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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. 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

  6. 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

  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. 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

  12. 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....

  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. 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

  16. 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

  17. 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

  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. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  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. 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...

  3. 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...

  4. 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...

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  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. 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

  15. 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

  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. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  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. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  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. 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

  12. 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

  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. 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

  16. 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

  17. 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

  18. 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

  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. 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

  1. 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

  2. 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

  3. [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

  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. 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

  7. 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

  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. 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

  14. 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

  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. 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

  1. 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

  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. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  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 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

  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. 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

  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. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  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 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. Poly[(6-carboxy-picolinato-κO,N,O)(μ(3)-pyridine-2,6-dicarboxyl-ato-κO,N,O:O:O)dysprosium(III)].

    PubMed

    Li, Xu; Lian, Qing-Yang; Meng, Qiu-Hui; Luo, Yi-Fan; Zeng, Rong-Hua

    2009-01-01

    In the title complex, [Dy(C(7)H(3)NO(4))(C(7)H(4)NO(4))](n), one of the ligands is fully deprotonated while the second has lost only one H atom. Each Dy(III) ion is coordinated by six O atoms and two N atoms from two pyridine-2,6-dicarboxyl-ate and two 6-carboxy-picolinate ligands, displaying a bicapped trigonal-prismatic geometry. The average Dy-O bond distance is 2.40 Å, some 0.1Å longer than the corresponding Ho-O distance in the isotypic holmium complex. Adjacent Dy(III) ions are linked by the pyridine-2,6-dicarboxyl-ate ligands, forming a layer in (100). These layers are further connected by π-π stacking inter-actions between neighboring pyridyl rings [centroid-centroid distance = 3.827 (3) Å] and C-H⋯O hydrogen-bonding inter-actions, assembling a three-dimensional supra-molecular network. Within each layer, there are other π-π stacking inter-actions between neighboring pyridyl rings [centroid-centroid distance = 3.501 (2) Å] and O-H⋯O and C-H⋯O hydrogen-bonding inter-actions, which further stabilize the structure. PMID:21578057

  12. Carboxy-terminal modulator protein attenuated extracellular matrix deposit by inhibiting phospho-Akt, TGF-β1 and α-SMA in kidneys of diabetic mice.

    PubMed

    Chen, Ning; Hao, Jun; Li, Lisha; Li, Fan; Liu, Shuxia; Duan, Huijun

    2016-06-10

    Glomerulosclerosis and tubular interstitial extracellular matrix deposit and fibrosis are the main features of diabetic nephropathy, which are mediated by activation of PI3K/Akt signal pathway. Carboxy-terminal modulator protein (CTMP) is known as a negative regulator of PI3K/Akt pathway. Whether CTMP regulates renal extracellular matrix metabolism of diabetic nephropathy is still not known. Here, renal decreased CTMP, enhanced phospho-Akt (Ser 473), TGF-β1, α-SMA and extracellular matrix deposit are found in diabetic mice. Furthermore, high glucose decreases CTMP expression accompanied by enhanced phospho-Akt (Ser 473), TGF-β1 and α-SMA in cultured human renal proximal tubular epithelial cells (HKC), which are effectively prevented by transfection of pYr-ads-4-musCTMP vector. Moreover, delivery of pYr-ads-4-musCTMP vector into kidneys via tail vein of diabetic mice increases CTMP expression by 8.84 times followed by 60.00%, 76.50% and 24.37% decreases of phospho-Akt (Ser 473), TGF-β1 and α-SMA compared with diabetic mice receiving pYr-adshuttle-4 vector. Again, increased renal extracellular matrix accumulation of diabetic mice is also inhibited with delivery of pYr-ads-4-musCTMP vector. Our results indicate that CTMP attenuates renal extracellular matrix deposit by regulating the phosphorylation of Akt, TGF-β1 and α-SMA expression in diabetic mice. PMID:27166156

  13. Studies on the growth, spectral, structural, electrical, optical and mechanical properties of Uronium 3-carboxy-4-hydroxybenzenesulfonate single crystal for third-order nonlinear optical applications

    NASA Astrophysics Data System (ADS)

    Silambarasan, A.; Krishna Kumar, M.; Thirunavukkarasu, A.; Md Zahid, I.; Mohan Kumar, R.; Umarani, P. R.

    2015-05-01

    Organic Uronium 3-carboxy-4-hydroxybenzenesulfonate (UCHBS) nonlinear optical single crystal was grown by solution growth technique. The solubility and nucleation studies were performed for UCHBS at different temperatures 30, 35, 40, 45, 50 and 55 °C. The crystal structure of UCHBS was elucidated from single crystal X-ray diffraction study. High resolution X-ray diffraction technique was employed to study the perfection and internal defects of UCHBS crystal. Infrared and Raman spectra were recorded to analyze the vibrational behavior of chemical bonds and its functional groups. The physico-chemical changes, stability and decomposition stages of the UCHBS compound were established by TG-DTA studies. The dielectric phenomenon of UCHBS crystal was studied at different temperatures with respect to frequency. Linear optical properties of transmittance, cut-off wavelength, band gap of UCHBS were found from UV-visible spectral studies. Third-order nonlinear optical susceptibility, nonlinear refractive index, nonlinear optical absorption coefficient values were measured by Z-scan technique. The mechanical properties of UCHBS crystal was studied by using Vicker's microhardness test. The growth features of UCHBS crystal were analyzed from etching studies.

  14. Recombinant forms of M13 procoat with an OmpA leader sequence or a large carboxy-terminal extension retain their independence of secY function.

    PubMed Central

    Kuhn, A; Kreil, G; Wickner, W

    1987-01-01

    The assembly of phage M13 procoat protein into the plasma membrane of Escherichia coli is independent of the secY protein. To test whether this is caused by the unusually small size of procoat, we fused DNA encoding 103 amino acids to the carboxy-terminal end of the procoat gene. The resulting fusion protein, which attains the same membrane-spanning conformation as mature coat protein, still does not require the secY function for membrane assembly. To determine whether the leader sequence governs interaction with the secY protein, we genetically exchanged the leader peptides between procoat and pro-OmpA, a protein which does require secY for its membrane assembly. Each of the resulting hybrid proteins assembles across the plasma membrane, though at a reduced rate. Membrane assembly of the fusion of procoat leader and OmpA required secY function, whereas assembly of the pro-OmpA leader/coat protein fusion was independent of secY. Properties of the entire procoat molecule, rather than its small size or a specific property of its leader peptide determines its mode of membrane assembly. Images Fig. 1. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3034592

  15. The Predictive Power of Serum α-Fetoprotein and Des-γ-Carboxy Prothrombin for Survival Varies by Tumor Size in Hepatocellular Carcinoma.

    PubMed

    Tsugawa, Daisuke; Fukumoto, Takumi; Kido, Masahiro; Takebe, Atsushi; Tanaka, Motofumi; Kuramitsu, Kaori; Matsumoto, Ippei; Ajiki, Tetsuo; Koyama, Tatsuki; Ku, Yonson

    2016-01-01

    Alpha-fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP) are frequently used as tumor markers in hepatocellular carcinoma (HCC). The authors hypothesized different patient populations with varying tumor sizes would influence the predictive power of tumor markers for survival in HCC patients. The authors investigated the influence of tumor size on predictive powers of AFP and DCP. 181 patients underwent hepatectomy for HCC from 2003 to 2008 at Kobe University Hospital. Tumor markers were measured before and at 1 month post-hepatectomy. The Cox proportional-hazards model revealed that preoperative serum AFP was associated with survival; its effects depended on tumor size. Hazard ratios (HRs) for preoperative AFP were maximum for medium-sized HCC, and for DCP, HRs were maximum in small-sized tumors. Post-hepatectomy, both tumor markers were associated with survival, revealing significant interactions with tumor size. HRs for postoperative AFP were greater than 1 for relatively wide range tumors (3-11 cm). HRs for postoperative DCP increased with tumor size, with a strong prognostic predictive power for tumors >5 cm. The predictive power of serum tumor markers varied by tumor size in HCC patients. By selecting the appropriate tumor marker, its predictive power can be improved. PMID:27363395

  16. Quantification of 11-nor-9-carboxy-δ9-tetrahydrocannabinol in human oral fluid by gas chromatography-tandem mass spectrometry.

    PubMed

    Barnes, Allan J; Scheidweiler, Karl B; Huestis, Marilyn A

    2014-04-01

    A sensitive and specific method for the quantification of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) in oral fluid collected with the Quantisal and Oral-Eze devices was developed and fully validated. Extracted analytes were derivatized with hexafluoroisopropanol and trifluoroacetic anhydride and quantified by gas chromatography-tandem mass spectrometry with negative chemical ionization. Standard curves, using linear least-squares regression with 1/x weighting were linear from 10 to 1000 ng/L with coefficients of determination >0.998 for both collection devices. Bias was 89.2%-112.6%, total imprecision 4.0%-5.1% coefficient of variation, and extraction efficiency >79.8% across the linear range for Quantisal-collected specimens. Bias was 84.6%-109.3%, total imprecision 3.6%-7.3% coefficient of variation, and extraction efficiency >92.6% for specimens collected with the Oral-Eze device at all 3 quality control concentrations (10, 120, and 750 ng/L). This effective high-throughput method reduces analysis time by 9 minutes per sample compared with our current 2-dimensional gas chromatography-mass spectrometry method and extends the capability of quantifying this important oral fluid analyte to gas chromatography-tandem mass spectrometry. This method was applied to the analysis of oral fluid specimens collected from individuals participating in controlled cannabis studies and will be effective for distinguishing passive environmental contamination from active cannabis smoking. PMID:24622724

  17. Quantification of 11-Nor-9-Carboxy-Δ9-Tetrahydrocannabinol in Human Oral Fluid by Gas Chromatography–Tandem Mass Spectrometry

    PubMed Central

    Barnes, Allan J.; Scheidweiler, Karl B.; Huestis, Marilyn A.

    2015-01-01

    A sensitive and specific method for the quantification of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) in oral fluid collected with the Quantisal and Oral-Eze devices was developed and fully validated. Extracted analytes were derivatized with hexafluoroisopropanol and trifluoroacetic anhydride and quantified by gas chromatography–tandem mass spectrometry with negative chemical ionization. Standard curves, using linear least-squares regression with 1/x2 weighting were linear from 10 to 1000 ng/L with coefficients of determination >0.998 for both collection devices. Bias was 89.2%–112.6%, total imprecision 4.0%–5.1% coefficient of variation, and extraction efficiency >79.8% across the linear range for Quantisal-collected specimens. Bias was 84.6%–109.3%, total imprecision 3.6%–7.3% coefficient of variation, and extraction efficiency >92.6% for specimens collected with the Oral-Eze device at all 3 quality control concentrations (10, 120, and 750 ng/L). This effective high-throughput method reduces analysis time by 9 minutes per sample compared with our current 2-dimensional gas chromatography–mass spectrometry method and extends the capability of quantifying this important oral fluid analyte to gas chromatography–tandem mass spectrometry. This method was applied to the analysis of oral fluid specimens collected from individuals participating in controlled cannabis studies and will be effective for distinguishing passive environmental contamination from active cannabis smoking. PMID:24622724

  18. Distribution of ∆(9)-Tetrahydrocannabinol and 11-Nor-9-Carboxy-∆(9)-Tetrahydrocannabinol Acid in Postmortem Biological Fluids and Tissues From Pilots Fatally Injured in Aviation Accidents.

    PubMed

    Kemp, Philip M; Cardona, Patrick S; Chaturvedi, Arvind K; Soper, John W

    2015-07-01

    Little is known of the postmortem distribution of ∆(9)-tetrahydrocannabinol (THC) and its major metabolite, 11-nor-9-carboxy-∆(9)-tetrahydrocannabinol (THCCOOH). Data from 55 pilots involved in fatal aviation accidents are presented in this study. Gas chromatography/mass spectrometry analysis obtained mean THC concentrations in blood from multiple sites, liver, lung, and kidney of 15.6 ng/mL, 92.4 ng/g, 766.0 ng/g, 44.1 ng/g and mean THCCOOH concentrations of 35.9 ng/mL, 322.4 ng/g, 42.6 ng/g, 138.5 ng/g, respectively. Heart THC concentrations (two cases) were 184.4 and 759.3 ng/g, and corresponding THCCOOH measured 11.0 and 95.9 ng/g, respectively. Muscle concentrations for THC (two cases) were 16.6 and 2.5 ng/g; corresponding THCCOOH, "confirmed positive" and 1.4 ng/g. The only brain tested in this study showed no THC detected and 2.9 ng/g THCCOOH, low concentrations that correlated with low values in other specimens from this case. This research emphasizes the need for postmortem cannabinoid testing and demonstrates the usefulness of a number of tissues, most notably lung, for these analyses. PMID:25800046

  19. A conserved carboxy-terminal domain in the major tegument structural protein VP22 facilitates virion packaging of a chimeric protein during productive herpes simplex virus 1 infection

    SciTech Connect

    Schlegel, Elisabeth F.M.; Blaho, John A.

    2009-05-10

    Recombinant virus HSV-1(RF177) was previously generated to examine tegument protein VP22 function by inserting the GFP gene into the gene encoding VP22. During a detailed analysis of this virus, we discovered that RF177 produces a novel fusion protein between the last 15 amino acids of VP22 and GFP, termed GCT-VP22. Thus, the VP22 carboxy-terminal specific antibody 22-3 and two anti-GFP antibodies reacted with an approximately 28 kDa protein from RF177-infected Vero cells. GCT-VP22 was detected at 1 and 3 hpi. Examination of purified virions indicated that GCT-VP22 was incorporated into RF177 virus particles. These observations imply that at least a portion of the information required for virion targeting is located in this domain of VP22. Indirect immunofluorescence analyses showed that GCT-VP22 also localized to areas of marginalized chromatin during RF177 infection. These results indicate that the last fifteen amino acids of VP22 participate in virion targeting during HSV-1 infection.

  20. Quantification of 11-Carboxy-Delta-9-Tetrahydrocannabinol (THC-COOH) in Meconium Using Gas Chromatography/Mass Spectrometry (GC/MS).

    PubMed

    Peat, Judy; Davis, Brehon; Frazee, Clint; Garg, Uttam

    2016-01-01

    Maternal substance abuse is an ongoing concern and detecting drug use during pregnancy is an important component of neonatal care when drug abuse is suspected. Meconium is the preferred specimen for drug testing because it is easier to collect than neonatal urine and it provides a much broader time frame of drug exposure. We describe a method for quantifying 11-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in meconium. After adding a labeled internal standard (THC-COOH D9) and acetonitrile, samples are sonicated to release both free and conjugated THC-COOH. The acetonitrile/aqueous layer is removed and mixed with a strong base to hydrolyze the conjugated THC-COOH. The samples are then extracted with an organic solvent mixture as part of a sample "cleanup." The organic solvent layer is discarded and the remaining aqueous sample is acidified. Following extraction with a second organic mixture, the organic layer is removed and concentrated to dryness. The resulting residue is converted to a trimethylsilyl (TMS) derivative and analyzed using gas chromatography/mass spectrometry (GC/MS) in selective ion monitoring (SIM) mode. PMID:26660178

  1. Neuroprotective effects of inositol 1,4,5-trisphosphate receptor carboxy-terminal fragment in Huntington’s disease mouse model

    PubMed Central

    Tang, Tie-Shan; Guo, Caixia; Wang, Hongyu; Chen, Xi; Bezprozvanny, Ilya

    2009-01-01

    Huntington’s disease (HD) is a dominantly inherited, progressive neurodegenerative disease caused by an expanded polyglutamine tract in Huntingtin protein (Htt). Medium spiny striatal neurons (MSN) are primarily affected in HD. Mutant Huntingtin protein (Httexp) specifically binds to and activates type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1), an intracellular Ca2+ release channel. Httexp-InsP3R1 association is mediated by a cytosolic carboxy-terminal tail of InsP3R1 (a 122 amino-acid long IC10 fragment). To evaluate an importance of Httexp association with InsP3R1 for HD pathology, we generated lentiviral and adeno-associated viruses expressing GFP-IC10 fusion protein and performed a series of experiments with YAC128 HD transgenic mouse. Infection with Lenti-GFP-IC10 protein stabilized Ca2+ signaling in cultured YAC128 MSN and protected YAC128 MSN from glutamate-induced apoptosis. Intra-striatal injections of AAV1-GFP-IC10 significantly alleviated motor deficits and reduced MSN loss and shrinkage in YAC128 mice. Our results demonstrate an importance of InsP3R1-Httexp association for HD pathogenesis and suggested that InsP3R1 is a potential therapeutic target for HD. Our data also support potential use of IC10 peptide as a novel HD therapeutic agent. PMID:19193873

  2. Seasonal and Interannual Glacier Terminus Fluctuations in Northwest Greenland and Links to Sea Ice and Velocity Trends during the 21st Century

    NASA Astrophysics Data System (ADS)

    Moon, T. A.; Joughin, I. R.; Smith, B. E.

    2014-12-01

    Current ice loss from the Greenland Ice Sheet is a significant component of global sea-level rise. Observations suggest that both increasing ice flow speeds and sustained terminus retreat on most Greenland glaciers have increased mass loss via ice discharge over the last several decades. However, our understanding of the mechanisms causing retreat and how well connected terminus fluctuations are to other dynamic changes in the ice sheet remains limited. We examined terminus position, sea ice and ice mélange conditions, and seasonal velocity patterns for 16 northwestern glaciers during 2009-2012, with extended 1999-2012 records for 4 glaciers. On a seasonal scale, there is strong correspondence between terminus advance and retreat and sea ice/ice mélange conditions, with a distinct seasonal signature. Longer sea-ice-free periods and reductions in rigid mélange formation appear to induce sustained multi-year retreat outside of the seasonal signal. While seasonal terminus retreat is not clearly linked to seasonal velocity patterns, multi-year retreat is accompanied by interannual speedup on most glaciers. Projections of continued warming and longer sea-ice-free periods around Greenland indicate that notable retreat over wide areas may continue. This sustained retreat likely will contribute to multi-year speedup. Longer melt seasons and earlier breakup of mélange may also alter the timing of seasonal ice-dynamic patterns.

  3. The N-terminus of Prp1 (Prp6/U5-102 K) is essential for spliceosome activation in vivo

    PubMed Central

    Lützelberger, Martin; Bottner, Claudia A.; Schwelnus, Wiebke; Zock-Emmenthal, Susanne; Razanau, Aleh; Käufer, Norbert F.

    2010-01-01

    The spliceosomal protein Prp1 (Prp6/U5-102 K) is necessary for the integrity of pre-catalytic spliceosomal complexes. We have identified a novel regulatory function for Prp1. Expression of mutations in the N-terminus of Prp1 leads to the accumulation of pre-catalytic spliceosomal complexes containing the five snRNAs U1, U2, U5 and U4/U6 and pre-mRNAs. The mutations in the N-terminus, which prevent splicing to occur, include in vitro and in vivo identified phosphorylation sites of Prp4 kinase. These sites are highly conserved in the human ortholog U5-102 K. The results presented here demonstrate that structural integrity of the N-terminus is required to mediate a splicing event, but is not necessary for the assembly of spliceosomes. PMID:20007600

  4. Pan-ice-sheet glacier terminus change in East Antarctica reveals sensitivity of Wilkes Land to sea-ice changes.

    PubMed

    Miles, Bertie W J; Stokes, Chris R; Jamieson, Stewart S R

    2016-05-01

    The dynamics of ocean-terminating outlet glaciers are an important component of ice-sheet mass balance. Using satellite imagery for the past 40 years, we compile an approximately decadal record of outlet-glacier terminus position change around the entire East Antarctic Ice Sheet (EAIS) marine margin. We find that most outlet glaciers retreated during the period 1974-1990, before switching to advance in every drainage basin during the two most recent periods, 1990-2000 and 2000-2012. The only exception to this trend was in Wilkes Land, where the majority of glaciers (74%) retreated between 2000 and 2012. We hypothesize that this anomalous retreat is linked to a reduction in sea ice and associated impacts on ocean stratification, which increases the incursion of warm deep water toward glacier termini. Because Wilkes Land overlies a large marine basin, it raises the possibility of a future sea level contribution from this sector of East Antarctica. PMID:27386519

  5. Pan–ice-sheet glacier terminus change in East Antarctica reveals sensitivity of Wilkes Land to sea-ice changes

    PubMed Central

    Miles, Bertie W. J.; Stokes, Chris R.; Jamieson, Stewart S. R.

    2016-01-01

    The dynamics of ocean-terminating outlet glaciers are an important component of ice-sheet mass balance. Using satellite imagery for the past 40 years, we compile an approximately decadal record of outlet-glacier terminus position change around the entire East Antarctic Ice Sheet (EAIS) marine margin. We find that most outlet glaciers retreated during the period 1974–1990, before switching to advance in every drainage basin during the two most recent periods, 1990–2000 and 2000–2012. The only exception to this trend was in Wilkes Land, where the majority of glaciers (74%) retreated between 2000 and 2012. We hypothesize that this anomalous retreat is linked to a reduction in sea ice and associated impacts on ocean stratification, which increases the incursion of warm deep water toward glacier termini. Because Wilkes Land overlies a large marine basin, it raises the possibility of a future sea level contribution from this sector of East Antarctica. PMID:27386519

  6. Characterization of the promoter, signal sequence, and amino terminus of a secreted beta-galactosidase from "Streptomyces lividans".

    PubMed Central

    Eckhardt, T; Strickler, J; Gorniak, L; Burnett, W V; Fare, L R

    1987-01-01

    The gene for a secreted 130-kilodalton beta-galactosidase from "Streptomyces lividans" has been cloned, its promoter, signal sequence, and amino terminal region have been localized, and their nucleotide sequence has been determined. The signal sequence extends over 56 amino acids and shows the characteristic-features of signal sequences, including a hydrophilic amino terminus followed by a hydrophobic core near the signal cleavage site. The secretion of beta-galactosidase depends on the presence of the signal sequence. beta-Galactosidase is the major protein in culture supernatants and extracts of strains expressing the cloned beta-galactosidase gene and represents a valuable tool in the study of protein secretion in Streptomyces spp. Images PMID:2442141

  7. Evaluations of new and existing methods for the quantification of tidewater glacier terminus change, and their comparability to numerical model output.

    NASA Astrophysics Data System (ADS)

    Lea, James M.; Mair, Douglas WF; Rea, Brice R.

    2014-05-01

    Several different methodologies have previously been employed in the tracking of glacier terminus change, though a systematic comparison of these has not been undertaken. Similarly, the suitability of using the resulting data for the calibration/validation of numerical models has not been evaluated. This could be especially significant for flowline modelling of tidewater glaciers, where discrepancies between the different terminus tracking methods could potentially introduce bias into model calibrations. The choice of method for quantifying terminus change of tidewater glaciers is therefore significant from both glacier monitoring, and numerical modelling viewpoints. In this study we evaluate three existing methodologies that have been widely used to track terminus change (the centreline, bow and box methods) against a full range of idealised glaciological scenarios, and examples of 6 real glaciers in Greenland. We also evaluate two new methodologies that aim to reduce measurement error compared to the existing methodologies, and allow direct comparison of results to those of flowline models. These are (1) a modification to the box method, that can account for termini retreating through fjords that change orientation (termed the curvilinear box method [CBM]), and (2) a method that determines the average terminus position relative to the glacier centreline using an inverse distance weighting extrapolation (termed the extrapolated centreline method [ECM]). No single method achieved complete accuracy for all scenarios though the ECM was best, being able to successfully account for variable fjord orientation, width and terminus geometry. Only results from the centreline, CBM and ECM will be directly comparable to flowline model output, though the CBM and ECM are likely to be the most accurate when applied to real world scenarios.

  8. Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Ghospurkar, Padmaja L; Wilson, Timothy M; Liu, Shengqin; Herauf, Anna; Steffes, Jenna; Mueller, Erica N; Oakley, Gregory G; Haring, Stuart J

    2015-02-01

    Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms. PMID:25499885

  9. The N Terminus of Andes Virus L Protein Suppresses mRNA and Protein Expression in Mammalian Cells

    PubMed Central

    Heinemann, Patrick; Schmidt-Chanasit, Jonas

    2013-01-01

    Little is known about the structure and function of the 250-kDa L protein of hantaviruses, although it plays a central role in virus genome transcription and replication. When attempting to study Andes virus (ANDV) L protein in mammalian cells, we encountered difficulties. Even in a strong overexpression system, ANDV L protein could not be detected by immunoblotting. Deletion analysis revealed that the 534 N-terminal amino acid residues determine the low-expression phenotype. Inhibition of translation due to RNA secondary structures around the start codon, rapid proteasomal degradation, and reduced half-life time were excluded. However, ANDV L protein expression could be rescued upon mutation of the catalytic PD-E-K motif and further conserved residues of the putative endonuclease at the N terminus of the protein. In addition, wild-type ANDV L rather than expressible L mutants suppressed the level of L mRNA, as well as reporter mRNAs. Wild-type L protein also reduced the synthesis of cellular proteins in the high-molecular-weight range. Using expressible ANDV L mutants as a tool for localization studies, we show that L protein colocalizes with ANDV N and NSs but not Gc protein. A fraction of L protein also colocalized with the cellular processing (P) body component DCP1a. Overall, these data suggest that ANDV L protein possesses a highly active endonuclease at the N terminus suppressing the level of its own as well as heterologous mRNAs upon recombinant expression in mammalian cells. PMID:23576516

  10. Structure and interactions of the carboxyl terminus of striated muscle alpha-tropomyosin: it is important to be flexible.

    PubMed Central

    Greenfield, Norma J; Palm, Thomas; Hitchcock-DeGregori, Sarah E

    2002-01-01

    Tropomyosin (TM) binds to and regulates the actin filament. We used circular dichroism and heteronuclear NMR to investigate the secondary structure and interactions of the C terminus of striated muscle alpha-TM, a major functional determinant, using a model peptide, TM9a(251-284). The (1)H(alpha) and (13)C(alpha) chemical shift displacements show that residues 252 to 277 are alpha-helical but residues 278 to 284 are nonhelical and mobile. The (1)H(N) and (13)C' displacements suggest that residues 257 to 269 form a coiled coil. Formation of an "overlap" binary complex with a 33-residue N-terminal chimeric peptide containing residues 1 to 14 of alpha-TM perturbs the (1)H(N) and (15)N resonances of residues 274 to 284. Addition of a fragment of troponin T, TnT(70-170), to the binary complex perturbs most of the (1)H(N)-(15)N cross-peaks. In addition, there are many new cross-peaks, showing that the binding is asymmetric. Q263, in a proposed troponin T binding site, shows two sets of side-chain (15)N-(1)H cross-peaks, indicating conformational flexibility. The conformational equilibrium of the side chain changes upon formation of the binary and ternary complexes. Replacing Q263 with leucine greatly increases the stability of TM9a(251-284) and reduces its ability to form the binary and ternary complexes, showing that conformational flexibility is crucial for the binding functions of the C terminus. PMID:12414708

  11. Phosphorylation and Cellular Function of the Human Rpa2 N-Terminus in the Budding Yeast Saccharomyces cerevisiae

    PubMed Central

    Ghospurkar, Padmaja L.; Wilson, Timothy M.; Liu, Shengqin; Herauf, Anna; Steffes, Jenna; Mueller, Erica N.; Oakley, Gregory G.; Haring, Stuart J.

    2015-01-01

    Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3–4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms. PMID:25499885

  12. Anti-inflammatory and anti-endotoxin properties of peptides derived from the carboxy-terminal region of a defensin from the tick Ornithodoros savignyi.

    PubMed

    Malan, Melissa; Serem, June C; Bester, Megan J; Neitz, Albert W H; Gaspar, Anabella R M

    2016-01-01

    Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti-inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)-induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti-inflammatory and anti-endotoxin activities of Os and Os-C, peptides derived from the carboxy-terminal of a tick defensin, were investigated. Both Os and Os-C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin-binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os-C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os-C showed no scavenging activity. Os and Os-C inhibited LPS/IFN-γ induced NO and TNF-α production in RAW 264.7 cells in a concentration-dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF-α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os-C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os-C, both peptides have in addition anti-inflammatory and anti-endotoxin properties. PMID:26662999

  13. In Vitro and in Vivo Characterization of MOD-4023, a Long-Acting Carboxy-Terminal Peptide (CTP)-Modified Human Growth Hormone.

    PubMed

    Hershkovitz, Oren; Bar-Ilan, Ahuva; Guy, Rachel; Felikman, Yana; Moschcovich, Laura; Hwa, Vivian; Rosenfeld, Ron G; Fima, Eyal; Hart, Gili

    2016-02-01

    MOD-4023 is a novel long-acting version of human growth hormone (hGH), containing the carboxy-terminal peptide (CTP) of human chorionic gonadotropin (hCG). MOD-4023 is being developed as a treatment for adults and children with growth hormone deficiency (GHD), which would require fewer injections than currently available GH formulations and thus reduce patient discomfort and increase compliance. This study characterizes MOD-4023's binding affinities for the growth hormone receptor, as well as the pharmacokinetic and pharmacodynamics, toxicology, and safety profiles of repeated dosing of MOD-4023 in Sprague-Dawley rats and Rhesus monkeys. Although MOD-4023 exhibited reduced in vitro potency and lower affinity to the GH receptor than recombinant hGH (rhGH), administration of MOD-4023 every 5 days in rats and monkeys resulted in exposure comparable to daily rhGH, and the serum half-life of MOD-4023 was significantly longer. Repeated administration of MOD-4023 led to elevated levels of insulin-like growth factor 1 (IGF-1), and twice-weekly injections of MOD-4023 resulted in larger increase in weight gain with fewer injections and a lower accumulative hGH dose. Thus, the increased half-life of MOD-4023 in comparison to hGH may increase the frequency of protein-receptor interactions and compensate for its decreased in vitro potency. MOD-4023 was found to be well-tolerated in rats and monkeys, with minimal adverse events, suggesting an acceptable safety profile. These results provide a basis for the continued clinical development of MOD-4023 as a novel treatment of GHD in children and adults. PMID:26713839

  14. Ubiquitin Carboxy-Terminal Hydrolase-L1 as a Serum Neurotrauma Biomarker for Exposure to Occupational Low-Level Blast

    PubMed Central

    Carr, Walter; Yarnell, Angela M.; Ong, Ricardo; Walilko, Timothy; Kamimori, Gary H.; da Silva, Uade; McCarron, Richard M.; LoPresti, Matthew L.

    2015-01-01

    Repeated exposure to low-level blast is a characteristic of a few select occupations and there is concern that such occupational exposures present risk for traumatic brain injury. These occupations include specialized military and law enforcement units that employ controlled detonation of explosive charges for the purpose of tactical entry into secured structures. The concern for negative effects from blast exposure is based on rates of operator self-reported headache, sleep disturbance, working memory impairment, and other concussion-like symptoms. A challenge in research on this topic has been the need for improved assessment tools to empirically evaluate the risk associated with repeated exposure to blast overpressure levels commonly considered to be too low in magnitude to cause acute injury. Evaluation of serum-based neurotrauma biomarkers provides an objective measure that is logistically feasible for use in field training environments. Among candidate biomarkers, ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) has some empirical support and was evaluated in this study. We used daily blood draws to examine acute change in UCH-L1 among 108 healthy military personnel who were exposed to repeated low-level blast across a 2-week period. These research volunteers also wore pressure sensors to record blast exposures, wrist actigraphs to monitor sleep patterns, and completed daily behavioral assessments of symptomology, postural stability, and neurocognitive function. UCH-L1 levels were elevated as a function of participating in the 2-week training with explosives, but the correlation of UCH-L1 elevation and blast magnitude was weak and inconsistent. Also, UCH-L1 elevations did not correlate with deficits in behavioral measures. These results provide some support for including UCH-L1 as a measure of central nervous system effects from exposure to low-level blast. However, the weak relation observed suggests that additional indicators of blast effect are needed

  15. Alpha-fetoprotein and des-gamma-carboxy-prothrombin at twenty-four weeks after interferon-based therapy predict hepatocellular carcinoma development

    PubMed Central

    Shakado, Satoshi; Sakisaka, Shotaro; Chayama, Kazuaki; Okanoue, Takeshi; Toyoda, Joji; Izumi, Namiki; Matsumoto, Akihiro; Takehara, Tetsuo; Ido, Akio; Hiasa, Yoichi; Yoshioka, Kentaro; Nomura, Hideyuki; Ueno, Yoshiyuki; Seike, Masataka; Kumada, Hiromitsu

    2015-01-01

    AIM: To investigate risk factors for development of hepatocellular carcinoma (HCC) in patients with hepatitis C virus-related liver cirrhosis (LC-C). METHODS: To evaluate the relationship between clinical factors including virological response and the development of HCC in patients with LC-C treated with interferon (IFN) and ribavirin, we conducted a multicenter, retrospective study in 14 hospitals in Japan. All patients had compensated LC-C with clinical or histological data available. HCC was diagnosed by the presence of typical hypervascular characteristics on computed tomography and/or magnetic resonance imaging. RESULTS: HCC was diagnosis in 50 (21.6%) of 231 LC-C patients during a median observation period of 3.8 years after IFN and ribavirin therapy. Patients who developed HCC were older (P = 0.018) and had higher serum levels of pretreatment alpha-fetoprotein (AFP) (P = 0.038). Multivariate analysis revealed the following independent risk factors for HCC development: history of treatment for HCC [P < 0.001, odds ratio (OR) = 15.27, 95%CI: 4.98-59.51], AFP levels of ≥ 10 ng/mL (P = 0.009, OR = 3.89, 95%CI: 1.38-11.94), and des-γ-carboxy prothrombin (DCP) levels of ≥ 40 mAU/mL at 24 wk after the completion of IFN and ribavirin therapy (P < 0.001, OR = 24.43, 95%CI: 4.11-238.67). CONCLUSION: We suggested that the elevation of AFP and DCP levels at 24 wk after the completion of IFN and ribavirin therapy were strongly associated with the incidence of HCC irrespective of virological response among Japanese LC-C patients. PMID:26644819

  16. Activation of p53 mediated glycolytic inhibition-oxidative stress-apoptosis pathway in Dalton's lymphoma by a ruthenium (II)-complex containing 4-carboxy N-ethylbenzamide.

    PubMed

    Koiri, Raj Kumar; Trigun, Surendra Kumar; Mishra, Lallan

    2015-03-01

    There is a general agreement that most of the cancer cells switch over to aerobic glycolysis (Warburg effect) and upregulate antioxidant enzymes to prevent oxidative stress induced apoptosis. Thus, there is an evolving view to target these metabolic alterations by novel anticancer agents to restrict tumor progression in vivo. Previously we have reported that when a non toxic dose (10 mg/kg bw i.p.) of a novel anticancer ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide; Ru(II)-CNEB, was administered to the Dalton's lymphoma (DL) bearing mice, it regressed DL growth by inducing apoptosis in the DL cells. It also inactivated M4-LDH (M4-lactate dehydrogenase), an enzyme that drives anaerobic glycolysis in the tumor cells. In the present study we have investigated whether this compound is able to modulate regulation of glycolytic inhibition-apoptosis pathway in the DL cells in vivo. We observed that Ru(II)-CNEB could decline expression of the inducible form of 6-phosphofructo-2-kinase (iPFK2: PFKFB3), the master regulator of glycolysis in the DL cells. The complex also activated superoxide dismutase (the H2O2 producing enzyme) but declined the levels of catalase and glutathione peroxidase (the two H2O2 degrading enzymes) to impose oxidative stress in the DL cells. This was consistent with the enhanced p53 level, decline in Bcl2/Bax ratio and activation of caspase 9 in those DL cells. The findings suggest that Ru(II)-CNEB is able to activate oxidative stress-apoptosis pathway via p53 (a tumor supressor protein) mediated repression of iPFK2, a key glycolytic regulator, in the DL cells in vivo. PMID:25576833

  17. The Contributions of the Amino and Carboxy Terminal Domains of Flightin to the Biomechanical Properties of Drosophila Flight Muscle Thick Filaments.

    PubMed

    Gasek, Nathan S; Nyland, Lori R; Vigoreaux, Jim O

    2016-01-01

    Flightin is a myosin binding protein present in Pancrustacea. In Drosophila, flightin is expressed in the indirect flight muscles (IFM), where it is required for the flexural rigidity, structural integrity, and length determination of thick filaments. Comparison of flightin sequences from multiple Drosophila species revealed a tripartite organization indicative of three functional domains subject to different evolutionary constraints. We use atomic force microscopy to investigate the functional roles of the N-terminal domain and the C-terminal domain that show different patterns of sequence conservation. Thick filaments containing a C-terminal domain truncated flightin (fln(ΔC44)) are significantly shorter (2.68 ± 0.06 μm; p < 0.005) than thick filaments containing a full length flightin (fln⁺; 3.21 ± 0.05 μm) and thick filaments containing an N-terminal domain truncated flightin (fln(ΔN62); 3.21 ± 0.06 μm). Persistence length was significantly reduced in fln(ΔN62) (418 ± 72 μm; p < 0.005) compared to fln⁺ (1386 ± 196μm) and fln(ΔC44)(1128 ± 193 μm). Statistical polymer chain analysis revealed that the C-terminal domain fulfills a secondary role in thick filament bending propensity. Our results indicate that the flightin amino and carboxy terminal domains make distinct contributions to thick filament biomechanics. We propose these distinct roles arise from the interplay between natural selection and sexual selection given IFM's dual role in flight and courtship behaviors. PMID:27128952

  18. Development of a direct ELISA based on carboxy-terminal of penicillin-binding protein BlaR for the detection of β-lactam antibiotics in foods.

    PubMed

    Peng, Juan; Cheng, Guyue; Huang, Lingli; Wang, Yulian; Hao, Haihong; Peng, Dapeng; Liu, Zhenli; Yuan, Zonghui

    2013-11-01

    β-Lactam antibiotics, including penicillins and cephalosporins, are commonly used in veterinary medicine. Illegal use and abuse of β-lactams could cause allergy and selected bacterial resistance. BlaR-CTD, the carboxy-terminal of penicillin-recognizing protein BlaR from Bacillus licheniformis ATCC 14580, was utilized in this study to develop a receptor-based ELISA for detection and determination of β-lactam antibiotics in milk, beef, and chicken. This assay was based on directly competitive inhibition of binding of horseradish peroxidase-labeled ampicillin to the immobilized BlaR-CTD by β-lactams. The assay was developed as screening test with the option as semiquantitative assay, when the identity of a single type of residual β-lactam was known. The IC50 values of 15 β-lactam antibiotics, including benzylpenicillin, ampicillin, amoxicillin, dicloxacillin, oxacillin, nafcillin, cefapirin, cefoperazone, cefalotin, cefazolin, cefquinome, ceftriaxone, cefotaxime, cefalexin, ceftiofur and its metabolite desfuroylceftiofur were evaluated and ranged from 0.18 to 170.81 μg L(-1). Simple sample extraction method was carried out with only phosphate-buffered saline, and the recoveries of selected β-lactam antibiotics in milk, beef, and chicken were in the range of 53.27 to 128.29 %, most ranging from 60 to 120 %. The inter-assay variability was below 30 %. Limits of detection in milk, beef, and chicken muscles with cefquinome matrix calibration were 2.10, 30.68, and 31.13 μg kg(-1), respectively. This study firstly established a rapid, simple, and accurate method for simultaneous detection of 15 β-lactams in edible tissues, among which 11 β-lactams controlled by European Union could be detected below maximum residue limits. PMID:24013636

  19. Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440.

    PubMed

    Mazurkewich, Scott; Brott, Ashley S; Kimber, Matthew S; Seah, Stephen Y K

    2016-04-01

    The bacterial catabolism of lignin and its breakdown products is of interest for applications in industrial processing of ligno-biomass. The gallate degradation pathway ofPseudomonas putidaKT2440 requires a 4-carboxy-2-hydroxymuconate (CHM) hydratase (GalB), which has a 12% sequence identity to a previously identified CHM hydratase (LigJ) fromSphingomonassp. SYK-6. The structure of GalB was determined and found to be a member of the PIG-LN-acetylglucosamine deacetylase family; GalB is structurally distinct from the amidohydrolase fold of LigJ. LigJ has the same stereospecificity as GalB, providing an example of convergent evolution for catalytic conversion of a common metabolite in bacterial aromatic degradation pathways. Purified GalB contains a bound Zn(2+)cofactor; however the enzyme is capable of using Fe(2+)and Co(2+)with similar efficiency. The general base aspartate in the PIG-L deacetylases is an alanine in GalB; replacement of the alanine with aspartate decreased the GalB catalytic efficiency for CHM by 9.5 × 10(4)-fold, and the variant enzyme did not have any detectable hydrolase activity. Kinetic analyses and pH dependence studies of the wild type and variant enzymes suggested roles for Glu-48 and His-164 in the catalytic mechanism. A comparison with the PIG-L deacetylases led to a proposed mechanism for GalB wherein Glu-48 positions and activates the metal-ligated water for the hydration reaction and His-164 acts as a catalytic acid. PMID:26867578

  20. The Contributions of the Amino and Carboxy Terminal Domains of Flightin to the Biomechanical Properties of Drosophila Flight Muscle Thick Filaments

    PubMed Central

    Gasek, Nathan S.; Nyland, Lori R.; Vigoreaux, Jim O.

    2016-01-01

    Flightin is a myosin binding protein present in Pancrustacea. In Drosophila, flightin is expressed in the indirect flight muscles (IFM), where it is required for the flexural rigidity, structural integrity, and length determination of thick filaments. Comparison of flightin sequences from multiple Drosophila species revealed a tripartite organization indicative of three functional domains subject to different evolutionary constraints. We use atomic force microscopy to investigate the functional roles of the N-terminal domain and the C-terminal domain that show different patterns of sequence conservation. Thick filaments containing a C-terminal domain truncated flightin (flnΔC44) are significantly shorter (2.68 ± 0.06 μm; p < 0.005) than thick filaments containing a full length flightin (fln+; 3.21 ± 0.05 μm) and thick filaments containing an N-terminal domain truncated flightin (flnΔN62; 3.21 ± 0.06 μm). Persistence length was significantly reduced in flnΔN62 (418 ± 72 μm; p < 0.005) compared to fln+ (1386 ± 196μm) and flnΔC44(1128 ± 193 μm). Statistical polymer chain analysis revealed that the C-terminal domain fulfills a secondary role in thick filament bending propensity. Our results indicate that the flightin amino and carboxy terminal domains make distinct contributions to thick filament biomechanics. We propose these distinct roles arise from the interplay between natural selection and sexual selection given IFM’s dual role in flight and courtship behaviors. PMID:27128952

  1. Simplified analysis of 11-hydroxy-delta-9-tetrahydrocannabinol and 11-carboxy-delta-9-tetrahydrocannabinol in human meconium: method development and validation.

    PubMed

    Tynon, Marykathryn; Porto, Marcellino; Logan, Barry K

    2015-01-01

    We describe the development of a sensitive analytical method for the analysis of 11-hydroxy-delta-9-tetrahydrocannabinol (11-OH-THC) and 11-carboxy-delta-9-tetrahydrocannabinol (THCC) in meconium using a gas chromatography-mass spectrometry (GC/MS) platform. The method was validated according to protocols, which included assessment of accuracy, precision, robustness, stability in meconium and in-process stability, interference and sensitivity and specificity. The method consists of a solid phase extraction with alkaline hydrolysis and derivatization of the analytes with N, O-Bis(trimethylsilyl)trifluoroacteamide, followed by GC/MS analysis using selected ion monitoring. The method uses deuterated internal standards for both analytes. Calibration curves had r(2) values >0.998, and extraction efficiency was determined to be 84.7% for THCC and 78.6% for 11-OH-THC. The detection limit for both analytes was 5 ng/g. This confirmatory method was successfully applied to 183 meconium samples that had screened positive by enzyme-linked immunosorbent assay, and 67.2% were confirmed for THCC, and 2.2% were confirmed positive for 11-OH-THC. The mean (SD) and median (range) THCC (n = 123) concentrations detected were 55.0 ng/g (±59.0) and 33.75 ng/g (5-265 ng/g), while the mean and median (range) for 11-OH-THC (n = 4) concentrations were 8.25 ng/g (±4.71) and 6.5 ng/g (5-15 ng/g). PMID:25315472

  2. Structure of the Receptor-Binding Carboxy-Terminal Domain of the Bacteriophage T5 L-Shaped Tail Fibre with and without Its Intra-Molecular Chaperone

    PubMed Central

    Garcia-Doval, Carmela; Castón, José R.; Luque, Daniel; Granell, Meritxell; Otero, José M.; Llamas-Saiz, Antonio L.; Renouard, Madalena; Boulanger, Pascale; van Raaij, Mark J.

    2015-01-01

    Bacteriophage T5, a Siphovirus belonging to the order Caudovirales, has a flexible, three-fold symmetric tail, to which three L-shaped fibres are attached. These fibres recognize oligo-mannose units on the bacterial cell surface prior to infection and are composed of homotrimers of the pb1 protein. Pb1 has 1396 amino acids, of which the carboxy-terminal 133 residues form a trimeric intra-molecular chaperone that is auto-proteolyzed after correct folding. The structure of a trimer of residues 970–1263 was determined by single anomalous dispersion phasing using incorporated selenomethionine residues and refined at 2.3 Å resolution using crystals grown from native, methionine-containing, protein. The protein inhibits phage infection by competition. The phage-distal receptor-binding domain resembles a bullet, with the walls formed by partially intertwined beta-sheets, conferring stability to the structure. The fold of the domain is novel and the topology unique to the pb1 structure. A site-directed mutant (Ser1264 to Ala), in which auto-proteolysis is impeded, was also produced, crystallized and its 2.5 Å structure solved by molecular replacement. The additional chaperone domain (residues 1263–1396) consists of a central trimeric alpha-helical coiled-coil flanked by a mixed alpha-beta domain. Three long beta-hairpin tentacles, one from each chaperone monomer, extend into long curved grooves of the bullet-shaped domain. The chaperone-containing mutant did not inhibit infection by competition. PMID:26670244

  3. Simultaneous analysis of Δ(9)-tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol in hair without different sample preparation and derivatization by gas chromatography-tandem mass spectrometry.

    PubMed

    Han, Eunyoung; Park, Yonghoon; Kim, Eunmi; In, Sangwhan; Yang, Wonkyung; Lee, Sooyeun; Choi, Hwakyung; Lee, Sangki; Chung, Heesun; Song, Joon Myong

    2011-07-15

    The present study describes a gas chromatography/tandem mass spectrometry-negative ion chemical ionization assay (GC/MS/MS-NCI) for simultaneous analysis of Δ(9)-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) in hair. Each hair sample, of approximately 20mg, was weighed and the sample was dissolved in 1ml of 1M sodium hydroxide (30min at 85°C) in the presence of THC-d(3) and THCCOOH-d(3). For the analysis of THC, hair samples were extracted with n-hexane:ethyl acetate (9:1) two times; acetic acid and sodium acetate buffer were added for the analysis of THCCOOH, and hair samples were re-extracted with n-hexane:ethyl acetate (9:1) two times. The extracts were then derivatized with pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH). This method allowed the analysis of THC and THCCOOH using the GC/MS/MS-NCI assay. This method was also fully validated and applied to hair specimens (n=54) collected from known cannabis users whose urine test results were positive. The concentrations of THC and THCCOOH in hair ranged from 7.52 to 60.41ng/mg and from 0.10 to 11.68pg/mg, respectively. In this paper, we simultaneously measured THC and THCCOOH in human hair using GC/MS/MS-NCI without requiring different sample preparation and derivatization procedures. The analytical sensitivity for THCCOOH in hair was good, while that for THC in hair needs to be improved in further study. PMID:21497038

  4. Serum carboxy-terminal telopeptide of type I collagen levels are associated with carotid atherosclerosis in patients with cardiovascular risk factors.

    PubMed

    Kondo, Takeshi; Endo, Itsuro; Aihara, Ken-Ichi; Onishi, Yukiyo; Dong, Bingzi; Ohguro, Yukari; Kurahashi, Kiyoe; Yoshida, Sumiko; Fujinaka, Yuichi; Kuroda, Akio; Matsuhisa, Munehide; Fukumoto, Seiji; Matsumoto, Toshio; Abe, Masahiro

    2016-04-25

    Carboxy-terminal telopeptide of type I collagen (ICTP) is generated through matrix metalloproteinase (MMP)-dependent type I collagen digestion, and has been widely utilized as a biomarker for bone turnover. The fact that atherosclerotic lesions are rich in both type I collagen and MMP-producing macrophages led to the hypothesis that serum ICTP concentrations may serve as a non-invasive clinical biomarker for atherosclerosis. Therefore, the association of serum ICTP concentrations with the maximum intima-media thickness (IMT) of carotid arteries, a surrogate index of systemic atherosclerosis, or brachial-ankle pulse wave velocity (baPWV) in patients with atherosclerotic risk factors was evaluated. A total of 52 male and 65 female (mean age: 62.8 yrs) patients without renal failure, malignancies or bone diseases known to affect serum ICTP concentrations were recruited. Patients with max IMTs ≥1.1 mm showed significantly higher serum ICTP concentrations compared with patients with max IMTs <1.1 mm (3.33 ± 0.97 vs 2.82 ± 0.65 ng/mL, p<0.05). Serum ICTP concentration was also positively correlated with max IMT (p<0.001) or baPWV values (p<0.05). Multivariate analyses also revealed that serum ICTP concentrations were correlated with max IMT (p<0.001; 95% CI 0.200 to 0.454). These results suggest that serum ICTP concentrations can be used as a non-invasive biomarker for systemic atherosclerosis. PMID:26877258

  5. Diagnostic Evaluation of Des-Gamma-Carboxy Prothrombin versus α-Fetoprotein for Hepatitis B Virus-Related Hepatocellular Carcinoma in China: A Large-Scale, Multicentre Study.

    PubMed

    Ji, Jun; Wang, Hao; Li, Yan; Zheng, Lei; Yin, Yuepeng; Zou, Zhenzhen; Zhou, Feiguo; Zhou, Weiping; Shen, Feng; Gao, Chunfang

    2016-01-01

    An efficient serum marker for hepatocellular carcinoma (HCC) is currently lacking and requires intensive exploration. We aimed to evaluate the performance of des-gamma-carboxy prothrombin (DCP) for identifying hepatitis B virus-related HCC in a large, multicentre study in China. A total of 1034 subjects in three cohorts (A, B, and C) including HCC and various non-HCC controls were enrolled from 4 academic medical centers in China from January 2011 to February 2014. Blind parallel detections were conducted for DCP and AFP. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic efficacies. In cohort A, which comprised 521 subjects, including patients with HCC, liver metastasis, liver cirrhosis (LC), and liver hemangiomas as well as healthy controls (HCs), the accuracy of DCP for distinguishing HCC from various controls was 6.2-9.7% higher than that of AFP. In cohort B, which comprised 447 subjects, including patients with HCC, LC, and chronic hepatitis B as well as HC, the accuracy of DCP was further elevated (12.3-20.67% higher than that of AFP). The superiority of DCP to AFP was more profound in the surveillance of early HCC [AUC 0.837 (95% CI: 0.771-0.903) vs. 0.650 (0.555-0.745)] and AFP-negative HCC [AUC: 0.856 (0.798-0.914)] and in discriminating HCC from LC (accuracy: 92.9% vs.64.71%). Higher DCP levels were associated with worse clinical behaviors and shorter disease-free survival. DCP not only is complementary to AFP in identifying AFP-negative HCC and in excluding AFP-positive non-HCC (liver cirrhosis), but also demonstrates improved performance in HCC surveillance, early diagnosis, treatment response and recurrence monitoring in the HBV-related population. PMID:27070780

  6. Diagnostic Evaluation of Des-Gamma-Carboxy Prothrombin versus α-Fetoprotein for Hepatitis B Virus-Related Hepatocellular Carcinoma in China: A Large-Scale, Multicentre Study

    PubMed Central

    Zheng, Lei; Yin, Yuepeng; Zou, Zhenzhen; Zhou, Feiguo; Zhou, Weiping; Shen, Feng; Gao, Chunfang

    2016-01-01

    An efficient serum marker for hepatocellular carcinoma (HCC) is currently lacking and requires intensive exploration. We aimed to evaluate the performance of des-gamma-carboxy prothrombin (DCP) for identifying hepatitis B virus-related HCC in a large, multicentre study in China. A total of 1034 subjects in three cohorts (A, B, and C) including HCC and various non-HCC controls were enrolled from 4 academic medical centers in China from January 2011 to February 2014. Blind parallel detections were conducted for DCP and AFP. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic efficacies. In cohort A, which comprised 521 subjects, including patients with HCC, liver metastasis, liver cirrhosis (LC), and liver hemangiomas as well as healthy controls (HCs), the accuracy of DCP for distinguishing HCC from various controls was 6.2–9.7% higher than that of AFP. In cohort B, which comprised 447 subjects, including patients with HCC, LC, and chronic hepatitis B as well as HC, the accuracy of DCP was further elevated (12.3–20.67% higher than that of AFP). The superiority of DCP to AFP was more profound in the surveillance of early HCC [AUC 0.837 (95% CI: 0.771–0.903) vs. 0.650 (0.555–0.745)] and AFP-negative HCC [AUC: 0.856 (0.798–0.914)] and in discriminating HCC from LC (accuracy: 92.9% vs.64.71%). Higher DCP levels were associated with worse clinical behaviors and shorter disease-free survival. DCP not only is complementary to AFP in identifying AFP-negative HCC and in excluding AFP-positive non-HCC (liver cirrhosis), but also demonstrates improved performance in HCC surveillance, early diagnosis, treatment response and recurrence monitoring in the HBV-related population. PMID:27070780

  7. Hormonogenic donor Tyr2522 of bovine thyroglobulin. Insight into preferential T3 formation at thyroglobulin carboxyl terminus at low iodination level

    SciTech Connect

    Cetrangolo, Giovanni Paolo; Arcaro, Alessia; Lepore, Alessio; Graf, Maria; Mamone, Gianfranco; Ferranti, Pasquale; Palumbo, Giuseppe; Gentile, Fabrizio

    2014-07-18

    Highlights: • A carboxy-terminal fragment (residues 2515–2750) was isolated from a low-iodine bTg. • Post-translational status of 8 tyrosines in bTg region 2515–2750 was assessed by MS. • Tyr2522 of bovine Tg is an interspecifically conserved hormonogenic donor site. • Propensities of Tyr residues to mono or diiodination optimize T3 yield from Tyr2748. - Abstract: A tryptic fragment (b5{sub TR,NR}), encompassing residues 2515–2750, was isolated from a low-iodine (0.26% by mass) bovine thyroglobulin, by limited proteolysis with trypsin and preparative, continuous-elution SDS–PAGE. The fragment was digested with Asp-N endoproteinase and analyzed by reverse-phase HPLC electrospray ionization quadrupole time-of-flight mass spectrometry, revealing the formation of: 3-monoiodotyrosine and dehydroalanine from Tyr2522; 3-monoiodotyrosine from Tyr2555 and Tyr2569; 3-monoiodotyrosine and 3,5-diiodotyrosine from Tyr2748. The data presented document, by direct mass spectrometric identifications, efficient iodophenoxyl ring transfer from monoiodinated hormonogenic donor Tyr2522 and efficient mono- and diiodination of hormonogenic acceptor Tyr2748, under conditions which permitted only limited iodination of Tyr2555 and Tyr2569, in low-iodine bovine thyroglobulin. The present study thereby provides: (1) a rationale for the preferential synthesis of T3 at the carboxy-terminal end of thyroglobulin, at low iodination level; (2) confirmation for the presence of an interspecifically conserved hormonogenic donor site in the carboxy-terminal domain of thyroglobulin; (3) solution for a previous uncertainty, concerning the precise location of such donor site in bovine thyroglobulin.

  8. A d-Amino Acid at the N-Terminus of a Protein Abrogates Its Degradation by the N-End Rule Pathway

    PubMed Central

    2015-01-01

    Eukaryotes have evolved the ubiquitin (Ub)/proteasome system to degrade polypeptides. The Ub/proteasome system is one way that cells regulate cytosolic protein and amino acids levels through the recognition and ubiquitination of a protein’s N-terminus via E1, E2, and E3 enzymes. The process by which the N-terminus stimulates intracellular protein degradation is referred to as the N-end rule. Characterization of the N-end rule has been limited to only the natural l-amino acids. Using a cytosolic delivery platform derived from anthrax lethal toxin, we probed the stability of mixed chirality proteins, containing one d-amino acid on the N-terminus of otherwise all l-proteins. In all cases, we observed that one N-terminal d-amino acid stabilized the cargo protein to proteasomal degradation with respect to the N-end rule. We found that since the mixed chirality proteins were not polyubiquitinated, they evaded N-end-mediated proteasomal degradation. Evidently, a subtle change on the N-terminus of a natural protein can enhance its intracellular lifetime. PMID:26807441

  9. A Unique Primer with an Inosine Chain at the 5′-Terminus Improves the Reliability of SNP Analysis Using the PCR-Amplified Product Length Polymorphism Method

    PubMed Central

    Shojo, Hideki; Tanaka, Mayumi; Takahashi, Ryohei; Kakuda, Tsuneo; Adachi, Noboru

    2015-01-01

    Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3′-terminus of each primer. To use this method at least two allele-specific primers and one “counter-primer”, which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3′-terminus, and another primer should have a few non-complementary flaps at the 5′-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5′-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method. PMID:26381262

  10. Enhancing the Predictive Power of Mutations in the C-Terminus of the KCNQ1-Encoded Kv7.1 Voltage-Gated Potassium Channel

    PubMed Central

    Kapplinger, Jamie D.; Tseng, Andrew S.; Salisbury, Benjamin A.; Tester, David J.; Callis, Thomas E.; Alders, Marielle; Wilde, Arthur A.M.; Ackerman, Michael J.

    2016-01-01

    Despite the overrepresentation of Kv7.1 mutations among patients with a robust diagnosis of LQTS, a background rate of innocuous Kv7.1 missense variants observed in healthy controls creates ambiguity in the interpretation of LQTS genetic test results. A recent study showed the probability of pathogenicity for rare missense mutations depends in part on the topological location of the variant in Kv7.1’s various structure-function domains. Since the Kv7.1 C-terminus accounts for nearly 50% of the overall protein and nearly 50% of the overall background rate of rare variants falls within the C-terminus, further enhancement in mutation calling may provide guidance in distinguishing pathogenic LQT1-causing mutations from non-disease causing rare variants in Kv7.1’s C-terminus. Therefore, we have used conservation analysis and a large case/control study to generate topology-based estimative predictive values to aid in interpretation; identifying three regions of high conservation within the Kv7.1 C-terminus which have a high probability of LQT1 pathogenicity. PMID:25854863

  11. Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen.

    PubMed Central

    Lucher, L A; Brackmann, K H; Symington, J S; Green, M

    1986-01-01

    The adenovirus E1A transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S. Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications. The adenovirus type 12 (Ad12) 13S and 12S mRNAs encode E1A T antigens of 266 and 235 amino acid residues (266R and 235R), respectively. To study possible posttranslational processing at the N and C termini and to distinguish between the Ad12 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206). The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli. Immunoprecipitation analysis of the E1A T antigens synthesized in Ad12-infected KB cells revealed the following. Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000, 42,000, and 39,000 (43K, 42K, and 39K). All three forms were phosphorylated and were present in both the nucleus and the cytoplasm. The 43K and 42K T antigens were rapidly synthesized during a 10-min pulse with [35S]methionine in Ad12-infected cells. The 43K T antigen had a half-life of 20 min, the 42K T antigen had a longer half-life of about 40 min, and the 39K T antigen became the predominant E1A T antigen. Antibodies to the unique region immunoprecipitated the 43K T antigen but not the 42K and 39K T antigens. Antibody to the N terminus immunoprecipitated the 43K and 42K T antigens but not the 39K T antigen, suggesting that the 39K T antigen possessed a modified N terminus. Partial N-terminal amino acid sequence analysis showed that the 43K and 42K T antigens contain methionine at residues 1 and 5, as predicted from the

  12. Detection of synenkephalin, the amino-terminal portion of proenkephalin, by antisera directed against its carboxyl terminus.

    PubMed

    Stell, W K; Chaminade, M; Metters, K M; Rougeot, C; Dray, F; Rossier, J

    1990-02-01

    Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of

  13. Three families with autosomal dominant nephrogenic diabetes insipidus caused by aquaporin-2 mutations in the C-terminus.

    PubMed

    Kuwahara, M; Iwai, K; Ooeda, T; Igarashi, T; Ogawa, E; Katsushima, Y; Shinbo, I; Uchida, S; Terada, Y; Arthus, M F; Lonergan, M; Fujiwara, T M; Bichet, D G; Marumo, F; Sasaki, S

    2001-10-01

    The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetramerize in the apical membrane of the renal tubular cells and contributes to urine concentration. We identified three novel mutations, each in a single allele of exon 4 of the AQP2 gene, in three families showing autosomal dominant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a deletion of 10 nucleotides starting at nucleotide 763 (763-772del), and a deletion of 7 nucleotides starting at nucleotide 812 (812-818del). The wild-type AQP2 is predicted to be a 271-amino acid protein, whereas these mutant genes are predicted to encode proteins that are 330-333 amino acids in length, because of the frameshift mutations. Interestingly, these three mutant AQP2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes injected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was much smaller than that of oocytes with the AQP2 wild-type (14%-17%). Immunoblot analysis of the lysates of the oocytes expressing the mutant AQP2s detected a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surface expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decreased the oocyte Pf in parallel with the surface expression of the wild-type AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQP2 indicated the formation of mixed oligomers composed of wild-type and mutant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant-negative effect is attributed to oligomerization of the wild-type and mutant AQP2s. Segregation of the mutations in the C-terminus of AQP2 with dominant-type NDI underlies the importance of this

  14. The N-Terminus of the Floral Arabidopsis TGA Transcription Factor PERIANTHIA Mediates Redox-Sensitive DNA-Binding

    PubMed Central

    Gutsche, Nora; Zachgo, Sabine

    2016-01-01

    The Arabidopsis TGA transcription factor (TF) PERIANTHIA (PAN) regulates the formation of the floral organ primordia as revealed by the pan mutant forming an abnormal pentamerous arrangement of the outer three floral whorls. The Arabidopsis TGA bZIP TF family comprises 10 members, of which PAN and TGA9/10 control flower developmental processes and TGA1/2/5/6 participate in stress-responses. For the TGA1 protein it was shown that several cysteines can be redox-dependently modified. TGA proteins interact in the nucleus with land plant-specific glutaredoxins, which may alter their activities posttranslationally. Here, we investigated the DNA-binding of PAN to the AAGAAT motif under different redox-conditions. The AAGAAT motif is localized in the second intron of the floral homeotic regulator AGAMOUS (AG), which controls stamen and carpel development as well as floral determinacy. Whereas PAN protein binds to this regulatory cis-element under reducing conditions, the interaction is strongly reduced under oxidizing conditions in EMSA studies. The redox-sensitive DNA-binding is mediated via a special PAN N-terminus, which is not present in other Arabidopsis TGA TFs and comprises five cysteines. Two N-terminal PAN cysteines, Cys68 and Cys87, were shown to form a disulfide bridge and Cys340, localized in a C-terminal putative transactivation domain, can be S-glutathionylated. Comparative land plant analyses revealed that the AAGAAT motif exists in asterid and rosid plant species. TGA TFs with N-terminal extensions of variable length were identified in all analyzed seed plants. However, a PAN-like N-terminus exists only in the rosids and exclusively Brassicaceae homologs comprise four to five of the PAN N-terminal cysteines. Redox-dependent modifications of TGA cysteines are known to regulate the activity of stress-related TGA TFs. Here, we show that the N-terminal PAN cysteines participate in a redox-dependent control of the PAN interaction with a highly conserved

  15. Virus-specific interaction between the human cytomegalovirus major capsid protein and the C terminus of the assembly protein precursor.

    PubMed Central

    Beaudet-Miller, M; Zhang, R; Durkin, J; Gibson, W; Kwong, A D; Hong, Z

    1996-01-01

    We previously identified a minimal 12-amino-acid domain in the C terminus of the herpes simplex virus type 1 (HSV-1) scaffolding protein which is required for interaction with the HSV-1 major capsid protein. An alpha-helical structure which maximizes the hydropathicity of the minimal domain is required for the interaction. To address whether cytomegalovirus (CMV) utilizes the same strategy for capsid assembly, several glutathione S-transferase fusion proteins to the C terminus of the CMV assembly protein precursor were produced and purified from bacterial cells. The study showed that the glutathione S-transferase fusion containing 16 amino acids near the C-terminal end was sufficient to interact with the major capsid protein. Interestingly, no cross-interaction between HSV-1 and CMV could be detected. Mutation analysis revealed that a three-amino-acid region at the N-terminal side of the central Phe residue of the CMV interaction domain played a role in determining the viral specificity of the interaction. When this region was converted so as to correspond to that of HSV-1, the CMV assembly protein domain lost its ability to interact with the CMV major capsid protein but gained full interaction with the HSV-1 major capsid protein. To address whether the minimal interaction domain of the CMV assembly protein forms an alpha-helical structure similar to that in HSV-1, peptide competition experiments were carried out. The results showed that a cyclic peptide derived from the interaction domain with a constrained (alpha-helical structure competed for interaction with the major capsid protein much more efficiently than the unconstrained linear peptide. In contrast, a cyclic peptide containing an Ala substitution for the critical Phe residue did not compete for the interaction at all. The results of this study suggest that (i) CMV may have developed a strategy similar to that of HSV-1 for capsid assembly; (ii) the minimal interaction motif in the CMV assembly protein

  16. Effect of adding amino acids residues in N- and C-terminus of Vip3Aa16 (L121I) toxin.

    PubMed

    Sellami, Sameh; Cherif, Marwa; Jamoussi, Kaïs

    2016-06-01

    To study the importance of N- and C-terminus of Bacillus thuringiensis Vip3Aa16 (L121I) toxin (88 kDa), a number of mutants were generated. The addition of two (2R: RS) or eleven (11R: RSRPGHHHHHH) amino acid residues at the Vip3Aa16 (L121I) C-terminus allowed to an unappropriated folding illustrated by the abundant presence of the 62 kDa proteolytic form. The produced Vip3Aa16 (L121I) full length form was less detected when increasing the number of amino acids residues in the C-terminus. Bioassays demonstrated that the growth of the lepidopteran Ephestia kuehniella was slightly affected by Vip3Aa16 (L121I)-2R and not affected by Vip3Aa16 (L121I)-11R. Additionally, the fusion at the Vip3Aa16 (L121I) N-terminus of 39 amino acids harboring the E. coli OmpA leader peptide and the His-tag sequence allowed to the increase of protease sensitivity of Vip3Aa16 (L121I) full length form, as only the 62 kDa proteolysis form was detected. Remarkably, this fused protein produced in Escherichia coli (E. coli) was biologically inactive toward Ephestia kuehniella larvae. Thus, the N-terminus of the protein is required to the accomplishment of the insecticidal activity of Vip3 proteins. This report serves as guideline for the study of Vip3Aa16 (L121I) protein stability and activity. PMID:26876111

  17. Displacement of the C Terminus of Herpes Simplex Virus gD Is Sufficient To Expose the Fusion-Activating Interfaces on gD

    PubMed Central

    Gallagher, John R.; Saw, Wan Ting; Atanasiu, Doina; Lou, Huan; Eisenberg, Roselyn J.

    2013-01-01

    Viral entry by herpes simplex virus (HSV) is executed and tightly regulated by four glycoproteins. While several viral glycoproteins can mediate viral adhesion to host cells, only binding of gD to cellular receptor can activate core fusion proteins gB and gH/gL to execute membrane fusion and viral entry. Atomic structures of gD bound to receptor indicate that the C terminus of the gD ectodomain must be displaced before receptor can bind to gD, but it is unclear which conformational changes in gD activate membrane fusion. We rationally designed mutations in gD to displace the C terminus and observe if fusion could be activated without receptor binding. Using a cell-based fusion assay, we found that gD V231W induced cell-cell fusion in the absence of receptor. Using recombinant gD V231W protein, we observed binding to conformationally sensitive antibodies or HSV receptor and concluded that there were changes proximal to the receptor binding interface, while the tertiary structure of gD V231W was similar to that of wild-type gD. We used a biosensor to analyze the kinetics of receptor binding and the extent to which the C terminus blocks binding to receptor. We found that the C terminus of gD V231W was enriched in the open or displaced conformation, indicating a mechanism for its function. We conclude that gD V231W triggers fusion through displacement of its C terminus and that this motion is indicative of how gD links receptor binding to exposure of interfaces on gD that activate fusion via gH/gL and gB. PMID:24049165

  18. The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity.

    PubMed

    Patil, Nitin A; Bathgate, Ross A D; Kocan, Martina; Ang, Sheng Yu; Tailhades, Julien; Separovic, Frances; Summers, Roger; Grosse, Johannes; Hughes, Richard A; Wade, John D; Hossain, Mohammed Akhter

    2016-04-01

    Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops. PMID:26661035

  19. The C Terminus of Na+,K+-ATPase Controls Na+ Affinity on Both Sides of the Membrane through Arg935*♦

    PubMed Central

    Toustrup-Jensen, Mads S.; Holm, Rikke; Einholm, Anja Pernille; Schack, Vivien Rodacker; Morth, J. Preben; Nissen, Poul; Andersen, Jens Peter; Vilsen, Bente

    2009-01-01

    The Na+,K+-ATPase C terminus has a unique location between transmembrane segments, appearing to participate in a network of interactions. We have examined the functional consequences of amino acid substitutions in this region and deletions of the C terminus of varying lengths. Assays revealing separately the mutational effects on internally and externally facing Na+ sites, as well as E1-E2 conformational changes, have been applied. The results pinpoint the two terminal tyrosines, Tyr1017 and Tyr1018, as well as putative interaction partners, Arg935 in the loop between transmembrane segments M8 and M9 and Lys768 in transmembrane segment M5, as crucial to Na+ activation of phosphorylation of E1, a partial reaction reflecting Na+ interaction on the cytoplasmic side of the membrane. Tyr1017, Tyr1018, and Arg935 are furthermore indispensable to Na+ interaction on the extracellular side of the membrane, as revealed by inability of high Na+ concentrations to drive the transition from E1P to E2P backwards toward E1P and inhibit Na+-ATPase activity in mutants. Lys768 is not important for Na+ binding from the external side of the membrane but is involved in stabilization of the E2 form. These data demonstrate that the C terminus controls Na+ affinity on both sides of the membrane and suggest that Arg935 constitutes an important link between the C terminus and the third Na+ site, involving an arginine-π stacking interaction between Arg935 and the C-terminal tyrosines. Lys768 may interact preferentially with the C terminus in E1 and E1P forms and with the loop between transmembrane segments M6 and M7 in E2 and E2P forms. PMID:19416970

  20. Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus.

    PubMed

    Meera, P; Wallner, M; Song, M; Toro, L

    1997-12-01

    Large conductance voltage- and Ca2+-dependent K+ (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers beta subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and "in vivo" reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus. PMID:9391153

  1. Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus

    PubMed Central

    Meera, Pratap; Wallner, Martin; Song, Min; Toro, Ligia

    1997-01-01

    Large conductance voltage- and Ca2+-dependent K+ (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers β subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and “in vivo” reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus. PMID:9391153

  2. Differential effects of carboxy-terminal sequence deletions on platelet-derived growth factor receptor signaling activities and interactions with cellular substrates.

    PubMed Central

    Seedorf, K; Millauer, B; Kostka, G; Schlessinger, J; Ullrich, A

    1992-01-01

    Chimeric receptors composed of the human epidermal growth factor receptor (EGF-R) extracellular domain fused to wild-type and truncated platelet-derived growth factor receptor (PDGF-R) intracellular sequences were stably expressed in NIH 3T3 cells devoid of endogenous EGF-Rs. This experimental system allowed us to investigate the biological activity of PDGF-R cytoplasmic-domain mutants in PDGF-R-responsive NIH 3T3 cells by activating PDGF-specific signaling pathways with EGF. Deletion of 74 carboxy-terminal amino acids severely impaired the ability of the PDGF-R cytoplasmic domain to associate with cellular substrates in vitro. This deletion also inhibited receptor and substrate phosphorylation, reduced the receptor's mitogenic activity, and completely abolished its oncogenic signaling potential. Surprisingly, removal of only six additional amino acids, including Tyr-989, restored substantial receptor and substrate phosphorylation capacity as well as transforming potential and yielded a receptor with wild-type levels of ligand-induced mitogenic activity. However, the ability of this chimera to bind phospholipase C gamma was severely impaired in comparison with the ability of the wild-type receptor, while the association with other cellular proteins was not affected. Further deletion of 35 residues, including Tyr-977, nearly abolished all PDGF-R cytoplasmic-domain biological signaling activities. None of the three C-terminal truncations completely abolished the mitogenic potential of the receptors or had any influence on ligand binding or receptor down regulation. Together, these data implicate the 80 C-terminal-most residues of the PDGF-R, and possibly Tyr-989, in phospholipase C gamma binding, while receptor sequences upstream from Asp-988 appear to be essential for specific interactions with other cellular polypeptides such as ras GTPase-activating protein and phosphatidylinositol 3-kinase. Thus, the mutants described here allow the separation of distinct PDGF

  3. Delta(9)-tetrahydrocannabinol, 11-hydroxy-delta(9)-tetrahydrocannabinol and 11-nor-9-carboxy-delta(9)-tetrahydrocannabinol in human plasma after controlled oral administration of cannabinoids.

    PubMed

    Goodwin, Robert S; Gustafson, Richard A; Barnes, Allan; Nebro, Wesenyalsh; Moolchan, Eric T; Huestis, Marilyn A

    2006-08-01

    A clinical study to investigate the pharmacokinetics and pharmacodynamics of oral tetrahydrocannabinol was performed. This randomized, double-blind, placebo-controlled, within-subject, inpatient study compared the effects of THC-containing hemp oils in liquid and capsule form to dronabinol (synthetic THC) in doses used for appetite stimulation. The National Institute on Drug Abuse Institutional Review Board approved the protocol and each participant provided informed consent. Detection times and concentrations of THC, 11-hydroxy-Delta-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Delta-tetrahydrocannabinol (THCCOOH) in plasma were determined by gas chromatography-mass spectrometry [limits of quantification (LOQ)=0.5, 0.5, and 1.0 ng/mL, respectively] after oral THC administration. Six volunteers ingested liquid hemp oil (0.39 and 14.8 mg THC/d), hemp oil in capsules (0.47 mg THC/d), dronabinol capsules (7.5 mg THC/d), and placebo. Plasma specimens were collected during and after each dosing condition. THC and 11-OH-THC concentrations were low and never exceeded 6.1 ng/mL. Analytes were detectable 1.5 hour after initiating dosing with the 7.5 mg THC/d regimen and 4.5 hour after starting the 14.8 mg THC/d sessions. THCCOOH was detected 1.5 hour after the first dose, except for the 0.47 mg THC/d session, which required 4.5 hour for concentrations to reach the LOQ. THCCOOH concentrations peaked at 3.1 ng/mL during dosing with the low-dose hemp oils. Plasma THC and 11-OH-THC concentrations were negative for all participants at all doses within 15.5 hours after the last THC dose. Plasma THCCOOH persisted for at least 39.5 hours after the end of dosing and at much higher concentrations (up to 43.0 ng/mL). This study demonstrated that subjects who used high THC content hemp oil (347 mug/mL) as a dietary supplement had THC and metabolites in plasma in quantities comparable to those of patients using dronabinol for appetite stimulation. There was a significant

  4. Identification and characterization of the carboxy-terminal region of Sip-1, a novel autoantigen in Behçet's disease

    PubMed Central

    Delunardo, Federica; Conti, Fabrizio; Margutti, Paola; Alessandri, Cristiano; Priori, Roberta; Siracusano, Alessandra; Riganò, Rachele; Profumo, Elisabetta; Valesini, Guido; Sorice, Maurizio; Ortona, Elena

    2006-01-01

    Given the lack of a serological test specific for Behçet's disease, its diagnosis rests upon clinical criteria. The clinical diagnosis is nevertheless difficult because the disease manifestations vary widely, especially at the onset of disease. The aim of this study was to identify molecules specifically recognized by serum autoantibodies in patients with Behçet's disease and to evaluate their diagnostic value. We screened a cDNA library from human microvascular endothelial cells with serum IgG from two patients with Behçet's disease and isolated a reactive clone specific to the carboxy-terminal subunit of Sip1 (Sip1 C-ter). Using ELISA, we measured IgG, IgM and IgA specific to Sip1 C-ter in patients with various autoimmune diseases characterized by the presence of serum anti-endothelial cell antibodies, such as Behçet's disease, systemic lupus erythematosus, systemic sclerosis and various forms of primary vasculitis, as well as in patients with diseases that share clinical features with Behçet's disease, such as inflammatory bowel disease and uveitis. IgM immunoreactivity to Sip1 C-ter was significantly higher in patients with Behçet's disease and in patients with primary vasculitis than in the other groups of patients and healthy subjects tested (P < 10-4 by Mann-Whitney test). ELISA detected IgG specific to Sip1 C-ter in sera from 11/56 (20%) patients with Behçet's disease, IgM in 23/56 (41%) and IgA in 9/54 (17%). No sera from patients with systemic lupus erythematosus, systemic sclerosis, inflammatory bowel disease, uveitis or healthy subjects but 45% of sera from patients with primary vasculitis contained IgM specific to Sip1 C-ter. Serum levels of soluble E-selectin, a marker of endothelial activation and inflammation, correlated with levels of serum IgM anti Sip-1 C-ter in patients with Behçet's disease (r = 0.36, P = 0.023). In conclusion, Sip1 C-ter is a novel autoantigen in Behçet's disease. IgM specific to Sip1 C-ter might be useful in clinical

  5. Pharmacological characterization of recombinant NR1/NR2A NMDA receptors with truncated and deleted carboxy termini expressed in Xenopus laevis oocytes

    PubMed Central

    Puddifoot, CA; Chen, PE; Schoepfer, R; Wyllie, DJA

    2009-01-01

    Background and purpose: The carboxy terminal domain (CTD) of NR2 N-methyl-d-aspartate receptor (NMDAR) subunits interacts with numerous scaffolding and signal transduction proteins. Mutations of this region affect trafficking and downstream signalling of NMDARs. This study determines to what extent characteristic pharmacological properties of NR2A-containing NMDARs are influenced by this key functional domain. Experimental approach: Using recombinant receptor expression in Xenopus laevis oocytes and two electrode voltage clamp recordings we characterized pharmacological properties of rat NR1/NR2A NMDARs with altered CTDs. We assessed the effects of truncating [at residue Iso1098; NR2A(trunC)] and deleting [from residue Phe822; NR2A(delC)] the CTD of NR2A NMDAR subunits on agonist potencies, channel block by Mg2+ and memantine and potentiation of NMDAR-mediated responses by chelating contaminating divalent cations. Key results: Truncation or deletion of the CTD of NR2A NMDAR subunits did not affect glutamate potency [EC50 = 2.2 µmol·L−1, NR2A(trunC); 2.7 µmol·L−1, NR2A(delC) compared with 3.3 µmol·L−1, NR2A(WT)] but did significantly increase glycine potency [EC50 = 500 nmol·L−1, NR2A(trunC); 900 nmol·L−1, NR2A(delC) compared with 1.3 µmol·L−1, NR2A(WT)]. Voltage-dependent Mg2+ block of NR2A(WT)- and NR2A(trunC)-containing NMDARs was similar but low concentrations of Mg2+ (1 µmol·L−1) potentiated NR1/NR2A(delC) NMDARs. Memantine block was not affected by changes to the structure of the NR2A CTD. EDTA-induced potentiation was similar at each of the three NMDAR constructs. Conclusions and implications: Of the parameters studied only minor influences of the CTD were observed; these are unlikely to compromise interpretation of studies that make use of CTD-mutated recombinant receptors or transgenic mice in investigations of the role of the CTD in NMDAR signalling. PMID:19154422

  6. Highly sensitive quantification of unconjugated 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol in a cannabis user's hair using micropulverized extraction.

    PubMed

    Kuwayama, Kenji; Miyaguchi, Hajime; Yamamuro, Tadashi; Tsujikawa, Kenji; Kanamori, Tatsuyuki; Iwata, Yuko T; Inoue, Hiroyuki

    2016-05-01

    We previously developed a simple and highly sensitive analytical method for 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) in spiked hair using micropulverized extraction (MPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Using this method, we were able to quantify THC-COOH at 0.2pg/mg, which is the cut-off level recommended by the Society of Hair Testing. However, it was impossible to prove the validity of the method and the presence of THC-COOH conjugated with glucuronide in hair because we did not have authentic hair containing THC-COOH at the cut-off levels at that time. In this study, the previously developed method was verified using recently obtained hair from a cannabis user. The concentrations of THC-COOH quantified using the method were 0.36±0.01pg/mg without hydrolyzation for glucuronide and 0.49±0.05pg/mg with hydrolyzation after MPE, whereas the concentration quantified using the conventional alkaline dissolution and gas chromatography/tandem mass spectrometry with negative ion chemical ionization was 0.50±0.02pg/mg. The results proved that THC-COOH could be extracted completely from authentic hair containing THC-COOH at the recommended cut-off level using MPE. In addition, MPE with and without hydrolyzation, unlike alkaline dissolution of hair, enabled the measurement of the percentage of the conjugate form in total THC-COOH. The percentage of conjugated THC-COOH in hair measured using the MPE was approximately 26%, which was greatly different from previously reported data (>75%). The discrimination between conjugated and unconjugated compounds in hair is important to understand the mechanism of drug uptakes into hair. More data obtained with our simple and highly sensitive method from the hair of cannabis users would help to understand the relationship of concentrations between THC-COOH and its conjugate in hair. PMID:27020617

  7. Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication.

    PubMed

    Cannon, P M; Byles, E D; Kingsman, S M; Kingsman, A J

    1996-01-01

    We have previously identified a residue in the carboxyl terminus of human immunodeficiency virus type 1 integrase (HIV-1 IN), W-235, the requirement for which is only revealed in viral assays for integrase function (P. M. Cannon, W. Wilson, E. Byles, S. M. Kingsman, and A. J. Kingsman, J. Virol. 68:4768-4775, 1994). Our further analysis of this region of retroviral IN has now identified several sequence motifs which are conserved in all the retroviruses we examined, apart from human spumaretrovirus. We have made mutations within these motifs in HIV-1 IN and examined their phenotypes when reintroduced into an infectious proviral clone. The deleterious effects of several of these mutations demonstrate the importance of these regions for IN function in vivo. We observed a further discrepancy, at a motif that is only conserved in the lentiviruses, in the ability of mutants to function in in vitro and in vivo assays. Substitutions both in this region and at W-235 abolish HIV-1 infectivity but do not affect particle production, morphology, reverse transcription, or nuclear import in T-cell lines. Taken together with the in vitro data suggesting that neither of these residues is directly involved in the catalytic reactions of IN, it seems likely that we have identified regions of IN that are essential for interactions with other components of the integration machinery. PMID:8523588

  8. Inhibiting bladder tumor growth with a cell penetrating R11 peptide derived from the p53 C-terminus.

    PubMed

    Zhang, Tingting; Wu, Kaijie; Ding, Chen; Sun, Kangwei; Guan, Zhenfeng; Wang, Xinyang; Hsieh, Jer-Tsong; He, Dalin; Fan, Jinhai

    2015-11-10

    Urothelial carcinoma of the bladder (UCB) is the most common malignancy of the urinary tract, nearly half of which contains a mutation in TP53 gene. Hence, therapeutic approach by restoring functional p53 protein in cancer cells will be beneficial. Recent studies have demonstrated the inhibition of cancer cell growth by p53 reactivation using a peptide derived from the p53 C-terminus (p53C). However, the outcome of reactivating p53 in controlling bladder cancer development is limited by its efficiency and specificity of peptide delivery, especially in metastatic animal models. Herein, we report that the cell penetrating peptide (polyarginine, R11)-conjugated p53C can exhibit a preferential uptake and growth inhibit of UCB cells expressing either mutant or wild-type TP53 by the activation of p53-dependent pathway. R11-p53C peptide treatment of preclinical orthotopic and metastatic bladder cancer models significantly decreased the tumor burden and increased the lifespan without a significant cytotoxicity. Based on these results, we believe that R11-p53C peptide has therapeutic potential for primary and metastatic bladder cancer, and R11-mediated transduction may be a useful strategy for the therapeutic delivery of large tumor suppressor molecules to tumor cells in vitro and in vivo. PMID:26462022

  9. Native N-terminus nitrophorin 2 from the kissing bug: similarities to and differences from NP2(D1A).

    PubMed

    Berry, Robert E; Muthu, Dhanasekaran; Shokhireva, Tatiana K; Garrett, Sarah A; Zhang, Hongjun; Walker, F Ann

    2012-09-01

    The first amino acid of mature native nitrophorin 2 is aspartic acid, and when expressed in E. coli, the wild-type gene of the mature protein retains the methionine-0, which is produced by translation of the start codon. This form of NP2, (M0)NP2, has been found to have different properties from its D1A mutant, for which the Met0 is cleaved by the methionine aminopeptidase of E. coli (R. E. Berry, T. K. Shokhireva, I. Filippov, M. N. Shokhirev, H. Zhang, F. A. Walker, Biochemistry 2007, 46, 6830). Native N-terminus nitrophorin 2 ((ΔM0)NP2) has been prepared by employing periplasmic expression of NP2 in E. coli using the pelB leader sequence from Erwinia carotovora, which is present in the pET-26b expression plasmid (Novagen). This paper details the similarities and differences between the three different N-terminal forms of nitrophorin 2, (M0)NP2, NP2(D1A), and (ΔM0)NP2. It is found that the NMR spectra of high- and low-spin (ΔM0)NP2 are essentially identical to those of NP2(D1A), but the rate and equilibrium constants for histamine and NO dissociation/association of the two are different. PMID:22976966

  10. GAIP Interacting Protein C-Terminus Regulates Autophagy and Exosome Biogenesis of Pancreatic Cancer through Metabolic Pathways

    PubMed Central

    Bhattacharya, Santanu; Pal, Krishnendu; Sharma, Anil K.; Dutta, Shamit K.; Lau, Julie S.; Yan, Irene K.; Wang, Enfeng; Elkhanany, Ahmed; Alkharfy, Khalid M.; Sanyal, Arunik; Patel, Tushar C.; Chari, Suresh T.; Spaller, Mark R.; Mukhopadhyay, Debabrata

    2014-01-01

    GAIP interacting protein C terminus (GIPC) is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer. PMID:25469510

  11. The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist

    SciTech Connect

    Madauss, Kevin P.; Burkhart, William A.; Consler, Thomas G.; Cowan, David J.; Gottschalk, William K.; Miller, Aaron B; Short, Steven A.; Tran, Thuy B.; Williams, Shawn P.

    2009-06-15

    Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain-CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined -helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

  12. Cutting Edge: Novel Tmem173 Allele Reveals Importance of STING N Terminus in Trafficking and Type I IFN Production.

    PubMed

    Surpris, Guy; Chan, Jennie; Thompson, Mikayla; Ilyukha, Vladimir; Liu, Beiyun C; Atianand, Maninjay; Sharma, Shruti; Volkova, Tatyana; Smirnova, Irina; Fitzgerald, Katherine A; Poltorak, Alexander

    2016-01-15

    With the stimulator of IFN genes (STING) C terminus being extensively studied, the role of the N-terminal domain (NTD) of STING remains an important subject of investigation. In this article, we identify novel mutations in NTD of Sting of the MOLF strain in response to HSV and Listeria monocytogenes both in vitro and in vivo. These mutations are responsible for low levels of IFN-β caused by failure of MOLF STING to translocate from the endoplasmic reticulum. These data provide evidence that the NTD of STING affects DNA responses via control of trafficking. They also show that the genetic diversity of wild-derived mice resembles the diversity observed in humans. Several human alleles of STING confer attenuated IFN-I production similar to what we observe with the MOLF Sting allele, a crucial functional difference not apparent in classical inbred mice. Thus, understanding the functional significance of polymorphisms in MOLF STING can provide basic mechanistic insights relevant to humans. PMID:26685207

  13. Reciprocal and nonreciprocal recombination in diploid clones from Bacillus subtilis protoplast fusion: Association with the replication origin and terminus

    PubMed Central

    Gabor, Magda H.; Hotchkiss, Rollin D.

    1983-01-01

    The primary heterodiploid bacteria regenerated after Bacillus subtilis fusion, although generally noncomplementing diploids, behave in pedigree analysis as multipotential systems. Individual diploid colonies yielding complete reciprocal recombinant (RR) progeny—often accompanied by one or both parents—constitute 10-30% of the total recombinant-forming units. The RR (reciprocal for 8-11 genes) usually occur in equivalent numbers both among and within individual colonies. Novel for bacteria, they demonstrate that entire parental genomes brought together within a diploid protoplast are retained as two independent replicons able to undergo classical recombination characteristic of eukaryotic gametogenesis. Parental or recombinant genomes are also subject to multiple rounds of recombination without obligate segregation and often not reciprocal. Diploid recombinant clones, sharing streptomycin resistance but reciprocal for auxotrophic markers, have displayed a partial ability to make a facultative shift in chromosome expression. They have also produced two types of prototrophs: a stable one (presumably haploid and recombinant) and an unstable one, (diploid and temporarily complementing at low frequency). It follows that chromosome extinction may affect both parental and recombinant chromosomes and does not interfere with recombination. Analysis of the number and chromosomal distribution of crossovers in all recombinants and those from single diploid clones shows increased frequency of exchange in the regions of the replication origin and terminus, possibly a result of the association of these sites with the cell wall or membrane. PMID:16593292

  14. Localized remodeling of the Escherichia coli chromosome: the patchwork of segments refractory and tolerant to inversion near the replication terminus.

    PubMed Central

    Guijo, M I; Patte, J; del Mar Campos, M; Louarn, J M; Rebollo, J E

    2001-01-01

    The behavior of chromosomal inversions in Escherichia coli depends upon the region they affect. Regions flanking the replication terminus have been termed nondivisible zones (NDZ) because inversions ending in the region were either deleterious or not feasible. This regional phenomenon is further analyzed here. Thirty segments distributed between 23 and 29 min on the chromosome map have been submitted to an inversion test. Twenty-five segments either became deleterious when inverted or were noninvertible, but five segments tolerated inversion. The involvement of polar replication pause sites in this distribution was investigated. The results suggest that the Tus/pause site system may forbid some inversion events, but that other constraints to inversion, unrelated to this system, exist. Our current model for deleterious inversions is that the segments involved carry polar sequences acting in concert with other polar sequences located outside the segments. The observed patchwork of refractory and tolerant segments supports the existence of several NDZs in the 23- to 29-min region. Microscopic observations revealed that deleterious inversions are associated with high frequencies of abnormal nucleoid structure and distribution. Combined with other information, the data suggest that NDZs participate in the organization of the terminal domain of the nucleoid. PMID:11290700

  15. Novel P2 promoter-derived HNF4{alpha} isoforms with different N-terminus generated by alternate exon insertion

    SciTech Connect

    Huang, Jianmin; Levitsky, Lynne L.; Rhoads, David B.

    2009-04-15

    Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a critical transcription factor for pancreas and liver development and functions in islet {beta} cells to maintain glucose homeostasis. Mutations in the human HNF4A gene lead to maturity onset diabetes of the young (MODY1) and polymorphisms are associated with increased risk for type 2 diabetes mellitus (T2DM). Expression of six HNF4{alpha} variants, three each from two developmentally regulated promoters, has been firmly established. We have now detected a new set of HNF4{alpha} variants designated HNF4{alpha}10-12 expressed from distal promoter P2. These variants, generated by inclusion of previously undetected exon 1E (human = 222 nt, rodent = 136 nt) following exon 1D have an altered N-terminus but identical remaining reading frame. HNF4{alpha}10-{alpha}12 are expressed in pancreatic islets (and liver) and exhibit transactivation potentials similar to the corresponding {alpha}7-{alpha}9 isoforms. DNA-binding analyses implied much higher protein levels of HNF4{alpha}10-{alpha}12 in liver than expected from the RT-PCR data. Our results provide evidence for a more complex expression pattern of HNF4{alpha} than previously appreciated. We recommend inclusion of exon 1E and nearby DNA sequences in screening for HNF4{alpha} mutations and polymorphisms in genetic analyses of MODY1 and T2DM.

  16. The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist

    SciTech Connect

    Madauss, Kevin P.; Burkhart, William A.; Consler, Thomas G.; Cowan, David J.; Gottschalk, William K.; Miller, Aaron B.; Short, Steven A.; Tran, Thuy B.; Williams, Shawn P.

    2009-05-01

    The use of biophysical assays permitted the identification of a specific human ACC2 carboxyl transferase (CT) domain mutant that binds inhibitors and crystallizes in their presence. This mutant led to determination of the human ACC2 CT domain–CP-640186 complex crystal structure, which revealed differences in the inhibitor conformation from the yeast protein complex that are caused by differing residues in the binding pocket. Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain–CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined α-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

  17. Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

    NASA Technical Reports Server (NTRS)

    Schauer-Vukasinovic, Vesna; Deo, Sapna K.; Daunert, Sylvia

    2002-01-01

    Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

  18. The RXR{alpha} C-terminus T462 is a NMR sensor for coactivator peptide binding

    SciTech Connect

    Lu Jianyun Chen Minghe; DeKoster, Gregory T.; Cistola, David P.; Li, Ellen

    2008-02-22

    The C-terminal activation function-2 (AF-2) helix plays a crucial role in retinoid X receptor alpha (RXR{alpha})-mediated gene expression. Here, we report a nuclear magnetic resonance (NMR) study of the RXR{alpha} ligand-binding domain complexed with 9-cis-retinoic acid and a glucocorticoid receptor-interacting protein 1 peptide. The AF-2 helix and most of the C-terminal residues were undetectable due to a severe line-broadening effect. Due to its outstanding signal-to-noise ratio, the C-terminus residue, threonine 462 (T462) exhibited two distinct crosspeaks during peptide titration, suggesting that peptide binding was in a slow exchange regime on the chemical shift timescale. Consistently, the K{sub d} derived from T462 intensity decay agreed with that derived from isothermal titration calorimetry. Furthermore, the exchange contribution to the {sup 15}N transverse relaxation rate was measurable in either T462 or the bound peptide. These results suggest that T462 is a sensor for coactivator binding and is a potential probe for AF-2 helix mobility.

  19. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor.

    PubMed

    Watson, Michael J; Lee, Shernita L; Marklew, Abigail J; Gilmore, Rodney C; Gentzsch, Martina; Sassano, Maria F; Gray, Michael A; Tarran, Robert

    2016-01-01

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR's function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR's PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs. PMID:27278076

  20. ENHANCEMENT OF SPHINGOSINE KINASE 1 CATALYTIC ACTIVITY BY DELETION OF 21 AMINO ACIDS FROM THE COOH-TERMINUS*

    PubMed Central

    Hengst, Jeremy A.; Guilford, Jacquelyn M.; Conroy, Elizabeth J.; Wang, Xujun; Yun, Jong K.

    2009-01-01

    Sphingosine kinase 1 (SphK1) responds to a variety of growth factor signals by increasing catalytic activity as it translocates to the plasma membrane (PM). Several studies have identified amino acids residues involved in translocation yet how SphK1 increases its catalytic activity remains to be elucidated. Herein, we report that deletion of 21 amino acids from the COOH terminus of SphK1 (1-363) results in increased catalytic activity relative to wild-type SphK1 (1-384) which is independent of the phosphorylation state of Serine 225 and PMA stimulation. Importantly, HEK293 cells stably expressing the 1-363 protein exhibit enhanced cell growth under serum-deprived cell culture conditions. Together the evidence indicates that the COOH-terminal region of SphK1 encompasses a structural element that is necessary for the increase in catalytic activity in response to PMA treatment and that its deletion renders SphK1 constitutively active with respect to PMA treatment. PMID:19914200

  1. The biliverdin chromophore binds covalently to a conserved cysteine residue in the N-terminus of Agrobacterium phytochrome Agp1.

    PubMed

    Lamparter, Tilman; Carrascal, Montserrat; Michael, Norbert; Martinez, Enriqueta; Rottwinkel, Gregor; Abian, Joaquin

    2004-03-30

    Phytochromes are widely distributed biliprotein photoreceptors. Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction. Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain. For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20. This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven. We therefore performed mass spectrometry studies on proteolytic holopeptide fragments. For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag. Following proteolysis, the chromophore co-purified with a ca. 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus. Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site. We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis. The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome. We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction. PMID:15035636

  2. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor

    PubMed Central

    Watson, Michael J.; Lee, Shernita L.; Marklew, Abigail J.; Gilmore, Rodney C.; Gentzsch, Martina; Sassano, Maria F.; Gray, Michael A.; Tarran, Robert

    2016-01-01

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR’s function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR’s PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs. PMID:27278076

  3. Three Surface Layer Homology Domains at the N Terminus of the Clostridium cellulovorans Major Cellulosomal Subunit EngE

    PubMed Central

    Tamaru, Yutaka; Doi, Roy H.

    1999-01-01

    The gene engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been isolated and sequenced. engE is comprised of an open reading frame (ORF) of 3,090 bp and encodes a protein of 1,030 amino acids with a molecular weight of 111,796. The amino acid sequence derived from engE revealed a structure consisting of catalytic and noncatalytic domains. The N-terminal-half region of EngE consisted of a signal peptide of 31 amino acid residues and three repeated surface layer homology (SLH) domains, which were highly conserved and homologous to an S-layer protein from the gram-negative bacterium Caulobacter crescentus. The C-terminal-half region, which is necessary for the enzymatic function of EngE and for binding of EngE to the scaffolding protein CbpA, consisted of a catalytic domain homologous to that of family 5 of the glycosyl hydrolases, a domain of unknown function, and a duplicated sequence (DS or dockerin) at its C terminus. engE is located downstream of an ORF, ORF1, that is homologous to the Bacillus subtilis phosphomethylpyrimidine kinase (pmk) gene. The unique presence of three SLH domains and a DS suggests that EngE is capable of binding both to CbpA to form a CbpA-EngE cellulosome complex and to the surface layer of C. cellulovorans. PMID:10322032

  4. USP47 and C Terminus of Hsp70-Interacting Protein (CHIP) Antagonistically Regulate Katanin-p60-Mediated Axonal Growth

    PubMed Central

    Yang, Seung Wook; Oh, Kyu Hee; Park, Esther; Chang, Hyun Min; Park, Jung Mi; Seong, Min Woo; Ka, Seung Hyeun; Song, Woo Keun; Park, Dong Eun; Baas, Peter W.

    2013-01-01

    Katanin is a heterodimeric enzyme that severs and disassembles microtubules. While the p60 subunit has the enzyme activity, the p80 subunit regulates the p60 activity. The microtubule-severing activity of katanin plays an essential role in axonal growth. However, the mechanisms by which neuronal cells regulate the expression of katanin-p60 remains unknown. Here we showed that USP47 and C terminus of Hsp70-interacting protein (CHIP) antagonistically regulate the stability of katanin-p60 and thereby axonal growth. USP47 was identified as a katanin-p60-specific deubiquitinating enzyme for its stabilization. We also identified CHIP as a ubiquitin E3 ligase that promotes proteasome-mediated degradation of katanin-p60. Moreover, USP47 promoted axonal growth of cultured rat hippocampal neurons, whereas CHIP inhibited it. Significantly, treatment with basic fibroblast growth factor (bFGF), an inducer of axonal growth, increased the levels of USP47 and katanin-p60, but not CHIP. Consistently, bFGF treatment resulted in a marked decrease in the level of ubiquitinated katanin-p60 and thereby in the promotion of axonal growth. On the other hand, the level of USP47, but not CHIP, decreased concurrently with that of katanin-p60 as axons reached their target cells. These results indicate that USP47 plays a crucial role in the control of axonal growth during neuronal development by antagonizing CHIP-mediated katanin-p60 degradation. PMID:23904609

  5. The characterization of a novel S100A1 binding site in the N-terminus of TRPM1.

    PubMed

    Jirku, Michaela; Lansky, Zdenek; Bednarova, Lucie; Sulc, Miroslav; Monincova, Lenka; Majer, Pavel; Vyklicky, Ladislav; Vondrasek, Jiri; Teisinger, Jan; Bousova, Kristyna

    2016-09-01

    Transient receptor potential melastatin-1 channel (TRPM1) is an important mediator of calcium influx into the cell that is expressed in melanoma and ON-bipolar cells. Similar to other members of the TRP channel family, the intracellular N- and C- terminal domains of TRPM1 are expected to play important roles in the modulation of TRPM1 receptor function. Among the most commonly occurring modulators of TRP channels are the cytoplasmically expressed calcium binding proteins calmodulin and S100 calcium-binding protein A1 (S100A1), but the interaction of TRPM1 with S100A1 has not been described yet. Here, using a combination of biophysical and bioinformatics methods, we have determined that the N-terminal L242-E344 region of TRPM1 is a S100A1 binding domain. We show that formation of the TRPM1/S100A1 complex is calcium-dependent. Moreover, our structural model of the complex explained data obtained from fluorescence spectroscopy measurements revealing that the complex formation is facilitated through interactions of clusters positively charged (K271A, R273A, R274A) and hydrophobic (L263A, V270A, L276A) residues at the N-terminus of TRPM1. Taken together, our data suggest a molecular mechanism for the potential regulation of TRPM1 by S100A1. PMID:27435061

  6. Regulation of Estrogen Receptor α N-Terminus Conformation and Function by Peptidyl Prolyl Isomerase Pin1

    PubMed Central

    Rajbhandari, Prashant; Finn, Greg; Solodin, Natalia M.; Singarapu, Kiran K.; Sahu, Sarata C.; Markley, John L.; Kadunc, Kelley J.; Ellison-Zelski, Stephanie J.; Kariagina, Anastasia; Haslam, Sandra Z.; Lu, Kun Ping

    2012-01-01

    Estrogen receptor alpha (ERα), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ERα AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifen-inducible activity of ERα and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ERα-positive rat mammary tumors. These results establish phosphorylation-coupled proline isomerization as a mechanism modulating AF1 functional activity and provide insight into the role of a conformational switch in the functional regulation of the intrinsically disordered transactivation domain of ERα. PMID:22064478

  7. Analysis of an origin of DNA replication located at the L terminus of the genome of pseudorabies virus.

    PubMed Central

    Kupershmidt, S; DeMarchi, J M; Lu, Z Q; Ben-Porat, T

    1991-01-01

    We have localized an origin of DNA replication at the L terminus of the pseudorabies virus genome. This origin differs in location as well as in general structure from the origins of replication of other herpesviruses that have been identified. The 600 leftmost nucleotides of the genome that were found to include origin function have been analyzed. This sequence is composed of an 82-bp palindrome whose center of symmetry is separated by 352 unique bp (UL2). Within the UL2, a sequence that fits the consensus sequence of the NF1 binding site, as well as one that has partial homology to the binding site of UL9 of herpes simplex virus, is present. Using truncated fragments of DNA, sequences essential for minimal origin function were delimited to within a fragment that includes the terminal 104 bp of the left end of the genome. Within these 104 bp, two elements essential to origin function have been identified. One of these elements is present within the terminal 64 bp of the L component (within one of the palindromic arms). The other is present within the 22 bp of the UL2 adjacent to this palindromic arm. Other auxiliary elements, although not essential for origin function, contribute to more efficient replication. The NF1 and UL9 binding site homologies were found to be nonessential to origin function. Images PMID:1656095

  8. Computational modeling of the N-terminus of the human dopamine transporter and its interaction with PIP2-containing membranes

    PubMed Central

    Khelashvili, George; Doktorova, Milka; Sahai, Michelle A.; Johner, Niklaus; Shi, Lei; Weinstein, Harel

    2015-01-01

    The dopamine transporter (DAT) is a transmembrane protein belonging to the family of Neurotransmitter:Sodium Symporters (NSS). Members of the NSS are responsible for the clearance of neurotransmitters from the synaptic cleft, and for their translocation back into the presynaptic nerve terminal. The DAT contains long intracellular N- and C-terminal domains that are strongly implicated in the transporter function. The N-terminus (N-term), in particular, regulates the reverse transport (efflux) of the substrate through DAT. Currently, the molecular mechanisms of the efflux remain elusive in large part due to lack of structural information on the N-terminal segment. Here we report a computational model of the N-term of the human DAT (hDAT), obtained through an ab initio structure prediction, in combination with extensive atomistic molecular dynamics (MD) simulations in the context of a lipid membrane. Our analysis reveals that whereas the N-term is a highly dynamic domain, it contains secondary structure elements that remain stable in the long MD trajectories of interactions with the bilayer (totaling >2.2 µs). Combining MD simulations with continuum mean-field modeling we found that the N-term engages with lipid membranes through electrostatic interactions with the charged lipids PIP2 (phosphatidylinositol 4,5-Biphosphate) or PS (phosphatidylserine) that are present in these bilayers. We identify specific motifs along the N-term implicated in such interactions and show that differential modes of N-term/membrane association result in differential positioning of the structured segments on the membrane surface. These results will inform future structure-based studies that will elucidate the mechanistic role of the N-term in DAT function. PMID:25739722

  9. Interactions of the C-terminus of lung surfactant protein B with lipid bilayers are modulated by acyl chain saturation.

    PubMed

    Antharam, Vijay C; Farver, R Suzanne; Kuznetsova, Anna; Sippel, Katherine H; Mills, Frank D; Elliott, Douglas W; Sternin, Edward; Long, Joanna R

    2008-11-01

    Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and (31)P and (2)H solid-state NMR spectroscopy. SP-B(59-80) forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B(59-80) in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B(59-80); in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B(59-80) penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL(4), a peptide mimetic of SP-B which was originally designed using SP-B(59-80) as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment. PMID:18694722

  10. Assessment of Vascular Geometry for Bilateral Carotid Artery Ligation to Induce Early Basilar Terminus Aneurysmal Remodeling in Rats.

    PubMed

    Tutino, Vincent Matthew; Liaw, Nicholas; Spernyak, Joseph Andrew; Ionita, Ciprian Nicolae; Siddiqui, Adnan Hussain; Kolega, John; Meng, Hui

    2016-01-01

    Bilateral common carotid artery (CCA) ligation in rabbits is a model for basilar terminus (BT) aneurysm formation. We asked if this model could be replicated in rats. Fourteen female Sprague Dawley rats underwent bilateral CCA ligation (n=8) or sham surgery (n=6). After 7 days, 5 ligated and 3 sham rats were euthanized for histological evaluation of BT aneurysm formation, while the remaining rats were imaged with magnetic resonance angiography, euthanized, and subjected to corrosion casting of the Circle of Willis (CoW). 3D micro computed tomography images of CoW casts were used for flow simulations at the rat BT, and electron micrographs of the casts were analyzed for aneurysmal and morphological changes. Results from these analyses were compared to rabbit model data (n=10 ligated and n=6 sham). Bilateral CCA ligation did not produce aneurysmal damage at the rat BT. While the surgical manipulation increased rat basilar artery flow, fluid dynamics simulations showed that the initial hemodynamic stress at the rat BT was significantly less than in rabbits. Rats also exhibited fewer morphological and pathological changes (minor changes only occurred in the posterior CoW) than rabbits, which had drastic changes throughout the CoW. A comparison of CoW anatomies demonstrated a greater number of branching arteries at the BT, larger CoW arteries in relation to basilar artery, and a steeper BT bifurcation angle in the rat. These differences could account for the lower hemodynamic stress at the BT and in the cerebrovasculature of the rat. In conclusion, bilateral CCA ligation in rats does not recapitulate the rabbit model of early flow-induced BT aneurysm. We suspect that the different CoW morphology of the rat lessens hemodynamic insults, thereby diminishing flow-induced aneurysmal remodeling. PMID:26503026

  11. A variant in the carboxyl-terminus of connexin 40 alters GAP junctions and increases risk for tetralogy of Fallot

    PubMed Central

    Guida, Valentina; Ferese, Rosangela; Rocchetti, Marcella; Bonetti, Monica; Sarkozy, Anna; Cecchetti, Serena; Gelmetti, Vania; Lepri, Francesca; Copetti, Massimiliano; Lamorte, Giuseppe; Cristina Digilio, Maria; Marino, Bruno; Zaza, Antonio; den Hertog, Jeroen; Dallapiccola, Bruno; De Luca, Alessandro

    2013-01-01

    GJA5 gene (MIM no. 121013), localized at 1q21.1, encodes for the cardiac gap junction protein connexin 40. In humans, copy number variants of chromosome 1q21.1 have been associated with variable phenotypes comprising congenital heart disease (CHD), including isolated TOF. In mice, the deletion of Gja5 can cause a variety of complex CHDs, in particular of the cardiac outflow tract, corresponding to TOF in many cases. In the present study, we screened for mutations in the GJA5 gene 178 unrelated probands with isolated TOF. A heterozygous nucleotide change (c.793C>T) in exon 2 of the gene leading to the p.Pro265Ser variant at the carboxyl-terminus of the protein was found in two unrelated sporadic patients, one with classic anatomy and one with pulmonary atresia. This GJA5 missense substitution was not observed in 1568 ethnically-matched control chromosomes. Immunofluorescent staining and confocal microscopy revealed that cells expressing the mutant protein form sparse or no visible gap-junction plaques in the region of cell–cell contact. Moreover, analysis of the transfer of the gap junction permanent tracer lucifer yellow showed that cells expressing the mutant protein have a reduced rate of dye transfer compared with wild-type cells. Finally, use of a zebrafish model revealed that microinjection of the GJA5-p.Pro265Ser mutant disrupts overall morphology of the heart tube in the 37% (22/60) of embryos, compared with the 6% (4/66) of the GJA5 wild-type-injected embryos. These findings implicate GJA5 gene as a novel susceptibility gene for TOF. PMID:22713807

  12. Crystal structures of tubulin acetyltransferase reveal a conserved catalytic core and the plasticity of the essential N terminus.

    PubMed

    Kormendi, Vasilisa; Szyk, Agnieszka; Piszczek, Grzegorz; Roll-Mecak, Antonina

    2012-12-01

    Tubulin acetyltransferase (TAT) acetylates Lys-40 of α-tubulin in the microtubule lumen. TAT is inefficient, and its activity is enhanced when tubulin is incorporated in microtubules. Acetylation is associated with stable microtubules and regulates the binding of microtubule motors and associated proteins. TAT is important in neuronal polarity and mechanosensation, and decreased tubulin acetylation levels are associated with axonal transport defects and neurodegeneration. We present the first structure of TAT in complex with acetyl-CoA (Ac-CoA) at 2.7 Å resolution. The structure reveals a conserved stable catalytic core shared with other GCN5 superfamily acetyltransferases consisting of a central β-sheet flanked by α-helices and a C-terminal β-hairpin unique to TAT. Structure-guided mutagenesis establishes the molecular determinants for Ac-CoA and tubulin substrate recognition. The wild-type TAT construct is a monomer in solution. We identify a metastable interface between the conserved core and N-terminal domain that modulates the oligomerization of TAT in solution and is essential for activity. The 2.45 Å resolution structure of an inactive TAT construct with an active site point mutation near this interface reveals a domain-swapped dimer in which the functionally essential N terminus shows evidence of marked structural plasticity. The sequence segment corresponding to this structurally plastic region in TAT has been implicated in substrate recognition in other GCN5 superfamily acetyltransferases. Our structures provide a rational platform for the mechanistic dissection of TAT activity and the design of TAT inhibitors with therapeutic potential in neuronal regeneration. PMID:23105108

  13. Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

    PubMed

    Fernández-García, Yaiza; Reguera, Juan; Busch, Carola; Witte, Gregor; Sánchez-Ramos, Oliberto; Betzel, Christian; Cusack, Stephen; Günther, Stephan; Reindl, Sophia

    2016-06-01

    Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening. PMID:27300328

  14. Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein

    PubMed Central

    Fernández-García, Yaiza; Reguera, Juan; Busch, Carola; Witte, Gregor; Sánchez-Ramos, Oliberto; Betzel, Christian; Cusack, Stephen; Günther, Stephan; Reindl, Sophia

    2016-01-01

    Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1–200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure–function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening. PMID:27300328

  15. Differential activation of yeast adenylyl cyclase by Ras1 and Ras2 depends on the conserved N terminus.

    PubMed

    Hurwitz, N; Segal, M; Marbach, I; Levitzki, A

    1995-11-21

    Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-RAS2 or RAS2-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts ras1 ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2 delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1 delta, in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 delta, Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements. PMID:7479926

  16. Effects of residue 5-point mutation and N-terminus hydrophobic residues on temporin-SHc physicochemical and biological properties.

    PubMed

    Abbassi, Feten; Piesse, Christophe; Foulon, Thierry; Nicolas, Pierre; Ladram, Ali

    2014-09-01

    Temporin-SHc (FLSHIAGFLSNLFamide) first isolated from skin extraction of the Tunisian frog Pelophylax saharica, which shows potent antimicrobial activity against Gram-positive bacteria and is highly active against yeasts and fungi without hemolytic activity at antimicrobial concentrations. The peptide adopts well-defined α-helical conformation when bound to SDS micelles. In this study, we explored the effects of residue at position 5 and the N-terminus hydrophobic character on the hydrophilic/polar face of temp-SHc, on its biological activities (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity and helicity). Antibacterial and hemolytic properties of temporin-SHc derivatives depend strongly on physicochemical properties. Therefore, slight decreasing amphipathicity together with hydrophobicity and helicity by the substitution Ile(5) → Leu decreased antimicrobial potency approximately twofold without changing of hemolytic activity. It is noteworthy that a conservative amino acid substitution decreases the antimicrobial activity, underlining the differences between Leu/Ile side chains insertion into the lipid bilayer. While the modification of N-terminal hydrophobic character by four residue inversion decreased amphipathicity (twofold) of (4-1)L5temp-SHc and resulted in an increase in antibacterial activity against E. coli, E. faecalis and C. parapsilosis of at least fourfold, its therapeutic potential is limited by its drastic increase of hemolysis (LC₅₀ = 2 μM). We found that the percentage of helicity of temp-SHc analog is directly correlated to its hemolytic activity. Last, the hydrophobic N-terminal character is an important determinant of antimicrobial activity. PMID:24842084

  17. Structural Studies of the Nedd4 WW Domains and Their Selectivity for the Connexin43 (Cx43) Carboxyl Terminus.

    PubMed

    Spagnol, Gaelle; Kieken, Fabien; Kopanic, Jennifer L; Li, Hanjun; Zach, Sydney; Stauch, Kelly L; Grosely, Rosslyn; Sorgen, Paul L

    2016-04-01

    Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) was the first ubiquitin protein ligase identified to interact with connexin43 (Cx43), and its suppressed expression results in accumulation of gap junction plaques at the plasma membrane. Nedd4-mediated ubiquitination of Cx43 is required to recruit Eps15 and target Cx43 to the endocytic pathway. Although the Cx43 residues that undergo ubiquitination are still unknown, in this study we address other unresolved questions pertaining to the molecular mechanisms mediating the direct interaction between Nedd4 (WW1-3 domains) and Cx43 (carboxyl terminus (CT)). All three WW domains display a similar three antiparallel β-strand structure and interact with the same Cx43CT(283)PPXY(286)sequence. Although Tyr(286)is essential for the interaction, MAPK phosphorylation of the preceding serine residues (Ser(P)(279)and Ser(P)(282)) increases the binding affinity by 2-fold for the WW domains (WW2 > WW3 ≫ WW1). The structure of the WW2·Cx43CT(276-289)(Ser(P)(279), Ser(P)(282)) complex reveals that coordination of Ser(P)(282)with the end of β-strand 3 enables Ser(P)(279)to interact with the back face of β-strand 3 (Tyr(286)is on the front face) and loop 2, forming a horseshoe-shaped arrangement. The close sequence identity of WW2 with WW1 and WW3 residues that interact with the Cx43CT PPXY motif and Ser(P)(279)/Ser(P)(282)strongly suggests that the significantly lower binding affinity of WW1 is the result of a more rigid structure. This study presents the first structure illustrating how phosphorylation of the Cx43CT domain helps mediate the interaction with a molecular partner involved in gap junction regulation. PMID:26841867

  18. Structural and Functional Studies of the Rap1 C-Terminus Reveal Novel Separation-of-Function Mutants

    SciTech Connect

    Feeser, Elizabeth A.; Wolberger, Cynthia

    2010-02-19

    The yeast Rap1 protein plays an important role in transcriptional silencing and in telomere length homeostasis. Rap1 mediates silencing at the HM loci and at telomeres by recruiting the Sir3 and Sir4 proteins to chromatin via a Rap1 C-terminal domain, which also recruits the telomere length regulators, Rif1 and Rif2. We report the 1.85 {angstrom} resolution crystal structure of the Rap1 C-terminus, which adopts an all-helical fold with no structural homologues. The structure was used to engineer surface mutations in Rap1, and the effects of these mutations on silencing and telomere length regulation were assayed in vivo. Our surprising finding was that there is no overlap between mutations affecting mating-type and telomeric silencing, suggesting that Rap1 plays distinct roles in silencing at the silent mating-type loci and telomeres. We also found novel Rap1 phenotypes and new separation-of-function mutants, which provide new tools for studying Rap1 function. Yeast two-hybrid studies were used to determine how specific mutations affect recruitment of Sir3, Rif1, and Rif2. A comparison of the yeast two-hybrid and functional data reveals patterns of protein interactions that correlate with each Rap1 phenotype. We find that Sir3 interactions are important for telomeric silencing, but not mating type silencing, and that Rif1 and Rif2 interactions are important in different subsets of telomeric length mutants. Our results show that the role of Rap1 in silencing differs between the HM loci and the telomeres and offer insight into the interplay between HM silencing, telomeric silencing, and telomere length regulation. These findings suggest a model in which competition and multiple recruitment events modulate silencing and telomere length regulation.

  19. Computational modeling of the N-terminus of the human dopamine transporter and its interaction with PIP2 -containing membranes.

    PubMed

    Khelashvili, George; Doktorova, Milka; Sahai, Michelle A; Johner, Niklaus; Shi, Lei; Weinstein, Harel

    2015-05-01

    The dopamine transporter (DAT) is a transmembrane protein belonging to the family of neurotransmitter:sodium symporters (NSS). Members of the NSS are responsible for the clearance of neurotransmitters from the synaptic cleft, and for their translocation back into the presynaptic nerve terminal. The DAT contains long intracellular N- and C-terminal domains that are strongly implicated in the transporter function. The N-terminus (N-term), in particular, regulates the reverse transport (efflux) of the substrate through DAT. Currently, the molecular mechanisms of the efflux remain elusive in large part due to lack of structural information on the N-terminal segment. Here we report a computational model of the N-term of the human DAT (hDAT), obtained through an ab initio structure prediction, in combination with extensive atomistic molecular dynamics (MD) simulations in the context of a lipid membrane. Our analysis reveals that whereas the N-term is a highly dynamic domain, it contains secondary structure elements that remain stable in the long MD trajectories of interactions with the bilayer (totaling >2.2 μs). Combining MD simulations with continuum mean-field modeling we found that the N-term engages with lipid membranes through electrostatic interactions with the charged lipids PIP2 (phosphatidylinositol 4,5-Biphosphate) or PS (phosphatidylserine) that are present in these bilayers. We identify specific motifs along the N-term implicated in such interactions and show that differential modes of N-term/membrane association result in differential positioning of the structured segments on the membrane surface. These results will inform future structure-based studies that will elucidate the mechanistic role of the N-term in DAT function. PMID:25739722

  20. A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter*

    PubMed Central

    Koban, Florian; El-Kasaby, Ali; Häusler, Cornelia; Stockner, Thomas; Simbrunner, Benedikt M.; Sitte, Harald H.; Freissmuth, Michael; Sucic, Sonja

    2015-01-01

    The folding trajectory of solute carrier 6 (SLC6) family members is of interest because point mutations result in misfolding and thus cause clinically relevant phenotypes in people. Here we examined the contribution of the C terminus in supporting folding of the serotonin transporter (SERT; SLC6A4). Our working hypothesis posited that the amphipathic nature of the C-terminal α-helix (Thr603–Thr613) was important for folding of SERT. Accordingly, we disrupted the hydrophobic moment of the α-helix by replacing Phe604, Ile608, or Ile612 by Gln. The bulk of the resulting mutants SERT-F604Q, SERT-I608Q, and SERT-I612Q were retained in the endoplasmic reticulum, but their residual delivery to the cell surface still depended on SEC24C. This indicates that the amphipathic nature of the C-terminal α-helix was dispensable to endoplasmic reticulum export. The folding trajectory of SERT is thought to proceed through the inward facing conformation. Consistent with this conjecture, cell surface expression of the misfolded mutants was restored by (i) introducing second site suppressor mutations, which trap SERT in the inward facing state, or (ii) by the pharmacochaperone noribogaine, which binds to the inward facing conformation. Finally, mutation of Glu615 at the end of the C-terminal α-helix to Lys reduced surface expression of SERT-E615K. A charge reversal mutation in intracellular loop 1 restored surface expression of SERT-R152E/E615K to wild type levels. These observations support a mechanistic model where the C-terminal amphipathic helix is stabilized by an intramolecular salt bridge between residues Glu615 and Arg152. This interaction acts as a pivot in the conformational search associated with folding of SERT. PMID:25869136

  1. Interactions of the C-terminus of pulmonary surfactant B with lipid bilayers are modulated by acyl chain saturation

    PubMed Central

    Antharam, Vijay C.; Farver, R. Suzanne; Kuznetsova, Anna; Sippel, Katherine H.; Mills, Frank D.; Elliott, Douglas W.; Sternin, Edward; Long, Joanna R.

    2009-01-01

    Summary Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and 31P and 2H solid-state NMR spectroscopy. SP-B59-80 forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B59-80 in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B59-80; in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B59-80 penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL4, a peptide mimetic of SP-B which was originally designed using SP-B59-80 as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment. PMID:18694722

  2. Revisiting Mitochondrial pH with an Improved Algorithm for Calibration of the Ratiometric 5(6)-carboxy-SNARF-1 Probe Reveals Anticooperative Reaction with H+ Ions and Warrants Further Studies of Organellar pH

    PubMed Central

    Żurawik, Tomasz Michał; Pomorski, Adam; Belczyk-Ciesielska, Agnieszka; Goch, Grażyna; Niedźwiedzka, Katarzyna; Kucharczyk, Róża; Krężel, Artur; Bal, Wojciech

    2016-01-01

    Fluorescence measurements of pH and other analytes in the cell rely on accurate calibrations, but these have routinely used algorithms that inadequately describe the properties of indicators. Here, we have established a more accurate method for calibrating and analyzing data obtained using the ratiometric probe 5(6)-carboxy-SNARF-1. We tested the implications of novel approach to measurements of pH in yeast mitochondria, a compartment containing a small number of free H+ ions. Our findings demonstrate that 5(6)-carboxy-SNARF-1 interacts with H+ ions inside the mitochondria in an anticooperative manner (Hill coefficient n of 0.5) and the apparent pH inside the mitochondria is ~0.5 unit lower than had been generally assumed. This result, at odds with the current consensus on the mechanism of energy generation in the mitochondria, is in better agreement with theoretical considerations and warrants further studies of organellar pH. PMID:27557123

  3. Revisiting Mitochondrial pH with an Improved Algorithm for Calibration of the Ratiometric 5(6)-carboxy-SNARF-1 Probe Reveals Anticooperative Reaction with H+ Ions and Warrants Further Studies of Organellar pH.

    PubMed

    Żurawik, Tomasz Michał; Pomorski, Adam; Belczyk-Ciesielska, Agnieszka; Goch, Grażyna; Niedźwiedzka, Katarzyna; Kucharczyk, Róża; Krężel, Artur; Bal, Wojciech

    2016-01-01

    Fluorescence measurements of pH and other analytes in the cell rely on accurate calibrations, but these have routinely used algorithms that inadequately describe the properties of indicators. Here, we have established a more accurate method for calibrating and analyzing data obtained using the ratiometric probe 5(6)-carboxy-SNARF-1. We tested the implications of novel approach to measurements of pH in yeast mitochondria, a compartment containing a small number of free H+ ions. Our findings demonstrate that 5(6)-carboxy-SNARF-1 interacts with H+ ions inside the mitochondria in an anticooperative manner (Hill coefficient n of 0.5) and the apparent pH inside the mitochondria is ~0.5 unit lower than had been generally assumed. This result, at odds with the current consensus on the mechanism of energy generation in the mitochondria, is in better agreement with theoretical considerations and warrants further studies of organellar pH. PMID:27557123

  4. Mutations in the catalytic core or the C-terminus of murine leukemia virus (MLV) integrase disrupt virion infectivity and exert diverse effects on reverse transcription

    SciTech Connect

    Steinrigl, Adolf; Nosek, Dagmara; Ertl, Reinhard; Guenzburg, Walter H.; Salmons, Brian; Klein, Dieter . E-mail: dieter.klein@vu-wien.ac.at

    2007-05-25

    Understanding of the structures and functions of the retroviral integrase (IN), a key enzyme in the viral replication cycle, is essential for developing antiretroviral treatments and facilitating the development of safer gene therapy vehicles. Thus, four MLV IN-mutants were constructed in the context of a retroviral vector system, harbouring either a substitution in the catalytic centre, deletions in the C-terminus, or combinations of both modifications. IN-mutants were tested for their performance in different stages of the viral replication cycle: RNA-packaging; RT-activity; transient and stable infection efficiency; dynamics of reverse transcription and nuclear entry. All mutant vectors packaged viral RNA with wild-type efficiencies and displayed only slight reductions in RT-activity. Deletion of either the IN C-terminus alone, or in addition to part of the catalytic domain exerted contrasting effects on intracellular viral DNA levels, implying that IN influences reverse transcription in more than one direction.

  5. Evidence for a role of the (alpha)-tubulin C terminus in the regulation of cyclin B synthesis in developing oocytes.

    PubMed

    Vée, S; Lafanechère, L; Fisher, D; Wehland, J; Job, D; Picard, A

    2001-03-01

    Microinjected mAb YL1/2, an (alpha)-tubulin antibody specific for the tyrosinated form of the protein, blocks the cell cycle in developing oocytes. Here, we have investigated the mechanism involved in the mAb effect. Both developing starfish and Xenopus oocytes were injected with two different (alpha)-tubulin C terminus antibodies. The injected antibodies blocked cell entry into mitosis through specific inhibition of cyclin B synthesis. The antibody effect was independent of the presence or absence of polymerized microtubules and was mimicked by injected synthetic peptides corresponding to the tyrosinated (alpha)-tubulin C terminus, whereas peptides lacking the terminal tyrosine were ineffective. These results indicate that tyrosinated (alpha)-tubulin, or another protein sharing the same C-terminal epitope, is involved in specific regulation of cyclin B synthesis in developing oocytes. PMID:11181172

  6. A direct interaction between the RAG2 C terminus and the core histones is required for efficient V(D)J recombination.

    PubMed

    West, Kelly L; Singha, Netai C; De Ioannes, Pablo; Lacomis, Lynne; Erdjument-Bromage, Hediye; Tempst, Paul; Cortes, Patricia

    2005-08-01

    V(D)J recombination is a tightly controlled process of somatic recombination whose regulation is mediated in part by chromatin structure. Here, we report that RAG2 binds directly to the core histone proteins. The interaction with histones is observed in developing lymphocytes and within the RAG1/RAG2 recombinase complex in a manner that is dependent on the RAG2 C terminus. Amino acids within the plant homeo domain (PHD)-like domain as well as a conserved acidic stretch of the RAG2 C terminus that is considered to be a linker region are important for this interaction. Point mutations that disrupt the RAG2-histone association inhibit the efficiency of the V(D)J recombination reaction at the endogenous immunoglobulin locus, with the most dramatic effect in the V to DJ(H) rearrangement. PMID:16111638

  7. Potentiation of Neuronal Nicotinic Receptors by 17β-Estradiol: Roles of the Carboxy-Terminal and the Amino-Terminal Extracellular Domains

    PubMed Central

    Jin, Xiaochun; Steinbach, Joe Henry

    2015-01-01

    The endogenous steroid 17β-estradiol (βEST) potentiates activation of neuronal nicotinic receptors containing α4 subunits. Previous work has shown that the final 4 residues of the α4 subunit are required for potentiation. However, receptors containing the α2 subunit are not potentiated although it has these 4 residues, and only one amino acid difference in the C-terminal tail (FLAGMI vs. WLAGMI). Previous work had indicated that the tryptophan residue was involved in binding an analog of βEST, but not in potentiation by βEST. To determine the structural basis for the loss of potentiation we analyzed data from chimeric subunits, which indicated that the major factor underlying the difference between α2 and α4 is the tryptophan/phenylalanine difference, while the N-terminal extracellular domain is a less significant factor. When the tryptophan in α4 was mutated, both phenylalanine and tyrosine conferred lower potentiation while lysine and leucine did not. The reduction reflected a reduced maximal magnitude of potentiation, indicating that the tryptophan is involved in transduction of steroid effects. The regions of the α4 N-terminal extracellular domain involved in potentiation lie near the agonist-binding pocket, rather than close to the membrane or the C-terminal tail, and appear to be involved in transduction rather than binding. These observations indicate that the C-terminal region is involved in both steroid binding (AGMI residues) and transduction (W). The role of the N-terminus appears to be independent of the C-terminal tryptophan and likely reflects an influence on conformational changes caused during channel activation by agonist and potentiation by estradiol. PMID:26684647

  8. The N Terminus of Type III Secretion Needle Protein YscF from Yersinia pestis Functions To Modulate Innate Immune Responses

    PubMed Central

    Osei-Owusu, Patrick; Jessen Condry, Danielle L.; Toosky, Melody; Roughead, William; Bradley, David S.

    2015-01-01

    The type III secretion system is employed by many pathogens, including the genera Yersinia, Shigella, Pseudomonas, and Salmonella, to deliver effector proteins into eukaryotic cells. The injectisome needle is formed by the polymerization of a single protein, e.g., YscF (Yersinia pestis), PscF (Pseudomonas aeruginosa), PrgI (Salmonella enterica SPI-1), SsaG (Salmonella enterica SPI-2), or MxiH (Shigella flexneri). In this study, we demonstrated that the N termini of some needle proteins, particularly the N terminus of YscF from Yersinia pestis, influences host immune responses. The N termini of several needle proteins were truncated and tested for the ability to induce inflammatory responses in a human monocytic cell line (THP-1 cells). Truncated needle proteins induced proinflammatory cytokines to different magnitudes than the corresponding wild-type proteins, except SsaG. Notably, N-terminally truncated YscF induced significantly higher activation of NF-κB and/or AP-1 and higher induction of proinflammatory cytokines, suggesting that a function of the N terminus of YscF is interference with host sensing of YscF, consistent with Y. pestis pathogenesis. To directly test the ability of the N terminus of YscF to suppress cytokine induction, a YscF-SsaG chimera with 15 N-terminal amino acids from YscF added to SsaG was constructed. The chimeric YscF-SsaG induced lower levels of cytokines than wild-type SsaG. However, the addition of 15 random amino acids to SsaG had no effect on NF-κB/AP-1 activation. These results suggest that the N terminus of YscF can function to decrease cytokine induction, perhaps contributing to a favorable immune environment leading to survival of Y. pestis within the eukaryotic host. PMID:25644012

  9. Determinant for β-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: An additional transmembrane region at the N terminus

    PubMed Central

    Wallner, Martin; Meera, Pratap; Toro, Ligia

    1996-01-01

    The pore-forming α subunit of large conductance voltage- and Ca2+-sensitive K (MaxiK) channels is regulated by a β subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming α subunit necessary for β-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to β-subunit modulation, and analyzed the topology of the α subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel α subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1–S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation experiments indicate that S0 leads to an exoplasmic N terminus. Therefore, we propose a new model where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homologue transfers β-subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function. PMID:8962157

  10. Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca(2+)-sensitive K+ channels: an additional transmembrane region at the N terminus.

    PubMed

    Wallner, M; Meera, P; Toro, L

    1996-12-10

    The pore-forming alpha subunit of large conductance voltage- and Ca(2+)-sensitive K (MaxiK) channels is regulated by a beta subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming alpha subunit necessary for beta-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to beta-subunit modulation, and analyzed the topology of the alpha subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel alpha subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1-S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation experiments indicate that S0 leads to an exoplasmic N terminus. Therefore, we propose a new model where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homologue transfers beta-subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function. PMID:8962157

  11. In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation

    PubMed Central

    Gao, Weiping; Liu, Wenge; Christensen, Trine; Zalutsky, Michael R.; Chilkoti, Ashutosh

    2010-01-01

    This paper reports a general in situ method to grow a polymer conjugate solely from the C terminus of a recombinant protein. GFP was fused at its C terminus with an intein; cleavage of the intein provided a unique thioester moiety at the C terminus of GFP that was used to install an atom transfer radical polymerization (ATRP) initiator. Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) yielded a site-specific (C-terminal) and stoichiometric conjugate with high yield and good retention of protein activity. A GFP-C-poly(OEGMA) conjugate (hydrodynamic radius (Rh): 21 nm) showed a 15-fold increase in its blood exposure compared to the protein (Rh: 3.0 nm) after intravenous administration to mice. This conjugate also showed a 50-fold increase in tumor accumulation, 24 h after intravenous administration to tumor-bearing mice, compared to the unmodified protein. This approach for in situ C-terminal polymer modification of a recombinant protein is applicable to a large subset of recombinant protein and peptide drugs and provides a general methodology for improvement of their pharmacological profiles. PMID:20810920

  12. Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation

    SciTech Connect

    Ambrose, R.L.; Mackenzie, J.M.

    2015-07-15

    The West Nile virus strain Kunjin virus (WNV{sub KUN}) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNV{sub KUN} replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNV{sub KUN} replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. - Highlights: • Mutation of Proline13 of the WNV NS4A protein is lethal to replication. • 1st TMB helix of NS4A contributes to protein stability and membrane remodelling. • Unstable mutants of NS4A can be rescued with a proteasome inhibitor. • This study (and of others) contributes to a functional mapping of the NS4A protein.

  13. Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

    PubMed

    Casal, J I; Langeveld, J P; Cortés, E; Schaaper, W W; van Dijk, E; Vela, C; Kamstrup, S; Meloen, R H

    1995-11-01

    The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine. PMID:7474152

  14. The N-terminus zinc finger domain of Xenopus SIP1 is important for neural induction, but not for suppression of Xbra expression.

    PubMed

    Nitta, Kazuhiro R; Takahashi, Shuji; Haramoto, Yoshikazu; Fukuda, Masakazu; Tanegashima, Kousuke; Onuma, Yasuko; Asashima, Makoto

    2007-01-01

    Smad-interacting protein-1 (SIP1), also known as deltaEF2, ZEB2 and zfhx1b, is essential for the formation of the neural tube and the somites. Overexpression of Xenopus SIP1 causes ectopic neural induction via inhibition of bone morphogenetic protein (BMP) signaling and inhibition of Xbra expression. Here, we report the functional analyses of 4 domain-deletion mutants of XSIP1. Deletion of the N-terminus zinc finger domain suppressed neural induction and BMP inhibition, but these were not affected by deletion of the other domains (the Smad binding domain, the DNA-binding homeodomain together with the CtBP binding site and the C-terminus zinc finger). Therefore SIP1 does not inhibit BMP signaling by binding to Smad proteins. In contrast, all of the deletion constructs inhibited Xbra expression. These results suggest that the N-terminus zinc finger domain of XSIP1 has an important role in neural induction and that Xbra suppression occurs via a mechanism separate from the neural inducing activity. PMID:17554684

  15. Cross-functional E3 ligases Parkin and C-terminus Hsp70-interacting protein in neurodegenerative disorders.

    PubMed

    Kumar, Pravir; Pradhan, Kaveri; Karunya, R; Ambasta, Rashmi K; Querfurth, Henry W

    2012-02-01

    The study of neurodegenerative disorders has had a major impact on our understanding of more fundamental mechanisms underlying neurobiology. Breakthroughs in the genetics of Alzheimer's (AD) and Parkinson's diseases (PD) has resulted in new knowledge in the areas of axonal transport, energy metabolism, protein trafficking/clearance and synaptic physiology. The major neurodegenerative diseases have in common a regional or network pathology associated with abnormal protein accumulation(s) and various degrees of motor or cognitive decline. In AD, β-amyloids are deposited in extracellular diffuse and compacted plaques as well as intracellularly. There is a major contribution to the disease by the co-existence of an intraneuronal tauopathy. Additionally, PD-like Lewy Bodies (LBs) bearing aggregated α-synuclein is present in 40-60% of all AD cases, especially involving amygdala. Amyloid deposits can be degraded or cleared by several mechanisms, including immune-mediated and transcytosis across the blood-brain barrier. Another avenue for disposal involves the lysosome pathway via autophagy. Enzymatic pathways include insulin degradative enzyme and neprilysin. Finally, the co-operative actions of C-terminus Hsp70 interacting protein (CHIP) and Parkin, components of a multiprotein E3 ubiquitin ligase complex, may be a portal to proteasome-mediated degradation. Mutations in the Parkin gene are the most common genetic link to autosomal recessive Parkinson's disease. Parkin catalyzes the post-translational modification of proteins with polyubiquitin, targeting them to the 26S proteasome. Parkin reduces intracellular Aβ(1-42) peptide levels, counteracts its effects on cell death, and reverses its effect to inhibit the proteasome. Additionally, Parkin has intrinsic cytoprotective activity to promote proteasome function and defend against oxidative stress to mitochondria. Parkin and CHIP are also active in amyloid clearance and cytoprotection in vivo. Parkin has cross

  16. Separation of actinides(III) from lanthanides(III) by extraction chromatography using new n,n'-dialkyl-n,n'-diphenyl-pyridine-2,6-di-carboxy-amides

    SciTech Connect

    Arisaka, Makoto; Watanabe, Masayuki; Kimura, Takaumi

    2007-07-01

    Four N,N'-dialkyl-N,N'-diphenyl-pyridine-2,6- di-carboxy-amides (R-PDA; R butyl, octyl, decyl, dodecyl) were newly synthesized and were applied to extraction chromatography as extractant to attain the separation of actinides(III) from high level radioactive waste containing lanthanides(III). R-PDA was successfully impregnated into XAD-4 resin. It was found that (i) the leakage of R-PDA from XAD-4 resin was suppressed with an increase of the length of the alkyl groups in R-PDA, while the leakage for each adsorbent resin was promoted with an increase of HNO{sub 3} concentration in the aqueous phase and (ii) Oc-PDA or De-PDA/XAD-4 resin exhibits moderate separation ability of actinides(III) from lanthanides(III) at relatively high HNO{sub 3} concentration. (authors)

  17. fac-Aqua­(2-carboxy­ethyl-κ2 C,O)trichlorido­tin(IV)–1,4,7,10,13-penta­oxacyclo­penta­deca­ne–water (1/1/2)

    PubMed Central

    Tiekink, Edward R. T.; Wardell, James L.; Wardell, Solange M. S. V.

    2010-01-01

    In the title compound, [Sn(C3H5O2)Cl3(H2O)]·C10H20O5·2H2O, the SnIV atom is octa­hedrally coordinated within a fac-CO2Cl3 donor set, arising from the C,O-bidentate carboxy­ethyl ligand, a water mol­ecule and three chloride ligands. In the crystal, supra­molecular chains linked by O—H⋯O hydrogen bonds propagate along the c axis These chains are connected into layers in the ac plane via C—H⋯O inter­actions. PMID:21578998

  18. Crystallization and preliminary X-ray crystallographic analysis of the Escherichia coli outer membrane cobalamin transporter BtuB in complex with the carboxy-terminal domain of TonB

    SciTech Connect

    Shultis, David D.; Purdy, Michael D.; Banchs, Christian N.; Wiener, Michael C.

    2006-07-01

    Crystals of a complex of the E. coli proteins BtuB (outer membrane cobalamin transporter) and TonB (carboxy-terminal domain) diffracting to 2.1 Å resolution have been obtained. The energy-dependent uptake of organometallic compounds and other micronutrients across the outer membranes of Gram-negative bacteria is carried out by outer membrane active-transport proteins that utilize the proton-motive force of the inner membrane via coupling to the TonB protein. The Escherichia coli outer membrane cobalamin transporter BtuB and a carboxy-terminal domain of the TonB protein, residues 147–239 of the wild-type protein, were expressed and purified individually. A complex of BtuB and TonB{sup 147–239} was formed in the presence of the substrate cyanocobalamin (CN-Cbl; vitamin B{sub 12}) and calcium and was crystallized. BtuB was purified in the detergent LDAO (n-dodecyl-N,N-dimethylamine-N-oxide) and the complex was formed in a detergent mixture of LDAO and C{sub 8}E{sub 4} (tetraethylene glycol monooctylether). Crystals were obtained by sitting-drop vapor diffusion, with the reservoir containing 30%(v/v) polyethylene glycol (PEG 300) and 100 mM sodium acetate pH 5.2. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1} (unit-cell parameters a = 74.3, b = 82.4, c = 122.6 Å). The asymmetric unit consists of a single BtuB–TonB complex. Data sets have been collected to 2.1 Å resolution at a synchrotron beamline (APS SER-CAT 22-ID)

  19. Investigating the Cause of the 2012 Acceleration of Jakobshavn Isbræ, Greenland Using High Resolution Observations of the Glacier Terminus

    NASA Astrophysics Data System (ADS)

    Cassotto, R.; Fahnestock, M. A.; Boettcher, M. S.; Amundson, J. M.; Truffer, M.

    2014-12-01

    After decades of relative stability, Jakobshavn Isbræ, a tidewater glacier in West Greenland, started to destabilize at the turn of the century. The glacier thinned, the perennial tongue disintegrated, velocities doubled, and the terminus retreated. The glacier evolved over the next several years as it showed large seasonal variations in speed and a progressive kilometer-scale retreat of its calving front. Then, during the 2012 summer, Jakobshavn set a new record when its speed increased to rates more than four times the 1990s values, and consequently became the fastest glacier recorded by satellite yet. A 2-week field study was conducted along the terminus at that time; ground portable radar interferometers (GPRI), time-lapse cameras, GPS, and a tide gauge were deployed to characterize glacier dynamics along the ice-ocean boundary. We use >10,000 interferograms recorded with the terrestrial interferometers to probe the cause of this acceleration. We observe a 33% increase in glacier speed and a 250% increase in the amplitude of response to tidal forcing during our study period. We explore how the location of the terminus along the reverse bed slope contributed to the observed speedup, and we compare our findings with the long-term record of satellite observations. Our data show that understanding tidewater glacier dynamics requires knowledge of short-term variations along glacier termini that is currently not available from satellites. This study provides insight into such short-term dynamics on spatial scales comparable to satellite InSAR but with temporal resolution similar to GPS.

  20. Alternative splicing at C terminus of Ca(V)1.4 calcium channel modulates calcium-dependent inactivation, activation potential, and current density.

    PubMed

    Tan, Gregory Ming Yeong; Yu, Dejie; Wang, Juejin; Soong, Tuck Wah

    2012-01-01

    The Ca(V)1.4 voltage-gated calcium channel is predominantly expressed in the retina, and mutations to this channel have been associated with human congenital stationary night blindness type-2. The L-type Ca(V)1.4 channel displays distinct properties such as absence of calcium-dependent inactivation (CDI) and slow voltage-dependent inactivation (VDI) due to the presence of an autoinhibitory domain (inhibitor of CDI) in the distal C terminus. We hypothesized that native Ca(V)1.4 is subjected to extensive alternative splicing, much like the other voltage-gated calcium channels, and employed the transcript scanning method to identify alternatively spliced exons within the Ca(V)1.4 transcripts isolated from the human retina. In total, we identified 19 alternative splice variations, of which 16 variations have not been previously reported. Characterization of the C terminus alternatively spliced exons using whole-cell patch clamp electrophysiology revealed a splice variant that exhibits robust CDI. This splice variant arose from the splicing of a novel alternate exon (43*) that can be found in 13.6% of the full-length transcripts screened. Inclusion of exon 43* inserts a stop codon that truncates half the C terminus. The Ca(V)1.4 43* channel exhibited robust CDI, a larger current density, a hyperpolarized shift in activation potential by ∼10 mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible for modulating channel activation and VDI, in addition to CDI. Calcium currents in the photoreceptors were observed to exhibit CDI and are more negatively activated as compared with currents elicited from heterologously expressed full-length Ca(V)1.4. Naturally occurring alternative splice variants may in part contribute to the properties of the native Ca(V)1.4 channels. PMID:22069316

  1. The cytosolic C-terminus of the glucose transporter GLUT4 contains an acidic cluster endosomal targeting motif distal to the dileucine signal.

    PubMed Central

    Shewan, A M; Marsh, B J; Melvin, D R; Martin, S; Gould, G W; James, D E

    2000-01-01

    The insulin-responsive glucose transporter GLUT4 is targeted to a post-endocytic compartment in adipocytes, from where it moves to the cell surface in response to insulin. Previous studies have identified two cytosolic targeting motifs that regulate the intracellular sequestration of this protein: FQQI(5-8) in the N-terminus and LL(489,490) (one-letter amino acid notation) in the C-terminus. In the present study we show that a GLUT4 chimaera in which the C-terminal 12 amino acids in GLUT4 have been replaced with the same region from human GLUT3 is constitutively targeted to the plasma membrane when expressed in 3T3-L1 adipocytes. To further dissect this domain it was divided into three regions, each of which was mutated en bloc to alanine residues. Analysis of these constructs revealed that the targeting information is contained within the residues TELEYLGP(498-505). Using the transferrin-horseradish peroxidase endosomal ablation technique in 3T3-L1 adipocytes, we show that mutants in which this C-terminal domain has been disrupted are more sensitive to chemical ablation than wild-type GLUT4. These data indicate that GLUT4 contains a targeting signal in its C-terminus, distal to the dileucine motif, that regulates its sorting into a post-endosomal compartment. Similar membrane-distal, acidic-cluster-based motifs are found in the cytosolic tails of the insulin-responsive aminopeptidase IRAP (insulin-regulated aminopeptidase) and the proprotein convertase PC6B, indicating that this type of motif may play an important role in the endosomal sequestration of a number of different proteins. PMID:10926832

  2. TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA.

    PubMed

    Qin, Haina; Lim, Liang-Zhong; Wei, Yuanyuan; Song, Jianxing

    2014-12-30

    Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) is the principal component of ubiquitinated inclusions characteristic of most forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia-frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), as well as an increasing spectrum of other neurodegenerative diseases. Previous structural and functional studies on TDP-43 have been mostly focused on its recognized domains. Very recently, however, its extreme N terminus was identified to be a double-edged sword indispensable for both physiology and proteinopathy, but thus far its structure remains unknown due to the severe aggregation. Here as facilitated by our previous discovery that protein aggregation can be significantly minimized by reducing salt concentrations, by circular dichroism and NMR spectroscopy we revealed that the TDP-43 N terminus encodes a well-folded structure in concentration-dependent equilibrium with its unfolded form. Despite previous failure in detecting any sequence homology to ubiquitin, the folded state was determined to adopt a novel ubiquitin-like fold by the CS-Rosetta program with NMR chemical shifts and 78 unambiguous long-range nuclear Overhauser effect (NOE) constraints. Remarkably, this ubiquitin-like fold could bind ssDNA, and the binding shifted the conformational equilibrium toward reducing the unfolded population. To the best of our knowledge, the TDP-43 N terminus represents the first ubiquitin-like fold capable of directly binding nucleic acid. Our results provide a molecular mechanism rationalizing the functional dichotomy of TDP-43 and might also shed light on the formation and dynamics of cellular ribonucleoprotein granules, which have been recently linked to ALS pathogenesis. As a consequence, one therapeutic strategy for TDP-43-causing diseases might be to stabilize its ubiquitin-like fold by ssDNA or designed molecules. PMID:25503365

  3. The heptad repeats region is essential for AcMNPV P10 filament formation and not the proline-rich or the C-terminus basic regions

    SciTech Connect

    Dong Chunsheng; Deng Fei; Li Dan; Wang Hualin; Hu Zhihong

    2007-09-01

    Baculovirus P10 protein is a small conserved protein and is expressed as bundles of filaments in the host cell during the late phase of virus infection. So far the published results on the domain responsible for filament structural formation have been contradictory. Electron microscopy revealed that the C-terminus basic region was involved in filament structural formation in the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) [van Oers, M.M., Flipsen, J.T., Reusken, C.B., Sliwinsky, E.L., Vlak, J.M., 1993. Functional domains of the p10 protein of Autographa californica nuclear polyhedorsis virus. J. Gen. Virol. 74, 563-574.]. While in the Helicoverpa armigera nucleopolyhedrovirus (HearNPV), the heptad repeats region but not the C-terminus domain was proven to be responsible for filament formation [Dong, C., Li, D., Long, G., Deng, F., Wang, H., Hu, Z., 2005. Identification of functional domains required for HearNPV P10 filament formation. Virology 338, 112-120.]. In this manuscript, fluorescence confocal microscopy was applied to study AcMNPV P10 filament formation. A set of plasmids containing different P10 structural domains fused with a fluorescent protein were constructed and transfected into Sf-9 cells. The data indicated that the heptad repeats region, but not the proline-rich region or the C-terminus basic region, is essential for AcMNPV P10 filament formation. Co-transfection of P10s tagged with different fluorescent revealed that P10s with defective heptad repeats region could not interact with intact heptad repeats region or even full-length P10s to form filament structure. Within the heptad repeats region, deletion of the three amino acids spacing of AcMNPV P10 appeared to have no significant impact on the formation of filament structures, but the content of the heptad repeats region appeared to play a role in the morphology of the filaments.

  4. The long N-terminus of the human monocarboxylate transporter 8 is a target of ubiquitin-dependent proteasomal degradation which regulates protein expression and oligomerization capacity.

    PubMed

    Zwanziger, Denise; Schmidt, Mathias; Fischer, Jana; Kleinau, Gunnar; Braun, Doreen; Schweizer, Ulrich; Moeller, Lars Christian; Biebermann, Heike; Fuehrer, Dagmar

    2016-10-15

    Monocarboxylate transporter 8 (MCT8) equilibrates thyroid hormones between the extra- and the intracellular sides. MCT8 exists either with a short or a long N-terminus, but potential functional differences between both variants are yet not known. We, therefore, generated MCT8 constructs which are different in N-terminal length: MCT8(1-613), MCT8(25-613), MCT8(49-613) and MCT8(75-613). The M75G substitution prevents translation of MCT8(75-613) and ensures expression of full-length MCT8 protein. The K56G substitution was made to prevent ubiquitinylation. Cell-surface expression, localization and proteasomal degradation were investigated using C-terminally GFP-tagged MCT8 constructs (HEK293 and MDCK1 cells) and oligomerization capacity was determined using N-terminally HA- and C-terminally FLAG-tagged MCT8 constructs (COS7 cells). MCT8(1-613)-GFP showed a lower protein expression than the shorter MCT8(75-613)-GFP protein. The proteasome inhibitor lactacystin increased MCT8(1-613)-GFP protein amount, suggesting proteasomal degradation of MCT8 with the long N-terminus. Ubiquitin conjugation of MCT8(1-613)-GFP was found by immuno-precipitation. A diminished ubiquitin conjugation caused by K56G substitution resulted in increased MCT8(1-613)-GFP protein expression. Sandwich ELISA was performed to investigate if the bands at higher molecular weight observed in Western blot analysis are due to MCT8 oligomerization, which was indeed shown. Our data imply a role of the long N-terminus of MCT8 as target of ubiquitin-dependent proteasomal degradation affecting MCT8 amount and subsequently oligomerization capacity. PMID:27222294

  5. Identification of adenovirus type 2 early region 1B proteins that share the same amino terminus as do the 495R and 155R proteins.

    PubMed Central

    Lewis, J B; Anderson, C W

    1987-01-01

    Adenovirus type 2 early region 1B (E1B) proteins synthesized in vitro were fractionated chromatographically and characterized by peptide and sequence analysis and by reaction with peptide-specific antisera targeted to either the N or C terminus of either of two overlapping E1B reading frames (175 or 495 codons). In addition to the previously identified E1B-495R, E1B-175R, and E1B-155R species, two other E1B proteins of similar electrophoretic mobility to the 175R protein were identified. E1B-82R is an abundant product in vitro and in vivo that has the same N terminus as that of the 495R and 155R proteins but a different C terminus. The structure of 82R is predicted by the structure of the abundant 13S (1.02-kilobase) E1B mRNA. E1B-168R is a novel minor species consisting of the 24 amino-terminal residues of the 495R protein fused to the entire polypeptide IX sequence. An additional, minor 16,000-molecular-weight polypeptide was detected that may correspond to a predicted 92R E1B protein, but definitive identification was not possible. These observations establish that the leftmost portion (78 codons) of the 495-codon reading frame, which overlaps the right half of the 175-codon reading frame, is expressed as an abundant protein that does not contain other 495R sequences. This region, which may participate in the regulation of region E1A expression, may thus constitute a functional domain distinct from the rightward portion of the 495R protein. Images PMID:2960832

  6. Barbiturates require the N terminus and first transmembrane domain of the delta subunit for enhancement of alpha1beta3delta GABAA receptor currents.

    PubMed

    Feng, Hua-Jun; Macdonald, Robert L

    2010-07-30

    GABA(A) receptors are composed predominantly of alphabetagamma receptors, which mediate primarily synaptic inhibition, and alphabetadelta receptors, which mediate primarily extrasynaptic inhibition. At saturating GABA concentrations, the barbiturate pentobarbital substantially increased the amplitude and desensitization of the alpha1beta3delta receptor but not the alpha1beta3gamma2L receptor currents. To explore the structural domains of the delta subunit that are involved in pentobarbital potentiation and increased desensitization of alpha1beta3delta currents, chimeric cDNAs were constructed by progressive replacement of gamma2L subunit sequence with a delta subunit sequence or a delta subunit sequence with a gamma2L subunit sequence, and HEK293T cells were co-transfected with alpha1 and beta3 subunits or alpha1 and beta3 subunits and a gamma2L, delta, or chimeric subunit. Currents evoked by a saturating concentration of GABA or by co-application of GABA and pentobarbital were recorded using the patch clamp technique. By comparing the extent of enhancement and changes in kinetic properties produced by pentobarbital among chimeric and wild type receptors, we concluded that although potentiation of alpha1beta3delta currents by pentobarbital required the delta subunit sequence from the N terminus to proline 241 in the first transmembrane domain (M1), increasing desensitization of alpha1beta3delta currents required a delta subunit sequence from the N terminus to isoleucine 235 in M1. These findings suggest that the delta subunit N terminus and N-terminal portion of the M1 domain are, at least in part, involved in transduction of the allosteric effect of pentobarbital to enhance alpha1beta3delta currents and that this effect involves a distinct but overlapping structural domain from that involved in altering desensitization. PMID:20525684

  7. Insight into the structural and biological relevance of the T/R transition of the N-terminus of the B-chain in human insulin.

    PubMed

    Kosinová, Lucie; Veverka, Václav; Novotná, Pavlína; Collinsová, Michaela; Urbanová, Marie; Moody, Nicholas R; Turkenburg, Johan P; Jiráček, Jiří; Brzozowski, Andrzej M; Žáková, Lenka

    2014-06-01

    The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin-insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1-B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the "classical" T-state and that a substantial flexibility of the B1-B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin-IR interaction. PMID:24819248

  8. Insight into the Structural and Biological Relevance of the T/R Transition of the N-Terminus of the B-Chain in Human Insulin

    PubMed Central

    2014-01-01

    The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin–insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1–B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the “classical” T-state and that a substantial flexibility of the B1–B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin–IR interaction. PMID:24819248

  9. Anionic Phospholipid Interactions of the Prion Protein N Terminus Are Minimally Perturbing and Not Driven Solely by the Octapeptide Repeat Domain*

    PubMed Central

    Boland, Martin P.; Hatty, Claire R.; Separovic, Frances; Hill, Andrew F.; Tew, Deborah J.; Barnham, Kevin J.; Haigh, Cathryn L.; James, Michael; Masters, Colin L.; Collins, Steven J.

    2010-01-01

    Although the N terminus of the prion protein (PrPC) has been shown to directly associate with lipid membranes, the precise determinants, biophysical basis, and functional implications of such binding, particularly in relation to endogenously occurring fragments, are unresolved. To better understand these issues, we studied a range of synthetic peptides: specifically those equating to the N1 (residues 23–110) and N2 (23–89) fragments derived from constitutive processing of PrPC and including those representing arbitrarily defined component domains of the N terminus of mouse prion protein. Utilizing more physiologically relevant large unilamellar vesicles, fluorescence studies at synaptosomal pH (7.4) showed absent binding of all peptides to lipids containing the zwitterionic headgroup phosphatidylcholine and mixtures containing the anionic headgroups phosphatidylglycerol or phosphatidylserine. At pH 5, typical of early endosomes, quartz crystal microbalance with dissipation showed the highest affinity binding occurred with N1 and N2, selective for anionic lipid species. Of particular note, the absence of binding by individual peptides representing component domains underscored the importance of the combination of the octapeptide repeat and the N-terminal polybasic regions for effective membrane interaction. In addition, using quartz crystal microbalance with dissipation and solid-state NMR, we characterized for the first time that both N1 and N2 deeply insert into the lipid bilayer with minimal disruption. Potential functional implications related to cellular stress responses are discussed. PMID:20679345

  10. Specificity of Baculovirus P6.9 Basic DNA-Binding Proteins and Critical Role of the C Terminus in Virion Formation▿ †

    PubMed Central

    Wang, Manli; Tuladhar, Era; Shen, Shu; Wang, Hualin; van Oers, Monique M.; Vlak, Just M.; Westenberg, Marcel

    2010-01-01

    The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 binds to DNA in a non-sequence-specific manner. By using a p6.9-null mutant of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), we demonstrate that P6.9 is not required for viral DNA replication but is essential for the production of infectious virus. Virion production was rescued by P6.9 homologs from a number of Alphabaculovirus species and one Gammabaculovirus species but not from the genus Betabaculovirus, comprising the granuloviruses, or by the P6.9 homolog VP15 from the unrelated white spot syndrome virus of shrimp. Mutational analyses demonstrated that AcMNPV P6.9 with a conserved 11-residue deletion of the C terminus was not capable of rescuing p6.9-null AcMNPV, while a chimeric Betabaculovirus P6.9 containing the P6.9 C-terminal region of an Alphabaculovirus strain was able to do so. This implies that the C terminus of baculovirus P6.9 contains sequence elements essential for virion formation. Such elements may possibly interact with species- or genus-specific domains of other nucleocapsid proteins during virus assembly. PMID:20519380

  11. Actinin-1 binds to the C-terminus of A2B adenosine receptor (A2BAR) and enhances A2BAR cell-surface expression.

    PubMed

    Sun, Ying; Hu, Wenbao; Yu, Xiaojie; Liu, Zhengzhao; Tarran, Robert; Ravid, Katya; Huang, Pingbo

    2016-07-15

    A2BAR (A2B adenosine receptor) has been implicated in several physiological conditions, such as allergic or inflammatory disorders, vasodilation, cell growth and epithelial electrolyte secretion. For mediating the protein-protein interactions of A2BAR, the receptor's C-terminus is recognized to be crucial. In the present study, we unexpectedly found that two point mutations in the A2BAR C-terminus (F297A and R298A) drastically impaired the expression of A2BAR protein by accelerating its degradation. Thus we tested the hypothesis that these two point mutations disrupt A2BAR's interaction with a protein essential for A2BAR stability. Our results show that both mutations disrupted the interaction of A2BAR with actinin-1, an actin-associated protein. Furthermore, actinin-1 binding stabilized the global and cell-surface expression of A2BAR. By contrast, actinin-4, another non-muscle actinin isoform, did not bind to A2BAR. Thus our findings reveal a previously unidentified regulatory mechanism of A2BAR abundance. PMID:27208173

  12. Identification of specific sites in the third intracellular loop and carboxyl terminus of the Bombyx mori pheromone biosynthesis activating neuropeptide receptor crucial for ligand-induced internalization.

    PubMed

    Hull, J J; Lee, J M; Matsumoto, S

    2011-12-01

    Sex pheromone production in most moths is mediated by the pheromone biosynthesis activating neuropeptide receptor (PBANR). Using fluorescent Bombyx mori PBANR (BmPBANR) chimeras to study PBANR regulation, we previously showed that BmPBANR undergoes rapid ligand-induced internalization, that the endocytotic motif resides between residues 358-367 of the BmPBANR C terminus, and that the internalization pathway is clathrin-dependent. Here, we sought to expand our understanding of the molecular mechanisms underlying BmPBANR function and regulation by transiently expressing a series of fluorescent BmPBANR chimeric constructs in cultured Spodoptera frugiperda (Sf9) cells and assaying for internalization of a fluorescently labelled ligand. Pharmacological inhibition of phospholipase C significantly reduced internalization, suggesting that BmPBANR regulation proceeds via a conventional G-protein-dependent pathway. This was further supported by impaired internalization following site-directed mutagenesis of R263 and R264, two basic residues at the transmembrane 6 intracellular junction that are thought to stabilize G-protein coupling via electrostatic interactions. Ala substitution of S333 and S366, two consensus protein kinase C sites in the C terminus, likewise impaired internalization, as did RNA interference-mediated knockdown of Sf9 protein kinase C. N-terminal truncations of BmPBANR indicate that the first 27 residues are not necessary for cell surface trafficking or receptor functionality. PMID:21955122

  13. The Golgi-Localized Arabidopsis Endomembrane Protein12 Contains Both Endoplasmic Reticulum Export and Golgi Retention Signals at Its C Terminus[C][W

    PubMed Central

    Gao, Caiji; Yu, Christine K.Y.; Qu, Song; San, Melody Wan Yan; Li, Kwun Yee; Lo, Sze Wan; Jiang, Liwen

    2012-01-01

    Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells. PMID:22570441

  14. A Lysine at the C-Terminus of an Odorant-Binding Protein is Involved in Binding Aldehyde Pheromone Components in Two Helicoverpa Species

    PubMed Central

    Sun, Ya-Lan; Huang, Ling-Qiao; Pelosi, Paolo; Wang, Chen-Zhu

    2013-01-01

    Odorant-binding proteins (OBPs) are soluble proteins, whose role in olfaction of insects is being recognized as more and more important. We have cloned, expressed and purified an OBP (HarmOBP7) from the antennae of the moth Helicoverpa armigera. Western blot experiments indicate specific expression of this protein in the antennae of adults. HarmOBP7 binds both pheromone components Z-11-hexadecenal and Z-9-hexadecenal with good affinity. We have also performed a series of binding experiments with linear aldehydes, alcohols and esters, as well as with other compounds and found a requirement of medium size for best affinity. The affinity of OBP7, as well as that of a mutant lacking the last 6 residues does not substantially decrease in acidic conditions, but increases at basic pH values with no significant differences between wild-type and mutant. Binding to both pheromone components, instead, is negatively affected by the lack of the C-terminus. A second mutant, where one of the three lysine residues in the C-terminus (Lys123) was replaced by methionine showed reduced affinity to both pheromone components, as well as to their analogues, thus indicating that Lys123 is involved in binding these compounds, likely forming hydrogen bonds with the functional groups of the ligands. PMID:23372826

  15. Exploring the structure of the 100 amino-acid residue long N-terminus of the plant antenna protein CP29.

    PubMed

    Shabestari, Maryam Hashemi; Wolfs, Cor J A M; Spruijt, Ruud B; van Amerongen, Herbert; Huber, Martina

    2014-03-18

    The structure of the unusually long (∼100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows α-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10-22 and 82-91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane. PMID:24655510

  16. Exploring the Structure of the 100 Amino-Acid Residue Long N-Terminus of the Plant Antenna Protein CP29

    PubMed Central

    Shabestari, Maryam Hashemi; Wolfs, Cor J.A.M.; Spruijt, Ruud B.; van Amerongen, Herbert; Huber, Martina

    2014-01-01

    The structure of the unusually long (∼100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows α-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10–22 and 82–91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane. PMID:24655510

  17. Allosteric coupling between proximal C-terminus and selectivity filter is facilitated by the movement of transmembrane segment 4 in TREK-2 channel

    PubMed Central

    Zhuo, Ren-Gong; Peng, Peng; Liu, Xiao-Yan; Yan, Hai-Tao; Xu, Jiang-Ping; Zheng, Jian-Quan; Wei, Xiao-Li; Ma, Xiao-Yun

    2016-01-01

    TREK-2, a member of two-pore-domain potassium channel family, regulates cellular excitability in response to diverse stimuli. However, how such stimuli control channel function remains unclear. Here, by characterizing the responses of cytosolic proximal C-terminus deletant (ΔpCt) and transmembrane segment 4 (M4)-glycine hinge mutant (G312A) to 2-Aminoethoxydiphenyl borate (2-APB), an activator of TREK-2, we show that the transduction initiated from pCt domain is allosterically coupled with the conformation of selectivity filter (SF) via the movements of M4, without depending on the original status of SF. Moreover, ΔpCt and G312A also exhibited blunted responses to extracellular alkalization, a model to induce SF conformational transition. These results suggest that the coupling between pCt domain and SF is bidirectional, and M4 movements are involved in both processes. Further mechanistic exploration reveals that the function of Phe316, a residue close to the C-terminus of M4, is associated with such communications. However, unlike TREK-2, M4-hinge of TREK-1 only controls the transmission from pCt to SF, rather than SF conformational changes triggered by pHo changes. Together, our findings uncover the unique gating properties of TREK-2, and elucidate the mechanisms for how the extracellular and intracellular stimuli harness the pore gating allosterically. PMID:26879043

  18. Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage–modified chromatin

    PubMed Central

    Melander, Fredrik; Bekker-Jensen, Simon; Falck, Jacob; Bartek, Jiri; Mailand, Niels; Lukas, Jiri

    2008-01-01

    DNA double-strand breaks (DSBs) trigger accumulation of the MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex, whose retention on the DSB-flanking chromatin facilitates survival. Chromatin retention of MRN requires the MDC1 adaptor protein, but the mechanism behind the MRN–MDC1 interaction is unknown. We show that the NBS1 subunit of MRN interacts with the MDC1 N terminus enriched in Ser-Asp-Thr (SDT) repeats. This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1. Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1–NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells. Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs. Together, these data suggest that phosphorylation of the SDT repeats in the MDC1 N terminus functions to recruit NBS1 and, thereby, increases the local concentration of MRN at the sites of chromosomal breakage. PMID:18411307

  19. Heat stability of maize endosperm ADP-glucose pyrophosphorylase is enhanced by insertion of a cysteine in the N terminus of the small subunit.

    PubMed

    Linebarger, Carla R Lyerly; Boehlein, Susan K; Sewell, Aileen K; Shaw, Janine; Hannah, L Curtis

    2005-12-01

    ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis. However, plant AGPases differ in several parameters, including spatial and temporal expression, allosteric regulation, and heat stability. AGPases of cereal endosperms are heat labile, while those in other tissues, such as the potato (Solanum tuberosum) tuber, are heat stable. Sequence comparisons of heat-stable and heat-labile AGPases identified an N-terminal motif unique to heat-stable enzymes. Insertion of this motif into recombinant maize (Zea mays) endosperm AGPase increased the half-life at 58 degrees C more than 70-fold. Km values for physiological substrates were unaffected, although Kcat was doubled. A cysteine within the inserted motif gives rise to small subunit homodimers not found in the wild-type maize enzyme. Placement of this N-terminal motif into a mosaic small subunit containing the N terminus from maize endosperm and the C terminus from potato tuber AGPase increases heat stability more than 300-fold. PMID:16299180

  20. Three-dimensional reconstruction of a co-complex of F-actin with antibody Fab fragments to actin's NH2 terminus.

    PubMed Central

    Orlova, A; Yu, X; Egelman, E H

    1994-01-01

    We have decorated F-actin with Fab fragments of antibodies to actin residues 1-7. These antibody fragments do not strongly affect the rigor binding of myosin S-1 to actin, but do affect the binding of S-1 to actin in the presence of nucleotide (DasGupta, G., and E. Reisler, 1989. J. Mol. Biol. 207:833-836; 1991. Biochemistry. 30:9961-9966; 1992. Biochemistry. 31:1836-1841). Although the binding constant is rather low, we estimate that we have achieved about 85% occupancy of the actin sites. Three-dimensional reconstructions from electron micrographs of both negatively stained and frozen-hydrated filaments show that the Fab fragment is bound at the location of the NH2 terminus in the model of Holmes et al. (Holmes, K.C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature. 347:37-44) for F-actin, excluding very different orientations of the actin subunit in the filament. Most of the mass of the antibody is not visualized, which is due to the large mobility of the NH2 terminus in F-actin, differences in binding angle within the polyclonal antibody population, or a combination of both of these possibilities. Images FIGURE 1 FIGURE 5 FIGURE 7 FIGURE 10 PMID:8161679

  1. Metalloprotease cleavage of the N terminus of the orphan G protein-coupled receptor GPR37L1 reduces its constitutive activity.

    PubMed

    Coleman, James L J; Ngo, Tony; Schmidt, Johannes; Mrad, Nadine; Liew, Chu Kong; Jones, Nicole M; Graham, Robert M; Smith, Nicola J

    2016-01-01

    Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gαswhen heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3',5'-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gαsor Gαisignaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue. PMID:27072655

  2. A C-terminal Hydrophobic, Solvent-protected Core and a Flexible N-terminus are Potentially Required for Human Papillomavirus 18 E7 Protein Functionality

    SciTech Connect

    Liu, S.; Tian, Y; Greenaway, F; Sun, M

    2010-01-01

    The oncogenic potential of the high-risk human papillomavirus (HPV) relies on the expression of genes specifying the E7 and E6 proteins. To investigate further the variation in oligomeric structure that has been reported for different E7 proteins, an HPV-18 E7 cloned from a Hispanic woman with cervical intraepithelial neoplasia was purified to homogeneity most probably as a stable monomeric protein in aqueous solution. We determined that one zinc ion is present per HPV-18 E7 monomer by amino acid and inductively coupled plasma-atomic emission spectroscopy analysis. Intrinsic fluorescence and circular dichroism spectroscopic results indicate that the zinc ion is important for the correct folding and thermal stability of HPV-18 E7. Hydroxyl radical mediated protein footprinting coupled to mass spectrometry and other biochemical and biophysical data indicate that near the C-terminus, the four cysteines of the two Cys-X{sub 2}-Cys motifs that are coordinated to the zinc ion form a solvent inaccessible core. The N-terminal LXCXE pRb binding motif region is hydroxyl radical accessible and conformationally flexible. Both factors, the relative flexibility of the pRb binding motif at the N-terminus and the C-terminal metal-binding hydrophobic solvent-protected core, combine together and facilitate the biological functions of HPV-18 E7.

  3. Enhanced Polysaccharide Binding and Activity on Linear β-Glucans through Addition of Carbohydrate-Binding Modules to Either Terminus of a Glucooligosaccharide Oxidase

    PubMed Central

    Foumani, Maryam; Vuong, Thu V.; MacCormick, Benjamin; Master, Emma R.

    2015-01-01

    The gluco-oligosaccharide oxidase from Sarocladium strictum CBS 346.70 (GOOX) is a single domain flavoenzyme that favourably oxidizes gluco- and xylo- oligosaccharides. In the present study, GOOX was shown to also oxidize plant polysaccharides, including cellulose, glucomannan, β-(1→3,1→4)-glucan, and xyloglucan, albeit to a lesser extent than oligomeric substrates. To improve GOOX activity on polymeric substrates, three carbohydrate binding modules (CBMs) from Clostridium thermocellum, namely CtCBM3 (type A), CtCBM11 (type B), and CtCBM44 (type B), were separately appended to the amino and carboxy termini of the enzyme, generating six fusion proteins. With the exception of GOOX-CtCBM3 and GOOX-CtCBM44, fusion of the selected CBMs increased the catalytic activity of the enzyme (kcat) on cellotetraose by up to 50%. All CBM fusions selectively enhanced GOOX binding to soluble and insoluble polysaccharides, and the immobilized enzyme on a solid cellulose surface remained stable and active. In addition, the CBM fusions increased the activity of GOOX on soluble glucomannan by up to 30 % and on insoluble crystalline as well as amorphous cellulose by over 50 %. PMID:25932926

  4. Role of Cysteine Residues in the Carboxyl-Terminus of the Follicle-Stimulating Hormone Receptor in Intracellular Traffic and Postendocytic Processing.

    PubMed

    Melo-Nava, Brenda; Casas-González, Patricia; Pérez-Solís, Marco A; Castillo-Badillo, Jean; Maravillas-Montero, José L; Jardón-Valadez, Eduardo; Zariñán, Teresa; Aguilar-Rojas, Arturo; Gallay, Nathalie; Reiter, Eric; Ulloa-Aguirre, Alfredo

    2016-01-01

    Posttranslational modifications occurring during the biosynthesis of G protein-coupled receptors include glycosylation and palmitoylation at conserved cysteine residues located in the carboxyl-terminus of the receptor. In a number of these receptors, these modifications play an important role in receptor function and particularly, in intracellular trafficking. In the present study, the three cysteine residues present in the carboxyl-terminus of the human FSHR were replaced with glycine by site-directed mutagenesis. Wild-type and mutant (Cys627/629/655Gly) FSHRs were then transiently expressed in HEK-293 cells and analyzed for cell-surface plasma membrane expression, agonist-stimulated signaling and internalization, and postendocytic processing in the absence and presence of lysosome and/or proteasome inhibitors. Compared with the wild-type FSHR, the triple mutant FSHR exhibited ~70% reduction in plasma membrane expression as well as a profound attenuation in agonist-stimulated cAMP production and ERK1/2 phosphorylation. Incubation of HEK-293 cells expressing the wild-type FSHR with 2-bromopalmitate (palmitoylation inhibitor) for 6 h, decreased plasma membrane expression of the receptor by ~30%. The internalization kinetics and β-arrestin 1 and 2 recruitment were similar between the wild-type and triple mutant FSHR as disclosed by assays performed in non-equilibrium binding conditions and by confocal microscopy. Cells expressing the mutant FSHR recycled the internalized FSHR back to the plasma membrane less efficiently than those expressing the wild-type FSHR, an effect that was counteracted by proteasome but not by lysosome inhibition. These results indicate that replacement of the cysteine residues present in the carboxyl-terminus of the FSHR, impairs receptor trafficking from the endoplasmic reticulum/Golgi apparatus to the plasma membrane and its recycling from endosomes back to the cell surface following agonist-induced internalization. Since in the FSHR these

  5. Role of Cysteine Residues in the Carboxyl-Terminus of the Follicle-Stimulating Hormone Receptor in Intracellular Traffic and Postendocytic Processing

    PubMed Central

    Melo-Nava, Brenda; Casas-González, Patricia; Pérez-Solís, Marco A.; Castillo-Badillo, Jean; Maravillas-Montero, José L.; Jardón-Valadez, Eduardo; Zariñán, Teresa; Aguilar-Rojas, Arturo; Gallay, Nathalie; Reiter, Eric; Ulloa-Aguirre, Alfredo

    2016-01-01

    Posttranslational modifications occurring during the biosynthesis of G protein-coupled receptors include glycosylation and palmitoylation at conserved cysteine residues located in the carboxyl-terminus of the receptor. In a number of these receptors, these modifications play an important role in receptor function and particularly, in intracellular trafficking. In the present study, the three cysteine residues present in the carboxyl-terminus of the human FSHR were replaced with glycine by site-directed mutagenesis. Wild-type and mutant (Cys627/629/655Gly) FSHRs were then transiently expressed in HEK-293 cells and analyzed for cell-surface plasma membrane expression, agonist-stimulated signaling and internalization, and postendocytic processing in the absence and presence of lysosome and/or proteasome inhibitors. Compared with the wild-type FSHR, the triple mutant FSHR exhibited ~70% reduction in plasma membrane expression as well as a profound attenuation in agonist-stimulated cAMP production and ERK1/2 phosphorylation. Incubation of HEK-293 cells expressing the wild-type FSHR with 2-bromopalmitate (palmitoylation inhibitor) for 6 h, decreased plasma membrane expression of the receptor by ~30%. The internalization kinetics and β-arrestin 1 and 2 recruitment were similar between the wild-type and triple mutant FSHR as disclosed by assays performed in non-equilibrium binding conditions and by confocal microscopy. Cells expressing the mutant FSHR recycled the internalized FSHR back to the plasma membrane less efficiently than those expressing the wild-type FSHR, an effect that was counteracted by proteasome but not by lysosome inhibition. These results indicate that replacement of the cysteine residues present in the carboxyl-terminus of the FSHR, impairs receptor trafficking from the endoplasmic reticulum/Golgi apparatus to the plasma membrane and its recycling from endosomes back to the cell surface following agonist-induced internalization. Since in the FSHR these

  6. Atomistic Simulations of Complex DNA DSBs and the Interactions with Ku70/80 Heterodimer

    NASA Technical Reports Server (NTRS)

    Hu, Shaowen; Cucinotta, Francis A.

    2011-01-01

    Compared to DNA with simple DSBs, the complex lesions can enhance the hydrogen bonds opening rate at the DNA terminus, and increase the mobility of the whole duplex. Binding of Ku drastically reduces the structural disruption and flexibility caused by the complex lesions. In all complex DSBs systems, the binding of DSB terminus with Ku70 is softened while the binding of the middle duplex with Ku80 is tightened. Binding of Ku promotes the rigidity of DNA duplexes, due to the clamp structure of the inner surface of the rings of Ku70/80.

  7. Structural Characterization of the Loop at the Alpha-Subunit C-Terminus of the Mixed Lineage Leukemia Protein Activating Protease Taspase1.

    PubMed

    van den Boom, Johannes; Trusch, Franziska; Hoppstock, Lukas; Beuck, Christine; Bayer, Peter

    2016-01-01

    Type 2 asparaginases, a subfamily of N-terminal nucleophile (Ntn) hydrolases, are activated by limited proteolysis. This activation yields a heterodimer and a loop region at the C-terminus of the α-subunit is released. Since this region is unresolved in all type 2 asparaginase crystal structures but is close to the active site residues, we explored this loop region in six members of the type 2 asparaginase family using homology modeling. As the loop model for the childhood cancer-relevant protease Taspase1 differed from the other members, Taspase1 activation as well as the conformation and dynamics of the 56 amino acids loop were investigated by CD and NMR spectroscopy. We propose a helix-turn-helix motif, which can be exploited as novel anticancer target to inhibit Taspase1 proteolytic activity. PMID:26974973

  8. The Radical S-Adenosyl-l-methionine Enzyme MftC Catalyzes an Oxidative Decarboxylation of the C-Terminus of the MftA Peptide.

    PubMed

    Bruender, Nathan A; Bandarian, Vahe

    2016-05-24

    Ribosomally synthesized post-translationally modified peptides (RiPPs) are encoded in the genomes of a wide variety of microorganisms, in the proximity of open reading frames that encode enzymes that conduct extensive modifications, many of which are novel. Recently, members of the radical S-adenosyl-l-methionine (SAM) superfamily have been identified in these biosynthetic clusters. Herein, we demonstrate the putative radical SAM enzyme, MftC, oxidatively decarboxylates the C-terminus of the MftA peptide in the presence of the accessory protein MftB. The reaction catalyzed by MftC expands the repertoire of peptide-based radical SAM chemistry beyond the intramolecular cross-links. PMID:27158836

  9. Structural Characterization of the Loop at the Alpha-Subunit C-Terminus of the Mixed Lineage Leukemia Protein Activating Protease Taspase1

    PubMed Central

    van den Boom, Johannes; Trusch, Franziska; Hoppstock, Lukas; Beuck, Christine; Bayer, Peter

    2016-01-01

    Type 2 asparaginases, a subfamily of N-terminal nucleophile (Ntn) hydrolases, are activated by limited proteolysis. This activation yields a heterodimer and a loop region at the C-terminus of the α-subunit is released. Since this region is unresolved in all type 2 asparaginase crystal structures but is close to the active site residues, we explored this loop region in six members of the type 2 asparaginase family using homology modeling. As the loop model for the childhood cancer-relevant protease Taspase1 differed from the other members, Taspase1 activation as well as the conformation and dynamics of the 56 amino acids loop were investigated by CD and NMR spectroscopy. We propose a helix-turn-helix motif, which can be exploited as novel anticancer target to inhibit Taspase1 proteolytic activity. PMID:26974973

  10. Nanopore detachment kinetics of poly(A) binding proteins from RNA molecules reveals the critical role of C-terminus interactions.

    PubMed

    Lin, Jianxun; Fabian, Marc; Sonenberg, Nahum; Meller, Amit

    2012-03-21

    The ubiquitous and abundant cytoplasmic poly(A) binding protein (PABP) is a highly conserved multifunctional protein, many copies of which bind to the poly(A) tail of eukaryotic mRNAs to promote translation initiation. The N-terminus of PABP is responsible for the high binding specificity and affinity to poly(A), whereas the C-terminus is known to stimulate PABP multimerization on poly(A). Here, we use single-molecule nanopore force spectroscopy to directly measure interactions between poly(A) and PABPs. Both electrical and biochemical results show that the C-C domain interaction between two consecutive PABPs promotes cooperative binding. Up to now, investigators have not been able to probe the detailed polarity configuration (i.e., the internal arrangement of two PABPs on a poly(A) streak in which the C-termini face toward or away from each other). Our nanopore force spectroscopy system is able to distinguish the cooperative binding conformation from the noncooperative one. The ∼50% cooperative binding conformation of wild-type PABPs indicates that the C-C domain interaction doubles the cooperative binding probability. Moreover, the longer dissociation time of a cooperatively bound poly(A)/PABP complex as compared with a noncooperatively bound one indicates that the cooperative mode is the most stable conformation for PABPs binding onto the poly(A). However, ∼50% of the poly(A)/PABP complexes exhibit a noncooperative binding conformation, which is in line with previous studies showing that the PABP C-terminal domain also interacts with additional protein cofactors. PMID:22455926

  11. Multisite tyrosine phosphorylation of the N-terminus of Mint1/X11α by Src kinase regulates the trafficking of amyloid precursor protein.

    PubMed

    Dunning, Christopher J R; Black, Hannah L; Andrews, Katie L; Davenport, Elizabeth C; Conboy, Michael; Chawla, Sangeeta; Dowle, Adam A; Ashford, David; Thomas, Jerry R; Evans, Gareth J O

    2016-05-01

    Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (APP) and are linked with its cleavage to generate β-amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2-binding motif ((202) YEEI) was identified in the N-terminus of Mint1 that is phosphorylated on tyrosine by C-Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N-terminus inhibits C-Src binding and tyrosine phosphorylation. Previous studies observed that co-expression of wild-type Mint1 and APP causes accumulation of APP in the trans-Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans-Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild-type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP. The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C-Src kinase. Mint1 causes APP accumulation in the trans-Golgi network, whereas inhibition of Src or mutation of Mint1-Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease. PMID:26865271

  12. Crystal Structure of Acivicin-Inhibited [gamma]-Glutamyltranspeptidase Reveals Critical Roles for Its C-Terminus in Autoprocessing and Catalysis

    SciTech Connect

    Williams, Kristin; Cullati, Sierra; Sand, Aaron; Biterova, Ekaterina I.; Barycki, Joseph J.

    2009-03-27

    Helicobacter pylori {gamma}-glutamyltranspeptidase (HpGT) is a general {gamma}-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used {gamma}-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k{sub max} = 0.033 {+-} 0.006 s{sup -1} and K{sub I} = 19.7 {+-} 7.2 {micro}M. Structure determination of acivicin-modified HpGT (1.7 {angstrom}; R{sub factor} = 17.9%; R{sub free} = 20.8%) demonstrates that acivicin is accommodated within the {gamma}-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a >10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole.

  13. Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis1[OPEN

    PubMed Central

    Ueda, Haruko; Yokota, Etsuo; Kuwata, Keiko; Kutsuna, Natsumaro; Mano, Shoji; Shimada, Tomoo; Tamura, Kentaro; Fukao, Yoichiro; Brandizzi, Federica; Shimmen, Teruo; Nishimura, Mikio

    2016-01-01

    The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network. PMID:26684656

  14. Mass balance, meteorology, area altitude distribution, glacier-surface altitude, ice motion, terminus position, and runoff at Gulkana Glacier, Alaska, 1996 balance year

    USGS Publications Warehouse

    March, Rod S.

    2003-01-01

    The 1996 measured winter snow, maximum winter snow, net, and annual balances in the Gulkana Glacier Basin were evaluated on the basis of meteorological, hydrological, and glaciological data. Averaged over the glacier, the measured winter snow balance was 0.87 meter on April 18, 1996, 1.1 standard deviation below the long-term average; the maximum winter snow balance, 1.06 meters, was reached on May 28, 1996; and the net balance (from August 30, 1995, to August 24, 1996) was -0.53 meter, 0.53 standard deviation below the long-term average. The annual balance (October 1, 1995, to September 30, 1996) was -0.37 meter. Area-averaged balances were reported using both the 1967 and 1993 area altitude distributions (the numbers previously given in this abstract use the 1993 area altitude distribution). Net balance was about 25 percent less negative using the 1993 area altitude distribution than the 1967 distribution. Annual average air temperature was 0.9 degree Celsius warmer than that recorded with the analog sensor used since 1966. Total precipitation catch for the year was 0.78 meter, 0.8 standard deviations below normal. The annual average wind speed was 3.5 meters per second in the first year of measuring wind speed. Annual runoff averaged 1.50 meters over the basin, 1.0 standard deviation below the long-term average. Glacier-surface altitude and ice-motion changes measured at three index sites document seasonal ice-speed and glacier-thickness changes. Both showed a continuation of a slowing and thinning trend present in the 1990s. The glacier terminus and lower ablation area were defined for 1996 with a handheld Global Positioning System survey of 126 locations spread out over about 4 kilometers on the lower glacier margin. From 1949 to 1996, the terminus retreated about 1,650 meters for an average retreat rate of 35 meters per year.

  15. Cauliflower mosaic virus gene VI product N-terminus contains regions involved in resistance-breakage, self-association and interactions with movement protein

    PubMed Central

    Hapiak, Michael; Li, Yongzhong; Agama, Keli; Swade, Shaddy; Okenka, Genevieve; Falk, Jessica; Khandekar, Sushant; Raikhy, Gaurav; Anderson, Alisha; Pollock, Justin; Zellner, Wendy; Schoelz, James; Leisner, Scott M.

    2008-01-01

    Cauliflower mosaic virus (CaMV) gene VI encodes a multifunctional protein (P6) involved in the translation of viral RNA, the formation of inclusion bodies, and the determination of host range. Arabidopsis thaliana ecotype Tsu-0 prevents the systemic spread of most CaMV isolates, including CM1841. However, CaMV isolate W260 overcomes this resistance. In this paper, the N-terminal 110 amino acids of P6 (termed D1) were identified as the resistance-breaking region. D1 also bound full-length P6. Furthermore, binding of W260 D1 to P6 induced higher β-galactosidase activity and better leucine-independent growth in the yeast two-hybrid system than its CM1841 counterpart. Thus, W260 may evade Tsu-0 resistance by mediating P6 self-association in a manner different from that of CM1841. Because Tsu-0 resistance prevents virus movement, interaction of P6 with P1 (CaMV movement protein) was investigated. Both yeast two-hybrid analyses and maltose-binding protein pull-down experiments show that P6 interacts with P1. Although neither half of P1 interacts with P6, the N-terminus of P6 binds P1. Interestingly, D1 by itself does not interact with P1, indicating that different portions of the P6 N-terminus are involved in different activities. The P1-P6 interactions suggest a role for P6 in virus transport, possibly by regulating P1 tubule formation or the assembly of movement complexes. PMID:18851998

  16. Cytological evidence that the C-terminus of carnitine palmitoyltransferase I is on the cytosolic face of the mitochondrial outer membrane.

    PubMed Central

    van der Leij, F R; Kram, A M; Bartelds, B; Roelofsen, H; Smid, G B; Takens, J; Zammit, V A; Kuipers, J R

    1999-01-01

    Carnitine palmitoyltransferase I (CPT I) is a key enzyme in the regulation of beta-oxidation. The topology of this enzyme has been difficult to elucidate by biochemical methods. We studied the topology of a fusion protein of muscle-type CPT I (M-CPT I) and green fluorescent protein (GFP) by microscopical means. To validate the use of the fusion protein, designated CPT I-GFP, we checked whether the main characteristics of native CPT I were retained. CPT I-GFP was expressed in HeLa cells after stable transfection. Confocal laser scanning microscopy in living cells revealed an extranuclear punctate distribution of CPT I-GFP, which coincided with a mitochondrial fluorescent marker. Immunogold electron microscopy localized CPT I-GFP almost exclusively to the mitochondrial periphery and showed that the C-terminus of CPT I must be on the cytosolic face of the mitochondrial outer membrane. Western analysis showed a protein that was 6 kDa smaller than predicted, which is consistent with previous results for the native M-CPT I. Mitochondria from CPT I-GFP-expressing cells showed an increased CPT activity that was inhibited by malonyl-CoA and was lost on solubilization with Triton X-100. We conclude that CPT I-GFP adopts the same topology as native CPT I and that its C-terminus is located on the cytosolic face of the mitochondrial outer membrane. The evidence supports a recently proposed model for the domain structure of CPT I based on biochemical evidence. PMID:10417344

  17. A mutation deleting sequences encoding the amino terminus of human cytomegalovirus UL84 impairs interaction with UL44 and capsid localization.

    PubMed

    Strang, Blair L; Bender, Brian J; Sharma, Mayuri; Pesola, Jean M; Sanders, Rebecca L; Spector, Deborah H; Coen, Donald M

    2012-10-01

    Protein-protein interactions are required for many biological functions. Previous work has demonstrated an interaction between the human cytomegalovirus DNA polymerase subunit UL44 and the viral replication factor UL84. In this study, glutathione S-transferase pulldown assays indicated that residues 1 to 68 of UL84 are both necessary and sufficient for efficient interaction of UL84 with UL44 in vitro. We created a mutant virus in which sequences encoding these residues were deleted. This mutant displayed decreased virus replication compared to wild-type virus. Immunoprecipitation assays showed that the mutation decreased but did not abrogate association of UL84 with UL44 in infected cell lysate, suggesting that the association in the infected cell can involve other protein-protein interactions. Further immunoprecipitation assays indicated that IRS1, TRS1, and nucleolin are candidates for such interactions in infected cells. Quantitative real-time PCR analysis of viral DNA indicated that the absence of the UL84 amino terminus does not notably affect viral DNA synthesis. Western blotting experiments and pulse labeling of infected cells with [(35)S]methionine demonstrated a rather modest downregulation of levels of multiple proteins and particularly decreased levels of the minor capsid protein UL85. Electron microscopy demonstrated that viral capsids assemble but are mislocalized in nuclei of cells infected with the mutant virus, with fewer cytoplasmic capsids detected. In sum, deletion of the sequences encoding the amino terminus of UL84 affects interaction with UL44 and virus replication unexpectedly, not viral DNA synthesis. Mislocalization of viral capsids in infected cell nuclei likely contributes to the observed decrease in virus replication. PMID:22855486

  18. The Carboxyl Terminus of Eremomycin Facilitates Binding to the Non-d-Ala-d-Ala Segment of the Peptidoglycan Pentapeptide Stem.

    PubMed

    Chang, James; Zhou, Hongyu; Preobrazhenskaya, Maria; Tao, Peng; Kim, Sung Joon

    2016-06-21

    Glycopeptide antibiotics inhibit cell wall biosynthesis in Gram-positive bacteria by targeting the peptidoglycan (PG) pentapeptide stem structure (l-Ala-d-iso-Gln-l-Lys-d-Ala-d-Ala). Structures of the glycopeptide complexed with a PG stem mimic have shown that the d-Ala-d-Ala segment is the primary drug binding site; however, biochemical evidence suggests that the glycopeptide-PG interaction involves more than d-Ala-d-Ala binding. Interactions of the glycopeptide with the non-d-Ala-d-Ala segment of the PG stem were investigated using solid-state nuclear magnetic resonance (NMR). LCTA-1421, a double (15)N-enriched eremomycin derivative with a C-terminal [(15)N]amide and [(15)N]Asn amide, was complexed with whole cells of Staphylococcus aureus grown in a defined medium containing l-[3-(13)C]Ala and d-[1-(13)C]Ala in the presence of alanine racemase inhibitor alaphosphin. (13)C{(15)N} and (15)N{(13)C} rotational-echo double-resonance (REDOR) NMR measurements determined the (13)C-(15)N internuclear distances between the [(15)N]Asn amide of LCTA-1421 and the (13)C atoms of the bound d-[1-(13)C]Ala-d-[1-(13)C]Ala to be 5.1 and 4.8 Å, respectively. These measurements also determined the distance from the C-terminal [(15)N]amide of LCTA-1421 to the l-[3-(13)C]Ala of PG to be 3.5 Å. The measured REDOR distance constraints position the C-terminus of the glycopeptide in the proximity of the l-Ala of the PG, suggesting that the C-terminus of the glycopeptide interacts near the l-Ala segment of the PG stem. In vivo REDOR measurements provided structural insight into how C-terminally modified glycopeptide antibiotics operate. PMID:27243469

  19. Mutations at the C-terminus of the simian immunodeficiency virus envelope glycoprotein affect gp120-gp41 stability on virions

    SciTech Connect

    Affranchino, Jose L.; Gonzalez, Silvia A. . E-mail: sigonzal@ub.edu.ar

    2006-03-30

    The transmembrane (TM) subunit of the envelope (Env) glycoprotein of the simian immunodeficiency virus (SIV) contains an unusually long cytoplasmic domain of 164 amino acids. Previously, we identified domains in the SIV TM cytoplasmic tail that are necessary for Env incorporation into virions and viral infectivity. In this study, we investigated the relevance to Env function of the highly conserved sequence comprising the immediate C-terminal 19 residues of TM. To this end, small in-frame deletions as well as a premature stop codon mutation were introduced into the coding region for the SIV TM C-terminus. All the mutant Env glycoproteins were expressed, processed and transported to the cell surface in an essentially wild-type manner. Moreover, the ability of the mutant Env proteins to mediate cell-to-cell fusion was similar to or slightly lower than that of the wild-type Env. However, viruses expressing the mutant Env glycoproteins were found to be poorly infectious in single-cycle infectivity assays. Further characterization of the TM mutant viruses revealed that while exhibiting wild-type levels of the TM protein, they contained significantly lower levels of the Env surface (SU) subunit, which is consistent with increased SU shedding from virions after Env incorporation. This phenotype was independent of Gag processing, since genetic inactivation of the viral protease did not increase SU retention by the resulting immature particles. Our findings indicate that deletions at the C-terminus of the SIV Env promote the instability of the SU-TM association on the virion surface and point to an important role for the TM cytoplasmic domain in modulating Env structure.

  20. Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling

    PubMed Central

    Mundell, SJ; Matharu, A-L; Nisar, S; Palmer, TM; Benovic, JL; Kelly, E

    2010-01-01

    Background and purpose: We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A2B adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5′-(N-ethylcarboxamido)-adenosine. Experimental approach: The trafficking of the wild type A2B adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. Key results: The wild type A2B adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln325-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu330-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln325-stop, Ser326-stop and Phe328-stop receptors. Following internalization, the wild type A2B adenosine receptor recycled rapidly to the cell surface, whereas the Gln325-stop receptor did not recycle. Conclusions and implications: Deletion of the COOH-terminus of the A2B adenosine receptor beyond Leu330 switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A2B adenosine receptor following prolonged agonist addition. PMID:20128803

  1. Conserved Residues in the N Terminus of Lipin-1 Are Required for Binding to Protein Phosphatase-1c, Nuclear Translocation, and Phosphatidate Phosphatase Activity*

    PubMed Central

    Kok, Bernard P. C.; Skene-Arnold, Tamara D.; Ling, Ji; Benesch, Matthew G. K.; Dewald, Jay; Harris, Thurl E.; Holmes, Charles F. B.; Brindley, David N.

    2014-01-01

    Lipin-1 is a phosphatidate phosphatase in glycerolipid biosynthesis and signal transduction. It also serves as a transcriptional co-regulator to control lipid metabolism and adipogenesis. These functions are controlled partly by its subcellular distribution. Hyperphosphorylated lipin-1 remains sequestered in the cytosol, whereas hypophosphorylated lipin-1 translocates to the endoplasmic reticulum and nucleus. The serine/threonine protein phosphatase-1 catalytic subunit (PP-1c) is a major protein dephosphorylation enzyme. Its activity is controlled by interactions with different regulatory proteins, many of which contain conserved RVXF binding motifs. We found that lipin-1 binds to PP-1cγ through a similar HVRF binding motif. This interaction depends on Mg2+ or Mn2+ and is competitively inhibited by (R/H)VXF-containing peptides. Mutating the HVRF motif in the highly conserved N terminus of lipin-1 greatly decreases PP-1cγ interaction. Moreover, mutations of other residues in the N terminus of lipin-1 also modulate PP-1cγ binding. PP-1cγ binds poorly to a phosphomimetic mutant of lipin-1 and binds well to the non-phosphorylatable lipin-1 mutant. This indicates that lipin-1 is dephosphorylated before PP-1cγ binds to its HVRF motif. Importantly, mutating the HVRF motif also abrogates the nuclear translocation and phosphatidate phosphatase activity of lipin-1. In conclusion, we provide novel evidence of the importance of the lipin-1 N-terminal domain for its catalytic activity, nuclear localization, and binding to PP-1cγ. PMID:24558042

  2. Ipomoelin, a Jacalin-Related Lectin with a Compact Tetrameric Association and Versatile Carbohydrate Binding Properties Regulated by Its N Terminus

    PubMed Central

    Chang, Wei-Chieh; Liu, Kai-Lun; Hsu, Fang-Ciao; Jeng, Shih-Tong; Cheng, Yi-Sheng

    2012-01-01

    Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (Ipomoea batatas cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (KA) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense. PMID:22808208

  3. Endopeptidase 3.4.24.11 converts N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide into a potent inhibitor of angiotensin-converting enzyme.

    PubMed Central

    Williams, C H; Yamamoto, T; Walsh, D M; Allsop, D

    1993-01-01

    It was reported recently that N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide (CPP-A-A-F-pAB), an inhibitor of endopeptidase 3.4.24.15 (E-24.15), also inhibits angiotensin-converting enzyme (ACE) from rabbit lung. We have found that this compound is without effect on ACE purified from pig kidney, at a concentration some 1000-fold greater than the Ki reported for inhibition of the enzyme from lung. However, preincubation of CPP-A-A-F-pAB with neutral endopeptidase 3.4.24.11 (E-24.11) does result in potent inhibitory effects on ACE. We have shown this to be due to formation of a fragment, CPP-A-A, the structure of which is closely related to ACE inhibitors such as enalaprilat. CPP-A-A was found to be a potent inhibitor of pig ACE. Under the conditions used it had an IC50 value of 1.6 x 10(-8) M, compared with the value obtained for captopril of 7.5 x 10(-10) M. These results have important implications for studies of E-24.15 when using CPP-A-A-F-pAB in vivo or in crude tissue extracts where E-24.11 might also be present. PMID:8379924

  4. Development and validation of a LC/MS method for the determination of Δ(9)-tetrahydrocannabinol and 11-carboxy-Δ(9)-tetrahydrocannabinol in the larvae of the blowfly Lucilia sericata: Forensic applications.

    PubMed

    Karampela, Sevasti; Pistos, Constantinos; Moraitis, Konstantinos; Stoukas, Vasilios; Papoutsis, Ioannis; Zorba, Eleni; Koupparis, Michalis; Spiliopoulou, Chara; Athanaselis, Sotiris

    2015-12-01

    In a number of forensic toxicological cases, Δ(9)-tetrahydrocannabinol (THC) and its metabolite 11-carboxy-delta-9-tetrahydrocannabinol (THCA) are frequently considered as contributor factors to the event. To that, a liquid chromatographic mass spectrometric method is described for the identification and quantitation of THC and its metabolite THCA in the forensically important larvae of L. sericata. Larvae of Lucilia sericata were fortified with varying concentrations of THC and THCA covering the calibration range between 10 and 500pg/mg. For the isolation of the analytes from larvae, several extraction techniques were evaluated and finally liquid-liquid extraction under acidic pH was selected using hexane-ethyl acetate (50:50, v/v) as extraction solvent. For the chromatographic separation, a Waters Symmetry® C18 analytical column was used while the mobile phase was acetonitrile-ammonium acetate (2mM) (30:70, v/v). The detection was performed using electrospray ionization source in negative mode (ESI-) and the selected ions monitored were m/z 313 for THC and m/z 343 for THCA. The proposed method which is simple and sufficiently sensitive for the detection of THC and THCA even in a single larva sampling, assisted the investigation of a forensic case. PMID:26654083

  5. Sequence analysis and structure prediction of 23S rRNA:m1G methyltransferases reveals a conserved core augmented with a putative Zn-binding domain in the N-terminus and family-specific elaborations in the C-terminus.

    PubMed

    Bujnicki, Janusz M; Blumenthal, Robert M; Rychlewski, Leszek

    2002-01-01

    N1-methylation of G748 within 23S ribosomal RNA results in resistance to the macrolide tylosin in Streptomyces. In contrast, the Escherichia coli mutant lacking N1-methylation of G745 exhibits increased resistance to viomycin, in addition to severe defects of growth characteristics. Both methylated guanines are located in hairpin 35, in domain II of prokaryotic 23S rRNA. G748 and G745 are modified by related S-adenosylmethionine-dependent methyltransferases (MTases), TlrB and RrmA respectively. Earlier sequence comparisons allowed identification of the AdoMet-binding site, however the catalytic site and the target-recognition region of these enzymes could not be delineated unambiguously. In this work, we carried out sequence-to-structure threading of the rRNA:m1G MTase family against the database of known structures to Identify those "missing regions". Our analysis confirms the earlier prediction of the AdoMet-binding site, but suggests a different location of the putative catalytic center than was previously postulated. We predict that RrmA and TlrB possess two regions that may be responsible for specific interactions with their target nucleic acid sequences: a putative Zn-finger domain in the N-terminus and the variable domain close to the C-terminus, which indicates that 23S rRNA MTases exhibit the primary structural organization distinct from other nucleic acid MTases, despite sharing the common catalytic domain. PMID:11763974

  6. Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L.

    PubMed

    Zhu, Shaotong; Canales, Alejandra; Bedair, Mai; Vik, Steven B

    2016-06-01

    Complex I is a multi-subunit enzyme of the respiratory chain with seven core subunits in its membrane arm (A, H, J, K, L, M, and N). In the enzyme from Escherichia coli the C-terminal ten amino acids of subunit K lie along the lateral helix of subunit L, and contribute to a junction of subunits K, L and N on the cytoplasmic surface. Using double cysteine mutagenesis, the cross-linking of subunit K (R99C) to either subunit L (K581C) or subunit N (T292C) was attempted. A partial yield of cross-linked product had no effect on the activity of the enzyme, or on proton translocation, suggesting that the C-terminus of subunit K has no dynamic role in function. To further elucidate the role of subunit K genetic deletions were constructed at the C-terminus. Upon the serial deletion of the last 4 residues of the C-terminus of subunit K, various results were obtained. Deletion of one amino acid had little effect on the activity of Complex I, but deletions of 2 or more amino acids led to total loss of enzyme activity and diminished levels of subunits L, M, and N in preparations of membrane vesicles. Together these results suggest that while the C-terminus of subunit K has no dynamic role in energy transduction by Complex I, it is vital for the correct assembly of the enzyme. PMID:26931547

  7. Crystal structure of the karyopherin Kap121p bound to the extreme C-terminus of the protein phosphatase Cdc14p

    SciTech Connect

    Kobayashi, Junya; Hirano, Hidemi; Matsuura, Yoshiyuki

    2015-07-31

    In Saccharomyces cerevisiae, the protein phosphatase Cdc14p is an antagonist of mitotic cyclin-dependent kinases and is a key regulator of late mitotic events such as chromosome segregation, spindle disassembly and cytokinesis. The activity of Cdc14p is controlled by cell-cycle dependent changes in its association with its competitive inhibitor Net1p (also known as Cfi1p) in the nucleolus. For most of the cell cycle up to metaphase, Cdc14p is sequestered in the nucleolus in an inactive state. During anaphase, Cdc14p is released from Net1p, spreads into the nucleus and cytoplasm, and dephosphorylates key mitotic targets. Although regulated nucleocytoplasmic shuttling of Cdc14p has been suggested to be important for exit from mitosis, the mechanism underlying Cdc14p nuclear trafficking remains poorly understood. Here we show that the C-terminal region (residues 517–551) of Cdc14p can function as a nuclear localization signal (NLS) in vivo and also binds to Kap121p (also known as Pse1p), an essential nuclear import carrier in yeast, in a Gsp1p-GTP-dependent manner in vitro. Moreover we report a crystal structure, at 2.4 Å resolution, of Kap121p bound to the C-terminal region of Cdc14p. The structure and structure-based mutational analyses suggest that either the last five residues at the extreme C-terminus of Cdc14p (residues 547–551; Gly-Ser-Ile-Lys-Lys) or adjacent residues with similar sequence (residues 540–544; Gly-Gly-Ile-Arg-Lys) can bind to the NLS-binding site of Kap121p, with two residues (Ile in the middle and Lys at the end of the five residues) of Cdc14p making key contributions to the binding specificity. Based on comparison with other structures of Kap121p-ligand complexes, we propose “IK-NLS” as an appropriate term to refer to the Kap121p-specific NLS. - Highlights: • The C-terminus of Cdc14p binds to Kap121p in a Gsp1p-GTP-dependent manner. • The crystal structure of Kap121p-Cdc14p complex is determined. • The structure reveals how

  8. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex

    PubMed Central

    Bondage, Devanand D.; Lin, Jer-Sheng; Ma, Lay-Sun; Kuo, Chih-Horng; Lai, Erh-Min

    2016-01-01

    Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone–effector complex (Tap-1–Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1–PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1–Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1–Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity. PMID:27313214

  9. Avulsion processes at the terminus of low-gradient semi-arid fluvial systems: Lessons from the Río Colorado, Altiplano endorheic basin, Bolivia

    NASA Astrophysics Data System (ADS)

    Donselaar, M. E.; Cuevas Gozalo, M. C.; Moyano, S.

    2013-01-01

    The Río Colorado dryland river system in the southeast of the endorheic Altiplano Basin (Bolivia) terminates on a very flat coastal plain at the edge of the Salar de Uyuni, the world's largest salt pan with an area of ca. 12,500 km2. Since the Pleistocene the basin has experienced several lake expansion and contraction cycles in response to wetter and drier climate periods, respectively. At present the basin is in a dry climate period which results in a lake level lowstand and progradation of fluvial systems such as the Río Colorado onto the former lake bottom. The present field study of the terminus of the Río Colorado shows that the river experiences a gradual downstream decrease of bankfull width and depth. This bankfull decrease is caused by the combined effects of: (1) extremely low gradient of the lake bottom and, hence, loss of flow energy, and (2) downstream transmission losses due to high evaporation potential and river water percolation through the channel floor. Peak water discharge in seasonal, short-duration rain periods causes massive overbank flooding and floodplain inundation. On satellite images the morphology of the river terminus has a divergent pattern and resembles a network of coeval sinuous distributary channels. However, field observations show that only one channel is active at low flow stage, and at high-flow stage an abandoned, partially infilled channel may be active as well. The active channel at its termination splits into narrow and shallow anastomosing streams before its demise on the lacustrine coastal plain. The rest of the channels which form the divergent network are older sediment-filled abandoned sinuous river courses with multiple random avulsion points. These channel deposits, together with extensive amalgamated crevasse-splay deposits, form an intricate network of fluvial sand deposits. Successive stages of progressively deeper crevasse-channel incision into the floodplain are the result of waning-stage return flow of

  10. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex.

    PubMed

    Bondage, Devanand D; Lin, Jer-Sheng; Ma, Lay-Sun; Kuo, Chih-Horng; Lai, Erh-Min

    2016-07-01

    Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity. PMID:27313214

  11. The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion

    PubMed Central

    Yen, Li-Chen; Liao, Jia-Teh; Lee, Hwei-Jen; Chou, Wei-Yuan; Chen, Chun-Wei; Lin, Yi-Ling

    2015-01-01

    ABSTRACT NS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using a trans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core β-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core β-ladder domain. IMPORTANCE The positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core β-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice

  12. Deletion of a Predicted β-Sheet Domain within the Amino Terminus of Herpes Simplex Virus Glycoprotein K Conserved among Alphaherpesviruses Prevents Virus Entry into Neuronal Axons

    PubMed Central

    Jambunathan, Nithya; Charles, Anu-Susan; Subramanian, Ramesh; Saied, Ahmad A.; Naderi, Misagh; Rider, Paul; Brylinski, Michal; Chouljenko, Vladimir N.

    2015-01-01

    ABSTRACT We have shown previously that herpes simplex virus 1 (HSV-1) lacking expression of the entire glycoprotein K (gK) or expressing gK with a 38-amino-acid deletion (gKΔ31–68 mutation) failed to infect ganglionic neurons after ocular infection of mice. We constructed a new model for the predicted three-dimensional structure of gK, revealing that the gKΔ31–68 mutation spans a well-defined β-sheet structure within the amino terminus of gK, which is conserved among alphaherpesviruses. The HSV-1(McKrae) gKΔ31–68 virus was tested for the ability to enter into ganglionic neuronal axons in cell culture of explanted rat ganglia using a novel virus entry proximity ligation assay (VEPLA). In this assay, cell surface-bound virions were detected by the colocalization of gD and its cognate receptor nectin-1 on infected neuronal surfaces. Capsids that have entered into the cytoplasm were detected by the colocalization of the virion tegument protein UL37, with dynein required for loading of virion capsids onto microtubules for retrograde transport to the nucleus. HSV-1(McKrae) gKΔ31–68 attached to cell surfaces of Vero cells and ganglionic axons in cell culture as efficiently as wild-type HSV-1(McKrae). However, unlike the wild-type virus, the mutant virus failed to enter into the axoplasm of ganglionic neurons. This work suggests that the amino terminus of gK is a critical determinant for entry into neuronal axons and may serve similar conserved functions for other alphaherpesviruses. IMPORTANCE Alphaherpesviruses, unlike beta- and gammaherpesviruses, have the unique ability to infect and establish latency in neurons. Glycoprotein K (gK) and the membrane protein UL20 are conserved among all alphaherpesviruses. We show here that a predicted β-sheet domain, which is conserved among alphaherpesviruses, functions in HSV-1 entry into neuronal axons, suggesting that it may serve similar functions for other herpesviruses. These results are in agreement with our

  13. Processing time-series point clouds to reveal strain conditions of the Helheim Glacier terminus and its adjacent mélange

    NASA Astrophysics Data System (ADS)

    Byers, L. C.; Stearns, L. A.; Finnegan, D. C.; LeWinter, A. L.; Gadomski, P. J.; Hamilton, G. S.

    2014-12-01

    Flow near the termini of tidewater glaciers varies over short time-scales due to mechanisms that are poorly understood. Repeat observations with high temporal and spatial resolution, recorded around the terminus, are required to better understand the processes that control flow variability. Progress in light detection and ranging (LiDAR) technology permit such observations of the near-terminus and the pro-glacial ice mélange, though standard workflows for quantifying deformation from point clouds currently do not exist. Here, we test and develop methods for processing displacements from LiDAR data of complexly deforming bodies. We use data collected at 30-minute intervals over three-days in August 2013 at Helheim Glacier, Greenland by a long-range (6-10 km), 1064 nm wavelength Terrestrial LiDAR Scanner (TLS). The total area of coverage was ~25 km2. Distributed shear in glaciers prevents a simple transformation for aligning repeat point clouds, but within small regions (~100 m2) strain is assumed to be minor between scans. Registering a large number of these individual regions, subset from the full point-cloud, results in reduced alignment errors. By subsetting in a regular grid, rasters of velocities between scans are created. However, using data-dependent properties such as point density causes the generation of unevenly spaced velocity estimations, which can locally improve resolution or decrease registration errors. The choice of subsets therefore controls the output product's resolution and accuracy. We test how the spatial segmentation scheme affects the displacement results and alignment errors, finding that displacements can be quantified with limited assumption of the true value of displacement for the subset, barring great morphological changes. By identifying areas that do not deform over the temporal domain of the dataset, and using these as the subsets to align, it should be possible to deduce which structures are accommodating strain. This allows for

  14. Role of N–epsilon- carboxy methyl lysine, advanced glycation end products and reactive oxygen species for the development of nonproliferative and proliferative retinopathy in type 2 diabetes mellitus

    PubMed Central

    Choudhuri, Subhadip; Dutta, Deep; Sen, Aditi; Chowdhury, Imran Hussain; Mitra, Bhaskar; Mondal, Lakshmi Kanta; Saha, Avijit; Bhadhuri, Gautam

    2013-01-01

    Purpose The aim of the present study was to evaluate the collective role of N-epsilon–carboxy methyl lysine (Nε-CML), advanced glycation end-products (AGEs), and reactive oxygen species (ROS) for the development of retinopathy among type 2 diabetic subjects. Methods Seventy type 2 diabetic subjects with nonproliferative diabetic retinopathy (NPDR), 105 subjects with proliferative diabetic retinopathy (PDR), and 102 patients with diabetes but without retinopathy (DNR) were enrolled in this study. In addition, 95 normal individuals without diabetes were enrolled as healthy controls in this study. Serum and vitreous Nε-CML and AGEs were measured by enzyme-linked immunosorbent assay. The peripheral blood mononuclear cell (PBMC) ROS level was measured by flow cytometric analysis. Serum and PBMC total thiols were measured by spectrophotometry. Results Serum AGEs and Nε-CML levels were significantly elevated in subjects with PDR (p<0.0001) and NPDR (p=0.0297 and p<0.0001, respectively) compared to DNR subjects. Further vitreous AGEs and Nε-CML levels were found to be significantly high among PDR subjects compared to the control group (p<0.0001). PBMC ROS production was found to be strikingly high among NPDR (p<0.0001) and PDR (p<0.0001) subjects as compared to the DNR group. Serum and PBMC total thiol levels were remarkably decreased in NPDR (p<0.0001 and p=0.0043, respectively) and PDR (p=0.0108 and p=0.0332 respectively) subjects than those were considered as DNR. Conclusions Our findings suggest that Nε-CML and ROS are the key modulators for the development of nonproliferative retinopathy among poorly controlled type 2 diabetic subjects. Furthermore, AGEs under persistent oxidative stress and the deprived antioxidant state might instigate the pathogenic process of retinopathy from the nonproliferative to the proliferative state. PMID:23378723

  15. Early Decreases in α-Fetoprotein and Des-γ-carboxy Prothrombin Predict the Antitumor Effects of Hepatic Transarterial Infusion Chemotherapy with Cisplatin (CDDP) Powder in Patients with Advanced Hepatocellular Carcinoma.

    PubMed

    Hatanaka, Takeshi; Kakizaki, Satoru; Shimada, Yasushi; Takizawa, Daichi; Katakai, Kenji; Yamazaki, Yuichi; Sato, Ken; Kusano, Motoyasu; Yamada, Masanobu

    2016-01-01

    Objective We retrospectively investigated the relationship between the tumor response and serial changes in α-fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP) during hepatic arterial infusion of a cisplatin powder formulation (CDDP powder) in patients with advanced hepatocellular carcinoma (HCC). Methods Seventy-six advanced HCC patients were analyzed. All HCC patients received high-concentration cisplatin (1.43 mg/mL) via the haptic artery at a dose of 65 mg/m(2). AFP and DCP were measured at baseline and four to eight weeks after treatment, and the antitumor responses were evaluated according to the response evaluation criteria in solid tumours (RECIST) criteria after one or two courses of treatment. The patients were classified into two groups, a decreased group and a non-decreased group, according to the change in the serum levels of AFP and DCP at four to eight weeks compared to baseline. Results The response to treatment of the decreased group (n=16) and non-decreased group (n=60) was complete response/partial response/stable disease/progressive disease (CR/PR/SD/PD) in 4/4/5/3 and 1/11/8/40 patients, respectively. The response rate and disease control rate of the decreased group were significantly higher than those of the non-decreased group (p=0.016 and p<0.001, respectively). The median survival time (MST) of the decreased/non-decreased groups were 25.9/10.6 months, respectively. The cumulative survival rates for the decreased group were significantly higher than those of the non-decreased group (p=0.042). In the multivariate analysis, vascular invasion and the decreased group were significant factors that affected the therapeutic efficacy. Conclusion A decrease in the levels of AFP and DCP after the first treatment with CDDP powder is a good predictor for the antitumor effect and the prognosis. PMID:27522991

  16. Validation of a Two-Dimensional Gas Chromatography Mass Spectrometry Method for the Simultaneous Quantification of Cannabidiol, Δ9-Tetrahydrocannabinol (THC), 11-Hydroxy-THC and 11-nor-9-Carboxy-THC in Plasma

    PubMed Central

    Karschner, Erin L.; Barnes, Allan J.; Lowe, Ross H.; Scheidweiler, Karl B.; Huestis, Marilyn A.

    2011-01-01

    A sensitive analytical method for simultaneous quantification of cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) in plasma is presented for monitoring cannabinoid pharmacotherapy and illicit cannabis use. Analytes were extracted from 1 mL plasma by solid phase extraction, derivatized with N, O,-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane, and analyzed by two-dimensional gas chromatography mass spectrometry (2D-GCMS) with cryofocusing. The lower calibration curve was linear from 0.25–25 ng/mL for CBD and THC, 0.125–25 ng/mL for 11-OH-THC and 0.25–50 ng/mL for THCCOOH. A second higher linear range from 5–100 ng/mL, achieved through modification of injection parameters, was validated for THC, 11-OH-THC and THCCOOH and was only implemented if concentrations exceeded the lower curve upper limit of linearity. This procedure prevented laborious re-extraction by allowing the same specimen to be re-injected for quantification on the high calibration curve. Intra- and inter-assay imprecision, determined at four quality control concentrations, were <7.8% CV. Analytical bias was within ±9.2% of target and extraction efficiencies were >72.9% for all analytes. Analytes were stable when stored at 22°C for 16h, 4°C for 48h, after three freeze-thaw cycles at −20°C and when stored on the autosampler for 48h. This sensitive and specific 2D-GCMS assay provides a new means of simultaneously quantifying CBD, THC and metabolite biomarkers in clinical medicine, forensic toxicology, workplace drug testing, and driving under the influence of drugs programs. PMID:20306180

  17. Clinical utility of simultaneous measurement of alpha-fetoprotein and des-γ-carboxy prothrombin for diagnosis of patients with hepatocellular carcinoma in China: A multi-center case-controlled study of 1,153 subjects.

    PubMed

    Song, Peipei; Feng, Xiaobin; Inagaki, Yoshinori; Song, Tianqiang; Zhang, Keming; Wang, Zhigang; Zheng, Shuguo; Ma, Kuansheng; Li, Qiang; Kong, Dalu; Wu, Qiang; Zhang, Ti; Zhao, Xin; Hasegawa, Kiyoshi; Sugawara, Yasuhiko; Kokudo, Norihiro; Tang, Wei

    2014-10-01

    This study aimed to investigate the clinical utility of simultaneous measurement of alphafetoprotein (AFP) and des-γ-carboxy prothrombin (DCP) for hepatocellular carcinoma (HCC) diagnosis in Chinese patients predominantly caused by hepatitis B virus infection by a multi-center case-controlled study. Subjects were 1,153 individuals from three major hospitals in China, including 550 cases in HCC group, 164 in Malignant disease group, 182 in Benign disease group, 85 in Chronic liver disease group, and 173 in Normal group. Serum levels of AFP and DCP were measured and clinicopathological features were determined for all subjects. Results showed that the levels of DCP and AFP were significantly higher in HCC group (550 patients, 74.18% with HBV infection) than that in other four groups (P < 0.001). Receiver operating curves (ROC) indicated the optimal cut-off value was 86 mAU/mL for DCP with a sensitivity of 71.50% and specificity of 86.30%, and 21 ng/mL for AFP with a sensitivity of 68.00% and specificity of 93.20%. The area under ROC curve was 0.846 for DCP, 0.832 for AFP, and 0.890 for the combination of DCP and AFP. The combination of DCP and AFP resulted in a higher Youden index and a sensitivity of approximately 90%, even for small tumors. The simultaneous measurement of AFP and DCP could achieve a better sensitivity in diagnosing Chinese HCC patients, even for small tumors. PMID:25382443

  18. A non-canonical di-acidic signal at the C-terminus of Kv1.3 determines anterograde trafficking and surface expression.

    PubMed

    Martínez-Mármol, Ramón; Pérez-Verdaguer, Mireia; Roig, Sara R; Vallejo-Gracia, Albert; Gotsi, Pelagia; Serrano-Albarrás, Antonio; Bahamonde, María Isabel; Ferrer-Montiel, Antonio; Fernández-Ballester, Gregorio; Comes, Núria; Felipe, Antonio

    2013-12-15

    Impairment of Kv1.3 expression at the cell membrane in leukocytes and sensory neuron contributes to the pathophysiology of autoimmune diseases and sensory syndromes. Molecular mechanisms underlying Kv1.3 channel trafficking to the plasma membrane remain elusive. We report a novel non-canonical di-acidic signal (E483/484) at the C-terminus of Kv1.3 essential for anterograde transport and surface expression. Notably, homologous motifs are conserved in neuronal Kv1 and Shaker channels. Biochemical analysis revealed interactions with the Sec24 subunit of the coat protein complex II. Disruption of this complex retains the channel at the endoplasmic reticulum. A molecular model of the Kv1.3-Sec24a complex suggests salt-bridges between the di-acidic E483/484 motif in Kv1.3 and the di-basic R750/752 sequence in Sec24. These findings identify a previously unrecognized motif of Kv channels essential for their expression on the cell surface. Our results contribute to our understanding of how Kv1 channels target to the cell membrane, and provide new therapeutic strategies for the treatment of pathological conditions. PMID:24144698

  19. UvrD303, a hyperhelicase mutant that antagonizes RecA-dependent SOS expression by a mechanism that depends on its C terminus.

    PubMed

    Centore, Richard C; Leeson, Michael C; Sandler, Steven J

    2009-03-01

    Genomic integrity is critical for an organism's survival and ability to reproduce. In Escherichia coli, the UvrD helicase has roles in nucleotide excision repair and methyl-directed mismatch repair and can limit reactions by RecA under certain circumstances. UvrD303 (D403A D404A) is a hyperhelicase mutant, and when expressed from a multicopy plasmid, it results in UV sensitivity (UV(s)), recombination deficiency, and antimutability. In order to understand the molecular mechanism underlying the UV(s) phenotype of uvrD303 cells, this mutation was transferred to the E. coli chromosome and studied in single copy. It is shown here that uvrD303 mutants are UV sensitive, recombination deficient, and antimutable and additionally have a moderate defect in inducing the SOS response after UV treatment. The UV-sensitive phenotype is epistatic with recA and additive with uvrA and is partially suppressed by removing the LexA repressor. Furthermore, uvrD303 is able to inhibit constitutive SOS expression caused by the recA730 mutation. The ability of UvrD303 to antagonize SOS expression was dependent on its 40 C-terminal amino acids. It is proposed that UvrD303, via its C terminus, can decrease the levels of RecA activity in the cell. PMID:19074381

  20. C11449. Native N-Terminus Nitrophorin 2 from the Kissing Bug: Similarities to and Differences from NP2(D1A)

    PubMed Central

    Berry, Robert E.; Muthu, Dhanasekaran; Shokhireva, Tatiana K.; Garrett, Sarah A.; Zhang, Hongjun; Walker, F. Ann

    2012-01-01

    The first amino acid of mature native nitrophorin 2 is aspartic acid, and when expressed in E. coli the wild-type gene of the mature protein retains the methionine-0 which is produced by translation of the start codon. This form of NP2, (M0)NP2, has been found to have different properties from its D1A mutant, for which the Met0 is cleaved by the methionine aminopeptidase of E. coli [R. E. Berry, T. K. Shokhireva, I. Filippov, M. N. Shokhirev, H. Zhang, F. A. Walker, Biochemistry 2007, 46, 6830]. Native N-terminus nitrophorin 2 ((ΔM0)NP2) has been prepared by employing periplasmic expression of NP2 in E. coli using the pelB leader sequence from Erwinia carotovora, which is present in the pET-26b expression plasmid (Novagen). This paper details the similarities and differences between the three different N-terminal forms of nitrophorin 2, (M0)NP2, NP2(D1A), and (ΔM0)NP2. It is found that the NMR spectra of high- and low-spin (ΔM0)NP2 are essentially identical to those of NP2(D1A), but the rate and equilibrium constants for histamine and NO dissociation/association of the two are different. PMID:22976966

  1. The relationship between truncation and phosphorylation at the C-terminus of tau protein in the paired helical filaments of Alzheimer's disease

    PubMed Central

    Flores-Rodríguez, Paola; Ontiveros-Torres, Miguel A.; Cárdenas-Aguayo, María C.; Luna-Arias, Juan P.; Meraz-Ríos, Marco A.; Viramontes-Pintos, Amparo; Harrington, Charles R.; Wischik, Claude M.; Mena, Raúl; Florán-Garduño, Benjamin; Luna-Muñoz, José

    2015-01-01

    We previously demonstrated that, in the early stages of tau processing in Alzheimer's disease, the N-terminal part of the molecule undergoes a characteristic cascade of phosphorylation and progressive misfolding of the proteins resulting in a structural conformation detected by Alz-50. In this immunohistochemical study of AD brain tissue, we have found that C-terminal truncation of tau at Asp-421 was an early event in tau aggregation and analyzed the relationship between phospho-dependent tau epitopes located at the C-terminus with truncation at Glu-391. The aim of this study was to determine whether C-terminal truncation may trigger events leading to the assembly of insoluble PHFs from soluble tau aggregates present in pre-tangle cells. Our findings suggest that there is a complex interaction between phosphorylated and truncated tau species. A model is presented here in which truncated tau protein represents an early neurotoxic species while phosphorylated tau species may provide a neuroprotective role in Alzheimer's disease. PMID:25717290

  2. The structural organization of the N-terminus domain of SopB, a virulence factor of Salmonella, depends on the nature of its protein partners.

    PubMed

    Roblin, Pierre; Lebrun, Pierre; Rucktooa, Prakash; Dewitte, Frederique; Lens, Zoe; Receveur-Brechot, Véronique; Raussens, Vincent; Villeret, Vincent; Bompard, Coralie

    2013-12-01

    The TTSS is used by Salmonella and many bacterial pathogens to inject virulence factors directly into the cytoplasm of target eukaryotic cells. Once translocated these so-called effector proteins hijack a vast array of crucial cellular functions to the benefit of the bacteria. In the bacterial cytoplasm, some effectors are stabilized and maintained in a secretion competent state by interaction with specific type III chaperones. In this work we studied the conformation of the Chaperone Binding Domain of the effector named Salmonella Outer protein B (SopB) alone and in complex with its cognate chaperone SigE by a combination of biochemical, biophysical and structural approaches. Our results show that the N-terminus part of SopB is mainly composed by α-helices and unfolded regions whose organization/stabilization depends on their interaction with the different partners. This suggests that the partially unfolded state of this N-terminal region, which confers the adaptability of the effector to bind very different partners during the infection cycle, allows the bacteria to modulate numerous host cells functions limiting the number of translocated effectors. PMID:24075929

  3. Specific Sites in the C Terminus of CTCF Interact with the SA2 Subunit of the Cohesin Complex and Are Required for Cohesin-Dependent Insulation Activity ▿

    PubMed Central

    Xiao, Tiaojiang; Wallace, Julie; Felsenfeld, Gary

    2011-01-01

    Recent studies have shown that the protein CTCF, which plays an important role in insulation and in large-scale organization of chromatin within the eukaryotic nucleus, depends for both activities on recruitment of the cohesin complex. We show here that the interaction of CTCF with the cohesin complex involves direct contacts between the cohesin subunit SA2 and specific regions of the C-terminal tail of CTCF. All other cohesin components are recruited through their interaction with SA2. Expression in vivo of CTCF mutants lacking the C-terminal domain, or with mutations at sites within it required for SA2 binding, disrupts the normal expression profile of the imprinted genes IGF2-H19 and also results in a loss of insulation activity. Taken together, our results demonstrate that specific sites on the C terminus of CTCF are essential for cohesin binding and insulator function. The only direct interaction between CTCF and cohesin involves contact with SA2, which is external to the cohesin ring. This suggests that in recruiting cohesin to CTCF, SA2 could bind first and the ring could assemble subsequently. PMID:21444719

  4. Turnip yellow mosaic virus forms infectious particles without the native beta-annulus structure and flexible coat protein N-terminus.

    PubMed

    Powell, Joshua D; Barbar, Elisar; Dreher, Theo W

    2012-01-20

    Structural studies have implicated the TYMV N-terminal amino acids of the coat protein (CP) in both static (virion stabilization) and dynamic (RNA encapsidation and disencapsidation) roles. We have deleted residues 2-5, 2-10 and 2-26 from the N-terminus and expressed the mutant CPs in E. coli to assess assembly in the absence of genomic RNA and in plant infections to assess infectivity and virion properties. In E. coli, the deletion constructs formed virus-like particles, but in decreased yield. All mutants were infectious in Chinese cabbage, producing normal symptoms but with a slight delay and decreased viral yields. Virions were progressively less stable with increasing deletion size and also more accessible to small molecules. These results show that the N-terminal 26 amino acids are not essential for viral processes in vivo, although removal of these residues decreases stability and increases porosity, both important factors for virion integrity and survival outside the host. PMID:22078163

  5. The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells.

    PubMed

    Caldas, Gina V; Lynch, Tina R; Anderson, Ryan; Afreen, Sana; Varma, Dileep; DeLuca, Jennifer G

    2015-11-01

    The spindle assembly checkpoint is a surveillance mechanism that blocks anaphase onset until all chromosomes are properly attached to microtubules of the mitotic spindle. Checkpoint activity requires kinetochore localization of Mad1/Mad2 to inhibit activation of the anaphase promoting complex/cyclosome in the presence of unattached kinetochores. In budding yeast and Caenorhabditis elegans, Bub1, recruited to kinetochores through KNL1, recruits Mad1/Mad2 by direct linkage with Mad1. However, in human cells it is not yet established which kinetochore protein(s) function as the Mad1/Mad2 receptor. Both Bub1 and the RZZ complex have been implicated in Mad1/Mad2 kinetochore recruitment; however, their specific roles remain unclear. Here, we investigate the contributions of Bub1, RZZ and KNL1 to Mad1/Mad2 kinetochore recruitment. We find that the RZZ complex localizes to the N-terminus of KNL1, downstream of Bub1, to mediate robust Mad1/Mad2 kinetochore localization. Our data also point to the existence of a KNL1-, Bub1-independent mechanism for RZZ and Mad1/Mad2 kinetochore recruitment. Based on our results, we propose that in humans, the primary mediator for Mad1/Mad2 kinetochore localization is the RZZ complex. PMID:26581576

  6. Protection against doxorubicin-induced myocardial dysfunction in mice by cardiac-specific expression of carboxyl terminus of hsp70-interacting protein.

    PubMed

    Wang, Lei; Zhang, Tian-Peng; Zhang, Yuan; Bi, Hai-Lian; Guan, Xu-Min; Wang, Hong-Xia; Wang, Xia; Du, Jie; Xia, Yun-Long; Li, Hui-Hua

    2016-01-01

    Carboxyl terminus of Hsp70-interacting protein (CHIP) is a critical ubiquitin ligase/cochaperone to reduce cardiac oxidative stress, inflammation, cardiomyocyte apoptosis and autophage etc. However, it is unclear whether overexpression of CHIP in the heart would exert protective effects against DOX-induced cardiomyopathy. Cardiac-specific CHIP transgenic (CHIP-TG) mice and the wild-type (WT) littermates were treated with DOX or saline. DOX-induced cardiac atrophy, dysfunction, inflammation, oxidative stress and cardiomyocyte apoptosis were significantly attenuated in CHIP-TG mice. CHIP-TG mice also showed higher survival rate than that of WT mice (40% versus 10%) after 10-day administration of DOX. In contrast, knockdown of CHIP by siRNA in vitro further enhanced DOX-induced cardiotoxic effects. Global gene microarray assay revealed that after DOX-treatment, differentially expressed genes between WT and CHIP-TG mice were mainly involved in apoptosis, atrophy, immune/inflammation and oxidative stress. Mechanistically, CHIP directly promotes ubiquitin-mediated degradation of p53 and SHP-1, which results in activation of ERK1/2 and STAT3 pathways thereby ameliorating DOX-induced cardiac toxicity. PMID:27323684

  7. A Proline-Rich Domain in the Genotype 4 Hepatitis E Virus ORF3 C-Terminus Is Crucial for Downstream V105DLP108 Immunoactivity.

    PubMed

    Wang, Heng; Ji, Fangxiao; Liang, Huanbin; Gu, Honglang; Ning, Zhangyong; Liu, Rongchang; Zhang, Guihong

    2015-01-01

    The hepatitis E virus (HEV) is responsible for serious viral hepatitis worldwide. Animals are considered a reservoir of HEV, particularly pigs. While HEV infection in pigs and dogs is always asymptomatic, the virus causes high death rates in patients with pre-existing chronic liver disease and pregnant women in developing countries. HEV open reading frame 2 (ORF2) has been used as a diagnostic target to detect specific antibodies against HEV in serum samples. Recent research has additionally supported the potential utility of the ORF3 protein as a target in serum anti-HEV detection. However, the epitope distribution of ORF3 protein remains ambiguous. In the current study, we showed that continuous amino acid motif, VDLP, at the C-terminus of genotype 4 HEV ORF3 is a core sequence of the ORF3 protein epitope. Moreover, cooperative interaction with upstream elements is essential for its immunoactivity. Three proline residues (P99, P102 and P103) in the upstream proline-rich domain exerted significant effects on the immunocompetence of VDLP. ELISA results revealed that SAPPLPPVVDLP and SAPPLPPVVDLPQLGL peptides containing the identified VDLP epitope display weaker reactions with anti-HEV serum than the commercial ELISA kit. Our collective findings provide valuable information on the epitope distribution characteristics of HEV ORF3 and improve our understanding of the influence of the proline-rich domain on the immunoactivity of downstream amino acids in the C-terminal region. PMID:26177202

  8. Protection against doxorubicin-induced myocardial dysfunction in mice by cardiac-specific expression of carboxyl terminus of hsp70-interacting protein

    PubMed Central

    Wang, Lei; Zhang, Tian-Peng; Zhang, Yuan; Bi, Hai-Lian; Guan, Xu-Min; Wang, Hong-Xia; Wang, Xia; Du, Jie; Xia, Yun-Long; Li, Hui-Hua

    2016-01-01

    Carboxyl terminus of Hsp70-interacting protein (CHIP) is a critical ubiquitin ligase/cochaperone to reduce cardiac oxidative stress, inflammation, cardiomyocyte apoptosis and autophage etc. However, it is unclear whether overexpression of CHIP in the heart would exert protective effects against DOX-induced cardiomyopathy. Cardiac-specific CHIP transgenic (CHIP-TG) mice and the wild-type (WT) littermates were treated with DOX or saline. DOX-induced cardiac atrophy, dysfunction, inflammation, oxidative stress and cardiomyocyte apoptosis were significantly attenuated in CHIP-TG mice. CHIP-TG mice also showed higher survival rate than that of WT mice (40% versus 10%) after 10-day administration of DOX. In contrast, knockdown of CHIP by siRNA in vitro further enhanced DOX-induced cardiotoxic effects. Global gene microarray assay revealed that after DOX-treatment, differentially expressed genes between WT and CHIP-TG mice were mainly involved in apoptosis, atrophy, immune/inflammation and oxidative stress. Mechanistically, CHIP directly promotes ubiquitin-mediated degradation of p53 and SHP-1, which results in activation of ERK1/2 and STAT3 pathways thereby ameliorating DOX-induced cardiac toxicity. PMID:27323684

  9. In vivo suppression of polyglutamine neurotoxicity by C-terminus of Hsp70-interacting protein (CHIP) supports an aggregation model of pathogenesis.

    PubMed

    Williams, Aislinn J; Knutson, Tina M; Colomer Gould, Veronica F; Paulson, Henry L

    2009-03-01

    Perturbations in neuronal protein homeostasis likely contribute to disease pathogenesis in polyglutamine (polyQ) neurodegenerative disorders. Here we provide evidence that the co-chaperone and ubiquitin ligase, CHIP (C-terminus of Hsp70-interacting protein), is a central component to the homeostatic mechanisms countering toxic polyQ proteins in the brain. Genetic reduction or elimination of CHIP accelerates disease in transgenic mice expressing polyQ-expanded ataxin-3, the disease protein in Spinocerebellar Ataxia Type 3 (SCA3). In parallel, CHIP reduction markedly increases the level of ataxin-3 microaggregates, which partition in the soluble fraction of brain lysates yet are resistant to dissociation with denaturing detergent, and which precede the appearance of inclusions. The level of microaggregates in the CNS, but not of ataxin-3 monomer, correlates with disease severity. Additional cell-based studies suggest that either of two quality control ubiquitin ligases, CHIP or E4B, can reduce steady state levels of expanded, but not wild-type, ataxin-3. Our results support an aggregation model of polyQ disease pathogenesis in which ataxin-3 microaggregates are a neurotoxic species, and suggest that enhancing CHIP activity is a possible route to therapy for SCA3 and other polyQ diseases. PMID:19084066

  10. N terminus determinants of MinC from Neisseria gonorrhoeae mediate interaction with FtsZ but do not affect interaction with MinD or homodimerization.

    PubMed

    Greco-Stewart, V; Ramirez-Arcos, S; Liao, M; Dillon, J R

    2007-06-01

    While bacterial cell division has been widely studied in rod-shaped bacteria, the mechanism of cell division in round (coccal) bacteria remains largely enigmatic. In the present study, interaction between the cell division inhibitor MinC from Neisseria gonorrhoeae (MinC(Ng)) and the gonococcal cell division proteins MinD(Ng) and FtsZ(Ng) are demonstrated. Protein truncation and site-directed mutagenic approaches determined which N-terminal residues were essential for cell division inhibition by MinC(Ng) using cell morphology as an indicator of protein functionality. Truncation from or mutation at the 13th amino acid of the N terminus of MinC(Ng) resulted in loss of protein function. Bioinformatic analyses predicted that point mutations of L35P and L68P would affect the alpha-helical conformation of the protein and we experimentally showed that these mutations alter the functionality of MinC(Ng). The bacterial two-hybrid system showed that interaction of MinC(Ng) with FtsZ(Ng) is abrogated upon truncation of 13 N-terminal residues while MinC(Ng)-MinD(Ng) interaction or MinC(Ng) homodimerization is unaffected. These data confirm interactions among gonococcal cell division proteins and determine the necessity of the 13th amino acid for MinC(Ng) function. PMID:17287984

  11. Identification of a novel domain at the N terminus of caveolin-1 that controls rear polarization of the protein and caveolae formation.

    PubMed

    Sun, Xing-Hui; Flynn, Daniel C; Castranova, Vincent; Millecchia, Lyndell L; Beardsley, Andrew R; Liu, Jun

    2007-03-01

    When cells are migrating, caveolin-1, the principal protein component of caveolae, is excluded from the leading edge and polarized at the cell rear. The dynamic feature depends on a specific sequence motif that directs intracellular trafficking of the protein. Deletion mutation analysis revealed a putative polarization domain at the N terminus of caveolin-1, between amino acids 32-60. Alanine substitution identified a minimal sequence of 10 residues ((46)TKEIDLVNRD(55)) necessary for caveolin-1 rear polarization. Interestingly, deletion of amino acids 1-60 did not prevent the polarization of caveolin-1 in human umbilical vein endothelial cells or wild-type mouse embryonic fibroblasts because of an interaction of Cav(61-178) mutant with endogenous caveolin-1. Surprisingly, expression of the depolarization mutant in caveolin-1 null cells dramatically impeded caveolae formation. Furthermore, knockdown of caveolae formation by methyl-beta-cyclodextrin failed to prevent wild-type caveolin-1 rear polarization. Importantly, genetic depletion of caveolin-1 led to disoriented migration, which can be rescued by full-length caveolin-1 but not the depolarization mutant, indicating a role of caveolin-1 polarity in chemotaxis. Thus, we have identified a sequence motif that is essential for caveolin-1 rear polarization and caveolae formation. PMID:17213184

  12. A single amino acid in the C-terminus of VP3 protein influences the replication of attenuated infectious bursal disease virus in vitro and in vivo.

    PubMed

    Wang, Yongqiang; Qi, Xiaole; Kang, Zhonghui; Yu, Fei; Qin, Liting; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

    2010-08-01

    The very virulent infectious bursal disease virus (vvIBDV) Gx strain causes over 60% mortality in chickens but cannot replicate in CEF cultures. The attenuated Gt strain, however, is not virulent in chickens and replicates well in CEF cultures. The two strains display differences in 6 amino acids in VP4 and 4 amino acids in VP3. To determine whether VP4 and VP3 are involved in the virulence and replication of IBDV, three chimeric viruses, in which the VP4/VP3/3'UTR, VP3/3'UTR or VP4 region of Gt were replaced by the corresponding region of Gx, were constructed and characterized in vitro and in vivo. The substituted regions in VP4 or VP3 did not affect virulence of Gt. While the substituted region in VP4 had no effect on viral replication of Gt in CEF cultures, substitution of the VP3/3'UTR region did reduce the replicative capacity of the virus. Through site-directed mutagenesis, three rescued recombinant viruses with a single amino acid substitution in the C-terminus of VP3 of the Gt strain (L981P, A990V and T1005A) were characterized in a similar manner. Amino acid substitution at position 990 reduced viral replication of Gt and reduced its efficacy of protection against vvIBDV Gx challenge in vivo. This study provides important information for the design and development of more effective IBDV vaccines using reverse genetics. PMID:20471998

  13. Intrinsic local destabilization of the C-terminus predisposes integrin α1 I domain to a conformational switch induced by collagen binding.

    PubMed

    Nunes, Ana Monica; Zhu, Jie; Jezioro, Jacqueline; Minetti, Conceição A S A; Remeta, David P; Farndale, Richard W; Hamaia, Samir W; Baum, Jean

    2016-09-01

    Integrin-collagen interactions play a critical role in a myriad of cellular functions that include immune response, and cell development and differentiation, yet their mechanism of binding is poorly understood. There is increasing evidence that conformational flexibility assumes a central role in the molecular mechanisms of protein-protein interactions and here we employ NMR hydrogen-deuterium exchange (HDX) experiments to explore the impact of slower timescale dynamic events. To gain insight into the mechanisms underlying collagen-induced conformational switches, we have undertaken a comparative study between the wild type integrin α1 I and a gain-of-function E317A mutant. NMR HDX results suggest a relationship between regions exhibiting a reduced local stability in the unbound I domain and those that undergo significant conformational changes upon binding. Specifically, the αC and α7 helices within the C-terminus are at the center of such major perturbations and present reduced local stabilities in the unbound state relative to other structural elements. Complementary isothermal titration calorimetry experiments have been performed to derive complete thermodynamic binding profiles for association of the collagen-like triple-helical peptide with wild type α1 I and E317A mutant. The differential energetics observed for E317A are consistent with the HDX experiments and support a model in which intrinsically destabilized regions predispose conformational rearrangement in the integrin I domain. This study highlights the importance of exploring different timescales to delineate allosteric and binding events. PMID:27342747

  14. The C terminus of NS1 protein of influenza A/WSN/1933(H1N1) virus modulates antiviral responses in infected human macrophages and mice.

    PubMed

    Anastasina, Maria; Schepens, Bert; Söderholm, Sandra; Nyman, Tuula A; Matikainen, Sampsa; Saksela, Kalle; Saelens, Xavier; Kainov, Denis E

    2015-08-01

    Non-structural protein NS1 of influenza A viruses interacts with cellular factors through its N-terminal RNA-binding, middle effector and C-terminal non-structured domains. NS1 attenuates antiviral responses in infected cells and thereby secures efficient virus replication. Some influenza strains express C-terminally truncated NS1 proteins due to nonsense mutations in the NS1 gene. To understand the role of the NS1 C-terminal region in regulation of antiviral responses, we engineered influenza viruses expressing C-terminally truncated NS1 proteins using A/WSN/33(H1N1) reverse genetics and tested them in human macrophages and in mice. We showed that a WSN virus expressing NS1 with a 28 aa deletion from its C terminus is a more powerful inducer of antiviral responses than the virus expressing full-length NS1, or one with a 10 aa truncation of NS1 in vitro. Thus, our findings suggest that the C-terminal region of NS1 is essential for regulation of antiviral responses. Moreover, viruses expressing truncated NS1 proteins could be good vaccine candidates. PMID:25934792

  15. Mutational analysis reveals a role for the C terminus of the proteasome subunit Rpt4p in spindle pole body duplication in Saccharomyces cerevisiae.

    PubMed Central

    McDonald, Heather B; Helfant, Astrid Hoes; Mahony, Erin M; Khosla, Shaun K; Goetsch, Loretta

    2002-01-01

    The ubiquitin/proteasome pathway plays a key role in regulating cell cycle progression. Previously, we reported that a conditional mutation in the Saccharomyces cerevisiae gene RPT4/PCS1, which encodes one of six ATPases in the proteasome 19S cap complex/regulatory particle (RP), causes failure of spindle pole body (SPB) duplication. To improve our understanding of Rpt4p, we created 58 new mutations, 53 of which convert clustered, charged residues to alanine. Virtually all mutations that affect the N-terminal region, which contains a putative nuclear localization signal and coiled-coil motif, result in a wild-type phenotype. Nine mutations that affect the central ATPase domain and the C-terminal region confer recessive lethality. The two conditional mutations identified, rpt4-145 and rpt4-150, affect the C terminus. After shift to high temperature, these mutations generally cause cells to progress slowly through the first cell cycle and to arrest in the second cycle with large buds, a G2 content of DNA, and monopolar spindles, although this phenotype can vary depending on the medium. Additionally, we describe a genetic interaction between RPT4 and the naturally polymorphic gene SSD1, which in wild-type form modifies the rpt4-145 phenotype such that cells arrest in G2 of the first cycle with complete bipolar spindles. PMID:12399382

  16. The C-terminus of nisin is important for the ABC transporter NisFEG to confer immunity in Lactococcus lactis

    PubMed Central

    AlKhatib, Zainab; Lagedroste, Marcel; Zaschke, Julia; Wagner, Manuel; Abts, André; Fey, Iris; Kleinschrodt, Diana; Smits, Sander H J

    2014-01-01

    The lantibiotic nisin is a small 3.4 kDa antimicrobial peptide, which acts against Gram-positive bacteria in the nmol/L range. Nisin is produced and secreted by several Lactococcus lactis strains to ensure advantages against other bacteria in their habitat. Nisin contains five specific lanthionine rings of which the first two are important for Lipid II binding and the last two are crucial for the pore formation in the membrane. To gain immunity against nisin, the producing strain is expressing an ABC transporter called NisFEG, which expels nisin from the membrane. As a result six to eightfold more nisin is needed to affect the cells. The hydrolysis of ATP by NisFEG is required for this immunity as shown by a mutant, where the ATP hydrolysis is disrupted (NisFH181AEG). Furthermore, NisFEG recognizes the C-terminus of nisin, since deletion of the last six amino acids as well as of the last ring lowered the fold of immunity displayed by NisFEG. PMID:25176038

  17. The C-terminus of nisin is important for the ABC transporter NisFEG to confer immunity in Lactococcus lactis.

    PubMed

    AlKhatib, Zainab; Lagedroste, Marcel; Zaschke, Julia; Wagner, Manuel; Abts, André; Fey, Iris; Kleinschrodt, Diana; Smits, Sander H J

    2014-10-01

    The lantibiotic nisin is a small 3.4 kDa antimicrobial peptide, which acts against Gram-positive bacteria in the nmol/L range. Nisin is produced and secreted by several Lactococcus lactis strains to ensure advantages against other bacteria in their habitat. Nisin contains five specific lanthionine rings of which the first two are important for Lipid II binding and the last two are crucial for the pore formation in the membrane. To gain immunity against nisin, the producing strain is expressing an ABC transporter called NisFEG, which expels nisin from the membrane. As a result six to eightfold more nisin is needed to affect the cells. The hydrolysis of ATP by NisFEG is required for this immunity as shown by a mutant, where the ATP hydrolysis is disrupted (NisFH181A EG). Furthermore, NisFEG recognizes the C-terminus of nisin, since deletion of the last six amino acids as well as of the last ring lowered the fold of immunity displayed by NisFEG. PMID:25176038

  18. Vaccination of Cattle with the N Terminus of LppQ of Mycoplasma mycoides subsp. mycoides Results in Type III Immune Complex Disease upon Experimental Infection

    PubMed Central

    Frey, Joachim; Smith, Ken; Schnier, Christian; Wesonga, Hezron; Naessens, Jan; McKeever, Declan

    2015-01-01

    Contagious bovine pleuropneumonia (CBPP) is a serious respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides. Current vaccines against CBPP induce short-lived immunity and can cause severe postvaccine reactions. Previous studies have identified the N terminus of the transmembrane lipoprotein Q (LppQ-N′) of M. mycoides subsp. mycoides as the major antigen and a possible virulence factor. We therefore immunized cattle with purified recombinant LppQ-N′ formulated in Freund's adjuvant and challenged them with M. mycoides subsp. mycoides. Vaccinated animals showed a strong seroconversion to LppQ, but they exhibited significantly enhanced postchallenge glomerulonephritis compared to the placebo group (P = 0.021). Glomerulonephritis was characterized by features that suggested the development of antigen-antibody immune complexes. Clinical signs and gross pathological scores did not significantly differ between vaccinated and placebo groups. These findings reveal for the first time the pathogenesis of enhanced disease as a result of antibodies against LppQ during challenge and also argue against inclusion of LppQ-N′ in a future subunit vaccine for CBPP. PMID:25733516

  19. A Transit Peptide–Like Sorting Signal at the C Terminus Directs the Bienertia sinuspersici Preprotein Receptor Toc159 to the Chloroplast Outer Membrane[C][W

    PubMed Central

    Lung, Shiu-Cheung; Chuong, Simon D.X.

    2012-01-01

    Although Toc159 is known to be one of the key GTPase receptors for selective recognition of chloroplast preproteins, the mechanism for its targeting to the chloroplast surface remains unclear. To compare the targeting of these GTPase receptors, we identified two Toc159 isoforms and a Toc34 from Bienertia sinuspersici, a single-cell C4 species with dimorphic chloroplasts in individual chlorenchyma cells. Fluorescent protein tagging and immunogold studies revealed that the localization patterns of Toc159 were distinctive from those of Toc34, suggesting different targeting pathways. Bioinformatics analyses indicated that the C-terminal tails (CTs) of Toc159 possess physicochemical and structural properties of chloroplast transit peptides (cTPs). These results were further confirmed by fluorescent protein tagging, which showed the targeting of CT fusion proteins to the chloroplast surface. The CT of Bs Toc159 in reverse orientation functioned as a cleavable cTP that guided the fluorescent protein to the stroma. Moreover, a Bs Toc34 mutant protein was retargeted to the chloroplast envelope using the CTs of Toc159 or reverse sequences of other cTPs, suggesting their conserved functions. Together, our data show that the C terminus and the central GTPase domain represent a novel dual domain–mediated sorting mechanism that might account for the partitioning of Toc159 between the cytosol and the chloroplast envelope for preprotein recognition. PMID:22517318

  20. Evidence for auto-inhibition by the N terminus of hADAR2 and activation by dsRNA binding

    PubMed Central

    MACBETH, MARK R.; LINGAM, ARUNTH T.; BASS, BRENDA L.

    2004-01-01

    Adenosine deaminases that act on RNA (ADARs) catalyze adenosine to inosine conversion in RNA that is largely double stranded. Human ADAR2 (hADAR2) contains two double-stranded RNA binding motifs (dsRBMs), separated by a 90-amino acid linker, and these are followed by the C-terminal catalytic domain. We assayed enzymatic activity of N-terminal deletion constructs of hADAR2 to determine the role of the dsRBMs and the intervening linker peptide. We found that a truncated protein consisting of one dsRBM and the deaminase domain was capable of deaminating a short 15-bp substrate. In contrast, full-length hADAR2 was inactive on this short substrate. In addition, we observed that the N terminus, which was deleted from the truncated protein, inhibits editing activity when added in trans. We propose that the N-terminal domain of hADAR2 contains sequences that cause auto-inhibition of the enzyme. Our results suggest activation requires binding to an RNA substrate long enough to accommodate interactions with both dsRBMs. PMID:15383678

  1. Bovine herpesvirus 1 tegument protein VP22 interacts with histones, and the carboxyl terminus of VP22 is required for nuclear localization.

    PubMed

    Ren, X; Harms, J S; Splitter, G A

    2001-09-01

    The bovine herpesvirus 1 (BHV-1) UL49 gene encodes a viral tegument protein termed VP22. UL49 homologs are conserved among alphaherpesviruses. Interestingly, the BHV-1 VP22 deletion mutant virus is asymptomatic and avirulent in infected cattle but produces only a slight reduction in titer in vitro. Attenuation of the BHV-1 VP22 deletion mutant virus in vivo suggests that VP22 plays a functional role in BHV-1 replication. In herpes simplex virus type 1, the VP22 homolog was previously shown to interact with another tegument protein,VP16, the alpha-transinducing factor in vitro. In this report, we show that (i) the nuclear targeting of VP22 is independent of other viral factors, (ii) the carboxyl terminus of VP22 is required for its nuclear localization, (iii) VP22 associates with histones and nucleosomes, (iv) an antihistone monoclonal antibody cross-reacts with VP22, and (v) acetylation of histone H4 is decreased in VP22-expressing cells as well as virus-infected cells. Our data suggest that VP22 may have a modulatory function during BHV-1 infection. PMID:11483770

  2. A Conserved Hydrophobic Tetrad near the C Terminus of the Secretory Na+-K+-2Cl- Cotransporter (NKCC1) Is Required for Its Correct Intracellular Processing*

    PubMed Central

    Nezu, Akihiro; Parvin, Most. Nahid; Turner, R. James

    2009-01-01

    Little is known about the intracellular folding and trafficking of integral membrane proteins. Here we identify a hydrophobic amino acid tetrad (ILLV) close to the C terminus of the secretory Na+-K+-2Cl- cotransporter (NKCC1) that is important for the proper intracellular processing of this protein. This tetrad appears in a C-terminal sequence pattern that is conserved across species in a number of members of the NKCC1 gene family (slc12) of electroneutral salt transporters. We studied the effects of various mutations of these amino acids on NKCC1 transiently transfected into HEK-293 cells. Our results show that mutation of two of these residues to alanine leads to a >50% reduction in expression and complex glycosylation levels and that multiple mutations to alanine have cumulative effects. By contrast, scrambling of these amino acids, or mutation of other nearby conserved C-terminal residues, has little effect on these parameters. Mutation of ILLV to AAAA reduces complex glycosylation of NKCC1 by ∼90% and results in a protein that does not form stable dimers and is retained in the endoplasmic reticulum in a highly aggregated state. Our results are consistent with the hypothesis that mutation of the hydrophobic tetrad ILLV to AAAA leads to the ab initio misfolding and concomitant aggregation of this NKCC1 mutant, resulting in its retention in the endoplasmic reticulum. PMID:19129177

  3. Impact of familial hypertrophic cardiomyopathy-linked mutations in the NH2 terminus of the RLC on β-myosin cross-bridge mechanics.

    PubMed

    Farman, Gerrie P; Muthu, Priya; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R

    2014-12-15

    Familial hypertrophic cardiomyopathy (HCM) is associated with mutations in sarcomeric proteins, including the myosin regulatory light chain (RLC). Here we studied the impact of three HCM mutations located in the NH2 terminus of the RLC on the molecular mechanism of β-myosin heavy chain (MHC) cross-bridge mechanics using the in vitro motility assay. To generate mutant β-myosin, native RLC was depleted from porcine cardiac MHC and reconstituted with mutant (A13T, F18L, and E22K) or wild-type (WT) human cardiac RLC. We characterized the mutant myosin force and motion generation capability in the presence of a frictional load. Compared with WT, all three mutants exhibited reductions in maximal actin filament velocity when tested under low or no frictional load. The actin-activated ATPase showed no significant difference between WT and HCM-mutant-reconstituted myosins. The decrease in velocity has been attributed to a significantly increased duty cycle, as was measured by the dependence of actin sliding velocity on myosin surface density, for all three mutant myosins. These results demonstrate a mutation-induced alteration in acto-myosin interactions that may contribute to the pathogenesis of HCM. PMID:25324513

  4. Vaccination of cattle with the N terminus of LppQ of Mycoplasma mycoides subsp. mycoides results in type III immune complex disease upon experimental infection.

    PubMed

    Mulongo, Musa; Frey, Joachim; Smith, Ken; Schnier, Christian; Wesonga, Hezron; Naessens, Jan; McKeever, Declan

    2015-05-01

    Contagious bovine pleuropneumonia (CBPP) is a serious respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides. Current vaccines against CBPP induce short-lived immunity and can cause severe postvaccine reactions. Previous studies have identified the N terminus of the transmembrane lipoprotein Q (LppQ-N') of M. mycoides subsp. mycoides as the major antigen and a possible virulence factor. We therefore immunized cattle with purified recombinant LppQ-N' formulated in Freund's adjuvant and challenged them with M. mycoides subsp. mycoides. Vaccinated animals showed a strong seroconversion to LppQ, but they exhibited significantly enhanced postchallenge glomerulonephritis compared to the placebo group (P = 0.021). Glomerulonephritis was characterized by features that suggested the development of antigen-antibody immune complexes. Clinical signs and gross pathological scores did not significantly differ between vaccinated and placebo groups. These findings reveal for the first time the pathogenesis of enhanced disease as a result of antibodies against LppQ during challenge and also argue against inclusion of LppQ-N' in a future subunit vaccine for CBPP. PMID:25733516

  5. The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage

    PubMed Central

    Alver, Robert C.; Zhang, Tianji; Josephrajan, Ajeetha; Fultz, Brandy L.; Hendrix, Chance J.; Das-Bradoo, Sapna; Bielinsky, Anja-Katrin

    2014-01-01

    Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-α and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair. PMID:24972833

  6. Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye.

    PubMed

    Hnedzko, Dziyana; McGee, Dennis W; Rozners, Eriks

    2016-09-15

    Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines. PMID:27430566

  7. Modular elements of the TPR domain in the Mps1 N terminus differentially target Mps1 to the centrosome and kinetochore.

    PubMed

    Marquardt, Joseph R; Perkins, Jennifer L; Beuoy, Kyle J; Fisk, Harold A

    2016-07-12

    Faithful segregation of chromosomes to two daughter cells is regulated by the formation of a bipolar mitotic spindle and the spindle assembly checkpoint, ensuring proper spindle function. Here we show that the proper localization of the kinase Mps1 (monopolar spindle 1) is critical to both these processes. Separate elements in the Mps1 N-terminal extension (NTE) and tetratricopeptide repeat (TPR) domains govern localization to either the kinetochore or the centrosome. The third TPR (TPR3) and the TPR-capping helix (C-helix) are each sufficient to target Mps1 to the centrosome. TPR3 binds to voltage-dependent anion channel 3, but although this is sufficient for centrosome targeting of Mps1, it is not necessary because of the presence of the C-helix. A version of Mps1 lacking both elements cannot localize to or function at the centrosome, but maintains kinetochore localization and spindle assembly checkpoint function, indicating that TPR3 and the C-helix define a bipartite localization determinant that is both necessary and sufficient to target Mps1 to the centrosome but dispensable for kinetochore targeting. In contrast, elements required for kinetochore targeting (the NTE and first two TPRs) are dispensable for centrosomal localization and function. These data are consistent with a separation of Mps1 function based on localization determinants within the N terminus. PMID:27339139

  8. The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells

    PubMed Central

    Caldas, Gina V.; Lynch, Tina R.; Anderson, Ryan; Afreen, Sana; Varma, Dileep; DeLuca, Jennifer G.

    2015-01-01

    The spindle assembly checkpoint is a surveillance mechanism that blocks anaphase onset until all chromosomes are properly attached to microtubules of the mitotic spindle. Checkpoint activity requires kinetochore localization of Mad1/Mad2 to inhibit activation of the anaphase promoting complex/cyclosome in the presence of unattached kinetochores. In budding yeast and Caenorhabditis elegans, Bub1, recruited to kinetochores through KNL1, recruits Mad1/Mad2 by direct linkage with Mad1. However, in human cells it is not yet established which kinetochore protein(s) function as the Mad1/Mad2 receptor. Both Bub1 and the RZZ complex have been implicated in Mad1/Mad2 kinetochore recruitment; however, their specific roles remain unclear. Here, we investigate the contributions of Bub1, RZZ and KNL1 to Mad1/Mad2 kinetochore recruitment. We find that the RZZ complex localizes to the N-terminus of KNL1, downstream of Bub1, to mediate robust Mad1/Mad2 kinetochore localization. Our data also point to the existence of a KNL1-, Bub1-independent mechanism for RZZ and Mad1/Mad2 kinetochore recruitment. Based on our results, we propose that in humans, the primary mediator for Mad1/Mad2 kinetochore localization is the RZZ complex. PMID:26581576

  9. The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1.

    PubMed

    Kahra, Dana; Kovermann, Michael; Wittung-Stafshede, Pernilla

    2016-01-01

    Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, KD, of ∼10(-14) M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones. PMID:26745413

  10. The N-terminus of histone H2B, but not that of histone H3 or its phosphorylation, is essential for chromosome condensation

    PubMed Central

    de la Barre, Anne-Elisabeth; Angelov, Dimitri; Molla, Annie; Dimitrov, Stefan

    2001-01-01

    We have studied the role of individual histone N-termini and the phosphorylation of histone H3 in chromosome condensation. Nucleosomes, reconstituted with histone octamers containing different combinations of recombinant full-length and tailless histones, were used as competitors for chromosome assembly in Xenopus egg extracts. Nucleosomes reconstituted with intact octamers inhibited chromosome condensation as efficiently as the native ones, while tailless nucleosomes were unable to affect this process. Importantly, the addition to the extract of particles containing only intact histone H2B strongly interfered with chromosome formation while such an effect was not observed with particles lacking the N-terminal tail of H2B. This demonstrates that the inhibition effect observed in the presence of competitor nucleosomes is mainly due to the N-terminus of this histone, which, therefore, is essential for chromosome condensation. Nucleosomes in which all histones but H3 were tailless did not impede chromosome formation. In addition, when competitor nucleosome particles were reconstituted with full-length H2A, H2B and H4 and histone H3 mutated at the phosphorylable serine 10 or serine 28, their inhibiting efficiency was identical to that of the native particles. Hence, the tail of H3, whether intact or phosphorylated, is not important for chromosome condensation. A novel hypothesis, termed ‘the ready production label’ was suggested to explain the role of histone H3 phosphorylation during cell division. PMID:11707409

  11. Targeting of protein phosphatases PP2A and PP2B to the C-terminus of the L-type calcium channel Ca v1.2.

    PubMed

    Xu, Hui; Ginsburg, Kenneth S; Hall, Duane D; Zimmermann, Maike; Stein, Ivar S; Zhang, Mingxu; Tandan, Samvit; Hill, Joseph A; Horne, Mary C; Bers, Donald; Hell, Johannes W

    2010-12-01

    The L-type Ca(2+) channel Ca(v)1.2 forms macromolecular signaling complexes that comprise the β(2) adrenergic receptor, trimeric G(s) protein, adenylyl cyclase, and cAMP-dependent protein kinase (PKA) for efficient signaling in heart and brain. The protein phosphatases PP2A and PP2B are part of this complex. PP2A counteracts increase in Ca(v)1.2 channel activity by PKA and other protein kinases, whereas PP2B can either augment or decrease Ca(v)1.2 currents in cardiomyocytes depending on the precise experimental conditions. We found that PP2A binds to two regions in the C-terminus of the central, pore-forming α(1) subunit of Ca(v)1.2: one region spans residues 1795-1818 and the other residues 1965-1971. PP2B binds immediately downstream of residue 1971. Injection of a peptide that contained residues 1965-1971 and displaced PP2A but not PP2B from endogenous Ca(v)1.2 increased basal and isoproterenol-stimulated L-type Ca(2+) currents in acutely isolated cardiomyocytes. Together with our biochemical data, these physiological results indicate that anchoring of PP2A at this site of Ca(v)1.2 in the heart negatively regulates cardiac L-type currents, likely by counterbalancing basal and stimulated phosphorylation that is mediated by PKA and possibly other kinases. PMID:21053940

  12. Specific sites in the C terminus of CTCF interact with the SA2 subunit of the cohesin complex and are required for cohesin-dependent insulation activity.

    PubMed

    Xiao, Tiaojiang; Wallace, Julie; Felsenfeld, Gary

    2011-06-01

    Recent studies have shown that the protein CTCF, which plays an important role in insulation and in large-scale organization of chromatin within the eukaryotic nucleus, depends for both activities on recruitment of the cohesin complex. We show here that the interaction of CTCF with the cohesin complex involves direct contacts between the cohesin subunit SA2 and specific regions of the C-terminal tail of CTCF. All other cohesin components are recruited through their interaction with SA2. Expression in vivo of CTCF mutants lacking the C-terminal domain, or with mutations at sites within it required for SA2 binding, disrupts the normal expression profile of the imprinted genes IGF2-H19 and also results in a loss of insulation activity. Taken together, our results demonstrate that specific sites on the C terminus of CTCF are essential for cohesin binding and insulator function. The only direct interaction between CTCF and cohesin involves contact with SA2, which is external to the cohesin ring. This suggests that in recruiting cohesin to CTCF, SA2 could bind first and the ring could assemble subsequently. PMID:21444719

  13. Public service user terminus study compendium of terminus equipment

    NASA Technical Reports Server (NTRS)

    1979-01-01

    General descriptions and specifications are given for equipments which facilitate satellite and terrestrial communications delivery by acting as interfaces between a human, mechanical, or electrical information generator (or source) and the communication system. Manufactures and suppliers are given as well as the purchase, service, or lease costs of various products listed under the following cateories: voice/telephony/facsimile equipment; data/graphics terminals; full motion and processes video equipment; and multiple access equipment.

  14. Comparative analysis of SV40 17kT and LT function in vivo demonstrates that LT's C-terminus re-programs hepatic gene expression and is necessary for tumorigenesis in the liver

    PubMed Central

    Comerford, S A; Schultz, N; Hinnant, E A; Klapproth, S; Hammer, R E

    2012-01-01

    Transformation by Simian Virus 40 (SV40) large T antigen (LT) is mediated in large part by its interaction with a variety of cellular proteins at distinct binding domains within LT. While the interaction of LT's N-terminus with the tumor suppressor Rb is absolutely required for LT-dependent transformation, the requirement for the interaction of LT's C-terminus with p53 is less clear and cell- and context-dependent. Here, we report a line of transgenic mice expressing a doxycycline-inducible liver-specific viral transcript that produces abundant 17kT, a naturally occurring SV40 early product that is co-linear with LT for the first 131 amino acids and that binds to Rb, but not p53. Comparative analysis of livers of transgenic mice expressing either 17kT or full length LT demonstrates that 17kT stimulates cell proliferation and induces hepatic hyperplasia but is incapable of inducing hepatic dysplasia or promoting hepatocarcinogenesis. Gene expression profiling demonstrates that 17kT and LT invoke a set of shared molecular signatures consistent with the action of LT's N-terminus on Rb-E2F-mediated control of hepatocyte transcription. However, 17kT also induces a unique set of genes, many of which are known transcriptional targets of p53, while LT actively suppresses them. LT also uniquely deregulates the expression of a subset of genes within the imprinted network and rapidly re-programs hepatocyte gene expression to a more fetal-like state. Finally, we provide evidence that the LT/p53 complex provides a gain-of-function for LT-dependent transformation in the liver, and confirm the absolute requirement for LT's C-terminus for liver tumor development by demonstrating that phosphatase and tensin homolog (PTEN)-deficiency readily cooperates with LT, but not 17kT, for tumorigenesis. These results confirm independent and inter-dependent functions for LT's N- and C-terminus and emphasize differences in the requirements for LT's C-terminus in cell-type dependent

  15. NMR structures of anti-HIV D-peptides derived from the N-terminus of viral chemokine vMIP-II

    SciTech Connect

    Mori, Mayuko; Liu Dongxiang; Kumar, Santosh; Huang Ziwei; E-mail: ziweihuang@burnham.org

    2005-09-30

    The viral macrophage inflammatory protein-II (vMIP-II) encoded by Kaposi's sarcoma-associated herpesvirus has unique biological activities in that it blocks the cell entry by several different human immunodeficiency virus type 1 (HIV-1) strains via chemokine receptors including CXCR4 and CCR5. In this paper, we report the solution structure of all-D-amino acid peptides derived from the N-terminus of vMIP-II, which have been shown to have strong CXCR4 binding activity and potently inhibit HIV-1 entry via CXCR4, by using long mixing time two-dimensional nuclear Overhauser enhancement spectroscopy experiments. Both of all-D-peptides vMIP-II (1-10) and vMIP-II (1-21), which are designated as DV3 and DV1, respectively, have higher CXCR4 binding ability than their L-peptide counterparts. They are partially structured in aqueous solution, displaying a turn-like structure over residues 5-8. The small temperature coefficients of His-6 amide proton for both peptides also suggest the formation of a small hydrophobic pocket centered on His-6. The structural features of DV3 are very similar to the reported solution structure of all-L-peptide vMIP-II (1-10) [M.P. Crump, E. Elisseeva, J. Gong, I. Clark-Lewis, B.D. Sykes, Structure/function of human herpesvirus-8 MIP-II (1-71) and the antagonist N-terminal segment (1-10), FEBS Lett. 489 (2001) 171], which is consistent with the notion that D- and L-enantiomeric peptides can adopt mirror image conformations. The NMR structures of the D-peptides provide a structural basis to understand their mechanism of action and design new peptidomimetic analogs to further explore the structure-activity relationship of D-peptide ligand binding to CXCR4.

  16. A Nonviral Peptide Can Replace the Entire N Terminus of Zucchini Yellow Mosaic Potyvirus Coat Protein and Permits Viral Systemic Infection

    PubMed Central

    Arazi, T.; Shiboleth, Y. M.; Gal-On, A.

    2001-01-01

    Systematic deletion and peptide tagging of the amino-terminal domain (NT, ∼43 amino acids) of an attenuated zucchini yellow mosaic potyvirus (ZYMV-AGII) coat protein (CP) were used to elucidate its role in viral systemic infection. Deletion mutants truncated by 8, 13, and 33 amino acid residues from the CP-NT 5′ end were systemically infectious and produced symptoms similar to those of the AGII virus. Tagging these deletion mutants with either human c-Myc (Myc) or hexahistidine peptides maintained viral infectivity. Similarly, addition of these peptides to the intact AGII CP-NT did not affect viral life cycle. To determine which parts, if any, of the CP-NT are essential for viral systemic infection, a series of Myc-tagged mutants with 8 to 43 amino acids removed from the CP-NT were constructed. All Myc-tagged CP-NT deletion mutants, including those from which virtually all the viral CP-NT had been eliminated, were able to encapsidate and cause systemic infection. Furthermore, chimeric viruses with deletions of up to 33 amino acids from CP-NT produced symptoms indistinguishable from those caused by the parental AGII virus. In contrast to CP-NT Myc fusion, addition of the foot-and-mouth disease virus (FMDV) immunogenic epitope to AGII CP-NT did not permit systemic infection. However, fusion of the Myc peptide to the N terminus of the FMDV peptide restored the capability of the virus to spread systemically. We have demonstrated that all CP-NT fused peptides were exposed on the virion surface, masking natural CP immunogenic determinants. Our findings demonstrate that CP-NT is not essential for ZYMV spread and that it can be replaced by an appropriate foreign peptide while maintaining systemic infectivity. PMID:11413299

  17. The CENP-T C-Terminus Is Exclusively Proximal to H3.1 and not to H3.2 or H3.3

    PubMed Central

    Abendroth, Christian; Hofmeister, Antje; Hake, Sandra B.; Kamweru, Paul K.; Miess, Elke; Dornblut, Carsten; Küffner, Isabell; Deng, Wen; Leonhardt, Heinrich; Orthaus, Sandra; Hoischen, Christian; Diekmann, Stephan

    2015-01-01

    The kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. The centromere–kinetochore complex contains specific nucleosomes and nucleosomal particles. CENP-A replaces canonical H3 in centromeric nucleosomes, defining centromeric chromatin. Next to CENP-A, the CCAN multi-protein complex settles which contains CENP-T/W/S/X. These four proteins are described to form a nucleosomal particle at centromeres. We had found the CENP-T C-terminus and the CENP-S termini next to histone H3.1 but not to CENP-A, suggesting that the Constitutive Centromere-Associated Network (CCAN) bridges a CENP-A- and a H3-containing nucleosome. Here, we show by in vivo FRET that this proximity between CENP-T and H3 is specific for H3.1 but neither for the H3.1 mutants H3.1C96A and H3.1C110A nor for H3.2 or H3.3. We also found CENP-M next to H3.1 but not to these H3.1 mutants. Consistently, we detected CENP-M next to CENP-S. These data elucidate the local molecular neighborhood of CCAN proteins next to a H3.1-containing centromeric nucleosome. They also indicate an exclusive position of H3.1 clearly distinct from H3.2, thus documenting a local, and potentially also functional, difference between H3.1 and H3.2. PMID:25775162

  18. Engineering the Expression and Characterization of Two Novel Laccase Isoenzymes from Coprinus comatus in Pichia pastoris by Fusing an Additional Ten Amino Acids Tag at N-Terminus

    PubMed Central

    Gu, Chunjuan; Zheng, Fei; Long, Liangkun; Wang, Jing; Ding, Shaojun

    2014-01-01

    The detail understanding of physiological/biochemical characteristics of individual laccase isoenzymes in fungi is necessary for fundamental and application purposes, but our knowledge is still limited for most of fungi due to difficult to express laccases heterologously. In this study, two novel laccase genes, named lac3 and lac4, encoding proteins of 547 and 532-amino acids preceded by 28 and 16-residue signal peptides, respectively, were cloned from the edible basidiomycete Coprinus comatus. They showed 70% identity but much lower homology with other fungal laccases at protein level (less than 58%). Two novel laccase isoenzymes were successfully expressed in Pichia pastoris by fusing an additional 10 amino acids (Thr-Pro-Phe-Pro-Pro-Phe-Asn-Thr-Asn-Ser) tag at N-terminus, and the volumetric activities could be dramatically enhanced from undetectable level to 689 and 1465 IU/l for Lac3 and Lac4, respectively. Both laccases possessed the lowest Km and highest kcat/Km value towards syringaldazine, followed by ABTS, guaiacol and 2,6-dimethylphenol similar as the low redox potential laccases from other microorganisms. Lac3 and Lac4 showed resistant to SDS, and retained 31.86% and 43.08% activity in the presence of 100 mM SDS, respectively. Lac3 exhibited higher decolorization efficiency than Lac4 for eleven out of thirteen different dyes, which may attribute to the relatively higher catalytic efficiency of Lac3 than Lac4 (in terms of kcat/Km) towards syringaldazine and ABTS. The mild synergistic decolorization by two laccases was observed for triphenylmethane dyes but not for anthraquinone and azo dyes. PMID:24710109

  19. Structure–activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation

    PubMed Central

    Hay, Debbie L; Harris, Paul WR; Kowalczyk, Renata; Brimble, Margaret A; Rathbone, Dan L; Barwell, James; Conner, Alex C; Poyner, David R

    2014-01-01

    Background and Purpose: The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1–7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3–6 and 8–9, excluding Cys-2 and Cys-7. Experimental Approach: CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and β-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key Results: Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at β-arrestin translocation was reduced by ninefold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and β-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and Implications: Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1–7 ring also contribute to agonist activity. PMID:24125506

  20. Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti-trp RNA-binding attenuation protein.

    PubMed

    Sachleben, Joseph R; McElroy, Craig A; Gollnick, Paul; Foster, Mark P

    2010-08-31

    Anti-TRAP (AT) is a small zinc-binding protein that regulates tryptophan biosynthesis in Bacillus subtilis by binding to tryptophan-bound trp RNA-binding attenuation protein (TRAP), thereby preventing it from binding RNA, and allowing transcription and translation of the trpEDCFBA operon. Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT(12), composed of a tetramer of trimers, AT(3). Structural and biochemical studies suggest that only trimeric AT is active for binding to TRAP. Our chromatographic and spectroscopic data revealed that a large fraction of recombinantly overexpressed AT retains the N-formyl group (fAT), presumably due to incomplete N-formyl-methionine processing by peptide deformylase. Hydrodynamic parameters from NMR relaxation and diffusion measurements showed that fAT is exclusively trimeric (AT(3)), while (deformylated) AT exhibits slow exchange between both trimeric and dodecameric forms. We examined this equilibrium using NMR spectroscopy and found that oligomerization of active AT(3) to form inactive AT(12) is linked to protonation of the amino terminus. Global analysis of the pH dependence of the trimer-dodecamer equilibrium revealed a near physiological pK(a) for the N-terminal amine of AT and yielded a pH-dependent oligomerization equilibrium constant. Estimates of excluded volume effects due to molecular crowding suggest the oligomerization equilibrium may be physiologically important. Because deprotonation favors "active" trimeric AT and protonation favors "inactive" dodecameric AT, our findings illuminate a possible mechanism for sensing and responding to changes in cellular pH. PMID:20713740

  1. The amino terminus of the F1-ATPase beta-subunit precursor functions as an intramolecular chaperone to facilitate mitochondrial protein import.

    PubMed Central

    Hájek, P; Koh, J Y; Jones, L; Bedwell, D M

    1997-01-01

    Mitochondrial import signals have been shown to function in many steps of mitochondrial protein import. Previous studies have shown that the F1-ATPase beta-subunit precursor (pre-F1beta) of the yeast Saccharomyces cerevisiae contains an extended, functionally redundant mitochondrial import signal at its amino terminus. However, the full significance of this functionally redundant targeting sequence has not been determined. We now report that the extended pre-F1beta signal acts to maintain the precursor in an import-competent conformation prior to import, in addition to its previously characterized roles in mitochondrial targeting and translocation. We found that this extended signal is required for the efficient posttranslational mitochondrial import of pre-F1beta both in vivo and in vitro. To determine whether the pre-F1beta signal directly influences precursor conformation, fusion proteins that contain wild-type and mutant forms of the pre-F1beta import signal attached to the model passenger protein dihydrofolate reductase (DHFR) were constructed. Deletions that reduced the import signal to a minimal functional unit decreased both the half-time of precursor folding and the efficiency of mitochondrial import. To confirm that the reduced mitochondrial import associated with this truncated signal was due to a defect in its ability to maintain DHFR in a loosely folded conformation, we introduced structurally destabilizing missense mutations into the DHFR passenger to block precursor folding independently of the import signal. We found that the truncated signal imported this destabilized form of DHFR as efficiently as the intact targeting signal, indicating that the primary defect associated with the minimal signal is an inability to maintain the precursor in a loosely folded conformation. Our results suggest that the loss of this intramolecular chaperone function leads to defects in the early stages of the import process. PMID:9372949

  2. Tectonic problems revisited: The eastern terminus of the Miocene Garlock fault and the amount of slip on the southern Death Valley fault zone

    SciTech Connect

    Davis, G.A. . Dept. of Geological Sciences); Burchfiel, B.C. . Dept. of Earth, Atmospheric, and Planetary Science)

    1993-04-01

    Prior to 1973, the eastern end of the sinistral Garlock fault (GF) was generally assumed to lie at its junction with the southern Death Valley fault zone (SDVFZ). Although there seems little doubt that the Quaternary GF ends there in a complicated zone of interaction with the dextral SDVFZ, the location of the eastern terminus of a late Miocene GF has been more controversial. Davis and Burchfiel (1973) analyzed the geometry of geologic terranes and features offset > 50 km along the eastern half of the present GF (several within 15 km of the SDVFZ), that it had been offset dextrally [approximately] 8 km along the younger zone, and that the GF was an intracontinental transform structure separating a more extended northern terrane (Basin-and-Range) from a less extended southern terrane (Mojave Desert). USC field studies in areas east of the SDVFZ/GF intersection support the original contention of Davis and Burchfiel that the Miocene GF lies beneath alluvial deposits of Kingston Wash. A left-slip fault with a displacement of [approximately]3 km has been identified beneath upper reaches of the Wash north of Kingston Spring. It lies above the older (and coeval ) west-rooting, mid- to Late Miocene Kingston Range detachment fault, and it appears to bound the southern margin of a distributed breakaway zone of N-S-striking normal faults that distends the Kingston Peak pluton (ca 12.5 Ma). The authors believe that the cumulative effects of pre- and post-12.5 Ma east-west extension north of this buried fault may explain the geometry of offset terranes along the GF in areas west of the SDVFZ. If so, total dextral slip on the younger, cross-cutting SDVFZ must be 10 km or less.

  3. The co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation decisions between proteasomal and lysosomal pathways.

    PubMed

    Shin, Youngah; Klucken, Jochen; Patterson, Cam; Hyman, Bradley T; McLean, Pamela J

    2005-06-24

    Alpha-synuclein is a major component of Lewy bodies, the pathological hallmark of Parkinson disease, dementia with Lewy bodies, and related disorders. Misfolding and aggregation of alpha-synuclein is thought to be a critical cofactor in the pathogenesis of certain neurodegenerative diseases. In the current study, we investigate the role of the carboxyl terminus of Hsp70-interacting protein (CHIP) in alpha-synuclein aggregation. We demonstrate that CHIP is a component of Lewy bodies in the human brain, where it colocalizes with alpha-synuclein and Hsp70. In a cell culture model, endogenous CHIP colocalizes with alpha-synuclein and Hsp70 in intracellular inclusions, and overexpression of CHIP inhibits alpha-synuclein inclusion formation and reduces alpha-synuclein protein levels. We demonstrate that CHIP can mediate alpha-synuclein degradation by two discrete mechanisms that can be dissected using deletion mutants; the tetratricopeptide repeat domain is critical for proteasomal degradation, whereas the U-box domain is sufficient to direct alpha-synuclein toward the lysosomal degradation pathway. Furthermore, alpha-synuclein, synphilin-1, and Hsp70 all coimmunoprecipitate with CHIP, raising the possibility of a direct alpha-synuclein-CHIP interaction. The fact that the tetratricopeptide repeat domain is required for the effects of CHIP on alpha-synuclein inclusion morphology, number of inclusions, and proteasomal degradation as well as the direct interaction of CHIP with Hsp70 implicates a cooperation of CHIP and Hsp70 in these processes. Taken together, these data suggest that CHIP acts a molecular switch between proteasomal and lysosomal degradation pathways. PMID:15845543

  4. Twenty amino acids at the C-terminus of PA-X are associated with increased influenza A virus replication and pathogenicity

    PubMed Central

    Gao, Huijie; Sun, Honglei; Hu, Jiao; Qi, Lu; Wang, Jinliang; Xiong, Xin; Wang, Yu; He, Qiming; Lin, Yang; Kong, Weili; Seng, Lai-Giea; Pu, Juan; Chang, Kin-Chow; Liu, Xiufan; Liu, Jinhua

    2015-01-01

    The PA-X protein, arising from ribosomal frameshift during PA translation, was recently discovered in influenza A virus (IAV). The C-terminal domain ‘X’ of PA-X proteins in IAVs can be classified as full-length (61 aa) or truncated (41 aa). In the main, avian influenza viruses express full-length PA-X proteins, whilst 2009 pandemic H1N1 (pH1N1) influenza viruses harbour truncated PA proteins. The truncated form lacks aa 232–252 of the full-length PA-X protein. The significance of PA-X length in virus function remains unclear. To address this issue, we constructed a set of contemporary influenza viruses (pH1N1, avian H5N1 and H9N2) with full and truncated PA-X by reverse genetics to compare their replication and host pathogenicity. All full-length PA-X viruses in human A549 cells conferred 10- to 100-fold increase in viral replication and 5–8 % increase in apoptosis relative to corresponding truncated PA-X viruses. Full-length PA-X viruses were more virulent and caused more severe inflammatory responses in mice. Furthermore, aa 233–252 at the C terminus of PA-X strongly suppressed co-transfected gene expression by ∼50 %, suggesting that these terminal 20 aa could play a role in enhancing viral replication and contribute to virulence. PMID:25877935

  5. A critical role for the C-terminus of Nej1 protein in Lif1p association, DNA binding and non-homologous end-joining.

    PubMed

    Sulek, M; Yarrington, R; McGibbon, G; Boeke, J D; Junop, M

    2007-12-01

    A predominant pathway implicated in repair of DNA double-strand breaks (DSBs) is the evolutionarily conserved non-homologous end-joining (NHEJ) pathway. Among the major constituents of this pathway in Saccharomyces cerevisiae is Nej1p, for which a biochemical function has yet to be determined. In this work we demonstrate that Nej1p exhibits a DNA binding activity (KD approximately 1.8 microM) comparable to Lif1p. Although binding is enhanced with larger substrates (>300 bp), short approximately 20 bp substrates can suffice. This DNA binding activity is the first biochemical evidence supporting the idea that Nej1p plays a direct role in the repair of double-strand breaks. The C-terminus of Nej1p is required for interaction with Lif1p and is sufficient for DNA binding. Structural characterization reveals that Nej1p exists as a dimer, and that residues 1-244 are sufficient for dimer formation. Nej1p (aa 1-244) is shown to be defective in end-joining in vivo. Preliminary functional and structural studies on the Nej1p-Lif1p complex suggest that the proteins stably co-purify and the complex binds DNA with a higher affinity than each independent component. The significance of these results is discussed with reference to current literature on Nej1p and other end-joining factors (mammalian and yeast), specifically the recently identified putative mammalian homologue of Nej1p, XLF/Cernunnos. PMID:17765666

  6. Regulation of fragile X mental retardation 1 protein by C-terminus of Hsc70-interacting protein depends on its phosphorylation status.

    PubMed

    Choi, Ye Na; Jeong, Dar Heum; Lee, Ji Sun; Yoo, Soon Ji

    2014-10-10

    The fragile X mental retardation 1 (FMR1) protein binds mRNA and acts as a negative regulator of translation. Lack of FMR1 causes the most common neurological disorder, fragile X syndrome, while its overexpression is associated with metastasis of breast cancer. Its activity has been well-studied in nervous tissue, but recent evidence as well as its role in cancer indicates that it also acts in other tissues. We have investigated the expression of FMR1 in brain and other tissues of mouse and examined its regulation. We detected expression of FMR1 in liver and heart tissues of mice as well as in brain tissue, supporting other contentions that it acts in non-nervous tissue. Expression of FMR1 inversely correlated with expression of the C-terminus of Hsc70-interacting protein (CHIP) and, based on the known activity of CHIP in protein homeostasis, we suggest that CHIP regulates expression of FMR1. CHIP ubiquitinated FMR1 for proteasomal degradation in a molecular chaperone-independent manner. FMR1 expression was reduced following treatment with okadaic acid, a phosphatase inhibitor, but not in CHIP-depleted cells. Also, a non-phospho FMR1 mutant was much less efficiently ubiquitinated by CHIP and had a longer half-life compared to either wild-type FMR or a phospho-mimic mutant. Taken together, our results demonstrate that CHIP regulates the levels of FMR1 as an E3 ubiquitin ligase in phosphorylation-dependent manner, suggesting that CHIP regulates FMR1-mediated translational repression by regulating the levels of FMR1. PMID:25268320

  7. Carboxyl terminus of HSC70-interacting protein (CHIP) down-regulates NF-κB-inducing kinase (NIK) and suppresses NIK-induced liver injury.

    PubMed

    Jiang, Bijie; Shen, Hong; Chen, Zheng; Yin, Lei; Zan, Linsen; Rui, Liangyou

    2015-05-01

    Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease. PMID:25792747

  8. The cytoplasmic C-terminus of polycystin-1 increases cell proliferation in kidney epithelial cells through serum-activated and Ca{sup 2+}-dependent pathway(s)

    SciTech Connect

    Manzati, Elisa; Aguiari, Gianluca; Banzi, Manuela; Manzati, Michele; Selvatici, Rita; Falzarano, Sofia; Maestri, Iva; Pinton, Paolo; Rizzuto, Rosario; Senno, Laura del . E-mail: sen@unife.it

    2005-04-01

    Polycystin-1 (PC1) is a large transmembrane protein important in renal differentiation and defective in most cases of autosomal dominant polycystic kidney disease (ADPKD), a common cause of renal failure in adults. Although the genetic basis of ADPKD has been elucidated, molecular and cellular mechanisms responsible for the dysregulation of epithelial cell growth in ADPKD cysts are still not well defined. We approached this issue by investigating the role of the carboxyl cytoplasmic domain of PC1 involved in signal transduction on the control of kidney cell proliferation. Therefore, we generated human HEK293 cells stably expressing the PC1 cytoplasmic tail as a membrane targeted TrkA-PC1 chimeric receptor protein (TrkPC1). We found that TrkPC1 increased cell proliferation through an increase in cytoplasmic Ca{sup 2+} levels and activation of PKC{alpha}, thereby upregulating D1 and D3 cyclin, downregulating p21{sup waf1} and p27{sup kip1} cyclin inhibitors, and thus inducing cell cycle progression from G0/G1 to the S phase. Interestingly, TrkPC1-dependent Ca{sup 2+} increase and PKC{alpha} activation are not constitutive, but require serum factor(s) as parallel component. In agreement with this observation, a significant increase in ERK1/2 phosphorylation was observed. Consistently, inhibitors specifically blocking either PKC{alpha} or ERK1/2 prevented the TrkPC1-dependent proliferation increase. NGF, the TrkA ligand, blocked this increase. We propose that in kidney epithelial cells the overexpression of PC1 C-terminus upregulates serum-evoked intracellular Ca{sup 2+} by counteracting the growth-suppression activity of endogenous PC1 and leading to an increase in cell proliferation.

  9. In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus α-subunit from Synechocystis sp. PCC 6803.

    PubMed

    Luján, María A; Martínez, Jesús I; Alonso, Pablo J; Torrado, Alejandro; Roncel, Mercedes; Ortega, José M; Sancho, Javier; Picorel, Rafael

    2015-11-01

    The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) cytochrome b559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit cytochrome b559 like structure. The features of the new structure were determined by UV-Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane. PMID:26183783

  10. Gβγ Binds to the Extreme C Terminus of SNAP25 to Mediate the Action of Gi/o-Coupled G Protein-Coupled Receptors.

    PubMed

    Zurawski, Zack; Rodriguez, Shelagh; Hyde, Karren; Alford, Simon; Hamm, Heidi E

    2016-01-01

    Gi/o-coupled G protein-coupled receptors can exert an inhibitory effect on vesicle release through several G protein-driven mechanisms, more than one of which may be concurrently present in individual presynaptic terminals. The synaptosomal-associated protein of 25 kDa (SNAP25) is a key downstream effector of Gβγ subunits. It has previously been shown that proteolytic cleavage of SNAP25 by botulinum toxin A reduces the ability of Gβγ to compete with the calcium sensor synaptotagmin 1 (Syt1) for binding to SNAP25 in a calcium-dependent manner. These truncated SNAP25 proteins sustain a low level of exocytosis but are unable to support serotonin-mediated inhibition of exocytosis in lamprey spinal neurons. Here, we generate a SNAP25 extreme C-terminal mutant that is deficient in its ability to bind Gβγ while retaining normal calcium-dependent Syt1 binding to soluble N-ethylmaleimide attachment protein receptor (SNARE) and vesicle release. The SNAP25Δ3 mutant, in which residue G204 is replaced by a stop codon, features a partial reduction in Gβ1γ2 binding in vitro as well as a partial reduction in the ability of the lamprey 5-hydroxytryptamine1b-type serotonin receptor to reduce excitatory postsynaptic current amplitudes, an effect previously shown to be mediated through the interaction of Gβγ with SNAP25. Syt1 calcium-dependent binding to SNAP25Δ3 was reduced by a small extent compared with the wild type. We conclude that the extreme C terminus of SNAP25 is a critical region for the Gβγ-SNARE interaction. PMID:26519224

  11. Gabaculine-resistant glutamate 1-semialdehyde aminotransferase of Synechococcus. Deletion of a tripeptide close to the NH2 terminus and internal amino acid substitution.

    PubMed

    Grimm, B; Smith, A J; Kannangara, C G; Smith, M

    1991-07-01

    Glutamate 1-semialdehyde aminotransferase (GSA-AT) is the last enzyme in the C5 pathway converting glutamate into the tetrapyrrole precursor delta-aminolevulinate in plants, algae, and several bacteria. Sequence analysis of the genes encoding GSA-AT in barley, Synechococcus, and Escherichia coli revealed 50-70% similarity in the primary structures of the proteins. The enzyme is inhibited rapidly by gabaculine when added in approximately stoichiometric amounts with the enzyme. A gabaculine-tolerant Synechococcus strain, GR6, was found to produce a GSA-AT less sensitive to the inhibitor. Accordingly, the mutant gene was isolated and sequenced. In comparison with the wild-type gene it contains a deletion of nine nucleotides (position 12-20) and a guanine to adenine substitution (position 743). This resulted in the loss of the amino acids serine, proline, and phenylalanine (position 5-7) close to the NH2 terminus of the enzyme and an exchange of Met-248 for isoleucine in the middle of the polypeptide chain. Wild-type and mutant GSA-AT were expressed in E. coli and purified close to homogeneity. Although the specific activity of the mutant GSA-AT was only one-fifth of the wild type, it displayed a 100-fold increased resistance to gabaculine. Peaks in the absorption spectrum of the purified recombinant GSA-ATs at 335 and 417 nm are typical of a transaminase containing a B6 cofactor. Incubation with substrate and with inhibitor induced spectral changes characteristic of other gabaculine-sensitive, B6-requiring enzymes. PMID:1905724

  12. Role of the cystine-knot motif at the C-terminus of rat mucin protein Muc2 in dimer formation and secretion.

    PubMed Central

    Bell, S L; Xu, G; Forstner, J F

    2001-01-01

    DNA constructs based on the 534-amino-acid C-terminus of rat mucin protein Muc2 (RMC), were transfected into COS cells and the resultant (35)S-labelled dimers and monomers were detected by SDS/PAGE of immunoprecipitates. The cystine-knot construct, encoding the C-terminal 115 amino acids, appeared in cell lysates as a 45 kDa dimer, but was not secreted. A construct, devoid of the cystine knot, failed to form dimers. Site-specific mutagenesis within the cystine knot was performed on a conserved unpaired cysteine (designated Cys-X), which has been implicated in some cystine-knot-containing growth factors as being important for intermolecular disulphide-bond formation. Dimerization of RMC was effectively abolished. Each cysteine (Cys-1-Cys-6) comprising the three intramolecular disulphide bonds of the cystine knot was then mutated. Dimer formation was impaired in each case, although much less so for the Cys-3 mutant than the others. Abnormal high-molecular-mass, disulphide-dependent aggregates formed with mutations Cys-1, Cys-2, Cys-4 and Cys-5(,) and were poorly secreted. It is concluded that the intact cystine-knot domain is essential for dimerization of the C-terminal domain of rat Muc2, and that residue Cys-X in the knot plays a key role. Th