in DNA double-strand break (DSB) repair and exhibits impaired V(D)J recombination. The molecular defect in this line is in the 80-kDa subunit of the Ku autoantigen, a protein that binds to DNA ends and recruits the DNA-dependent protein kinase to DNA. Using an I-SceI endonuclease expression system, chromosomal DSB repair was examined in xrs-6 and parental CHO-K1 cell lines. A DSB in chromosomal DNA increased the yield of recombinants several thousand-fold above background in both the xrs-6 and CHO-K1 cells, with recombinational repair of DSBs occurring in as many as 1 of 100 cells electroporated with the endonuclease expression vector. Thus, recombinational repair of chromosomal DSBs can occur at substantial levels in mammalian cells and it is not grossly affected in our assay by a deficiency of the Ku autoantigen. Rejoining of broken chromosome ends (end-joining) near the site of the DSB was also examined. In contrast to recombinational repair, end-joining was found to be severely impaired in the xrs-6 cells. Thus, the Ku protein appears to play a critical role in only one of the chromosomal DSB repair pathways. Images Fig. 1 Fig. 2 PMID:8799130

  1. Mechanical ventilation with positive end-expiratory pressure decreases the circulating concentrations of the N-terminus and C-terminus of the atrial natriuretic factor prohormone.

    PubMed

    Vesely, D L; Salmon, J S

    1990-01-01

    Mechanical ventilation with positive end-expiratory pressure (PEEP) decreases urine output and urinary sodium excretion. The influence of PEEP during controlled mechanical ventilation on the circulating concentrations of the N-terminus and C-terminus of the atrial natriuretic factor (ANF) prohormone which both contain natriuretic and diuretic peptides was investigated in 7 patients with acute respiratory failure. The 98 amino acid (aa) N-terminus, the midportion of the N-terminus consisting of aa 31-67 of the 126 aa ANF prohormone (i.e., pro ANF 31-67) and the C-terminus (aa 99-126; ANF) were found to be significantly (p less than 0.05; ANOVA) elevated compared to 54 healthy volunteers during acute respiratory failure prior to institution of PEEP. With institution of 10 cm of H2O of PEEP all 7 patients had a significant (p less than 0.05) decrease in the circulating concentrations of pro ANFs 1-98, 31-67 and ANF. These findings suggest that the increased thoracic pressure secondary to PEEP which reduces venous return and lowers atrial filling pressure results in a decreased release of the N-terminus and C-terminus of the ANF prohormone. This decrease in the N-terminus and C-terminus of the ANF prohormone appears to represent a physiologic mechanism for restoration of intravascular volume, secondary to decreased sodium excretion. PMID:2151585

  2. Marine Geophysical Surveying Along the Hubbard Glacier Terminus, Southeast Alaska

    NASA Astrophysics Data System (ADS)

    Goff, J. A.; Davis, M.; Gulick, S. P.; Lawson, D. E.; Willems, B. A.

    2010-12-01

    Tidewater glaciers are a challenging environment for marine investigations, owing to the dangers associated with calving and restrictions on operations due to dense floating ice. We report here on recent efforts to conduct marine geophysical surveys proximal to the ice face of Hubbard Glacier, in Disenchantment Bay, Alaska. Hubbard is an advancing tidewater glacier that has twice recently (1986 and 2002) impinged on Gilbert Point, which separates Russell Fiord from Disenchantment Bay, thereby temporarily creating a glacially-dammed Russell Lake. Continued advance will likely form a more permanent dam, rerouting brackish outflow waters into the Situk River, near Yakutat, Alaska. Our primary interest is in studying the development and motion of the morainal bank which, for an advancing tidewater glacier, stabilizes it against rapid retreat. For survey work, we operated with a small, fast, aluminum-hulled vessel and a captain experienced in operating in ice-bound conditions, providing a high margin of safety and maneuverability. Differencing of multibeam bathymetric data acquired in different years can identify and quantify areas of deposition and erosion on the morainal bank front and in Disenchantment Bay proper, where accumulation rates are typically > 1 m/yr within 1 km of the glacier terminus. The advance or retreat rate of the morainal bank can be determined by changes in the bed elevation through time; we document advance rates that average > 30 m/yr in Disenchantment Bay, but which vary substantially over different time periods and at different positions along the ice face. Georeferencing of available satellite imagery allows us to directly compare the position of the glacial terminus with the position of the morainal bank. From 1978 to 1999, and then to 2006, the advances in terminus and morainal bank positions were closely synchronized along the length of the glacier face. In the shallower Russell Fiord side of the terminus, a sediment ridge was mapped both

  3. Interaction of T4 UvsW helicase and single-stranded DNA binding protein gp32 through its carboxy terminal acidic tail

    PubMed Central

    Perumal, Senthil K.; Nelson, Scott W.; Benkovic, Stephen J.

    2013-01-01

    Bacteriophage T4 UvsW helicase contains both unwinding and annealing activities and displays some functional similarities to bacterial RecG and RecQ helicases. UvsW is involved in several DNA repair pathways, playing important roles in recombination-dependent DNA repair and the reorganization of stalled replication forks. The T4 single-stranded DNA binding protein, gp32, is a central player in nearly all DNA replication and repair processes and is thought to facilitate their coordination by recruiting and regulating the various proteins involved. Here, we show that the activities of the UvsW protein are modulated by gp32. UvsW catalyzed unwinding of recombination intermediates such as D-loops and static X-DNA (Holliday junction mimic) to ssDNA products is enhanced by the gp32 protein. The enhancement requires the presence of the protein interaction domain of gp32 (the acidic carboxy terminus), suggesting that a specific interaction between UvsW and gp32 is required. In the absence of this interaction, the ssDNA annealing and ATP-dependent translocation activities of UvsW are severely inhibited when gp32 coats the ssDNA lattice. However, when UvsW and gp32 do interact, UvsW is able to efficiently displace the gp32 protein from the ssDNA. This ability of UvsW to remove gp32 from ssDNA may explain its ability to enhance the strand invasion activity of the T4 recombinase (UvsX) and suggests a possible new role for UvsW in gp32-mediated DNA transactions. PMID:23732982

  4. Pegylated peptides. V. Carboxy-terminal PEGylated analogs of growth hormone-releasing factor (GRF) display enhanced duration of biological activity in vivo.

    PubMed

    Campbell, R M; Heimer, E P; Ahmad, M; Eisenbeis, H G; Lambros, T J; Lee, Y; Miller, R W; Stricker, P R; Felix, A M

    1997-06-01

    In the present study, human growth hormone-releasing factor (hGRF) and analogs were successfully pegylated at the carboxy-terminus using a novel solid- and solution-phase strategy. Following synthesis, these pegylated hGRF analogs were evaluated for in vitro and in vivo biological activity. Specifically, hGRF (1-29)-NH2, [Ala15]-hGRF (1-29)-NH2, [desNH2Tyr1, D-Ala2, Ala15]-hGRF(1-29)-NH2 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-OH were each C-terminally extended using a Gly-Gly-Cys-NH2 spacer (previously demonstrated not to alter intrinsic biological activity), and then monopegylated via coupling to an activated dithiopyridyl-PEG reagent. PEG moieties of 750, 2000, 5000 or 10,000 molecular weight (MW) were examined to determine the effect of polymer weight on activity. Initial biological evaluations in vitro revealed that all C-terminally pegylated hGRF analogs retained high growth hormone (GH)-releasing potencies, regardless of the MW of PEG polymer employed. Two of these pegylated hGRF analogs, [desNH2Tyr1, D-Ala2, Ala15]-hGRF (1-29)-Gly-Gly-Cys(NH2)-S-Nle-PEG5000 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-Gly-Cys(NH2)-S-Nle-PEG5000, were subsequently evaluated in both pig and mouse models and found to be highly potent (in vivo potency range = 12-55-fold that of native hGRF). Relative to their non-pegylated counterparts, these two pegylated hGRF analogs exhibited enhanced duration of activity. PMID:9266480

  5. Carboxy-THC in Washed Hair: Still the Reliable Indicator of Marijuana Ingestion.

    PubMed

    Hill, Virginia A; Schaffer, Michael I; Stowe, G Neil

    2016-06-01

    The presence of the metabolite 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (C-THC) in hair is generally accepted as the definitive proof of delta-9-tetrahydrocannabinol (THC) ingestion. During hair analysis, the removal of any potential C-THC external contamination that could result from marijuana smoke or close personal contact via a wash procedure is critical. Here, we performed a series of experiments to demonstrate that C-THC is the reliable indicator of marijuana ingestion when paired with the correct washing procedure to remove potential external contamination. PMID:27185816

  6. Bis[3-(2-carboxy­ethen­yl)pyridinium-1-acetato]dichloridozinc(II)

    PubMed Central

    Jing, Xue-Hui; Sun, Wei-Wei; Gao, En-Qing

    2009-01-01

    In the title complex, [ZnCl2(C10H9NO4)2], the ZnII ion lies on a twofold rotation axis and is four-coordinated by two carboxyl­ate O atoms from two 3-(2-carboxy­ethen­yl)pyridinium-1-acetate ligands in a monodentate mode and two Cl atoms in a distorted tetra­hedral geometry. In the crystal structure, inter­molecular O—H⋯O hydrogen bonds link the mol­ecules into a double-chain structure extending parallel to [101]. PMID:21578626

  7. Interpreting Terminus Fluctuations at Helheim Glacier, Southeast Greenland, through Modeling and Observations

    NASA Astrophysics Data System (ADS)

    Kehrl, L. M.; Joughin, I. R.; Shapero, D.

    2014-12-01

    Marine-terminating outlet glaciers are highly sensitive to changes at the ice-ocean boundary. Changes at the ice-ocean boundary (calving events, submarine melting) can alter the terminus position and thereby the stress balance. If the terminus retreats into deeper water, more of the driving stress must then be balanced by longitudinal stress gradients, which cause the glacier to speed up. This study combines satellite observations and modeling (Elmer/Ice) to investigate the relationship between glacier dynamics and terminus position at Helheim Glacier, southeast Greenland, from 2000-2014. Helheim Glacier retreated about 7 km from 2001-2005 as warm ocean water entered the nearby fjord. From 2005-2006, the glacier re-advanced by 3 km as a floating or near-floating ice tongue formed over the basal overdeepening in front of the glacier terminus. Since then, Helheim's terminus position has remained relatively stable, with terminus fluctuations of < 2 km. Our model experiments consider both large terminus fluctuations (> 2 km) associated with rapid retreat and small terminus fluctuations (< 500 m) associated with individual calving events. We run the model simulations with both a flowline and three-dimensional model to better constrain our uncertainties. Our results show that Helheim Glacier responds rapidly to changes in terminus position of more than a few hundred meters. Small terminus fluctuations can cause velocity variations that extend up to 30 km inland, which roughly corresponds with the spatial extent of the weak bed (20-40 kPa) underneath Helheim Glacier.

  8. Simple colorimetric method for quantification of surface carboxy groups on polymer particles.

    PubMed

    Hennig, Andreas; Hoffmann, Angelika; Borcherding, Heike; Thiele, Thomas; Schedler, Uwe; Resch-Genger, Ute

    2011-06-15

    We present a novel, simple, and fast colorimetric method to quantify the total number of carboxy groups on polymer microparticle and nanoparticle surfaces. This method exploits that small divalent transition metal cations (M(2+) = Ni(2+), Co(2+), Cd(2+)) are efficiently bound to these surface functional groups, which allows their extraction by a single centrifugation step. Remaining M(2+) in the supernatant is subsequently quantified spectrophotometrically after addition of the metal ion indicator pyrocatechol violet, for which Ni(2+) was identified to be the most suitable transition metal cation. We demonstrate that the difference between added and detected M(2+) is nicely correlated to the number of surface carboxy groups as determined by conductometry, thereby affording a validated measure for the trueness of this procedure. The variation coefficient of ~5% found in reproducibility studies underlines the potential of this novel method that can find conceivable applications for the characterization of different types of poly(carboxylic acid)-functionalized materials, e.g., for quality control by manufacturers of such materials. PMID:21561064

  9. 5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation

    PubMed Central

    Aik, WeiShen; Che, Ka Hing; Li, Xuan Shirley; Kristensen, Jan B. L.; King, Oliver N. F.; Chan, Mun Chiang; Yeoh, Kar Kheng; Choi, Hwanho; Walport, Louise J.; Thinnes, Cyrille C.; Bush, Jacob T.; Lejeune, Clarisse; Rydzik, Anna M.; Rose, Nathan R.; Bagg, Eleanor A.; McDonough, Michael A.; Krojer, Tobias; Yue, Wyatt W.; Ng, Stanley S.; Olsen, Lars; Brennan, Paul E.; Oppermann, Udo; Muller-Knapp, Susanne; Klose, Robert J.; Ratcliffe, Peter J.; Schofield, Christopher J.; Kawamura, Akane

    2015-01-01

    2-Oxoglutarate and iron dependent oxygenases are therapeutic targets for human diseases. Using a representative 2OG oxygenase panel, we compare the inhibitory activities of 5-carboxy-8-hydroxyquinoline (IOX1) and 4-carboxy-8-hydroxyquinoline (4C8HQ) with that of two other commonly used 2OG oxygenase inhibitors, N-oxalylglycine (NOG) and 2,4-pyridinedicarboxylic acid (2,4-PDCA). The results reveal that IOX1 has a broad spectrum of activity, as demonstrated by the inhibition of transcription factor hydroxylases, representatives of all 2OG dependent histone demethylase subfamilies, nucleic acid demethylases and γ-butyrobetaine hydroxylase. Cellular assays show that, unlike NOG and 2,4-PDCA, IOX1 is active against both cytosolic and nuclear 2OG oxygenases without ester derivatisation. Unexpectedly, crystallographic studies on these oxygenases demonstrate that IOX1, but not 4C8HQ, can cause translocation of the active site metal, revealing a rare example of protein ligand-induced metal movement PMID:26682036

  10. Negatively-charged residues in the polar carboxy-terminal region in FSP27 are indispensable for expanding lipid droplets.

    PubMed

    Tamori, Yoshikazu; Tateya, Sanshiro; Ijuin, Takeshi; Nishimoto, Yuki; Nakajima, Shinsuke; Ogawa, Wataru

    2016-03-01

    FSP27 has an important role in large lipid droplet (LD) formation because it exchanges lipids at the contact site between LDs. In the present study, we clarify that the amino-terminal domain of FSP27 (amino acids 1-130) is dispensable for LD enlargement, although it accelerates LD growth. LD expansion depends on the carboxy-terminal domain of FSP27 (amino acids 131-239). Especially, the negative charge of the acidic residues (D215, E218, E219 and E220) in the polar carboxy-terminal region (amino acids 202-239) is essential for the enlargement of LD. We propose that the carboxy-terminal domain of FSP27 has a crucial role in LD expansion, whereas the amino-terminal domain only has a supportive role. PMID:26921608

  11. Novel Archaeal Adhesion Pilins with a Conserved N Terminus

    PubMed Central

    Esquivel, Rianne N.; Xu, Rachel

    2013-01-01

    Type IV pili play important roles in a wide array of processes, including surface adhesion and twitching motility. Although archaeal genomes encode a diverse set of type IV pilus subunits, the functions for most remain unknown. We have now characterized six Haloferax volcanii pilins, PilA[1-6], each containing an identical 30-amino-acid N-terminal hydrophobic motif that is part of a larger highly conserved domain of unknown function (Duf1628). Deletion mutants lacking up to five of the six pilin genes display no significant adhesion defects; however, H. volcanii lacking all six pilins (ΔpilA[1-6]) does not adhere to glass or plastic. Consistent with these results, the expression of any one of these pilins in trans is sufficient to produce functional pili in the ΔpilA[1-6] strain. PilA1His and PilA2His only partially rescue this phenotype, whereas ΔpilA[1-6] strains expressing PilA3His or PilA4His adhere even more strongly than the parental strain. Most surprisingly, expressing either PilA5His or PilA6His in the ΔpilA[1-6] strain results in microcolony formation. A hybrid protein in which the conserved N terminus of the mature PilA1His is replaced with the corresponding N domain of FlgA1 is processed by the prepilin peptidase, but it does not assemble functional pili, leading us to conclude that Duf1628 can be annotated as the N terminus of archaeal PilA adhesion pilins. Finally, the pilin prediction program, FlaFind, which was trained primarily on archaeal flagellin sequences, was successfully refined to more accurately predict pilins based on the in vivo verification of PilA[1-6]. PMID:23794623

  12. On the computational ability of the RNA polymerase II carboxy terminal domain

    PubMed Central

    Karagiannis, Jim

    2014-01-01

    The RNA polymerase II carboxy terminal domain has long been known to play an important role in the control of eukaryotic transcription. This role is mediated, at least in part, through complex post-translational modifications that take place on specific residues within the heptad repeats of the domain. In this addendum, a speculative, but formal mathematical conceptualization of this biological phenomenon (in the form of a semi-Thue string rewriting system) is presented. Since the semi-Thue formalism is known to be Turing complete, this raises the possibility that the CTD – in association with the regulatory pathways controlling its post-translational modification – functions as a biological incarnation of a universal computing machine. PMID:25371772

  13. E. coli QueD is a 6-carboxy-5,6,7,8-tetrahydropterin synthase†

    PubMed Central

    McCarty, Reid M.; Somogyi, Árpád; Bandarian, Vahe

    2009-01-01

    To elucidate the early steps required during biosynthesis of a broad class of 7-deazapurine containing natural products, we have studied the reaction catalyzed by Escherichia coli QueD, a 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase (PTPS) homolog possibly involved in queuosine biosynthesis. While mammalian PTPS homologs convert 7,8-dihydroneopterin triphosphate (H2NTP) to 6-pyruvoyltetrahydropterin (PPH4) in biopterin biosynthesis, E. coli QueD catalyzes the conversion of H2NTP to 6-carboxy-5,6,7,8-tetrahydropterin (CPH4). E. coli QueD can also convert PPH4 and sepiapterin to CPH4, allowing a mechanism to be proposed. PMID:19231875

  14. Investigation of a recently detected 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol isomer: Studies on the degradation of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol glucuronide.

    PubMed

    Hanisch, Stephanie; Paulke, Alexander; Toennes, Stefan W

    2016-09-10

    An isomer of the tetrahydrocannabinol (THC) metabolite 11-nor-9-carboxy-Δ(9)-THC (THCCOOH) had been detected in blood of cannabis users. The present study was initiated to elucidate whether the labile metabolite THCCOOH-glucuronide could be the precursor. THCCOOH-glucuronide was incubated in human serum and albumin (HSA) solution at various temperatures (-18, 4.5, 22 and 37°C) and pH values (pH 7.4 and 8.3) for seven days in the presence or absence of the esterase inhibitor sodium fluoride. Analysis of incubation samples was performed using LC-MS/MS. Marked degradation of THCCOOH-glucuronide was observed at 37°C. It was found that not only THCCOOH, but also the isomer is a degradation product of THCCOOH-glucuronide and its in-vivo production is assumed. Degradation to THCCOOH and the isomer occurred at alkaline pH, in the presence of fluoride-sensitive esterases and of HSA alone. To inhibit isomer formation during sample storage, refrigeration and controlling of the pH are recommended. However, THCCOOH and the isomer exhibit similar properties during incubations in serum, but differ in their interaction with HSA. The present study confirmed the nature of the isomer as degradation product of the abundant THC metabolite THCCOOH-glucuronide. Serum albumin and esterases are obviously involved. The isomer is formed not only during storage, but also under physiological conditions, suggesting that it can be considered an in-vivo metabolite. However, the chemical structure of the isomer remains unknown and further research is necessary. PMID:27448313

  15. High CO2-capture ability of a porous organic polymer bifunctionalized with carboxy and triazole groups.

    PubMed

    Xie, Lin-Hua; Suh, Myunghyun Paik

    2013-08-26

    A new porous organic polymer, SNU-C1, incorporating two different CO2 -attracting groups, namely, carboxy and triazole groups, has been synthesized. By activating SNU-C1 with two different methods, vacuum drying and supercritical-CO2 treatment, the guest-free phases, SNU-C1-va and SNU-C1-sca, respectively, were obtained. Brunauer-Emmett-Teller (BET) surface areas of SNU-C1-va and SNU-C1-sca are 595 and 830 m(2) g(-1), respectively, as estimated by the N2-adsorption isotherms at 77 K. At 298 K and 1 atm, SNU-C1-va and SNU-C1-sca show high CO2 uptakes, 2.31 mmol  g(-1) and 3.14 mmol  g(-1), respectively, the high level being due to the presence of abundant polar groups (carboxy and triazole) exposed on the pore surfaces. Five separation parameters for flue gas and landfill gas in vacuum-swing adsorption were calculated from single-component gas-sorption isotherms by using the ideal adsorbed solution theory (IAST). The data reveal excellent CO2-separation abilities of SNU-C1-va and SNU-C1-sca, namely high CO2-uptake capacity, high selectivity, and high regenerability. The gas-cycling experiments for the materials and the water-treated samples, experiments that involved treating the samples with a CO2-N2 gas mixture (15:85, v/v) followed by a pure N2 purge, further verified the high regenerability and water stability. The results suggest that these materials have great potential applications in CO2 separation. PMID:23881821

  16. Solid Phase Formylation of N-Terminus Peptides.

    PubMed

    Tornesello, Anna Lucia; Sanseverino, Marina; Buonaguro, Franco Maria

    2016-01-01

    Formylation of amino groups is a critical reaction involved in several biological processes including post-translational modification of histones. The addition of a formyl group (CHO) to the N-terminal end of a peptide chain generates biologically active molecules. N-formyl-peptides can be produced by different methods. We performed the N-formylation of two chemotactic hexapetides, Met1-Leu2-Lys3-Leu4-Ile5-Val6 and Met1-Met2-Tyr3-Ala4-Leu5-Phe6, carrying out the reaction directly on peptidyl-resin following pre-activation of formic acid with N,N-dicyclohexylcarbodiimmide (DCC) in liquid phase. The overnight incubation at 4 °C resulted in a significant increase in production yields of formylated peptides compared to the reaction performed at room temperature. The method is consistently effective, rapid, and inexpensive. Moreover, the synthetic strategy can be applied for the formylation of all primary amines at N-terminus of peptide chains or amino groups of lysine side-chains in solid phase. PMID:27271589

  17. Confirmation of a blocked amino terminus of sulfhydryl oxidase

    SciTech Connect

    Janolino, V.G.; Morrison-Rowe, S.J.; Swaisgood, H.E. )

    1990-09-01

    The isolation of sulfhydryl oxidase from bovine milk in a suitably pure form for sequencing was carried out by transient covalent affinity chromatography of diafiltered whey using cysteinylsuccinamidopropyl-glass as matrix. The glutathione-eluted proteins were separated by SDS-PAGE. By radiolabeling the affinity chromatography-purified enzyme with ({sup 14}C)iodoacetate before subjecting to SDS-PAGE, the sulfhydryl oxidase band was identified, because sulfhydryl oxidase is known to be inactivated by alkylation of one sulfhydryl group per mole. The results confirmed that sulfhydryl oxidase corresponds to the 85 ({plus minus} 5)-kDa band observed on SDS-PAGE. The protein band corresponding to radiolabeled sulfhydryl oxidase was recovered from SDS-PAGE gels by electrophoretic elution and by electroblotting on polyvinylidene difluoride membrane and subjected to gas phase sequencing. Precautions were taken during electrophoretic elution to prevent reactions that result in N-terminal blocking. Both methods of protein recovery yielded negative results when subjected to sequence analysis indicating that the N-terminus of sulfhydryl oxidase is blocked.

  18. The variable C-terminus of cysteine string proteins modulates exocytosis and protein-protein interactions.

    PubMed

    Boal, Frédéric; Zhang, Hui; Tessier, Céline; Scotti, Pier; Lang, Jochen

    2004-12-28

    Cysteine string proteins (Csps) are vesicle proteins involved in neurotransmission and hormone exocytosis. They are composed of distinct domains: a variable N-terminus, a J-domain followed by a linker region, a cysteine-rich string, and a C-terminus which diverges among isoforms. Their precise function and interactions are not fully understood. Using insulin exocytosis as a model, we show that the linker region and the C-terminus, but not the variable N-terminus, regulate overall secretion. Moreover, endogenous Csp1 binds in a calcium-dependent manner to monomeric VAMP2, and this interaction requires the C-terminus of Csp. The interaction is isoform specific as recombinant Csp1 binds VAMP1 and VAMP7, but not VAMP3. Cross-linking in permeabilized clonal beta-cells revealed homodimerization of Csp which is stimulated by Ca(2+) and again modulated by the variant C-terminus. Our data suggest that both interactions of Csp occur during exocytosis and may explain the effect of the variant C-terminus of this chaperon protein on peptide hormone secretion. PMID:15610015

  19. Functions of the conserved thrombospondin carboxy-terminal cassette in cell-extracellular matrix interactions and signaling.

    PubMed

    Adams, Josephine C

    2004-06-01

    Thrombospondins (TSPs) are extracellular, multidomain, calcium-binding glycoproteins that function at cell surfaces, in extracellular matrix (ECM) and as bridging molecules in cell-cell interactions. TSPs are multifunctional and modulate cell behavior during development, wound-healing, immune response, tumor growth and in the homeostasis of adult tissues. TSPs are assembled as oligomers that are composed of homologous polypeptides. In all the TSP polypeptides, the most highly-conserved region is the carboxyl-region, which contains a characteristic set of domains comprising EGF domains, TSP type 3 repeats and a globular carboxy-terminal domain. This large region is termed here the thrombospondin carboxy-terminal cassette (TSP-CTC). The strong conservation of the TSP-CTC suggests that it may mediate ancestral functions that are shared by all TSPs. This review summarizes the current knowledge of the TSP-CTC and areas of future interest. PMID:15094125

  20. Functional Studies of the Carboxy-Terminal Repeat Domain of Drosophila RNA Polymerase II in Vivo

    PubMed Central

    Brickey, W. J.; Greenleaf, A. L.

    1995-01-01

    To understand the in vivo function of the unique and conserved carboxy-terminal repeat domain (CTD) of RNA polymerase II largest subunit (RpII215), we have studied RNA polymerase II biosynthesis, activity and genetic function in Drosophila RpII215 mutants that possessed all (C4), half (W81) or none (IIt) of the CTD repeats. We have discovered that steady-state mRNA levels from transgenes encoding a fully truncated, CTD-less subunit (IIt) are essentially equal to wild-type levels, whereas the levels of the CTD-less subunit itself and the amount of polymerase harboring it (Pol IIT) are significantly lower than wild type. In contrast, for the half-CTD mutant (W81), steady-state mRNA levels are somewhat lower than for wild type or IIt, while W81 subunit and polymerase amounts are much less than wild type. Finally, we have tested genetically the ability of CTD mutants to complement (rescue) partially functional RpII215 alleles and have found that IIt fails to complement whereas W81 complements partially to completely. These results suggest that removal of the entire CTD renders polymerase completely defective in vivo, whereas eliminating half of the CTD results in a polymerase with significant in vivo activity. PMID:7498740

  1. Conformational Analysis of the Carboxy-Terminal Tails of Human β-Tubulin Isotypes

    PubMed Central

    Luchko, Tyler; Huzil, J. Torin; Stepanova, Maria; Tuszynski, Jack

    2008-01-01

    Several isotypes of the structural protein tubulin have been characterized. Their expression offers a plausible explanation for differences regarding microtubule function. Although sequence variation between tubulin isotypes occurs throughout the entire protein, it is the extreme carboxy-terminal tails (CTTs) that exhibit the greatest concentration of differences. In humans, the CTTs range in length from 9 to 25 residues and because of a considerable number of glutamic acid residues, contain over 1/3 of tubulin's total electrostatic charge. The CTTs are believed to be highly disordered and their precise function has yet to be determined. However, their absence has been shown to result in altered microtubule stability and a reduction in the interaction with several microtubule-associated proteins (MAPs). To characterize the role that CTTs play in microtubule function, we examined the global conformational differences within a set of nine human β-tubulin isotypes using replica exchange molecular dynamics simulations. Through the analysis of the resulting configuration ensembles, we quantified differences such as the CTTs sequence influence on overall flexibility and average secondary structure. Although only minor variations between each CTT were observed, we suggest that these differences may be significant enough to affect interactions with MAPs, thereby influencing important properties such as microtubule assembly and stability. PMID:17993481

  2. Simultaneous identification of 2-carboxy-tetrahydrocannabinol, tetrahydrocannabinol, cannabinol and cannabidiol in oral fluid.

    PubMed

    Moore, Christine; Rana, Sumandeep; Coulter, Cynthia

    2007-06-01

    Tetrahydrocannabinol (THC) is an important psychoactive ingredient in marijuana, which is the most widely used illegal recreational drug in the USA. Since it is generally smoked, the constituents of the plant material, as well as THC may be present in oral fluid specimens collected for the purposes of drug testing. We present an analytical procedure for the simultaneous determination of the pyrolytic precursor Delta(9)-tetrahydrocannabinolic acid A, tetrahydrocannabinol, cannabinol and cannabidiol in human oral fluid specimens using gas chromatography mass spectrometry (GC/MS). Solid phase extraction and GC/MS/EI with selected ion monitoring were used, and the linearity of the method ranged from 0-16 ng/mL of neat oral fluid. The recovery of the cannabinoids from the collection pad into the transportation buffer was greater than 70% for all cannabinoids tested at 4 ng/mL, and the intra- and inter-day precision was less than 10.3 and 15.2% for all analytes. The stability of the drugs in oral fluid and of the extracted derivatives was investigated. The procedure was applied to oral fluid specimens taken from habitual marijuana smokers. We have previously reported the presence of the metabolite 11-nor-Delta(9)-tetra-hydrocannabinol-9-carboxylic acid in oral fluid, but this is the first report of the plant constituent 2-carboxy-THC being detected in saliva. PMID:17321807

  3. The negatively charged carboxy-terminal tail of β-tubulin promotes proper chromosome segregation

    PubMed Central

    Fees, Colby P.; Aiken, Jayne; O’Toole, Eileen T.; Giddings, Thomas H.; Moore, Jeffrey K.

    2016-01-01

    Despite the broadly conserved role of microtubules in chromosome segregation, we have a limited understanding of how molecular features of tubulin proteins contribute to the underlying mechanisms. Here we investigate the negatively charged carboxy-terminal tail domains (CTTs) of α- and β-tubulins, using a series of mutants that alter or ablate CTTs in budding yeast. We find that ablating β-CTT causes elevated rates of chromosome loss and cell cycle delay. Complementary live-cell imaging and electron tomography show that β-CTT is necessary to properly position kinetochores and organize microtubules within the assembling spindle. We identify a minimal region of negatively charged amino acids that is necessary and sufficient for proper chromosome segregation and provide evidence that this function may be conserved across species. Our results provide the first in vivo evidence of a specific role for tubulin CTTs in chromosome segregation. We propose that β-CTT promotes the ordered segregation of chromosomes by stabilizing the spindle and contributing to forces that move chromosomes toward the spindle poles. PMID:27053662

  4. Carboxy alkyl esters of Uncaria tomentosa augment recovery of sensorineural functions following noise injury.

    PubMed

    Guthrie, O'neil W; Gearhart, Caroline A; Fulton, Sherry; Fechter, Laurence D

    2011-08-17

    This study tested the hypothesis that hydrophilic chemotypes of the medicinal vine Uncaria tomentosa (UT) would facilitate recovery of sensorineural functions following exposure to a damaging level of noise. The particular chemotypes investigated were carboxy alkyl esters (CAE) which are known to exhibit multifunctional cytoprotective properties that include: enhanced cellular DNA repair, antioxidation and anti-inflammation. Long-Evans rats were divided into four treatment groups: vehicle-control, noise-only, CAE-only and CAE+noise. The noise exposure was an 8kHz octave band of noise at 105dB SPL for 4h. Outer hair cell (OHC) function was measured with the cubic 2f(1)-f(2) distortion product otoacoustic emissions (DPOAE) at the start of the study (baseline) and at time-points that corresponded to 1day, 1week and 4weeks post-noise exposure to determine within-group effects. Compound action potentials to puretone stimuli were recorded from the VIIIth craniofacial nerve at 4weeks post-noise exposure to determine between-group effects. Additionally, cytocochleograms were constructed for each row of OHCs from each group. Noise exposure produced significant sensorineural impairments. However, CAE treatment facilitated almost complete recovery of OHC function and limited the magnitude of cell loss. The loss of neural sensitivity to puretone stimuli was inhibited with CAE treatment. Therefore, it appears that the multifunctional cytoprotective capacity of CAE from UT may generalize to otoprotection from acoustic over-exposure. PMID:21762882

  5. Structural and Mechanistic Studies on Klebsiella pneumoniae 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2010-11-12

    The stereospecific oxidative degradation of uric acid to (S)-allantoin was recently shown to proceed via three enzymatic steps. The final conversion is a decarboxylation of the unstable intermediate 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and is catalyzed by OHCU decarboxylase. Here we present the structures of Klebsiella pneumoniae OHCU decarboxylase in unliganded form and with bound allantoin. These structures provide evidence that ligand binding organizes the active site residues for catalysis. Modeling of the substrate and intermediates provides additional support for this hypothesis. In addition we characterize the steady state kinetics of this enzyme and report the first OHCU decarboxylase inhibitor, allopurinol, a structural isomer of hypoxanthine. This molecule is a competitive inhibitor of K. pneumoniae OHCU decarboxylase with a K{sub i} of 30 {+-} 2 {micro}m. Circular dichroism measurements confirm structural observations that this inhibitor disrupts the necessary organization of the active site. Our structural and biochemical studies also provide further insights into the mechanism of catalysis of OHCU decarboxylation.

  6. SERCaMP: a carboxy-terminal protein modification that enables monitoring of ER calcium homeostasis

    PubMed Central

    Henderson, Mark J.; Wires, Emily S.; Trychta, Kathleen A.; Richie, Christopher T.; Harvey, Brandon K.

    2014-01-01

    Endoplasmic reticulum (ER) calcium homeostasis is disrupted in diverse pathologies, including neurodegeneration, cardiovascular diseases, and diabetes. Temporally defining calcium dysregulation during disease progression, however, has been challenging. Here we describe secreted ER calcium-monitoring proteins (SERCaMPs), which allow for longitudinal monitoring of ER calcium homeostasis. We identified a carboxy-terminal modification that is sufficient to confer release of a protein specifically in response to ER calcium depletion. A Gaussia luciferase (GLuc)–based SERCaMP provides a simple and sensitive method to monitor ER calcium homeostasis in vitro or in vivo by analyzing culture medium or blood. GLuc-SERCaMPs revealed ER calcium depletion in rat primary neurons exposed to various ER stressors. In vivo, ER calcium disruption in rat liver was monitored over several days by repeated sampling of blood. Our results suggest that SERCaMPs will have broad applications for the long-term monitoring of ER calcium homeostasis and the development of therapeutic approaches to counteract ER calcium dysregulation. PMID:25031430

  7. A Naturally Occurring HER2 Carboxy-Terminal Fragment Promotes Mammary Tumor Growth and Metastasis▿ †

    PubMed Central

    Pedersen, Kim; Angelini, Pier-Davide; Laos, Sirle; Bach-Faig, Alba; Cunningham, Matthew P.; Ferrer-Ramón, Cristina; Luque-García, Antonio; García-Castillo, Jesús; Parra-Palau, Josep Lluis; Scaltriti, Maurizio; y Cajal, Santiago Ramón; Baselga, José; Arribas, Joaquín

    2009-01-01

    HER2 is a tyrosine kinase receptor causally involved in cancer. A subgroup of breast cancer patients with particularly poor clinical outcomes expresses a heterogeneous collection of HER2 carboxy-terminal fragments (CTFs). However, since the CTFs lack the extracellular domain that drives dimerization and subsequent activation of full-length HER2, they are in principle expected to be inactive. Here we show that at low expression levels one of these fragments, 611-CTF, activated multiple signaling pathways because of its unanticipated ability to constitutively homodimerize. A transcriptomic analysis revealed that 611-CTF specifically controlled the expression of genes that we found to be correlated with poor prognosis in breast cancer. Among the 611-CTF-regulated genes were several that have previously been linked to metastasis, including those for MET, EPHA2, matrix metalloproteinase 1, interleukin 11, angiopoietin-like 4, and different integrins. It is thought that transgenic mice overexpressing HER2 in the mammary glands develop tumors only after acquisition of activating mutations in the transgene. In contrast, we show that expression of 611-CTF led to development of aggressive and invasive mammary tumors without the need for mutations. These results demonstrate that 611-CTF is a potent oncogene capable of promoting mammary tumor progression and metastasis. PMID:19364815

  8. Immunomagnetic Reduction Assay on Des-Gamma-Carboxy Prothrombin for Screening of Hepatocellular Carcinoma.

    PubMed

    Chieh, Jen-Jie; Huang, K W; Chuang, C P; Wei, W C; Dong, J J; Lee, Y Y

    2016-08-01

    The accredited biomarker alpha-fetoprotein (AFP) offers limited sensitivity and specificity in the early detection of hepatocellular carcinoma (HCC). To improve the screening performance, des-gamma-carboxy prothrombin (DCP) has been identified as another promising biomarker of HCC, combined with AFP biomarkers. The results of the commercial optical enzyme-linked immunosorbent assay (ELISA) kit easily have the interference problem due to the optical methodology. The immunomagnetic reduction (IMR) assay based on the magnetic measurement was utilized to assay DCP biomarkers without the excellent antiinterference performances. A DCP magnetic reagent, composed of iron-oxide (Fe3O4 ) magnetic nanoparticles coated with anti-DCP antibodies solved in phosphoryl-buffer solution, was synthesized and characterized. In the test of standard DCP antigens, superior antiinterference and sensitivity than optical ELISA were proved. In the animal test, the results indicate good agreement between the IMR assay findings and the tumor sizes of HCC rats at all time points after the HCC implantation. The feasibility of the developed DCP magnetic reagent with the IMR for the detection of DCP is verified, and demonstrates the high potential for future clinical applications. PMID:26415145

  9. A Systematic Review of Des-γ-Carboxy Prothrombin for the Diagnosis of Primary Hepatocellular Carcinoma

    PubMed Central

    De, Ji; Shen, Yi; Qin, Jinyu; Feng, Li; Wang, Yiping; Yang, Li

    2016-01-01

    Abstract Determining the serum des-γ-carboxy-prothrombin (DCP) level is of great importance for the diagnosis of primary hepatocellular carcinoma (PHC). Although several studies have investigated the accuracy of diagnostic DCP tests for PHC, the results have been inconsistent. The aim of this study was to systematically evaluate DCP as a diagnostic standard for PHC. Several databases, including PubMed, EMBASE, MEDLINE (Ovid), the Chinese National Knowledge Infrastructure (CNKI), the VIP Database for Chinese Technical Periodicals (VIP), WanFang Data, and the China Biological Medicine Database (CBM), were searched from the date of database inception until July 1, 2015 to collect published international and domestic studies of DCP in the diagnosis of PHC. Two investigators screened the literature according to the inclusion and exclusion criteria, extracted the data, and assessed the methodological quality of the included studies. A total of 38 studies involving 11,124 cases were included (5298 cases in the PHC group and 5826 cases in the control group). A meta-analysis was then performed using Meta-Disc 1.4 and RevMan 5.2 software. The overall sensitivity, specificity, positive likelihood ratio (+LR), and negative likelihood ratio (−LR) of DCP for the detection of PHC were 0.66 (95% confidence interval [CI]: 0.65–0.68), 0.88 (95% CI: 0.87–0.90), 7.13 (95% CI: 5.73–8.87), and 0.33 (95% CI: 0.29–0.38), respectively. The area under the curve (AUC) of the summary receiver-operating characteristic curve (SROC) was 0.9002. In conclusion, DCP has moderate diagnostic utility for PHC. Owing to the heterogeneity and limitations of the included studies, the above conclusion requires further support from additional high-quality studies. PMID:27124038

  10. Contribution of active-site glutamine to rate enhancement in ubiquitin carboxy terminal hydrolases

    PubMed Central

    Boudreaux, David; Chaney, Joseph; Maiti, Tushar K.; Das, Chittaranjan

    2012-01-01

    Ubiquitin carboxy terminal hydrolases (UCHs) are cysteine proteases featuring a classical cysteine-histidine-aspartate catalytic triad, also a highly conserved glutamine thought to be a part of the oxyanion hole. However, the contribution of this side chain to the catalysis by UCH enzymes is not known. Herein, we demonstrate that the glutamine side chain contributes to rate enhancement in UCHL1, UCHL3 and UCHL5. Mutation of the glutamine to alanine in these enzymes impairs the catalytic efficiency mainly due to a 16 to 30-fold reduction in kcat, which is consistent with a loss of approximately 2 kcal/mol in transition-state stabilization. However, the contribution to transition-state stabilization observed here is rather modest for the side chain’s role in oxyanion stabilization. Interestingly, we discovered that the carbonyl oxygen of this side chain is engaged in a C—H•••O hydrogen-bonding contact with the CεH group of the catalytic histidine. Upon further analysis, we found that this interaction is a common active-site structural feature in most cysteine proteases, including papain, belonging to families with the QCH(N/D) type of active-site configuration. It is possible that removal of the glutamine side chain might have abolished the C—H•••O interaction, which typically accounts for 2 kcal/mol of stabilization, leading to the effect on catalysis observed here. Additional studies performed on UCHL3 by mutating the glutamine to glutamate (strong C—H•••O acceptor but oxyanion destabilizer) and to lysine (strong oxyanion stabilizer but lacking C—H•••O hydrogen-bonding property) suggest that the C—H•••O hydrogen bond could contribute to catalysis. PMID:22284438

  11. A Systematic Review of Des-γ-Carboxy Prothrombin for the Diagnosis of Primary Hepatocellular Carcinoma.

    PubMed

    De, Ji; Shen, Yi; Qin, Jinyu; Feng, Li; Wang, Yiping; Yang, Li

    2016-04-01

    Determining the serum des-γ-carboxy-prothrombin (DCP) level is of great importance for the diagnosis of primary hepatocellular carcinoma (PHC). Although several studies have investigated the accuracy of diagnostic DCP tests for PHC, the results have been inconsistent.The aim of this study was to systematically evaluate DCP as a diagnostic standard for PHC.Several databases, including PubMed, EMBASE, MEDLINE (Ovid), the Chinese National Knowledge Infrastructure (CNKI), the VIP Database for Chinese Technical Periodicals (VIP), WanFang Data, and the China Biological Medicine Database (CBM), were searched from the date of database inception until July 1, 2015 to collect published international and domestic studies of DCP in the diagnosis of PHC. Two investigators screened the literature according to the inclusion and exclusion criteria, extracted the data, and assessed the methodological quality of the included studies.A total of 38 studies involving 11,124 cases were included (5298 cases in the PHC group and 5826 cases in the control group). A meta-analysis was then performed using Meta-Disc 1.4 and RevMan 5.2 software. The overall sensitivity, specificity, positive likelihood ratio (+LR), and negative likelihood ratio (-LR) of DCP for the detection of PHC were 0.66 (95% confidence interval [CI]: 0.65-0.68), 0.88 (95% CI: 0.87-0.90), 7.13 (95% CI: 5.73-8.87), and 0.33 (95% CI: 0.29-0.38), respectively. The area under the curve (AUC) of the summary receiver-operating characteristic curve (SROC) was 0.9002. In conclusion, DCP has moderate diagnostic utility for PHC. Owing to the heterogeneity and limitations of the included studies, the above conclusion requires further support from additional high-quality studies. PMID:27124038

  12. Bursts of calving activity and controls on the terminus position of Yahtse Glacier, Alaska

    NASA Astrophysics Data System (ADS)

    Bartholomaus, T. C.; Larsen, C. F.; West, M. E.; Oneel, S.

    2011-12-01

    The tidewater glacier terminus is the interface that links oceanic and glaciological processes. Tidewater glaciers contribute large amounts of cold, fresh water to their fjords. Ocean heat exerts a significant control on glacier mass balance. On the Gulf of Alaska, the terminus of tidewater Yahtse Glacier has advanced slowly since its 1990 post-Little Ice Age minimum. At Yahtse's terminus, ice flowing at 18 m/d encounters water with temperatures of up to 10.5°C (measured 1.5 km from the terminus). Profiles of temperature and salinity in Icy Bay, in which Yahtse Glacier terminates, have revealed a strongly stratified, single-cell circulation pattern. Fresh, glacier outflow exits the bay atop warm, saline Gulf of Alaska water. The Alaska Coastal Current, a major source of Icy Bay water, has warmed by 1°C over the last 40 years. These observations prompt the question of how a tidewater advance may be sustained in spite of warming ocean and atmosphere temperatures. Superimposed on Yahtse Glacier's longer-term advance have been smaller-scale summer retreats and winter-spring re-advances. These smaller fluctuations indicate that factors that change on short timescales, such as ocean conditions and weather, also have an important control on terminus position. Observed bursts in calving frequency are a further reflection of the unsteady conditions at the glacier terminus. In the present study, we use seismograms recorded on bedrock within 500 m of the glacier terminus as a calving counter. The epicenters of a significant majority of glacier-generated seismic events within the St. Elias Mountains have been located to within 15 km of the terminus of Yahtse Glacier. Previous study at Yahtse Glacier has revealed that at least 75% of these seismic events originate from calving processes, most notably through the interactions between iceberg and water. Calving frequency is characterized by a relatively steady rate of background events, punctuated by bursts of calving activity

  13. Structural features of the C-terminus from the human neurokinin-1 receptor.

    PubMed

    Orel, Mikhail; Padrós, Esteve; Manyosa, Joan

    2012-07-01

    The neurokinin-1 receptor (NK1R) is a G-protein coupled receptor found in the central and peripheral nervous systems of vertebrates, and is responsible for many physiological processes. The C-terminus domain seems to be essential for coupling to the corresponding G-protein and β-arrestin, and is important for receptor desensitization, internalization and recycling. We have focused our study on expression of the human NK1R (hNK1R) C-terminus in Escherichia coli, and its purification and characterization, in order to elucidate its structural properties. CD and Fourier transform infrared spectroscopy showed that the hNK1R C-terminus, rather than having a random structure, has well-defined secondary-structure patterns. The presence of three tyrosine residues in the primary sequence of the hNK1R C-terminus facilitated the use of UV and fluorescence spectroscopy techniques which revealed tyrosine fluorescence and UV absorption at anomalous wavelengths. In their entirety, the results show that the hNK1R C-terminus has clearly defined secondary (25% α-helix, 27% unordered structure and 48% β-sheets and β-turns) and tertiary structures which, it is believed, are tightly related to its multiple functions. PMID:22530884

  14. Two-step biocatalytic route to biobased functional polyesters from omega-carboxy fatty acids and diols.

    PubMed

    Yang, Yixin; Lu, Wenhua; Zhang, Xiaoyan; Xie, Wenchun; Cai, Minmin; Gross, Richard A

    2010-01-11

    Biobased omega-carboxy fatty acid monomers 1,18-cis-9-octadecenedioic, 1,22-cis-9-docosenedioic, and 1,18-cis-9,10-epoxy-octadecanedioic acids were synthesized in high conversion yields from oleic, erucic and epoxy stearic acids by whole-cell biotransformations catalyzed by C. tropicalis ATCC20962. Maximum volumetric yields in shake-flasks were 17.3, 14.2, and 19.1 g/L after 48 h conversion for oleic acid and 72 h conversions for erucic and epoxy stearic acids, respectively. Studies in fermentor with better control of pH and glucose feeding revealed that conversion of oleic acid to 1,18-cis-9-octadecenedioic acid by C. tropicalis ATCC20962 occurred with productivities up to 0.5 g/L/h. The conversion of omega-carboxy fatty acid monomers to polyesters was then studied using immobilized Candida antarctica Lipase B (N435) as catalyst. Polycondensations with diols were performed in bulk as well as in diphenyl ether. The retension of functionality from fatty acid, to omega-carboxy fatty acid monomer and to corresponding polyesters resulted in polymers with with unsaturated and epoxidized repeat units and M(w) values ranging from 25000 to 57000 g/mol. These functional groups along chains disrupted crystallization giving materials that are low melting (23-40 degrees C). In contrast, saturated polyesters prepared from 1,18-octadecanedioic acid and 1,8-octanediol have correspondingly higher melting transitions (88 degrees C). TGA results indicated that all synthesized polyesters showed high thermal stabilities. Thus, the preparation of functional monomers from C. tropicalis omega-oxidation of fatty acids provides a wide range of new monomer building blocks to construct functional polymers. PMID:20000460

  15. Fibrinogen {alpha} genes: Conservation of bipartite transcripts and carboxy-terminal-extended {alpha} subunits in vertebrates

    SciTech Connect

    Fu, Y.; Cao, Y.; Hertzberg, K.M.; Grieninger, G.

    1995-11-01

    All three well-studied subunits of the clotting protein fibrinogen ({alpha}, {beta}, {gamma}) share N-terminal structural homologies, but until recently only the {beta} and {gamma} chains were recognized as having similar globular C-termini. With the discovery of an extra exon in the human fibrinogen {alpha} gene (exon VI), a minor form of the {alpha} subunit ({alpha}{sub E}) with an extended {beta}- and {gamma}-like C-terminus has been identified. In the present study, the polymerase chain reaction has been used to identify sequences that encode counterparts to {alpha}{sub E} in chicken, rabbit, rat, and baboon. The basic six-exon structure of the fibrinogen {alpha} genes is shown to be conserved among mammals and birds, as are the intron positions. Bipartite transcripts - still bearing an intron prior to the last exon - are found among the products of the various vertebrate fibrinogen {alpha} genes. The last exon represents the largest conserved segment of the gene and, in each species examined, encodes exactly 236 amino acids. The C-termini of these {alpha}{sub E} chains align without a single gap and are between 76 and 99% identical. Since the exon VI-encoded domain of {alpha}{sub E} is as well conserved as the corresponding regions of the {beta} and {gamma} chains, it follows that it is equally important and that {alpha}{sub E}-fibrinogen plays a vital, if as-yet unrecognized physiological role. 21 refs., 7 figs., 1 tab.

  16. Differentiating Carotid Terminus Occlusions into Two Distinct Populations Based on Willisian Collateral Status

    PubMed Central

    Lee, Sun-Uk; Hong, Ji Man; Kim, Sun Yong; Bang, Oh Young; Demchuk, Andrew M.; Lee, Jin Soo

    2016-01-01

    Background and Purpose The outcomes of acute internal carotid artery (ICA) terminus occlusions are poor. We classified ICA terminus occlusions into 2 groups according to the occlusion pattern of the circle of Willis and hypothesized that clinical outcomes would significantly differ between them. Methods Consecutive patients with acute ICA terminus occlusions evaluated by baseline computed tomographic angiography were enrolled. We investigated the occlusion patterns in the circle of Willis, retrospectively classified patients into simple ICA terminus occlusion (STO; with good Willisian collaterals from neighboring cerebral circulation) and complex ICA terminus occlusion (CTO; with one or more of A2 anterior cerebral artery, fetal posterior cerebral artery occlusion, or hypoplastic/absent contralateral A1; or with poor collaterals from anterior communicating artery) groups, and compared their baseline characteristics and outcomes. Results The STO group (n=58) showed smaller infarct volumes at 72 hours than the CTO group (n=34) (median, 81 mL [interquartile range, 38-192] vs. 414 mL [193-540], P<0.001) and more favorable outcomes (3-month modified Rankin Scale 0-3, 44.8% vs. 8.8%, P<0.001; 3-month mortality, 24.1% vs. 67.6%, P<0.001). In multivariable analyses, STO remained an independent predictor for favorable outcomes (odds ratio 6.1, P=0.010). Conclusions Favorable outcomes in STO group suggested that the outcomes of acute ICA terminus occlusions depend on Willisian collateral status. Documenting the subtypes on computed tomographic angiography would help predict patient outcome. PMID:26915505

  17. Regioselective Syntheses of [13C]4-Labelled Sodium 1-Carboxy-2-(2-ethylhexyloxycarbonyl)ethanesulfonate and Sodium 2-Carboxy-1-(2-ethylhexyloxycarbonyl)ethanesulfonate from [13C]4-Maleic Anhydride

    PubMed Central

    Barsamian, Adam L.; Perkins, Matt J.; Field, Jennifer A.; Blakemore, Paul R.

    2014-01-01

    The entitled monohydrolysis products, also known as α- and β-ethylhexyl sulfosuccinate ('EHSS'), of the surfactant diisooctyl sulfosuccinate ('DOSS') were synthesized in stable isotope labelled form from [13C]4-maleic anhydride. Sodium [13C]4-1-carboxy-2-(2-ethylhexyloxycarbonyl)ethanesulfonate (α-EHSS) was prepared by the method of Larpent by reaction of 2-ethylhexan-1-ol with [13C]4-maleic anhydride followed by regioselective conjugate addition of sodium bisulfite to the resulting monoester (38% overall yield). The regiochemical outcome of bisulfite addition was confirmed by a combination of 13C/13C (INADEQUATE) and 1H/13C (HMBC) NMR spectral correlation experiments. Sodium [13C]4-2-carboxy-1-(2-ethylhexyloxycarbonyl)ethanesulfonate (β-EHSS) was prepared in four steps by reaction of 4-methoxybenzyl alcohol (PMBOH) with [13C]4-maleic anhydride, regioselective sodium bisulfite addition, DCC mediated esterification with 2-ethylhexan-1-ol, and PMB ester deprotection with trifluoroacetic acid (13% overall yield). The regiochemical outcome of the second synthesis was confirmed by a combination of 1JCC scalar coupling constant analysis and 1H/13C (HMBC) NMR spectral correlation. The materials prepared are required as internal standards for the LC-MS/MS trace analysis of the degradation products of DOSS, the anionic surfactant found in Corexit, the oil dispersant used during emergency response efforts connected to the Deepwater Horizon oil spill of April 2010. PMID:24700711

  18. Regulation of Nuclear Positioning and Dynamics of the Silent Mating Type Loci by the Yeast Ku70/Ku80 Complex▿

    PubMed Central

    Bystricky, Kerstin; Van Attikum, Haico; Montiel, Maria-Dolores; Dion, Vincent; Gehlen, Lutz; Gasser, Susan M.

    2009-01-01

    We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLα or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and α cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLα, unlike HMRa and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLα donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLα creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus. PMID:19047366

  19. Removal of uranium (VI) from aqueous systems by nanoscale zero-valent iron particles suspended in carboxy-methyl cellulose

    NASA Astrophysics Data System (ADS)

    Popescu (Hoştuc), Ioana-Carmen; Filip, Petru; Humelnicu, Doina; Humelnicu, Ionel; Scott, Thomas Bligh; Crane, Richard Andrew

    2013-11-01

    Carboxy-methyl-cellulose (CMC), a common "delivery vehicle" for the subsurface deployment of iron nanoparticles (INP) has been tested in the current work for the removal of aqueous uranium from synthetic water samples. A comparison of the removal of aqueous uranium from solutions using carboxy-methyl-cellulose with and without iron nanoparticles (CMC-INP and CMC, respectively) was tested over a 48 h reaction period. Analysis of liquid samples using spectrophotometry determined a maximum sorption capacity of uranium, Qmax, of 185.18 mg/g and 322.58 mg/g for CMC and CMC-INP respectively, providing strong evidence of an independent aqueous uranium removal ability exhibited by CMC. The results point out that CMC provides an additional capacity for aqueous uranium removal. Further tests are required to determine whether similar behaviour will be observed for other aqueous contaminant species and if the presence of CMC within a INP slurry inhibits or aids the reactivity, reductive capacity and affinity of INP for aqueous contaminant removal.

  20. Carboxy-terminal sequence divergence and processing of the polyprotein antigen from Dirofilaria immitis.

    PubMed

    Poole, C B; Hornstra, L J; Benner, J S; Fink, J R; McReynolds, L A

    1996-11-12

    A polyprotein composed of multiple units arranged in direct tandem arrays has been identified in parasitic and free living nematodes. Analysis of previously cloned units from the Dirofilaria immitis polyprotein antigen (DiPA) indicated the units were nearly identical but here we demonstrate that they segregate into two related families. The consensus repeats, DiPA-CR1 and CR2, derived for each family are 80% identical. However, the repeats at the C-terminus of the polyprotein have diverged from DiPA-CR1 and CR2. This was shown by DNA sequence and Southern blot analysis of a 1.9 kb cDNA clone that encodes 4.4 C-terminal repeats (DiPA-TR1 through TR5). DiPA-TR3 through TR5 show 27-52% amino acid identity with the consensus repeats and 31-35% amino acid identity with one another. Metabolic labeling studies have shown that cleavage of DiPA generates a protein "ladder' from 14 to > 200 kDa. RRKR, a cleavage motif of subtilisin-like proprotein convertases, was identified as the natural cleavage site. In vitro digestion experiments with proteinase K suggest a structural model for DiPA consisting of protease resistant cores joined by protease sensitive linkers containing the RRKR site. This motif is absent between DiPA-TR3 and TR4 and has been altered to KR between DiPA-TR4 and TR5. An immunoblot of D. immitis extract probed with anti-DiPA-TR4/5 serum demonstrates the absence of cleavage at these sites. These divergent repeats provide an opportunity to investigate processing of the D. immitis polyprotein in vivo. PMID:8943150

  1. Role of cardiac troponin I carboxy terminal mobile domain and linker sequence in regulating cardiac contraction.

    PubMed

    Meyer, Nancy L; Chase, P Bryant

    2016-07-01

    Inhibition of striated muscle contraction at resting Ca(2+) depends on the C-terminal half of troponin I (TnI) in thin filaments. Much focus has been on a short inhibitory peptide (Ip) sequence within TnI, but structural studies and identification of disease-associated mutations broadened emphasis to include a larger mobile domain (Md) sequence at the C-terminus of TnI. For Md to function effectively in muscle relaxation, tight mechanical coupling to troponin's core-and thus tropomyosin-is presumably needed. We generated recombinant, human cardiac troponins containing one of two TnI constructs: either an 8-amino acid linker between Md and the rest of troponin (cTnILink8), or an Md deletion (cTnI1-163). Motility assays revealed that Ca(2+)-sensitivity of reconstituted thin filament sliding was markedly increased with cTnILink8 (∼0.9 pCa unit leftward shift of speed-pCa relation compared to WT), and increased further when Md was missing entirely (∼1.4 pCa unit shift). Cardiac Tn's ability to turn off filament sliding at diastolic Ca(2+) was mostly (61%), but not completely eliminated with cTnI1-163. TnI's Md is required for full inhibition of unloaded filament sliding, although other portions of troponin-presumably including Ip-are also necessary. We also confirm that TnI's Md is not responsible for superactivation of actomyosin cycling by troponin. PMID:26971468

  2. N-terminus regulation of VMAT2 mediates methamphetamine stimulated efflux

    PubMed Central

    Torres, Brian; Ruoho, Arnold E.

    2014-01-01

    The 20 amino acid N-terminus of the vesicular monoamine transporter 2 (VMAT2) was examined as a regulator of VMAT2 function. Removal of the first 16 or 19 amino acids of the N-terminus resulted in a molecule with reduced ability to sequester [3H]-5HT. A GST-construct of the N-terminus underwent phosphorylation in the presence of PKC at serines 15 and 18. These putative phosphorylation sites were examined for effects on function. Phospho-mimetic substitution of serines 15 and 18 with aspartate in the full-length VMAT2 resulted in reduced [3H]-5HT sequestration and reduced methamphetamine-stimulated efflux of preloaded [3H]-5HT. In contrast, mutation of serines 15 and 18 to alanines maintained intact net substrate sequestration but eliminated methamphetamine-stimulated efflux of pre-accumulated [3H]-5HT. In summary, these data suggest a model in which the VMAT2 N-terminus regulates monoamine sequestration. PMID:24321511

  3. Glaciological and marine geological controls on terminus dynamics of Hubbard Glacier, southeast Alaska

    NASA Astrophysics Data System (ADS)

    Stearns, L. A.; Hamilton, G. S.; van der Veen, C. J.; Finnegan, D. C.; O'Neel, S.; Scheick, J. B.; Lawson, D. E.

    2015-06-01

    Hubbard Glacier, located in southeast Alaska, is the world's largest nonpolar tidewater glacier. It has been steadily advancing since it was first mapped in 1895; occasionally, the advance creates an ice or sediment dam that blocks a tributary fjord (Russell Fiord). The sustained advance raises the probability of long-term closure in the near future, which will strongly impact the ecosystem of Russell Fiord and the nearby community of Yakutat. Here, we examine a 43 year record of flow speeds and terminus position to understand the large-scale dynamics of Hubbard Glacier. Our long-term record shows that the rate of terminus advance has increased slightly since 1895, with the exception of a slowed advance between approximately 1972 and 1984. The short-lived closure events in 1986 and 2002 were not initiated by perturbations in ice velocity or environmental forcings but were likely due to fluctuations in sedimentation patterns at the terminus. This study points to the significance of a coupled system where short-term velocity fluctuations and morainal shoal development control tidewater glacier terminus position.

  4. 29 CFR Appendix C to Subpart R of... - Illustrations of Bridging Terminus Points: Non-mandatory

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 8 2013-07-01 2013-07-01 false Illustrations of Bridging Terminus Points: Non-mandatory C Appendix C to Subpart R of Part 1926 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY... CONSTRUCTION Steel Erection Pt. 1926, Subpt. R, App. C Appendix C to Subpart R of Part 1926—Illustrations...

  5. 29 CFR Appendix C to Subpart R of... - Illustrations of Bridging Terminus Points: Non-mandatory

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 8 2012-07-01 2012-07-01 false Illustrations of Bridging Terminus Points: Non-mandatory C Appendix C to Subpart R of Part 1926 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY... CONSTRUCTION Steel Erection Pt. 1926, Subpt. R, App. C Appendix C to Subpart R of Part 1926—Illustrations...

  6. 29 CFR Appendix C to Subpart R of... - Illustrations of Bridging Terminus Points: Non-mandatory

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 8 2011-07-01 2011-07-01 false Illustrations of Bridging Terminus Points: Non-mandatory C Appendix C to Subpart R of Part 1926 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY... CONSTRUCTION Steel Erection Pt. 1926, Subpt. R, App. C Appendix C to Subpart R of Part 1926—Illustrations...

  7. The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity

    SciTech Connect

    Chang, Chung-ke; Wu, Tzong-Huah; Wu, Chu-Ya; Chiang, Ming-hui; Toh, Elsie Khai-Woon; Hsu, Yin-Chih; Lin, Ku-Feng; Liao, Yu-heng; Huang, Tai-huang; Huang, Joseph Jen-Tse

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer The N-terminus of TDP-43 contains an independently folded structural domain (NTD). Black-Right-Pointing-Pointer The structural domains of TDP-43 are arranged in a beads-on-a-string fashion. Black-Right-Pointing-Pointer The NTD promotes TDP-43 oligomerization in a concentration-dependent manner. Black-Right-Pointing-Pointer The NTD may assist nucleic acid-binding activity of TDP-43. -- Abstract: TDP-43 is a DNA/RNA-binding protein associated with different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). Here, the structural and physical properties of the N-terminus on TDP-43 have been carefully characterized through a combination of nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence anisotropy studies. We demonstrate for the first time the importance of the N-terminus in promoting TDP-43 oligomerization and enhancing its DNA-binding affinity. An unidentified structural domain in the N-terminus is also disclosed. Our findings provide insights into the N-terminal domain function of TDP-43.

  8. Glaciological and marine geological controls on terminus dynamics of Hubbard Glacier, southeast Alaska

    USGS Publications Warehouse

    Stearns, Leigh A.; Hamilton, Gordon S.; van der Veen, C. J.; Finnegan, D. C.; O'Neel, Shad; Scheick, J. B.; Lawson, D. E.

    2015-01-01

    Hubbard Glacier, located in southeast Alaska, is the world's largest non-polar tidewater glacier. It has been steadily advancing since it was first mapped in 1895; occasionally, the advance creates an ice or sediment dam that blocks a tributary fjord (Russell Fiord). The sustained advance raises the probability of long-term closure in the near-future, which will strongly impact the ecosystem of Russell Fiord and the nearby community of Yakutat. Here, we examine a 43-year record of flow speeds and terminus position to understand the large-scale dynamics of Hubbard Glacier. Our long-term record shows that the rate of terminus advance has increased slightly since 1895, with the exception of a slowed advance between approximately 1972 and 1984. The short-lived closure events in 1986 and 2002 were not initiated by perturbations in ice velocity or environmental forcings, but were likely due to fluctuations in sedimentation patterns at the terminus. This study points to the significance of a coupled system where short-term velocity fluctuations and morainal shoal development control tidewater glacier terminus position.

  9. Subaqueous terminus evolution at Tasman Glacier, New Zealand, as determined by remote-controlled survey

    NASA Astrophysics Data System (ADS)

    Purdie, Heather; Bealing, Paul; Tidey, Emily; Harrison, Justin

    2016-04-01

    The presence of subaqueous ice ramps at the terminus of calving glaciers result from a combination of subaerial and subaqueous processes. These ice ramps eventually buoyantly calve, an event that can be hazardous to companies operating boat tours on proglacial lakes. However our knowledge of ice ramp forming processes, and feedbacks associated with their evolution, is sparse. We are using a remote controlled jet boat to survey bathymetry at an active calving margin. This vessel, mounted with both depth and side-scan sonar, can map subaqueous portions of the terminus right up to the active calving face at no risk to the operators. Surveys at the Tasman Glacier terminus over three consecutive years have revealed that subaqueous ice ramps are ephemeral features. In 2015 multiple ice ramps extended out into the lake from the terminus by 100-200 m, with the ramp surface being as much as 60 m below the water line at its outer perimeter. The maximum depth of the Tasman Lake at this time was 240 m. Within one month of the survey taking place, the largest of these ice ramps had calved and disintegrated. The consistent location of ice ramps between surveys indicates that other factors, like subglacial hydrology, may influence ice ramp evolution.

  10. The Inhibitory Helix Controls the Intramolecular Conformational Switching of the C-Terminus of STIM1

    PubMed Central

    Lin, Zhijie; Wang, Zheng; Dong, Cheng; Shen, Yuequan

    2013-01-01

    Store-operated Ca2+ entry (SOCE) is a critical Ca2+ signaling pathway in many cell types. After sensing Ca2+ store depletion in the endoplasmic reticulum (ER) lumen, STIM1 (STromal Interaction Molecule 1) oligomerizes and then interacts with and activates the Orai1 calcium channel. Our previous research has demonstrated that the inhibitory helix (IH) adjacent to the first coiled-coil region (CC1) of STIM1 may keep the whole C-terminus of STIM1 in an inactive state. However, the specific conformational change of CC1-IH that drives the transition of STIM1 from the resting state to the active state remains elusive. Herein, we report the structural analysis of CC1-IH, which revealed that the entire CC1-IH molecule forms a very long helix. Structural and biochemical analyses indicated that IH, and not the CC1 region, contributes to the oligomerization of STIM1. Small-angle X-ray scattering (SAXS) analysis suggested that the C-terminus of STIM1 including the IH region displays a collapsed conformation, whereas the construct without the IH region has an extended conformation. These two conformations may correspond to the conformational states of the C-terminus of STIM1 before and after activation. Taken together, our results provide direct biochemical evidence that the IH region controls the conformational switching of the C-terminus of STIM1. PMID:24069340

  11. Controls on interannual and seasonal terminus velocity and position of Yahtse Glacier in SE Alaska

    NASA Astrophysics Data System (ADS)

    Durkin, W. J., IV; Melkonian, A. K.; Pritchard, M. E.; Willis, M. J.; Bartholomaus, T.

    2015-12-01

    We construct a 30 year velocity time-series for comparison with recent studies on the submarine melt rate (Bartholomaus et al., 2013), calving rate (Bartholomaus et al., 2013b), velocities (McNabb et al., 2014), and subglacial discharge (Bartholomaus et al., 2015) of Yahtse Glacier in southeast Alaska. Velocities are constructed from feature tracking on Landsat, ALOS, and ASTER satellite imagery spanning 1985-2015. Yahtse is undergoing an interannual advance of ~82 m yr-1 that is concurrent with deceleration between 1996 and 2015 of -0.55 m day-1yr-1 measured 2.5km down-glacier from the icefall. We estimate that up to 35% of the slowdown is due to divergence associated with thickening near the terminus of ~7 m yr-1measured by differencing WorldView and SRTM DEMs. Much of the remaining deceleration may be due to greater basal and lateral drag as ongoing advance increases the contact area between the terminus and bedrock. We observe a seasonal cycle in centerline terminus speeds superimposed on the interannual deceleration. Terminus speeds climb from a minimum in October to a maximum in May, then decline until October. The timing of this cycle is in phase with the seasonality of subglacial discharge at the front of Yahtse and salinity levels measured in the Gulf of Alaska, which agrees with models of subglacial channel development proposed for many glaciers. Seasonal speed changes measured 1 km up-glacier from the front are associated with terminus advance and retreat. The terminus is in a retracted position following the deceleration to a minimum speed in October and elevated submarine melt rates in summer and early autumn. The front holds this position from November through February as speeds there accelerate to their seasonal maximum and submarine melt is reduced. Yahtse Glacier then advances between 200 and 500 m during the spring as frontal speeds decrease by ~10% from their highest level. This slowdown may be caused by a decrease in buoyancy due to the terminus

  12. Individual Substitution Mutations in the AID C Terminus That Ablate IgH Class Switch Recombination

    PubMed Central

    Kadungure, Tatenda; Ucher, Anna J.; Linehan, Erin K.; Schrader, Carol E.; Stavnezer, Janet

    2015-01-01

    Activation-induced cytidine deaminase (AID) is essential for class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The C terminus of AID is required for CSR but not for SHM, but the reason for this is not entirely clear. By retroviral transduction of mutant AID proteins into aid-/- mouse splenic B cells, we show that 4 amino acids within the C terminus of mouse AID, when individually mutated to specific amino acids (R190K, A192K, L196S, F198S), reduce CSR about as much or more than deletion of the entire C terminal 10 amino acids. Similar to ΔAID, the substitutions reduce binding of UNG to Ig Sμ regions and some reduce binding of Msh2, both of which are important for introducing S region DNA breaks. Junctions between the IgH donor switch (S)μ and acceptor Sα regions from cells expressing ΔAID or the L196S mutant show increased microhomology compared to junctions in cells expressing wild-type AID, consistent with problems during CSR and the use of alternative end-joining, rather than non-homologous end-joining (NHEJ). Unlike deletion of the AID C terminus, 3 of the substitution mutants reduce DNA double-strand breaks (DSBs) detected within the Sμ region in splenic B cells undergoing CSR. Cells expressing these 3 substitution mutants also have greatly reduced mutations within unrearranged Sμ regions, and they decrease with time after activation. These results might be explained by increased error-free repair, but as the C terminus has been shown to be important for recruitment of NHEJ proteins, this appears unlikely. We hypothesize that Sμ DNA breaks in cells expressing these C terminus substitution mutants are poorly repaired, resulting in destruction of Sμ segments that are deaminated by these mutants. This could explain why these mutants cannot undergo CSR. PMID:26267846

  13. Regioselective syntheses of [13C]4-labelled sodium 1-carboxy-2-(2-ethylhexyloxycarbonyl)ethanesulfonate and sodium 2-carboxy-1-(2-ethylhexyloxycarbonyl)ethanesulfonate from [13C]4-maleic anhydride.

    PubMed

    Barsamian, Adam L; Perkins, Matt J; Field, Jennifer A; Blakemore, Paul R

    2014-05-15

    The entitled monohydrolysis products, also known as α-ethylhexyl and β-ethylhexyl sulfosuccinate (EHSS), of the surfactant diisooctyl sulfosuccinate (DOSS) were synthesized in stable isotope-labelled form from [(13)C]4 -maleic anhydride. Sodium [(13)C]4 -1-carboxy-2-(2-ethylhexyloxycarbonyl)ethanesulfonate (α-EHSS) was prepared by the method of Larpent by reaction of 2-ethylhexan-1-ol with [(13)C]4 -maleic anhydride followed by regioselective conjugate addition of sodium bisulfite to the resulting monoester (38% overall yield). The regiochemical outcome of bisulfite addition was confirmed by a combination of (13)C/(13)C (incredible natural abundance double quantum transfer) and (1)H/(13)C (heteronuclear multiple-bond correlation (HMBC)) NMR spectral correlation experiments. Sodium [(13)C]4 -2-carboxy-1-(2-ethylhexyloxycarbonyl)ethanesulfonate (β-EHSS) was prepared in four steps by reaction of 4-methoxybenzyl alcohol with [(13)C]4 -maleic anhydride, regioselective sodium bisulfite addition, N,N'-dicyclohexylcarbodiimide-mediated esterification with 2-ethylhexan-1-ol, and p-methoxybenzyl ester deprotection with trifluoroacetic acid (13% overall yield). The regiochemical outcome of the second synthesis was confirmed by a combination of (1)JCC scalar coupling constant analysis and (1)H/(13)C (HMBC) NMR spectral correlation. The materials prepared are required as internal standards for the liquid chromatography-mass spectrometry (LC-MS)/MS trace analysis of the degradation products of DOSS, the anionic surfactant found in Corexit, the oil dispersant used during emergency response efforts connected to the Deepwater Horizon oil spill of April 2010. PMID:24700711

  14. Upregulation of des-gamma-carboxy-prothrombin after portal vein embolization in a cirrhotic patient with hepatocellular carcinoma.

    PubMed

    Sohda, Tetsuro; Iwata, Kaoru; Anan, Akira; Kunimoto, Hideo; Yotsumoto, Kaoru; Yokoyama, Keiji; Morihara, Daisuke; Takeyama, Yasuaki; Shakado, Satoshi; Osame, Akinobu; Kora, Shinichi; Ohishi, Jun; Yamauchi, Yasushi; Noritomi, Tomoaki; Yoshimitsu, Kengo; Yamashita, Yuichi; Sakisaka, Shotaro

    2015-10-01

    A 73-year-old female with hepatocellular carcinoma (HCC) received percutaneous transhepatic portal vein embolization (PTPE) before extensive right lobe hepatectomy. Serum levels of des-gamma-carboxy-prothrombin (DCP) were increased and remained at a high level until hepatectomy. Immunohistochemical examination revealed that an increased expression of DCP was demonstrated not only in HCC tissues, but also in the non-cancerous liver of the right lobe, where portal blood flow was blocked off as a result of PTPE. The serum level of DCP is known to be greatly increased in patients with HCC accompanied by portal vein invasion. We speculate that this increased DCP level is caused by both increased DCP production in HCC tissue and the surrounding non-cancerous liver, where portal flow is blocked off as a result of portal invasion by HCC. PMID:26374567

  15. A key role for the carboxy-terminal tail of the murine coronavirus nucleocapsid protein in coordination of genome packaging.

    PubMed

    Kuo, Lili; Koetzner, Cheri A; Masters, Paul S

    2016-07-01

    The prototype coronavirus mouse hepatitis virus (MHV) exhibits highly selective packaging of its genomic positive-stranded RNA into assembled virions, despite the presence in infected cells of a large excess of subgenomic viral mRNAs. One component of this selectivity is the MHV packaging signal (PS), an RNA structure found only in genomic RNA and not in subgenomic RNAs. It was previously shown that a major determinant of PS recognition is the second of the two RNA-binding domains of the viral nucleocapsid (N) protein. We have now found that PS recognition additionally depends upon a segment of the carboxy-terminal tail (domain N3) of the N protein. Since domain N3 is also the region of N protein that interacts with the membrane (M) protein, this finding suggests a mechanism by which selective genome packaging is accomplished, through the coupling of genome encapsidation to virion assembly. PMID:27105451

  16. Identification of functionally important negatively charged residues in the carboxy end of mouse hepatitis coronavirus A59 nucleocapsid protein.

    PubMed

    Verma, Sandhya; Bednar, Valerie; Blount, Andrew; Hogue, Brenda G

    2006-05-01

    The coronavirus nucleocapsid (N) protein is a multifunctional viral gene product that encapsidates the RNA genome and also plays some as yet not fully defined role in viral RNA replication and/or transcription. A number of conserved negatively charged amino acids are located within domain III in the carboxy end of all coronavirus N proteins. Previous studies suggested that the negatively charged residues are involved in virus assembly by mediating interaction between the membrane (M) protein carboxy tail and nucleocapsids. To determine the importance of these negatively charged residues, a series of alanine and other charged-residue substitutions were introduced in place of those in the N gene within a mouse hepatitis coronavirus A59 infectious clone. Aspartic acid residues 440 and 441 were identified as functionally important. Viruses could not be isolated when both residues were replaced by positively charged amino acids. When either amino acid was replaced by a positively charged residue or both were changed to alanine, viruses were recovered that contained second-site changes within N, but not in the M or envelope protein. The compensatory role of the new changes was confirmed by the construction of new viruses. A few viruses were recovered that retained the D441-to-arginine change and no compensatory changes. These viruses exhibited a small-plaque phenotype and produced significantly less virus. Overall, results from our analysis of a large panel of plaque-purified recovered viruses indicate that the negatively charged residues at positions 440 and 441 are key residues that appear to be involved in virus assembly. PMID:16611893

  17. Structure of the human MLH1 N-terminus: implications for predisposition to Lynch syndrome

    SciTech Connect

    Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Kerr, Iain D.; Min, Jinrong

    2015-07-28

    The crystal structure of the human MLH1 N-terminus is reported at 2.30 Å resolution. The overall structure is described along with an analysis of two clinically important mutations. Mismatch repair prevents the accumulation of erroneous insertions/deletions and non-Watson–Crick base pairs in the genome. Pathogenic mutations in the MLH1 gene are associated with a predisposition to Lynch and Turcot’s syndromes. Although genetic testing for these mutations is available, robust classification of variants requires strong clinical and functional support. Here, the first structure of the N-terminus of human MLH1, determined by X-ray crystallography, is described. The structure shares a high degree of similarity with previously determined prokaryotic MLH1 homologs; however, this structure affords a more accurate platform for the classification of MLH1 variants.

  18. Cellular location of origin and terminus of replication in Bacillus subtilis.

    PubMed Central

    Sonnenfeld, E M; Koch, A L; Doyle, R J

    1985-01-01

    The origin of replication of Bacillus subtilis 168 trp thy dna-1 (temperature-sensitive initiation mutant) was labeled with [3H]thymidine. Analysis of labeled cells by autoradiography revealed that most of the radioactivity was associated with cell pole areas. To label the terminus, cells that had initiated were treated with chloramphenicol to inhibit cell growth and division but to allow continued DNA synthesis. These cells were then labeled with [3H]thymidine at a time when chromosome replication was nearly complete. The distribution of radioactivity was similar to that observed in origin-labeled cells. In contrast, exponentially growing cells that were labeled for a brief time at the permissive temperature showed a random distribution of radioactivity. These data indicate that the origin and terminus of replication are located at cell poles. Images PMID:3928600

  19. Short Period Velocity Response to Tides and Calving Near the Terminus of Jakobshavn Isbrae

    NASA Astrophysics Data System (ADS)

    Podrasky, D. B.; Truffer, M.; Fahnestock, M. A.; Luethi, M. P.

    2010-12-01

    The loss of the floating ice tongue on Jakobshavn Isbrae in the early 2000s has been concurrent with a pattern of thinning, retreat, and acceleration. In addition to these longer term changes, we are trying to understand the glacier response on shorter time scales, such as tidal forcing and calving events. During a twelve day period in August 2009, we used optical surveys, GPS techniques, cameras, and seismometers to document changes in terminus geometry and speed. Optical survey targets were placed 1-2 km from the ice front and single and dual frequency GPS were deployed 2-4 km upstream of the terminus. We observe how ice flow responds to tidal forcing at the front, particularly how the strength of the response decays upstream with a characteristic length scale of a few ice thicknesses. Also captured in the time series is a step increase in velocity of the glacier during a large calving event.

  20. C-Terminus of the B-Chain of Relaxin-3 Is Important for Receptor Activity

    PubMed Central

    Shabanpoor, Fazel; Bathgate, Ross A. D.

    2013-01-01

    Human relaxin-3 is a neuropeptide that is structurally similar to human insulin with two chains (A and B) connected by three disulfide bonds. It is expressed primarily in the brain and has modulatory roles in stress and anxiety, feeding and metabolism, and arousal and behavioural activation. Structure-activity relationship studies have shown that relaxin-3 interacts with its cognate receptor RXFP3 primarily through its B-chain and that its A-chain does not have any functional role. In this study, we have investigated the effect of modification of the B-chain C-terminus on the binding and activity of the peptide. We have chemically synthesised and characterized H3 relaxin as C-termini acid (both A and B chains having free C-termini; native form) and amide forms (both chains’ C-termini were amidated). We have confirmed that the acid form of the peptide is more potent than its amide form at both RXFP3 and RXFP4 receptors. We further investigated the effects of amidation at the C-terminus of individual chains. We report here for the first time that amidation at the C-terminus of the B-chain of H3 relaxin leads to significant drop in the binding and activity of the peptide at RXFP3/RXFP4 receptors. However, modification of the A-chain C-terminus does not have any effect on the activity. We have confirmed using circular dichroism spectroscopy that there is no secondary structural change between the acid and amide form of the peptide, and it is likely that it is the local C-terminal carboxyl group orientation that is crucial for interacting with the receptors. PMID:24349312

  1. The C-terminus of p53 binds the N-terminal domain of MDM2

    PubMed Central

    Poyurovsky, Masha V.; Katz, Chen; Laptenko, Oleg; Beckerman, Rachel; Lokshin, Maria; Ahn, Jinwoo; Byeon, In-Ja L.; Gabizon, Ronen; Mattia, Melissa; Zupnick, Andrew; Brown, Lewis M.; Friedler, Assaf; Prives, Carol

    2010-01-01

    The p53 tumor suppressor interacts with its negative regulator Mdm2 via the former’s N-terminal region and core domain. Yet the extreme p53 C-terminal region contains lysine residues ubiquitinated by Mdm2 and can bear post-translational modifications that inhibit Mdm2–p53 association. We show that, the Mdm2–p53 interaction is decreased upon deletion, mutation or acetylation of the p53 C-terminus. Mdm2 decreases the association of full-length but not C-terminally deleted p53 with a DNA target sequence in vitro and in cells. Further, using multiple approaches we demonstrate that a peptide from p53 C-terminus directly binds Mdm2 N-terminus in vitro. We also show that p300-acetylated p53 binds inefficiently to Mdm2 in vitro, and Nutlin-3 treatment induces C-terminal modification(s) of p53 in cells, explaining the low efficiency of Nutlin-3 in dissociating p53-MDM2 in vitro. PMID:20639885

  2. Cadherin adhesion depends on a salt bridge at the N-terminus.

    PubMed

    Harrison, Oliver J; Corps, Elaine M; Kilshaw, Peter J

    2005-09-15

    There is now considerable evidence that cell adhesion by cadherins requires a strand exchange process in which the second amino acid at the N-terminus of the cadherin molecule, Trp2, docks into a hydrophobic pocket in the domain fold of the opposing cadherin. Here we show that strand exchange depends on a salt bridge formed between the N-terminal amino group of one cadherin molecule and the acidic side chain of Glu89 of the other. Prevention of this bond in N-cadherin by introducing the mutation Glu89Ala or by extending the N-terminus with additional amino acids strongly inhibited strand exchange. But when the two modifications were present in opposing cadherin molecules respectively, they acted in a complementary manner, lowering activation energy for strand exchange and greatly increasing the strength of the adhesive interaction. N-cadherin that retained an uncleaved prodomain or lacked Trp2 adhered strongly to the Glu89Ala mutant but not to wild-type molecules. Similarly, N-cadherin in which the hydrophobic acceptor pocket was blocked by an isoleucine side chain adhered to a partner that had an extended N-terminus. We explain these results in terms of the free energy changes that accompany strand exchange. Our findings provide new insight into the mechanism of adhesion and demonstrate the feasibility of greatly increasing cadherin affinity. PMID:16118243

  3. Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus.

    PubMed

    Li, R; Liu, T; Wong, B S; Pan, T; Morillas, M; Swietnicki, W; O'Rourke, K; Gambetti, P; Surewicz, W K; Sy, M S

    2000-08-18

    We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP(C)) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP. The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP(23-145), indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP. The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP(90-231). Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP(23-145) and PrP(90-231), suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145. By Western blotting with PepSpot(TM), only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes. Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein. PMID:10966770

  4. Location of the Bacteriophage P22 Coat Protein C-terminus Provides Opportunities for the Design of Capsid Based Materials

    PubMed Central

    Servid, Amy; Jordan, Paul; O’Neil, Alison; Prevelige, Peter; Douglas, Trevor

    2013-01-01

    Rational design of modifications to the interior and exterior surfaces of virus-like particles (VLPs) for future therapeutic and materials applications is based on structural information about the capsid. Existing cryo-electron microscopy based models suggest that the C-terminus of the bacteriophage P22 coat protein (CP) extends towards the capsid exterior. Our biochemical analysis through genetic manipulations of the C-terminus supports the model where the CP C-terminus is exposed on the exterior of the P22 capsid. Capsids displaying a 6xHis tag appended to the CP C-terminus bind to a Ni affinity column, and the addition of positively or negatively charged coiled coil peptides to the capsid results in association of these capsids upon mixing. Additionally, a single cysteine appended to the CP C-terminus results in the formation of intercapsid disulfide bonds and can serve as a site for chemical modifications. Thus, the C-terminus is a powerful location for multivalent display of peptides that facilitate nanoscale assembly and capsid modification. PMID:23957641

  5. Discovery of pyrazole as C-terminus of selective BACE1 inhibitors.

    PubMed

    Zou, Yiquan; Xu, Lei; Chen, Wuyan; Zhu, Yiping; Chen, Tiantian; Fu, Yan; Li, Li; Ma, Lanping; Xiong, Bing; Wang, Xin; Li, Jian; He, Jianhua; Zhang, Haiyan; Xu, Yechun; Li, Jia; Shen, Jingkang

    2013-10-01

    We recently discovered and reported dual inhibitor 5 of AChE and BACE1 with N-benzylpiperidine ethyl as C-terminus. Compound 5 showed potent inhibitory activities for BACE1, and could reduce endogenous Aβ1-40 production in APP transgenic mice. In present work, we rapidly identified substituted triazole as the C-terminus of compound 5 by replacing the benzylpiperidine ethyl group with click chemistry and tested these synthesized compounds by in situ screening assay. As revealed by the crystal structures of BACE1 in complex with our triazole compound 12, we found that Pro70 and Thr72 located in the flap region were the critical components for binding with these inhibitors. With the aid of the crystal structure, a new series of five-membered heterocyclic compounds was prepared in order to explore the structure-activity relationship (SAR) of this class of molecules. From these efforts, pyrazole was discovered as a novel C-terminus of BACE1 inhibitors. After further modification of pyrazole with variable substituents, compound 37 exhibited good potency in enzyme inhibition assay (IC50=0.025 μM) and compound 33 showed moderate inhibition effects on Aβ production of APP transfected HEK293 cells. Moreover, these pyrazole derivatives demonstrated good selectivity versus cathepsin D. Our results indicated that the vicinity of Pro70 and Thr72 might be utilized as a subsite, and the discovered pyrazole derivatives might provide useful hints for developing novel BACE1 inhibitors as anti-AD drugs. PMID:23988410

  6. C-Terminus Glycans with Critical Functional Role in the Maturation of Secretory Glycoproteins

    PubMed Central

    Petrescu, Andrei-Jose; Petrescu, Stefana M.

    2011-01-01

    The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs - one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I. PMID:21625599

  7. Synthesis of 3-amino-1-carboxy-o-carborane and an improved, general method for the synthesis of all three C-amino-C-carboxycarboranes

    SciTech Connect

    Kasar, R.A.; Knudsen, G.M.; Kahl, S.B.

    1999-06-14

    Amino acids of the polyhedral carboranes have potential applications in boron neutron capture therapy and in other areas of bioorganic chemistry, but simple, general methods for their synthesis are nonexistent. A general method for synthesis of C-amino-C-carboxy derivatives of o-, m-, and p-carborane is reported, starting from their respective monoacids and proceeding through nucleophilic attack by an alcohol on the intermediate C-isocyanates. Deprotection of the resulting carbamates provides a simple method for access to the C-amines. Alternatively, the C-isocyanates can be isolated for further reactions. Carbonylation of the carbamates at the remaining carboranyl CH results in high-yield production of the carbamate-protected amino acid. Another related method for the high yield preparation of the isomeric 3-amino-1-carboxy-o-carborane is also described which makes available for the first time all four reasonably accessible members of the series.

  8. Tool for Insertion of a Fiber-Optic Terminus in a Connector

    NASA Technical Reports Server (NTRS)

    King, Wes; Domonoske, Donald J.; Krier, John; White, John

    2004-01-01

    A tool has been developed for the special purpose of inserting the terminus of an optical fiber in a cable connector that conforms to NASA Specification SSQ- 21635. What prompted the development of the tool was the observation that because of some aspects of the designs of fiber-optic termini and of springs, sealing rings, and a grommet inside the shell of such a connector, there is a tendency for the grommet to become damaged and detached from the sealing rings during installation. It is necessary to ensure the integrity of the grommet for proper sealing and proper functioning of the connector. The special-purpose tool provides the needed protection for the grommet. The grommet-protection tool resembles a funnel into which an axial slit has been cut (see figure). Prior to insertion, the grommet-protection tool is rolled so that one side of the slit overlaps the other side. The rolled-up grommet-protection tool is inserted in one of the connector holes that accommodate the fiber-optic termini and is pushed in until the flange (the wider of the two conical portions) of the tool becomes seated on the connector grommet. Then a special-purpose installation tool is inserted in the flange of the grommet-protection tool and pressed in until it becomes seated in the flange. This operation expands the narrower of the two conical portions of the grommet-protection tool. The installation tool is removed and the grommet-protection tool remains expanded due to the flat surfaces on the axial slit. By use of a standard contact-insertion tool, a fiber-optic terminus is inserted, through the grommet-protection tool, into the connector cavity. By use of a pair of forceps or needle-nose pliers, the grommet-protection tool is then pulled out of the cavity. Finally, the grommet-protection tool is removed from around the installed fiber-optic cable by pulling the cable through the axial slit. Unlike in some prior procedures for installing the fiber-optic termini in the connector, the

  9. The role of the Cx43 C-terminus in GJ plaque formation and internalization

    SciTech Connect

    Wayakanon, Praween; Bhattacharjee, Rajib; Nakahama, Ken-ichi; Morita, Ikuo

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Cx43-GFP or -DsRed fusion proteins were expressed in HeLa cells. Black-Right-Pointing-Pointer Roles of C-terminus were examined using various mutants. Black-Right-Pointing-Pointer Gap junction plaque size was dependent on the length of C-terminus. Black-Right-Pointing-Pointer C-terminus dependent gap junction plaque internalization was observed. -- Abstract: Connexin 43 (Cx43) is a major gap junction (GJ) protein found in many mammalian cell types. The C-terminal (CT) domain of Cx43 has unique characteristics in terms of amino acid (aa) sequence and its length differs from other connexins. This CT domain can be associated with protein partners to regulate GJ assembly and degradation, which results in the direct control of gap junction intercellular communication (GJIC). However, the essential roles of the CT regions involved in these mechanisms have not been fully elucidated. In this study, we aimed to investigate the specific regions of Cx43CT involved in GJ formation and internalization. Wild type Cx43{sub (382aa)} and 10 CT truncated mutants were stably expressed in HeLa cells as GFP or DsRed tagged proteins. First, we found that the deletion of 235-382aa from Cx43 resulted in failure to make GJ and establish GJIC. Second, the Cx43 with 242-382aa CT deletion could form functional GJs and be internalized as annular gap junctions (AGJs). However, the plaques consisting of Cx43 with CT deletions ({Delta}242-382aa to {Delta}271-382aa) were longer than the plaques consisting of Cx43 with CT deletions ({Delta}302-382aa). Third, co-culture experiments of cells expressing wild type Cx43{sub (382)} with cells expressing Cx43CT mutants revealed that the directions of GJ internalization were dependent on the length of the respective CT. Moreover, a specific region, 325-342aa residues of Cx43, played an important role in the direction of GJ internalization. These results showed the important roles of the Cx43 C-terminus in GJ

  10. Elevated Expression of Carboxy-Terminal Modulator Protein (CTMP) Aggravates Brain Ischemic Injury in Diabetic db/db Mice.

    PubMed

    Chen, Yu; Cai, Min; Deng, Jiao; Tian, Li; Wang, Shiquan; Tong, Li; Dong, Hailong; Xiong, Lize

    2016-09-01

    Deregulation of Akt signaling is important in the brain injuries caused by cerebral ischemia in diabetic animals, and the underlying mechanism is not fully understood. We investigated the role of carboxy-terminal modulator protein (CTMP), an endogenous Akt inhibitor, in brain injury following focal cerebral ischemia in type 2 diabetic db/db mice and their control littermates non-diabetic db/+ mice. db/db mice showed a significant elevation in the expression of CTMP compared to db/+ mice under normal physiological conditions. After ischemia, db/db mice exhibit higher levels of CTMP expression, decreased Akt kinase activity, adverse neurological deficits and cerebral infarction than db/+ mice. To further certain the effectiveness of Akt signaling to the final outcome of cerebral ischemia, the animals were treated with LY294002, an inhibitor of the Akt pathway, which aggravated the ischemic injury in db/+ mice but not in db/db mice. RNA interference-mediated depletion of CTMP were finally applied in db/db mice, which restored Akt activity, improved neurological scores and reduced infarct volume. These results suggest that elevation of CTMP in diabetic mice suppresses Akt activity and ultimately negatively affects the outcome of ischemia. Inhibitors specifically targeting CTMP may be beneficial in the treatment of cerebral ischemia in patients with diabetes. PMID:27161366

  11. Des-γ-carboxy prothrombin (DCP) as a potential autologous growth factor for the development of hepatocellular carcinoma.

    PubMed

    Zhang, Yu-Sheng; Chu, Jia-Hui; Cui, Shu-Xiang; Song, Zhi-Yu; Qu, Xian-Jun

    2014-01-01

    Des-γ-carboxy prothrombin (DCP) is a prothrombin precursor produced in hepatocellular carcinoma (HCC). Because of deficiency of vitamin K or γ-glutamyl carboxylase in HCC cells, the 10 glutamic acid (Glu) residues in prothrombin precursor did not completely carboxylate to γ-carboxylated glutamic acid (Gla) residues, leaving some Glu residues remained in N-terminal domain. These prothrombin precursors with Glu residues are called DCPs. DCP displays insufficient coagulation activity. Since Liebman reported an elevated plasma DCP in patients with HCC, DCP has been used in the diagnosis of HCC. Recently, its biological malignant potential has been specified to describe DCP as an autologous growth factor to stimulate HCC growth and a paracrine factor to integrate HCC with vascular endothelial cells. DCP was found to stimulate HCC growth through activation of the DCP-Met-JAK1-STAT3 signaling pathway. DCP might increase HCC invasion and metastasis through activation of matrix metalloproteinase (MMPs) and the ERK1/2 MAPK signaling pathway. DCP has also been found to play a crucial role in the formation of angiogenesis. DCP could increase the angiogenic factors released from HCC and vascular endothelial cells. These effects of DCP in angiogenesis might be related to activation of the DCP-KDR-PLC-γ-MAPK signaling pathway. In this article, we summarized recent studies on DCP in biological roles related to cancer progression and angiogenesis in HCC. PMID:25200250

  12. Diagnostic performance of des-γ-carboxy prothrombin (DCP) for hepatocellular carcinoma: a bivariate meta-analysis.

    PubMed

    Gao, P; Li, M; Tian, Q B; Liu, Dian-Wu

    2012-01-01

    Serum markers are needed to be developed to specifically diagnose Hepatocellular carcinoma (HCC). Des-γ-carboxy prothrombin (DCP) is a promising tool with limited expense and widely accessibility, but the reported results have been controversial. In order to review the performance of DCP for the diagnosis of HCC, the meta-analysis was performed. After a systematic review of relevant studies, the sensitivity, specificity, positive and negative likelihood ratios (PLR and NLR, respectively) were pooled using a bivariate meta-analysis. Potential between-study heterogeneity was explored by meta-regression model. The post-test probability and the likelihood ratio scattergram to evaluate clinical usefulness were calculated. Based on literature review of 20 publications, the overall sensitivity, specificity, PLR and NLR of DCP for the detection of HCC were 67% (95%CI, 58%-74%), 92% (95%CI, 88%-94%), 7.9 (95%CI, 5.6-11.2) and 0.36 (95%CI, 0.29-0.46), respectively. The area under the bivariate summary receiving operating characteristics curve was 0.89 (95%CI, 0.85-0.92). Significant heterogeneity was present. In conclusion, the major role of DCP is the moderate confirmation of HCC. More prospective studies of DCP are needed in future. PMID:22248272

  13. The Carboxy-Terminal Domain of Erb1 Is a Seven-Bladed ß-Propeller that Binds RNA

    PubMed Central

    Marcin, Wegrecki; Neira, Jose Luis; Bravo, Jeronimo

    2015-01-01

    Erb1 (Eukaryotic Ribosome Biogenesis 1) protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other β-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis. PMID:25880847

  14. Pharmacokinetic and milk penetration of a difloxacin long-acting poloxamer gel formulation with carboxy-methylcellulose in lactating goats.

    PubMed

    Escudero, Elisa; Marín, Pedro; Cárceles, Carlos M; Ramírez, María J; Fernández-Varón, Emilio

    2011-04-01

    The single-dose disposition kinetics of difloxacin were determined in clinically normal lactating goats (n=6) after subcutaneous administration of a long-acting poloxamer 407 gel formulation with carboxy-methylcellulose (P407-CMC). Difloxacin concentrations were determined by high performance liquid chromatography with fluorescence detection. The concentration-time data were analysed by non-compartmental kinetic methods. Plasma and milk elimination half-lives after P407-CMC dosing were 35.19 h and 33.93 h, respectively. With this formulation, difloxacin achieved maximum plasma concentrations of 2.67±0.34 mg/L at 2.92±1.20 h and maximum milk concentrations of 2.31±0.35 mg/L at 4.00±0.00 h. The area under the curve (AUC) ratio AUC(milk)/AUC(plasma) was 0.89 after P407-CMC administration. It was concluded that a 15 mg/kg dose of difloxacin within P407-CMC would be effective against mastitis pathogens with a minimum inhibitory concentration (MIC)≤0.12 mg/L. PMID:20359917

  15. Nucleation kinetics, growth, crystalline perfection, mechanical, thermal, optical and electrical characterization of brucinium 2-carboxy-6-nitrophthalate dihydrate single crystal

    NASA Astrophysics Data System (ADS)

    Krishnan, P.; Gayathri, K.; Sivakumar, N.; Gunasekaran, S.; Anbalagan, G.

    2014-06-01

    Single crystals of brucinium 2-carboxy-6-nitrophthalate dihydrate (B2C6ND) have been grown by the slow evaporation solution technique at room temperature using water-ethanol (1:1) mixed solvent. The metastable zone width and induction period have been experimentally determined for the growth conditions. Nucleation kinetics and fundamental growth parameters such as surface free energy, critical radius and critical free energy change are also evaluated according to the experimental data. The crystal system and the lattice parameters have been confirmed by single crystal X-ray diffraction. The crystalline perfection of the grown B2C6ND crystals has been characterized by HRXRD method. Optical band gap and Urbach tail width of the sample have been studied employing UV-Vis absorption spectroscopy. The Vickers microhardness number (Hv), yield strength (σv) and stiffness constant (C11) of the grown crystal have been evaluated. The dielectric permittivity and dielectric loss of the grown B2C6ND crystal have been investigated as a function of frequency in the temperature range 313-353 K. The laser damage threshold value of B2C6ND crystal was estimated to be 2.8 GW/cm2 using a Nd:YAG laser.

  16. Growth, spectral, optical, thermal, and mechanical behaviour of an organic single crystal: Quinolinium 2-carboxy 6-nitrophthalate monohydrate

    NASA Astrophysics Data System (ADS)

    Mohana, J.; Ahila, G.; Bharathi, M. Divya; Anbalagan, G.

    2016-09-01

    Organic single crystals of quinolinium 2-carboxy 6-nitrophthalate monohydrate (QN) were grown by slow evaporation solution growth technique using ethanol and water as a mixed solvent. X-ray powder diffraction analysis revealed that the crystal belongs to the monoclinic crystal system with space group of P21/c. The functional groups present in the crystallized material confirmed its molecular structure. The optical transparency range and the lower cutoff wavelength were identified from the UV-vis spectrum. The optical constants were determined by UV-visible transmission spectrum at normal incidence, measured over the 200-700 nm spectral range. The dispersion of the refractive index was discussed in terms of the single-oscillator Wemple and DiDomenico model. The calculated HOMO and LUMO energies show that the charge transfer occur within the molecule. Electronic excitation properties were discussed within the framework of two level model on the basis of an orbital analysis. The nonlinear optical absorption coefficient (β) and nonlinear refraction (n2) of QN was measured by Z-scan technique and reported here. Thermal stability of QN was determined using TGA/DSC curves. Vicker's microhardness studies were carried out on the (1 1 ̅0) plane to understand the mechanical properties of the grown crystal. The microhardness measurements showed a Vickers hardness value as 18.4 kg/mm2 which is comparable to well-known organic crystal, urea.

  17. Synthesis and structure-activity relationships of 2-amino-3-carboxy-4-phenylthiophenes as novel atypical protein kinase C inhibitors

    PubMed Central

    Titchenell, Paul M.; Hollis Showalter, H. D.; Pons, Jean-François; Barber, Alistair J.; Jin, Yafei

    2013-01-01

    Recent evidence suggests atypical protein kinase C (aPKC) isoforms are required for both TNF- and VEGF-induced breakdown of the blood-retinal barrier (BRB) and endothelial permeability to 70kDa dextran or albumin. A chemical library screen revealed a series of novel small molecule phenylthiophene based inhibitors of aPKC isoforms that effectively block permeability in cell culture and in vivo. In an effort to further elucidate the structural requirements of this series of inhibitors, we detail in this study a structure-activity relationship (SAR) built on screening hit 1, which expands on our initial pharmacophore model. The biological activity of our analogues was evaluated in models of bona fide aPKC-dependent signaling including NFκB driven-gene transcription as a marker for an inflammatory response and VEGF/TNF-induced vascular endothelial permeability. The EC50 for the most efficacious inhibitors (6, 32) was in the low nanomolar range in these two cellular assays. Our study demonstrates the key structural elements that confer inhibitory activity and highlights the requirement for electron-donating moieties off the C-4 aryl moiety of the 2-amino-3-carboxy-4-phenylthiophene backbone. These studies suggest that this class has potential for further development into small molecule aPKC inhibitors with therapeutic efficacy in a host of diseases involving increased vascular permeability and inflammation. PMID:23566515

  18. Synthesis of 5-carboxy-6-methyl-3,4-dihydro-2(1H)-pyridone derivatives and their electrochemical oxidation to 2-pyridones

    NASA Astrophysics Data System (ADS)

    Smits, Rufus; Turovska, Baiba; Belyakov, Sergey; Plotniece, Aiva; Duburs, Gunars

    2016-04-01

    A series of variously substituted 5-carboxy-6-methyl-3,4-dihydro-2(1H)-pyridone derivatives were synthesized and their oxidation potentials determined by cyclic voltammetry. The resulting 2-pyridone structure and a tricyclic heterocycle which was formed during an attempted synthesis of 4-(2-hydroxyphenyl) substituted 3,4-dihydro-2(1H)-pyridone were confirmed by single crystal X-ray crystallography.

  19. Conserved arginine residues in the carboxyl terminus of the equine arteritis virus E protein may play a role in heparin binding but may not affect viral infectivity in equine endothelial cells.

    PubMed

    Lu, Zhengchun; Sarkar, Sanjay; Zhang, Jianqiang; Balasuriya, Udeni B R

    2016-04-01

    Equine arteritis virus (EAV), the causative agent of equine viral arteritis, has relatively broad cell tropism in vitro. In horses, EAV primarily replicates in macrophages and endothelial cells of small blood vessels. Until now, neither the cellular receptor(s) nor the mechanism(s) of virus attachment and entry have been determined for this virus. In this study, we investigated the effect of heparin on EAV infection in equine endothelial cells (EECs). Heparin, but not other glycosaminoglycans, could reduce EAV infection up to 93 %. Sequence analysis of the EAV E minor envelope protein revealed a conserved amino acid sequence (52 RSLVARCSRGARYR 65) at the carboxy terminus of the E protein, which was predicted to be the heparin-binding domain. The basic arginine (R) amino acid residues were subsequently mutated to glycine by site-directed mutagenesis of ORF2a in an E protein expression vector and an infectious cDNA clone of EAV. Two single mutations in E (R52G and R57G) did not affect the heparin-binding capability, whereas the E double mutation (R52,60G) completely eliminated the interaction between the E protein and heparin. Although the mutant R52,60G EAV did not bind heparin, the mutations did not completely abolish infectivity, indicating that heparin is not the only critical factor for EAV infection. This also suggested that other viral envelope protein(s) might be involved in attachment through heparin or other cell-surface molecules, and this warrants further investigation. PMID:26739582

  20. The structure-function role of C-terminus in human bitter taste receptor T2R4 signaling.

    PubMed

    Upadhyaya, Jasbir; Singh, Nisha; Bhullar, Rajinder P; Chelikani, Prashen

    2015-07-01

    Bitter taste, in humans, is sensed by 25 G protein-coupled receptors, referred to as bitter taste receptors (T2Rs). The diverse roles of T2Rs in various extraoral tissues have implicated them as a potential target for therapeutic intervention. Structure-function studies have provided insights into the role of transmembrane and loop regions in the activation mechanism of T2Rs. However, studies aimed at deciphering the role of their carboxyl-terminus (C-terminus) are limited. In this study, we identified a KLK/R motif in the C-terminus that is conserved in 19 of the 25 T2Rs. Using site-directed mutagenesis we studied the role of 16 residues in the C-terminus of T2R4. The C-terminus of T2R4 is polybasic with 6 of the 16 residues consisting of lysines, constituting two separate KK motifs. We analyzed the effect of the C-terminus mutations on plasma membrane trafficking, and characterized their function in response to the T2R4 agonist quinine. The majority of the mutants showed defective receptor trafficking with ≤50% expression on the cell surface. Interestingly, mutation of the distal Lys296 of the KLK motif in T2R4 resulted in constitutive activity. The K296A mutant displayed five-fold basal activity over wild type T2R4, while the conservative substitution K296R showed wild type characteristics. The Lys294, Leu295 and Lys296 of the KLK motif in T2R4 were found to perform crucial roles, both, in receptor trafficking and function. Results from this study provide unique mechanistic insights into the structure-function role of the C-terminus in T2R signaling. PMID:25858111

  1. Xenopsin: the neurotensin-like octapeptide from Xenopus skin at the carboxyl terminus of its precursor.

    PubMed Central

    Sures, I; Crippa, M

    1984-01-01

    We have synthesized two oligodeoxyribonucleotide mixtures that contain sequences complementary to different parts of the hypothetical mRNA sequence of xenopsin, a biologically active octapeptide found in skin extracts from Xenopus laevis. The two primer pools were independently used to initiate reverse transcription on skin poly(A)+ RNA and the resulting cDNAs were then used to screen in parallel a cDNA library prepared from skin poly(A)+ RNA. One of the clones that hybridized with both probes was subjected to sequence analysis. It contains a nearly full-length DNA copy of a mRNA of approximately equal to 490 nucleotides that encodes a xenopsin precursor protein. The deduced precursor is 80 amino acids long, exhibits a putative signal sequence at the NH2 terminus, and contains the biologically active peptide at the COOH terminus. The region corresponding to the NH2-terminal portion of the xenopsin precursor shows a striking nucleotide and amino acid sequence homology with the precursor of PYLa, another recently described peptide from Xenopus skin. Images PMID:6582494

  2. The far C-terminus of MCAK regulates its conformation and spindle pole focusing

    PubMed Central

    Zong, Hailing; Carnes, Stephanie K.; Moe, Christina; Walczak, Claire E.; Ems-McClung, Stephanie C.

    2016-01-01

    To ensure proper spindle assembly, microtubule (MT) dynamics needs to be spatially regulated within the cell. The kinesin-13 MCAK is a potent MT depolymerase with a complex subcellular localization, yet how MCAK spatial regulation contributes to spindle assembly is not understood. Here we show that the far C-terminus of MCAK plays a critical role in regulating MCAK conformation, subspindle localization, and spindle assembly in Xenopus egg extracts. Alteration of MCAK conformation by the point mutation E715A/E716A in the far C-terminus increased MCAK targeting to the poles and reduced MT lifetimes, which induced spindles with unfocused poles. These effects were phenocopied by the Aurora A phosphomimetic mutation, S719E. Furthermore, addition of the kinesin-14 XCTK2 to spindle assembly reactions rescued the unfocused-pole phenotype. Collectively our work shows how the regional targeting of MCAK regulates MT dynamics, highlighting the idea that multiple phosphorylation pathways of MCAK cooperate to spatially control MT dynamics to maintain spindle architecture. PMID:26941326

  3. The N-terminus of survivin is a mitochondrial-targeting sequence and Src regulator.

    PubMed

    Dunajová, Lucia; Cash, Emily; Markus, Robert; Rochette, Sophie; Townley, Amelia R; Wheatley, Sally P

    2016-07-15

    Survivin (also known as BIRC5) is a cancer-associated protein that exists in several locations in the cell. Its cytoplasmic residence in interphase cells is governed by CRM1 (also known as XPO1)-mediated nuclear exportation, and its localisation during mitosis to the centromeres and midzone microtubules is that of a canonical chromosomal passenger protein. In addition to these well-established locations, survivin is also a mitochondrial protein, but how it gets there and its function therein is presently unclear. Here, we show that the first ten amino acids at the N-terminus of survivin are sufficient to target GFP to the mitochondria in vivo, and ectopic expression of this decapeptide decreases cell adhesion and accelerates proliferation. The data support a signalling mechanism in which this decapeptide regulates the tyrosine kinase Src, leading to reduced focal adhesion plaques and disruption of F-actin organisation. This strongly suggests that the N-terminus of survivin is a mitochondrial-targeting sequence that regulates Src, and that survivin acts in concert with Src to promote tumorigenesis. PMID:27246243

  4. A peptide with a cysteine terminus: probe for label-free fluorescent detection of thrombin activity.

    PubMed

    Feng, Jingjing; Zhuo, Caixia; Ma, Xuejuan; Li, Shuangqin; Zhang, Yaodong

    2016-07-21

    Thrombin has been implicated in atherosclerotic disease development. However, thrombin activity detection is currently limited because of the lack of convenient fluorescent probes. We developed a label-free fluorescent method to assay thrombin activity on the basis of a designed peptide probe with a thrombin-cleavable peptide sequence and a cysteine terminus. The peptide probe can be conjugated to DNA-templated silver nanoclusters (DNA-AgNCs) through Ag-S bonding; as a result, the fluorescence of DNA-AgNCs was enhanced. As the DNA-AgNCs-peptide conjugate was adsorbed to graphene oxide (GO), the enhanced fluorescence of DNA-AgNCs was quenched. Once the peptide probe was cleaved by thrombin, the resulting release of the DNA-AgNCs from the surface of GO restored the enhanced fluorescence. Thrombin can be determined with a linear range of 0.0-50.0 nM with a detection limit of 1 nM. The thrombin-sensitive probe with a cysteine terminus may be developed into probes to detect other proteases. PMID:27187619

  5. A molecular genetic dissection of the evolutionarily conserved N terminus of yeast Rad52.

    PubMed Central

    Mortensen, Uffe H; Erdeniz, Naz; Feng, Qi; Rothstein, Rodney

    2002-01-01

    Rad52 is a DNA-binding protein that stimulates the annealing of complementary single-stranded DNA. Only the N terminus of Rad52 is evolutionarily conserved; it contains the core activity of the protein, including its DNA-binding activity. To identify amino acid residues that are important for Rad52 function(s), we systematically replaced 76 of 165 amino acid residues in the N terminus with alanine. These substitutions were examined for their effects on the repair of gamma-ray-induced DNA damage and on both interchromosomal and direct repeat heteroallelic recombination. This analysis identified five regions that are required for efficient gamma-ray damage repair or mitotic recombination. Two regions, I and II, also contain the classic mutations, rad52-2 and rad52-1, respectively. Interestingly, four of the five regions contain mutations that impair the ability to repair gamma-ray-induced DNA damage yet still allow mitotic recombinants to be produced at rates that are similar to or higher than those obtained with wild-type strains. In addition, a new class of separation-of-function mutation that is only partially deficient in the repair of gamma-ray damage, but exhibits decreased mitotic recombination similar to rad52 null strains, was identified. These results suggest that Rad52 protein acts differently on lesions that occur spontaneously during the cell cycle than on those induced by gamma-irradiation. PMID:12072453

  6. Glacier terminus fluctuations on Mt. Baker, Washington, USA, 1940-1990, and climatic variations

    SciTech Connect

    Harper, J.T. )

    1993-11-01

    The terminus positions of six glaciers located on Mount Baker, Washington, were mapped by photogrammetric techniques at 2- to 7-yr intervals for the period 1940-1990. Although the timing varied slightly, each of the glaciers experienced a similar fluctuation sequence consisting of three phases: (1) rapid retreat, beginning prior to 1940 and lasting through the late 1940s to early 1950s; (2) approximately 30 yr of advance, ending in the late 1970s to early 1980s; (3) retreat though 1990. Terminus positions changed by up to 750 m during phases, with the advance phase increasing the lengths of glaciers by 13 to 24%. These fluctuations are well explained by variations in a smoothed time-series of accumulation-season precipitation and ablation-season mean temperature. The study glaciers appear to respond to interannual scale changes in climate within 20 yr or less. The glaciers on Mount Baker have a maritime location and a large percentage of area at high elevation, which may make their termini undergo greater fluctuations in response to climatic changes, especially precipitation variations, than most other glaciers in the North Cascades region. 40 refs., 6 figs., 2 tabs.

  7. The N-terminus of survivin is a mitochondrial-targeting sequence and Src regulator

    PubMed Central

    Dunajová, Lucia; Cash, Emily; Markus, Robert; Rochette, Sophie; Townley, Amelia R.

    2016-01-01

    ABSTRACT Survivin (also known as BIRC5) is a cancer-associated protein that exists in several locations in the cell. Its cytoplasmic residence in interphase cells is governed by CRM1 (also known as XPO1)-mediated nuclear exportation, and its localisation during mitosis to the centromeres and midzone microtubules is that of a canonical chromosomal passenger protein. In addition to these well-established locations, survivin is also a mitochondrial protein, but how it gets there and its function therein is presently unclear. Here, we show that the first ten amino acids at the N-terminus of survivin are sufficient to target GFP to the mitochondria in vivo, and ectopic expression of this decapeptide decreases cell adhesion and accelerates proliferation. The data support a signalling mechanism in which this decapeptide regulates the tyrosine kinase Src, leading to reduced focal adhesion plaques and disruption of F-actin organisation. This strongly suggests that the N-terminus of survivin is a mitochondrial-targeting sequence that regulates Src, and that survivin acts in concert with Src to promote tumorigenesis. PMID:27246243

  8. The far C-terminus of MCAK regulates its conformation and spindle pole focusing.

    PubMed

    Zong, Hailing; Carnes, Stephanie K; Moe, Christina; Walczak, Claire E; Ems-McClung, Stephanie C

    2016-05-01

    To ensure proper spindle assembly, microtubule (MT) dynamics needs to be spatially regulated within the cell. The kinesin-13 MCAK is a potent MT depolymerase with a complex subcellular localization, yet how MCAK spatial regulation contributes to spindle assembly is not understood. Here we show that the far C-terminus of MCAK plays a critical role in regulating MCAK conformation, subspindle localization, and spindle assembly in Xenopus egg extracts. Alteration of MCAK conformation by the point mutation E715A/E716A in the far C-terminus increased MCAK targeting to the poles and reduced MT lifetimes, which induced spindles with unfocused poles. These effects were phenocopied by the Aurora A phosphomimetic mutation, S719E. Furthermore, addition of the kinesin-14 XCTK2 to spindle assembly reactions rescued the unfocused-pole phenotype. Collectively our work shows how the regional targeting of MCAK regulates MT dynamics, highlighting the idea that multiple phosphorylation pathways of MCAK cooperate to spatially control MT dynamics to maintain spindle architecture. PMID:26941326

  9. Numerical Modeling of Climatic Change from the Terminus Record of Lewis Glacier, Mount Kenya.

    NASA Astrophysics Data System (ADS)

    Kruss, Phillip Donald

    Over the last 100 years, the glaciers and lakes of East Africa have undergone dramatic change in response to climatic forcing. However, the available conventional meterological series have not proven sufficient to explain these environmental events. The secular climatic change at Lewis Glacier, Mount Kenya (0(DEGREES)9'S, 37(DEGREES)19'E), is reconstructed from its terminus record documented since 1893. The short-time-step numerical model developed for this study consists of climate and ice dynamics segments. The climate segment directly computes the effect on the net balance of change in the four forcings: precipitation, albedo, cloudiness, and temperature. The flow segment calculates the dynamic glacier response to net balance variation. Climatic change occurs over a wide range of time scales. Each glacier responds in a unique fashion to this spectrum of climatic forcings. The response of the Lewis terminus extent to repeated sinusoidal fluctuation in the net balance is calculated. The net balance versus elevation profile is separately translated along the orthogonal balance and elevation axes. Net balance amplitudes of 0.1 to 0.5 m a('-1) of ice and 10 to 50 m elevation, respectively, and periods ranging from 20 to 1000 years are covered. Consideration of the Lewis response is perspective with similar results for Hintereisferner, Storglaciaren, and Berendon and South Cascade Glaciers identifies general characteristics of the time lag and amplitude of the terminus response. The magnitude and timing of the change in only one of the climatic forcings precipitation, albedo, cloudiness, or temperature necessary to produce the retreat of the Lewis terminus from its late 19th century maximum are computed. Equivalent changes for two scenarios of simultaneous variation, namely precipitation/albedo/cloudiness and temperature/albedo, are also estimated. These numerical results are interpreted in the light of long-term lake level, river flow, and instrumental information. A

  10. Bacillus licheniformis MC14 alkaline phosphatase I gene with an extended COOH-terminus.

    PubMed

    Kim, J W; Peterson, T; Bee, G; Hulett, F M

    1998-02-01

    Bacterial alkaline phosphatases (APases), except those isolated from Bacillus licheniformis, are approximately 45-kDa proteins while eucaryotic alkaline phosphatases are 60 kDa. To answer the question of whether the apparent 60-kDa alkaline phosphatase from Bacillus licheniformis accurately reflected the size of the protein, the entire gene was analyzed. DNA sequence analysis of the alkaline phosphatase I (APaseI) gene of B. licheniformis MC14 indicated that the gene could code for a 60-kDa protein of 553 amino acids. The deduced protein sequence of APaseI showed about 32% identity to those of B. subtilis APase III and IV and had apparent sequence homologies in the core structure and active sites that are conserved among APases of various sources. The extra carboxy-terminal sequence of APaseI, which made the enzyme bigger than other procaryotic APases, was not homologous to those of eucaryotic APases. The amino acid composition of APaseI was most similar to that of salt-dependent APase among the isozymes of B. licheniformis MC14. Another open reading frame of 261 amino acids was present 142 nucleotide upstream of the APaseI gene and its predicted amino acid sequence showed 68% identity to that of glucose dehydrogenase of B. megaterium. PMID:9485594

  11. N-terminus-mediated dimerization of ROCK-I is required for RhoE binding and actin reorganization.

    PubMed

    Garg, Ritu; Riento, Kirsi; Keep, Nicholas; Morris, Jonathan D H; Ridley, Anne J

    2008-04-15

    ROCK-I (Rho-associated kinase 1) is a serine/threonine kinase that can be activated by RhoA and inhibited by RhoE. ROCK-I has an N-terminal kinase domain, a central coiled-coil region and a RhoA-binding domain near the C-terminus. We have previously shown that RhoE binds to the N-terminal 420 amino acids of ROCK-I, which includes the kinase domain as well as N-terminal and C-terminal extensions. In the present study, we show that N-terminus-mediated dimerization of ROCK-I is required for RhoE binding. The central coiled-coil domain can also dimerize ROCK-I in cells, but this is insufficient in the absence of the N-terminus to allow RhoE binding. The kinase activity of ROCK-I(1-420) is required for dimerization and RhoE binding; however, inclusion of part of the coiled-coil domain compensates for lack of kinase activity, allowing RhoE to bind. N-terminus-mediated dimerization is also required for ROCK-I to induce the formation of stellate actin stress fibres in cells. These results indicate that dimerization via the N-terminus is critical for ROCK-I function in cells and for its regulation by RhoE. PMID:18215121

  12. The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion*

    PubMed Central

    Chong, Brandi M.; Russell, Tanya D.; Schaack, Jerome; Orlicky, David J.; Reigan, Philip; Ladinsky, Mark; McManaman, James L.

    2011-01-01

    Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion. PMID:21383012

  13. 11-Nor-9-carboxy-∆9-tetrahydrocannabinol quantification in human oral fluid by liquid chromatography-tandem mass spectrometry.

    PubMed

    Scheidweiler, Karl B; Himes, Sarah K; Chen, Xiaohong; Liu, Hua-Fen; Huestis, Marilyn A

    2013-07-01

    Currently, ∆9-tetrahydrocannabinol (THC) is the analyte quantified for oral fluid cannabinoid monitoring. The potential for false-positive oral fluid cannabinoid results from passive exposure to THC-laden cannabis smoke raises concerns for this promising new monitoring technology. Oral fluid 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THCCOOH) is proposed as a marker of cannabis intake since it is not present in cannabis smoke and was not measureable in oral fluid collected from subjects passively exposed to cannabis. THCCOOH concentrations are in the picogram per milliliter range in oral fluid and pose considerable analytical challenges. A liquid chromatography-tandem mass spectrometry (LCMSMS) method was developed and validated for quantifying THCCOOH in 1 mL Quantisal-collected oral fluid. After solid phase extraction, chromatography was performed on a Kinetex C18 column with a gradient of 0.01% acetic acid in water and 0.01% acetic acid in methanol with a 0.5-mL/min flow rate. THCCOOH was monitored in negative mode electrospray ionization and multiple reaction monitoring mass spectrometry. The THCCOOH linear range was 12-1,020 pg/mL (R(2) > 0.995). Mean extraction efficiencies and matrix effects evaluated at low and high quality control (QC) concentrations were 40.8-65.1 and -2.4-11.5%, respectively (n = 10). Analytical recoveries (bias) and total imprecision at low, mid, and high QCs were 85.0-113.3 and 6.6-8.4% coefficient of variation, respectively (n = 20). This is the first oral fluid THCCOOH LCMSMS triple quadrupole method not requiring derivatization to achieve a <15 pg/mL limit of quantification. The assay is applicable for the workplace, driving under the influence of drugs, drug treatment, and pain management testing. PMID:23681203

  14. Urinary excretion of 11-nor-9-carboxy-delta9-tetrahydrocannabinol and cannabinoids in frequent and infrequent drug users.

    PubMed

    Smith-Kielland, A; Skuterud, B; Mørland, J

    1999-09-01

    Urinary excretion of 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCCOOH) and cannabinoids was monitored in prison inmates. Urinary specimens were collected up to five times per day. EMIT (cutoff 20 ng/mL; EMIT20) and gas chromatography (GC) (cutoff 10.3 ng/mL, LOD 1.4 ng/mL) were used for cannabinoid screening and THCCOOH confirmation, respectively. Urinary creatinine concentrations were recorded. Of the samples with positive EMIT screens, 78% were confirmed by GC analysis. The plotting of THCCOOH/creatinine ratios (THCCOOH/C) versus time gave smoother excretion curves than THCCOOH concentrations alone. Based on THCCOOH/C the first 5 days after the last reported intake, the mean urinary excretion half-life was 1.3 days in infrequent users, and a median of 1.4 days was found in frequent users. In the latter group, apparent terminal urinary excretion half-lives up to 10.3 days were observed. The last positive specimens were found after 4 days for THCCOOH with cutoff 15.0 ng/mL (NIDA/SAMSHA), 5 days for THCCOOH with cutoff 10.3 ng/mL, and 12 days for cannabinoids (EMIT20) in infrequent users and after 17, 22, and 27 days, respectively, in frequent users. Increases in urinary cannabinoids were sometimes found without concomitant increase in THCCOOH or THCCOOH/C. One subject admitted new cannabis intake, after which marked increases in THCCOOH and THCCOOH/C were observed. In others, new intake was suspected. Considerable variations between consecutive specimens were also observed in THCCOOH concentration and THCCOOH/C ratio without suspicion of a new intake. PMID:10488918

  15. Structure and Sialyllactose Binding of the Carboxy-Terminal Head Domain of the Fibre from a Siadenovirus, Turkey Adenovirus 3

    PubMed Central

    Singh, Abhimanyu K.; Berbís, M. Álvaro; Ballmann, Mónika Z.; Kilcoyne, Michelle; Menéndez, Margarita; Nguyen, Thanh H.; Joshi, Lokesh; Cañada, F. Javier; Jiménez-Barbero, Jesús; Benkő, Mária; Harrach, Balázs; van Raaij, Mark J.

    2015-01-01

    The virulent form of turkey adenovirus 3 (TAdV-3), also known as turkey hemorrhagic enteritis virus (THEV), is an economically important poultry pathogen, while the avirulent form is used as a vaccine. TAdV-3 belongs to the genus Siadenovirus. The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure. Two amino acid sequence differences between virulent and avirulent TAdV-3 map on the fibre head: where virulent TAdV-3 contains Ile354 and Thr376, avirulent TAdV-3 contains Met354 and Met376. We determined the crystal structures of the trimeric virulent and avirulent TAdV-3 fibre head domains at 2.2 Å resolution. Each monomer contains a beta-sandwich, which, surprisingly, resembles reovirus fibre head more than other adenovirus fibres, although the ABCJ-GHID topology is conserved in all. A beta-hairpin insertion in the C-strand of each trimer subunit embraces its neighbouring monomer. The avirulent and virulent TAdV-3 fibre heads are identical apart from the exact orientation of the beta-hairpin insertion. In vitro, sialyllactose was identified as a ligand by glycan microarray analysis, nuclear magnetic resonance spectroscopy, and crystallography. Its dissociation constant was measured to be in the mM range by isothermal titration calorimetry. The ligand binds to the side of the fibre head, involving amino acids Glu392, Thr419, Val420, Lys421, Asn422, and Gly423 binding to the sialic acid group. It binds slightly more strongly to the avirulent form. We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component. PMID:26418008

  16. Quantitative determination of 11-nor-9-carboxy-tetrahydrocannabinol in hair by column switching LC-ESI-MS(3).

    PubMed

    Park, Meejung; Kim, Jihyun; Park, Yuran; In, Sanghwan; Kim, Eunmi; Park, Yonghoon

    2014-02-01

    Hair analysis has been regarded as an alternative method to urine analysis in forensic and criminal cases. Cannabis (marijuana) is one of the most widely used drugs in the world and it has been controlled in South Korea since 1976. Identification of 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) in hair can be an important proof of cannabis use because it can exclude the possibility of passive cannabis smoke exposure. In this study, we described a quantitative method of THCCOOH in hair using simple liquid-liquid extraction (LLE), selective column switching liquid chromatography with electrospray ionization (ESI)-MS(3). For the column switching system three columns (precolumn, trap column and analytical column) were used. Valve switch from the precolumn to the trap column was set from 3.0 to 4.0 min because THCCOOH appeared around 3.5 min with this precolumn. After 4.0 min the valve was switched to the original position and the analytes in the trap column were eluted onto the analytical column. Resolution occurred in this column and eluted into the ESI-MS(3) system. The internal standard was THCCOOH-d3. We used ESI-negative-MS(3) transition of ions at m/z 343 to 299 to 245 (343/299/245) and m/z 346 to 302 to 248 (346/302/248) for quantification of THCCOOH and THCCOOH-d3, respectively. The validation results of selectivity, matrix effect, recovery, linearity, precision and accuracy, and processed sample stability were satisfactory. The limit of detection (LOD) was 0.05 pg/mg and the limit of quantification (LOQ) was 0.10 pg/mg. The range of concentration of THCCOOH from 98 authentic human hair was 0.13-15.75 pg/mg. This method was successfully applied in the analysis of authentic human hair samples. PMID:24434565

  17. Structure and Sialyllactose Binding of the Carboxy-Terminal Head Domain of the Fibre from a Siadenovirus, Turkey Adenovirus 3.

    PubMed

    Singh, Abhimanyu K; Berbís, M Álvaro; Ballmann, Mónika Z; Kilcoyne, Michelle; Menéndez, Margarita; Nguyen, Thanh H; Joshi, Lokesh; Cañada, F Javier; Jiménez-Barbero, Jesús; Benkő, Mária; Harrach, Balázs; van Raaij, Mark J

    2015-01-01

    The virulent form of turkey adenovirus 3 (TAdV-3), also known as turkey hemorrhagic enteritis virus (THEV), is an economically important poultry pathogen, while the avirulent form is used as a vaccine. TAdV-3 belongs to the genus Siadenovirus. The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure. Two amino acid sequence differences between virulent and avirulent TAdV-3 map on the fibre head: where virulent TAdV-3 contains Ile354 and Thr376, avirulent TAdV-3 contains Met354 and Met376. We determined the crystal structures of the trimeric virulent and avirulent TAdV-3 fibre head domains at 2.2 Å resolution. Each monomer contains a beta-sandwich, which, surprisingly, resembles reovirus fibre head more than other adenovirus fibres, although the ABCJ-GHID topology is conserved in all. A beta-hairpin insertion in the C-strand of each trimer subunit embraces its neighbouring monomer. The avirulent and virulent TAdV-3 fibre heads are identical apart from the exact orientation of the beta-hairpin insertion. In vitro, sialyllactose was identified as a ligand by glycan microarray analysis, nuclear magnetic resonance spectroscopy, and crystallography. Its dissociation constant was measured to be in the mM range by isothermal titration calorimetry. The ligand binds to the side of the fibre head, involving amino acids Glu392, Thr419, Val420, Lys421, Asn422, and Gly423 binding to the sialic acid group. It binds slightly more strongly to the avirulent form. We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component. PMID:26418008

  18. 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol quantification in human oral fluid by liquid chromatography–tandem mass spectrometry

    PubMed Central

    Scheidweiler, Karl B.; Himes, Sarah K.; Chen, Xiaohong; Liu, Hua-Fen

    2013-01-01

    Currently, Δ9-tetrahydrocannabinol (THC) is the analyte quantified for oral fluid cannabinoid monitoring. The potential for false-positive oral fluid cannabinoid results from passive exposure to THC-laden cannabis smoke raises concerns for this promising new monitoring technology. Oral fluid 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) is proposed as a marker of cannabis intake since it is not present in cannabis smoke and was not measureable in oral fluid collected from subjects passively exposed to cannabis. THCCOOH concentrations are in the picogram per milliliter range in oral fluid and pose considerable analytical challenges. A liquid chromatography–tandem mass spectrometry (LCMSMS) method was developed and validated for quantifying THCCOOH in 1 mL Quantisal-collected oral fluid. After solid phase extraction, chromatography was performed on a Kinetex C18 column with a gradient of 0.01 % acetic acid in water and 0.01 % acetic acid in methanol with a 0.5-mL/min flow rate. THCCOOH was monitored in negative mode electrospray ionization and multiple reaction monitoring mass spectrometry. The THCCOOH linear range was 12–1,020 pg/mL (R2>0.995). Mean extraction efficiencies and matrix effects evaluated at low and high quality control (QC) concentrations were 40.8–65.1 and −2.4–11.5 %, respectively (n=10). Analytical recoveries (bias) and total imprecision at low, mid, and high QCs were 85.0–113.3 and 6.6–8.4 % coefficient of variation, respectively (n=20). This is the first oral fluid THCCOOH LCMSMS triple quadrupole method not requiring derivatization to achieve a <15 pg/mL limit of quantification. The assay is applicable for the workplace, driving under the influence of drugs, drug treatment, and pain management testing. PMID:23681203

  19. Phosphorylation and Ionic Strength Alter the LRAP-HAP Interface in the N-terminus

    SciTech Connect

    Lu, Junxia; Xu, Yimin; Shaw, Wendy J.

    2013-04-02

    The conditions present during enamel crystallite development change dramatically as a function of time, including the pH, protein concentration, surface type and ionic strength. In this work, we investigate the role that two of these changing conditions, pH and ionic strength, have in modulating the interaction of amelogenin, LRAP, with hydroxyapatite (HAP). Using solid state NMR dipolar recoupling and chemical shift data, we investigate the structure, orientation and dynamics of three regions in the N-terminus of the protein, L15 to V19, V19 to L23 and K24 to S28. These regions are also near the only phosphorylated residue in the protein, pS16, therefore, changes in the LRAP-HAP interaction as a function of phosphorylation (LRAP(-P) vs. LRAP(+P)) were also investigated. All of the regions and conditions studies for the surface immobilized proteins showed restricted motion, with more mobility under all conditions for L15(+P) and K24(-P). The structure and orientation of the LRAP-HAP interaction in the N-terminus of the phosphorylated protein is very stable to changing solution conditions. From REDOR dipolar recoupling data, the structure and orientation in the region L15V19(+P) did not change significantly as a function of pH or ionic strength. The structure and orientation of the region V19L23(+P) were also stable to changes in pH, with the only significant change observed at high ionic strength, where the region becomes extended, suggesting this may be an important region in regulating mineral development. Chemical shift studies also suggest minimal changes in all three regions studied for both LRAP(-P) and LRAP(+P) as a function of pH or ionic strength. Phosphorylation also alters the LRAP-HAP interface. All of the three residues investigated (L15, V19, and K24) are closer to the surface in LRAP(+P), but K24S28 also changes structure as a result of phosphorylation, from a random coil to a largely helical structure, and V19L23 becomes more extended at high ionic

  20. An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit.

    PubMed Central

    Conner, J; Cooper, J; Furlong, J; Clements, J B

    1992-01-01

    We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1). R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus. When the N terminus was separately expressed in E. coli and partially purified, a similar autophosphorylation reaction was observed. Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted. Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E. coli contaminant protein kinase. We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication. Images PMID:1331536

  1. Transferring the C-terminus of the chemokine CCL21 to CCL19 confers enhanced heparin binding.

    PubMed

    Barmore, Austin J; Castex, Sally M; Gouletas, Brittany A; Griffith, Alex J; Metz, Slater W; Muelder, Nicolas G; Populin, Michael J; Sackett, David M; Schuster, Abigail M; Veldkamp, Christopher T

    2016-09-01

    Chemokines direct the migration of cells during various immune processes and are involved in many disease states. For example, CCL19 and CCL21, through activation of the CCR7 receptor, recruit dendritic cells and naïve T-cells to the secondary lymphoid organs aiding in balancing immune response and tolerance. However, CCL19 and CCL21 can also direct the metastasis of CCR7 expressing cancers. Chemokine binding to glycosaminoglycans, such as heparin, is as important to chemokine function as receptor activation. CCL21 is unique in that it contains an extended C-terminus not found in other chemokines like CCL19. Deletion of this extended C-terminus reduces CCL21's affinity for heparin and transferring the CCL21 C-terminus to CCL19 enhances heparin binding mainly through non-specific, electrostatic interactions. PMID:27338641

  2. [Functions of carboxyl-terminus of Hsc70 interacting protein and its role in neurodegenerative disease].

    PubMed

    Yan, Wei-qian; Wang, Jun-ling; Tang, Bei-sha

    2012-08-01

    Neurodegenerative diseases are a group of chronic progressive neuronal damage disorders. The cause is unclear, most of them share a same pathological hallmark with misfold proteins accumulating in neurons. Carboxyl-terminus of Hsc70 interacting protein (CHIP) is a dual functional molecule, which has a N terminal tetratrico peptide repeat (TPR) domain that interacts with Hsc/Hsp70 complex and Hsp90 enabling CHIP to modulate the aberrant protein folding; and a C terminal U-box ubiquitin ligase domain that binds to the 26S subunit of the proteasome involved in protein degradation via ubiqutin-proteasome system. CHIP protein mediates interactions between the chaperone system and the ubiquitin-proteasome system, and plays an important role in maintaining the protein homeostasis in cells. This article reviews the molecular characteristics and physiological functions of CHIP, and its role in cellular metabolism and discusses the relationship between CHIP dysfunction and neurodegenerative diseases. PMID:22875499

  3. Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding

    PubMed Central

    Webb, Christopher J.; Zakian, Virginia A.

    2015-01-01

    The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex. PMID:26305931

  4. Biological and immunological properties of the carboxyl terminus of staphylococcal enterotoxin C1.

    PubMed

    Bohach, G A; Handley, J P; Schlievert, P M

    1989-01-01

    Comparisons of recently published primary sequences of staphylococcal and streptococcal pyrogenic toxins prompted an evaluation of biological and immunological properties of the C terminus of staphylococcal enterotoxin C1. The 59 N-terminal amino acids were deleted from the toxin by digestion with trypsin. The resulting fragment (Mr, 20,659) contained the remaining 180 C-terminal residues. This fragment (Trp F1) consisted of two polypeptide chains (Trp F1a and Trp F1b) linked by cysteine residues. Trp F1 was mitogenic, pyrogenic, and enhanced susceptibility of rabbits to lethal endotoxin shock. In addition, this fragment contained at least one antigenic epitope that cross-reacted with enterotoxin B. PMID:2909489

  5. Mutational analysis of the N terminus of the protein of maize transposable element Ac.

    PubMed Central

    Li, M G; Starlinger, P

    1990-01-01

    Mutations of transposable element Ac were tested for their capability to excise themselves from their location autonomously, to be excised by an active Ac, or to act in trans in the excision of an Ac delta element. Removal of 101 amino acids from the N terminus of the Ac protein does not decrease excision. A cis-acting site between base pairs 186 and 207 is important for excision by the wild-type protein but is not necessary for excision by the truncated protein. Improvement of the sequence context of the first AUG does not have a significant effect. Mutations in a small open reading frame of Ac encoding a 102-amino acid protein do not visibly alter excision frequency. Images PMID:2166942

  6. Localization of the Intracellular Activity Domain of Pasteurella multocida Toxin to the N Terminus

    PubMed Central

    Wilson, Brenda A.; Ponferrada, Virgilio G.; Vallance, Jefferson E.; Ho, Mengfei

    1999-01-01

    We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells. PMID:9864199

  7. Carboxyl terminus-truncated α1D-adrenoceptors inhibit the ERK pathway.

    PubMed

    Alfonzo-Méndez, Marco A; Castillo-Badillo, Jean A; Romero-Ávila, M Teresa; Rivera, Richard; Chun, Jerold; García-Sáinz, J Adolfo

    2016-08-01

    Human α1D-adrenoceptors are G protein-coupled receptors that mediate adrenaline/noradrenaline actions. There is a growing interest in identifying regulatory domains in these receptors and determining how they function. In this work, we show that the absence of the human α1D-adrenoceptor carboxyl tail results in altered ERK (extracellular signal-regulated kinase) and p38 phosphorylation states. Amino terminus-truncated and both amino and carboxyl termini-truncated α1D-adrenoceptors were transfected into Rat-1, HEK293, and B103 cells, and changes in the phosphorylation state of extracellular signal-regulated kinase was assessed using biochemical and biophysical approaches. The phosphorylation state of other protein kinases (p38, MEK1, and Raf-1) was also studied. Noradrenaline-induced ERK phosphorylation in Rat-1 fibroblasts expressing amino termini-truncated α1D-adrenoceptors. However, in cells expressing receptors with both amino and carboxyl termini truncations, noradrenaline-induced activation was abrogated. Interestingly, ERK phosphorylation that normally occurs through activation of endogenous G protein-coupled receptors, EGF receptors, and protein kinase C, was also decreased, suggesting that downstream steps in the mitogen-activated protein kinase pathway were affected. A similar effect was observed in B103 cells but not in HEK 293 cells. Phosphorylation of Raf-1 and MEK1 was also diminished in Rat-1 fibroblasts expressing amino- and carboxyl-truncated α1D-adrenoceptors. Our data indicate that expression of carboxyl terminus-truncated α1D-adrenoceptors alters ERK and p38 phosphorylation state. PMID:27146292

  8. The carboxy-terminal half of nonstructural protein 3A is not essential for foot-and-mouth disease virus replication in cultured cell lines.

    PubMed

    Behura, Mrutyunjay; Mohapatra, Jajati K; Pandey, Laxmi K; Das, Biswajit; Bhatt, Mukesh; Subramaniam, Saravanan; Pattnaik, Bramhadev

    2016-05-01

    In foot-and-mouth disease (FMD)-endemic parts of the globe, control is mainly implemented by preventive vaccination with an inactivated purified vaccine. ELISAs detecting antibodies to the viral nonstructural proteins (NSP) distinguish FMD virus (FMDV)-infected animals in the vaccinated population (DIVA). However, residual NSPs present in the vaccines are suspected to be a cause of occasional false positive results, and therefore, an epitope-deleted negative marker vaccine strategy is considered a more logical option. In this study, employing a serotype Asia 1 FMDV infectious cDNA clone, it is demonstrated that while large deletions differing in size and location in the carboxy-terminal half of 3A downstream of the putative hydrophobic membrane-binding domain (deletion of residues 86-110, 101-149, 81-149 and 81-153) are tolerated by the virus without affecting its infectivity in cultured cell lines, deletions in the amino-terminal half (residues 5-54, 21-50, 21-80, 55-80 and 5-149) containing the dimerization and the transmembrane domains are deleterious to its multiplication. Most importantly, the virus could dispense with the entire carboxy-terminal half of 3A (residues 81-153) including the residues involved in the formation of the 3A-3B1 cleavage junction. The rescue of a replication-competent FMDV variant carrying the largest deletion ever in 3A (residues 81-153) and the fact that the deleted region contains a series of linear B-cell epitopes inspired us to devise an indirect ELISA based on a recombinant 3A carboxy-terminal fragment and to evaluate its potential to serve as a companion diagnostic assay for differential serosurveillance if the 3A-truncated virus is used as a marker vaccine. PMID:26935917

  9. The function of the two-pore channel TPC1 depends on dimerization of its carboxy-terminal helix.

    PubMed

    Larisch, Nina; Kirsch, Sonja A; Schambony, Alexandra; Studtrucker, Tanja; Böckmann, Rainer A; Dietrich, Petra

    2016-07-01

    Two-pore channels (TPCs) constitute a family of intracellular cation channels with diverse permeation properties and functions in animals and plants. In the model plant Arabidopsis, the vacuolar cation channel TPC1 is involved in propagation of calcium waves and in cation homeostasis. Here, we discovered that the dimerization of a predicted helix within the carboxyl-terminus (CTH) is essential for the activity of TPC1. Bimolecular fluorescence complementation and co-immunoprecipitation demonstrated the interaction of the two C-termini and pointed towards the involvement of the CTH in this process. Synthetic CTH peptides dimerized with a dissociation constant of 3.9 µM. Disruption of this domain in TPC1 either by deletion or point mutations impeded the dimerization and cation transport. The homo-dimerization of the CTH was analyzed in silico using coarse-grained molecular dynamics (MD) simulations for the study of aggregation, followed by atomistic MD simulations. The simulations revealed that the helical region of the wild type, but not a mutated CTH forms a highly stable, antiparallel dimer with characteristics of a coiled-coil. We propose that the voltage- and Ca(2+)-sensitive conformation of TPC1 depends on C-terminal dimerization, adding an additional layer to the complex regulation of two-pore cation channels. PMID:26781468

  10. Degradation of carboxy-terminal-tagged cytoplasmic proteins by the Escherichia coli protease HflB (FtsH)

    PubMed Central

    Herman, Christophe; Thévenet, Danielle; Bouloc, Philippe; Walker, Graham C.; D’Ari, Richard

    1998-01-01

    Proteins with short nonpolar carboxyl termini are unstable in Escherichia coli. This proteolytic pathway is used to dispose of polypeptides synthesized from truncated mRNA molecules. Such proteins are tagged with an 11-amino-acid nonpolar destabilizing tail via a mechanism involving the 10Sa (SsrA) stable RNA and then degraded. We show here that the ATP-dependent zinc protease HflB (FtsH) is involved in the degradation of four unstable derivatives of the amino-terminal domain of the λcI repressor: three with nonpolar pentapeptide tails (cI104, cI105, cI108) and one with the SsrA tag (cI–SsrA). cI105 and cI-SsrA are also degraded by the ClpP-dependent proteases. Loss of ClpP can be compensated for by overproducing HflB. In an in vitro system, cI108 and cI–SsrA are degraded by HflB in an energy-dependent reaction, indicating that HflB itself recognizes the carboxyl terminus. These results establish a tail-specific pathway for removing abnormal cytoplasmic proteins via the HflB and Clp proteases. PMID:9573051

  11. The dual functions of the extreme N-terminus of TDP-43 in regulating its biological activity and inclusion formation.

    PubMed

    Zhang, Yong-Jie; Caulfield, Thomas; Xu, Ya-Fei; Gendron, Tania F; Hubbard, Jaime; Stetler, Caroline; Sasaguri, Hiroki; Whitelaw, Ena C; Cai, Shuyi; Lee, Wing Cheung; Petrucelli, Leonard

    2013-08-01

    TAR DNA-binding protein-43 (TDP-43) is the principal component of ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS) and the most common pathological subtype of frontotemporal dementia-frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). To date, the C-terminus of TDP-43, which is aggregation-prone and contains almost all ALS-associated mutations, has garnered much attention while the functions of the N-terminus of TDP-43 remain largely unknown. To bridge this gap in our knowledge, we utilized novel cell culture and computer-assisted models to evaluate which region(s) of TDP-43 regulate its folding, self-interaction, biological activity and aggregation. We determined that the extreme N-terminus of TDP-43, specifically the first 10 residues, regulates folding of TDP-43 monomers necessary for proper homodimerization and TDP-43-regulated splicing. Despite such beneficial functions, we discovered an interesting dichotomy: full-length TDP-43 aggregation, which is believed to be a pathogenic process, also requires the extreme N-terminus of TDP-43. As such, we provide new insight into the structural basis for TDP-43 function and aggregation, and we suggest that stabilization of TDP-43 homodimers, the physiologically active form of TDP-43, may be a promising therapeutic strategy for ALS and FTLD-TDP. PMID:23575225

  12. HSP90 Regulation of P2X7 Receptor Function Requires an Intact Cytoplasmic C-Terminus.

    PubMed

    Migita, Keisuke; Ozaki, Taku; Shimoyama, Shuji; Yamada, Junko; Nikaido, Yoshikazu; Furukawa, Tomonori; Shiba, Yuko; Egan, Terrance M; Ueno, Shinya

    2016-08-01

    P2X7 receptors (P2X7Rs) are ATP-gated ion channels that display the unusual property of current facilitation during long applications of agonists. Here we show that facilitation disappears in chimeric P2X7Rs containing the C-terminus of the P2X2 receptor (P2X2R), and in a truncated P2X7R missing the cysteine-rich domain of the C-terminus. The chimeric and truncated receptors also show an apparent decreased permeability to N-methyl-d-glucamine(+) (NMDG(+)). The effects of genetic modification of the C-terminus on NMDG(+) permeability were mimicked by preapplication of the HSP90 antagonist geldanamycin to the wild-type receptor. Further, the geldanamycin decreased the shift in the reversal potential of the ATP-gated current measured under bi-ionic NMDG(+)/Na(+) condition without affecting the ability of the long application of agonist to facilitate current amplitude. Taken together, the results suggest that HSP90 may be essential for stabilization and function of P2X7Rs through an action on the cysteine-rich domain of the cytoplasmic the C-terminus. PMID:27301716

  13. Sequence requirements for maturation of the 5' terminus of human 18 S rRNA in vitro.

    PubMed

    Yu, Y T; Nilsen, T W

    1992-05-01

    Creation of the mature 5' terminus of human 18 S rRNA in vitro occurs via a two-step processing reaction. In the first step, an endonucleolytic activity found in HeLa cell nucleolar extract cleaves an rRNA precursor spanning the external transcribed spacer-18 S boundary at a position 3 bases upstream from the mature 18 S terminus leaving 2',3'-cyclic phosphate, 5' hydroxyl termini. In the second step, a nucleolytic activity(s) found in HeLa cell cytoplasmic extract removes the 3 extra bases and creates the authentic 5'-phosphorylated terminus of 18 S rRNA. Here we have examined the sequence requirements for the trimming reaction. The trimming activity(s), in addition to requiring a 5' hydroxyl terminus, prefers the naturally occurring adenosine as the 5'-terminal base. By a combination of deletion, site-directed mutagenesis, and chemical modification interference approaches we have also identified a region of 18 S rRNA spanning bases +6 to +25 (with respect to the mature 5' end) which comprises a critical recognition sequence for the trimming activity(s). PMID:1577760

  14. Effects of N-terminus modifications on the conformation and permeation activities of the synthetic peptide L1A.

    PubMed

    Zanin, Luciana Puia Moro; de Araujo, Alexandre Suman; Juliano, Maria Aparecida; Casella, Tiago; Nogueira, Mara Correa Lelles; Ruggiero Neto, João

    2016-06-01

    We investigate the effect of the N-terminus modification of the L1A, a synthetic octadecapeptide, on its helical content, affinity and lytic action in model membranes and on its hemolytic and antibacterial activities. L1A and its acetylated analog displayed a selective antibacterial activity to Gram-negative bacteria without being hemolytic. The covalently linked 2-aminobezoic acid to the N-terminus impaired the antibacterial efficacy and increased hemolysis. Despite their lower net charge (+2), N-terminus modifications resulted in enhanced affinity and improved lytic efficiency in anionic vesicles. The analogs also showed higher helical content and consequently higher amphipathicity in these vesicles. The conformational analysis by molecular dynamics simulations in 30 % of TFE/water showed that the hydrophobic faces of the peptides are in close contact with CF3 groups of TFE while the hydrophilic faces with water molecules. Due to the loss of the amino charge, the N-termini of the analogs are buried in TFE molecules. The analysis of the pair distribution functions, obtained for the center of mass of the charged groups, has evidenced that the state of the N-terminus has influenced the possibility of different ion-pairing. The higher complexity of the bacterial cells compared with anionic vesicles hampers to establish correlations structure-function for the analogs. PMID:26920749

  15. Amyloid fibril formation of peptides derived from the C-terminus of CETP modulated by lipids

    SciTech Connect

    García-González, Victor; Mas-Oliva, Jaime

    2013-04-26

    Highlights: •The secondary structure of a C-terminal peptide derived from CETP was studied. •Lipids modulate secondary structure changes of a C-terminal peptide derived from CETP. •Lysophosphatidic acid maintains a functional α-helix and prevents fibril formation. •Transfer of lipids by CETP is related to the presence of an α-helix at its C-end. -- Abstract: Cholesteryl-ester transfer protein (CETP) is a plasmatic protein involved in neutral lipid transfer between lipoproteins. Focusing on the last 12 C-terminus residues we have previously shown that mutation D{sub 470}N promotes a conformational change towards a β-secondary structure. In turn, this modification leads to the formation of oligomers and fibrillar structures, which cause cytotoxic effects similar to the ones provoked by amyloid peptides. In this study, we evaluated the role of specific lipid arrangements on the structure of peptide helix-Z (D{sub 470}N) through the use of thioflavin T fluorescence, peptide bond absorbance, circular dichroism and electron microscopy. The results indicate that the use of micelles formed with lysophosphatidylcholine and lysophosphatidic acid (LPA) under neutral pH induce a conformational transition of peptide helix-Z containing a β-sheet conformation to a native α-helix structure, therefore avoiding the formation of amyloid fibrils. In contrast, incubation with phosphatidic acid does not change the profile for the β-sheet conformation. When the electrostatic charge at the surface of micelles or vesicles is regulated through the use of lipids such as phospholipid and LPA, minimal changes and the presence of β-structures were recorded. Mixtures with a positive net charge diminished the percentage of β-structure and the amount of amyloid fibrils. Our results suggest that the degree of solvation determined by the presence of a free hydroxyl group on lipids such as LPA is a key condition that can modulate the secondary structure and the consequent formation of

  16. Differences in activity of actinoporins are related with the hydrophobicity of their N-terminus.

    PubMed

    Ros, Uris; Rodríguez-Vera, Wendy; Pedrera, Lohans; Valiente, Pedro A; Cabezas, Sheila; Lanio, María E; García-Sáez, Ana J; Alvarez, Carlos

    2015-09-01

    Actinoporins are pore-forming toxins (PFT) produced by sea anemones with molecular mass around 20 kDa and high affinity for sphingomyelin. The most studied atinoporins are sticholysins I and II (StI/StII) from Stichodactyla helianthus, equinatoxin II (EqtII) from Actinia equina, and fragaceatoxin C (FraC) from Actinia fragacea. Their N-terminal sequences encompassing residues 1-30 seem to be the best candidates for pore formation. This segment comprises an amphipathic α-helix preceded by a more or less hydrophobic segment, depending on the toxin, of around 10 amino acid residues. Although it is clear that the N-terminal is the most variable sequence in this protein family, the role of their hydrophobic segment in not fully understood. Here we show a comparison of StI, StII, EqtII, and FraC activities with that of their respective N-terminal synthetic peptides. The hemolytic and permeabilizing activity of the peptides reproduce qualitatively the behavior of their respective parental proteins and are particularly related to the hydrophobicity of the corresponding 1-10 segment. Furthermore, the dendrogram analysis of actinoporins' N-terminal sequence allows relating differences in alignment with differences in activity among the four toxins. We have also evaluated the penetration depth of the N-terminal segment of StI and StII by using Trp-containing peptide-analogs. Our data suggest that the N-terminus of StII is more deeply buried into the hydrophobic core of the bilayer than that of StI. We hypothesize that the highest activity of StII could be ascribed to a larger hydrophobic continuum, an uninterrupted sequence of non-charged mainly hydrophobic amino acid residues, of its N-terminus promoting a highest ability to partially insert in the membrane core. Moreover, as we show for four related peptides that a higher hydrophobicity contributes to increase the activity, we reinforce the notion that this property must be taken into account to design new potent

  17. Structural and dynamic evolution of the amphipathic N-terminus diversifies enzyme thermostability in the glycoside hydrolase family 12.

    PubMed

    Jiang, Xukai; Chen, Guanjun; Wang, Lushan

    2016-08-21

    Understanding the molecular mechanism underlying protein thermostability is central to the process of efficiently engineering thermostable cellulases, which can provide potential advantages in accelerating the conversion of biomass into clean biofuels. Here, we explored the general factors that diversify enzyme thermostability in the glycoside hydrolase family 12 (GH12) using comparative molecular dynamics (MD) simulations coupled to a bioinformatics approach. The results indicated that protein stability is not equally distributed over the whole structure: the N-terminus is the most thermal-sensitive region of the enzymes with a β-sandwich architecture and it tends to lose its secondary structure during the course of protein unfolding. Furthermore, we found that the total interaction energy within the N-terminus is appreciably correlated with enzyme thermostability. Interestingly, the internal interactions within the N-terminus are organized in a special amphipathic pattern in which a hydrophobic packing cluster and a hydrogen bonding cluster lie at the two ends of the N-terminus. Finally, bioinformatics analysis demonstrated that the amphipathic pattern is highly conserved in GH12 and besides that, the evolution of the amino acids in the N-terminal region is an inherent mechanism underlying the diversity of enzyme thermostability. Taken together, our results demonstrate that the N-terminus is generally the structure that determines enzyme thermostability in GH12, and thereby it is also an ideal engineering target. The dynameomics study of a protein family can give a general view of protein functions, which will offer a wide range of applications in future protein engineering. PMID:27425569

  18. Positive regulation of phenolic catabolism in Agrobacterium tumefaciens by the pcaQ gene in response to beta-carboxy-cis,cis-muconate.

    PubMed Central

    Parke, D

    1993-01-01

    An Escherichia coli system for generating a commercially unavailable catabolite in vivo was developed and was used to facilitate molecular genetic studies of phenolic catabolism. Introduction of the plasmid-borne Acinetobacter pcaHG genes, encoding the 3,4-dioxygenase which acts on protocatechuate, into E. coli resulted in bioconversion of exogenously supplied protocatechuate into beta-carboxy-cis,cis-muconate. This compound has been shown to be an inducer of the protocatechuate (pca) genes required for catabolism of protocatechuate to tricarboxylic acid cycle intermediates in Rhizobium leguminosarum biovar trifolii. The E. coli bioconversion system was used to explore regulation of the pca genes in a related bacterium, Agrobacterium tumefaciens. The pcaD gene, which encodes beta-ketoadipate enol-lactone hydrolase, from A. tumefaciens A348 was cloned and was shown to be adjacent to a regulatory region which responds strongly to beta-carboxy-cis,cis-muconate in E. coli. Site-specific insertional mutagenesis of the regulatory region eliminated expression of the pcaD gene in E. coli. When the mutation was incorporated into the A. tumefaciens chromosome, it eliminated expression of the pcaD gene and at least three other pca genes as well. The regulatory region was shown to activate gene expression in trans. The novel regulatory gene was termed pcaQ to differentiate it from pca regulatory genes identified in other microbes, which bind different metabolites. PMID:8501056

  19. Exploring the role of the α-carboxyphosphonate moiety in the HIV-RT activity of α-carboxy nucleoside phosphonates

    PubMed Central

    Mullins, Nicholas D.; Maguire, Nuala M.; Ford, Alan; Das, Kalyan; Arnold, Edward; Balzarini, Jan; Maguire, Anita R.

    2016-01-01

    As α-carboxy nucleoside phosphonates (α-CNPs) have demonstrated a novel mode of action of HIV-1 reverse transcriptase inhibition, structurally related derivatives were synthesized, namely the malonate 2, the unsaturated and saturated bisphosphonates 3 and 4, respectively and the amide 5. These compounds were evaluated for inhibition of HIV-1 reverse transcriptase in cell-free assays. The importance of the α-carboxy phosphonoacetic acid moiety for achieving reverse transcriptase inhibition, without the need for prior phosphorylation, was confirmed. The malonate derivative 2 was less active by two orders of magnitude than the original α-CNPs, while displaying the same pattern of kinetic behavior; interestingly the activity resides in the “L”-enantiomer of 2, as seen with the earlier series of α-CNPs. A crystal structure with an RT/DNA complex at 2.95 Å resolution revealed the binding of the “L”-enantiomer of 2, at the polymerase active site with a weaker metal ion chelation environment compared to 1a (T-α-CNP) which may explain the lower inhibitory activity of 2. PMID:26813581

  20. Hydroxy-1,2,5-oxadiazolyl moiety as bioisoster of the carboxy function. A computational study on gamma-aminobutyric acid (GABA) related compounds.

    PubMed

    Tosco, Paolo; Lolli, Marco L

    2008-04-01

    Recently, our research group has proposed the hydroxyfurazanyl (4-hydroxy-1,2,5-oxadiazole-3-yl) moiety as a new non-classical isoster of the carboxy function in the design of gamma-aminobutyric acid (GABA) analogues. Some compounds showed significant activity at the GABA(A) receptor, representing the only examples of pentatomic heterocycles bearing an omega-aminoalkyl flexible side chain in the position vicinal to the hydroxy group displaying agonist activity at this receptor subtype. In this work, an ab initio analysis of the structural and electronic features of furazan-3-ol is presented, in order to provide a theoretical basis to the claimed bioisosterism with the carboxy function. An ab initio conformational study with the C-PCM implicit solvent model was carried out to elucidate the reasons of the peculiar behaviour of the furazan models. Alongside, another conformational search through molecular dynamics in explicit solvent was accomplished, in order to validate the first method. The electronic features of the 4-hydroxy-1,2,5-oxadiazole-3-yl substructure seem to account for a marked stabilising effect of the putative bioactive conformation at the GABA(A) receptor subtype. The 1,2,5-thiadiazole analogue, which shares the same conformational preference of its oxygenated counterpart, was identified as a potential candidate for synthesis and pharmacological testing. Figure 4-(omega-aminoalkyl)-1,2,5-oxadiazole-3-ol analogues of GABA. PMID:18247067

  1. Characterization of the native form and the carboxy-terminally truncated halotolerant form of α-amylases from Bacillus subtilis strain FP-133.

    PubMed

    Takenaka, Shinji; Miyatake, Ayaka; Tanaka, Kosei; Kuntiya, Ampin; Techapun, Charin; Leksawasdi, Noppol; Seesuriyachan, Phisit; Chaiyaso, Thanongsak; Watanabe, Masanori; Yoshida, Ken-ichi

    2015-06-01

    Two amylases, amylase I and amylase II from Bacillus subtilis strain FP-133, were purified to homogeneity and characterized. Their stabilities toward temperature, pH, and organic solvents, and their substrate specificities toward polysaccharides and oligosaccharides were similar. Under moderately high salt conditions, both amylases were more stable than commercial B. licheniformis amylase, and amylase I retained higher amylase activity than amylase II. The N-terminal amino acid sequence, genomic southern blot analysis, and MALDI-TOFF-MS analysis indicated that the halotolerant amylase I was produced by limited carboxy-terminal truncation of the amylase II peptide. The deduced amino acid sequence of amylase II was >95% identical to that of previously reported B. subtilis α-amylases, but their carboxy-terminal truncation points differed. Three recombinant amylases--full-length amylase corresponding to amylase II, an artificially truncated amylase corresponding to amylase I, and an amylase with a larger artificial C-terminal truncation--were expressed in B. subtilis. The artificially truncated recombinant amylases had the same high amylase activity as amylase I under moderately high salt conditions. Sequence comparisons indicated that an increased ratio of Asp/Glu residues in the enzyme may be one factor responsible for increasing halotolerance. PMID:25689045

  2. Binding of heparin by type III domains and peptides from the carboxy terminal hep-2 region of fibronectin.

    PubMed

    Ingham, K C; Brew, S A; Migliorini, M M; Busby, T F

    1993-11-23

    The major sites of heparin binding by fibronectin are located in fragments of 30 or 40 kDa that contain type III modules 12 through 14 or 15. Various proteolytic or recombinant subfragments and several synthetic peptides derived from this region have been compared with respect to their binding to fluorescein-labeled heparin in solution. Binding was monitored by the change in fluorescence anisotropy at 25 degrees C and pH 7.4 in 0.02 M Tris buffer, alone (TB) or with 0.15M NaCl (TBS). A 23-kDa fragment containing III13 and III14 but lacking III12 had Kd values of 0.3 and 1.8 microM in TB, and TBS, respectively, indistinguishable from the 30-kDa parent. Fragments containing only module III13 bound 2-3-fold weaker than the parent while those containing only III14 bound 6-50-fold weaker depending on the ionic strength. Fragments containing only III12 or III15 failed to bind at all in TBS. A cationic peptide derived from the amino terminus of III13 and containing the Arg-Arg-Ala-Arg consensus sequence, whose integrity was shown by Barkalow and Schwarzbauer [Barkalow, F. J., & Schwarzbauer, J. E. (1991) J. Biol. Chem. 266, 7812-7818] to be critical, failed to bind in TBS but bound weakly in TB. Two additional cationic peptides derived from the middle and C-terminal regions of III14 showed similar behavior. Thus while the major determinant(s) of heparin binding are located in III13, those determinants are only active when part of a properly folded structure. Furthermore, module III13 when isolated had a slightly lower affinity than fragments containing both III13 and III14.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8241146

  3. Specificity studies on enteropeptidase substrates related to the N-terminus of trypsinogen.

    PubMed Central

    Jenö, P; Green, J R; Lentze, M J

    1987-01-01

    The specificity of the synthetic substrate Gly-[L-Asp]4-L-Lys 2-naphthylamide originally developed for the assay of enteropeptidase (EC 3.4.21.9), was investigated with partially purified aminopeptidase. Our results indicate that, not only enteropeptidase, but also the concerted action of the aminopeptidases of the rat small intestine, can rapidly release 2-naphthylamine from the substrate. A previously undescribed, highly active, dipeptidylaminopeptidase, which hydrolyses a Gly-Asp dipeptide from the N-terminus of the substrate, was detected in rat small intestine. The resulting [L-Asp]3-L-Lys 2-naphthylamide fragment is then degraded by a combination of aminopeptidase A and N to yield free 2-naphthylamine. Thus the present substrate cannot be regarded as being specific for enteropeptidase, and its use leads to an over-estimation of enteropeptidase activity in homogenates and extracts of intestinal tissue. In order to prevent this non-specific hydrolysis by aminopeptidases, stereoisomeric substrates with the sequence L-Ala-D-Asp-[L-Asp]3-L-Lys methyl ester, D-Ala-[L-Asp]4-L-Lys methyl ester and L-Ala-[Asp]4-L-Lys methyl ester were synthesized and tested as alternative substrates by their ability to inhibit the enteropeptidase-catalysed activation of trypsinogen. PMID:3297038

  4. Oxidative stress induces nuclear translocation of C-terminus of {alpha}-synuclein in dopaminergic cells

    SciTech Connect

    Xu Shengli; Zhou Ming; Yu Shun; Cai Yanning; Zhang Alex; Ueda, Kenji; Chan Piu . E-mail: pbchan@bjsap.org

    2006-03-31

    Growing evidence suggests that oxidative stress is involved in the neuronal degeneration and can promote the aggregation of {alpha}-synuclein. However, the role of {alpha}-synuclein under physiological and pathological conditions remains poorly understood. In the present study, we examined the possible interaction between the {alpha}-synuclein and oxidative stress. In a dopaminergic cell line MES23.5, we have found that the 200 {mu}M H{sub 2}O{sub 2} treatment induced the translocation of {alpha}-synuclein from cytoplasm to nuclei at 30 min post-treatment. The immunoactivity of {alpha}-synuclein became highly intensive in the nuclei after 2 h treatment. The protein translocated to nucleus was a 10 kDa fragment of C-terminus region of {alpha}-synuclein, while full-length {alpha}-synuclein remained in cytoplasm. Thioflavine-S staining suggested that the C-terminal fragment in the nuclei has no {beta}-sheet structures. Our present results indicated that 200 {mu}M H{sub 2}O{sub 2} treatment induces the intranuclear accumulation of the C-terminal fragment of {alpha}-synuclein in dopaminergic neurons, whose role remains to be investigated.

  5. Glacier-terminus fluctuations in the Wrangell and Chugach mountains resulting from non-climate controls

    SciTech Connect

    Sturm, M.; Hall, D.K.; Benson, C.S.; Field, W.O.

    1992-03-01

    Non-climatically controlled fluctuations of glacier termini were studied in two regions in Alaska. In the Wrangell Mountains, eight glaciers on Mt. Wrangell, an active volcano, have been monitored over the past 30 years using terrestrial surveys, aerial photogrammetry and digitally registered satellite images. Results, which are consistent between different methods of measurement, indicate that the termini of most glaciers were stationary or had retreated slightly. However, the termini of the 30-km-long Ahtna Glacier and the smaller Center and South MacKeith glaciers began to advance in the early 1960s and have advanced steadily at rates between 5 and 18 m yr-1 since then. These three glaciers flow from the summit caldera of ML Wrangell near the active North Crater, where increased volcanic heating since 1964 has melted over 7 x 107 M3 of ice. The authors suspect that volcanic meltwater has changed the basal conditions for the glaciers, resulting in their advance. In College Fjord, Prince William Sound, the terminus fluctuations of two tidewater glaciers have been monitored since 1931 by terrestrial surveying, photogrammetry, and most recently, from satellite imagery. Harvard Glacier, a 40-kmlong tidewater glacier, has been advancing steadily at nearly 20 m yr-1 since 1931, while the adjacent Yale Glacier has retreated at approximately 50 m yr-1 during the same period, though for short periods, both rates have been much higher.

  6. Interactions of antimicrobial peptide from C-terminus of myotoxin II with phospholipid mono- and bilayers.

    PubMed

    Won, Amy; Ianoul, Anatoli

    2009-10-01

    Comparative studies of the effect of a short synthetic cationic peptide, pEM-2 (KKWRWWLKALAKK), derived from the C-terminus of myotoxin II from the venom of the snake Bothrops asper on phospholipid mono- and bilayers were performed by means of Langmuir Blodgett (LB) monolayer technique, atomic force microscopy and calcein leakage assay. Phospholipid mono- and bilayers composed of single zwitterionic or anionic phospholipids as well as lipid mixtures mimicking bacterial cell membrane were used. LB measurements indicate that the peptide binds to both anionic and zwitterionic phospholipid monolayers at low surface pressure but only to anionic at high surface pressure. Preferential interaction of the peptide with anionic phospholipid monolayer is also supported by a more pronounced change of the monolayer pressure/area isotherms induced by the peptide. AFM imaging reveals the presence of nanoscale aggregates in lipid/peptide mixture monolayers. At the same time, calcein leakage experiment demonstrated that pEM-2 induces stronger disruption of zwitterionic than anionic bilayers. Results of the study indicate that electrostatic interactions play a significant role in the initial recognition and binding of pEM-2 to the cell membrane. However, membrane rupturing activity of the peptide depends on interactions other than simple ionic attraction. PMID:19632195

  7. Improved radioimmunoassay for thymosin. cap alpha. 1 recognizes the N-14 amino terminus

    SciTech Connect

    Naylor, P.H.; Goldstein, A.L.

    1986-03-01

    Thymosin ..cap alpha../sub 1/(T..cap alpha../sub 1/) is a biologically active thymic peptide currently undergoing trials as an immunomodulator in cancer patients and patients with immunodeficiencies. Abnormally elevated levels of T..cap alpha../sub 1/ have been found in the serum of individuals with or at risk for AIDS, with T-cell leukemias, and chronic progressive multiple sclerosis. Absorption of the current antibody with a synthetic C-14 fragment of T..cap alpha../sub 1/ results in an antisera specific for the N-14 amino terminus of T..cap alpha../sub 1/, which measures significantly higher levels of T..cap alpha../sub 1/ in serum from normal individuals and significantly increases the sensitivity of the assay. Ongoing studies indicate that this new RIA for T..cap alpha../sub 1/ will be useful in monitoring changes of immunoreactive T..cap alpha../sub 1/ in serum with age and in patients with known or suspected T-cell abnormalities.

  8. Landing at the terminus of Sabrina Vallis: A potential 2020 Mars rover landing site

    NASA Astrophysics Data System (ADS)

    Platz, T.; Hauber, E.; Le Deit, L.; Van Gasselt, S.; Kinch, K.; Madsen, M. B.; Rosenberg, H.

    2014-04-01

    For the upcoming 2020 Mars rover mission we selected a potential landing site that meets all geological criteria including the presence of Noachian/Early Hesperian aqueous sediments and associated hydrous mineral phases and access to unaltered igneous rocks. Our proposed landing site is located at the terminus of Sabrina Vallis in Magong crater. The 25 km × 20 km landing ellipse is centred at 11.990°N, 313.425°E. This site features deltaic sediments and distal lacustrine sediments. In central delta cliff sections weak signatures of Fe/Mg-bearing phyllosilicates are detected. Lacustrine sediments are cut by a partially exhumed igneous dyke. On the crater floor of Magong crater, remnants of an approximately 1 m thick dark deposit are observed, which is interpreted to be a tephra layer sourced from the adjacent volcanic field within Lederberg crater. Detailed terrain analysis of the landing site shows that engineering constraints are met with respect to slope and relief.

  9. Hpa1 harpin needs nitroxyl terminus to promote vegetative growth and leaf photosynthesis in Arabidopsis.

    PubMed

    Li, Xiaojie; Han, Liping; Zhao, Yanying; You, Zhenzhen; Dong, Hansong; Zhang, Chunling

    2014-03-01

    Hpa1 is a harpin protein produced by Xanthomonas oryzae, an important bacterial pathogen of rice, and has the growth-promoting activity in plants. To understand the molecular basis for the function of Hpa1, we generated an inactive variant protein, Hpa1 delta NT, by deleting the nitroxyl-terminal region of the Hpa1 sequence and compared Hpa1 delta NT with the full-length protein in terms of the effects on vegetative growth and related physiological responses in Arabidopsis. When Hpa1 was applied to plants, it acted to enhance the vegetative growth but did not affect the floral development. Enhanced plant growth was accompanied by induced expression of growth-promoting genes in plant leaves. The growth-promoting activity of Hpa1 was further correlated with a physiological consequence shown as promoted leaf photosynthesis as a result of facilitated CO2 conduction through leaf stomata and mesophyll cells. On the contrary, plant growth, growth-promoting gene expression, and the physiological consequence changed little in response to the Hpa1 delta NT treatment. These analyses suggest that Hpa1 requires the nitroxyl-terminus to facilitate CO2 transport inside leaf cells and promote leaf photosynthesis and vegetative growth of the plant. PMID:24499797

  10. Subcellular localization of the Streptococcus mutans P1 protein C terminus.

    PubMed

    Homonylo-McGavin, M K; Lee, S F; Bowden, G H

    1999-06-01

    To determine the subcellular location of the Streptococcus mutans P1 protein C-terminal anchor, cell envelope fractionation experiments were conducted in combination with Western immunoblotting, using monoclonal antibody MAb 6-8C specific for an epitope that maps near the C terminus of P1 protein and also a polyclonal antibody preparation directed against the P1 C-terminal 144 amino acids (P1COOH). P1 protein was detected in cell walls but not the membrane purified from S. mutans cells by the monoclonal antibody. In contrast, P1 protein was not detected in the same cell wall preparation using the anti-P1COOH polyclonal antibody. However, proteins released from the cell walls by treatment with mutanolysin contained antigen that was recognized by the anti-P1COOH antibody, suggesting that the epitopes recognized by the antibody were masked by peptidoglycan in the cell wall preparations. When cell walls were treated with boiling trichloroacetic acid to solubilize cell-wall-associated carbohydrate, P1 antigen could not be detected in either the solubilized carbohydrate, or in the remaining peptidoglycan, regardless of whether polyclonal or monoclonal antibody was used. However, when the peptidoglycan was treated with mutanolysin, P1 antigen could be detected in the mutanolysin solubilized fraction by MAb 6-8C. Collectively, these data suggest that the C-terminal 144 amino acids of the P1 protein are embedded within the cell wall, and associated exclusively with the peptidoglycan. Furthermore, the ability of the anti-P1COOH antibody to recognize P1 antigen only after mutanolysin treatment of cell walls suggests these C-terminal 144 amino acids are tightly intercalated within the peptidoglycan strands. PMID:10453480

  11. Structural studies of the tethered N-terminus of the Alzheimer's disease amyloid-β peptide.

    PubMed

    Nisbet, Rebecca M; Nuttall, Stewart D; Robert, Remy; Caine, Joanne M; Dolezal, Olan; Hattarki, Meghan; Pearce, Lesley A; Davydova, Natalia; Masters, Colin L; Varghese, Jose N; Streltsov, Victor A

    2013-10-01

    Alzheimer's disease is the most common form of dementia in humans and is related to the accumulation of the amyloid-β (Aβ) peptide and its interaction with metals (Cu, Fe, and Zn) in the brain. Crystallographic structural information about Aβ peptide deposits and the details of the metal-binding site is limited owing to the heterogeneous nature of aggregation states formed by the peptide. Here, we present a crystal structure of Aβ residues 1-16 fused to the N-terminus of the Escherichia coli immunity protein Im7, and stabilized with the fragment antigen binding fragment of the anti-Aβ N-terminal antibody WO2. The structure demonstrates that Aβ residues 10-16, which are not in complex with the antibody, adopt a mixture of local polyproline II-helix and turn type conformations, enhancing cooperativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aβ (residues, 10-16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13-metal-His14 coordination in the Aβ1-16 fragment implicated in Aβ metal binding. This novel structure, therefore, has the potential to provide a foundation for investigating the effect of metal ion binding to Aβ and illustrates a potential target for the development of future Alzheimer's disease therapeutics aimed at stabilizing the N-terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal-binding-induced neurotoxicity. PMID:23609990

  12. Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL.

    PubMed

    Yao, Yong; Nisan, Danielle; Fujimoto, Lynn M; Antignani, Antonella; Barnes, Ashley; Tjandra, Nico; Youle, Richard J; Marassi, Francesca M

    2016-06-01

    BCL-XL is a dominant inhibitor of apoptosis and a significant anti-cancer drug target. Endogenous BCL-XL is integral to the mitochondrial outer membrane (MOM). BCL-XL reconstituted in detergent-free lipid bilayer nanodiscs is anchored to the nanodisc lipid bilayer membrane by tight association of its C-terminal tail, while the N-terminal head retains the canonical structure determined for water-soluble, tail-truncated BCL-XL, with the surface groove solvent-exposed and available for BH3 ligand binding. To better understand the conformation and dynamics of this key region of BCL-XL we have developed methods for isolating the membrane-embedded C-terminal tail from its N-terminal head and for preparing protein suitable for structural and biochemical studies. Here, we outline the methods for sample preparation and characterization and describe previously unreported structural and dynamics features. We show that the C-terminal tail of BCL-XL forms a transmembrane α-helix that retains a significant degree of conformational dynamics. We also show that the presence of the intact C-terminus destabilizes the soluble state of the protein, and that the small fraction of soluble recombinant protein produced in Escherichia coli is susceptible to proteolytic degradation of C-terminal residues beyond M218. This finding impacts the numerous previous studies where recombinant soluble BCL-XL was presumed to be full-length. Nevertheless, the majority of recombinant BCL-XL produced in E. coli is insoluble and protected from proteolysis. This protein retains the complete C-terminal tail and can be reconstituted in lipid bilayers in a folded and active state. PMID:26923059

  13. Deconstructing honeybee vitellogenin: novel 40 kDa fragment assigned to its N terminus

    PubMed Central

    Havukainen, Heli; Halskau, Øyvind; Skjaerven, Lars; Smedal, Bente; Amdam, Gro V.

    2011-01-01

    Vitellogenin, an egg-yolk protein precursor common to oviparous animals, is found abundantly in honeybee workers – a caste of helpers that do not usually lay eggs. Instead, honeybee vitellogenin (180 kDa) participates in processes other than reproduction: it influences hormone signaling, food-related behavior, immunity, stress resistance and longevity. The molecular basis of these functions is largely unknown. Here, we establish and compare the molecular properties of vitellogenin from honeybee hemolymph (blood) and abdominal fat body, two compartments that are linked to vitellogenin functions. Our results reveal a novel 40 kDa vitellogenin fragment in abdominal fat body tissue, the main site for vitellogenin synthesis and storage. Using MALDI-TOF combined with MS/MS mass-spectroscopy, we assign the 40 kDa fragment to the N terminus of vitellogenin, whereas a previously observed 150 kDa fragment corresponded to the remainder of the protein. We show that both protein units are N glycosylated and phosphorylated. Focusing on the novel 40 kDa fragment, we present a homology model based on the structure of lamprey lipovitellin that includes a conserved β-barrel-like shape, with a lipophilic cavity in the interior and two insect-specific loops that have not been described before. Our data indicate that the honeybee fat body vitellogenin experiences cleavage unlike hemolymph vitellogenin, a pattern that can suggest a tissue-specific role. Our experiments advance the molecular understanding of vitellogenin, of which the multiple physiological and behavioral effects in honeybees are well established. PMID:21270306

  14. The C-terminus of the Hermes transposase contains a protein multimerization domain.

    PubMed

    Michel, K; O'Brochta, D A; Atkinson, P W

    2003-10-01

    Transposase activity that mediates the mobility of class II transposable elements, is most commonly initiated by the assembly of higher order synaptic complexes, called transpososomes. The formation of these complexes, that contain the transposable element's DNA as well as two or more molecules of the transposase, is dependent on interactions between transposase molecules. Using the yeast Two-Hybrid system, we were able to identify three regions mediating multimerization of the Hermes transposase, an element used for germline transformation of insects belonging to the hAT family of transposable elements. One region facilitating protein binding of Hermes transposase molecules was found within the first 252 amino acids of the transposase. The second region was located at the C-terminus of the transposase, and was found to be specific for Hermes transposase multimerization. Amino acids 551-569 were not only required for multimerization but were also necessary for transposition of the element. The third region was located between amino acids 253 and 380 and was found to eliminate the non-specific protein binding ability of the N-terminal protein interaction region but was required for the specific protein binding ability of the C-terminal region of the transposase. Five point mutations affecting the structural integrity of the C-terminal multimerization region abolished or significantly reduced transpositional activity. The same region had been previously identified to mediate dimerization in Activator (Ac), another hAT element, indicating that hAT transposase multimerization is likely to be a prerequisite for mobility of their elements. PMID:14505689

  15. The Rough Creek graben of western Kentucky: Some structures and hydrocarbon occurrences near the eastern terminus

    SciTech Connect

    Kuehn, K.W. . Dept. of Geography and Geology)

    1994-04-01

    The southeastern flank of the Illinois Basin in western kentucky contains numerous structures of interest both for their tectonic history and hydrocarbon potential. Recent research and exploration has been focused upon relations between Illinois Basin structures and basement structures in a region known as the Rough Creek Graben (RCG). The RCG extends eastward from the Illinois-Kentucky border approximately 140 miles and is about 30 miles at its widest point north-to-south. It is a wedge-shaped aulacogen attributed to opening of the proto-Atlantic Ocean near the start of the Cambrian Period. Mixed clastic and carbonate basin-fill sequences accumulated as much as 16,000 feet within the RCG before deposition of the Cambro-Ordovician Knox Group platform carbonates. Though most of the Paleozoic permitted quiescent, epeirogenic adjustments within the RCG, post-Knox tectonic episodes caused the reactivation of older faults. Today, surface expression of the northern RCG boundary is marked by the rough Creek Fault System, a complex zone up to five miles wide comprising numerous normal, reverse, and wrench faults which have potential as hydrocarbon traps. Only nine wells have tested sub-Knox sequences within the RCG including two recently completed deep tests (TD about 14,000[prime]) in McLean and Grayson counties. Most oil and gas production in the region is from wells less than 3,000 feet completed in Devonian through Pennsylvanian strata. This paper contributes structure sections from portions of Edmonson and Grayson counties near the eastern terminus of the fault system which illustrate stratigraphic throw, trapping mechanisms, and the influence of fractures on shallow hydrocarbon production in this area.

  16. N-terminus-modified Hec1 suppresses tumour growth by interfering with kinetochore-microtubule dynamics.

    PubMed

    Orticello, M; Fiore, M; Totta, P; Desideri, M; Barisic, M; Passeri, D; Lenzi, J; Rosa, A; Orlandi, A; Maiato, H; Del Bufalo, D; Degrassi, F

    2015-06-01

    Mitotic proteins are attractive targets to develop molecular cancer therapeutics due to the intimate interdependence between cell proliferation and mitosis. In this work, we have explored the therapeutic potential of the kinetochore (KT) protein Hec1 (Highly Expressed in Cancer protein 1) as a molecular target to produce massive chromosome missegregation and cell death in cancer cells. Hec1 is a constituent of the Ndc80 complex, which mediates KT-microtubule (MT) attachments at mitosis and is upregulated in various cancer types. We expressed Hec1 fused with enhanced green fluorescent protein (EGFP) at its N-terminus MT-interaction domain in HeLa cells and showed that expression of this modified Hec1, which localized at KTs, blocked cell proliferation and promoted apoptosis in tumour cells. EGFP-Hec1 was extremely potent in tumour cell killing and more efficient than siRNA-induced Hec1 depletion. In striking contrast, normal cells showed no apparent cell proliferation defects or cell death following EGFP-Hec1 expression. Live-cell imaging demonstrated that cancer cell death was associated with massive chromosome missegregation within multipolar spindles after a prolonged mitotic arrest. Moreover, EGFP-Hec1 expression was found to increase KT-MT attachment stability, providing a molecular explanation for the abnormal spindle architecture and the cytotoxic activity of this modified protein. Consistent with cell culture data, EGFP-Hec1 expression was found to strongly inhibit tumour growth in a mouse xenograft model by disrupting mitosis and inducing multipolar spindles. Taken together, these findings demonstrate that stimulation of massive chromosome segregation defects can be used as an anti-cancer strategy through the activation of mitotic catastrophe after a multipolar mitosis. Importantly, this study represents a clear proof of concept that targeting KT proteins required for proper KT-MT attachment dynamics constitutes a powerful approach in cancer therapy. PMID

  17. Molecular Basis for Membrane Pore Formation by Bax Protein Carboxyl Terminus

    PubMed Central

    Tatulian, Suren A.; Garg, Pranav; Nemec, Kathleen N.; Chen, Bo; Khaled, Annette R.

    2015-01-01

    Bax protein plays a key role in mitochondrial membrane permeabilization and cytochrome c release upon apoptosis. Our recent data have indicated that the 20-residue C-terminal peptide of Bax (BaxC-KK; VTIFVAGVL-TASLTIWKKMG), when expressed intracellularly, translocates to the mitochondria and exerts lethal effect on cancer cells. Moreover, the BaxC-KK peptide, as well as two mutants where the two lysines are replaced with glutamate (BaxC-EE) or leucine (BaxC-LL), have been shown to form relatively large pores in lipid membranes, composed of up to eight peptide molecules per pore. Here the pore structure is analyzed by polarized Fourier transform infrared, circular dichroism, and fluorescence experiments on the peptides reconstituted in phospholipid membranes. The peptides assume an α/β-type secondary structure within membranes. Both β-strands and α-helices are significantly (by 30–60 deg) tilted relative to the membrane normal. The tryptophan residue embeds into zwitterionic membranes at 8–9 Å from the membrane center. The membrane anionic charge causes a deeper insertion of tryptophan for BaxC-KK and BaxC-LL but not for BaxC-EE. Combined with the pore stoichiometry determined earlier, these structural constraints allow construction of a model of the pore where eight peptide molecules form an “α/β-ring” structure within the membrane. These results identify a strong membranotropic activity of Bax C-terminus and propose a new mechanism by which peptides can efficiently perforate cell membranes. Knowledge on the pore forming mechanism of the peptide may facilitate development of peptide-based therapies to kill cancer or other detrimental cells such as bacteria or fungi. PMID:23110300

  18. The insulin receptor C-terminus is involved in regulation of the receptor kinase activity.

    PubMed

    Kaliman, P; Baron, V; Alengrin, F; Takata, Y; Webster, N J; Olefsky, J M; Van Obberghen, E

    1993-09-21

    During the insulin receptor activation process, ligand binding and autophosphorylation induce two distinct conformational changes in the C-terminal domain of the receptor beta-subunit. To analyze the role of this domain and the involvement of the C-terminal autophosphorylation sites (Tyr1316 and Tyr1322) in receptor activation, we used (i) antipeptide antibodies against three different C-terminal sequences (1270-1281, 1294-1317, and 1309-1326) and (ii) an insulin receptor mutant (Y/F2) where Tyr1316 and Tyr1322 have been replaced by Phe. We show that the autophosphorylation-induced C-terminal conformational change is preserved in the Y/F2 receptor, indicating that this change is not induced by phosphorylation of the C-terminal sites but most likely by phosphorylation of the major sites in the kinase domain (Tyr1146, Tyr1150, and Tyr1151). Binding of antipeptide antibodies to the C-terminal domain modulated (activated or inhibited) both mutant and wild-type receptor-mediated phosphorylation of poly(Glu/Tyr). In contrast to the wild-type receptor, Y/F2 exhibited the same C-terminal configuration before and after insulin binding, evidencing that mutation of Tyr1316 and Tyr1322 introduced conformational changes in the C-terminus. Finally, the mutant receptor was 2-fold more active than the wild-type receptor for poly(Glu/Tyr) phosphorylation. In conclusion, the whole C-terminal region of the insulin receptor beta-subunit is likely to exert a regulatory influence on the receptor kinase activity. Perturbations of the C-terminal region, such as binding of antipeptides or mutation of Tyr1316 and Tyr1322, provoke alterations at the receptor kinase level, leading to activation or inhibition of the enzymic activity. PMID:7690586

  19. The N-terminus of VDAC: Structure, mutational analysis, and a potential role in regulating barrel shape.

    PubMed

    Shuvo, Sabbir R; Ferens, Fraser G; Court, Deborah A

    2016-06-01

    A novel feature of the voltage-dependent anion channel (VDAC, mitochondrial porin), is the barrel, comprising an odd number of β-strands and closed by parallel strands. Recent research has focused on the N-terminal segment, which in the available structures, resides in the lumen and is not part of the barrel. In this review, the structural data obtained from vertebrate VDAC are integrated with those from VDAC in artificial bilayers, emphasizing the array of native and tagged versions of VDAC used. The data are discussed with respect to a recent gating model (Zachariae et al. (2012) Structure 20:1-10), in which the N-terminus acts not as a gate on a stable barrel, but rather stabilizes the barrel, preventing its shift into a partially collapsed, low-conductance, closed state. Additionally, the role of the N-terminus in VDAC oligomerization, apoptosis through interactions with hexokinase and its interaction with ATP are discussed briefly. PMID:26997586

  20. DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes

    PubMed Central

    Choi, Si Ho; Gearhart, Micah D.; Cui, Ziyou; Bosnakovski, Darko; Kim, Minjee; Schennum, Natalie; Kyba, Michael

    2016-01-01

    Ectopic expression of the double homeodomain transcription factor DUX4 causes facioscapulohumeral muscular dystrophy (FSHD). Mechanisms of action of DUX4 are currently unknown. Using immortalized human myoblasts with a titratable DUX4 transgene, we identify by mass spectrometry an interaction between the DUX4 C-terminus and the histone acetyltransferases p300/CBP. Chromatin immunoprecipitation shows that DUX4 recruits p300 to its target gene, ZSCAN4, displaces histone H3 from the center of its binding site, and induces H3K27Ac in its vicinity, but C-terminal deleted DUX4 does not. We show that a DUX4 minigene, bearing only the homeodomains and C-terminus, is transcriptionally functional and cytotoxic, and that overexpression of a nuclear targeted C-terminus impairs the ability of WT DUX4 to interact with p300 and to regulate target genes. Genomic profiling of DUX4, histone H3, and H3 modifications reveals that DUX4 binds two classes of loci: DNase accessible H3K27Ac-rich chromatin and inaccessible H3K27Ac-depleted MaLR-enriched chromatin. At this latter class, it acts as a pioneer factor, recruiting H3K27 acetyltransferase activity and opening the locus for transcription. In concert with local increased H3K27Ac, the strong H3K27Ac peaks at distant sites are significantly depleted of H3K27Ac, thus DUX4 uses its C-terminus to induce a global reorganization of H3K27 acetylation. PMID:26951377

  1. DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes.

    PubMed

    Choi, Si Ho; Gearhart, Micah D; Cui, Ziyou; Bosnakovski, Darko; Kim, Minjee; Schennum, Natalie; Kyba, Michael

    2016-06-20

    Ectopic expression of the double homeodomain transcription factor DUX4 causes facioscapulohumeral muscular dystrophy (FSHD). Mechanisms of action of DUX4 are currently unknown. Using immortalized human myoblasts with a titratable DUX4 transgene, we identify by mass spectrometry an interaction between the DUX4 C-terminus and the histone acetyltransferases p300/CBP. Chromatin immunoprecipitation shows that DUX4 recruits p300 to its target gene, ZSCAN4, displaces histone H3 from the center of its binding site, and induces H3K27Ac in its vicinity, but C-terminal deleted DUX4 does not. We show that a DUX4 minigene, bearing only the homeodomains and C-terminus, is transcriptionally functional and cytotoxic, and that overexpression of a nuclear targeted C-terminus impairs the ability of WT DUX4 to interact with p300 and to regulate target genes. Genomic profiling of DUX4, histone H3, and H3 modifications reveals that DUX4 binds two classes of loci: DNase accessible H3K27Ac-rich chromatin and inaccessible H3K27Ac-depleted MaLR-enriched chromatin. At this latter class, it acts as a pioneer factor, recruiting H3K27 acetyltransferase activity and opening the locus for transcription. In concert with local increased H3K27Ac, the strong H3K27Ac peaks at distant sites are significantly depleted of H3K27Ac, thus DUX4 uses its C-terminus to induce a global reorganization of H3K27 acetylation. PMID:26951377

  2. Truncations of xyloglucan xylosyltransferase 2 provide insights into the roles of the N- and C-terminus.

    PubMed

    Culbertson, Alan T; Smith, Adrienne L; Cook, Matthew D; Zabotina, Olga A

    2016-08-01

    Xyloglucan is the most abundant hemicellulose in the primary cell wall of dicotyledonous plants. In Arabidopsis, three xyloglucan xylosyltransferases, XXT1, XXT2, and XXT5, participate in xylosylation of the xyloglucan backbone. Despite the importance of these enzymes, there is a lack of information on their structure and the critical residues required for substrate binding and transferase activity. In this study, the roles of different domains of XX2 in protein expression and catalytic activity were investigated by constructing a series of N- and C-terminal truncations. XXT2 with an N-terminal truncation of 31 amino acids after the predicted transmembrane domain showed the highest protein expression, but truncations of more than 31 residues decreased protein expression and catalytic activity. XXT2 constructs with C-terminal truncations showed increased protein expression but decreased activity, particularly for truncations of 44 or more amino acids. Site-directed mutagenesis was also used to investigate six positively charged residues near the C-terminus and found that four of the mutants showed decreased enzymatic activity. We conclude that the N- and C-termini of XXT2 have important roles in protein folding and enzymatic activity: the stem region (particularly the N-terminus of the catalytic domain) is critical for protein folding and the C-terminus is essential for enzymatic activity but not for protein folding. PMID:27193738

  3. The N-terminus of classical swine fever virus (CSFV) nonstructural protein 2 modulates viral genome RNA replication.

    PubMed

    Li, Ling; Wu, Rui; Zheng, Fengwei; Zhao, Cheng; Pan, Zishu

    2015-12-01

    Pestivirus nonstructural protein 2 (NS2) is a multifunctional, hydrophobic protein with an important but poorly understood role in viral RNA replication and infectious virus production. In the present study, based on sequence analysis, we mutated several representative conserved residues within the N-terminus of NS2 of classical swine fever virus (CSFV) and investigated how these mutations affected viral RNA replication and infectious virus production. Our results demonstrated that the mutation of two aspartic acids, NS2/D60A or NS2/D60K and NS2/D78K, in the N-terminus of NS2 abolished infectious virus production and that the substitution of arginine for alanine at position 100 (NS2/R100A) resulted in significantly decreased viral titer. The serial passage of cells containing viral genomic RNA molecules generated the revertants NS2/A60D, NS2/K60D and NS2/K78D, leading to the recovery of infectious virus. In the context of the NS2/R100A mutant, the NS2/I90L mutation compensated for infectious virus production. The regulatory roles of the indicated amino acid residues were identified to occur at the viral RNA replication level. These results revealed a novel function for the NS2 N-terminus of CSFV in modulating viral RNA replication. PMID:26232654

  4. The C-terminus of DSX(F5) protein acts as a novel regulatory domain in Bombyx mori.

    PubMed

    Duan, Jianping; Meng, Xianxin; Ma, Sanyuan; Wang, Feng; Guo, Huozhen; Zhang, Liying; Zhao, Ping; Kan, Yunchao; Yao, Lunguang; Xia, Qingyou

    2016-08-01

    The doublesex gene regulates the somatic sexual development of Bombyx mori by alternatively splicing into sex-specific splice forms. In our previous study, the splice form Bmdsx (F7) , which encodes the BmDSX(F5) protein, was found to be expressed in a female-specific manner and to contain a novel C-terminus. In this study, we aimed to investigate the role of this C-terminus. Two transgenic lines, L1 and L2, were constructed to ectopically express Bmdsx (F7) in males. Phenotype and W chromosome-specific polymerase chain reaction (PCR) analysis showed that developmental abnormalities and sex reversal did not occur. Moreover, the sex ratio was also normal. Quantitative PCR revealed that the expression levels of SP1 and Vg were upregulated in the fat body of transgenic males. Additionally, the expression level of PBP was downregulated in the antenna of transgenic males. The results suggested that the C-terminus of BmDSX(F5) functioned as a regulatory domain during regulation of downstream target gene expression and that BmDSX(F5) participated in the sexual development of somatic cells together with other DSX proteins in B. mori. PMID:26975733

  5. Structure-based design of 3-carboxy-substituted 1,2,3,4-tetrahydroquinolines as inhibitors of myeloid cell leukemia-1 (Mcl-1).

    PubMed

    Chen, L; Wilder, P T; Drennen, B; Tran, J; Roth, B M; Chesko, K; Shapiro, P; Fletcher, S

    2016-06-28

    Mcl-1 has recently emerged as an attractive target to expand the armamentarium in the war on cancer. Using structure-based design, 3-carboxy-substituted 1,2,3,4-tetrahydroquinolines were developed as a new chemotype to inhibit the Mcl-1 oncoprotein. The most potent compound inhibited Mcl-1 with a Ki of 120 nM, as determined by a fluorescence polarization competition assay. Direct binding was confirmed by 2D (1)H-(15)N HSQC NMR spectroscopy with (15)N-Mcl-1, which indicated that interactions with R263 and T266, and occupation of the p2 pocket are likely responsible for the potent binding affinity. The short and facile synthetic chemistry to access target molecules is expected to mediate lead optimization. PMID:26751150

  6. mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain

    PubMed Central

    Cho, Eun-Jung; Takagi, Toshimitsu; Moore, Christine R.; Buratowski, Stephen

    1997-01-01

    Capping of mRNA occurs shortly after transcription initiation, preceding other mRNA processing events such as mRNA splicing and polyadenylation. To determine the mechanism of coupling between transcription and capping, we tested for a physical interaction between capping enzyme and the transcription machinery. Capping enzyme is not stably associated with basal transcription factors or the RNA polymerase II (Pol II) holoenzyme. However, capping enzyme can directly and specifically interact with the phosphorylated form of the RNA polymerase carboxy-terminal domain (CTD). This association occurs in the context of the transcription initiation complex and is blocked by the CTD–kinase inhibitor H8. Furthermore, conditional truncation mutants of the Pol II CTD are lethal when combined with a capping enzyme mutant. Our results provide in vitro and in vivo evidence that capping enzyme is recruited to the transcription complex via phosphorylation of the RNA polymerase CTD. PMID:9407025

  7. The Ile93Met mutation in the ubiquitin carboxy-terminal-hydrolase-L1 gene is not observed in European cases with familial Parkinson's disease.

    PubMed

    Harhangi, B S; Farrer, M J; Lincoln, S; Bonifati, V; Meco, G; De Michele, G; Brice, A; Dürr, A; Martinez, M; Gasser, T; Bereznai, B; Vaughan, J R; Wood, N W; Hardy, J; Oostra, B A; Breteler, M M

    1999-07-23

    Recently an Ile93Met mutation in the ubiquitin-carboxy-terminal-hydrolase-L1 gene (UCH-L1) has been described in a German family with Parkinson's disease (PD). The authors showed that this mutation is responsible for an impaired proteolytic activity of the UCH-L1 protein and may lead to an abnormal aggregation of proteins in the brain. In order to determine the importance of this or any other mutation in the coding region of the UCH-L1 gene in PD, we performed mutation analysis on Caucasian families with at least two affected sibs. We did not detect any mutations in the UCH-L1 gene, however, we cannot exclude mutations in the regulatory or intronic regions of the UCH-L1 gene since these regions were not sequenced. We conclude that the UCH-L1 gene is not a major gene responsible for familial PD. PMID:10454131

  8. Thermochromism and structural change in polydiacetylenes including carboxy and 4-carboxyphenyl groups as the intermolecular hydrogen bond linkages in the side chain.

    PubMed

    Tanioku, Chiaki; Matsukawa, Kimihiro; Matsumoto, Akikazu

    2013-02-01

    We investigated the thermochromic behavior of polydiacetylenes including the carboxy and 4-carboxyphenyl groups as the side-chain substituents adjacent to the conjugated main chain, and then, the thermal stability and the thermochromism reversibility of the polymers were related to changes in the polymer conformations monitored by IR and Raman spectroscopies and powder X-ray diffractions. The polydiacetylenes with no or a phenylene spacer between the main chain and the carboxylic acid moiety were revealed to exhibit a thermal resistance for maintaining reversible thermochromism in a high temperature range, rather than polydiacetylenes with a conventional structure with a flexible alkylene spacer. The molecular stacking structures of the diacetylenes and the corresponding polymers in the crystals were discussed based on the results of an X-ray single-crystal structure analysis as well as the powder X-ray diffraction measurements. PMID:23276165

  9. The carboxy-terminal domain of Grp94 binds to protein kinase CK2 alpha but not to CK2 holoenzyme.

    PubMed

    Roher, N; Sarno, S; Miró, F; Ruzzene, M; Llorens, F; Meggio, F; Itarte, E; Pinna, L A; Plana, M

    2001-09-01

    Surface plasmon resonance analysis shows that the carboxy-terminal domain of Grp94 (Grp94-CT, residues 518-803) physically interacts with the catalytic subunit of protein kinase CK2 (CK2 alpha) under non-stressed conditions. A K(D) of 4 x 10(-7) was determined for this binding. Heparin competed with Grp94-CT for binding to CK2 alpha. CK2 beta also inhibited the binding of Grp94-CT to CK2 alpha, and CK2 holoenzyme reconstituted in vitro was unable to bind Grp94-CT. The use of CK2 alpha mutants made it possible to map the Grp94-CT binding site to the four lysine stretch (residues 74-77) present in helix C of CK2 alpha. Grp94-CT stimulated the activity of CK2 alpha wild-type but was ineffective on the CK2 alpha K74-77A mutant. PMID:11557039

  10. Biochemical and Structural Studies of 6-Carboxy-5,6,7,8-tetrahydropterin Synthase Reveal the Molecular Basis of Catalytic Promiscuity within the Tunnel-fold Superfamily*

    PubMed Central

    Miles, Zachary D.; Roberts, Sue A.; McCarty, Reid M.; Bandarian, Vahe

    2014-01-01

    6-Pyruvoyltetrahydropterin synthase (PTPS) homologs in both mammals and bacteria catalyze distinct reactions using the same 7,8-dihydroneopterin triphosphate substrate. The mammalian enzyme converts 7,8-dihydroneopterin triphosphate to 6-pyruvoyltetrahydropterin, whereas the bacterial enzyme catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin. To understand the basis for the differential activities we determined the crystal structure of a bacterial PTPS homolog in the presence and absence of various ligands. Comparison to mammalian structures revealed that although the active sites are nearly structurally identical, the bacterial enzyme houses a His/Asp dyad that is absent from the mammalian protein. Steady state and time-resolved kinetic analysis of the reaction catalyzed by the bacterial homolog revealed that these residues are responsible for the catalytic divergence. This study demonstrates how small variations in the active site can lead to the emergence of new functions in existing protein folds. PMID:24990950

  11. Optimal Replication Activity of Vesicular Stomatitis Virus RNA Polymerase Requires Phosphorylation of a Residue(s) at Carboxy-Terminal Domain II of Its Accessory Subunit, Phosphoprotein P

    PubMed Central

    Hwang, Leroy N.; Englund, Nathan; Das, Tapas; Banerjee, Amiya K.; Pattnaik, Asit K.

    1999-01-01

    The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites (Ser-226, Ser-227, and Ser-233) were altered to alanines either individually or in various combinations. Analyses of the mutant proteins for their ability to support replication of a VSV minigenomic RNA suggest that phosphorylation of either Ser-226 or Ser-227 is necessary for optimal replication activity of the protein. The mutant protein (P226/227) in which both of these residues were altered to alanines was only about 8% active in replication compared to the wild-type (wt) protein. Substitution of alanine for Ser-233 did not have any adverse effect on replication activity of the protein. In contrast, all the mutant proteins showed activities similar to that of the wt protein in transcription. These results indicate that phosphorylation of the carboxy-terminal domain II residues of P protein are required for optimal replication activity but not for transcription activity. Furthermore, substitution of glutamic acid residues for Ser-226 and Ser-227 resulted in a protein that was only 14% active in replication but almost fully active in transcription. Taken together, these results, along with our earlier studies, suggest that phosphorylation of residues at two different domains in the P protein regulates its activity in transcription and replication of the VSV genome. PMID:10364310

  12. Elevated carboxy terminal cross linked telopeptide of type I collagen in alcoholic cirrhosis: relation to liver and kidney function and bone metabolism

    PubMed Central

    Moller, S; Hansen, M; Hillingso, J; Jensen, J; Henriksen, J

    1999-01-01

    BACKGROUND—The carboxy terminal cross linked telopeptide of type I collagen (ICTP) has been put forward as a marker of bone resorption. Patients with alcoholic liver disease may have osteodystrophy. 
AIMS—To assess circulating and regional concentrations of ICTP in relation to liver dysfunction, bone metabolism, and fibrosis. 
METHODS—In 15 patients with alcoholic cirrhosis and 20 controls, hepatic venous, renal venous, and femoral arterial concentrations of ICTP, and bone mass and metabolism were measured. 
RESULTS—Circulating ICTP was higher in patients with cirrhosis than in controls. No overall significant hepatic disposal or production was found in the patient or control groups but slightly increased production was found in a subset of patients with advanced disease. Significant renal extraction was observed in the controls, whereas only a borderline significant extraction was observed in the patients. Measurements of bone mass and metabolism indicated only a mild degree of osteodystrophy in the patients with cirrhosis. ICTP correlated significantly in the cirrhotic patients with hepatic and renal dysfunction and fibrosis, but not with measurements of bone mass or metabolism. 
CONCLUSIONS—ICTP is highly elevated in patients with cirrhosis, with no detectable hepatic net production or disposal. No relation between ICTP and markers of bone metabolism was identified, but there was a relation to indicators of liver dysfunction and fibrosis. As the cirrhotic patients conceivably only had mild osteopenia, the elevated ICTP in cirrhosis may therefore primarily reflect liver failure and hepatic fibrosis. 

 Keywords: bone mineral density; carboxy terminal cross linked telopeptide of type I collagen; chronic liver disease; fibrosis; hepatic osteodystrophy; portal hypertension PMID:10026331

  13. Processing, surface expression, and immunogenicity of carboxy-terminally truncated mutants of G protein of human respiratory syncytial virus.

    PubMed Central

    Olmsted, R A; Murphy, B R; Lawrence, L A; Elango, N; Moss, B; Collins, P L

    1989-01-01

    Posttranslational processing and cell surface expression were examined for three C-terminally truncated mutants of the G protein of respiratory syncytial virus expressed from engineered cDNAs. The truncated mutants, encoded by cDNAs designated G71, G180, and G230, contained the N-terminal 71, 180, and 230 amino acids, respectively, of the 298-amino-acid G protein. To facilitate detection of G71, which reacted inefficiently with G-specific antisera, we constructed a parallel set of cDNAs, designated G71/13, G180/13, and G230/13, to encode the same truncated species with the addition of a C-terminal 13-amino-acid reporter peptide which could be detected efficiently with an antipeptide serum. G71, G180, and G230 were expressed as species of Mr 7,500, 48,000, and 51,000, respectively, compared with 84,000 for parental G protein. The proteins encoded by G180 and G230, like parental G protein, contained both N-linked and O-linked carbohydrate. Also, the protein encoded by G71/13 appeared to be O glycosylated, showing that even this highly truncated form contained the structural information required to target the protein for O glycosylation. As for parental G protein, the estimated Mrs of the proteins encoded by G180 and G230 were approximately twice the calculated molecular weight of the polypeptide chain. Experiments with monensin showed that most of this difference between the calculated and observed Mr was due to posttranslational processing in or beyond the trans-Golgi compartment, presumably owing to the addition of carbohydrate or aggregation into dimers or both. Like parental G protein, all three truncated forms accumulated abundantly at the cell surface, and in each case the C terminus was extracellular. Thus, the N-terminal 71 amino acids of the G protein contained all the structural information required for efficient membrane insertion and cell surface expression, whereas the extracellular domain was dispensable for these activities. Cotton rats were immunized

  14. Role of the C-terminus in the activity, conformation, and stability of interleukin-6.

    PubMed Central

    Ward, L. D.; Hammacher, A.; Zhang, J. G.; Weinstock, J.; Yasukawa, K.; Morton, C. J.; Norton, R. S.; Simpson, R. J.

    1993-01-01

    Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule. PMID:8401231

  15. Diffuse Crustal Accretion at the Southern Terminus of the Malaguana-Gadao Ridge, Mariana Trough

    NASA Astrophysics Data System (ADS)

    Sleeper, J. D.; Martinez, F.; Fryer, P. B.

    2014-12-01

    The mode of extension and crustal accretion in backarc basins is strongly affected by proximity to the arc volcanic front. The factor that likely has the strongest control on these processes is mantle water content. At Mid-Ocean Ridges, the small amount of water in the mantle is efficiently extracted into the melt, dehydrating the residual material and increasing the viscosity and strength of the lithosphere. This may aid in focusing melt generated over a broad (~200+ km wide) zone in the mantle toward a narrow zone of crustal accretion ~1-2 km wide. In the near-arc setting, the continuous flux of water into the mantle wedge should oppose lithospheric dehydration and inhibit strengthening of the lithosphere, which may allow deformation, volcanism, and crustal accretion to occur over a broad area instead of along a narrow axis. A possible example of this process can be observed at the southern terminus of the Malaguana-Gadao Ridge, a backarc spreading center in the Southern Mariana Trough, at the southern end of the Izu-Bonin-Mariana convergent margin. The spreading axis, which forms an axial high in this area, abruptly terminates at 143˚20'E, 12˚37'N and is replaced by a broad zone of active volcanism and tectonism characterized by short volcanic ridges, volcanic cones, and low-relief grabens. This study uses deep-towed and ship multibeam sonar, gravity, and magnetics data collected during an early 2012 cruise on R/V Thomas G. Thompson (TN273) along with available geophysical and geochemical data in the Southern Mariana Trough to gain insight into the nature of the diffuse crustal accretion process. Evidence of a similar transition from organized to "disorganized" spreading can also be observed at Valu Fa Ridge in the southern Lau basin and other backarc spreading centers. This suggests that this process is not unique to the Southern Mariana Trough, and may be an important mode of crustal accretion in a variety of backarc settings where there is extension in

  16. 29 CFR Appendix C to Subpart R of... - Illustrations of Bridging Terminus Points: Non-mandatory Guidelines for Complying With §§ 1926...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 8 2014-07-01 2014-07-01 false Illustrations of Bridging Terminus Points: Non-mandatory Guidelines for Complying With §§ 1926.757(a)(10) and § 1926.757(c)(5) C Appendix C to Subpart R of Part.... R, App. C Appendix C to Subpart R of Part 1926—Illustrations of Bridging Terminus Points:...

  17. Pannexin-1 Is Blocked by Its C-Terminus through a Delocalized Non-Specific Interaction Surface

    PubMed Central

    Dourado, Michelle; Wong, Evera; Hackos, David H.

    2014-01-01

    The Pannexin-1 (Panx1) channel is known to become activated under a variety of physiological conditions resulting in the release of medium-sized molecules such as ATP and amino acids from the cell. The detailed molecular mechanism of activation of the channel resulting in the opening of the Pannexin pore is poorly understood. The best-studied gating mechanism is caspase-3/7-mediated cleavage and truncation of the c-terminus. In the absence of caspase-cleavage, the c-terminal peptide maintains the channel in the closed state, possibly by directly plugging the pore from the intracellular side. We sought to understand in detail the part of the c-terminus necessary for this interaction by alanine-scanning and truncation mutagenesis of the c-terminal gating peptide. These experiments demonstrate that no single amino acid side-chain is necessary for this interaction. In fact, replacing blocks of 10–12 amino acids in different parts of the c-terminal peptide with alanines fails to disrupt the ability of the c-terminus to keep the channel closed. Surprisingly, even replacing the entire c-terminal gating peptide with a scrambled peptide of the same length maintains the interaction in some cases. Further analysis revealed that the interaction surface, while delocalized, is located within the amino-terminal two-thirds of the c-terminal peptide. Such a delocalized and potentially low-affinity interaction surface is allowed due to the high effective concentration of the c-terminal peptide near the inner vestibule of the pore and likely explains why this region is poorly conserved between species. This type of weak interaction with a tethered gating peptide may be required to maintain high-sensitivity to caspase-dependent activation. PMID:24911976

  18. Palmitoylation on the carboxyl terminus tail is required for the selective regulation of dopamine D2 versus D3 receptors.

    PubMed

    Zhang, Xiaowei; Le, Hang Thi; Zhang, Xiaohan; Zheng, Mei; Choi, Bo-Gil; Kim, Kyeong-Man

    2016-09-01

    Dopamine D2 receptor (D2R) and D3 receptor (D3R) possess highly conserved amino acid sequences but this study showed that D3R was more extensively palmitoylated than D2R. Based on this finding, the molecular basis of this selective palmitoylation of D3R was determined and the roles of palmitoylation in the regulation of D3R functions were investigated. D3R was palmitoylated on the cysteine residue on its carboxyl terminus tail, the last amino acid residue of D3R, and an exchange of the carboxyl terminus tail between D2R and D3R (D2R-D3C and D3R-D2C) resulted in the switching of the palmitoylation phenotype. When the consensus site for palmitoylation was mutated or the palmitoylation of D3R was inhibited by treatment with 2-bromopalmitate (2BP), a palmitoylation blocker, cell-surface expression, PKC-mediated endocytosis, agonist affinity, and agonist-induced tolerance of D3R were all inhibited. However, these changes were not observed when D3R palmitoylation was inhibited by replacing its carboxyl tail with that of D2R (D3R-D2C) or when the palmitoylation of D2R-D3C was inhibited by treatment with 2BP. Overall, this study shows that D3R is palmitoylated more extensively than D2R even though the carboxyl terminus tails of D2R and D3R are highly homologous, and thus provides a new clue regarding the consensus sequence for palmitoylation. This study also shows that palmitoylation controls various functionalities of D3R only when the receptor is in the intact D3R configuration. PMID:27349735

  19. Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase C Terminus Is Required for Nuclear Localization and Viral DNA Replication

    PubMed Central

    Feng, Guozhong

    2014-01-01

    ABSTRACT The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production. IMPORTANCE The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are

  20. Pathological conformations involving the amino terminus of tau occur early in Alzheimer's disease and are differentially detected by monoclonal antibodies.

    PubMed

    Combs, Benjamin; Hamel, Chelsey; Kanaan, Nicholas M

    2016-10-01

    Conformational changes involving the amino terminus of the tau protein are among the earliest alterations associated with tau pathology in Alzheimer's disease and other tauopathies. This region of tau contains a phosphatase-activating domain (PAD) that is aberrantly exposed in pathological forms of the protein, an event that is associated with disruptions in anterograde fast axonal transport. We utilized four antibodies that recognize the amino terminus of tau, TNT1, TNT2 (a novel antibody), Tau12, and Tau13, to further study this important region. Using scanning alanine mutations in recombinant tau proteins, we refined the epitopes of each antibody. We examined the antibodies' relative abilities to specifically label pathological tau in non-denaturing and denaturing assays to gain insight into some of the mechanistic details of PAD exposure. We then determined the pattern of tau pathology labeled by each antibody in human hippocampal sections at various disease stages in order to characterize PAD exposure in the context of disease progression. The characteristics of reactivity for the antibodies fell into two groups. TNT1 and TNT2 recognized epitopes within amino acids 7-12 and specifically identified recombinant tau aggregates and pathological tau from Alzheimer's disease brains in a conformation-dependent manner. These antibodies labeled early pre-tangle pathology from neurons in early Braak stages and colocalized with thiazine red, a marker of fibrillar pathology, in classic neurofibrillary tangles. However, late tangles were negative for TNT1 and TNT2 indicating a loss of the epitope in later stages of tangle evolution. In contrast, Tau12 and Tau13 both identified discontinuous epitopes in the amino terminus and were unable to differentiate between normal and pathological tau in biochemical and tissue immunohistological assays. Despite the close proximity of these epitopes, the antibodies demonstrated remarkably different abilities to identify pathological

  1. Identification and structure of a putative Ca2+-binding domain at the C terminus of AQP1.

    PubMed

    Fotiadis, Dimitrios; Suda, Kitaru; Tittmann, Peter; Jenö, Paul; Philippsen, Ansgar; Müller, Daniel J; Gross, Heinz; Engel, Andreas

    2002-05-17

    Aquaporin-1 (AQP1) is the first functionally identified aquaporin of a growing family of membrane water channels found in all forms of life. Recently, a possible secondary function as a cyclic guanosine monophosphate (cGMP) gated ion channel was attributed to AQP1. We have reconstituted purified protein from bovine and human red blood cell membranes into highly ordered 2D crystals. The topography of both AQP1s was determined by electron microscopy from freeze-dried, unidirectionally metal-shadowed 2D crystals as well as from surface topographs of native crystals recorded in buffer solution with the atomic force microscope (AFM). In spite of the high level of sequence homology between bovine and human AQP1, the surfaces showed distinct differences. Alignment of both sequences and comparison of the acquired surface topographies with the atomic model of human AQP1 revealed the topographic changes on the surface of bovine AQP1 to be induced by a few amino acid substitutions. A striking degree of sequence homology was found between the carboxyl-terminal domains of AQP1s from different organisms and EF-hands from Ca2+-binding proteins belonging to the calmodulin superfamily, suggesting the existence of a Ca2+-binding site at the C terminus of AQP1 instead of the putative cGMP-binding site reported previously. To unveil its position on the acquired surface topographies, 2D crystals of AQP1 were digested with carboxypeptidase Y, which cleaves off the intracellular C terminus. Difference maps of AFM topographs between the native and the peptidase-treated AQP1s showed the carboxylic tail to be close to the 4-fold symmetry axis of the tetramer. SDS-PAGE and matrix-assisted laser desorption/ionisation mass spectrometry of native and decarboxylated bovine and human AQP1 revealed that the EF-hand motif found at the C terminus of AQP1 was partially resistant to peptidase digestion. The importance of the C-terminal domain is implicated by structural instability of decarboxylated

  2. A Reduced Risk of Infection with Plasmodium vivax and Clinical Protection against Malaria Are Associated with Antibodies against the N Terminus but Not the C Terminus of Merozoite Surface Protein 1†

    PubMed Central

    Nogueira, Paulo Afonso; Piovesan Alves, Fabiana; Fernandez-Becerra, Carmen; Pein, Oliver; Rodrigues Santos, Neida; Pereira da Silva, Luiz Hildebrando; Plessman Camargo, Erney; del Portillo, Hernando A.

    2006-01-01

    Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic. The occurrence of clinical protection in P. vivax malaria in Brazil was first reported among residents of the riverine community of Portuchuelo, in Rondônia, western Amazon. We thus analyzed immune sera from this same human population to determine if naturally acquired humoral immune responses against the merozoite surface protein 1 of P. vivax, PvMSP1, could be associated with reduced risk of infection and/or clinical protection. Our results demonstrated that this association could be established with anti-PvMSP1 antibodies predominantly of the immunoglobulin G3 subclass directed against the N terminus but not against the C terminus, in spite of the latter being more immunogenic and capable of natural boosting. This is the first report of a prospective study of P. vivax malaria demonstrating an association of reduced risk of infection and clinical protection with antibodies against an antigen of this parasite. PMID:16622209

  3. A Functional Dissection of PTEN N-Terminus: Implications in PTEN Subcellular Targeting and Tumor Suppressor Activity

    PubMed Central

    Gil, Anabel; Rodríguez-Escudero, Isabel; Stumpf, Miriam; Molina, María; Cid, Víctor J.; Pulido, Rafael

    2015-01-01

    Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted S. cerevisiae defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations. PMID:25875300

  4. The role of N-terminus of Plasmodium falciparum ORC1 in telomeric localization and var gene silencing

    PubMed Central

    Deshmukh, Abhijit S.; Srivastava, Sandeep; Herrmann, Susann; Gupta, Ashish; Mitra, Pallabi; Gilberger, Tim Wolf; Dhar, Suman Kumar

    2012-01-01

    Plasmodium falciparum origin recognition complex 1 (ORC1) protein has been implicated in DNA replication and silencing var gene family. However, the mechanism and the domain structure of ORC1 related to the regulation of var gene family are unknown. Here we show that the unique N-terminus of PfORC1 (PfORC1N1–238) is targeted to the nuclear periphery in vivo and this region binds to the telomeric DNA in vitro due to the presence of a leucine heptad repeats. Like PfORC1N1–238, endogenous full length ORC1, was found to be associated with sub telomeric repeat regions and promoters of various var genes. Additionally, binding and propagation of ORC1 to telomeric and subtelomeric regions was severely compromised in PfSir2 deficient parasites suggesting the dependence of endogenous ORC1 on Sir2 for var gene regulation. This feature is not previously described for Plasmodium ORC1 and contrary to yeast Saccharomyces cerevisiae where ORC function as a landing pad for Sir proteins. Interestingly, the overexpression of ORC1N1–238 compromises the binding of Sir2 at the subtelomeric loci and var gene promoters consistent with de-repression of some var genes. These results establish role of the N-terminus of PfORC1 in heterochromatin formation and regulation of var gene expression in co-ordination with Sir2 in P. falciparum. PMID:22379140

  5. The cell-bound fructosyltransferase of Streptococcus salivarius: the carboxyl terminus specifies attachment in a Streptococcus gordonii model system.

    PubMed Central

    Rathsam, C; Giffard, P M; Jacques, N A

    1993-01-01

    The ftf gene, coding for the cell-bound beta-D-fructosyltransferase (FTF) of Streptococcus salivarius ATCC 25975, has been analyzed, and its deduced amino acid sequence has been compared with that of the secreted FTF of Streptococcus mutans and the levansucrases (SacBs) of Bacillus species. A unique proline-rich region detected at the C terminus of the FTF of S. salivarius preceded a hydrophobic terminal domain. This proline-rich region was shown to possess strong homology to the product of the prgC gene from pCF10 in Enterococcus faecalis, which encodes a pheromone-responsive protein of unknown function, as well as homology to the human proline-rich salivary protein PRP-4. A series of 3'-OH deletions of the S. salivarius ftf gene expressed in Streptococcus gordonii Challis LGR2 showed that the C terminus was required for cell surface attachment in this heterologous organism, as only the complete gene product was cell bound. This cell-bound activity was released in the presence of sucrose, suggesting that the mode of attachment and release of the S. salivarius FTF in S. gordonii was similar to that in its native host. PMID:8331080

  6. The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies.

    PubMed

    Wang, I-Fan; Chang, Hsiang-Yu; Hou, Shin-Chen; Liou, Gunn-Guang; Way, Tzong-Der; James Shen, C-K

    2012-01-01

    The degraded, misfolded C terminus of TAR DNA-binding protein-43 is associated with a wide spectrum of neurodegenerative diseases, particularly frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. However, the precise mechanism of pathological cleavage of the TAR DNA-binding protein-43 remains unknown. Here we show that the TAR DNA-binding protein-43 C-terminal protein physically interacts with itself or with the cellular-folded yeast prion domain of Sup35 forming dynamic aggregates. This prion-like nature governs known cellular functions of the TAR DNA-binding protein-43, including subcellular localisation and exon skipping of the cystic fibrosis transmembrane conductance regulator. Significantly, mutants with a failure to engage in prion-like interactions are processed into an ~24-kDa C-terminal fragment of the TAR DNA-binding protein-43. The estimated cleavage site of degraded TAR DNA-binding protein-43 fragments corresponds to the pathological cleavage site identified in patients with the TAR DNA-binding protein-43 proteinopathies. Consistently, epigallocatechin gallate constrains prion-like interactions, attenuating pathological-like degradation. Thus, the native folding of TAR DNA-binding protein-43 C terminus acts as a guardian of pathogenesis, which is directly associated with loss-of-function. PMID:22473010

  7. Role of the C-terminus of Pleurotus eryngii Ery4 laccase in determining enzyme structure, catalytic properties and stability.

    PubMed

    Bleve, Gianluca; Lezzi, Chiara; Spagnolo, Stefano; Tasco, Gianluca; Tufariello, Maria; Casadio, Rita; Mita, Giovanni; Rampino, Patrizia; Grieco, Francesco

    2013-01-01

    The ERY4 laccase gene of Pleurotus eryngii is not biologically active when expressed in yeast. To explain this finding, we analysed the role of the C-terminus of Ery4 protein by producing a number of its different mutant variants. Two different categories of ERY4 mutant genes were produced and expressed in yeast: (i) mutants carrying C-terminal deletions and (ii) mutants carrying different site-specific mutations at their C-terminus. Investigation of the catalytic properties of the recombinant enzymes indicated that each novel variant acquired different affinities and catalytic activity for various substrates. Our results highlight that C-terminal processing is fundamental for Ery4 laccase enzymatic activities allowing substrate accessibility to the enzyme catalytic core. Apparently, the last 18 amino acids in the C-terminal end of the Ery4 laccase play a critical role in enzyme activity, stability and kinetic and, in particular biochemical and structural data indicate that the K532 residue is fundamental for enzyme activation. These studies shed light on the structure/function relationships of fungal laccases and will enhance the development of biotechnological strategies for the industrial exploitation of these enzymes. PMID:22996391

  8. A model for the function of the bisphosphorylated heart-specific troponin-I N-terminus.

    PubMed

    Jaquet, K; Lohmann, K; Czisch, M; Holak, T; Gulati, J; Jaquet, R

    1998-08-01

    Bisphosphorylation of two adjacently located serine residues in the heart-specific N-terminus of the cTnl subunit reduces calcium affinity of the cTnC subunit. An interaction of the phosphorylation region of cTnI with acidic residues of another cTn subunit has been proposed formerly based on 31P nuclear magnetic resonance (NMR) data. A possible candidate is cTnC. Thus, an interaction model of cTnC with the bisphosphorylated cTnI N-terminus has been built using a homology model of hcTnC based on the crystal structure of tusTnC and the structure of the phosphorylation region of cTnI determined by 2D NMR. By computational search, five cluster of acidic residues of cTnC might interact with the cTnI phosphorylation region. Three sites could be excluded by 31P-NMR experiments. The two remaining sites are located in the N-terminal helix of cTnC and between calcium binding sites III and IV. Reorientation of the arginine and phosphoserine sidechains within the phosphorylation region as proposed by refined docking could explain the formerly measured changes in pKaapp values. Thus, local pKa changes might lead to the reduction of calcium affinity observed upon cTnI bisphosphorylation. PMID:9742449

  9. N-terminus conservation in the anchor polypeptide of a prokaryotic and eukaryotic alga. [Nostoc; Porphydium cruentum

    SciTech Connect

    Gantt, E.; Lipschultz, C.A.; Cunningham, F.X. Jr.; Mimuro, M.

    1987-04-01

    Energy flow between the extrinsic phycobilisomes and the photosystems within thylakoids, is probably mediated by a blue anchor polypeptide. Polypeptides in the 94 kD range, purified by LiDS-PAGE from phycobilisomes of Nostoc and Porphyrdium cruentum, crossreacted with anti-Nostoc-94 (although weakly with the latter). Though rich in ASP and GLU, the polypeptides were very hydrophobic, and low in MET, CYS, and HIS. Partial sequence of the N-terminus shows considerable homology 1 - 5 - 10 - 15 - 20 N: (S)-V-K-A-S-G-G-S-S-V-A-(R)-P-Q-L-Y-Q-(G)-L-(A)-V- P: V-()-K-A-S-G-G-S-P-V-V-K-P-Q-L-Y-(K)-()-A-(S)- between the species. There is a lack of homology when compared with ..cap alpha.. and ..beta.. polypeptides of allophycocyanin with rod linkers of phycobilisomes and other phycobiliproteins. Polypeptides of 94 and 92 kD from thylakoids of Nostoc, also immunoreactive with anti-94, were blocked at the N-terminus.

  10. Identification of a Tetrameric Assembly Domain in the C Terminus of Heat-activated TRPV1 Channels*

    PubMed Central

    Zhang, Feng; Liu, Shuang; Yang, Fan; Zheng, Jie; Wang, KeWei

    2011-01-01

    Transient receptor potential (TRP) channels as cellular sensors are thought to function as tetramers. Yet, the molecular determinants governing channel multimerization remain largely elusive. Here we report the identification of a segment comprising 21 amino acids (residues 752–772 of mouse TRPV1) after the known TRP-like domain in the channel C terminus that functions as a tetrameric assembly domain (TAD). Purified recombinant C-terminal proteins of TRPV1–4, but not the N terminus, mediated the protein-protein interaction in an in vitro pulldown assay. Western blot analysis combined with electrophysiology and calcium imaging demonstrated that TAD exerted a robust dominant-negative effect on wild-type TRPV1. When fused with the membrane-tethered peptide Gap43, the TAD blocked the formation of stable homomultimers. Calcium imaging and current recordings showed that deletion of the TAD in a poreless TRPV1 mutant subunit suppressed its dominant-negative phenotype, confirming the involvement of the TAD in assembly of functional channels. Our findings suggest that the C-terminal TAD in TRPV1 channels functions as a domain that is conserved among TRPV1–4 and mediates a direct subunit-subunit interaction for tetrameric assembly. PMID:21357419