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Sample records for c-terminal telopeptide concentration

  1. Efficacy of the C-terminal telopeptide test in predicting the development of bisphosphonate-related osteonecrosis of the jaw: a systematic review.

    PubMed

    Dal Prá, K J; Lemos, C A A; Okamoto, R; Soubhia, A M P; Pellizzer, E P

    2017-02-01

    This systematic review evaluated the efficacy of the morning fasting serum C-terminal telopeptide (CTX) test in predicting the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ). A comprehensive search of studies published up to March 2016, and listed in the PubMed/MEDLINE, Web of Science, and Cochrane Library databases, was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. This review has been registered in the PROSPERO international prospective register of systematic reviews (CRD42016036717). The search identified 542 publications; eight studies were finally deemed eligible for inclusion according to the study criteria. These studies included a total 1442 patients (mean age 66.7 years). The most prescribed drug was alendronate, with osteoporosis being the most frequent indication for the prescription of bisphosphonates. Tooth extraction was the most common trigger for BRONJ. Of all patients evaluated after bisphosphonate treatment, only 24 (1.7%) developed BRONJ. All eight of the selected studies found that CTX levels were not predictive of the development of BRONJ. In conclusion, this systematic review indicates that the CTX test has no predictive value in determining the risk of osteonecrosis in patients taking bisphosphonates.

  2. Study of the distribution by age group of serum cross-linked C-terminal telopeptide of type I collagen and procollagen type I N-propeptide in healthy Japanese women to establish reference values.

    PubMed

    Nomura, Yoshiyuki; Yoshizaki, Atsuo; Yoshikata, Hiromi; Kikuchi, Ritsuko; Sakakibara, Hideya; Chaki, Osamu; Fukunaga, Masao; Hirahara, Fumiki

    2013-11-01

    Osteoporosis prevention is an important public health goal. Bone turnover markers are clinically measured to assess bone strength. C-terminal telopeptide of type I collagen (CTX) is released when collagens degrade and serves as an indicator of bone resorption. Simple CTX immunoassays are now available. However, serum CTX (sCTX) reference ranges for Japanese women are lacking. Procollagen type I N-propeptide (intact P1NP) reflects osteoblast activity, serving as a marker of bone formation. Because sCTX and intact P1NP are clinically applied as bone turnover markers, we determined reference ranges for both sCTX and intact P1NP in healthy Japanese women. We collected 228 blood samples from healthy Japanese women aged 19-83 years, grouped by age and menopausal status. We measured sCTX and intact P1NP and examined their correlation. sCTX values differed significantly between the two consecutive decade groups encompassing 19-39 years of age, intact P1NP values between 20 and 30 s, between post-menopausal 50 and 60 s, and between pre-and post-menopausal women in their 50 s. The mean sCTX of 91 healthy pre-menopausal women was 0.255 (0.100-0.653) ng/mL, the intact P1NP in 90 women 33.2 (17.1-64.7) μg/L. Corresponding values for post-menopausal women were 0.345 (0.115-1.030) ng/mL and 41.6 (21.9-79.1) μg/L. sCTX correlated with intact P1NP. Bone resorption markers are measured to assess anti-resorption agents, bone formation markers to assess the effects of bone-forming agents. The sCTX and intact P1NP reference values determined herein, in healthy Japanese women, are expected to be useful for osteoporosis treatment, assessment of fracture risk, and other clinical applications.

  3. Kaposi's sarcoma herpesvirus C-terminal LANA concentrates at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes

    SciTech Connect

    Kelley-Clarke, Brenna; Ballestas, Mary E.; Komatsu, Takashi; Kaye, Kenneth M. . E-mail: kkaye@rics.bwh.harvard.edu

    2007-01-20

    The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) tethers KSHV terminal repeat (TR) DNA to mitotic chromosomes to efficiently segregate episomes to progeny nuclei. LANA contains N- and C-terminal chromosome binding regions. We now show that C-terminal LANA preferentially concentrates to paired dots at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes through residues 996-1139. Deletions within C-terminal LANA abolished both self-association and chromosome binding, consistent with a requirement for self-association to bind chromosomes. A deletion abolishing TR DNA binding did not affect chromosome targeting, indicating LANA's localization is not due to binding its recognition sequence in chromosomal DNA. LANA distributed similarly on human and non-human mitotic chromosomes. These results are consistent with C-terminal LANA interacting with a cell factor that concentrates at pericentromeric and peri-telomeric regions of mitotic chromosomes.

  4. Effects of a one year physical activity program on serum C Terminal Agrin Fragment (CAF) concentrations among mobility limited older adults

    Technology Transfer Automated Retrieval System (TEKTRAN)

    OBJECTIVES: C terminal Agrin Fragment (CAF) has been proposed as a potential circulating biomarker for predicting changes in physical function among older adults. To determine the effect of a one year PA intervention on changes in CAF concentrations and to evaluate baseline and longitudinal associat...

  5. Serum Concentrations of Ubiquitin C-Terminal Hydrolase-L1 and Glial Fibrillary Acidic Protein after Pediatric Traumatic Brain Injury

    PubMed Central

    Mondello, Stefania; Kobeissy, Firas; Vestri, Annarita; Hayes, Ronald L.; Kochanek, Patrick M.; Berger, Rachel P.

    2016-01-01

    Objective reliable markers to assess traumatic brain injury (TBI) and predict outcome soon after injury are a highly needed tool for optimizing management of pediatric TBI. We assessed serum concentrations of Glial Fibrillary Acidic Protein (GFAP) and Ubiquitin C-Terminal Hydrolase-L1 (UCH-L1) in a cohort of 45 children with clinical diagnosis of TBI (Glasgow Coma Scale [GCS] 3–15) and 40 healthy subjects, evaluated their associations with clinical characteristics and outcomes, and compared their performance to previously published data on two well-studied blood biomarkers, S100B and MBP. We observed higher serum levels of GFAP and UCH-L1 in brain-injured children compared with controls and also demonstrated a step-wise increase of biomarker concentrations over the continuum of severity from mild to severe TBI. Furthermore, while we found that only the neuronal biomarker UCH-L1 holds potential to detect acute intracranial lesions as assessed by computed tomography (CT), both markers were substantially increased in TBI patients even with a normal CT suggesting the presence of undetected microstructural injuries. Serum UCH-L1 and GFAP concentrations also strongly predicted poor outcome and performed better than S100B and MBP. Our results point to a role of GFAP and UCH-L1 as candidate biomarkers for pediatric TBI. Further studies are warranted. PMID:27319802

  6. Kinetic Analysis of the Digestion of Bovine Type I Collagen Telopeptides with Porcine Pepsin.

    PubMed

    Qian, Jun; Okada, Yukari; Ogura, Takayuki; Tanaka, Keisuke; Hattori, Shunji; Ito, Shinji; Satoh, Junko; Takita, Teisuke; Yasukawa, Kiyoshi

    2016-01-01

    Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N- and C-terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N- and in the C-terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N-terminal and in the C-terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS-PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the Km and kcat values increased with increasing temperature (30 to 65 °C), although the kcat /Km values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the Km and kcat values increased with decreasing pH, and the kcat /Km values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin.

  7. Serum N-terminal telopeptide of type I collagen as an early marker of fracture nonunion in rabbits

    PubMed Central

    Lin, Jian-Ping; Shi, Zhan-Jun; Shen, Ning-Jiang; Wang, Jian; Li, Zao-Min; Xiao, Jun

    2016-01-01

    The aim of the present study was to establish an experimental animal model of fracture nonunion, and to investigate the changes in serum biomarker concentrations in fracture nonunion. A total of 20 purebred New Zealand rabbits were divided into two group: A bone defect group and a bone fracture group. In the bone defect group, a 15-mm section of bone (including the periosteum) was removed from the mid-radius, and the medullary cavities were closed with bone wax. In the bone fracture group, the mid-radius was fractured. X-rays were taken and blood samples were collected preoperatively and at 2, 3, 4, 5, 6, 7, 8, 10 and 12 weeks after the surgical procedure. The serum concentrations of osteocalcin (OC) and bone-specific alkaline phosphatase (BSAP) served as markers of bone formation, and those of C-terminal telopeptide of type I collagen (CTX), N-terminal telopeptide of type I collagen (NTX) and tartrate-resistant acid phosphatase 5b (TRACP 5b) served as markers of bone resorption. The concentration levels of the markers were measured using a biotin double-antibody sandwich enzyme-linked immunosorbent assay. In the bone defect group, bone callus was observed on X-ray at 2 weeks in three rabbits and the bone calluses stabilized at 5 weeks; however, none of the bones had healed at 8 weeks. In the bone fracture group, the fracture line was distorted at 2 weeks and bone calluses formed at 6–8 weeks. In the bone defect group, the serum BSAP and TRACP 5b concentrations increased following the surgical procedure, peaked at 4 weeks, began to decrease at 5 weeks and stabilized after 6 weeks. The serum OC concentrations did not change significantly following the surgical procedure. The serum CTX concentrations fluctuated during the first 4 weeks, peaked at 5 weeks, then decreased and stabilized after 6 weeks. The serum NTX concentrations fluctuated during the first 4 weeks, were significantly lower at 5 weeks compared with the other time points and stabilized after 6 weeks

  8. Elevated Plasma C-Terminal Endothelin-1 Precursor Fragment Concentrations Are Associated with Less Anxiety in Patients with Cardiovascular Risk Factors. Results from the Observational DIAST-CHF Study

    PubMed Central

    Meyer, Thomas; Chavanon, Mira-Lynn; Herrrmann-Lingen, Christoph; Roggenthien, Maren; Nolte, Kathleen; Pieske, Burkert

    2015-01-01

    Background The role of endothelin-1 (ET-1) in the neurobiology of anxiety is unknown, therefore, we assessed in the observational multicenter DIAST-CHF study whether the C-terminal ET-1 precursor fragment (CT-proET-1) is linked to anxiety. Methods Plasma concentrations of CT-proET-1 were measured in a total of 1,410 patients presenting with cardiovascular risk factors (mean age 66.91±8.2 years, 49.3% males, mean left ventricular ejection fraction 60.0±8.2%) who had completed the Hospital Anxiety and Depression Scale (HADS) questionnaire. Results Among the total study cohort (n = 1,410), there were 118 subjects (8.4%) with an HADS anxiety score above the cut-off level of 11 suggestive of clinically relevant anxiety. Plasma CT-proET-1 levels were significantly lower in the group of anxious patients as compared to non-anxious patients (p = 0.013). In regression models adjusted for sex, age, systolic blood pressure, and diameters of left atrium and ventricle, plasma CT-proET-1 was again linked to anxiety (Exp(β) = 0.247, 95%-confidence interval [95%-CI] = 0.067–0.914, p = 0.036). Given the high prevalence of depressive disorders in anxious patients, we additionally included the HADS depression score as an independent variable in the models and found that CT-proET-1 remained a significant predictor of anxiety, independent of comorbid depression (Exp(β) = 0.114, 95%-CI = 0.023–0.566, p = 0.008). Conclusions Our data from a population-based study in outpatients with cardiovascular risk factors revealed that circulating CT-proET-1 levels are negatively associated with anxiety. Further investigations are required to clarify the putative anxiolytic effect of ET-1 or its precursor molecules in humans and to decipher its mechanistic pathways. PMID:26322793

  9. Collagen telopeptides (cross-linking sites) play a role in collagen gel lattice contraction

    NASA Technical Reports Server (NTRS)

    Woodley, D. T.; Yamauchi, M.; Wynn, K. C.; Mechanic, G.; Briggaman, R. A.

    1991-01-01

    Solubilized interstitial collagens will form a fibrillar, gel-like lattice when brought to physiologic conditions. In the presence of human dermal fibroblasts the collagen lattice will contract. The rate of contraction can be determined by computer-assisted planemetry. The mechanisms involved in contraction are as yet unknown. Using this system it was found that the rate of contraction was markedly decreased when collagen lacking telopeptides was substituted for native collagen. Histidinohydroxylysinonorleucine (HHL) is a major stable trifunctional collagen cross-link in mature skin that involves a carboxyl terminal, telopeptide site 16c, the sixteenth amino acid residue from the carboxy terminal of the telopeptide region of alpha 1 (I) in type I collagen. Little, if any, HHL was present in native, purified, reconstituted, soluble collagen fibrils from 1% acetic acid-extracted 2-year-old bovine skin. In contrast, HHL cross-links were present (0.22 moles of cross-link per mole of collagen) in lattices of the same collagen contracted by fibroblasts. However, rat tail tendon does not contain HHL cross-links, and collagen lattices made of rat tail tendon collagen are capable of contraction. This suggests that telopeptide sites, and not mature HHL cross-links per se, are essential for fibroblasts to contract collagen lattices. Beta-aminopropionitrile fumarate (BAPN), a potent lathyrogen that perturbs collagen cross-linking by inhibition of lysyl oxidase, also inhibited the rate of lattice cell contraction in lattices composed of native collagen. However, the concentrations of BAPN that were necessary to inhibit the contraction of collagen lattices also inhibited fibroblast growth suggestive of cellular toxicity. In accordance with other studies, we found no inhibition of the rate of lattice contraction when fibronectin-depleted serum was used. Electron microscopy of contracted gels revealed typical collagen fibers with a characteristic axial periodicity. The data

  10. Cross-link analysis of the C-telopeptide domain from type III collagen.

    PubMed Central

    Henkel, W

    1996-01-01

    Several peptides were isolated from tryptic digests of insoluble calf aorta matrix by chromatography. Reductive pyridylethylation of a tryptic 15 kDa pool released fragments deriving from the C-terminus of type III collagen. A 50-residue peptide Tc(III) was shown by sequence analysis to be the C-terminal peptide from the alpha 1(III)-chain, containing a helical and non-helical region of equal sizes. The peptide was further digested with collagenase to give Colc(III), comprising the complete C-terminal non-helical region of alpha 1(III) including a hydroxylysine in position 16c. The peptide Tc(III) x TN(III) was isolated, demonstrating covalent cross-linking between the C-terminal non-helical region of one type III molecule and the N-terminal helical cross-linking region of another. Its digestion with cyanogen bromide yielded the small fragments alpha 1(III)CB3B* and alpha 1(III)CB3C, confirming TN(III) as an N-terminal helical crosslink site. Sequence analysis of both Tc(III) x TN(III) and its collagenase-derived cross-linked peptide Colc(III) x TN(III) established the 4D-staggered alignment of adjacent collagen III molecules. The cross-link structure of both peptides was mainly dihydroxylysinonorleucine with a small amount of hydroxylysinonorleucine, indicating that the lysine residues involved in formation of the cross-links are both hydroxylated. No pyridinoline or histidinohydroxylysinonorleucine cross-links were found within the non-reduced C-telopeptide region of type III collagen. PMID:8809038

  11. Design, Synthesis and Biological Evaluation of Biphenylamide Derivatives as Hsp90 C-terminal Inhibitors

    PubMed Central

    Zhao, Huiping; Garg, Gaurav; Zhao, Jinbo; Moroni, Elisabetta; Girgis, Antwan; Franco, Lucas S.; Singh, Swapnil; Colombo, Giorgio; Blagg, Brian S. J.

    2015-01-01

    Modulation of Hsp90 C-terminal function represents a promising therapeutic approach for the treatment of cancer and neurodegenerative diseases. Current drug discovery efforts toward Hsp90 C-terminal inhibition focus on novobiocin, an antibiotic that was transformed into an Hsp90 inhibitor. Based on structural information obtained during the development of novobiocin derivatives and molecular docking studies, scaffolds containing a biphenyl moiety in lieu of the coumarin ring present in novobiocin were identified as new Hsp90 C-terminal inhibitors. Structure-activity relationship studies produced new derivatives that inhibit the proliferation of breast cancer cell lines at nanomolar concentrations, which corresponded directly with Hsp90 inhibition. PMID:25462258

  12. Measurement of urinary N-telopeptides and serum C-telopeptides from type I collagen using a lateral flow-based immunoassay.

    PubMed

    Lee, Kyoung Min; Lee, Min Ho; Chung, Chin Youb; Seong, Woo Kyeong; Lee, Sang Dae; Park, Moon Seok

    2012-12-24

    Measuring bone turnover markers could detect early stages of osteoporosis and early responses to anti-osteoporotic treatments. Currently, commonly used bone turnover markers, N-telopeptides (NTx) and C-telopeptides (CTx), are measured using ELISA tests, which demands time and increases cost. Bone turnover markers need to be measured more easily for general use. Lateral flow-based immunoassay would be an appropriate method for this context. This study was performed to investigate the precision of a newly developed lateral flow-based immunoassay for measuring the urinary NTx and serum CTx, and their correlations with ELISA measurements. Urine NTx and serum CTx concentrations were determined by photoscan of newly developed strips, using a lateral flow-based immunoassay for 36 subjects (mean age 66.2 years, SD 7.5 years; four males and 32 females). Repeated measurement of urinary NTx and serum CTx were performed three times, using this technology for a precision test. The correlation of the lateral flow-based immunoassay with the ELISA measurements was analyzed. Precision of the newly developed lateral flow based immunoassay was 0.974 (ICC, 95% confidence interval, 0.955 to 0.986) and 0.995 (ICC, 95% confidence interval, 0.991 to 0.997) for urinary NTx and serum CTx, respectively. The correlation of lateral flow based immunoassay with ELISA was 0.913 for urinary NTx and 0.872 for serum CTx. These results suggest that measuring the urinary NTx and serum CTx, using a lateral flow-based immunoassay, is a relevant method for point-of-care testing and screening of bone resorption markers.

  13. C-Terminal Modification and Multimerization Increase the Efficacy of a Proline-Rich Antimicrobial Peptide.

    PubMed

    Li, Wenyi; O'Brien-Simpson, Neil M; Yao, Shenggen; Tailhades, Julien; Reynolds, Eric C; Dawson, Raymond M; Otvos, Laszlo; Hossain, Mohammed Akhter; Separovic, Frances; Wade, John D

    2017-01-05

    Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL(-1) ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.

  14. Modules for C-terminal epitope tagging of Tetrahymena genes

    PubMed Central

    Kataoka, Kensuke; Schoeberl, Ursula E.; Mochizuki, Kazufumi

    2010-01-01

    Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies. PMID:20624430

  15. DNA aptamer beacon assay for C-telopeptide and handheld fluorometer to monitor bone resorption.

    PubMed

    Bruno, John Gordon; Carrillo, Maria P; Phillips, Taylor; Hanson, Douglas; Bohmann, Jonathan A

    2011-09-01

    A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.

  16. C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

    PubMed Central

    Nika, Heinz; Hawke, David H.; Angeletti, Ruth Hogue

    2014-01-01

    A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTipC18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2′-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures. PMID:24688319

  17. Development of Noviomimetics as C-Terminal Hsp90 Inhibitors

    PubMed Central

    2015-01-01

    KU-32 and KU-596 are novobiocin-derived, C-terminal heat shock protein 90 (Hsp90) modulators that induce Hsp70 levels and manifest neuroprotective activity. However, the synthetically complex noviose sugar requires 10 steps to prepare, which makes translational development difficult. In this study, we developed a series of “noviomimetic” analogues of KU-596, which contain noviose surrogates that can be easily prepared, while maintaining the ability to induce Hsp70 levels. Both sugar and sugar analogues were designed, synthesized, and evaluated in a luciferase reporter assay, which identified compound 37, a benzyl containing noviomimetic, as the most potent inducer of Hsp70. PMID:26819668

  18. Nonlinear dynamics of C-terminal tails in cellular microtubules

    NASA Astrophysics Data System (ADS)

    Sekulic, Dalibor L.; Sataric, Bogdan M.; Zdravkovic, Slobodan; Bugay, Aleksandr N.; Sataric, Miljko V.

    2016-07-01

    The mechanical and electrical properties, and information processing capabilities of microtubules are the permanent subject of interest for carrying out experiments in vitro and in silico, as well as for theoretical attempts to elucidate the underlying processes. In this paper, we developed a new model of the mechano-electrical waves elicited in the rows of very flexible C-terminal tails which decorate the outer surface of each microtubule. The fact that C-terminal tails play very diverse roles in many cellular functions, such as recruitment of motor proteins and microtubule-associated proteins, motivated us to consider their collective dynamics as the source of localized waves aimed for communication between microtubule and associated proteins. Our approach is based on the ferroelectric liquid crystal model and it leads to the effective asymmetric double-well potential which brings about the conditions for the appearance of kink-waves conducted by intrinsic electric fields embedded in microtubules. These kinks can serve as the signals for control and regulation of intracellular traffic along microtubules performed by processive motions of motor proteins, primarly from kinesin and dynein families. On the other hand, they can be precursors for initiation of dynamical instability of microtubules by recruiting the proper proteins responsible for the depolymerization process.

  19. Evolution of the RNA polymerase II C-terminal domain

    PubMed Central

    Stiller, John W.; Hall, Benjamin D.

    2002-01-01

    In recent years a great deal of biochemical and genetic research has focused on the C-terminal domain (CTD) of the largest subunit (RPB1) of DNA-dependent RNA polymerase II. This strongly conserved domain of tandemly repeated heptapeptides has been linked functionally to important steps in the initiation and processing of mRNA transcripts in both animals and fungi. Although they are absolutely required for viability in these organisms, C-terminal tandem repeats do not occur in RPB1 sequences from diverse eukaryotic taxa. Here we present phylogenetic analyses of RPB1 sequences showing that canonical CTD heptads are strongly conserved in only a subset of eukaryotic groups, all apparently descended from a single common ancestor. Moreover, eukaryotic groups in which the most complex patterns of ontogenetic development occur are descended from this CTD-containing ancestor. Consistent with the results of genetic and biochemical investigations of CTD function, these analyses suggest that the enhanced control over RNA polymerase II transcription conveyed by acquired CTD/protein interactions was an important step in the evolution of intricate patterns of gene expression that are a hallmark of large, developmentally complex eukaryotic organisms. PMID:11972039

  20. Binding of a C-terminal fragment (residues 369 to 435) of vitamin D-binding protein to actin.

    PubMed

    Buch, Stefan; Gremm, Dagmar; Wegner, Albrecht; Mannherz, Hans Georg

    2002-10-01

    The vitamin D-binding protein (DBP) binds to monomeric actin with high affinity. The variation in DBP isoforms is due to genetic polymorphism and varying glycosylation. To obtain a homogeneous preparation, the cDNA for human DBP and truncations thereof were cloned and various systems were applied for heterologous bacterial and yeast expression. The full-length protein and the N- and C-terminal halves of DBP remained insoluble probably because the protein did not fold to its native three-dimensional structure due to formation of accidental intra- and inter-molecular disulfide bonds during expression in bacteria or yeast. This problem was overcome by cloning of a C-terminal fragment comprising residues 369 to 435 that did not contain disulfide bonds and was completely soluble. Binding of the C-terminal fragment to monomeric actin was demonstrated by comigration with actin during native polyacrylamide gel electrophoresis and surface plasmon resonance, however, at considerably lower affinity than full-length DBP. This suggests that in addition to the C-terminal amino acid sequence other parts (amino acid residues or sugar moieties) of DBP participate in actin binding. The C-terminal fragment was found to inhibit denaturation of actin and to decrease the rate of actin polymerisation both at the barbed and at the pointed end in a concentration-dependent manner. According to a quantitative analysis of the polymerisation kinetics, association of actin monomers to nucleate filaments was not prevented by binding of the C-terminal fragment to actin. These data suggest that the sites on the surface of actin that are involved in actin nucleation and elongation are different.

  1. C-terminal functionalization of nylon-3 polymers: effects of C-terminal groups on antibacterial and hemolytic activities.

    PubMed

    Zhang, Jihua; Markiewicz, Matthew J; Mowery, Brendan P; Weisblum, Bernard; Stahl, Shannon S; Gellman, Samuel H

    2012-02-13

    Nylon-3 polymers contain β-amino-acid-derived subunits and can be viewed as higher homologues of poly(α-amino acids). This structural relationship raises the possibility that nylon-3 polymers offer a platform for development of new materials with a variety of biological activities, a prospect that has recently begun to receive experimental support. Nylon-3 homo- and copolymers can be prepared via anionic ring-opening polymerization of β-lactams, and use of an N-acyl-β-lactam as coinitiator in the polymerization reaction allows placement of a specific functional group, borne by the N-acyl-β-lactam, at the N-terminus of each polymer chain. Controlling the unit at the C-termini of nylon-3 polymer chains, however, has been problematic. Here we describe a strategy for specifying C-terminal functionality that is based on the polymerization mechanism. After the anionic ring-opening polymerization is complete, we introduce a new β-lactam, approximately 1 equiv relative to the expected number of polymer chains. Because the polymer chains bear a reactive imide group at their C-termini, this new β-lactam should become attached at this position. If the terminating β-lactam bears a distinctive functional group, that functionality should be affixed to most or all C-termini in the reaction mixture. We use the new technique to compare the impact of N- and C-terminal placement of a critical hydrophobic fragment on the biological activity profile of nylon-3 copolymers. The synthetic advance described here should prove to be generally useful for tailoring the properties of nylon-3 materials.

  2. Involvement of C-Terminal Histidines in Soybean PM1 Protein Oligomerization and Cu2+ Binding.

    PubMed

    Liu, Guobao; Liu, Ke; Gao, Yang; Zheng, Yizhi

    2017-04-06

    Late embryogenesis abundant (LEA) proteins are widely distributed among plant species, where they contribute to abiotic stress tolerance. LEA proteins can be classified into seven groups according to conserved sequence motifs. The PM1 protein from soybean, which belongs to the Pfam LEA_1 group, has been shown previously to be at least partially natively unfolded, to bind metal ions and potentially to stabilize proteins and membranes. Here, we investigated the role of the PM1 C-terminal domain and in particular the multiple histidine residues in this half of the protein. We constructed recombinant plasmids expressing full-length PM1 and two truncated forms, PM1-N and PM1-C, which represent the N- and C-terminal halves of the protein, respectively. Immunoblotting and cross-linking experiments showed that full-length PM1 forms oligomers and high molecular weight (HMW) complexes in vitro and in vivo, while PM1-C, but not PM1-N, also formed oligomers and HMW complexes in vitro. When the histidine residues in PM1 and PM1-C were chemically modified, oligomerization was abolished, suggesting that histidines play a key role in this process. Furthermore, we demonstrated that high Cu2+ concentrations promote oligomerization and induce PM1 and PM1-C to form HMW complexes. Therefore, we speculate that PM1 proteins not only maintain ion homeostasis in the cytoplasm, but also potentially stabilize and protect other proteins during abiotic stress by forming a large, oligomeric molecular shield around biological targets.

  3. FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D

    NASA Technical Reports Server (NTRS)

    Bruno, John G.

    2013-01-01

    Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is

  4. C-terminal domains of bacterial proteases: structure, function and the biotechnological applications.

    PubMed

    Huang, J; Wu, C; Liu, D; Yang, X; Wu, R; Zhang, J; Ma, C; He, H

    2017-01-01

    C-terminal domains widely exist in the C-terminal region of multidomain proteases. As a β-sandwich domain in multidomain protease, the C-terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C-terminal protease domains are described. Furthermore, the application prospects of C-terminal domains, including polycystic kidney disease, prepeptidase C-terminal and collagen-binding domain, in the area of medicine and biological artificial materials are also discussed.

  5. Mechanistic studies of ubiquitin C-terminal hydrolase L1.

    PubMed

    Case, April; Stein, Ross L

    2006-02-21

    Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.

  6. Protein splicing of inteins with atypical glutamine and aspartate C-terminal residues.

    PubMed

    Amitai, Gil; Dassa, Bareket; Pietrokovski, Shmuel

    2004-01-30

    Inteins are protein-splicing domains present in many proteins. They self-catalyze their excision from the host protein, ligating their former flanks by a peptide bond. The C-terminal residue of inteins is typically an asparagine (Asn). Cyclization of this residue to succinimide causes the final detachment of inteins from their hosts. We studied protein-splicing activity of two inteins with atypical C-terminal residues. One having a C-terminal glutamine (Gln), isolated from Chilo iridescent virus (CIV), and another unique intein, first reported here, with a C-terminal aspartate, isolated from Carboxydothermus hydrogenoformans (Chy). Protein-splicing activity was examined in the wild-type inteins and in several mutants with N- and C-terminal amino acid substitutions. We demonstrate that both wild-type inteins can protein splice, probably by new variations of the typical protein-splicing mechanism. Substituting the atypical C-terminal residue to the typical Asn retained protein-splicing only in the CIV intein. All diverse C-terminal substitutions in the Chy intein (Asp(345) to Asn, Gln, Glu, and Ala) abolished protein-splicing and generated N- and C-terminal cleavage. The observed C-terminal cleavage in the Chy intein ending with Ala cannot be explained by cyclization of this residue. We present and discuss several new models for reactions in the protein-splicing pathway.

  7. AMINO ACID COMPOSITION AND C-TERMINAL RESIDUES OF ALGAL BILIPROTEINS,

    DTIC Science & Technology

    R-phycoerythrin from Ceramium rubrum and C- phycocyanin from Nostoc nuscorum were obtained in purified form by fractional crystallization, followed by...as amino acids. Alanine was identified as the only C-terminal amino acid of R-phycoerythrin, each molecule of which contained about 12 terminal groups. Serine was identified as the only C-terminal group of C- phycocyanin . (Author)

  8. The ∼16 kDa C-Terminal Sequence of Clathrin Assembly Protein AP180 Is Essential for Efficient Clathrin Binding

    PubMed Central

    Chan, Ling-Shan; Moshkanbaryans, Lia; Xue, Jing; Graham, Mark E.

    2014-01-01

    Brain-specific AP180 is present in clathrin coats at equal concentration to the adapter complex, AP2, and assembles clathrin faster than any other protein in vitro. Both AP180 and its ubiquitously expressed homolog clathrin assembly lymphoid myeloid leukemia protein (CALM) control vesicle size and shape in clathrin mediated endocytosis. The clathrin assembly role of AP180 is mediated by a long disordered C-terminal assembly domain. Within this assembly domain, a central acidic clathrin and adapter binding (CLAP) sub-domain contains all of the known short binding motifs for clathrin and AP2. The role of the remaining ∼16 kDa C-terminal sequence has not been clear. We show that this sequence has a separate function in ensuring efficient binding of clathrin, based on in vitro binding and ex vivo transferrin uptake assays. Sequence alignment suggests the C-terminal sub-domain is conserved in CALM. PMID:25329427

  9. Role of the C-terminal domain of PCSK9 in degradation of the LDL receptors.

    PubMed

    Holla, Øystein L; Cameron, Jamie; Tveten, Kristian; Strøm, Thea Bismo; Berge, Knut Erik; Laerdahl, Jon K; Leren, Trond P

    2011-10-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and disrupts the normal recycling of the LDLR. In this study, we investigated the role of the C-terminal domain for the activity of PCSK9. Experiments in which conserved residues and histidines on the surface of the C-terminal domain were mutated indicated that no specific residues of the C-terminal domain, apart from those responsible for maintaining the overall structure, are required for the activity of PCSK9. Rather, the net charge of the C-terminal domain is important. The more positively charged the C-terminal domain, the higher the activity toward the LDLR. Moreover, replacement of the C-terminal domain with an unrelated protein of comparable size led to significant activity of the chimeric protein. We conclude that the role of the evolutionary, poorly conserved C-terminal domain for the activity of PCSK9 reflects its overall positive charge and size and not the presence of specific residues involved in protein-protein interactions.

  10. Cytokinin Response Factor 5 has transcriptional activity governed by its C-terminal domain.

    PubMed

    Striberny, Bernd; Melton, Anthony E; Schwacke, Rainer; Krause, Kirsten; Fischer, Karsten; Goertzen, Leslie R; Rashotte, Aaron M

    2017-02-01

    Cytokinin Response Factors (CRFs) are AP2/ERF transcription factors involved in cytokinin signal transduction. CRF proteins consist of a N-terminal dimerization domain (CRF domain), an AP2 DNA-binding domain, and a clade-specific C-terminal region of unknown function. Using a series of sequential deletions in yeast-2-hybrid assays, we provide evidence that the C-terminal region of Arabidopsis CRF5 can confer transactivation activity. Although comparative analyses identified evolutionarily conserved protein sequence within the C-terminal region, deletion experiments suggest that this transactivation domain has a partially redundant modular structure required for activation of target gene transcription.

  11. Predictors of intact and C-terminal fibroblast growth factor 23 in Gambian children

    PubMed Central

    Braithwaite, Vickie; Jones, Kerry S; Assar, Shima; Schoenmakers, Inez; Prentice, Ann

    2013-01-01

    Elevated C-terminal fibroblast growth factor 23 (C-FGF23) concentrations have been reported in Gambian children with and without putative Ca-deficiency rickets. The aims of this study were to investigate whether i) elevated C-FGF23 concentrations in Gambian children persist long term; ii) they are associated with higher intact FGF23 concentrations (I-FGF23), poor iron status and shorter 25-hydroxyvitamin D half-life (25OHD-t1/2); and iii) the persistence and predictors of elevated FGF23 concentrations differ between children with and without a history of rickets. Children (8–16 years, n=64) with a history of rickets and a C-FGF23 concentration >125 RU/ml (bone deformity (BD), n=20) and local community children with a previously measured elevated C-FGF23 concentration (LC+, n=20) or a previously measured C-FGF23 concentration within the normal range (LC−, n=24) participated. BD children had no remaining signs of bone deformities. C-FGF23 concentration had normalised in BD children, but remained elevated in LC+ children. All the children had I-FGF23 concentration within the normal range, but I-FGF23 concentration was higher and iron status poorer in LC+ children. 1,25-dihydroxyvitamin D was the strongest negative predictor of I-FGF23 concentration (R2=18%; P=0.0006) and soluble transferrin receptor was the strongest positive predictor of C-FGF23 concentration (R2=33%; P≤0.0001). C-FGF23 and I-FGF23 concentrations were poorly correlated with each other (R2=5.3%; P=0.07). 25OHD-t1/2 was shorter in BD children than in LC− children (mean (s.d.): 24.5 (6.1) and 31.5 (11.5) days respectively; P=0.05). This study demonstrated that elevated C-FGF23 concentrations normalised over time in Gambian children with a history of rickets but not in local children, suggesting a different aetiology; that children with resolved rickets had a shorter 25OHD-t1/2, suggesting a long-standing increased expenditure of 25OHD, and that iron deficiency is a predictor of elevated C

  12. C-Terminal Modification of Fully Unprotected Peptide Hydrazides via in Situ Generation of Isocyanates.

    PubMed

    Vinogradov, Alexander A; Simon, Mark D; Pentelute, Bradley L

    2016-03-18

    A method for chemo- and regioselective conjugation of nucleophiles to fully unprotected peptides and proteins via in situ generation of C-terminal isocyanates is reported. Oxidation of C-terminal peptide hydrazides in aqueous media followed by Curtius rearrangement of acyl azides reliably generates isocyanates, which react with a variety of external nucleophiles, such as hydrazines, hydrazides, aromatic thiols, and hydroxylamines. Multiple peptides and a 53 kDa protein hydrazide were conjugated to different nucleophiles using this reaction.

  13. Solid phase synthesis of a GHRP analog containing C-terminal thioamide group.

    PubMed

    Majer, Z; Zewdu, M; Hollósi, M; Sepródi, J; Vadász, Z; Teplán, I

    1988-02-15

    [Lyst6]GHRP, the C-terminally thionated analog of the highly potent growth hormone releasing hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 was prepared by using solid support. The success of the synthesis showed that Lawesson's reagent can be used for selective thionation of an amide group not only in solution but also on the surface of a resin. The C-terminal thioamide group proved to be stable under the conditions of the solid phase synthesis.

  14. The Impact of the Human DNA Topoisomerase II C-Terminal Domain on Activity

    PubMed Central

    Meczes, Emma L.; Gilroy, Kathryn L.; West, Katherine L.; Austin, Caroline A.

    2008-01-01

    Background Type II DNA topoisomerases (topos) are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, α and β, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD) of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity. Methodology/Principle Findings We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIα and β and topoIIα+β-tail and topoIIβ+α-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoIIα-CTD increasing activity, and the topoIIβ-CTD decreasing activity. Conclusions/Significance In vivo complementation data show that the topoIIα C-terminal domain is needed for growth, but the topoIIβ isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIβ has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoIIα or β C-terminal domain can affect strand passage activity. Data indicates that the topoIIβ-CTD may be a negative regulator. This is the first report of in vitro data with chimeric human topoIIs. PMID:18335031

  15. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    PubMed Central

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-01-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring. PMID:28155880

  16. Human Frataxin Folds Via an Intermediate State. Role of the C-Terminal Region

    PubMed Central

    Faraj, Santiago E.; González-Lebrero, Rodolfo M.; Roman, Ernesto A.; Santos, Javier

    2016-01-01

    The aim of this study is to investigate the folding reaction of human frataxin, whose deficiency causes the neurodegenerative disease Friedreich’s Ataxia (FRDA). The characterization of different conformational states would provide knowledge about how frataxin can be stabilized without altering its functionality. Wild-type human frataxin and a set of mutants, including two highly destabilized FRDA-associated variants were studied by urea-induced folding/unfolding in a rapid mixing device and followed by circular dichroism. The analysis clearly indicates the existence of an intermediate state (I) in the folding route with significant secondary structure content but relatively low compactness, compared with the native ensemble. However, at high NaCl concentrations I-state gains substantial compaction, and the unfolding barrier is strongly affected, revealing the importance of electrostatics in the folding mechanism. The role of the C-terminal region (CTR), the key determinant of frataxin stability, was also studied. Simulations consistently with experiments revealed that this stretch is essentially unstructured, in the most compact transition state ensemble (TSE2). The complete truncation of the CTR drastically destabilizes the native state without altering TSE2. Results presented here shed light on the folding mechanism of frataxin, opening the possibility of mutating it to generate hyperstable variants without altering their folding kinetics. PMID:26856628

  17. Human Frataxin Folds Via an Intermediate State. Role of the C-Terminal Region.

    PubMed

    Faraj, Santiago E; González-Lebrero, Rodolfo M; Roman, Ernesto A; Santos, Javier

    2016-02-09

    The aim of this study is to investigate the folding reaction of human frataxin, whose deficiency causes the neurodegenerative disease Friedreich's Ataxia (FRDA). The characterization of different conformational states would provide knowledge about how frataxin can be stabilized without altering its functionality. Wild-type human frataxin and a set of mutants, including two highly destabilized FRDA-associated variants were studied by urea-induced folding/unfolding in a rapid mixing device and followed by circular dichroism. The analysis clearly indicates the existence of an intermediate state (I) in the folding route with significant secondary structure content but relatively low compactness, compared with the native ensemble. However, at high NaCl concentrations I-state gains substantial compaction, and the unfolding barrier is strongly affected, revealing the importance of electrostatics in the folding mechanism. The role of the C-terminal region (CTR), the key determinant of frataxin stability, was also studied. Simulations consistently with experiments revealed that this stretch is essentially unstructured, in the most compact transition state ensemble (TSE2). The complete truncation of the CTR drastically destabilizes the native state without altering TSE2. Results presented here shed light on the folding mechanism of frataxin, opening the possibility of mutating it to generate hyperstable variants without altering their folding kinetics.

  18. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    NASA Astrophysics Data System (ADS)

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-02-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring.

  19. Human Frataxin Folds Via an Intermediate State. Role of the C-Terminal Region

    NASA Astrophysics Data System (ADS)

    Faraj, Santiago E.; González-Lebrero, Rodolfo M.; Roman, Ernesto A.; Santos, Javier

    2016-02-01

    The aim of this study is to investigate the folding reaction of human frataxin, whose deficiency causes the neurodegenerative disease Friedreich’s Ataxia (FRDA). The characterization of different conformational states would provide knowledge about how frataxin can be stabilized without altering its functionality. Wild-type human frataxin and a set of mutants, including two highly destabilized FRDA-associated variants were studied by urea-induced folding/unfolding in a rapid mixing device and followed by circular dichroism. The analysis clearly indicates the existence of an intermediate state (I) in the folding route with significant secondary structure content but relatively low compactness, compared with the native ensemble. However, at high NaCl concentrations I-state gains substantial compaction, and the unfolding barrier is strongly affected, revealing the importance of electrostatics in the folding mechanism. The role of the C-terminal region (CTR), the key determinant of frataxin stability, was also studied. Simulations consistently with experiments revealed that this stretch is essentially unstructured, in the most compact transition state ensemble (TSE2). The complete truncation of the CTR drastically destabilizes the native state without altering TSE2. Results presented here shed light on the folding mechanism of frataxin, opening the possibility of mutating it to generate hyperstable variants without altering their folding kinetics.

  20. Mobility and Core-Protein Binding Patterns of Disordered C-Terminal Tails in β-Tubulin Isotypes.

    PubMed

    Laurin, Yoann; Eyer, Joel; Robert, Charles H; Prevost, Chantal; Sacquin-Mora, Sophie

    2017-03-28

    Although they play a significant part in the regulation of microtubule structure, dynamics, and function, the disordered C-terminal tails of tubulin remain invisible to experimental structural methods and do not appear in the crystallographic structures that are currently available in the Protein Data Bank. Interestingly, these tails concentrate most of the sequence variability between tubulin isotypes and are the sites of the principal post-translational modifications undergone by this protein. Using homology modeling, we developed two complete models for the human αI/βI- and αI/βIII-tubulin isotypes that include their C-terminal tails. We then investigated the conformational variability of the two β-tails using long time-scale classical molecular dynamics simulations that revealed similar features, notably the unexpected presence of common anchoring regions on the surface of the tuulin dimer, but also distinctive mobility or interaction patterns, some of which could be related to the tail lengths and charge distributions. We also observed in our simulations that the C-terminal tail from the βI isotype, but not the βIII isotype, formed contacts in the putative binding site of a recently discovered peptide that disrupts microtubule formation in glioma cells. Hindering the binding site in the βI isotype would be consistent with this peptide's preferential disruption of microtubule formation in glioma, whose cells overexpress βIII, compared to normal glial cells. While these observations need to be confirmed with more intensive sampling, our study opens new perspectives for the development of isotype-specific chemotherapy drugs.

  1. C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

    PubMed Central

    Gong, Jihong; Lai, Ying; Li, Xiaohong; Wang, Mengxian; Leitz, Jeremy; Hu, Yachong; Zhang, Yunxiang; Choi, Ucheor B.; Cipriano, Daniel; Pfuetzner, Richard A.; Südhof, Thomas C.; Yang, Xiaofei; Brunger, Axel T.

    2016-01-01

    In presynaptic nerve terminals, complexin regulates spontaneous “mini” neurotransmitter release and activates Ca2+-triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using single-particle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca2+-independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents. PMID:27821736

  2. PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch.

    PubMed

    Espejo, Alexsandra B; Gao, Guozhen; Black, Karynne; Gayatri, Sitaram; Veland, Nicolas; Kim, Jeesun; Chen, Taiping; Sudol, Marius; Walker, Cheryl; Bedford, Mark T

    2017-02-10

    PRMT5 is the primary enzyme responsible for the deposition of the symmetric dimethylarginine in mammalian cells. In an effort to understand how PRMT5 is regulated, we identified a threonine phosphorylation site within a C-terminal tail motif, which is targeted by the Akt/serum- and glucocorticoid-inducible kinases. While investigating the function of this posttranslational modification, we serendipitously discovered that its free C-terminal tail binds PDZ domains (when unphosphorylated) and 14-3-3 proteins (when phosphorylated). In essence, a phosphorylation event within the last few residues of the C-terminal tail generates a posttranslational modification-dependent PDZ/14-3-3 interaction "switch." The C-terminal motif of PRMT5 is required for plasma membrane association, and loss of this switching capacity is not compatible with life. This signaling phenomenon was recently reported for the HPV E6 oncoprotein but has not yet been observed for mammalian proteins. To investigate the prevalence of PDZ/14-3-3 switching in signal transduction, we built a protein domain microarray that harbors PDZ domains and 14-3-3 proteins. We have used this microarray to interrogate the C-terminal tails of a small group of candidate proteins and identified ERBB4, PGHS2, and IRK1 (as well as E6 and PRMT5) as conforming to this signaling mode, suggesting that PDZ/14-3-3 switching may be a broad biological paradigm.

  3. Transmembrane signalling at the epidermal growth factor receptor. Positive regulation by the C-terminal phosphotyrosine residues.

    PubMed Central

    Magni, M; Pandiella, A; Helin, K; Meldolesi, J; Beguinot, L

    1991-01-01

    Mutant epidermal growth factor (EGF) receptors (obtained by substitution of one, two or three C-terminal autophosphorylable tyrosine residues with phenylalanine residues or by deletion of the C-terminal 19 amino acids, including the distal tyrosine) were expressed in mouse NIH-3T3 fibroblast clones at densities comparable (less than 25% difference) with those in control clones expressing the wild-type receptor. Total EGF-induced phosphorylation of the mutated receptors was not appreciably changed with respect to controls, whereas autophosphorylation at tyrosine residues was decreased, especially in the double and the triple mutants. In the latter mutant, expression of the EGF-receptor-activated lipolytic enzyme phospholipase C gamma was unchanged, whereas its tyrosine phosphorylation induced by the growth factor was lowered to approx. 25% of that in the controls. In all of the cell clones employed, the accumulation of inositol phosphates induced by treatment with fetal calf serum varied only slightly, whereas the same effect induced by EGF was consistently lowered in those lines expressing mutated receptors. This decrease was moderate for those receptors missing only the distal tyrosine (point and deletion mutants), intermediate in the dual mutants and almost complete in the triple mutants. Likewise, increases in intracellular Ca2+ concentrations [( Ca2+]i) induced by fibroblast growth factor were approximately the same in all of the clones, whereas those induced by EGF were decreased in the mutants, again in proportion to the loss of the phosphorylable C-terminal tyrosine residues. The same trend occurred with membrane hyperpolarization, an effect secondary to the increase in [Ca2+]i via the activation of Ca2(+)-dependent K+ channels. We conclude that C-terminal autophosphorylable tyrosine residues play a positive role in the regulation of transmembrane signalling at the EGF receptor. The stepwise decrease in signal generation observed in single, double and triple

  4. The VSG C-terminal domain is inaccessible to antibodies on live trypanosomes.

    PubMed

    Schwede, Angela; Jones, Nicola; Engstler, Markus; Carrington, Mark

    2011-02-01

    In the mammalian host, the Trypanosoma brucei cell surface is covered with a densely packed protein coat of a single protein, the variant surface glycoprotein (VSG). The VSG is believed to shield invariant surface proteins from host antibodies but there is limited information on how far antibodies can penetrate into the VSG monolayer. Here, the VSG surface coat was probed to determine whether it acts as a barrier to binding of antibodies to the membrane proximal VSG C-terminal domain. The binding of C-terminal domain antibodies to VSG221 or VSG118 was compared with antibodies recognising the cognate whole VSGs. The C-terminal VSG domain was inaccessible to antibodies on live cells but not on fixed cells. This provides further evidence that the VSG coat acts as a barrier and protects the cell from antibodies that would otherwise bind to some of the other externally disposed proteins.

  5. Structure and dynamics of the epidermal growth factor receptor C-terminal phosphorylation domain.

    PubMed

    Lee, Nam Y; Hazlett, Theodore L; Koland, John G

    2006-05-01

    The C-terminal phosphorylation domain of the epidermal growth factor receptor is believed to regulate protein kinase activity as well as mediate the assembly of signal transduction complexes. The structure and dynamics of this proposed autoregulatory domain were examined by labeling the extreme C terminus of the EGFR intracellular domain (ICD) with an extrinsic fluorophore. Fluorescence anisotropy decay analysis of the nonphosphorylated EGFR-ICD yielded two rotational correlation times: a longer time, consistent with the global rotational motion of a 60- to 70-kDa protein with an elongated globular conformation, and a shorter time, presumably contributed by segmental motion near the fluorophore. A C-terminally truncated form of EGFR-ICD yielded a slow component consistent with the rotational motion of the 38-kDa kinase core. These findings suggested a structural arrangement of the EGFR-ICD in which the C-terminal phosphorylation domain interacts with the kinase core to move as an extended structure. A marked reduction in the larger correlation time of EGFR-ICD was observed upon its autophosphorylation. This dynamic component was faster than predicted for the globular motion of the 62-kDa EGFR-ICD, suggesting an increase in the mobility of the C-terminal domain and a likely displacement of this domain from the kinase core. The interaction between the SH2 domain of c-Src and the phosphorylated EGFR C-terminal domain was shown to impede its mobility. Circular dichroism spectroscopy indicated that the EGFR C-terminal domain possessed a significant level of secondary structure in the form of alpha-helices and beta-sheets, with a marginal change in beta-sheet content occurring upon phosphorylation.

  6. Structure and dynamics of the epidermal growth factor receptor C-terminal phosphorylation domain

    PubMed Central

    Lee, Nam Y.; Hazlett, Theodore L.; Koland, John G.

    2006-01-01

    The C-terminal phosphorylation domain of the epidermal growth factor receptor is believed to regulate protein kinase activity as well as mediate the assembly of signal transduction complexes. The structure and dynamics of this proposed autoregulatory domain were examined by labeling the extreme C terminus of the EGFR intracellular domain (ICD) with an extrinsic fluorophore. Fluorescence anisotropy decay analysis of the nonphosphorylated EGFR-ICD yielded two rotational correlation times: a longer time, consistent with the global rotational motion of a 60- to 70-kDa protein with an elongated globular conformation, and a shorter time, presumably contributed by segmental motion near the fluorophore. A C-terminally truncated form of EGFR-ICD yielded a slow component consistent with the rotational motion of the 38-kDa kinase core. These findings suggested a structural arrangement of the EGFR-ICD in which the C-terminal phosphorylation domain interacts with the kinase core to move as an extended structure. A marked reduction in the larger correlation time of EGFR-ICD was observed upon its autophosphorylation. This dynamic component was faster than predicted for the globular motion of the 62-kDa EGFR-ICD, suggesting an increase in the mobility of the C-terminal domain and a likely displacement of this domain from the kinase core. The interaction between the SH2 domain of c-Src and the phosphorylated EGFR C-terminal domain was shown to impede its mobility. Circular dichroism spectroscopy indicated that the EGFR C-terminal domain possessed a significant level of secondary structure in the form of α-helices and β-sheets, with a marginal change in β-sheet content occurring upon phosphorylation. PMID:16597832

  7. New melanocortin 1 receptor binding motif based on the C-terminal sequence of alpha-melanocyte-stimulating hormone.

    PubMed

    Schiöth, Helgi B; Muceniece, Ruta; Mutule, Ilga; Wikberg, Jarl E S

    2006-10-01

    The C-terminal tripeptide of the alpha-melanocyte stimulating hormone (alpha-MSH11-13) possesses strong antiinflammatory activity without known cellular target. In order to better understand the structural requirements for function of such motif, we designed, synthesized and tested out Trp- and Tyr-containing analogues of the alpha-MSH11-13. Seven alpha-MSH11-13 analogues were synthesized and characterized for their binding to the melanocortin receptors recombinantly expressed in insect (Sf9) cells, infected with baculovirus carrying corresponding MC receptor DNA. We also tested these analogues on B16-F1 mouse melanoma cells endogenously expressing the MC1 receptor for binding and for ability to increase cAMP levels as well as on COS-7 cells transfected with the human MC receptors. The data indicate that HS401 (Ac-Tyr-Lys-Pro-Val-NH2) and HS402 (Ac-Lys-Pro-Val-Tyr-NH2) selectively bound to the MC1 receptor and stimulated cAMP generation in a concentration dependent way while the other Tyr- and Trp-containing alpha-MSH11-13 analogues neither bound to MC receptors nor stimulated cAMP. We have thus identified new MC receptor binding motif derived from the C-terminal sequence of alpha-MSH. The tetrapeptides have novel properties as the both act via MC-ergic pathways and also carry the anti-inflammatory alpha-MSH11-13 message sequence.

  8. Exploiting conformational dynamics in drug discovery: design of C-terminal inhibitors of Hsp90 with improved activities

    PubMed Central

    Moroni, Elisabetta; Zhao, Huiping; Blagg, Brian S.J.; Colombo, Giorgio

    2014-01-01

    The interaction that occurs between molecules is a dynamic process that impacts both structural and conformational properties of the ligand and the ligand binding site. Herein, we investigate the dynamic cross-talk between a protein and the ligand as a source for new opportunities in ligand design. Analysis of the formation/disappearance of protein pockets produced in response to a first-generation inhibitor assisted in the identification of functional groups that could be introduced onto scaffolds to facilitate optimal binding, which allowed for increased binding with previously uncharacterized regions. MD simulations were used to elucidate primary changes that occur in the Hsp90 C-terminal binding pocket in the presence of first-generation ligands. This data was then used to design ligands that adapt to these receptor conformations, which provides access to an energy landscape that is not visible in a static model. The newly synthesized compounds demonstrated anti-proliferative activity at ~150 nanomolar concentration. The method identified herein may be used to design chemical probes that provide additional information on structural variations of Hsp90 C-terminal binding site. PMID:24397468

  9. Chaperone-Like Effect of the Linker on the Isolated C-Terminal Domain of Rabbit Muscle Creatine Kinase

    PubMed Central

    Chen, Zhe; Chen, Xiang-Jun; Xia, Mengdie; He, Hua-Wei; Wang, Sha; Liu, Huihui; Gong, Haipeng; Yan, Yong-Bin

    2012-01-01

    Intramolecular chaperones (IMCs), which are specific domains/segments encoded in the primary structure of proteins, exhibit chaperone-like activity against the aggregation of the other domains in the same molecule. In this research, we found that the truncation of the linker greatly promoted the thermal aggregation of the isolated C-terminal domain (CTD) of rabbit muscle creatine kinase (RMCK). Either the existence of the linker covalently linked to CTD or the supply of the synthetic linker peptide additionally could successfully protect the CTD of RMCK against aggregation in a concentration-dependent manner. Truncated fragments of the linker also behaved as a chaperone-like effect with lower efficiency, revealing the importance of its C-terminal half in the IMC function of the linker. The aggregation sites in the CTD of RMCK were identified by molecular dynamics simulations. Mutational analysis of the three key hydrophobic residues resulted in opposing effects on the thermal aggregation between the CTD with intact or partial linker, confirming the role of linker as a lid to protect the hydrophobic residues against exposure to solvent. These observations suggested that the linkers in multidomain proteins could act as IMCs to facilitate the correct folding of the aggregation-prone domains. Furthermore, the intactness of the IMC linker after proteolysis modulates the production of off-pathway aggregates, which may be important to the onset of some diseases caused by the toxic effects of aggregated proteolytic fragments. PMID:22947872

  10. Specific inhibition of AGC protein kinases by antibodies against C-terminal epitopes.

    PubMed

    Traincard, François; Giacomoni, Véronique; Veron, Michel

    2004-08-13

    The sequences contributing to the catalytic site of protein kinases are not all comprised within the highly conserved catalytic core. Thus, in mammalian cAMP-dependent protein kinase (PKA), the C-terminal sequence participates in substrate binding. Using synthetic peptides mimicking the FxxF motif present at most C-termini of AGC kinases, we have raised highly specific antibodies which are potent and specific inhibitors of the catalytic activity of the cognate protein kinase. Taking into account the structure of PKA, these results point to the potential of the C-terminal region of protein kinases as a target for designing specific protein kinase inhibitors.

  11. A novel C-terminal truncating NR5A1 mutation in dizygotic twins.

    PubMed

    Hattori, Atsushi; Zukeran, Hiroaki; Igarashi, Maki; Toguchi, Suzuka; Toubaru, Yuji; Inoue, Takanobu; Katoh-Fukui, Yuko; Fukami, Maki

    2017-01-01

    Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is a nuclear receptor involved in gonadal and adrenal development. We identified a novel C-terminally truncating NR5A1 mutation, p.Leu423Trpfs*7, in dizygotic twins with 46,XY disorders of sex development. Our results highlight the functional importance of C-terminal region of NR5A1 and indicate that NR5A1 mutations can be associated with intrafamilial phenotypic variations, progressive testicular dysfunction, hypogonadotropic hypogonadism, and borderline adrenal dysfunction.

  12. A novel C-terminal truncating NR5A1 mutation in dizygotic twins

    PubMed Central

    Hattori, Atsushi; Zukeran, Hiroaki; Igarashi, Maki; Toguchi, Suzuka; Toubaru, Yuji; Inoue, Takanobu; Katoh-Fukui, Yuko; Fukami, Maki

    2017-01-01

    Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is a nuclear receptor involved in gonadal and adrenal development. We identified a novel C-terminally truncating NR5A1 mutation, p.Leu423Trpfs*7, in dizygotic twins with 46,XY disorders of sex development. Our results highlight the functional importance of C-terminal region of NR5A1 and indicate that NR5A1 mutations can be associated with intrafamilial phenotypic variations, progressive testicular dysfunction, hypogonadotropic hypogonadism, and borderline adrenal dysfunction. PMID:28326187

  13. The β(1a) subunit of the skeletal DHPR binds to skeletal RyR1 and activates the channel via its 35-residue C-terminal tail.

    PubMed

    Rebbeck, Robyn T; Karunasekara, Yamuna; Gallant, Esther M; Board, Philip G; Beard, Nicole A; Casarotto, Marco G; Dulhunty, Angela F

    2011-02-16

    Although it has been suggested that the C-terminal tail of the β(1a) subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca(2+) release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β(1a) bound to RyR1 in affinity chromatography. The full-length β(1a) subunit and the C-terminal peptide increased [(3)H]ryanodine binding and RyR1 channel activity with an AC(50) of 450-600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca(2+), ATP, and Mg(2+) concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg(2+) inhibition or addition of 100 nM Ca(2+) (without ATP). Maximum increases were seen with 1-10 μM Ca(2+), in the absence of Mg(2+) inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [(3)H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β(1a) subunit and RyR1 may support an in vivo function of β(1a) during voltage-activated Ca(2+) release.

  14. Structural characterization of two pore-forming peptides: consequences of introducing a C-terminal tryptophan.

    PubMed

    Herrera, Alvaro I; Al-Rawi, Ahlam; Cook, Gabriel A; Gao, Jian; Iwamoto, Takeo; Prakash, Om; Tomich, John M; Chen, Jianhan

    2010-08-01

    Synthetic channel-forming peptides that can restore chloride conductance across epithelial membranes could provide a novel treatment of channelopathies such as cystic fibrosis. Among a series of 22-residue peptides derived from the second transmembrane segment of the glycine receptor alpha(1)-subunit (M2GlyR), p22-S22W (KKKKP ARVGL GITTV LTMTT QW) is particularly promising with robust membrane insertion and assembly. The concentration to reach one-half maximal short circuit current is reduced to 45 +/- 6 microM from that of 210 +/- 70 microM of peptide p22 (KKKKP ARVGL GITTV LTMTT QS). However, this is accompanied with nearly 50% reduction in conductance. Toward obtaining a molecular level understanding of the channel activities, we combine information from solution NMR, existing biophysical data, and molecular modeling to construct atomistic models of the putative pentameric channels of p22 and p22-S22W. Simulations in membrane bilayers demonstrate that these structural models, even though highly flexible, are stable and remain adequately open for ion conductance. The membrane-anchoring tryptophan residues not only rigidify the whole channel, suggesting increased stability, but also lead to global changes in the pore profile. Specifically, the p22-S22W pore has a smaller opening on average, consistent with lower measured conductance. Direct observation of several incidences of chloride transport suggests several qualitative features of how these channels might selectively conduct anions. The current study thus helps to rationalize the functional consequences of introducing a single C-terminal tryptophan. Availability of these structural models also paves the way for future work to rationally modify and improve M2GlyR-derived peptides toward potential peptide-based channel replacement therapy.

  15. Alpha-A crystallin: quantitation of C-terminal modification during lens aging

    NASA Technical Reports Server (NTRS)

    Takemoto, L.; Gopalakrishnan, S.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Previous studies have demonstrated that the C-terminal region of alpha-A crystallin is susceptible to age-dependent, posttranslational modification. To quantitate the amount of modification, alpha-A crystallin was purified from total proteins of the aging bovine lens, then digested with lys-C endoproteinase. Reverse phase, high pressure liquid chromatography was used to resolve and quantitate the resulting peptides, to determine the amount of C-terminal peptide relative to peptides from other regions of the protein that have not been reported to undergo modification. The results indicate that relative to alpha-A crystallin from newborn lens, posttranslational modification has occurred in approximately 45-55% of the C-terminal region from mature lens. These results demonstrate extensive modification of the C-terminal region of alpha-A crystallin from the mature lens, indicating that during the aging process, posttranslational modifications in this region may make significant contributions to the aggregated state and/or molecular chaperone properties of the molecule.

  16. Kinetic and stability properties of Penicillium chrysogenum ATP sulfurylase missing the C-terminal regulatory domain.

    PubMed

    Hanna, Eissa; Ng, Kit Fai; MacRae, Ian J; Bley, Christopher J; Fisher, Andrew J; Segel, Irwin H

    2004-02-06

    ATP sulfurylase from Penicillium chrysogenum is a homohexameric enzyme that is subject to allosteric inhibition by 3'-phosphoadenosine 5'-phosphosulfate. In contrast to the wild type enzyme, recombinant ATP sulfurylase lacking the C-terminal allosteric domain was monomeric and noncooperative. All kcat values were decreased (the adenosine 5'-phosphosulfate (adenylylsulfate) (APS) synthesis reaction to 17% of the wild type value). Additionally, the Michaelis constants for MgATP and sulfate (or molybdate), the dissociation constant of E.APS, and the monovalent oxyanion dissociation constants of dead end E.MgATP.oxyanion complexes were all increased. APS release (the k6 step) was rate-limiting in the wild type enzyme. Without the C-terminal domain, the composite k5 step (isomerization of the central complex and MgPPi release) became rate-limiting. The cumulative results indicate that besides (a) serving as a receptor for the allosteric inhibitor, the C-terminal domain (b) stabilizes the hexameric structure and indirectly, individual subunits. Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more steps following the formation of the binary E.MgATP and E.SO4(2-) complexes and preceding the release of MgPPi are optimized. The more negative entropy of activation of the truncated enzyme for APS synthesis is consistent with a role of the C-terminal domain in promoting the effective orientation of MgATP and sulfate at the active site.

  17. Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

    PubMed Central

    Reulen, Sanne WA; van Baal, Ingrid; Raats, Jos MH; Merkx, Maarten

    2009-01-01

    Background Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging. Results Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb)-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA)-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step. Conclusion A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers. PMID:19619333

  18. Structural and functional characterization of the C-terminal transmembrane region of NBCe1-A.

    PubMed

    Zhu, Quansheng; Kao, Liyo; Azimov, Rustam; Abuladze, Natalia; Newman, Debra; Pushkin, Alexander; Liu, Weixin; Chang, Connie; Kurtz, Ira

    2010-11-26

    NBCe1-A and AE1 both belong to the SLC4 HCO(3)(-) transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala(800)-Lys(967). Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met(858) accessible to both biotin maleimide and TAMRA and Thr(926)-Ala(929) only to TAMRA labeling. The intracellular surface contains a highly exposed (Met(813)-Gly(828)) region and a cryptic (Met(887)-Arg(904)) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp(960). Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro(868)-Leu(967) (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.

  19. A summary of staphylococcal C-terminal SH3b_5 cell wall binding domains.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcal peptidoglycan hydrolases are a potential new source of antimicrobials. A large subset of these proteins contain a C-terminal SH3b_5 cell wall binding domain that has been shown for some to be essential for accurate cell wall recognition and subsequent staphylolytic activity, propert...

  20. Relationship between C-telopeptide pyridinoline cross-links (ICTP) and putative periodontal pathogens in periodontitis

    PubMed Central

    Palys, Michael D.; Haffajee, Anne D.; Socransky, Sigmund S.; Giannobile, William V.

    2008-01-01

    Crevicular fluid pyridinoline cross-linked carboxyterminal telopeptide of type 1 collagen (ICTP) is predictive for future alveolar bone loss in experimental periodontitis in dogs. The present study sought to relate ICTP to a panel of subgingival species in subjects exhibiting various clinical presentations such as health (n=7), gingivitis (n=8) and periodontitis (n=21). 28 subgingival plaque and GCF samples were taken from mesiobuccal sites in each of 36 subjects. The presence and levels of 40 subgingival taxa were determined in plaque samples using whole genomic DNA probes and checkerboard DNA-DNA hybridization. GCF ICTP levels were quantified using radioimmunoassay (RIA). Clinical assessments made at the same sites included: BOP, gingival redness, plaque, pocket depth, and attachment level. Differences among ICTP levels in the 3 subject groups were sought using the Kruskal-Wallis test. Relationships between ICTP levels and clinical parameters as well as subgingival species were determined by regression analysis. The results demonstrated significant differences among disease categories for GCF ICTP levels for healthy (1.1+0.6 pg/site (mean±SEM)) gingivitis (14.8±6.6 pg/site) and peridontitis subjects (30.3+5.7 pg/site) (p=0.0017). ICTP levels related modestly to several clinical parameters. Regression analysis indicated that ICTP levels correlated strongly with mean subject levels of several periodontal pathogens including B. forsythus, P. gingivalis, P. intermedia, P. nigrescens and T. denticola (p<0.01). The data indicate that there is a positive relationship between the putative bone resorptive marker ICTP and periodontal pathogens. PMID:9846794

  1. Comparative Analysis of the Biochemical and Functional Properties of C-Terminal Domains of Autotransporters ▿

    PubMed Central

    Marín, Elvira; Bodelón, Gustavo; Fernández, Luis Ángel

    2010-01-01

    Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the β-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal α-helix and a β-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved α-helix severely impairs β-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT β-barrel without its α-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed. PMID:20802036

  2. Conformational changes accompany phosphorylation of the epidermal growth factor receptor C-terminal domain

    PubMed Central

    Lee, Nam Y.; Koland, John G.

    2005-01-01

    The precise regulation of epidermal growth factor receptor (EGFR) signaling is crucial to its function in cellular growth control. Various studies have suggested that the C-terminal phosphorylation domain, itself a substrate for the EGFR kinase activity, exerts a regulatory influence upon it, although the molecular mechanism for this regulation is unknown. The fluorescence resonance energy transfer (FRET) technique was employed to examine how C-terminal domain conformational changes in the context of receptor activation and autophosphorylation might regulate EGFR enzymatic activity. A novel FRET reporter system was devised in which recombinant purified EGFR intracellular domain (ICD) proteins of varying C-terminal lengths were site-specifically labeled at their extreme C termini with blue fluorescent protein (BFP) and a fluorescent nucleotide analog, 2′(3′)-O-(2,4,6-trinitrophenyl)-adenosine 5′-triphosphate (TNP-ATP), binding at their active sites. This novel BFP/TNP-ATP FRET pair demonstrated efficient energy transfer as evidenced by appreciable BFP-donor quenching by bound TNP-ATP. In particular, a marked reduction in energy transfer was observed for the full-length BFP-labeled EGFR-ICD protein upon phosphorylation, likely reflecting its movement away from the active site. The estimated distances from the BFP module to the TNP-ATP-occupied active site for the full-length and C-terminally truncated proteins also reveal the possible folding geometry of this domain with respect to the kinase core. The present studies demonstrate the first use of BFP/TNP-ATP as a FRET reporter system. Furthermore, the results described here provide biophysical evidence for phosphorylation-dependent conformational changes in the C-terminal phosphorylation domain and its likely interaction with the kinase core. PMID:16199664

  3. Conformational changes accompany phosphorylation of the epidermal growth factor receptor C-terminal domain.

    PubMed

    Lee, Nam Y; Koland, John G

    2005-11-01

    The precise regulation of epidermal growth factor receptor (EGFR) signaling is crucial to its function in cellular growth control. Various studies have suggested that the C-terminal phosphorylation domain, itself a substrate for the EGFR kinase activity, exerts a regulatory influence upon it, although the molecular mechanism for this regulation is unknown. The fluorescence resonance energy transfer (FRET) technique was employed to examine how C-terminal domain conformational changes in the context of receptor activation and autophosphorylation might regulate EGFR enzymatic activity. A novel FRET reporter system was devised in which recombinant purified EGFR intracellular domain (ICD) proteins of varying C-terminal lengths were site-specifically labeled at their extreme C termini with blue fluorescent protein (BFP) and a fluorescent nucleotide analog, 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP), binding at their active sites. This novel BFP/TNP-ATP FRET pair demonstrated efficient energy transfer as evidenced by appreciable BFP-donor quenching by bound TNP-ATP. In particular, a marked reduction in energy transfer was observed for the full-length BFP-labeled EGFR-ICD protein upon phosphorylation, likely reflecting its movement away from the active site. The estimated distances from the BFP module to the TNP-ATP-occupied active site for the full-length and C-terminally truncated proteins also reveal the possible folding geometry of this domain with respect to the kinase core. The present studies demonstrate the first use of BFP/TNP-ATP as a FRET reporter system. Furthermore, the results described here provide biophysical evidence for phosphorylation-dependent conformational changes in the C-terminal phosphorylation domain and its likely interaction with the kinase core.

  4. GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

    SciTech Connect

    Singh, Pratibha; Savithri, H.S.

    2015-08-15

    Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

  5. Evolutionary bridges to new protein folds: design of C-terminal Cro protein chameleon sequences.

    PubMed

    Anderson, William J; Van Dorn, Laura O; Ingram, Wendy M; Cordes, Matthew H J

    2011-09-01

    Regions of amino-acid sequence that are compatible with multiple folds may facilitate evolutionary transitions in protein structure. In a previous study, we described a heuristically designed chameleon sequence (SASF1, structurally ambivalent sequence fragment 1) that could adopt either of two naturally occurring conformations (α-helical or β-sheet) when incorporated as part of the C-terminal dimerization subdomain of two structurally divergent transcription factors, P22 Cro and λ Cro. Here we describe longer chameleon designs (SASF2 and SASF3) that in the case of SASF3 correspond to the full C-terminal half of the ordered region of a P22 Cro/λ Cro sequence alignment (residues 34-57). P22-SASF2 and λ(WDD)-SASF2 show moderate thermal stability in denaturation curves monitored by circular dichroism (T(m) values of 46 and 55°C, respectively), while P22-SASF3 and λ(WDD)-SASF3 have somewhat reduced stability (T(m) values of 33 and 49°C, respectively). (13)C and (1)H NMR secondary chemical shift analysis confirms two C-terminal α-helices for P22-SASF2 (residues 36-45 and 54-57) and two C-terminal β-strands for λ(WDD)-SASF2 (residues 40-45 and 50-52), corresponding to secondary structure locations in the two parent sequences. Backbone relaxation data show that both chameleon sequences have a relatively well-ordered structure. Comparisons of (15)N-(1)H correlation spectra for SASF2 and SASF3-containing proteins strongly suggest that SASF3 retains the chameleonism of SASF2. Both Cro C-terminal conformations can be encoded in a single sequence, showing the plausibility of linking different Cro folds by smooth evolutionary transitions. The N-terminal subdomain, though largely conserved in structure, also exerts an important contextual influence on the structure of the C-terminal region.

  6. Rearrangement of the histone H2A C-terminal domain in the nucleosome

    SciTech Connect

    Usachenko, S.I.; Bavykin, S.G.; Gavin, I.M.; Bradbury, M. |

    1994-07-19

    Using zero-length covalent protein-DNA crosslinking, the authors have mapped the histone-DNA contacts in nucleosome core particles from which the C- and N-terminal domains of histone H2A were selectively trimmed by trypsin or clostripain. They found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axis, whereas its globular domain contacts the end of DNA in the nucleosome core particle. The appearance of the histone H2A contact at the dyad axis occurs only in the absence of linker DNA and does not depend on the absence of linker histones. The results show the ability of the histone H2A C-terminal domain to rearrange. This rearrangement might play a biological role in nucleosome disassembly and reassembly and the retention of the H2A-H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome.

  7. Crystallization of the C-terminal domain of the bacteriophage T7 fibre protein gp17

    PubMed Central

    Garcia-Doval, Carmela; van Raaij, Mark J.

    2012-01-01

    Bacteriophage T7 attaches to its host using the C-terminal domains of its six fibres, which are trimers of the gp17 protein. A C-terminal fragment of gp17 consisting of amino acids 371–553 has been expressed, purified and crystallized. Crystals of two forms were obtained, belonging to space group P212121 (unit-cell parameters a = 61.2, b = 86.0, c = 118.4 Å) and space group C2221 (unit-cell parameters a = 68.3, b = 145.6, c = 172.1 Å). They diffracted to 1.9 and 2.0 Å resolution, respectively. Both crystals are expected to contain one trimer in the asymmetric unit. Multiwavelength anomalous dispersion phasing with a mercury derivative is in progress. PMID:22297990

  8. Crystallization of the C-terminal domain of the bacteriophage T7 fibre protein gp17.

    PubMed

    Garcia-Doval, Carmela; van Raaij, Mark J

    2012-02-01

    Bacteriophage T7 attaches to its host using the C-terminal domains of its six fibres, which are trimers of the gp17 protein. A C-terminal fragment of gp17 consisting of amino acids 371-553 has been expressed, purified and crystallized. Crystals of two forms were obtained, belonging to space group P2(1)2(1)2(1) (unit-cell parameters a = 61.2, b = 86.0, c = 118.4 Å) and space group C222(1) (unit-cell parameters a = 68.3, b = 145.6, c = 172.1 Å). They diffracted to 1.9 and 2.0 Å resolution, respectively. Both crystals are expected to contain one trimer in the asymmetric unit. Multiwavelength anomalous dispersion phasing with a mercury derivative is in progress.

  9. TubZ filament assembly dynamics requires the flexible C-terminal tail

    PubMed Central

    Fuentes-Pérez, Maria E.; Núñez-Ramírez, Rafael; Martín-González, Alejandro; Juan-Rodríguez, David; Llorca, Oscar; Moreno-Herrero, Fernando; Oliva, Maria A.

    2017-01-01

    Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. TubZ is a tubulin-like protein that functions in extrachromosomal DNA movement within bacteria. TubZ filaments grow in a helical fashion following treadmilling or dynamic instability, although the underlying mechanism is unclear. We have unraveled the molecular basis for filament assembly and dynamics combining electron and atomic force microscopy and biochemical analyses. Our findings suggest that GTP caps retain the filament helical structure and hydrolysis triggers filament stiffening upon disassembly. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process. This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY. PMID:28230082

  10. TubZ filament assembly dynamics requires the flexible C-terminal tail.

    PubMed

    Fuentes-Pérez, Maria E; Núñez-Ramírez, Rafael; Martín-González, Alejandro; Juan-Rodríguez, David; Llorca, Oscar; Moreno-Herrero, Fernando; Oliva, Maria A

    2017-02-23

    Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. TubZ is a tubulin-like protein that functions in extrachromosomal DNA movement within bacteria. TubZ filaments grow in a helical fashion following treadmilling or dynamic instability, although the underlying mechanism is unclear. We have unraveled the molecular basis for filament assembly and dynamics combining electron and atomic force microscopy and biochemical analyses. Our findings suggest that GTP caps retain the filament helical structure and hydrolysis triggers filament stiffening upon disassembly. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process. This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY.

  11. Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A*

    PubMed Central

    Zhu, Quansheng; Kao, Liyo; Azimov, Rustam; Abuladze, Natalia; Newman, Debra; Pushkin, Alexander; Liu, Weixin; Chang, Connie; Kurtz, Ira

    2010-01-01

    NBCe1-A and AE1 both belong to the SLC4 HCO3− transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala800–Lys967. Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met858 accessible to both biotin maleimide and TAMRA and Thr926–Ala929 only to TAMRA labeling. The intracellular surface contains a highly exposed (Met813–Gly828) region and a cryptic (Met887–Arg904) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp960. Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro868–Leu967 (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1. PMID:20837482

  12. Dual Thermosensitive Hydrogels Assembled from the Conserved C-Terminal Domain of Spider Dragline Silk.

    PubMed

    Qian, Zhi-Gang; Zhou, Ming-Liang; Song, Wen-Wen; Xia, Xiao-Xia

    2015-11-09

    Stimuli-responsive hydrogels have great potentials in biomedical and biotechnological applications. Due to the advantages of precise control over molecular weight and being biodegradable, protein-based hydrogels and their applications have been extensively studied. However, protein hydrogels with dual thermosensitive properties are rarely reported. Here we present the first report of dual thermosensitive hydrogels assembled from the conserved C-terminal domain of spider dragline silk. First, we found that recombinant C-terminal domain of major ampullate spidroin 1 (MaSp1) of the spider Nephila clavipes formed hydrogels when cooled to approximately 2 °C or heated to 65 °C. The conformational changes and self-assembly of the recombinant protein were studied to understand the mechanism of the gelation processes using multiple methods. It was proposed that the gelation in the low-temperature regime was dominated by hydrogen bonding and hydrophobic interaction between folded protein molecules, whereas the gelation in the high-temperature regime was due to cross-linking of the exposed hydrophobic patches resulting from partial unfolding of the protein upon heating. More interestingly, genetic fusion of the C-terminal domain to a short repetitive region of N. clavipes MaSp1 resulted in a chimeric protein that formed a hydrogel with significantly improved mechanical properties at low temperatures between 2 and 10 °C. Furthermore, the formation of similar hydrogels was observed for the recombinant C-terminal domains of dragline silk of different spider species, thus demonstrating the conserved ability to form dual thermosensitive hydrogels. These findings may be useful in the design and construction of novel protein hydrogels with tunable multiple thermosensitivity for applications in the future.

  13. Study on the C-terminal beta-hairpin of protein G in AB heteropolymer model

    NASA Astrophysics Data System (ADS)

    Kim, Seung-Yeon

    2016-08-01

    The off-lattice AB heteropolymer model, consisting of the hydrophobic (A) and hydrophilic (B) polymers, is one of popular protein models. Its energy function includes the bending energy and the van der Waals interaction energy. The properties and the energy landscape of the C-terminal beta-hairpin of protein G are studied in the off-lattice AB heteropolymer model with conformational space annealing, a powerful global optimization method.

  14. Structural and functional comparisons of retroviral envelope protein C-terminal domains: still much to learn.

    PubMed

    Steckbeck, Jonathan D; Kuhlmann, Anne-Sophie; Montelaro, Ronald C

    2014-01-16

    Retroviruses are a family of viruses that cause a broad range of pathologies in animals and humans, from the apparently harmless, long-term genomic insertion of endogenous retroviruses, to tumors induced by the oncogenic retroviruses and acquired immunodeficiency syndrome (AIDS) resulting from human immunodeficiency virus infection. Disease can be the result of diverse mechanisms, including tumorigenesis induced by viral oncogenes or immune destruction, leading to the gradual loss of CD4 T-cells. Of the virally encoded proteins common to all retroviruses, the envelope (Env) displays perhaps the most diverse functionality. Env is primarily responsible for binding the cellular receptor and for effecting the fusion process, with these functions mediated by protein domains localized to the exterior of the virus. The remaining C-terminal domain may have the most variable functionality of all retroviral proteins. The C-terminal domains from three prototypical retroviruses are discussed, focusing on the different structures and functions, which include fusion activation, tumorigenesis and viral assembly and lifecycle influences. Despite these genetic and functional differences, however, the C-terminal domains of these viruses share a common feature in the modulation of Env ectodomain conformation. Despite their differences, perhaps each system still has information to share with the others.

  15. Conserved C-Terminal Domain of Spider Tubuliform Spidroin 1 Contributes to Extensibility in Synthetic Fibers

    SciTech Connect

    Gnesa, Eric; Hsia, Yang; Yarger, Jeffery L.; Weber, Warner; Lin-Cereghino, Joan; Lin-Cereghino, Geoff; Tang, Simon; Agari, Kimiko; Vierra, Craig

    2012-05-24

    Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that postdrawn fibers containing the C-terminal domain of TuSp1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to nontagged recombinant dragline silk proteins spun from equivalently sized proteins.

  16. Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response

    PubMed Central

    Chan, Tung O.; Zhang, Jin; Tiegs, Brian C.; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M.; Armen, Roger S.; Rodeck, Ulrich; Penn, Raymond B.

    2015-01-01

    The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr308 in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr308 dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser473) increased phosphatase resistance of the phosphorylated activation loop (pThr308) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr308 phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. PMID:26201515

  17. Electrostatic interactions at the C-terminal domain of nucleoplasmin modulate its chromatin decondensation activity.

    PubMed

    Hierro, Aitor; Arizmendi, Jesús M; Bañuelos, Sonia; Prado, Adelina; Muga, Arturo

    2002-05-21

    The chromatin decondensation activity, thermal stability, and secondary structure of recombinant nucleoplasmin, of two deletion mutants, and of the protein isolated from Xenopus oocytes have been characterized. As previously reported, the chromatin decondensation activity of recombinant, unphosphorylated nucleoplasmin is almost negligible. Our data show that deletion of 50 residues at the C-terminal domain of the protein, containing the positively charged nuclear localization sequence, activates its chromatin decondensation ability and decreases its stability. Interestingly, both the decondensation activity and thermal stability of this deletion mutant resemble those of the phosphorylated protein isolated from Xenopus oocytes. Deletion of 80 residues at the C-terminal domain, containing the above-mentioned positively charged region and a poly(Glu) tract, inactivates the protein and increases its thermal stability. These findings, along with the effect of salt on the thermal stability of these proteins, suggest that electrostatic interactions between the positive nuclear localization sequence and the poly(Glu) tract, at the C-terminal domain, modulate protein activity and stability.

  18. The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

    SciTech Connect

    Papazyan, Romeo; Rozengurt, Enrique; Rey, Osvaldo . E-mail: orey@mednet.ucla.edu

    2006-04-14

    We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.

  19. The role of the C-terminal region on the oligomeric state and enzymatic activity of Trypanosoma cruzi hypoxanthine phosphoribosyl transferase.

    PubMed

    Valsecchi, Wanda M; Cousido-Siah, Alexandra; Defelipe, Lucas A; Mitschler, André; Podjarny, Alberto; Santos, Javier; Delfino, José M

    2016-06-01

    Hypoxanthine phosphoribosyl transferase from Trypanosoma cruzi (TcHPRT) is a critical enzyme for the survival of the parasite. This work demonstrates that the full-length form in solution adopts a stable and enzymatically active tetrameric form, exhibiting large inter-subunit surfaces. Although this protein irreversibly aggregates during unfolding, oligomerization is reversible and can be modulated by low concentrations of urea. When the C-terminal region, which is predicted as a disordered stretch, is excised by proteolysis, TcHPRT adopts a dimeric state, suggesting that the C-terminal region acts as a main guide for the quaternary arrangement. These results are in agreement with X-ray crystallographic data presented in this work. On the other hand, the C-terminal region exhibits a modulatory role on the enzyme, as attested by the enhanced activity observed for the dimeric form. Bisphosphonates act as substrate-mimetics, uncovering long-range communications among the active sites. All in all, this work contributes to establish new ways applicable to the design of novel inhibitors that could eventually result in new drugs against parasitic diseases.

  20. Development of the sigma-1 receptor in C-terminals of motoneurons and colocalization with the N,N'-dimethyltryptamine forming enzyme, indole-N-methyl transferase.

    PubMed

    Mavlyutov, T A; Epstein, M L; Liu, P; Verbny, Y I; Ziskind-Conhaim, L; Ruoho, A E

    2012-03-29

    The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein-coupled receptors (GPCR). In the CNS, the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and S1Rs suggest that DMT is synthesized locally to effectively activate S1R in MN.

  1. Probing the Impact of the EchinT C-Terminal Domain on Structure and Catalysis

    SciTech Connect

    S Bardaweel; J Pace; T Chou; V Cody; C Wagner

    2011-12-31

    Histidine triad nucleotide binding protein (Hint) is considered as the ancestor of the histidine triad protein superfamily and is highly conserved from bacteria to humans. Prokaryote genomes, including a wide array of both Gram-negative bacteria and Gram-positive bacteria, typically encode one Hint gene. The cellular function of Hint and the rationale for its evolutionary conservation in bacteria have remained a mystery. Despite its ubiquity and high sequence similarity to eukaryote Hint1 [Escherichia coli Hint (echinT) is 48% identical with human Hint1], prokaryote Hint has been reported in only a few studies. Here we report the first conformational information on the full-length N-terminal and C-terminal residues of Hint from the E. coli complex with GMP. Structural analysis of the echinT-GMP complex reveals that it crystallizes in the monoclinic space group P2{sub 1} with four homodimers in the asymmetric unit. Analysis of electron density for both the N-terminal residues and the C-terminal residues of the echinT-GMP complex indicates that the loops in some monomers can adopt more than one conformation. The observation of conformational flexibility in terminal loop regions could explain the presence of multiple homodimers in the asymmetric unit of this structure. To explore the impact of the echinT C-terminus on protein structure and catalysis, we conducted a series of catalytic radiolabeling and kinetic experiments on the C-terminal deletion mutants of echinT. In this study, we show that sequential deletion of the C-terminus likely has no effect on homodimerization and a modest effect on the secondary structure of echinT. However, we observed a significant impact on the folding structure, as reflected by a significant lowering of the T{sub m} value. Kinetic analysis reveals that the C-terminal deletion mutants are within an order of magnitude less efficient in catalysis compared to wild type, while the overall kinetic mechanism that proceeds through a fast step

  2. Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

    PubMed

    Hu, Zhilan; Zhang, Henry; Haley, Benjamin; Macchi, Frank; Yang, Feng; Misaghi, Shahram; Elich, Joseph; Yang, Renee; Tang, Yun; Joly, John C; Snedecor, Bradley R; Shen, Amy

    2016-10-01

    Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

  3. Heterologous C-terminal sequences disrupt transcriptional activation and oncogenesis by p59v-rel.

    PubMed Central

    Diehl, J A; Hannink, M

    1993-01-01

    Members of the NF-kappa B/rel family of transcription factors are regulated through a trans association with members of a family of inhibitor proteins, collectively known as I kappa B proteins, that contain five to eight copies of a 33-amino-acid repeat sequence (ankyrin repeat). Certain NF-kappa B/rel proteins are also regulated by cis-acting ankyrin repeat-containing domains. The C terminus of p105NF-kappa B, the precursor of the 50-kDa subunit of NF-kappa B, contains a series of ankyrin repeats; proteolytic removal of this ankyrin domain is necessary for the manifestation of sequence-specific DNA binding and nuclear translocation of the N-terminal product. To investigate the structural requirements important for regulation of different NF-kappa B/rel family members by polypeptides containing ankyrin repeat domains, we have constructed a p59v-rel:p105NF-kappa B chimeric protein (p110v-rel-ank). The presence of C-terminal p105NF-kappa B-derived sequences in p110v-rel-ank inhibited nuclear translocation, sequence-specific DNA binding, pp40I kappa B-alpha association, and oncogenic transformation. Sequential truncation of the C-terminal ankyrin domain of p110v-rel-ank resulted in the restoration of nuclear translocation, DNA binding, and pp40I kappa B-alpha association but did not restore the oncogenic properties of p59v-rel. The presence of 67 C-terminal p105NF-kappa B-derived amino acids was sufficient to inhibit both transcriptional activation and oncogenic transformation by p59v-rel. These results support a model in which activation of gene expression by p59v-rel is required for its ability to induce oncogenic transformation. Images PMID:8230438

  4. C-terminal tail of FGF19 determines its specificity toward Klotho co-receptors.

    PubMed

    Wu, Xinle; Lemon, Bryan; Li, XiaoFan; Gupte, Jamila; Weiszmann, Jennifer; Stevens, Jennitte; Hawkins, Nessa; Shen, Wenyan; Lindberg, Richard; Chen, Jin-Long; Tian, Hui; Li, Yang

    2008-11-28

    FGF19 subfamily proteins (FGF19, FGF21, and FGF23) are unique members of fibroblast growth factors (FGFs) that regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis in an endocrine fashion. Their activities require the presence of alpha or betaKlotho, two related single-pass transmembrane proteins, as co-receptors in relevant target tissues. We previously showed that FGF19 can bind to both alpha and betaKlotho, whereas FGF21 and FGF23 can bind only to either betaKlotho or alphaKlotho, respectively in vitro. To determine the mechanism regulating the binding and specificity among FGF19 subfamily members to Klotho family proteins, chimeric proteins between FGF19 subfamily members or chimeric proteins between Klotho family members were constructed to probe the interaction between those two families. Our results showed that a chimera of FGF19 with the FGF21 C-terminal tail interacts only with betaKlotho and a chimera with the FGF23 C-terminal tail interacts only with alphaKlotho. FGF signaling assays also reflected the change of specificity we observed for the chimeras. These results identified the C-terminal tail of FGF19 as a region necessary for its recognition of Klotho family proteins. In addition, chimeras between alpha and betaKlotho were also generated to probe the regions in Klotho proteins that are important for signaling by this FGF subfamily. Both FGF23 and FGF21 require intact alpha or betaKlotho for signaling, respectively, whereas FGF19 can signal through a Klotho chimera consisting of the N terminus of alphaKlotho and the C terminus of betaKlotho. Our results provide the first glimpse of the regions that regulate the binding specificity between this unique family of FGFs and their co-receptors.

  5. The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins.

    PubMed

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-03-20

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (alpha, beta, and gamma), in which three laminin globular modules in the alpha chain and the Glu residue in the C-terminal tail of the gamma chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the beta chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the beta1 or beta2 chain toward a panel of laminin-binding integrins, and we found that beta2 chain-containing laminins (beta2-laminins) bound more avidly to alpha3beta1 and alpha7X2beta1 integrins than beta1 chain-containing laminins (beta1-laminins), whereas alpha6beta1, alpha6beta4, and alpha7X1beta1 integrins did not show any preference toward beta2-laminins. Because alpha3beta1 contains the "X2-type" variable region in the alpha3 subunit and alpha6beta1 and alpha6beta4 contain the "X1-type" region in the alpha6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between beta1-laminins and beta2-laminins. In support of this possibility, a putative X2-type variant of alpha6beta1 was produced and found to bind preferentially to beta2-laminins. Production of a series of swap mutants between the beta1 and beta2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by beta2-laminins. Taken together, the results provide evidence that the C-terminal region of beta chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.

  6. Crystallization of the C-terminal globular domain of avian reovirus fibre

    SciTech Connect

    Raaij, Mark J. van; Hermo Parrado, X. Lois; Guardado Calvo, Pablo; Fox, Gavin C.; Llamas-Saiz, Antonio L.; Costas, Celina; Martínez-Costas, José; Benavente, Javier

    2005-07-01

    Partial proteolysis of the avian reovirus cell-attachment protein σC yields a major homotrimeric C-terminal fragment that presumably contains the receptor-binding domain. This fragment has been crystallized in the presence and absence of zinc sulfate and cadmium sulfate. One of the crystal forms diffracts synchrotron X-rays to 2.2–2.3 Å. Avian reovirus fibre, a homotrimer of the σC protein, is responsible for primary host-cell attachment. Using the protease trypsin, a C-terminal σC fragment containing amino acids 156–326 has been generated which was subsequently purified and crystallized. Two different crystal forms were obtained, one grown in the absence of divalent cations and belonging to space group P6{sub 3}22 (unit-cell parameters a = 75.6, c = 243.1 Å) and one grown in the presence of either zinc or cadmium sulfate and belonging to space group P321 (unit-cell parameters a = 74.7, c = 74.5 Å and a = 73.1, c = 69.9 Å for the Zn{sup II}- and Cd{sup II}-grown crystals, respectively). The first crystal form diffracted synchrotron radiation to 3.0 Å resolution and the second form to 2.2–2.3 Å. Its closest related structure, the C-terminal fragment of mammalian reovirus fibre, has only 18% sequence identity and molecular-replacement attempts were unsuccessful. Therefore, a search is under way for suitable heavy-atom derivatives and attempts are being made to grow protein crystals containing selenomethionine instead of methionine.

  7. BS69/ZMYND11 C-Terminal Domains Bind and Inhibit EBNA2

    PubMed Central

    Shen, Chih-Lung; Gonzalez-Hurtado, Elsie; Zhang, Zhi-Min; Xu, Muyu; Martinez, Ernest; Peng, Chih-Wen; Song, Jikui

    2016-01-01

    Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) plays an important role in driving immortalization of EBV-infected B cells through regulating the expression of many viral and cellular genes. We report a structural study of the tumor suppressor BS69/ZMYND11 C-terminal region, comprised of tandem coiled-coil-MYND domains (BS69CC-MYND), in complex with an EBNA2 peptide containing a PXLXP motif. The coiled-coil domain of BS69 self-associates to bring two separate MYND domains in close proximity, thereby enhancing the BS69 MYND-EBNA2 interaction. ITC analysis of BS69CC-MYND with a C-terminal fragment of EBNA2 further suggests that the BS69CC-MYND homodimer synergistically binds to the two EBNA2 PXLXP motifs that are respectively located in the conserved regions CR7 and CR8. Furthermore, we showed that EBNA2 interacts with BS69 and down-regulates its expression at both mRNA and protein levels in EBV-infected B cells. Ectopic BS69CC-MYND is recruited to viral target promoters through interactions with EBNA2, inhibits EBNA2-mediated transcription activation, and impairs proliferation of lymphoblastoid cell lines (LCLs). Substitution of critical residues in the MYND domain impairs the BS69-EBNA2 interaction and abolishes the BS69 inhibition of the EBNA2-mediated transactivation and LCL proliferation. This study identifies the BS69 C-terminal domains as an inhibitor of EBNA2, which may have important implications in development of novel therapeutic strategies against EBV infection. PMID:26845565

  8. The role of the C-terminal region in phosphoglycerate mutase.

    PubMed Central

    Walter, R A; Nairn, J; Duncan, D; Price, N C; Kelly, S M; Rigden, D J; Fothergill-Gilmore, L A

    1999-01-01

    Removal of the C-terminal seven residues from phosphoglycerate mutase from Saccharomyces cerevisiae by limited proteolysis is associated with loss of mutase activity, but no change in phosphatase activity. The presence of the cofactor 2, 3-bisphosphoglycerate, or of the cofactor and substrate 3-phosphoglycerate together, confers protection against proteolysis. The substrate alone offers no protection. Replacement of either or both of the two lysines at the C-terminus by glycines has only limited effects on the kinetic properties of phosphoglycerate mutase, indicating that these residues are unlikely to be involved in crucial electrostatic interactions with the substrate, intermediate or product in the reaction. However, the double-mutant form of the enzyme is more sensitive to proteolysis and is no longer protected against proteolysis by the presence of cofactor. The proteolysed wild-type and two of the mutated forms of the enzyme show a reduced response to 2-phosphoglycollate, which enhances the instability of the phospho form of the native enzyme. The phosphoglycerate mutase from Schizosaccharomyces pombe, which lacks the analogous C-terminal tail, has an inherently lower mutase activity and is also less responsive to stimulation by 2-phosphoglycollate. It is proposed that the C-terminal region of phosphoglycerate mutase helps to maintain the enzyme in its active phosphorylated form and assists in the retention of the bisphosphoglycerate intermediate at the active site. However, its role seems not to be to contribute directly to ligand binding, but rather to exert indirect effects on the transfer of the phospho group between substrate, enzyme, intermediate and product. PMID:9854029

  9. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations

    PubMed Central

    Mehler, Vera J.; Mueller, Christina; Vonhoff, Fernando; White, Robin; Duch, Carsten

    2016-01-01

    Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X), a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80) and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2. PMID:27442528

  10. C-Terminal acetylene derivatized peptides via silyl-based alkyne immobilization.

    PubMed

    Strack, Martin; Metzler-Nolte, Nils; Albada, H Bauke

    2013-06-21

    A new Silyl-based Alkyne Modifying (SAM)-linker for the synthesis of C-terminal acetylene-derivatized peptides is reported. The broad scope of this SAM2-linker is illustrated by manual synthesis of peptides that are side-chain protected, fully deprotected, and disulfide-bridged. Synthesis of a 14-meric (KLAKLAK)2 derivative by microwave-assisted automated SPPS and a one-pot cleavage click procedure yielding protected 1,2,3-triazole peptide conjugates are also described.

  11. C-terminal dimerization of apo-cyclic AMP receptor protein validated in solution.

    PubMed

    Sim, Dae-Won; Choi, Jae Wan; Kim, Ji-Hun; Ryu, Kyoung-Seok; Kim, Myeongkyu; Yu, Hee-Wan; Jo, Ku-Sung; Kim, Eun-Hee; Seo, Min-Duk; Jeon, Young Ho; Lee, Bong-Jin; Kim, Young Pil; Won, Hyung-Sik

    2017-04-01

    Although cyclic AMP receptor protein (CRP) has long served as a typical example of effector-mediated protein allostery, mechanistic details into its regulation have been controversial due to discrepancy between the known crystal structure and NMR structure of apo-CRP. Here, we report that the recombinant protein corresponding to its C-terminal DNA-binding domain (CDD) forms a dimer. This result, together with structural information obtained in the present NMR study, is consistent with the previous crystal structure and validates its relevance also in solution. Therefore, our findings suggest that dissociation of the CDD may be critically involved in cAMP-induced allosteric activation of CRP.

  12. A C-terminal Aldehyde Analog of the Insect Kinins Inhibits Diuresis in the Housefly

    DTIC Science & Technology

    2006-09-21

    p e p t i d e s 2 8 ( 2 0 0 7 ) 1 4 6 – 1 5 2A C-terminal aldehyde analog of the insect kinins inhibits diuresis in the housefly Ronald J. Nachman a...secretion in crickets, but shows inhibition of both in vitro and in vivo diuresis in the housefly. R-LK-CHO reduced the total amount of urine voided over 3 h...to stimulate Malpighian tubule fluid secretion [2,25]. In the housefly, muscakinin has been implicated in the control of diuresis in response to

  13. [Research advances on ubiquitin C-terminal hydrolase in oncogenesis and progression].

    PubMed

    Yu, Juan; Chen, Wei-lin

    2015-03-01

    By regulating the ubiquitination and deubiquitination of key proteins, ubiquitin-proteasome system mediates a variety of cellular activities. Ubiquitin C-terminal hydrolase (UCH) is a deubiquitinating enzyme which can remove ubiquitin chains at the end of ubiquited proteins. The abnormal expression of UCH has been found in a variety of tumor tissues, indicating that it participates in the process of tumor development. Here we review the characteristics of UCH members and current understanding about the role of UCH in tumor development, and the potential target for cancer treatment.

  14. Role of the Cationic C-Terminal Segment of Melittin on Membrane Fragmentation.

    PubMed

    Therrien, Alexandre; Fournier, Alain; Lafleur, Michel

    2016-05-05

    The widespread distribution of cationic antimicrobial peptides capable of membrane fragmentation in nature underlines their importance to living organisms. In the present work, we determined the impact of the electrostatic interactions associated with the cationic C-terminal segment of melittin, a 26-amino acid peptide from bee venom (net charge +6), on its binding to model membranes and on the resulting fragmentation. In order to detail the role played by the C-terminal charges, we prepared a melittin analogue for which the four cationic amino acids in positions 21-24 were substituted with the polar residue citrulline, providing a peptide with the same length and amphiphilicity but with a lower net charge (+2). We compared the peptide bilayer affinity and the membrane fragmentation for bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS) mixtures. It is shown that neutralization of the C-terminal considerably increased melittin affinity for zwitterionic membranes. The unfavorable contribution associated with transferring the cationic C-terminal in a less polar environment was reduced, leaving the hydrophobic interactions, which drive the peptide insertion in bilayers, with limited counterbalancing interactions. The presence of negatively charged lipids (DPPS) in bilayers increased melittin binding by introducing attractive electrostatic interactions, the augmentation being, as expected, greater for native melittin than for its citrullinated analogue. The membrane fragmentation power of the peptide was shown to be controlled by electrostatic interactions and could be modulated by the charge carried by both the membrane and the lytic peptide. The analysis of the lipid composition of the extracted fragments from DPPC/DPPS bilayers revealed no lipid specificity. It is proposed that extended phase separations are more susceptible to lead to the extraction of a lipid species in a specific manner

  15. Synthesis and characterization of photoaffinity labelling reagents towards the Hsp90 C-terminal domain.

    PubMed

    Simon, Binto; Huang, Xuexia; Ju, Huangxian; Sun, Guoxuan; Yang, Min

    2017-02-21

    Glucosyl-novobiocin-based diazirine photoaffinity labelling reagents (PALs) were designed and synthesized to probe the Hsp90 C-terminal domain unknown binding pocket and the structure-activity relationship. Five PALs were successfully synthesized from novobiocin in six consecutive steps employing phase transfer catalytic glycosylation. Reactions were monitored and guided by analytical LC/MS which led to different strategies of adding either a PAL precursor or a sugar moiety first. The structures and bonding linkages of these compounds were characterised by various 2D-NMR spectroscopy and MS techniques. Synthetic techniques provide powerful probes for unknown protein binding pockets.

  16. Synthesis of Peptides Containing C-Terminal Esters Using Trityl Side-Chain Anchoring: Applications to the Synthesis of C-Terminal Ester Analogs of the Saccharomyces cerevisiae Mating Pheromone a-Factor.

    PubMed

    Diaz-Rodriguez, Veronica; Ganusova, Elena; Rappe, Todd M; Becker, Jeffrey M; Distefano, Mark D

    2015-11-20

    Peptides containing C-terminal esters are an important class of bioactive molecules that includes a-factor, a farnesylated dodecapeptide, involved in the mating of Saccharomyces cerevisiae. Here, results that expand the scope of solid-phase peptide synthetic methodology that uses trityl side-chain anchoring for the preparation of peptides with C-terminal cysteine alkyl esters are described. In this method, Fmoc-protected C-terminal cysteine esters are anchored to trityl chloride resin and extended by standard solid-phase procedures followed by acidolytic cleavage and HPLC purification. Analysis using a Gly-Phe-Cys-OMe model tripeptide revealed minimal epimerization of the C-terminal cysteine residue under basic conditions used for Fmoc deprotection. (1)H NMR analysis of the unfarnesylated a-factor precursor peptide confirmed the absence of epimerization. The side-chain anchoring method was used to produce wild-type a-factor that contains a C-terminal methyl ester along with ethyl-, isopropyl-, and benzyl-ester analogs in good yield. Activity assays using a yeast-mating assay demonstrate that while the ethyl and isopropyl esters manifest near-wild-type activity, the benzyl ester-containing analog is ca. 100-fold less active. This simple method opens the door to the synthesis of a variety of C-terminal ester-modified peptides that should be useful in studies of protein prenylation and other structurally related biological processes.

  17. Identification of Novel Short C-Terminal Transcripts of Human SERPINA1 Gene

    PubMed Central

    Matamala, Nerea; Aggarwal, Nupur; Iadarola, Paolo; Fumagalli, Marco; Gomez-Mariano, Gema; Lara, Beatriz; Martinez, Maria Teresa; Cuesta, Isabel; Stolk, Jan

    2017-01-01

    Human SERPINA1 gene is located on chromosome 14q31-32.3 and is organized into three (IA, IB, and IC) non-coding and four (II, III, IV, V) coding exons. This gene produces α1-antitrypsin (A1AT), a prototypical member of the serpin superfamily of proteins. We demonstrate that human peripheral blood leukocytes express not only a product corresponding to the transcript coding for the full-length A1AT protein but also two short transcripts (ST1C4 and ST1C5) of A1AT. In silico sequence analysis revealed that the last exon of the short transcripts contains an Open Reading Frame (ORF) and thus putatively can produce peptides. We found ST1C4 expression across different human tissues whereas ST1C5 was mainly restricted to leukocytes, specifically neutrophils. A high up-regulation (10-fold) of short transcripts was observed in isolated human blood neutrophils after activation with lipopolysaccharide. Parallel analyses by liquid chromatography-mass spectrometry identified peptides corresponding to C-terminal region of A1AT in supernatants of activated but not naïve neutrophils. Herein we report for the first time a tissue specific expression and regulation of short transcripts of SERPINA1 gene, and the presence of C-terminal peptides in supernatants from activated neutrophils, in vitro. This gives a novel insight into the studies on the transcription of SERPINA1 gene. PMID:28107454

  18. Structure of the RecQ C-terminal domain of human Bloom syndrome protein.

    PubMed

    Kim, Sun-Yong; Hakoshima, Toshio; Kitano, Ken

    2013-11-21

    Bloom syndrome is a rare genetic disorder characterized by genomic instability and cancer predisposition. The disease is caused by mutations of the Bloom syndrome protein (BLM). Here we report the crystal structure of a RecQ C-terminal (RQC) domain from human BLM. The structure reveals three novel features of BLM RQC which distinguish it from the previous structures of the Werner syndrome protein (WRN) and RECQ1. First, BLM RQC lacks an aromatic residue at the tip of the β-wing, a key element of the RecQ-family helicases used for DNA-strand separation. Second, a BLM-specific insertion between the N-terminal helices exhibits a looping-out structure that extends at right angles to the β-wing. Deletion mutagenesis of this insertion interfered with binding to Holliday junction. Third, the C-terminal region of BLM RQC adopts an extended structure running along the domain surface, which may facilitate the spatial positioning of an HRDC domain in the full-length protein.

  19. The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters

    PubMed Central

    Algarra, B.; Han, L.; Soriano-Úbeda, C.; Avilés, M.; Coy, P.; Jovine, L.; Jiménez-Movilla, M.

    2016-01-01

    OVGP1 is the major non-serum glycoprotein in the oviduct fluid at the time of fertilization and early embryo development. Its activity differs among species. Here, we show that the C-terminal region of recombinant OVGP1 regulates its binding to the extracellular zona pellucida and affects its activity during fertilization. While porcine OVGP1 penetrates two-thirds of the thickness of the zona pellucida, shorter OVGP1 glycoproteins, including rabbit OVGP1, are restricted to the outer one-third of the zona matrix. Deletion of the C-terminal region reduces the ability of the glycoprotein to penetrate through the zona pellucida and prevents OVGP1 endocytosis. This affects the structure of the zona matrix and increases its resistance to protease digestion. However, only full-length porcine OVGP1 is able to increase the efficiency rate of in vitro fertilization. Thus, our findings document that the presence or absence of conserved regions in the C-terminus of OVGP1 modify its association with the zona pellucida that affects matrix structure and renders the zona matrix permissive to sperm penetration and OVGP1 endocytosis into the egg. PMID:27601270

  20. Intrinsic Disorder of the C-Terminal Domain of Drosophila Methoprene-Tolerant Protein

    PubMed Central

    Kolonko, Marta; Ożga, Katarzyna; Hołubowicz, Rafał; Taube, Michał; Kozak, Maciej; Ożyhar, Andrzej; Greb-Markiewicz, Beata

    2016-01-01

    Methoprene tolerant protein (Met) has recently been confirmed as the long-sought juvenile hormone (JH) receptor. This protein plays a significant role in the cross-talk of the 20-hydroxyecdysone (20E) and JH signalling pathways, which are important for control of insect development and maturation. Met belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) family of transcription factors. In these proteins, bHLH domains are typically responsible for DNA binding and dimerization, whereas the PAS domains are crucial for the choice of dimerization partner and the specificity of target gene activation. The C-terminal region is usually responsible for the regulation of protein complex activity. The sequence of the Met C-terminal region (MetC) is not homologous to any sequence deposited in the Protein Data Bank (PDB) and has not been structurally characterized to date. In this study, we show that the MetC exhibits properties typical for an intrinsically disordered protein (IDP). The final averaged structure obtained with small angle X-ray scattering (SAXS) experiments indicates that intrinsically disordered MetC exists in an extended conformation. This extended shape and the long unfolded regions characterise proteins with high flexibility and dynamics. Therefore, we suggest that the multiplicity of conformations adopted by the disordered MetC is crucial for its activity as a biological switch modulating the cross-talk of different signalling pathways in insects. PMID:27657508

  1. C-terminal interactions of apolipoprotein E4 respond to the postprandial state.

    PubMed

    Tetali, Sarada D; Budamagunta, Madhu S; Voss, John C; Rutledge, John C

    2006-07-01

    Increased triglyceride-rich lipoproteins (TGRLs) in the postprandial state are associated with atherosclerosis. We investigated whether the postprandial state induced structural changes at the apolipoprotein E4 (apoE4) C terminus, its principal lipid binding domain, using electron paramagnetic resonance (EPR) spectroscopy of a site-directed spin label attached to the cysteine of apoE4-W264C. Spin coupling between labels located in the C termini was followed after mixing with preprandial and postprandial human plasma samples. Our results indicate that postprandial plasma triggers a reorganization of the protein such that the dipolar broadening is diminished, indicating a reduction in C-terminal interaction. The loss of spectral broadening was directly correlated with an increase in postprandial plasma triglycerides and was reduced with delipidated plasma. The spin-labeled apoE4 displayed a lipid preference of VLDL > LDL > HDL in the preprandial and postprandial states. The apoE4 shift to VLDL during the postprandial state was accompanied by a loss in spectral broadening of the protein. These findings suggest that apoE4 associated with LDL maintains self-association via its C terminus and that this association is diminished in VLDL-associated protein. Lipolyzed TGRL reflected a depletion of the C-terminal interaction of apoE4. Addition of palmitate to VLDL gave a similar response as lipolyzed TGRL, suggesting that lipolysis products play a major role in reorganizing apoE4 during the postprandial state.

  2. A C-terminal membrane association domain of phototropin 2 is necessary for chloroplast movement.

    PubMed

    Kong, Sam-Geun; Kagawa, Takatoshi; Wada, Masamitsu; Nagatani, Akira

    2013-01-01

    Phototropins (phot1 and phot2), plant-specific blue light receptor kinases, mediate a range of physiological responses in Arabidopsis, including phototropism, chloroplast photorelocation movement, stomatal opening and leaf flattening. Phototropins consist of two photoreceptive domains at their N-terminus, LOV1 (light, oxygen or voltage 1) and LOV2, and a serine/threonine kinase domain at their C-terminus. Here, we determined the molecular moiety for the membrane association of phototropins using the yeast CytoTrap and Arabidopsis protoplast systems. We then examined the physiological significance of the membrane association of phototropins. This detailed study with serial deletions narrowed down the association domain to a relatively small part of the C-terminal domain of phototropin. The functional analysis of phot2 deletion mutants in the phot2-deficient Adiantum and Arabidopsis mutants revealed that the ability to mediate the chloroplast avoidance response correlated well with phot2's membrane association, especially with the Golgi apparatus. Taken together, our data suggest that a small part of the C-terminal domain of phototropins is necessary not only for membrane association but also for the physiological activities that elicit phototropin-specific responses.

  3. PrPSc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains

    PubMed Central

    Saijo, Eri; Hughson, Andrew G.; Raymond, Gregory J.; Suzuki, Akio; Horiuchi, Motohiro

    2016-01-01

    ABSTRACT Understanding the structure of PrPSc and its strain variation has been one of the major challenges in prion disease biology. To study the strain-dependent conformations of PrPSc, we purified proteinase-resistant PrPSc (PrPRES) from mouse brains with three different murine-adapted scrapie strains (Chandler, 22L, and Me7) and systematically tested the accessibility of epitopes of a wide range of anti-PrP and anti-PrPSc specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). We found that epitopes of most anti-PrP antibodies were hidden in the folded structure of PrPRES, even though these epitopes are revealed with guanidine denaturation. However, reactivities to a PrPSc-specific conformational C-terminal antibody showed significant differences among the three different prion strains. Our results provide evidence for strain-dependent conformational variation near the C termini of molecules within PrPSc multimers. IMPORTANCE It has long been apparent that prion strains can have different conformations near the N terminus of the PrPSc protease-resistant core. Here, we show that a C-terminal conformational PrPSc-specific antibody reacts differently to three murine-adapted scrapie strains. These results suggest, in turn, that conformational differences in the C terminus of PrPSc also contribute to the phenotypic distinction between prion strains. PMID:26937029

  4. Structure of the C-terminal domain of nsp4 from feline coronavirus

    SciTech Connect

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh; Berglind, Hanna; Nordlund, Pär; Coutard, Bruno; Tucker, Paul A.

    2009-08-01

    The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4{sub 3}. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions.

  5. Crystallization and halide phasing of the C-terminal domain of human KIN17

    SciTech Connect

    Maire, Albane le; Schiltz, Marc; Braud, Sandrine; Gondry, Muriel; Charbonnier, Jean-Baptiste; Zinn-Justin, Sophie; Stura, Enrico

    2006-03-01

    Expression, purification, crystallization and phasing procedure are reported for the C-terminal domain of human KIN17. Here, the crystallization and initial phasing of the C-terminal domain of human KIN17, a 45 kDa protein mainly expressed in response to ionizing radiation and overexpressed in certain tumour cell lines, are reported. Crystals diffracting to 1.4 Å resolution were obtained from 10% ethylene glycol, 27% PEG 6000, 500 mM LiCl and 100 mM sodium acetate pH 6.3 in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 45.75, b = 46.31, c = 60.80 Å and one molecule in the asymmetric unit. Since this domain has a basic pI, heavy-atom derivatives were obtained by soaking the crystals with negatively charged ions such as tungstate and iodine. The replacement of LiCl by KI in the cryosolution allowed the determination of phases from iodide ions to give an interpretable electron-density map.

  6. Identification of Novel Short C-Terminal Transcripts of Human SERPINA1 Gene.

    PubMed

    Matamala, Nerea; Aggarwal, Nupur; Iadarola, Paolo; Fumagalli, Marco; Gomez-Mariano, Gema; Lara, Beatriz; Martinez, Maria Teresa; Cuesta, Isabel; Stolk, Jan; Janciauskiene, Sabina; Martinez-Delgado, Beatriz

    2017-01-01

    Human SERPINA1 gene is located on chromosome 14q31-32.3 and is organized into three (IA, IB, and IC) non-coding and four (II, III, IV, V) coding exons. This gene produces α1-antitrypsin (A1AT), a prototypical member of the serpin superfamily of proteins. We demonstrate that human peripheral blood leukocytes express not only a product corresponding to the transcript coding for the full-length A1AT protein but also two short transcripts (ST1C4 and ST1C5) of A1AT. In silico sequence analysis revealed that the last exon of the short transcripts contains an Open Reading Frame (ORF) and thus putatively can produce peptides. We found ST1C4 expression across different human tissues whereas ST1C5 was mainly restricted to leukocytes, specifically neutrophils. A high up-regulation (10-fold) of short transcripts was observed in isolated human blood neutrophils after activation with lipopolysaccharide. Parallel analyses by liquid chromatography-mass spectrometry identified peptides corresponding to C-terminal region of A1AT in supernatants of activated but not naïve neutrophils. Herein we report for the first time a tissue specific expression and regulation of short transcripts of SERPINA1 gene, and the presence of C-terminal peptides in supernatants from activated neutrophils, in vitro. This gives a novel insight into the studies on the transcription of SERPINA1 gene.

  7. Presynaptic C-terminal truncated tau is released from cortical synapses in Alzheimer's disease

    PubMed Central

    Sokolow, Sophie; Henkins, Kristen M.; Bilousova, Tina; Gonzalez, Bianca; Vinters, Harry V.; Miller, Carol A.; Cornwell, Lindsey; Poon, Wayne W.; Gylys, Karen H.

    2015-01-01

    The microtubule-associated protein tau has primarily been associated with axonal location and function; however, recent work shows tau release from neurons and suggests an important role for tau in synaptic plasticity. In our study, we measured synaptic levels of total tau using synaptosomes prepared from cryopreserved human postmortem Alzheimer's disease (AD) and control samples. Flow cytometry data show that a majority of synaptic terminals are highly immunolabeled with the total tau antibody (HT7) in both AD and control samples. Immunoblots of synaptosomal fractions reveal increases in a 20 kDa tau fragment and in tau dimers in AD synapses, and terminal-specific antibodies show that in many synaptosome samples tau lacks a C-terminus. Flow cytometry experiments to quantify the extent of C-terminal truncation reveal that only 15-25% of synaptosomes are positive for intact C-terminal tau. Potassium-induced depolarization demonstrates release of tau and tau fragments from presynaptic terminals, with increased release from AD compared to control samples. This study indicates that tau is normally highly localized to synaptic terminals in cortex where it is well-positioned to affect synaptic plasticity. Tau cleavage may facilitate tau aggregation as well as tau secretion and propagation of tau pathology from the presynaptic compartment in AD. PMID:25393609

  8. Activation of human prolegumain by cleavage at a C-terminal asparagine residue.

    PubMed Central

    Chen, J M; Fortunato, M; Barrett, A J

    2000-01-01

    The processing and activation of prolegumain were studied using the recombinant protein synthesized by cells that had been stably transfected with a human legumain cDNA construct. A cell line termed C13 was selected for the high-level expression of prolegumain. C13 cells produced primarily 56 kDa prolegumain. The 56 kDa form was enzymically inactive but stable at neutral pH, unlike the 35 kDa mature pig legumain; it could be converted into a 46 kDa active form by incubation at pH 4.5. The 56 kDa pro-form and the 46 kDa active form were found to have the same N-terminal amino acid sequence, indicating that cleavage at the N-terminus was not necessary for prolegumain activation, and that the decrease in molecular mass was due to a C-terminal cleavage. The C-terminal processing site was identified as Asn(323). Replacement of Asn(323) at the cleavage site with aspartate, serine, alanine or glutamate abolished the processing and activation of prolegumain. In contrast, mutation of other asparagine and aspartate residues near the cleavage site had no effect. These results demonstrate that Asn(323) is essential for prolegumain activation. PMID:11085925

  9. Activation of human prolegumain by cleavage at a C-terminal asparagine residue.

    PubMed

    Chen, J M; Fortunato, M; Barrett, A J

    2000-12-01

    The processing and activation of prolegumain were studied using the recombinant protein synthesized by cells that had been stably transfected with a human legumain cDNA construct. A cell line termed C13 was selected for the high-level expression of prolegumain. C13 cells produced primarily 56 kDa prolegumain. The 56 kDa form was enzymically inactive but stable at neutral pH, unlike the 35 kDa mature pig legumain; it could be converted into a 46 kDa active form by incubation at pH 4.5. The 56 kDa pro-form and the 46 kDa active form were found to have the same N-terminal amino acid sequence, indicating that cleavage at the N-terminus was not necessary for prolegumain activation, and that the decrease in molecular mass was due to a C-terminal cleavage. The C-terminal processing site was identified as Asn(323). Replacement of Asn(323) at the cleavage site with aspartate, serine, alanine or glutamate abolished the processing and activation of prolegumain. In contrast, mutation of other asparagine and aspartate residues near the cleavage site had no effect. These results demonstrate that Asn(323) is essential for prolegumain activation.

  10. Structure of the C-terminal Domain of Transcription Facto IIB from Trypanosoma brucei

    SciTech Connect

    Ibrahim, B.; Kanneganti, N; Rieckhof, G; Das, A; Laurents, D; Palenchar, J; Bellofatto, V; Wah, D

    2009-01-01

    In trypanosomes, the production of mRNA relies on the synthesis of the spliced leader (SL) RNA. Expression of the SL RNA is initiated at the only known RNA polymerase II promoter in these parasites. In the pathogenic trypanosome, Trypanosoma brucei, transcription factor IIB (tTFIIB) is essential for SL RNA gene transcription and cell viability, but has a highly divergent primary sequence in comparison to TFIIB in well-studied eukaryotes. Here we describe the 2.3 A resolution structure of the C-terminal domain of tTFIIB (tTFIIBC). The tTFIIBC structure consists of 2 closely packed helical modules followed by a C-terminal extension of 32 aa. Using the structure as a guide, alanine substitutions of basic residues in regions analogous to functionally important regions of the well-studied eukaryotic TFIIB support conservation of a general mechanism of TFIIB function in eukaryotes. Strikingly, tTFIIBC contains additional loops and helices, and, in contrast to the highly basic DNA binding surface of human TFIIB, contains a neutral surface in the corresponding region. These attributes probably mediate trypanosome-specific interactions and have implications for the apparent bidirectional transcription by RNA polymerase II in protein-encoding gene expression in these organisms.

  11. The spt5 C-terminal region recruits yeast 3' RNA cleavage factor I.

    PubMed

    Mayer, Andreas; Schreieck, Amelie; Lidschreiber, Michael; Leike, Kristin; Martin, Dietmar E; Cramer, Patrick

    2012-04-01

    During transcription elongation, RNA polymerase II (Pol II) binds the general elongation factor Spt5. Spt5 contains a repetitive C-terminal region (CTR) that is required for cotranscriptional recruitment of the Paf1 complex (D. L. Lindstrom et al., Mol. Cell. Biol. 23:1368-1378, 2003; Z. Zhang, J. Fu, and D. S. Gilmour, Genes Dev. 19:1572-1580, 2005). Here we report a new role of the Spt5 CTR in the recruitment of 3' RNA-processing factors. Chromatin immunoprecipitation (ChIP) revealed that the Spt5 CTR is required for normal recruitment of pre-mRNA cleavage factor I (CFI) to the 3' ends of Saccharomyces cerevisiae genes. RNA contributes to CFI recruitment, as RNase treatment prior to ChIP further decreases CFI ChIP signals. Genome-wide ChIP profiling detected occupancy peaks of CFI subunits around 100 nucleotides downstream of the polyadenylation (pA) sites of genes. CFI recruitment to this defined region may result from simultaneous binding to the Spt5 CTR, to nascent RNA containing the pA sequence, and to the elongating Pol II isoform that is phosphorylated at serine 2 (S2) residues in its C-terminal domain (CTD). Consistent with this model, the CTR interacts with CFI in vitro but is not required for pA site recognition and transcription termination in vivo.

  12. Molecular architecture of the nucleoprotein C-terminal domain from the Ebola and Marburg viruses.

    PubMed

    Baker, Laura E; Ellena, Jeffrey F; Handing, Katarzyna B; Derewenda, Urszula; Utepbergenov, Darkhan; Engel, Daniel A; Derewenda, Zygmunt S

    2016-01-01

    The Filoviridae family of negative-sense, single-stranded RNA (ssRNA) viruses is comprised of two species of Marburgvirus (MARV and RAVV) and five species of Ebolavirus, i.e. Zaire (EBOV), Reston (RESTV), Sudan (SUDV), Taï Forest (TAFV) and Bundibugyo (BDBV). In each of these viruses the ssRNA encodes seven distinct proteins. One of them, the nucleoprotein (NP), is the most abundant viral protein in the infected cell and within the viral nucleocapsid. It is tightly associated with the viral RNA in the nucleocapsid, and during the lifecycle of the virus is essential for transcription, RNA replication, genome packaging and nucleocapsid assembly prior to membrane encapsulation. The structure of the unique C-terminal globular domain of the NP from EBOV has recently been determined and shown to be structurally unrelated to any other known protein [Dziubańska et al. (2014), Acta Cryst. D70, 2420-2429]. In this paper, a study of the C-terminal domains from the NP from the remaining four species of Ebolavirus, as well as from the MARV strain of Marburgvirus, is reported. As expected, the crystal structures of the BDBV and TAFV proteins show high structural similarity to that from EBOV, while the MARV protein behaves like a molten globule with a core residual structure that is significantly different from that of the EBOV protein.

  13. The Atlastin C-terminal Tail Is an Amphipathic Helix That Perturbs the Bilayer Structure during Endoplasmic Reticulum Homotypic Fusion

    PubMed Central

    Faust, Joseph E.; Desai, Tanvi; Verma, Avani; Ulengin, Idil; Sun, Tzu-Lin; Moss, Tyler J.; Betancourt-Solis, Miguel A.; Huang, Huey W.; Lee, Tina; McNew, James A.

    2015-01-01

    Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel α helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion. PMID:25555915

  14. Loss of Type I Collagen Telopeptide Lysyl Hydroxylation Causes Musculoskeletal Abnormalities in a Zebrafish Model of Bruck Syndrome

    PubMed Central

    Gistelinck, Charlotte; Witten, Paul Eckhard; Huysseune, Ann; Symoens, Sofie; Malfait, Fransiska; Larionova, Daria; Simoens, Pascal; Dierick, Manuel; Van Hoorebeke, Luc; De Paepe, Anne; Kwon, Ronald Y; Weis, MaryAnn; Eyre, David R; Willaert, Andy; Coucke, Paul J

    2017-01-01

    Bruck syndrome (BS) is a disorder characterized by joint flexion contractures and skeletal dysplasia that shows strong clinical overlap with the brittle bone disease Osteogenesis Imperfecta (OI). BS is caused by bi-allelic mutations in either the FKBP10 or the PLOD2 gene. PLOD2 encodes the lysyl hydroxylase 2 (LH2) enzyme, which is responsible for the hydroxylation of lysine residues in fibrillar collagen telopeptides. This hydroxylation directs cross-linking of collagen fibrils in the extracellular matrix, which is necessary to provide stability and tensile integrity to the collagen fibrils. To further elucidate the function of LH2 in vertebrate skeletal development, we created a zebrafish model harboring a homozygous plod2 nonsense mutation resulting in reduced telopeptide hydroxylation and cross-linking of bone type I collagen. Adult plod2 mutants present with a shortened body axis and severe skeletal abnormalities with evidence of bone fragility and fractures. The vertebral column of plod2 mutants is short and scoliotic with compressed vertebrae that show excessive bone formation at the vertebral end plates, and increased tissue mineral density in the vertebral centra. The muscle fibers of mutant zebrafish have a reduced diameter near the horizontal myoseptum. The endomysium, a layer of connective tissue ensheathing the individual muscle fibers, is enlarged. Transmission electron microscopy of mutant vertebral bone shows type I collagen fibrils that are less organized with loss of the typical plywood-like structure. In conclusion, plod2 mutant zebrafish show molecular and tissue abnormalities in the musculoskeletal system that are concordant with clinical findings in BS patients. Therefore, the plod2 zebrafish mutant is a promising model for the elucidation of the underlying pathogenetic mechanisms leading to BS and the development of novel therapeutic avenues in this syndrome. PMID:27541483

  15. The C-terminal Domain Supports a Novel Function for CETPI as a New Plasma Lipopolysaccharide-Binding Protein

    PubMed Central

    García-González, Victor; Gutiérrez-Quintanar, Nadia; Mas-Oliva, Jaime

    2015-01-01

    Described by our group a few years ago, the cholesteryl-ester transfer protein isoform (CETPI), exclusively expressed in the small intestine and present in human plasma, lacked a functional identification for a role of physiological relevance. Now, this study introduces CETPI as a new protein with the potential capability to recognise, bind and neutralise lipopolysaccharides (LPS). Peptides derived from the C-terminal domain of CETPI showed that CETPI not only might interact with several LPS serotypes but also might displace LPS bound to the surface of cells. Peptide VSAK, derived from the last 18 residues of CETPI, protected against the cytotoxic effect of LPS on macrophages. At high concentrations, when different cell types were tested in culture, it did not exhibit cytotoxicity by itself and it did prevent the expression of pro-inflammatory cytokines as well as the generation of oxidative stress conditions. In a rabbit model of septic shock, the infusion of peptide VSAK exerted a protective effect against the effects of LPS and reduced the presence of tumor necrosis factor-alpha (TNFα) in plasma. Therefore, CETPI is proposed as a new protein with the capability to advance the possibilities for better understanding and treatment of the dangerous effects of LPS in vivo. PMID:26537318

  16. Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading*

    PubMed Central

    Felczak, Magdalena M.; Sage, Jay M.; Hupert-Kocurek, Katarzyna; Aykul, Senem; Kaguni, Jon M.

    2016-01-01

    The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity in DNA binding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and by UV cross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaB-DnaC complex at a DnaA box. Because a decreased ATP concentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature. PMID:26728455

  17. Diverse C-terminal sequences involved in Flavobacterium johnsoniae protein secretion.

    PubMed

    Kulkarni, Surashree S; Zhu, Yongtao; Brendel, Colton J; McBride, Mark J

    2017-04-10

    Flavobacterium johnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to family TIGR04183 (type-A CTDs). We functionally characterized diverse CTDs for secretion by the F. johnsoniae T9SS. Attachment of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign protein sfGFP that had a signal peptide at the amino terminus resulted in secretion across the outer membrane. In each case approximately 80 to 100 amino acids from the extreme carboxy-termini was needed for efficient secretion. Several type-A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting secretion of sfGFP by the F. johnsoniae T9SS. F. johnsoniae SprB requires the T9SS for secretion but lacks a type-A CTD. It has a conserved C-terminal domain belonging to family TIGR04131, which we refer to as a type-B CTD. The CTD of SprB was required for its secretion, but attachment of C-terminal regions of SprB of up to 1182 amino acids to sfGFP failed to result in secretion. Additional features outside of the C-terminal region of SprB may be required for its secretion.Importance Type IX protein secretion systems (T9SSs) are common in but limited to members of the phylum Bacteroidetes Most proteins that are secreted by T9SSs have conserved carboxy-terminal domains that belong to either protein domain family TIGR04183 (type-A CTDs) or TIGR04131 (type-B CTDs). Here we identify features of T9SS CTDs of F. johnsoniae that are required for protein secretion and demonstrate that type-A CTDs from distantly related members of the phylum function with the F. johnsoniae T9SS to secrete the foreign protein sfGFP. In contrast, type-B CTDs failed

  18. C-terminal amide to alcohol conversion changes the cardiovascular effects of endomorphins in anesthetized rats.

    PubMed

    Yu, Ye; Wang, Chang-lin; Cui, Yun; Fan, Ying-zhe; Liu, Jing; Shao, Xuan; Liu, Hong-mei; Wang, Rui

    2006-01-01

    Endomorphin1-ol (Tyr-Pro-Trp-Phe-ol, EM1-ol) and endomorphin2-ol (Tyr-Pro-Phe-Phe-ol, EM2-ol), with C-terminal alcohol (-ol) containing, have been shown to exhibit higher affinity and lower intrinsic efficacy in vitro than endomorphins. In the present study, in order to investigate the alterations of systemic hemodynamic effects induced by C-terminal amide to alcohol conversion, responses to intravenous (i.v.) or intracerebroventricular (i.c.v.) injection of EM1-ol, EM2-ol and their parents were compared in the system arterial pressure (SAP) and heart rate (HR) of anesthetized rats. Both EM1-ol and EM2-ol induced dose-related decrease in SAP and HR when injected in doses of 3-100 nmol/kg, i.v. In terms of relative vasodepressor activity, it is interesting to note that EM2-ol was more potent than endomorphin2 [the dose of 25% decrease in SAP (DD25) = 6.01+/-3.19 and 13.99+/-1.56 nmol/kg, i.v., respectively] at a time when responses to EM1-ol were less potent than endomorphin1. Moreover, decreases in SAP in response to EM1-ol and EM2-ol were reduced by naloxone, atropine sulfate, L-NAME and bilateral vagotomy. It indicated that the vasodepressor responses were possibly mediated by a naloxone-sensitive, nitric oxide release, vagus-activated mechanism. It is noteworthy that i.c.v. injections of -ol derivatives produced dose-related decreases in SAP and HR, which were significantly less potent than endomorphins and were attenuated by naloxone and atropine sulfate. In summary, the results of the present study indicated that the C-terminal amide to alcohol conversion produced different effects on the vasodepressor activity of endomorphin1 and endomorphin2 and endowed EM2-ol distinctive hypotension characters in peripheral (i.v.) and central (i.c.v.) tissues. Moreover, these results provided indirect evidence that amidated C-terminus might play an important role in the regulation of the cardiovascular system.

  19. C-Terminal-oriented Immobilization of Enzymes Using Sortase A-mediated Technique.

    PubMed

    Hata, Yuto; Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko

    2015-10-01

    In the present study, sortase A-mediated immobilization of enzymes was used for the preparation of immobilized enzymes. Thermobifida fusca YX β-glucosidase (BGL) or Streptococcus bovis 148 α-amylase (AmyA) were produced with C-terminal sortase A recognition sequences. The resulting fusion proteins were successfully immobilized on nanoparticle surfaces using sortase A. Some properties (activity, stability, and reusability) of the immobilized fusion proteins were evaluated. Both immobilized BGL and immobilized AmyA prepared by the sortase A-mediated technique retained their catalytic activity, exhibiting activities 3.0- or 1.5-fold (respectively) of those seen with the same enzymes immobilized by chemical crosslinking. Immobilized enzymes prepared by the sortase A-mediated technique did not undergo dramatic changes in stability compared with the respective free enzymes. Thus, the sortase A-mediated technique provides a promising method for immobilization of active, stable enzymes.

  20. Folding of the C-terminal bacterial binding domain in statherin upon adsorption onto hydroxyapatite crystals

    PubMed Central

    Goobes, Gil; Goobes, Rivka; Schueler-Furman, Ora; Baker, David; Stayton, Patrick S.; Drobny, Gary P.

    2006-01-01

    Statherin is an enamel pellicle protein that inhibits hydroxyapatite (HAP) nucleation and growth, lubricates the enamel surface, and is recognized by oral bacteria in periodontal diseases. We report here from solid-state NMR measurements that the protein's C-terminal region folds into an α-helix upon adsorption to HAP crystals. This region contains the binding sites for bacterial fimbriae that mediate bacterial cell adhesion to the surface of the tooth. The helical segment is shown through long-range distance measurements to fold back onto the intermediate region (residues Y16–P28) defining the global fold of the protein. Statherin, previously shown to be unstructured in solution, undergoes conformation selection on its substrate mineral surface. This surface-induced folding of statherin can be related to its functionality in inhibiting HAP crystal growth and can explain how oral pathogens selectively recognize HAP-bound statherin. PMID:17060618

  1. C-terminal truncation of GSK-3β enhances its dephosphorylation by PP2A.

    PubMed

    Jin, Nana; Wu, Yue; Xu, Wen; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2017-03-07

    Glycogen synthase kinase-3β (GSK-3β) is the major tau kinase. Its phosphorylation at Ser9 suppresses the activity. In Alzheimer's disease (AD) brain, GSK-3β is truncated at the C-terminus by over-activated calpain I, leading to an increase in its activity. However, the effect of truncation on its phosphorylation is unknown. We found here that in AD brain and in cultured cells, C-terminally truncated GSK-3β is less phosphorylated at Ser9 than the full-length enzyme. The truncation promotes GSK-3β nuclear translocation and enhances its interaction with protein phosphatase 2A (PP2A), leading to dephosphorylation. Thus, the truncation of GSK-3β may enhance its activity through Ser9 dephosphorylation by PP2A. Our findings shed new light onto the role of calpain - GSK-3β - PP2A in tau pathogenesis of AD. This article is protected by copyright. All rights reserved.

  2. The C-terminal region of E1A: a molecular tool for cellular cartography.

    PubMed

    Yousef, Ahmed F; Fonseca, Gregory J; Cohen, Michael J; Mymryk, Joe S

    2012-04-01

    The adenovirus E1A proteins function via protein-protein interactions. By making many connections with the cellular protein network, individual modules of this virally encoded hub reprogram numerous aspects of cell function and behavior. Although many of these interactions have been thoroughly studied, those mediated by the C-terminal region of E1A are less well understood. This review focuses on how this region of E1A affects cell cycle progression, apoptosis, senescence, transformation, and conversion of cells to an epithelial state through interactions with CTBP1/2, DYRK1A/B, FOXK1/2, and importin-α. Furthermore, novel potential pathways that the C-terminus of E1A influences through these connections with the cellular interaction network are discussed.

  3. Crystal structure of the C-terminal domain of mouse TLR9

    PubMed Central

    Collins, Bernard; Wilson, Ian A.

    2014-01-01

    Toll-like receptors (TLRs) are important pattern recognition receptors that function in innate immunity. Elucidating the structure and signaling mechanisms of TLR9, a sensor of foreign and endogenous DNA, is essential for understanding its critical roles in immunity and autoimmunity. Abundant evidence suggests that the TLR9-CTD (C-terminal domain) by itself is capable of DNA-binding and signaling. We present the crystal structure of unliganded mouse TLR9-CTD. TLR9-CTD exhibits one unique feature, a cluster of stacked aromatic and arginine side chains on its concave face. Overall, its structure is most related to the TLR8-CTD, suggesting a similar mode of ligand binding and signaling. PMID:24888966

  4. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    PubMed Central

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  5. Affinity labelling of proteinases with tryptic specificity by peptides with C-terminal lysine chloromethyl ketone

    PubMed Central

    Coggins, John R.; Kray, William; Shaw, Elliott

    1974-01-01

    Methods are described for the synthesis of peptides terminating in Lys-CH2Cl. The products were examined as affinity labels for several enzymes of trypsin-like specificity which are resistant to Tos-Lys-CH2Cl. In part, the inertness of the latter may be due to the sulphonamide group, since Z-Lys-CH2Cl was more effective. However, a number of tripeptides with C-terminal Lys-CH2Cl were superior in their ability to inactivate subtilisin, thrombin and plasma kallikrein. The possibility of developing enzyme-specific reagents selective for members within the trypsin-like group is demonstrated by Ala-Phe-Lys-CH2Cl, which readily inactivates plasma kallikrein but not thrombin. PMID:4422496

  6. Ubiquitin C-terminal hydrolase L1 deficiency decreases bone mineralization.

    PubMed

    Shim, Sehwan; Kwon, Young-Bae; Yoshikawa, Yasuhiro; Kwon, Jungkee

    2008-06-01

    Ubiquitin C-terminal hydrolase L1 is a component of the ubiquitin proteasome system, which evidences unique biological activities. In this study, we report the pattern of UCH-L1 expression, and show that it regulates bone mineralization in osteogenesis. UCH-L1 was expressed in osteoblasts, osteoclasts, and hematopoietic precursor cells of bone marrow in the metaphysis and diaphysis of the femora. To further assess the involvement of UCH-L1 in the regulation of bone mineralization, we evaluated the bone mineral density (BMD) rate of gad mice, using the Latheta computed tomography system. Male gad mice evidenced a significantly decreased BMD rate in the metaphysis and diaphysis of the femora. These findings of decreased BMD rate in the bones of gad mice may suggest that UCH-L1 function regulates bone mineralization during osteogenesis.

  7. Control of cytoplasmic dynein force production and processivity by its C-terminal domain

    PubMed Central

    Nicholas, Matthew P.; Höök, Peter; Brenner, Sibylle; Wynne, Caitlin L.; Vallee, Richard B.; Gennerich, Arne

    2015-01-01

    Cytoplasmic dynein is a microtubule motor involved in cargo transport, nuclear migration and cell division. Despite structural conservation of the dynein motor domain from yeast to higher eukaryotes, the extensively studied S. cerevisiae dynein behaves distinctly from mammalian dyneins, which produce far less force and travel over shorter distances. However, isolated reports of yeast-like force production by mammalian dynein have called interspecies differences into question. We report that functional differences between yeast and mammalian dynein are real and attributable to a C-terminal motor element absent in yeast, which resembles a ‘cap’ over the central pore of the mammalian dynein motor domain. Removal of this cap increases the force generation of rat dynein from 1 pN to a yeast-like 6 pN and greatly increases its travel distance. Our findings identify the CT-cap as a novel regulator of dynein function. PMID:25670086

  8. Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein

    PubMed Central

    Martinez, Nicolas; Ribeiro, Euripedes A.; Leyrat, Cédric; Tarbouriech, Nicolas; Ruigrok, Rob W. H.

    2013-01-01

    Lettuce necrotic yellows virus (LNYV) is a prototype of the plant-adapted cytorhabdoviruses. Through a meta-prediction of disorder, we localized a folded C-terminal domain in the amino acid sequence of its phosphoprotein. This domain consists of an autonomous folding unit that is monomeric in solution. Its structure, solved by X-ray crystallography, reveals a lollipop-shaped structure comprising five helices. The structure is different from that of the corresponding domains of other Rhabdoviridae, Filoviridae, and Paramyxovirinae; only the overall topology of the polypeptide chain seems to be conserved, suggesting that this domain evolved under weak selective pressure and varied in size by the acquisition or loss of functional modules. PMID:23785215

  9. Conformationally restricted C-terminal peptides of substance P. Synthesis, mass spectral analysis and pharmacological properties.

    PubMed

    Theodoropoulos, D; Poulos, C; Gatos, D; Cordopatis, P; Escher, E; Mizrahi, J; Regoli, D; Dalietos, D; Furst, A; Lee, T D

    1985-10-01

    Four cyclic analogues of the C-terminal hepta- or hexapeptide of substance P were prepared by the solution method. The cyclizations were obtained by substituting with cysteine the residues normally present in positions 5 or 6 or 11 of substance P and by subsequent disulfide bond formation. The final products were identified by ordinary analytical procedures and advanced mass spectroscopy. The biological activities were determined on three bioassays: the guinea pig ileum, the guinea pig trachea and the rabbit mesenteric vein. Results obtained with these assays indicate that all peptides with a disulfide bridgehead in position 11 are inactive and that a cycle between positions 5 and 6 already strongly reduces the biological activity. The acyclic precursors containing thiol protection groups display weak biological activities. These results further underline the importance of the side chain in position 11 of substance P and suggest that optimal biological activities may require a linear peptide sequence.

  10. Structure-activity relationships of C-terminal tri- and tetrapeptide fragments that inhibit gastrin activity.

    PubMed

    Martinez, J; Bali, J P; Magous, R; Laur, J; Lignon, M F; Briet, C; Nisato, D; Castro, B

    1985-03-01

    A series of tri- and tetrapeptide derivatives, analogues of the gastrin C-terminal region with no phenylalanine residue, were synthesized. These peptides were tested for their ability to inhibit gastrin-stimulated acid secretion in vivo as well as binding of [125I]-(Nle11)-HG-13 to gastric mucosal cell receptors in vitro. Most of the peptides tested exhibited gastrin antagonist activity in vivo and in vitro. Most active derivatives were 20-30 times more potent than the well-known gastrin antagonist derivatives proglumide and benzotript and had 20-200 times more binding affinity. The smallest fragment exhibiting antagonist activity was the tripeptide Boc-L-tryptophyl-L-methionyl-L-aspartic acid amide.

  11. Control of cytoplasmic dynein force production and processivity by its C-terminal domain

    NASA Astrophysics Data System (ADS)

    Nicholas, Matthew P.; Höök, Peter; Brenner, Sibylle; Wynne, Caitlin L.; Vallee, Richard B.; Gennerich, Arne

    2015-02-01

    Cytoplasmic dynein is a microtubule motor involved in cargo transport, nuclear migration and cell division. Despite structural conservation of the dynein motor domain from yeast to higher eukaryotes, the extensively studied S. cerevisiae dynein behaves distinctly from mammalian dyneins, which produce far less force and travel over shorter distances. However, isolated reports of yeast-like force production by mammalian dynein have called interspecies differences into question. We report that functional differences between yeast and mammalian dynein are real and attributable to a C-terminal motor element absent in yeast, which resembles a ‘cap’ over the central pore of the mammalian dynein motor domain. Removal of this cap increases the force generation of rat dynein from 1 pN to a yeast-like 6 pN and greatly increases its travel distance. Our findings identify the CT-cap as a novel regulator of dynein function.

  12. Turn structures in CGRP C-terminal analogues promote stable arrangements of key residue side chains.

    PubMed

    Carpenter, K A; Schmidt, R; von Mentzer, B; Haglund, U; Roberts, E; Walpole, C

    2001-07-27

    The 37-amino acid calcitonin gene-related peptide (CGRP) is a potent endogenous vasodilator thought to be implicated in the genesis of migraine attack. CGRP antagonists may thus have therapeutic value for the treatment of migraine. The CGRP C-terminally derived peptide [D(31),P(34),F(35)]CGRP(27-37)-NH(2) was recently identified as a high-affinity hCGRP(1) receptor selective antagonist. Reasonable CGRP(1) affinity has also been demonstrated for several related analogues, including [D(31),A(34),F(35)]CGRP(27-37)-NH(2). In the study presented here, conformational and structural features in CGRP(27-37)-NH(2) analogues that are important for hCGRP(1) receptor binding were explored. Structure-activity studies carried out on [D(31),P(34),F(35)]CGRP(27-37)-NH(2) resulted in [D(31),P(34),F(35)]CGRP(30-37)-NH(2), the shortest reported CGRP C-terminal peptide analogue exhibiting reasonable hCGRP(1) receptor affinity (K(i) = 29.6 nM). Further removal of T(30) from the peptide's N-terminus greatly reduced receptor affinity from the nanomolar to micromolar range. Additional residues deemed critical for hCGRP(1) receptor binding were identified from an alanine scan of [A(34),F(35)]CGRP(28-37)-NH(2) and included V(32) and F(37). Replacement of the C-terminal amide in this same peptide with a carboxyl, furthermore, resulted in a greater than 50-fold reduction in hCGRP(1) affinity, thus suggesting a direct role for the amide moiety in receptor binding. The conformational properties of two classes of CGRP(27-37)-NH(2) peptides, [D(31),X(34),F(35)]CGRP(27-37)-NH(2) (X is A or P), were examined by NMR spectroscopy and molecular modeling. A beta-turn centered on P(29) was a notable feature consistently observed among active peptides in both series. This turn led to exposure of the critical T(30) residue to the surrounding environment. Peptides in the A(34) series were additionally characterized by a stable C-terminal helical turn that resulted in the three important residues (T(30), V

  13. C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast Schizosaccharomyces pombe.

    PubMed

    Singh, Gaurav Kumar; Karade, Sharanbasappa Shrimant; Ranjan, Rajeev; Ahamad, Nafees; Ahmed, Shakil

    2017-02-01

    The mitotic arrest deficiency 2 (Mad2) protein is an essential component of the spindle assembly checkpoint that interacts with Cdc20/Slp1 and inhibit its ability to activate anaphase promoting complex/cyclosome (APC/C). In bladder cancer cell line the C-terminal residue of the mad2 gene has been found to be deleted. In this study we tried to understand the role of the C-terminal region of mad2 on the spindle checkpoint function. To envisage the role of C-terminal region of Mad2, we truncated 25 residues of Mad2 C-terminal region in fission yeast S.pombe and characterized its effect on spindle assembly checkpoint function. The cells containing C-terminal truncation of Mad2 exhibit sensitivity towards microtubule destabilizing agent suggesting perturbation of spindle assembly checkpoint. Further, the C-terminal truncation of Mad2 exhibit reduced viability in the nda3-KM311 mutant background at non-permissive temperature. Truncation in mad2 gene also affects its foci forming ability at unattached kinetochore suggesting that the mad2-∆CT mutant is unable to maintain spindle checkpoint activation. However, in response to the defective microtubule, only brief delay of mitotic progression was observed in Mad2 C-terminal truncation mutant. In addition we have shown that the deletion of two β strands of Mad2 protein abolishes its ability to interact with APC activator protein Slp1/Cdc20. We purpose that the truncation of two β strands (β7 and β8) of Mad2 destabilize the safety belt and affect the Cdc20-Mad2 interaction leading to defects in the spindle checkpoint activation.

  14. Temperature dependence of C-terminal carboxylic group IR absorptions in the amide I' region.

    PubMed

    Anderson, Benjamin A; Literati, Alex; Ball, Borden; Kubelka, Jan

    2015-01-05

    Studies of structural changes in peptides and proteins using IR spectroscopy often rely on subtle changes in the amide I' band as a function of temperature. However, these changes can be obscured by the overlap with other absorptions, namely the side-chain and terminal carboxylic groups. The former were the subject of our previous report (Anderson et al., 2014). In this paper we investigate the IR spectra of the asymmetric stretch of α-carboxylic groups for amino acids representing all major types (Gly, Ala, Val, Leu, Ser, Thr, Asp, Glu, Lys, Asn, His, Trp, Pro) as well as the C-terminal groups of three dipeptides (Gly-Gly, Gly-Ala, Ala-Gly) in D₂O at neutral pH. Experimental temperature dependent IR spectra were analyzed by fitting of both symmetric and asymmetric pseudo-Voigt functions. Qualitatively the spectra exhibit shifts to higher frequency, loss in intensity and narrowing with increased temperature, similar to that observed previously for the side-chain carboxylic groups of Asp. The observed dependence of the band parameters (frequency, intensity, width and shape) on temperature is in all cases linear: simple linear regression is therefore used to describe the spectral changes. The spectral parameters vary between individual amino acids and show systematic differences between the free amino acids and dipeptides, particularly in the absolute peak frequencies, but the temperature variations are comparable. The relative variations between the dipeptide spectral parameters are most sensitive to the C-terminal amino acid, and follow the trends observed in the free amino acid spectra. General rules for modeling the α-carboxylic IR absorption bands in peptides and proteins as the function of temperature are proposed.

  15. Screening for Small Molecule Inhibitors of Statin-Induced APP C-terminal Toxic Fragment Production.

    PubMed

    Poksay, Karen S; Sheffler, Douglas J; Spilman, Patricia; Campagna, Jesus; Jagodzinska, Barbara; Descamps, Olivier; Gorostiza, Olivia; Matalis, Alex; Mullenix, Michael; Bredesen, Dale E; Cosford, Nicholas D P; John, Varghese

    2017-01-01

    Alzheimer's disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds - identified here using cells and tissues expressing wt human APP - in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects.

  16. Insulin resistance uncoupled from dyslipidemia due to C-terminal PIK3R1 mutations

    PubMed Central

    Huang-Doran, Isabel; Tomlinson, Patsy; Payne, Felicity; Gast, Alexandra; Sleigh, Alison; Bottomley, William; Harris, Julie; Daly, Allan; Rocha, Nuno; Rudge, Simon; Clark, Jonathan; Kwok, Albert; Romeo, Stefano; McCann, Emma; Müksch, Barbara; Dattani, Mehul; Zucchini, Stefano; Wakelam, Michael; Foukas, Lazaros C.; Savage, David B.; Murphy, Rinki; O’Rahilly, Stephen; Semple, Robert K.

    2016-01-01

    Obesity-related insulin resistance is associated with fatty liver, dyslipidemia, and low plasma adiponectin. Insulin resistance due to insulin receptor (INSR) dysfunction is associated with none of these, but when due to dysfunction of the downstream kinase AKT2 phenocopies obesity-related insulin resistance. We report 5 patients with SHORT syndrome and C-terminal mutations in PIK3R1, encoding the p85α/p55α/p50α subunits of PI3K, which act between INSR and AKT in insulin signaling. Four of 5 patients had extreme insulin resistance without dyslipidemia or hepatic steatosis. In 3 of these 4, plasma adiponectin was preserved, as in insulin receptor dysfunction. The fourth patient and her healthy mother had low plasma adiponectin associated with a potentially novel mutation, p.Asp231Ala, in adiponectin itself. Cells studied from one patient with the p.Tyr657X PIK3R1 mutation expressed abundant truncated PIK3R1 products and showed severely reduced insulin-stimulated association of mutant but not WT p85α with IRS1, but normal downstream signaling. In 3T3-L1 preadipocytes, mutant p85α overexpression attenuated insulin-induced AKT phosphorylation and adipocyte differentiation. Thus, PIK3R1 C-terminal mutations impair insulin signaling only in some cellular contexts and produce a subphenotype of insulin resistance resembling INSR dysfunction but unlike AKT2 dysfunction, implicating PI3K in the pathogenesis of key components of the metabolic syndrome. PMID:27766312

  17. Temperature dependence of C-terminal carboxylic group IR absorptions in the amide I‧ region

    NASA Astrophysics Data System (ADS)

    Anderson, Benjamin A.; Literati, Alex; Ball, Borden; Kubelka, Jan

    2015-01-01

    Studies of structural changes in peptides and proteins using IR spectroscopy often rely on subtle changes in the amide I‧ band as a function of temperature. However, these changes can be obscured by the overlap with other absorptions, namely the side-chain and terminal carboxylic groups. The former were the subject of our previous report (Anderson et al., 2014). In this paper we investigate the IR spectra of the asymmetric stretch of α-carboxylic groups for amino acids representing all major types (Gly, Ala, Val, Leu, Ser, Thr, Asp, Glu, Lys, Asn, His, Trp, Pro) as well as the C-terminal groups of three dipeptides (Gly-Gly, Gly-Ala, Ala-Gly) in D2O at neutral pH. Experimental temperature dependent IR spectra were analyzed by fitting of both symmetric and asymmetric pseudo-Voigt functions. Qualitatively the spectra exhibit shifts to higher frequency, loss in intensity and narrowing with increased temperature, similar to that observed previously for the side-chain carboxylic groups of Asp. The observed dependence of the band parameters (frequency, intensity, width and shape) on temperature is in all cases linear: simple linear regression is therefore used to describe the spectral changes. The spectral parameters vary between individual amino acids and show systematic differences between the free amino acids and dipeptides, particularly in the absolute peak frequencies, but the temperature variations are comparable. The relative variations between the dipeptide spectral parameters are most sensitive to the C-terminal amino acid, and follow the trends observed in the free amino acid spectra. General rules for modeling the α-carboxylic IR absorption bands in peptides and proteins as the function of temperature are proposed.

  18. Screening for Small Molecule Inhibitors of Statin-Induced APP C-terminal Toxic Fragment Production

    PubMed Central

    Poksay, Karen S.; Sheffler, Douglas J.; Spilman, Patricia; Campagna, Jesus; Jagodzinska, Barbara; Descamps, Olivier; Gorostiza, Olivia; Matalis, Alex; Mullenix, Michael; Bredesen, Dale E.; Cosford, Nicholas D. P.; John, Varghese

    2017-01-01

    Alzheimer’s disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds – identified here using cells and tissues expressing wt human APP – in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects. PMID:28261092

  19. The 60-kilodalton protein encoded by orf2 in the cry19A operon of Bacillus thuringiensis subsp. jegathesan functions like a C-terminal crystallization domain.

    PubMed

    Barboza-Corona, J Eleazar; Park, Hyun-Woo; Bideshi, Dennis K; Federici, Brian A

    2012-03-01

    The cry19A operon of Bacillus thuringiensis subsp. jegathesan encodes two proteins, mosquitocidal Cry19A (ORF1; 75 kDa) and an ORF2 (60 kDa) of unknown function. Expression of the cry19A operon in an acrystalliferous strain of B. thuringiensis (4Q7) yielded one small crystal per cell, whereas no crystals were produced when cry19A or orf2 was expressed alone. To determine the function of the ORF2 protein, different combinations of Cry19A, ORF2, and the N- or C-terminal half of Cry1C were synthesized in strain 4Q7. Stable crystalline inclusions of these fusion proteins similar in shape to those in the strain harboring the wild-type operon were observed in sporulating cells. Comparative analysis showed that ORF2 shares considerable amino acid sequence identity with the C-terminal region of large Cry proteins. Together, these results suggest that ORF2 assists in synthesis and crystallization of Cry19A by functioning like the C-terminal domain characteristic of Cry protein in the 130-kDa mass range. In addition, to determine whether overexpression of the cry19A operon stabilized its shape and increased Cry19A yield, it was expressed under the control of the strong chimeric cyt1A-p/STAB-SD promoter. Interestingly, in contrast to the expression seen with the native promoter, overexpression of the operon yielded uniform bipyramidal crystals that were 4-fold larger on average than the wild-type crystal. In bioassays using the 4th instar larvae of Culex quinquefasciatus, the strain producing the larger Cry19A crystal showed moderate larvicidal activity that was 4-fold (95% lethal concentration [LC(95)] = 1.9 μg/ml) more toxic than the activity produced in the strain harboring the wild-type operon (LC(95) = 8.2 μg/ml).

  20. Enhanced responses in matrix-assisted laser desorption/ionization mass spectrometry of peptides derivatized with arginine via a C-terminal oxazolone.

    PubMed

    Nakazawa, Takashi; Yamaguchi, Minoru; Nishida, Kimiko; Kuyama, Hiroki; Obama, Takashi; Ando, Eiji; Okamura, Taka-Aki; Ueyama, Norikazu; Tanaka, Koichi; Norioka, Shigemi

    2004-01-01

    We have developed a novel method for enhancing the response of a peptide in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) by activating the C-terminal carboxyl group through an oxazolone with which is coupled an amine containing a functional group to help ionize the peptide. The reactions consist of dehydration with acetic anhydride to give an oxazolone, followed by aminolysis with an appropriate amino acid derivative such as arginine methyl ester. The MALDI signal of Ac-Tyr-Gly-Gly-Phe-Leu-Arg-OMe, thus converted from leucine-enkephalin, was detected while completely excluding the responses of arginine-deficient peptides coexisting in the reaction mixture. Some less intense peaks corresponding to a few sequential degradation products, also terminated with the arginine derivative, were also observed. The side-chain groups potentially that are reactive were conveniently protected by acetylation simultaneous with the C-terminal activation, and those that remained unprotected were reduced to virtually negligible proportions when the reaction was conducted in a peptide solution of concentration less than 1 mM. The greatly increased responses of such arginine-terminated peptides could possibly be exploited to discern the C-terminal tryptic peptide of a protein that is otherwise almost insensitive to MALDI-MS in general. The simplicity of the post-source decay spectrum of enkephalin derivatized by arginine methyl ester characteristically accentuated z- and b-type ions, and this should facilitate sequencing of such derivatized peptides. Remaining problems with practical applications of this approach are discussed.

  1. Human Gpn1 purified from bacteria binds guanine nucleotides and hydrolyzes GTP as a protein dimer stabilized by its C-terminal tail.

    PubMed

    González-González, Rogelio; Guerra-Moreno, José A; Cristóbal-Mondragón, Gema R; Romero, Violeta; Peña-Gómez, Sonia G; Montero-Morán, Gabriela M; Lara-González, Samuel; Hernández-Arana, Andrés; Fernández-Velasco, Daniel A; Calera, Mónica R; Sánchez-Olea, Roberto

    2017-04-01

    The essential GTPase Gpn1 mediates RNA polymerase II nuclear targeting and controls microtubule dynamics in yeast and human cells by molecular mechanisms still under investigation. Here, we purified human HisGpn1 expressed as a recombinant protein in bacteria E. coli BL-21 (DE3). Affinity purified HisGpn1 eluted from a size exclusion column as a protein dimer, a state conserved after removing the hexa-histidine tail and confirmed by separating HisGpn1 in native gels, and in dynamic light scattering experiments. Human HisGpn1 purity was higher than 95%, molecularly monodisperse and could be concentrated to more than 10 mg/mL without aggregating. Circular dichroism spectra showed that human HisGpn1 was properly folded and displayed a secondary structure rich in alpha helices. HisGpn1 effectively bound GDP and the non-hydrolyzable GTP analogue GMPPCP, and hydrolyzed GTP. We next tested the importance of the C-terminal tail, present in eukaryotic Gpn1 but not in the ancestral archaeal Gpn protein, on HisGpn1 dimer formation. C-terminal deleted human HisGpn1 (HisGpn1ΔC) was also purified as a protein dimer, indicating that the N-terminal GTPase domain contains the interaction surface needed for dimer formation. In contrast to HisGpn1, however, HisGpn1ΔC dimer spontaneously dissociated into monomers. In conclusion, we have developed a method to purify properly folded and functionally active human HisGpn1 from bacteria, and showed that the C-terminal tail, universally conserved in all eukaryotic Gpn1 orthologues, stabilizes the GTPase domain-mediated Gpn1 protein dimer. The availability of recombinant human Gpn1 will open new research avenues to unveil the molecular and pharmacological properties of this essential GTPase.

  2. The 60-Kilodalton Protein Encoded by orf2 in the cry19A Operon of Bacillus thuringiensis subsp. jegathesan Functions Like a C-Terminal Crystallization Domain

    PubMed Central

    Barboza-Corona, J. Eleazar; Park, Hyun-Woo; Bideshi, Dennis K.

    2012-01-01

    The cry19A operon of Bacillus thuringiensis subsp. jegathesan encodes two proteins, mosquitocidal Cry19A (ORF1; 75 kDa) and an ORF2 (60 kDa) of unknown function. Expression of the cry19A operon in an acrystalliferous strain of B. thuringiensis (4Q7) yielded one small crystal per cell, whereas no crystals were produced when cry19A or orf2 was expressed alone. To determine the function of the ORF2 protein, different combinations of Cry19A, ORF2, and the N- or C-terminal half of Cry1C were synthesized in strain 4Q7. Stable crystalline inclusions of these fusion proteins similar in shape to those in the strain harboring the wild-type operon were observed in sporulating cells. Comparative analysis showed that ORF2 shares considerable amino acid sequence identity with the C-terminal region of large Cry proteins. Together, these results suggest that ORF2 assists in synthesis and crystallization of Cry19A by functioning like the C-terminal domain characteristic of Cry protein in the 130-kDa mass range. In addition, to determine whether overexpression of the cry19A operon stabilized its shape and increased Cry19A yield, it was expressed under the control of the strong chimeric cyt1A-p/STAB-SD promoter. Interestingly, in contrast to the expression seen with the native promoter, overexpression of the operon yielded uniform bipyramidal crystals that were 4-fold larger on average than the wild-type crystal. In bioassays using the 4th instar larvae of Culex quinquefasciatus, the strain producing the larger Cry19A crystal showed moderate larvicidal activity that was 4-fold (95% lethal concentration [LC95] = 1.9 μg/ml) more toxic than the activity produced in the strain harboring the wild-type operon (LC95 = 8.2 μg/ml). PMID:22247140

  3. The C-terminal region of alpha-crystallin: involvement in protection against heat-induced denaturation

    NASA Technical Reports Server (NTRS)

    Takemoto, L.; Emmons, T.; Horwitz, J.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Recent studies have demonstrated that the alpha-crystallins can protect other proteins against heat-induced denaturation and aggregation. To determine the possible involvement of the C-terminal region in this activity, the alpha-crystallins were subjected to limited tryptic digestion, and the amount of cleavage from the N-terminal and C-terminal regions of the alpha-A and alpha-B crystallin chains was assessed using antisera specific for these regions. Limited tryptic digestion resulted in cleavage only from the C-terminal region of alpha-A crystallin. This trypsin-treated alpha-A crystallin preparation showed a decreased ability to protect proteins from heat-induced aggregation using an in vitro assay. Together, these results demonstrate that the C-terminal region of alpha-A crystallin is important for its ability to protect against heat-induced aggregation, which is consistent with the hypothesis that post-translational changes that are known to occur at the C-terminal region may have significant effects on the ability of alpha-A crystallin to protect against protein denaturation in vivo.

  4. Trypanosoma evansi: identification and characterization of a variant surface glycoprotein lacking cysteine residues in its C-terminal domain.

    PubMed

    Jia, Yonggen; Zhao, Xinxin; Zou, Jingru; Suo, Xun

    2011-01-01

    African trypanosomes are flagellated unicellular parasites which proliferate extracellularly in the mammalian host blood-stream and tissue spaces. They evade the hosts' antibody-mediated lyses by sequentially changing their variant surface glycoprotein (VSG). VSG tightly coats the entire parasite body, serving as a physical barrier. In Trypanosoma brucei and the closely related species Trypanosoma evansi, Trypanosoma equiperdum, each VSG polypeptide can be divided into N- and C-terminal domains, based on cysteine distribution and sequence homology. N-terminal domain, the basis of antigenic variation, is hypervariable and contains all the exposed epitopes; C-terminal domain is relatively conserved and a full set of four or eight cysteines were generally observed. We cloned two genes from two distinct variants of T. evansi, utilizing RT-PCR with VSG-specific primers. One contained a VSG type A N-terminal domain followed a C-terminal domain lacking cysteine residues. To confirm that this gene is expressed as a functional VSG, the expression and localization of the corresponding gene product were characterized using Western blotting and immunofluorescent staining of living trypanosomes. Expression analysis showed that this protein was highly expressed, variant-specific, and had a ubiquitous cellular surface localization. All these results indicated that it was expressed as a functional VSG. Our finding showed that cysteine residues in VSG C-terminal domain were not essential; the conserved C-terminal domain generally in T. brucei like VSGs would possibly evolve for regulating the VSG expression.

  5. The C-terminal region of the RNA helicase CshA is required for the interaction with the degradosome and turnover of bulk RNA in the opportunistic pathogen Staphylococcus aureus.

    PubMed

    Giraud, Caroline; Hausmann, Stéphane; Lemeille, Sylvain; Prados, Julien; Redder, Peter; Linder, Patrick

    2015-01-01

    Staphylococcus aureus is a versatile opportunistic pathogen that adapts readily to a variety of different growth conditions. This adaptation requires a rapid regulation of gene expression including the control of mRNA abundance. The CshA DEAD-box RNA helicase was previously shown to be required for efficient turnover of the agr quorum sensing mRNA. Here we show by transcriptome-wide RNA sequencing and microarray analyses that CshA is required for the degradation of bulk mRNA. Moreover a subset of mRNAs is significantly stabilised in absence of CshA. Deletion of the C-terminal extension affects RNA turnover similar to the full deletion of the cshA gene. In accordance with RNA decay data, the C-terminal region of CshA is required for an RNA-independent interaction with components of the RNA degradation machinery. The C-terminal truncation of CshA reduces its ATPase activity and this reduction cannot be compensated at high RNA concentrations. Finally, the deletion of the C-terminal extension does affect growth at low temperatures, but to a significantly lesser degree than the full deletion, indicating that the core of the helicase can assume a partial function and opening the possibility that CshA is involved in different cellular processes.

  6. Mre11 nuclease and C-terminal tail-mediated DDR functions are required for initiating yeast telomere healing.

    PubMed

    Bhattacharyya, M K; Matthews, K M; Lustig, A J

    2008-08-01

    Mre11 is a central factor in creating an optimal substrate for telomerase loading and elongation. We have used a G2/M synchronized telomere-healing assay as a tool to separate different functions of Mre11 that are not apparent in null alleles. An analysis of healing efficiencies of several mre11 alleles revealed that both nuclease and C-terminal mutations led to a loss of healing. Interestingly, trans-complementation of the 49 amino acid C-terminal deletion (DeltaC49) and the D16A mutant, deficient in nuclease activity and partially defective in MRX complex formation, restores healing. DeltaC49 provokes Rad53 phosphorylation after treatment with the radiomimetic agent MMS exclusively through the Tel1 pathway, suggesting that a Tel1-mediated function is initiated through the C-terminal tail.

  7. Correct processing of the kiwifruit protease actinidin in transgenic tobacco requires the presence of the C-terminal propeptide.

    PubMed Central

    Paul, W; Amiss, J; Try, R; Praekelt, U; Scott, R; Smith, H

    1995-01-01

    A 355 cauliflower mosaic virus promoter and a tapetum-specific promoter were used to direct the synthesis in tobacco of preproactinidin and a derivative that lacked a C-terminal extension. Preproactinidin was processed into a form that migrated identically on protein gels with mature actinidin extracted from kiwifruit. This protein was proteolytically active in vitro, and high-level accumulation of this protein appeared to be detrimental to plant growth. Plants expressing an actinidin cDNA construct that lacked the sequence encoding the C-terminal propeptide were phenotypically normal but accumulated N-proactinidin, which was proteolytically active in vitro but did not self-cleave to mature actinidin. In transgenic tobacco, the C-terminal extension of actinidin is therefore required for correct processing. PMID:7784505

  8. Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells.

    PubMed

    Chen, Chun; Zhuang, Yingting; Chen, Xianling; Chen, Xiaole; Li, Ding; Fan, Yingjuan; Xu, Jianhua; Chen, Yuanzhong; Wu, Lixian

    2017-02-07

    Heat shock protein 90 (Hsp90) contains amino (N)-terminal domain, carboxyl(C)-terminal domain, and middle domains, which activate Hsp90 chaperone function cooperatively in tumor cells. One terminal occupancy might influence another terminal binding with inhibitor. The Bcr-Abl kinase is one of the Hsp90 clients implicated in the pathogenesis of chronic myeloid leukemia (CML). Present studies demonstrate that double inhibition of the N- and C-terminal termini can disrupt Hsp90 chaperone function synergistically, but not antagonistically, in Bcr-Abl-positive human leukemia cells. Furthermore, both the N-terminal inhibitor 17-AAG and the C-terminal inhibitor cisplatin (CP) have the capacity to suppress progenitor cells; however, only CP is able to inhibit leukemia stem cells (LSCs) significantly, which implies that the combinational treatment is able to suppress human leukemia in different mature states.

  9. Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

    PubMed Central

    Dar, Mohd J; Monel, Blandine; Krishnan, Lavanya; Shun, Ming-Chieh; Di Nunzio, Francesca; Helland, Dag E; Engelman, Alan

    2009-01-01

    Background The 18 residue tail abutting the SH3 fold that comprises the heart of the C-terminal domain is the only part of HIV-1 integrase yet to be visualized by structural biology. To ascertain the role of the tail region in integrase function and HIV-1 replication, a set of deletion mutants that successively lacked three amino acids was constructed and analyzed in a variety of biochemical and virus infection assays. HIV-1/2 chimers, which harbored the analogous 23-mer HIV-2 tail in place of the HIV-1 sequence, were also studied. Because integrase mutations can affect steps in the replication cycle other than integration, defective mutant viruses were tested for integrase protein content and reverse transcription in addition to integration. The F185K core domain mutation, which increases integrase protein solubility, was furthermore analyzed in a subset of mutants. Results Purified proteins were assessed for in vitro levels of 3' processing and DNA strand transfer activities whereas HIV-1 infectivity was measured using luciferase reporter viruses. Deletions lacking up to 9 amino acids (1-285, 1-282, and 1-279) displayed near wild-type activities in vitro and during infection. Further deletion yielded two viruses, HIV-11-276 and HIV-11-273, that displayed approximately two and 5-fold infectivity defects, respectively, due to reduced integrase function. Deletion mutant HIV-11-270 and the HIV-1/2 chimera were non-infectious and displayed approximately 3 to 4-fold reverse transcription in addition to severe integration defects. Removal of four additional residues, which encompassed the C-terminal β strand of the SH3 fold, further compromised integrase incorporation into virions and reverse transcription. Conclusion HIV-11-270, HIV-11-266, and the HIV-1/2 chimera were typed as class II mutant viruses due to their pleiotropic replication defects. We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as

  10. The C-terminal 42 residues of the Tula virus Gn protein regulate interferon induction.

    PubMed

    Matthys, Valery; Gorbunova, Elena E; Gavrilovskaya, Irina N; Pepini, Timothy; Mackow, Erich R

    2011-05-01

    Hantaviruses primarily infect the endothelial cell lining of capillaries and cause two vascular permeability-based diseases. The ability of pathogenic hantaviruses to regulate the early induction of interferon determines whether hantaviruses replicate in endothelial cells. Tula virus (TULV) and Prospect Hill virus (PHV) are hantaviruses which infect human endothelial cells but fail to cause human disease. PHV is unable to inhibit early interferon (IFN) responses and fails to replicate within human endothelial cells. However, TULV replicates successfully in human endothelial cells, suggesting that TULV is capable of regulating cellular IFN responses. We observed a >300-fold reduction in the IFN-stimulated genes (ISGs) MxA and ISG56 following TULV versus PHV infection of endothelial cells 1 day postinfection. Similar to results with pathogenic hantaviruses, expressing the TULV Gn protein cytoplasmic tail (Gn-T) blocked RIG-I- and TBK1-directed transcription from IFN-stimulated response elements (ISREs) and IFN-β promoters (>90%) but not transcription directed by constitutively active IFN regulatory factor-3 (IRF3). In contrast, expressing the PHV Gn-T had no effect on TBK1-induced transcriptional responses. Analysis of Gn-T truncations demonstrated that the C-terminal 42 residues of the Gn-T (Gn-T-C42) from TULV, but not PHV, inhibited IFN induction >70%. These findings demonstrate that the TULV Gn-T inhibits IFN- and ISRE-directed responses upstream of IRF3 at the level of the TBK1 complex and further define a 42-residue domain of the TULV Gn-T that inhibits IFN induction. In contrast to pathogenic hantavirus Gn-Ts, the TULV Gn-T lacks a C-terminal degron domain and failed to bind tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), a TBK1 complex component required for IRF3 activation. These findings indicate that the nonpathogenic TULV Gn-T regulates IFN induction but accomplishes this via unique interactions with cellular TBK1 complexes. These

  11. Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

    SciTech Connect

    Yun, Ji-Hye; Kim, Heeyoun; Park, Jung Eun; Lee, Jung Sup; Lee, Weontae

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We have determined solution structures of vEP C-terminal regulatory domain. Black-Right-Pointing-Pointer vEP C-ter100 has a compact {beta}-barrel structure with eight anti-parallel {beta}-strands. Black-Right-Pointing-Pointer Solution structure of vEP C-ter100 shares its molecular topology with that of the collagen-binding domain of collagenase. Black-Right-Pointing-Pointer Residues in the {beta}3 region of vEP C-ter100 might be important in putative ligand/receptor binding. Black-Right-Pointing-Pointer vEP C-ter100 interacts strongly with iron ion. -- Abstract: An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel {beta}-strands with a unique fold that has a compact {beta}-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe{sup 3+}). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates

  12. Protein identification with N and C-terminal sequence tags in proteome projects.

    PubMed

    Wilkins, M R; Gasteiger, E; Tonella, L; Ou, K; Tyler, M; Sanchez, J C; Gooley, A A; Walsh, B J; Bairoch, A; Appel, R D; Williams, K L; Hochstrasser, D F

    1998-05-08

    Genome sequences are available for increasing numbers of organisms. The proteomes (protein complement expressed by the genome) of many such organisms are being studied with two-dimensional (2D) gel electrophoresis. Here we have investigated the application of short N-terminal and C-terminal sequence tags to the identification of proteins separated on 2D gels. The theoretical N and C termini of 15, 519 proteins, representing all SWISS-PROT entries for the organisms Mycoplasma genitalium, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and human, were analysed. Sequence tags were found to be surprisingly specific, with N-terminal tags of four amino acid residues found to be unique for between 43% and 83% of proteins, and C-terminal tags of four amino acid residues unique for between 74% and 97% of proteins, depending on the species studied. Sequence tags of five amino acid residues were found to be even more specific. To utilise this specificity of sequence tags for protein identification, we created a world-wide web-accessible protein identification program, TagIdent (http://www.expasy.ch/www/tools.html), which matches sequence tags of up to six amino acid residues as well as estimated protein pI and mass against proteins in the SWISS-PROT database. We demonstrate the utility of this identification approach with sequence tags generated from 91 different E. coli proteins purified by 2D gel electrophoresis. Fifty-one proteins were unambiguously identified by virtue of their sequence tags and estimated pI and mass, and a further 11 proteins identified when sequence tags were combined with protein amino acid composition data. We conlcude that the TagIdent identification approach is best suited to the identification of proteins from prokaryotes whose complete genome sequences are available. The approach is less well suited to proteins from eukaryotes, as many eukaryotic proteins are not amenable to sequencing via Edman degradation, and tag protein

  13. Tetramerization Dynamics of C-terminal Domain Underlies Isoform-specific cAMP Gating in Hyperpolarization-activated Cyclic Nucleotide-gated Channels*

    PubMed Central

    Lolicato, Marco; Nardini, Marco; Gazzarrini, Sabrina; Möller, Stefan; Bertinetti, Daniela; Herberg, Friedrich W.; Bolognesi, Martino; Martin, Holger; Fasolini, Marina; Bertrand, Jay A.; Arrigoni, Cristina; Thiel, Gerhard; Moroni, Anna

    2011-01-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually activated by hyperpolarization and binding of cAMP to their cyclic nucleotide binding domain (CNBD). HCN isoforms respond differently to cAMP; binding of cAMP shifts activation of HCN2 and HCN4 by 17 mV but shifts that of HCN1 by only 2–4 mV. To explain the peculiarity of HCN1, we solved the crystal structures and performed a biochemical-biophysical characterization of the C-terminal domain (C-linker plus CNBD) of the three isoforms. Our main finding is that tetramerization of the C-terminal domain of HCN1 occurs at basal cAMP concentrations, whereas those of HCN2 and HCN4 require cAMP saturating levels. Therefore, HCN1 responds less markedly than HCN2 and HCN4 to cAMP increase because its CNBD is already partly tetrameric. This is confirmed by voltage clamp experiments showing that the right-shifted position of V½ in HCN1 is correlated with its propensity to tetramerize in vitro. These data underscore that ligand-induced CNBD tetramerization removes tonic inhibition from the pore of HCN channels. PMID:22006928

  14. Species-specific functioning of the Pseudomonas XcpQ secretin: role for the C-terminal homology domain and lipopolysaccharide.

    PubMed

    Bitter, Wilbert; van Boxtel, Ria; Groeneweg, Mathijs; Carballo, Patricia Sánchez; Zähringer, Ulrich; Tommassen, Jan; Koster, Margot

    2007-04-01

    Secretins are oligomeric proteins that mediate the export of macromolecules across the bacterial outer membrane. The members of the secretin superfamily possess a C-terminal homology domain that is important for oligomerization and channel formation, while their N-terminal halves are thought to be involved in system-specific interactions. The XcpQ secretin of Pseudomonas spp. is a component of the type II secretion pathway. XcpQ from Pseudomonas alcaligenes is not able to functionally replace the secretin of the closely related species Pseudomonas aeruginosa. By analysis of chimeric XcpQ proteins, a region important for species-specific functioning was mapped between amino acid residues 344 and 478 in the C-terminal homology domain. Two chromosomal suppressor mutations were obtained that resulted in the proper functioning in P. aeruginosa of P. alcaligenes XcpQ and inactive hybrids. These mutations caused a defect in the synthesis of the lipopolysaccharide (LPS) outer core region. Subsequent analysis of different LPS mutants showed that changes in the outer core and not the loss of O antigen caused the suppressor phenotype. High concentrations of divalent cations in the growth medium also allowed P. alcaligenes XcpQ and inactive hybrids to function properly in P. aeruginosa. Since divalent cations are known to affect the structure of LPS, this observation supports the hypothesis that LPS has a role in the functioning of secretins.

  15. [Covalent C-terminal fixation of cyanogen bromide peptides in the liquid-phase-sequenator (author's transl)].

    PubMed

    Braunitzer, G; Pfletschinger, J

    1978-08-01

    This paper describes the covalent fixation and hydrophilisation of homoserin lactone peptides enabling complete C-terminal sequencing in the squenator. Dimethylformamide, dimethylsulfoxide and 6M guanidine hydrochloride in water were used as solvents, ethylendiamine, hexamethylendiamine and histamine base as amino components. The diamine peptide derivative was reacted with the hydrophilic isothiocyanates I and IV, the fixed peptide was sequenced to the C-terminal amino acid, Histamine reacted particularly well and the program with 0.1N quadrol and the hydrophobic buffers was especially suitable for this derivative. The phenylthiohydantoin derivative of homoserine was proven in good yields. The application of this method is suggested.

  16. Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

    SciTech Connect

    Amand, Helene L.

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. Black-Right-Pointing-Pointer Dimer formation enhances peptiplex stability, resulting in increased transfection. Black-Right-Pointing-Pointer By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ('peptiplexes') enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the 'chelate effect' and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from

  17. The C-terminal Ca2+-binding domain of SPARC confers anti-spreading activity to human urothelial cells.

    PubMed

    Delostrinos, Catherine F; Hudson, Amber E; Feng, Waldo C; Kosman, Jeffrey; Bassuk, James A

    2006-01-01

    The anti-spreading activity of secreted protein acidic and rich in cysteine (SPARC) has been assigned to the C-terminal third domain, a region rich in alpha-helices. This "extracellular calcium-binding" (EC) domain contains two EF-hands that each coordinates one Ca2+ ion, forming a helix-loop-helix structure that not only drives the conformation of the protein but is also necessary for biological activity. Recombinant (r) EC, expressed in E. coli, was fused at the C-terminus to a His hexamer and isolated under denaturing conditions by nickel-chelate affinity chromatography. rEC-His was renatured by procedures that simultaneously (i) removed denaturing conditions, (ii) catalyzed disulfide bond isomerization, and (iii) initiated Ca2+-dependent refolding. Intrinsic tryptophan fluorescence and circular dichroism spectroscopies demonstrated that rEC-His exhibited a Ca2+-dependent conformation that was consistent with the known crystal structure. Spreading assays confirmed that rEC-His was biologically active through its ability to inhibit the spreading of freshly plated human urothelial cells propagated from transitional epithelium. rEC-His and rSPARC-His exhibited highly similar anti-spreading activities when measured as a function of concentration or time. In contrast to the wild-type and EC recombinant proteins, rSPARC(E268F)-His, a point substitution mutant at the Z position of EF-hand 2, failed to exhibit both Ca2+-dependent changes in alpha-helical secondary structure and anti-spreading activity. The collective data provide evidence that the motif of SPARC responsible for anti-spreading activity was dependent on the coordination of Ca2+ by a Glu residue at the Z position of EF-hand 2 and provide insights into how adhesive forces are balanced within the extracellular matrix of urothelial cells. .

  18. Location of smooth-muscle myosin and tropomyosin binding sites in the C-terminal 288 residues of human caldesmon.

    PubMed Central

    Huber, P A; Fraser, I D; Marston, S B

    1995-01-01

    We have produced nine recombinant fragments, H1 to H9, from a human cDNA that codes for the C-terminal 288 residues of caldesmon. The fragment H1, encompassing the 288 residues, is equivalent to domains 3 and 4 of caldesmon (amino acids 506-793 in human, 476-737 in the chicken gizzard sequence). It has been shown [Huber, Redwood, Avent, Tanner and Marston (1993) J. Muscle Res. Cell Motil. 14, 385-391] to bind to actin, Ca(2+)-calmodulin, tropomyosin and myosin. The fragments, H2 to H9, differ in length between 60 and 176 residues and cover the whole of domains 3 and 4 with many of the fragments overlapping. We have characterized the myosin and tropomyosin binding of these fragments. The binding of both tropomyosin and myosin is highly dependent on salt concentration, indicating the ionic nature of these interactions. The location of the myosin binding is an extended region encompassing the junction of domains 3/4 and domain 4a (residues 622-714, human; 566-657, chicken gizzard). Tropomyosin binds in a smaller region within domain 4a of caldesmon (residues 663-714, human; 606-657 chicken gizzard). We confirmed predictions based on sequence similarities of a tropomyosin binding site in domain 3 of caldesmon; however, this site bound to skeletal-muscle tropomyosin and had little affinity for the smooth-muscle tropomyosin isoform. None of the protein fragments H2-H9 retained the affinity of the parent fragment H1 for either myosin or tropomyosin. This indicates the need for several interaction sites scattered over an extended region to attain higher affinity. The regions interacting with caldesmon in both tropomyosin and myosin are coiled-coil structures. This is probably the reason for their shared interaction sites on caldesmon and their similar natures of binding. Images Figure 1 Figure 2 Figure 9 PMID:8526878

  19. The Rrp6 C-terminal domain binds RNA and activates the nuclear RNA exosome

    PubMed Central

    Wasmuth, Elizabeth V.; Lima, Christopher D.

    2017-01-01

    The eukaryotic RNA exosome is an essential, multi-subunit complex that catalyzes RNA turnover, maturation, and quality control processes. Its non-catalytic donut-shaped core includes 9 subunits that associate with the 3′ to 5′ exoribonucleases Rrp6, and Rrp44/Dis3, a subunit that also catalyzes endoribonuclease activity. Although recent structures and biochemical studies of RNA bound exosomes from S. cerevisiae revealed that the Exo9 central channel guides RNA to either Rrp6 or Rrp44 using partially overlapping and mutually exclusive paths, several issues related to RNA recruitment remain. Here, we identify activities for the highly basic Rrp6 C-terminal tail that we term the ‘lasso’ because it binds RNA and stimulates ribonuclease activities associated with Rrp44 and Rrp6 within the 11-subunit nuclear exosome. Stimulation is dependent on the Exo9 central channel, and the lasso contributes to degradation and processing activities of exosome substrates in vitro and in vivo. Finally, we present evidence that the Rrp6 lasso may be a conserved feature of the eukaryotic RNA exosome. PMID:27899565

  20. The E. coli thioredoxin folding mechanism: the key role of the C-terminal helix.

    PubMed

    Vazquez, Diego S; Sánchez, Ignacio E; Garrote, Ana; Sica, Mauricio P; Santos, Javier

    2015-02-01

    In this work, the unfolding mechanism of oxidized Escherichia coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against global unfolding while leaving the native structure unchanged. Global analysis of the unfolding kinetics of all variants revealed a linear unfolding route with a high-energy on-pathway intermediate state flanked by two transition state ensembles TSE1 and TSE2. The experiments show that CTH is mainly unfolded in TSE1 and the intermediate and becomes structured in TSE2. Structure-based molecular dynamics are in agreement with these experiments and provide protein-wide structural information on transient states. In our model, EcTRX folding starts with structure formation in the β-sheet, while the protein helices coalesce later. As a whole, our results indicate that the CTH is a critical module in the folding process, restraining a heterogeneous intermediate ensemble into a biologically active native state and providing the native protein with thermodynamic and kinetic stability.

  1. The C-terminal region of Trypanosoma cruzi MASPs is antigenic and secreted via exovesicles

    PubMed Central

    De Pablos, Luis Miguel; Díaz Lozano, Isabel María; Jercic, Maria Isabel; Quinzada, Markela; Giménez, Maria José; Calabuig, Eva; Espino, Ana Margarita; Schijman, Alejandro Gabriel; Zulantay, Inés; Apt, Werner; Osuna, Antonio

    2016-01-01

    Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and emerging tropical disease, endemic to South America and present in non-endemic regions due to human migration. The MASP multigene family is specific to T. cruzi, accounting for 6% of the parasite’s genome and plays a key role in immune evasion. A common feature of MASPs is the presence of two conserved regions: an N-terminal region codifying for signal peptide and a C-terminal (C-term) region, which potentially acts as GPI-addition signal peptide. Our aim was the analysis of the presence of an immune response against the MASP C-term region. We found that this region is highly conserved, released via exovesicles (EVs) and has an associated immune response as revealed by epitope affinity mapping, IFA and inhibition of the complement lysis assays. We also demonstrate the presence of a fast IgM response in Balb/c mice infected with T. cruzi. Our results reveal the presence of non-canonical secreted peptides in EVs, which can subsequently be exposed to the immune system with a potential role in evading immune system targets in the parasite. PMID:27270330

  2. Structural characterization of a C-terminally truncated E5 oncoprotein from papillomavirus in lipid bilayers.

    PubMed

    Windisch, Dirk; Ziegler, Colin; Bürck, Jochen; Ulrich, Anne S

    2014-12-01

    E5 is the major transforming oncoprotein of bovine papillomavirus, which activates the platelet-derived growth factor receptor β in a highly specific manner. The short transmembrane protein E5 with only 44 residues interacts directly with the transmembrane segments of the receptor, but structural details are not available. Biophysical investigations are challenging, because the hydrophobic E5 protein tends to aggregate and get cross-linked non-specifically via two Cys residues near its C-terminus. Here, we demonstrate that a truncation by 10 amino acids creates a more manageable protein that can be conveniently used for structure analysis. Synchrotron radiation circular dichroism and solid-state (15)N- and (31)P-nuclear magnetic resonance spectroscopy show that this E5 variant serves as a representative model for the wild-type protein. The helical conformation of the transmembrane segment, its orientation in the lipid bilayer, and the ability to form homodimers in the membrane are not affected by the C-terminal truncation.

  3. Polycomb Group Targeting through Different Binding Partners of RING1B C-Terminal Domain

    PubMed Central

    Wang, Renjing; Taylor, Alexander B.; Leal, Belinda Z.; Chadwell, Linda V.; Ilangovan, Udayar; Robinson, Angela K.; Schirf, Virgil; Hart, P. John; Lafer, Eileen M.; Demeler, Borries; Hinck, Andrew P.; McEwen, Donald G.; Kim, Chongwoo A.

    2015-01-01

    SUMMARY RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure. PMID:20696397

  4. Sub1 Globally Regulates RNA Polymerase II C-Terminal Domain Phosphorylation ▿

    PubMed Central

    García, Alicia; Rosonina, Emanuel; Manley, James L.; Calvo, Olga

    2010-01-01

    The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3′-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle. PMID:20823273

  5. Solution structure of the RecQ C-terminal domain of human Bloom syndrome protein.

    PubMed

    Park, Chin-Ju; Ko, Junsang; Ryu, Kyoung-Seok; Choi, Byong-Seok

    2014-02-01

    RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1-α2 loop and α3 region), and the aromatic side chain on the top of the β-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.

  6. Intrinsic ssDNA annealing activity in the C-terminal region of WRN.

    PubMed

    Muftuoglu, Meltem; Kulikowicz, Tomasz; Beck, Gad; Lee, Jae Wan; Piotrowski, Jason; Bohr, Vilhelm A

    2008-09-30

    Werner syndrome (WS) is a rare autosomal recessive disorder in humans characterized by premature aging and genetic instability. WS is caused by mutations in the WRN gene, which encodes a member of the RecQ family of DNA helicases. Cellular and biochemical studies suggest that WRN plays roles in DNA replication, DNA repair, telomere maintenance, and homologous recombination and that WRN has multiple enzymatic activities including 3' to 5' exonuclease, 3' to 5' helicase, and ssDNA annealing. The goal of this study was to map and further characterize the ssDNA annealing activity of WRN. Enzymatic studies using truncated forms of WRN identified a C-terminal 79 amino acid region between the RQC and the HRDC domains (aa1072-1150) that is required for ssDNA annealing activity. Deletion of the region reduced or eliminated ssDNA annealing activity of the WRN protein. Furthermore, the activity appears to correlate with DNA binding and oligomerization status of the protein.

  7. Physical association of GPR54 C-terminal with protein phosphatase 2A

    SciTech Connect

    Evans, Barry J.; Wang Zixuan; Mobley, La'Tonya; Khosravi, Davood; Fujii, Nobutaka; Navenot, Jean-Marc; Peiper, Stephen C.

    2008-12-26

    KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.

  8. Piezo1 ion channel pore properties are dictated by C-terminal region

    NASA Astrophysics Data System (ADS)

    Coste, Bertrand; Murthy, Swetha E.; Mathur, Jayanti; Schmidt, Manuela; Mechioukhi, Yasmine; Delmas, Patrick; Patapoutian, Ardem

    2015-05-01

    Piezo1 and Piezo2 encode mechanically activated cation channels that function as mechanotransducers involved in vascular system development and touch sensing, respectively. Structural features of Piezos remain unknown. Mouse Piezo1 is bioinformatically predicted to have 30-40 transmembrane (TM) domains. Here, we find that nine of the putative inter-transmembrane regions are accessible from the extracellular side. We use chimeras between mPiezo1 and dPiezo to show that ion-permeation properties are conferred by C-terminal region. We further identify a glutamate residue within a conserved region adjacent to the last two putative TM domains of the protein, that when mutated, affects unitary conductance and ion selectivity, and modulates pore block. We propose that this amino acid is either in the pore or closely associates with the pore. Our results describe important structural motifs of this channel family and lay the groundwork for a mechanistic understanding of how Piezos are mechanically gated and conduct ions.

  9. Structure of the C-terminal domain of nsp4 from feline coronavirus

    PubMed Central

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh; Snijder, Eric J.; Gorbalenya, Alexander E.; Berglind, Hanna; Nordlund, Pär; Coutard, Bruno; Tucker, Paul A.

    2009-01-01

    Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions. PMID:19622868

  10. PrP106-126 peptide disrupts lipid membranes: Influence of C-terminal amidation

    SciTech Connect

    Zheng Wenfu; Wang Lijun; Hong Yuankai; Sha Yinlin

    2009-02-06

    PrP106-126 is located within the important domain concerning membrane related conformational conversion of human Prion protein (from cellular isoform PrP{sup C} to scrapie isoform PrP{sup Sc}). Recent advances reveal that the pathological and physicochemical properties of PrP106-126 peptide are very sensitive to its N-terminal amidation, however, the detailed mechanism remains unclear. In this work, we studied the interactions of the PrP106-126 isoforms (PrP106-126{sub CONH2} and PrP106-126{sub COOH}) with the neutral lipid bilayers by atomic force microscopy, surface plasmon resonance and fluorescence spectroscopy. The membrane structures were disturbed by the two isoforms in a similarly stepwise process. The distinct morphological changes of the membrane were characterized by formation of semi-penetrated defects and sigmoidal growth of flat high-rise domains on the supported lipid bilayers. However, PrP106-126{sub COOH} displayed a higher peptide-lipid binding affinity than PrP106-126{sub CONH2} ({approx}2.9 times) and facilitated the peptide-lipid interactions by shortening the lag time. These results indicate that the C-terminal amidation may influence the pathological actions of PrP106-126 by lowering the interaction potentials with lipid membranes.

  11. Dynein's C-terminal Domain Plays a Novel Role in Regulating Force Generation

    NASA Astrophysics Data System (ADS)

    Gennerich, Arne; Nicholas, Matthew; Brenner, Sibylle; Lazar, Caitlin; Weil, Sarah; Vallee, Richard; Hook, Peter; Gennerich Lab Collaboration; Vallee Lab Collaboration

    2014-03-01

    Cytoplasmic dynein is a microtubule motor involved in a wide range of low and high force requiring functions in metazoans. In contrast, yeast dynein is involved in a single, nonessential function, nuclear positioning. Interestingly, the single-molecule function of yeast dynein is also unique: whereas mammalian dyneins generate forces of 1-2 pN, S. cerevisiae dynein stalls at 5-7 pN. The basis for this functional difference is unknown. However, the major structural difference between mammalian and yeast dyneins is a ~30 kDa C-terminal extension (CT) present in higher eukaryotic dyneins, but missing in yeast. To test whether the CT accounts for the differences in function, we produced recombinant rat dynein motor domains (MD) with (WT-MD) and without (ΔCT-MD) the CT, using baculovirus expression. To define motor function, we performed single-molecule optical trapping studies. Dimerized WT-MD stalls at ~1 pN and detaches from microtubules after brief stalls, in agreement with previous studies on native mammalian dynein. In sharp contrast, but similar to yeast dynein, ΔCT-MD stalls at ~6 pN, with stall durations up to minutes. These results identify the CT as a new regulatory element for controlling dynein force generation. Supported by NIH GM094415 (A.G.) and GM102347 (R.B.V.)

  12. C-terminal Amidation of an Osteocalcin-derived Peptide Promotes Hydroxyapatite Crystallization*

    PubMed Central

    Hosseini, Samaneh; Naderi-Manesh, Hossein; Mountassif, Driss; Cerruti, Marta; Vali, Hojatollah; Faghihi, Shahab

    2013-01-01

    Genesis of natural biocomposite-based materials, such as bone, cartilage, and teeth, involves interactions between organic and inorganic systems. Natural biopolymers, such as peptide motif sequences, can be used as a template to direct the nucleation and crystallization of hydroxyapatite (HA). In this study, a natural motif sequence consisting of 13 amino acids present in the first helix of osteocalcin was selected based on its calcium binding ability and used as substrate for nucleation of HA crystals. The acidic (acidic osteocalcin-derived peptide (OSC)) and amidic (amidic osteocalcin-derived peptide (OSN)) forms of this sequence were synthesized to investigate the effects of different C termini on the process of biomineralization. Electron microscopy analyses show the formation of plate-like HA crystals with random size and shape in the presence of OSN. In contrast, spherical amorphous calcium phosphate is formed in the presence of OSC. Circular dichroism experiments indicate conformational changes of amidic peptide to an open and regular structure as a consequence of interaction with calcium and phosphate. There is no conformational change detectable in OSC. It is concluded that HA crystal formation, which only occurred in OSN, is attributable to C-terminal amidation of a natural peptide derived from osteocalcin. It is also proposed that natural peptides with the ability to promote biomineralization have the potential to be utilized in hard tissue regeneration. PMID:23362258

  13. Sub1 globally regulates RNA polymerase II C-terminal domain phosphorylation.

    PubMed

    García, Alicia; Rosonina, Emanuel; Manley, James L; Calvo, Olga

    2010-11-01

    The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3'-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

  14. Amyloid β-Protein C-Terminal Fragments: Formation of Cylindrins and β-Barrels.

    PubMed

    Do, Thanh D; LaPointe, Nichole E; Nelson, Rebecca; Krotee, Pascal; Hayden, Eric Y; Ulrich, Brittany; Quan, Sarah; Feinstein, Stuart C; Teplow, David B; Eisenberg, David; Shea, Joan-Emma; Bowers, Michael T

    2016-01-20

    In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer's disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using diglycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins.

  15. Amyloid β-Protein C-terminal Fragments: Formation of Cylindrins and β-barrels

    PubMed Central

    Do, Thanh D.; LaPointe, Nichole E.; Nelson, Rebecca; Krotee, Pascal; Hayden, Eric Y.; Ulrich, Brittany; Quan, Sarah; Feinstein, Stuart C.; Teplow, David B.; Eisenberg, David; Shea, Joan-Emma; Bowers, Michael T.

    2016-01-01

    In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer’s disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross-sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using di-glycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins. PMID:26700445

  16. Identification and characterization of the role of c-terminal Src kinase in dengue virus replication

    PubMed Central

    Kumar, Rinki; Agrawal, Tanvi; Khan, Naseem Ahmed; Nakayama, Yuji; Medigeshi, Guruprasad R.

    2016-01-01

    We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication. PMID:27457684

  17. C-Terminal Alpha-1 Antitrypsin Peptide: A New Sepsis Biomarker with Immunomodulatory Function.

    PubMed

    Blaurock, Nancy; Schmerler, Diana; Hünniger, Kerstin; Kurzai, Oliver; Ludewig, Katrin; Baier, Michael; Brunkhorst, Frank Martin; Imhof, Diana; Kiehntopf, Michael

    2016-01-01

    Systemic inflammatory response syndrome (SIRS) is a life threatening condition and the leading cause of death in intensive care units. Although single aspects of pathophysiology have been described in detail, numerous unknown mediators contribute to the progression of this complex disease. The aim of this study was to elucidate the pathophysiological role of CAAP48, a C-terminal alpha-1 antitrypsin fragment, that we found to be elevated in septic patients and to apply this peptide as diagnostic marker for infectious and noninfectious etiologies of SIRS. Incubation of human polymorphonuclear neutrophils with synthetic CAAP48, the SNP-variant CAAP47, and several control peptides revealed intense neutrophil activation, induction of neutrophil chemotaxis, reduction of neutrophil viability, and release of cytokines. We determined the abundance of CAAP48 in patients with severe sepsis, severe SIRS of noninfectious origin, and viral infection. CAAP48 levels were 3-4-fold higher in patients with sepsis compared to SIRS of noninfectious origin and allowed discrimination of those patients with high sensitivity and specificity. Our results suggest that CAAP48 is a promising discriminatory sepsis biomarker with immunomodulatory functions, particularly on human neutrophils, supporting its important role in the host response and pathophysiology of sepsis.

  18. C-terminal motif of human neuropeptide Y4 receptor determines internalization and arrestin recruitment.

    PubMed

    Wanka, Lizzy; Babilon, Stefanie; Burkert, Kerstin; Mörl, Karin; Gurevich, Vsevolod V; Beck-Sickinger, Annette G

    2017-01-01

    The human neuropeptide Y4 receptor is a rhodopsin-like G protein-coupled receptor (GPCR), which contributes to anorexigenic signals. Thus, this receptor is a highly interesting target for metabolic diseases. As GPCR internalization and trafficking affect receptor signaling and vice versa, we aimed to investigate the molecular mechanism of hY4R desensitization and endocytosis. The role of distinct segments of the hY4R carboxyl terminus was investigated by fluorescence microscopy, binding assays, inositol turnover experiments and bioluminescence resonance energy transfer assays to examine the internalization behavior of hY4R and its interaction with arrestin-3. Based on results of C-terminal deletion mutants and substitution of single amino acids, the motif (7.78)EESEHLPLSTVHTEVSKGS(7.96) was identified, with glutamate, threonine and serine residues playing key roles, based on site-directed mutagenesis. Thus, we identified the internalization motif for the human neuropeptide Y4 receptor, which regulates arrestin-3 recruitment and receptor endocytosis.

  19. Crystallization of the C-terminal globular domain of avian reovirus fibre

    PubMed Central

    van Raaij, Mark J.; Hermo Parrado, X. Lois; Guardado Calvo, Pablo; Fox, Gavin C.; Llamas-Saiz, Antonio L.; Costas, Celina; Martínez-Costas, José; Benavente, Javier

    2005-01-01

    Avian reovirus fibre, a homotrimer of the σC protein, is responsible for primary host-cell attachment. Using the protease trypsin, a C-terminal σC fragment containing amino acids 156–326 has been generated which was subsequently purified and crystallized. Two different crystal forms were obtained, one grown in the absence of divalent cations and belonging to space group P6322 (unit-cell parameters a = 75.6, c = 243.1 Å) and one grown in the presence of either zinc or cadmium sulfate and belonging to space group P321 (unit-cell parameters a = 74.7, c = 74.5 Å and a = 73.1, c = 69.9 Å for the ZnII- and CdII-grown crystals, respectively). The first crystal form diffracted synchrotron radiation to 3.0 Å resolution and the second form to 2.2–2.3 Å. Its closest related structure, the C-­terminal fragment of mammalian reovirus fibre, has only 18% sequence identity and molecular-replacement attempts were unsuccessful. Therefore, a search is under way for suitable heavy-atom derivatives and attempts are being made to grow protein crystals containing selenomethionine instead of methionine. PMID:16511119

  20. A rule-based kinetic model of RNA polymerase II C-terminal domain phosphorylation

    PubMed Central

    Aitken, Stuart; Alexander, Ross D.; Beggs, Jean D.

    2013-01-01

    The complexity of many RNA processing pathways is such that a conventional systems modelling approach is inadequate to represent all the molecular species involved. We demonstrate that rule-based modelling permits a detailed model of a complex RNA signalling pathway to be defined. Phosphorylation of the RNA polymerase II (RNAPII) C-terminal domain (CTD; a flexible tail-like extension of the largest subunit) couples pre-messenger RNA capping, splicing and 3′ end maturation to transcriptional elongation and termination, and plays a central role in integrating these processes. The phosphorylation states of the serine residues of many heptapeptide repeats of the CTD alter along the coding region of genes as a function of distance from the promoter. From a mechanistic perspective, both the changes in phosphorylation and the location at which they take place on the genes are a function of the time spent by RNAPII in elongation as this interval provides the opportunity for the kinases and phosphatases to interact with the CTD. On this basis, we synthesize the available data to create a kinetic model of the action of the known kinases and phosphatases to resolve the phosphorylation pathways and their kinetics. PMID:23804443

  1. The C-terminal region of Trypanosoma cruzi MASPs is antigenic and secreted via exovesicles.

    PubMed

    De Pablos, Luis Miguel; Díaz Lozano, Isabel María; Jercic, Maria Isabel; Quinzada, Markela; Giménez, Maria José; Calabuig, Eva; Espino, Ana Margarita; Schijman, Alejandro Gabriel; Zulantay, Inés; Apt, Werner; Osuna, Antonio

    2016-06-08

    Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and emerging tropical disease, endemic to South America and present in non-endemic regions due to human migration. The MASP multigene family is specific to T. cruzi, accounting for 6% of the parasite's genome and plays a key role in immune evasion. A common feature of MASPs is the presence of two conserved regions: an N-terminal region codifying for signal peptide and a C-terminal (C-term) region, which potentially acts as GPI-addition signal peptide. Our aim was the analysis of the presence of an immune response against the MASP C-term region. We found that this region is highly conserved, released via exovesicles (EVs) and has an associated immune response as revealed by epitope affinity mapping, IFA and inhibition of the complement lysis assays. We also demonstrate the presence of a fast IgM response in Balb/c mice infected with T. cruzi. Our results reveal the presence of non-canonical secreted peptides in EVs, which can subsequently be exposed to the immune system with a potential role in evading immune system targets in the parasite.

  2. Functional Characteristics of C-terminal Lysine to Cysteine Mutant Form of CTLA-4Ig

    PubMed Central

    Kim, Bongi; Shin, Jun-Seop

    2013-01-01

    CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation. PMID:23559896

  3. Structure of the C-terminal domain of Tup1, a corepressor of transcription in yeast.

    PubMed

    Sprague, E R; Redd, M J; Johnson, A D; Wolberger, C

    2000-06-15

    The Tup1-Ssn6 corepressor complex regulates the expression of several sets of genes, including genes that specify mating type in the yeast Saccharomyces cerevisiae. Repression of mating-type genes occurs when Tup1-Ssn6 is brought to the DNA by the Matalpha2 DNA-binding protein and assembled upstream of a- and haploid-specific genes. We have determined the 2.3 A X-ray crystal structure of the C-terminal domain of Tup1 (accesion No. 1ERJ), a 43 kDa fragment that contains seven copies of the WD40 sequence motif and binds to the Matalpha2 protein. Moreover, this portion of the protein can partially substitute for full-length Tup1 in bringing about transcriptional repression. The structure reveals a seven-bladed beta propeller with an N-terminal subdomain that is anchored to the side of the propeller and extends the beta sheet of one of the blades. Point mutations in Tup1 that specifically affect the Tup1-Matalpha2 interaction cluster on one surface of the propeller. We identified regions of Tup1 that are conserved among the fungal Tup1 homologs and may be important in protein-protein interactions with additional components of the Tup1-mediated repression pathways.

  4. C-Terminal Alpha-1 Antitrypsin Peptide: A New Sepsis Biomarker with Immunomodulatory Function

    PubMed Central

    Blaurock, Nancy; Schmerler, Diana; Hünniger, Kerstin; Kurzai, Oliver; Ludewig, Katrin; Baier, Michael; Brunkhorst, Frank Martin; Imhof, Diana; Kiehntopf, Michael

    2016-01-01

    Systemic inflammatory response syndrome (SIRS) is a life threatening condition and the leading cause of death in intensive care units. Although single aspects of pathophysiology have been described in detail, numerous unknown mediators contribute to the progression of this complex disease. The aim of this study was to elucidate the pathophysiological role of CAAP48, a C-terminal alpha-1 antitrypsin fragment, that we found to be elevated in septic patients and to apply this peptide as diagnostic marker for infectious and noninfectious etiologies of SIRS. Incubation of human polymorphonuclear neutrophils with synthetic CAAP48, the SNP-variant CAAP47, and several control peptides revealed intense neutrophil activation, induction of neutrophil chemotaxis, reduction of neutrophil viability, and release of cytokines. We determined the abundance of CAAP48 in patients with severe sepsis, severe SIRS of noninfectious origin, and viral infection. CAAP48 levels were 3-4-fold higher in patients with sepsis compared to SIRS of noninfectious origin and allowed discrimination of those patients with high sensitivity and specificity. Our results suggest that CAAP48 is a promising discriminatory sepsis biomarker with immunomodulatory functions, particularly on human neutrophils, supporting its important role in the host response and pathophysiology of sepsis. PMID:27382189

  5. Role of ubiquitin C-terminal hydrolase-L1 in antipolyspermy defense of mammalian oocytes.

    PubMed

    Susor, Andrej; Liskova, Lucie; Toralova, Tereza; Pavlok, Antonin; Pivonkova, Katerina; Karabinova, Pavla; Lopatarova, Miloslava; Sutovsky, Peter; Kubelka, Michal

    2010-06-01

    The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

  6. The region-specific functions of two ubiquitin C-terminal hydrolase isozymes along the epididymis.

    PubMed

    Kwon, Jungkee; Sekiguchi, Satoshi; Wang, Yu-Lai; Setsuie, Rieko; Yoshikawa, Yasuhiro; Wada, Keiji

    2006-01-01

    We previously showed that gad mice, which are deficient for ubiquitin C-terminal hydrolase L1 (UCH-L1), have a significantly increased number of defective spermatozoa, suggesting that UCH-L1 functions in sperm quality control during epididymal maturation. The epididymis is the site of spermatozoa maturation, transport and storage. Region-specific functions along the epididymis are essential for establishing the environment required for sperm maturation. We analyzed the region-specific expression of UCH-L1 and UCH-L3 along the epididymis, and also assessed the levels of ubiquitin, which has specificity for UCH-L1. In wild-type mice, western blot analysis demonstrated a high level of UCH-L1 expression in the caput epididymis, consistent with ubiquitin expression, whereas UCH-L3 expression was high in the cauda epididymis. We also investigated the function of UCH-L1 and UCH-L3 in epididymal apoptosis induced by efferent duct ligation. The caput epididymides of gad mice were resistant to apoptotic stress induced by efferent duct ligation, whereas Uchl3 knockout mice showed a marked increase in apoptotic cells following ligation. In conclusion, the response of gad and Uchl3 knockout mice to androgen withdrawal suggests a reciprocal function of the two UCH enzymes in the caput epididymis.

  7. Effects of ubiquitin C-terminal hydrolase L1 deficiency on mouse ova.

    PubMed

    Koyanagi, Sayaka; Hamasaki, Hiroko; Sekiguchi, Satoshi; Hara, Kenshiro; Ishii, Yoshiyuki; Kyuwa, Shigeru; Yoshikawa, Yasuhiro

    2012-03-01

    Maternal proteins are rapidly degraded by the ubiquitin-proteasome system during oocyte maturation in mice. Ubiquitin C-terminal hydrolase L1 (UCHL1) is highly and specifically expressed in mouse ova and is involved in the polyspermy block. However, the role of UCHL1 in the underlying mechanism of polyspermy block is poorly understood. To address this issue, we performed a comprehensive proteomic analysis to identify maternal proteins that were relevant to the role of UCHL1 in mouse ova using UCHL1-deficient gad. Furthermore, we assessed morphological features in gad mouse ova using transmission electron microscopy. NACHT, LRR, and PYD domain-containing (NALP) family proteins and endoplasmic reticulum (ER) chaperones were identified by proteomic analysis. We also found that the 'maternal antigen that embryos require' (NLRP5 (MATER)) protein level increased significantly in gad mouse ova compared with that in wild-type mice. In an ultrastructural study, gad mouse ova contained less ER in the cortex than in wild-type mice. These results provide new insights into the role of UCHL1 in the mechanism of polyspermy block in mouse ova.

  8. Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

    PubMed

    Cartier, Anna E; Djakovic, Stevan N; Salehi, Afshin; Wilson, Scott M; Masliah, Eliezer; Patrick, Gentry N

    2009-06-17

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

  9. Crystal structure of Ralstonia eutropha polyhydroxyalkanoate synthase C-terminal domain and reaction mechanisms.

    PubMed

    Kim, Jieun; Kim, Yeo-Jin; Choi, So Young; Lee, Sang Yup; Kim, Kyung-Jin

    2017-01-01

    Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by numerous microorganisms as energy and reducing power storage materials, and have attracted much attention as substitutes for petroleum-based plastics. Here, we report the first crystal structure of Ralstonia eutropha PHA synthase at 1.8 Å resolution and structure-based mechanisms for PHA polymerization. RePhaC1 contains two distinct domains, the N-terminal (RePhaC1ND ) and C-terminal domains (RePhaC1CD ), and exists as a dimer. RePhaC1CD catalyzes polymerization via non-processive ping-pong mechanism using a Cys-His-Asp catalytic triad. Molecular docking simulation of 3-hydroxybutyryl-CoA to the active site of RePhaC1CD reveals residues involved in the formation of 3-hydroxybutyryl-CoA binding pocket and substrate binding tunnel. Comparative analysis with other polymerases elucidates how different classes of PHA synthases show different substrate specificities. Furthermore, we attempted structure-based protein engineering and developed a RePhaC1 mutant with enhanced PHA synthase activity.

  10. Immunometric assay of BN 52080, a heptapeptide C-terminal analogue of sorbin.

    PubMed

    Ezan, E; Tarrade, T; Cazenave, C; Ardouin, T; Genet, R; Grassi, J; Grognet, J M; Pradelles, P

    1995-01-01

    A novel type of enzyme immunometric assay has been developed for a heptapeptide, BN 52080. This compound is a short C-terminal analogue of sorbin and is under clinical evaluation for treatment of chronic diarrhea. In this solid-phase immobilized epitope immunoassay (SPIE-IA), the peptide is first immunologically bound to polyclonal antibodies adsorbed to a solid phase and then, after covalent immobilization with glutaraldehyde, is released from the antibody paratope by NaOH. The peptide linked to the solid phase is further quantified with a tracer consisting of the same antibodies purified by affinity chromatography and coupled to acetylcholinesterase. This assay has a detection limit of 10 pg/ml and is therefore five times more sensitive than competitive enzyme immunoassay using the same antibodies and BN 52080 coupled to acetylcholinesterase as tracer. The assay is specific and allows direct measurement of peptide in human plasma after subcutaneous or intravenous administration of 200 micrograms of BN 52080 to volunteers.

  11. Impedance Analysis of Ovarian Cancer Cells upon Challenge with C-terminal Clostridium Perfringens Enterotoxin

    NASA Astrophysics Data System (ADS)

    Gordon, Geoffrey; Lo, Chun-Min

    2007-03-01

    Both in vitro and animal studies in breast, prostate, and ovarian cancers have shown that clostridium perfringens enterotoxin (CPE), which binds to CLDN4, may have an important therapeutic benefit, as it is rapidly cytotoxic in tissues overexpressing CLDN4. This study sought to evaluate the ability of C-terminal clostridium perfringens enterotoxin (C-CPE), a CLDN4-targetting molecule, to disrupt tight junction barrier function. Electric cell-substrate impedance sensing (ECIS) was used to measure both junctional resistance and average cell-substrate separation of ovarian cancer cell lines after exposure to C-CPE. A total of 14 ovarian cancer cell lines were used, and included cell lines derived from serous, mucinous, and clear cells. Our results showed that junctional resistance increases as CLDN4 expression increases. In addition, C-CPE is non-cytotoxic in ovarian cancer cells expressing CLDN4. However, exposure to C-CPE results in a significant (p<0.05) dose- and CLDN4-dependent decrease in junctional resistance and an increase in cell-substrate separation. Treatment of ovarian cancer cell lines with C-CPE disrupts tight junction barrier function.

  12. Role of the teneurins, teneurin C-terminal associated peptides (TCAP) in reproduction: clinical perspectives.

    PubMed

    Lovejoy, David A; Pavlović, Téa

    2015-11-01

    In humans, the teneurin gene family consists of four highly conserved paralogous genes that are the result of early vertebrate gene duplications arising from a gene introduced into multicellular organisms from a bacterial ancestor. In vertebrates and humans, the teneurins have become integrated into a number of critical physiological systems including several aspects of reproductive physiology. Structurally complex, these genes possess a sequence in their terminal exon that encodes for a bioactive peptide sequence termed the 'teneurin C-terminal associated peptide' (TCAP). The teneurin/TCAP protein forms an intercellular adhesive unit with its receptor, latrophilin, an Adhesion family G-protein coupled receptor. It is present in numerous cell types and has been implicated in gamete migration and gonadal morphology. Moreover, TCAP is highly effective at reducing the corticotropin-releasing factor (CRF) stress response. As a result, TCAP may also play a role in regulating the stress-associated inhibition of reproduction. In addition, the teneurins and TCAP have been implicated in tumorigenesis associated with reproductive tissues. Therefore, the teneurin/TCAP system may offer clinicians a novel biomarker system upon which to diagnose some reproductive pathologies.

  13. HGF signaling regulates Claudin-3 dynamics through its C-terminal tyrosine residues.

    PubMed

    Twiss, Floor; Oldenkamp, Michiel; Hiemstra, Annemieke; Zhou, Houjiang; Matheron, Lucrèce; Mohammed, Shabaz; de Rooij, Johan

    2013-10-01

    The hormone HGF regulates morphogenesis and regeneration of multiple organs and increased HGF signaling is strongly associated with metastatic cancer. At the cellular level, one of the distinct effects of HGF is the de-stabilization of cell-cell junctions. Several molecular mechanisms have been shown to be involved that mostly culminate at the E-cadherin adhesion complex. One of the key determinants in HGF-driven morphological changes is the actomyosin cytoskeleton whose organization and physical parameters changes upon stimulation. Here we have investigated how HGF affects the different actomyosin-associated cell-cell junction complexes, Nectin Junctions, Adherens Junctions and Tight Junctions in MDCK cells. We find that components of all complexes stay present at cell-cell contacts until their physical dissociation. We find that at cell-cell junctions, the mobility of Claudin-3, but not that of other cell-cell adhesion receptors, is affected by HGF. This depends on tyrosine residues that likely affect PDZ-domain interactions at the C-terminal tail of Claudin-3, although their phosphorylation is not directly regulated by HGF. Thus we uncovered Claudins as novel targets of HGF signaling at cell-cell junctions.

  14. Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability

    PubMed Central

    Ebersole, Brittany; Petko, Jessica; Woll, Matthew; Murakami, Shoko; Sokolina, Kate; Wong, Victoria; Stagljar, Igor; Lüscher, Bernhard; Levenson, Robert

    2015-01-01

    We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH) screen, we identified the palmitoyl acyltransferase (PAT) zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R. PMID:26535572

  15. Mutational analysis of the C-terminal domain of the Rhodobacter sphaeroides response regulator PrrA

    PubMed Central

    Jones, Denise F.; Stenzel, Rachelle A.; Donohue, Timothy J.

    2009-01-01

    The Rhodobacter sphaeroides response regulator PrrA directly activates transcription of genes necessary for energy conservation at low O2 tensions and under anaerobic conditions. It is proposed that PrrA homologues contain a C-terminal DNA-binding domain (PrrA-CTD) that lacks significant amino acid sequence similarity to those found in other response regulators. To test this hypothesis, single amino acid substitutions were created at 12 residues in the PrrA-CTD. These mutant PrrA proteins were purified and tested for the ability to be phosphorylated by the low-molecular-mass phosphate donor acetyl phosphate, to activate transcription and to bind promoter DNA. Each mutant PrrA protein accepted phosphate from 32P-labelled acetyl phosphate. At micromolar concentrations of acetyl phosphate-treated wild-type PrrA, a single 20 bp region in the PrrA-dependent cycA P2 promoter was protected from DNase I digestion. Of the mutant PrrA proteins tested, only acetyl phosphate-treated PrrA-N168A and PrrA-I177A protected cycA P2 from DNase I digestion at similar protein concentrations compared to wild-type PrrA. The use of in vitro transcription assays with the PrrA-dependent cycA P2 and puc promoters showed that acetyl phosphate-treated PrrA-N168A produced transcript levels similar to that of wild-type PrrA at comparable protein concentrations. Using concentrations of acetyl phosphate-treated PrrA that are saturating for the wild-type protein, PrrA-H170A and PrrA-I177A produced<45%as much transcript as wild-type PrrA. Under identical conditions, the remaining mutant PrrA proteins produced little or no detectable transcripts from either promoter in vitro. Explanations are presented for why these amino acid side chains in the PrrA-CTD are important for its ability to activate transcription. PMID:16339955

  16. Topology and dynamics of the 10 kDa C-terminal domain of DnaK in solution.

    PubMed Central

    Bertelsen, E. B.; Zhou, H.; Lowry, D. F.; Flynn, G. C.; Dahlquist, F. W.

    1999-01-01

    Hsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N-terminal ATPase domain, a 17 kDa peptide-binding domain, and a 10 kDa C-terminal domain. The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized. The function of the 10 kDa variable C-terminal domain is less well understood. We have characterized the secondary structure and dynamics of the C-terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high-resolution NMR. The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C-terminal subdomain of approximately 33 amino acids. The mobility of the flexible region is maintained in the context of the full-length protein and does not appear to be modulated by the nucleotide state. The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications. We also developed a method to analyze 15N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis. The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle-like structure in solution. PMID:10048327

  17. NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide.

    PubMed

    Mikami, Suzuka; Kanaba, Teppei; Ito, Yutaka; Mishima, Masaki

    2013-10-01

    The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.

  18. SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity

    PubMed Central

    Muramatsu, Tomonari; Takemoto, Chie; Kim, Yong-Tae; Wang, Hongfei; Nishii, Wataru; Terada, Takaho; Shirouzu, Mikako

    2016-01-01

    The 3C-like protease (3CLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV) cleaves 11 sites in the polyproteins, including its own N- and C-terminal autoprocessing sites, by recognizing P4–P1 and P1′. In this study, we determined the crystal structure of 3CLpro with the C-terminal prosequence and the catalytic-site C145A mutation, in which the enzyme binds the C-terminal prosequence of another molecule. Surprisingly, Phe at the P3′ position [Phe(P3′)] is snugly accommodated in the S3′ pocket. Mutations of Phe(P3′) impaired the C-terminal autoprocessing, but did not affect N-terminal autoprocessing. This difference was ascribed to the P2 residue, Phe(P2) and Leu(P2), in the C- and N-terminal sites, as follows. The S3′ subsite is formed by Phe(P2)-induced conformational changes of 3CLpro and the direct involvement of Phe(P2) itself. In contrast, the N-terminal prosequence with Leu(P2) does not cause such conformational changes for the S3′ subsite formation. In fact, the mutation of Phe(P2) to Leu in the C-terminal autoprocessing site abolishes the dependence on Phe(P3′). These mechanisms explain why Phe is required at the P3' position when the P2 position is occupied by Phe rather than Leu, which reveals a type of subsite cooperativity. Moreover, the peptide consisting of P4–P1 with Leu(P2) inhibits protease activity, whereas that with Phe(P2) exhibits a much smaller inhibitory effect, because Phe(P3′) is missing. Thus, this subsite cooperativity likely exists to avoid the autoinhibition of the enzyme by its mature C-terminal sequence, and to retain the efficient C-terminal autoprocessing by the use of Phe(P2). PMID:27799534

  19. Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Dupré, Mathieu; Cantel, Sonia; Martinez, Jean; Enjalbal, Christine

    2012-02-01

    By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (bn-1 + H2O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H2O and NH3) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in

  20. Structure predictions of two Bauhinia variegata lectins reveal patterns of C-terminal properties in single chain legume lectins.

    PubMed

    Moreira, Gustavo M S G; Conceição, Fabricio R; McBride, Alan J A; Pinto, Luciano da S

    2013-01-01

    Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.

  1. Structure Predictions of Two Bauhinia variegata Lectins Reveal Patterns of C-Terminal Properties in Single Chain Legume Lectins

    PubMed Central

    Moreira, Gustavo M. S. G.; Conceição, Fabricio R.; McBride, Alan J. A.; Pinto, Luciano da S.

    2013-01-01

    Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and –II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins. PMID:24260572

  2. Autoinhibition of the Nuclease ARTEMIS Is Mediated by a Physical Interaction between Its Catalytic and C-terminal Domains.

    PubMed

    Niewolik, Doris; Peter, Ingrid; Butscher, Carmen; Schwarz, Klaus

    2017-02-24

    The nuclease ARTEMIS is essential for the development of B and T lymphocytes. It is required for opening DNA hairpins generated during antigen receptor gene assembly from variable (V), diversity (D), and joining (J) subgenic elements (V(D)J recombination). As a member of the non-homologous end-joining pathway, it is also involved in repairing a subset of pathological DNA double strand breaks. Loss of ARTEMIS function therefore results in radiosensitive severe combined immunodeficiency (RS-SCID). The hairpin opening activity is dependent on the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which can bind to and phosphorylate ARTEMIS. The ARTEMIS C terminus is dispensable for cellular V(D)J recombination and in vitro nuclease assays with C-terminally truncated ARTEMIS showing DNA-PKcs-independent hairpin opening activity. Therefore, it has been postulated that ARTEMIS is regulated via autoinhibition by its C terminus. To obtain evidence for the autoinhibition model, we performed co-immunoprecipitation experiments with combinations of ARTEMIS mutants. We show that an N-terminal fragment comprising the catalytic domain can interact both with itself and with a C-terminal fragment. Amino acid exchanges N456A+S457A+E458Q in the C terminus of full-length ARTEMIS resulted in unmasking of the N terminus and in increased ARTEMIS activity in cellular V(D)J recombination assays. Mutations in ARTEMIS-deficient patients impaired the interaction with the C terminus and also affected protein stability. The interaction between the N- and C-terminal domains was not DNA-PKcs-dependent, and phosphomimetic mutations in the C-terminal domain did not result in unmasking of the catalytic domain. Our experiments provide strong evidence that a physical interaction between the C-terminal and catalytic domains mediates ARTEMIS autoinhibition.

  3. The N- and C-terminal domains of MecA recognize different partners in the competence molecular switch.

    PubMed

    Persuh, M; Turgay, K; Mandic-Mulec, I; Dubnau, D

    1999-08-01

    ComK is a transcription factor required for the expression of competence genes in Bacillus subtilis. Binding to MecA targets ComK for degradation by the ClpCP protease. MecA therefore acts as an adapter protein recruiting a regulatory protein for proteolysis. However, when ComS is synthesized, ComK is released from binding by MecA and thereby protected from degradation. MecA binds to three protein partners during these processes: ComK, ClpC and ComS. Using limited proteolysis, we have defined N- and C-terminal structural domains of MecA and evaluated the interactions of these domains with the protein partners of MecA. Using surface plasmon resonance, we have determined that the N-terminal domain of MecA interacts with ComK and ComS and the C-terminal domain with ClpC. MecA is shown to exist as a dimer with dimerization sites on both the N- and C-terminal domains. The C-terminal domain stimulates the ATPase activity of ClpC and is degraded by the ClpCP protease, while the N-terminal domain is inactive in both of these assays. In vivo data were consistent with these findings, as comG-lacZ expression was decreased in a strain overproducing the N-terminal domain, indicating reduced ComK activity. We propose a model in which binding of ClpC to the C-terminal domain of MecA induces a conformational change enabling the N-terminal domain to bind ComK with enhanced affinity. MecA is widespread among Gram-positive organisms and may act generally as an adapter protein, targeting proteins for regulated degradation.

  4. The C- terminal region of the Major Outer Sheath Protein (Msp) of Treponema denticola inhibits neutrophil chemotaxis.

    PubMed

    Jones, Megan M; Vanyo, Stephen T; Visser, Michelle B

    2017-03-13

    Treponema denticola is an oral spirochete strongly associated with severe periodontal disease. A prominent virulence factor, the major outer sheath protein (Msp), disorients neutrophil chemotaxis by altering the cellular phosphoinositide balance, leading to impairment of downstream chemotactic events including actin rearrangement, Rac1 activation and Akt activation in response to chemoattractant stimulation. The specific regions of Msp responsible for interactions with neutrophils remain unknown. In this study, we investigated the inhibitory effect of truncated Msp regions on neutrophil chemotaxis and associated signaling pathways. Murine neutrophils were treated with recombinant protein truncations followed by assessment of chemotaxis and associated signal pathway activation. Chemotaxis assays indicate sequences within the C-terminal region; particularly the first 130 amino acids, have the strongest inhibitory effect on neutrophil chemotaxis. Neutrophils incubated with the C-terminal region protein also demonstrated the greatest inhibition of Rac1 activation, increased phosphoinositide phosphatase activity, and decreased Akt activation; orchestrating impairment of chemotaxis. Furthermore, incubation with antibodies specific to only the C-terminal region blocked the Msp induced inhibition of chemotaxis and denaturing the protein restored Rac1 activation. Msp from the strain OTK, with numerous amino acid substitutions throughout the polypeptide, including the C-terminal region compared to strain 35405, showed increased ability to impair neutrophil chemotaxis. Collectively, these results indicate the C-terminal region of Msp is the most potent region to modulate neutrophil chemotactic signaling and that specific sequences and structure is likely required. Knowledge of how spirochetes dampen neutrophil response is limited and Msp may represent a novel therapeutic target for periodontal disease. This article is protected by copyright. All rights reserved.

  5. New proangiogenic activity on vascular endothelial cells for C-terminal mechano growth factor.

    PubMed

    Deng, Moyuan; Wang, Yuanliang; Zhang, Bingbing; Liu, Peng; Xiao, Hualiang; Zhao, Jianhua

    2012-04-01

    Angiogenesis is crucial in wound healing. The administration of the C-terminal 24-a.a. peptide of mechano growth factor (MGF24E) has been previously demonstrated to induce more blood vessels in regenerating bone around defective areas compared with the control. Accordingly, this study aims to determine whether MGF24E promotes bone defect healing through MGF24E-increased angiogenesis and whether MGF24E has positive effects on angiogenesis in vitro. The roles of MGF24E on angiogenesis and the underlying mechanisms were investigated. The cell proliferation, migration, and tubulogenesis of the human vascular endothelial EA.hy926 cells co-treated with 2% serum and MGF24E were determined to assess angiogenesis in comparison with 100 ng/ml of vascular endothelial growth factor 165 (VEGF(165))-positive control or vehicle control (phosphate-buffered saline). MGF24E treatment (10 ng/ml) significantly promoted the biological processes of angiogenesis on EA.hy926 cells compared with the vehicle control. The suppression of vascular endothelial growth factor and angiopoietin-I expressions by 2% serum starvation was reversed by the addition of 10 ng/ml of MGF24E in 2% serum medium. This result suggests that MGF24E has a protective effect on angiogenesis. Moreover, the inhibition of ERK due to PD98050 pretreatment completely abolished and mostly blocked MGF24E-induced proliferation and migration, respectively, whereas the MGF24-induced tubulogenesis and the angiogenic factor expression were only partially inhibited. These new findings suggest that MGF24E promotes angiogenesis by enhancing the expression of angiogenic cytokines which involves the MAPK/ERK-signaling pathway.

  6. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins.

    PubMed

    Enz, Ralf

    2012-01-01

    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

  7. A C-terminal di-leucine motif controls plasma membrane expression of PMCA4b.

    PubMed

    Antalffy, Géza; Pászty, Katalin; Varga, Karolina; Hegedűs, Luca; Enyedi, Agnes; Padányi, Rita

    2013-12-01

    Recent evidences show that the localization of different plasma membrane Ca(2+) ATPases (PMCAs) is regulated in various complex, cell type-specific ways. Here we show that in low-density epithelial and endothelial cells PMCA4b localized mostly in intracellular compartments and its plasma membrane localization was enhanced upon increasing density of cells. In good correlation with the enhanced plasma membrane localization a significantly more efficient Ca(2+) clearance was observed in confluent versus non-confluent HeLa cell cultures expressing mCherry-PMCA4b. We analyzed the subcellular localization and function of various C-terminally truncated PMCA4b variants and found that a truncated mutant PMCA4b-ct24 was mostly intracellular while another mutant, PMCA4b-ct48, localized more to the plasma membrane, indicating that a protein sequence corresponding to amino acid residues 1158-1181 contained a signal responsible for the intracellular retention of PMCA4b in non-confluent cultures. Alteration of three leucines to alanines at positions 1167-1169 resulted in enhanced cell surface expression and an appropriate Ca(2+) transport activity of both wild type and truncated pumps, suggesting that the di-leucine-like motif (1167)LLL was crucial in targeting PMCA4b. Furthermore, upon loss of cell-cell contact by extracellular Ca(2+) removal, the wild-type pump was translocated to the early endosomal compartment. Targeting PMCA4b to early endosomes was diminished by the L(1167-69)A mutation, and the mutant pump accumulated in long tubular cytosolic structures. In summary, we report a di-leucine-like internalization signal at the C-tail of PMCA4b and suggest an internalization-mediated loss of function of the pump upon low degree of cell-cell contact.

  8. New de-ubiquitinating enzyme, ubiquitin C-terminal hydrolase 8, in chick skeletal muscle.

    PubMed Central

    Baek, S H; Woo, S K; Lee, J I; Yoo, Y J; Cho, C M; Kang, M S; Tanaka, K; Chung, C H

    1997-01-01

    We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). Here we report the purification and characterization of one of the UCHs, called UCH-8, with 125I-labelled ubiquitin-alpha-NH-MHISPPEPESEEEEEHYC as a substrate. The purified UCH-8 behaved as a 240 kDa protein on a Superdex-200 column under non-denaturing conditions but as a 130 kDa polypeptide on analysis by PAGE under denaturing conditions, suggesting that the enzyme consists of two identical subunits. Thus this enzyme seems to be distinct in its dimeric nature from other purified UCHs that consist of a single polypeptide, except that UCH-6 is also a homodimer of 27 kDa subunits. UCH-8 was maximally active between pH 7.5 and 8, but showed little or no activity below pH 7 and above pH 9. Like other UCHs it was sensitive to inhibition by thiol-blocking agents such as N-ethylmaleimide, and by ubiquitin aldehyde. The purified UCH-8 hydrolysed not only ubiquitin-alpha-NH-protein extensions, including ubiquitin-alpha-NH-carboxy extension protein of 80 amino acid residues and ubiquitin-alpha-NH-dihydrofolate reductase, but also branched poly-ubiquitin that are ligated to proteins through epsilon-NH-isopeptide bonds. However, it showed little or no activity against poly-His-tagged di-ubiquitin, suggesting that UCH-8 is not involved in the generation of free ubiquitin from the linear poly-ubiquitin precursors. These results suggest that UCH-8 might have an important role in the production of free ubiquitin and ribosomal proteins from their conjugates as well as in the recycling of ubiquitin molecules after the degradation of poly-ubiquitinated protein conjugates by the 26 S proteasome. PMID:9230110

  9. Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis.

    PubMed

    Singh, Amit Kumar; Rastogi, Shivangi; Shukla, Harish; Asalam, Mohd; Rath, Srikanta Kumar; Akhtar, Md Sohail

    2017-03-31

    In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1, and Ctk1) and phosphatases (Ssu72, Rtr1, and Fcp1), which act through the post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD comprises the repeated Tyr-Ser-Pro-Thr-Ser-Pro-Ser motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase(s) and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser-2 and Ser-5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation(s). The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript levels peak during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser-5 at the promoter and Ser-2 toward the 3' end of the gene. The observation that Cdc15 could phosphorylate Ser-5, as well as Ser-2, during transcription in mitosis is in contrast to the phosphorylation marks put by the kinases in interphase (G1, S, and G2), where Cdck7/Kin28 phosphorylates Ser-5 at promoter and Bur1/Ctk1 phosphorylates Ser-2 at the 3' end of the genes.

  10. Cloning of a C-terminally truncated NK-1 receptor from guinea-pig nervous system.

    PubMed

    Baker, Sarah J; Morris, Judy L; Gibbins, Ian L

    2003-03-17

    In order to examine the possibility that some actions of substance P may be mediated by a variant of the neurokinin-1 (NK-1) receptor, we isolated and sequenced the cDNA encoding a truncated NK-1 receptor from guinea-pig celiac ganglion and brain mRNA by two-step RT-PCR based on the 3'RACE method. The truncated NK-1 receptor sequence corresponded to a splice variant missing the final exon 5, and encoded a 311-amino acid protein that was truncated just after transmembrane domain 7, in an identical position to a truncated variant of the human NK-1 receptor. Thus, the truncated NK-1 receptor lacked the intracellular C-terminus sequence required for the phosphorylation and internalisation of the full-length NK-1 receptor. Using a sensitive one-step semi-quantitative RT-PCR assay, we detected mRNA for both the full length and truncated NK-1 receptors throughout the brain, spinal cord, sensory and autonomic ganglia, and viscera. Truncated NK-1 receptor mRNA was present in lower quantities than mRNA for the full-length NK-1R in all tissues. Highest levels of mRNA for the truncated NK-1 receptor were detected in coeliac ganglion, spinal cord, basal ganglia and hypothalamus. An antiserum to the N-terminus of the NK-1 receptor labelled dendrites of coeliac ganglion neurons that were not labelled with antisera to the C-terminus of the full length NK-1 receptor. These results show that a C-terminally truncated variant of the NK-1 receptor is likely to be widespread in central and peripheral nervous tissue. We predict that this receptor will mediate actions of substance P on neurons where immunohistochemical evidence for a full-length NK-1 receptor is lacking.

  11. Interaction of CheY with the C-terminal peptide of CheZ

    SciTech Connect

    Guhaniyogi,J.; Wu, T.; Patel, S.; Stock, A.

    2008-01-01

    Chemotaxis, a means for motile bacteria to sense the environment and achieve directed swimming, is controlled by flagellar rotation. The primary output of the chemotaxis machinery is the phosphorylated form of the response regulator CheY (P{approx}CheY). The steady-state level of P{approx}CheY dictates the direction of rotation of the flagellar motor. The chemotaxis signal in the form of P{approx}CheY is terminated by the phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two distinct protein-protein interfaces: one involving the strongly conserved C-terminal helix of CheZ (CheZC) tethering the two proteins together and the other constituting an active site for catalytic dephosphorylation. In a previous work, we presented high-resolution crystal structures of CheY in complex with the CheZC peptide that revealed alternate binding modes subject to the conformational state of CheY. In this study, we report biochemical and structural data that support the alternate-binding-mode hypothesis and identify key recognition elements in the CheY-CheZC interaction. In addition, we present kinetic studies of the CheZC-associated effect on CheY phosphorylation with its physiologically relevant phosphodonor, the histidine kinase CheA. Our results indicate mechanistic differences in phosphotransfer from the kinase CheA versus that from small-molecule phosphodonors, explaining a modest twofold increase of CheY phosphorylation with the former, observed in this study, relative to a 10-fold increase previously documented with the latter.

  12. Reduced ubiquitin C-terminal hydrolase-1 expression levels in dementia with Lewy bodies.

    PubMed

    Barrachina, Marta; Castaño, Esther; Dalfó, Esther; Maes, Tamara; Buesa, Carlos; Ferrer, Isidro

    2006-05-01

    Parkinson disease (PD) and dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal alpha-synuclein and ubiquitin in protein aggregates conforming Lewy bodies and Lewy neurites. Ubiquitin C-terminal hydrolase-1 (UCHL-1) disassembles polyubiquitin chains to increase the availability of free monomeric ubiquitin to the ubiquitin proteasome system (UPS) thus favoring protein degradation. Since mutations in the UCHL-1 gene, reducing UPS activity by 50%, have been reported in autosomal dominant PD, and UCHL-1 inhibition results in the formation of alpha-synuclein aggregates in mesencephalic cultured neurons, the present study was initiated to test UCHL-1 mRNA and protein levels in post-mortem frontal cortex (area 8) of PD and DLB cases, compared with age-matched controls. TaqMan PCR assays, and Western blots demonstrated down-regulation of UCHL-1 mRNA and UCHL-1 protein in the cerebral cortex in DLB (either in pure forms, not associated with Alzheimer disease: AD, and in common forms, with accompanying AD changes), but not in PD, when compared with age-matched controls. Interestingly, UCHL-1 mRNA and protein expressions were reduced in the medulla oblongata in the same PD cases. Moreover, UCHL-1 protein was decreased in the substantia nigra in cases with Lewy body pathology. UCHL-1 down-regulation was not associated with reduced protein levels of several proteasomal subunits, including 20SX, 20SY, 19S and 11Salpha. Yet UCHL-3 expression was reduced in the cerebral cortex of PD and DLB patients. Together, these observations show reduced UCHL-1 expression as a contributory factor in the abnormal protein aggregation in DLB, and points UCHL-1 as a putative therapeutic target in the treatment of DLB.

  13. The potential role of ubiquitin c-terminal hydrolases in oncogenesis.

    PubMed

    Fang, Ying; Fu, Da; Shen, Xi-Zhong

    2010-08-01

    Deubiquitinating enzymes (DUBs), capable of removing ubiquitin (Ub) from protein substrates, are involved in numerous biological processes. The ubiquitin C-terminal hydrolases (UCHs) subfamily of DUBs consists of four members: UCH-L1, UCH-L3, UCH37 and BRCA1-associated protein-1 (BAP1). UCH-L1 possesses deubiquitinating activity and dimerization-dependent ubiquitin ligase activity, and functions as a mono-ubiquitin stabilizer; UCH-L3 does both deubiquitinating and deneddylating activity, except dimerization or ligase activity, and unlike UCH-L1, can interact with Lys48-linked Ub dimers to protect it from degradation and in the meanwhile to inhibit its hydrolase activity; UCH37 is responsible for the deubiquitinating activity in the 19S proteasome regulatory complex, and as indicated by the recent study, UCH37 is also associated with the human Ino80 chromatin-remodeling complex (hINO80) in the nucleus and can be activated via transient association of 19S regulatory particle- or proteasome-bound hRpn13 with hINO80; BAP1, binding to the wild-type BRCA1 RING finger domain, is regarded as a tumor suppressor, but for such suppressing activity, as demonstrated otherwise, both deubiquitinating activity and nucleus localization are required. There is growing evidence that UCH enzymes and human malignancies are closely correlated. Previous studies have shown that UCH enzymes play a crucial role in some signalings and cell-cycle regulation. In this review, we provided an insight into the relation between UCH enzymes and oncogenesis.

  14. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach

    PubMed Central

    Tirupula, Kalyan C.; Zhang, Dongmei; Osbourne, Appledene; Chatterjee, Arunachal; Desnoyer, Russ; Willard, Belinda; Karnik, Sadashiva S.

    2015-01-01

    Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A ‘cardiac-specific finger print’ of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a ‘MAS-signalosome’ model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of ‘signalosome’ components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor. PMID

  15. A helix-turn motif in the C-terminal domain of histone H1.

    PubMed Central

    Vila, R.; Ponte, I.; Jiménez, M. A.; Rico, M.; Suau, P.

    2000-01-01

    The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1(o) (residues 99-121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i,i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C(alpha) protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the alpha-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) beta-turn conformation, a sigma-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs. PMID:10794405

  16. Dandelion PPO-1/PPO-2 domain-swaps: the C-terminal domain modulates the pH optimum and the linker affects SDS-mediated activation and stability.

    PubMed

    Leufken, Christine M; Moerschbacher, Bruno M; Dirks-Hofmeister, Mareike E

    2015-02-01

    Plant polyphenol oxidases (PPOs) have a conserved three-domain structure: (i) the N-terminal domain (containing the active site) is connected via (ii) a linker to (iii) the C-terminal domain. The latter covers the active site, thereby maintaining the enzyme in a latent state. Activation can be achieved with SDS but little is known about the mechanism. We prepared domain-swap variants of dandelion PPO-1 and PPO-2 to test the specific functions of individual domains and their impact on enzyme characteristics. Our experiments revealed that the C-terminal domain modulates the pH optimum curve and has a strong influence on the optimal pH value. The linker determines the SDS concentration required for full activation. It also influences the SDS concentration required for half maximal activation (kSDS) and the stability of the enzyme during prolonged incubation in buffers containing SDS, but the N-terminal domain has the strongest effect on these parameters. The N-terminal domain also determines the IC50 of SDS and the stability in buffers containing or lacking SDS. We propose that the linker and C-terminal domain fine-tune the activation of plant PPOs. The C-terminal domain adjusts the pH optimum and the linker probably contains an SDS-binding/interaction site that influences inactivation and determines the SDS concentration required for activation. For the first time, we have determined the influence of the three PPO domains on enzyme activation and stability providing insight into the regulation and activation mechanisms of type-3 copper proteins in general.

  17. Citrate-capped gold nanoparticles for the label-free detection of ubiquitin C-terminal hydrolase-1.

    PubMed

    Agarwal, Srishti; Mishra, Priyanka; Shivange, Gururaj; Kodipelli, Naveena; Moros, María; de la Fuente, Jesús M; Anindya, Roy

    2015-02-21

    Ubiquitin C-terminal hydrolase-1 (UCH-L1) is a specific neuronal endoprotease that cleaves the specific peptide bond between ubiquitin molecules. UCH-L1 is released in serum and cerebrospinal fluid after severe brain injury and is considered to be an important biomarker of brain injury. A common polymorphism of UCH-L1 (S18Y) is also linked to a reduced risk of Parkinson's disease. In addition to its function in neuronal tissues, UCH-L1 may also play a part in the progression of certain non-neuronal cancers. UCH-L1 is highly expressed in primary lung tumors and colo-rectal cancers, suggesting a role in tumorigenesis. We report here the development of a sensitive and accurate UCH-L1 assay based on the surface plasmon resonance (SPR) absorbance of gold nanoparticles. We created a unique UCH-L1 substrate containing a ubiquitin molecule with two terminal thiol groups. This UCH-L1 substrate interacted with gold nanoparticles via the terminal thiol groups and induced clustering of the nanoparticles, which was detected by SPR absorbance at 650 nm. UCH-L1 proteolytically cleaved the substrate and the clustered gold nanoparticles were dispersed and could be detected by a shift in the SPR absorbance to 530 nm. This change in absorbance was proportional to the concentration of UCH-L1 and can be used for the quantification of functional UCH-L1. The currently available fluorescence-based UCH-L1 assay is affected by a high background signal and a poor detection limit, especially in the presence of serum. The assay reported here can detect concentrations of UCH-L1 as low as 20 ng ml(-1) (0.8 nM) and the presence of serum had no effect on the detection limit. This assay could be adapted for the rapid determination of the severity of brain injury and could also be applied to high-throughput screening of inhibitors of UCH-L1 enzymatic activity in Parkinson's disease and cancer.

  18. The structure of the C-terminal domain of the pro-apoptotic protein Bak and its interaction with model membranes.

    PubMed Central

    Martínez-Senac, María del Mar; Corbalán-García, Senena; Gómez-Fernández, Juan C

    2002-01-01

    Bak is a pro-apoptotic protein widely distributed in different cell types that is associated with the mitochondrial outer membrane, apparently through a C-terminal hydrophobic domain. We used infrared spectroscopy to study the secondary structure of a synthetic peptide ((+)(3)HN-(188)ILNVLVVLGVVLLGQFVVRRFFKS(211)-COO(-)) with the same sequence as the C-terminal domain of Bak. The spectrum of this peptide in D(2)O buffer shows an amide I' band with a maximum at 1636 cm(-1), which clearly indicates the predominance of an extended beta-structure in aqueous solvent. However, the peptide incorporated in multilamellar dimyristoylphosphatidylcholine (DMPC) membranes shows a different amide I' band spectrum, with a maximum at 1658 cm(-1), indicating a predominantly alpha-helical structure induced by its interaction with the membrane. It was observed that through differential scanning calorimetry the transition of the phospholipid model membrane was broadened in the presence of the peptide. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) in fluid DMPC vesicles showed that increasing concentrations of the peptide produced increased polarization values, which is compatible with the peptide being inserted into the membrane. High concentrations of the peptide considerably broaden the phase transition of DMPC multilamellar vesicles, and DPH polarization increased, especially at temperatures above the T(c) transition temperature of the pure phospholipid. The addition of peptide destabilized unilamellar vesicles and released encapsulated carboxyfluorescein. These results indicate that this domain is able to insert itself into membranes, where it adopts an alpha-helical structure and considerably perturbs the physical properties of the membrane. PMID:11751312

  19. The pH-sensitive structure of the C-terminal domain of voltage-gated proton channel and the thermodynamic characteristics of Zn²⁺ binding to this domain.

    PubMed

    Zhao, Qing; Li, Chuanyong; Li, Shu Jie

    2015-01-02

    The voltage-gated proton channel Hv1 is strongly sensitive to Zn(2+). The H(+) conduction is decreased at a high concentration of Zn(2+) and Hv1 channel closing is slowed by the internal application of Zn(2+). Although the recent studies demonstrated that Zn(2+) interacts with the intracellular C-terminal domain, the binding sites and details of the interaction remain unknown. Here, we studied the pH-dependent structural stability of the intracellular C-terminal domain of human Hv1 and showed that Zn(2+) binds to His(244) and His(266) residues. The thermodynamics signature of Zn(2+) binding to the two sites was investigated by isothermal titration calorimetry. The binding of Zn(2+) to His(244) (mutant H266A) and His(266) (mutant H244A) were an endothermic heat reaction and an exothermic heat reaction, respectively.

  20. N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

    PubMed Central

    Lei, Yan-Chang; Tian, Yong-Jun; Ding, Hong-Hui; Wang, Bao-Ju; Yang, Yan; Hao, You-Hua; Zhao, Xi-Ping; Lu, Meng-Ji; Gong, Fei-Li; Yang, Dong-Liang

    2006-01-01

    AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA. The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the

  1. Differential Contributions of Tacaribe Arenavirus Nucleoprotein N-Terminal and C-Terminal Residues to Nucleocapsid Functional Activity

    PubMed Central

    D'Antuono, Alejandra; Loureiro, Maria Eugenia; Foscaldi, Sabrina; Marino-Buslje, Cristina

    2014-01-01

    ABSTRACT The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCE The mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be

  2. Generating recombinant C-terminal prion protein fragments of exact native sequence.

    PubMed

    Johanssen, V A; Barnham, K J; Masters, C L; Hill, A F; Collins, S J

    2012-02-01

    Transmissibility and distinctive neuropathology are hallmark features of prion diseases differentiating them from other neurodegenerative disorders, with pathogenesis and transmission appearing closely linked to misfolded conformers (PrP(Sc)) of the ubiquitously expressed cellular form of the prion protein (PrP(C)). Given the apparent pathogenic primacy of misfolded PrP, the utilisation of peptides based on the prion protein has formed an integral approach for providing insights into misfolding pathways and pathogenic mechanisms. In parallel with studies employing prion peptides, similar approaches in other neurodegenerative disorders such as Alzheimer Disease, have demonstrated that differential processing of parent proteins and quite minor variations in the primary sequence of cognate peptides generated from the same constitutive processing (such as Aβ1-40 versus Aβ1-42 produced from γ-secretase activity) can be associated with very different pathogenic consequences. PrP(C) also undergoes constitutive α- or β-cleavage yielding C1 (residues 112-231 human sequence) or C2 (residues 90-231), respectively, with the full cell biological significance of such processing unresolved; however, it is noteworthy that in prion diseases, such as Creutzfeldt-Jakob disease (CJD) and murine models, the moderately extended C2 fragment predominates in the brain suggesting that the two cleavage events and the consequent C-terminal fragments may differ in their pathogenic significance. Accordingly, studies characterising biologically relevant peptides like C1 and C2, would be most valid if undertaken using peptides completely free of any inherent non-native sequence that arises as a by-product of commonly employed recombinant production techniques. To achieve this aim and thereby facilitate more representative biophysical and neurotoxicity studies, we adapted the combination of high fidelity Taq TA cloning with a SUMO-Hexa-His tag-type approach, incorporating the SUMO protease

  3. Crystallization and preliminary X-ray analysis of the C-terminal fragment of Ski7 from Saccharomyces cerevisiae

    PubMed Central

    Lee, Ji-Young; Park, Si Hoon; Jeong, Byung-Cheon; Song, Hyun Kyu

    2014-01-01

    Ski7 (superkiller protein 7) plays a critical role in the mRNA surveillance pathway. The C-terminal fragment of Ski7 (residues 520–747) from Saccharo­myces cerevisiae was heterologously expressed in Escherichia coli and purified to homogeneity. It was successfully crystallized and preliminary X-ray data were collected to 2.0 Å resolution using synchrotron radiation. The crystal belonged to a trigonal space group, either P3121 or P3221, with unit-cell parameters a = b = 73.5, c = 83.6 Å. The asymmetric unit contains one molecule of the C-terminal fragment of Ski7 with a corresponding crystal volume per protein mass (V M) of 2.61 Å3 Da−1 and a solvent content of 52.8% by volume. The merging R factor is 6.6%. Structure determination by MAD phasing is under way. PMID:25195903

  4. Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

    SciTech Connect

    Kamada, Katsuhiko; De Angelis, Jacqueline; Roeder, Robert G.; Burley, Stephen K.

    2012-12-13

    The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-{angstrom} resolution. The {alpha}/{beta} structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3{gamma} (HNF-3{gamma}), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3{gamma} and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the {beta} subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

  5. Solution conformation of the C-terminal domain of skeletal troponin C. Cation, trifluoperazine and troponin I binding effects.

    PubMed

    Drabikowski, W; Dalgarno, D C; Levine, B A; Gergely, J; Grabarek, Z; Leavis, P C

    1985-08-15

    Proton magnetic resonance spectroscopy has been used to study the cation (Mg2+, Ca2+)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the cation binding reaction. Complex formation with troponin I and the drug trifluoperazine is observed to adjust the solution conformation of the C-terminal domain of troponin C. The interactive conformational response to cation coordination and the binding of the drug and troponin I are discussed.

  6. Crystal Structure in the Vivo-Assembled Bacillus subtilis Spx/RNA Polymerase alpha Subunit C-Terminal Domain Complex

    SciTech Connect

    Lamour, V.; Westblade, L; Campbell, E; Darst, S

    2009-01-01

    The Bacillus subtilis Spx protein is a global transcription factor that interacts with the C-terminal domain of the RNA polymerase {alpha} subunit ({alpha}CTD) and regulates transcription of genes involved in thiol-oxidative stress, sporulation, competence, and organosulfur metabolism. Here we determined the X-ray crystal structure of the Spx/{alpha}CTD complex from an entirely new crystal form than previously reported [Newberry, K.J., Nakano, S., Zuber, P., Brennan, R.G., 2005. Crystal structure of the Bacillus subtilis anti-alpha, global transcriptional regulator, Spx, in complex with the alpha C-terminal domain of RNA polymerase. Proc. Natl. Acad. Sci. USA 102, 15839-15844]. Comparison of the previously reported sulfate-bound complex and our sulfate-free complex reveals subtle conformational changes that may be important for the role of Spx in regulating organosulfur metabolism.

  7. Improved synthesis of C-terminal peptide thioesters on "safety-catch" resins using LiBr/THF.

    PubMed

    Quaderer, R; Hilvert, D

    2001-10-04

    [reaction: see text] The alkanesulfonamide "safety-catch" resin has proven useful for Fmoc-based synthesis of C-terminal peptide thioesters. We now report that the yield of isolated thioester can increase significantly when the cleavage reaction is carried out in 2 M LiBr/THF rather than DMF or THF. The largest effects are seen with problematic peptides that aggregate or form secondary structures on the resin.

  8. Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+-binding properties.

    PubMed

    Yang, Yifei; Keeler, Camille; Kuo, Ivana Y; Lolis, Elias J; Ehrlich, Barbara E; Hodsdon, Michael E

    2015-04-17

    Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca(2+)-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca(2+)-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca(2+) bound, although TRP channels are believed to be tetramers. We found that there is only one Ca(2+)-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca(2+) binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca(2+) binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca(2+)-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca(2+)-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca(2+)-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca(2+)-binding proteins.

  9. XRCC1 interaction with the REV1 C-terminal domain suggests a role in post replication repair.

    PubMed

    Gabel, Scott A; DeRose, Eugene F; London, Robert E

    2013-12-01

    The function of X-ray cross complementing group 1 protein (XRCC1), a scaffold that binds to DNA repair enzymes involved in single-strand break and base excision repair, requires that it be recruited to sites of damaged DNA. However, structural insights into this recruitment are currently limited. Sequence analysis of the first unstructured linker domain of XRCC1 identifies a segment consistent with a possible REV1 interacting region (X1RIR) motif. The X1RIR motif is present in translesion polymerases that can be recruited to the pol /REV1 DNA repair complex via a specific interaction with the REV1 C-terminal domain. NMR and fluorescence titration studies were performed on XRCC1-derived peptides containing this putative RIR motif in order to evaluate the binding affinity for the REV1 C-terminal domain. These studies demonstrate an interaction of the XRCC1-derived peptide with the human REV1 C-terminal domain characterized by dissociation constants in the low micromolar range. Ligand competition studies comparing the XRCC1 RIR peptide with previously studied RIR peptides were found to be inconsistent with the NMR based Kd values. These discrepancies were resolved using a fluorescence assay for which the RIR–REV1 system is particularly well suited. The structure of a REV1-XRCC1 peptide complex was determined by using NOE restraints to dock the unlabeled XRCC1 peptide with a labeled REV1 C-terminal domain. The structure is generally homologous with previously determined complexes with the pol κ and pol η RIR peptides, although the helical segment in XRCC1 is shorter than was observed in these cases. These studies suggest the possible involvement of XRCC1 and its associated repair factors in post replication repair.

  10. Fertilization in C. elegans requires an intact C-terminal RING finger in sperm protein SPE-42

    PubMed Central

    2011-01-01

    Background The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure. Results We made an in silico structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants. Conclusions The results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain. PMID:21345212

  11. The C-terminal dimerization motif of cyclase-associated protein is essential for actin monomer regulation.

    PubMed

    Iwase, Shohei; Ono, Shoichiro

    2016-12-01

    Cyclase-associated protein (CAP) is a conserved actin-regulatory protein that functions together with actin depolymerizing factor (ADF)/cofilin to enhance actin filament dynamics. CAP has multiple functional domains, and the function to regulate actin monomers is carried out by its C-terminal half containing a Wiskott-Aldrich Syndrome protein homology 2 (WH2) domain, a CAP and X-linked retinitis pigmentosa 2 (CARP) domain, and a dimerization motif. WH2 and CARP are implicated in binding to actin monomers and important for enhancing filament turnover. However, the role of the dimerization motif is unknown. Here, we investigated the function of the dimerization motif of CAS-2, a CAP isoform in the nematode Caenorhabditis elegans, in actin monomer regulation. CAS-2 promotes ATP-dependent recycling of ADF/cofilin-bound actin monomers for polymerization by enhancing exchange of actin-bound nucleotides. The C-terminal half of CAS-2 (CAS-2C) has nearly as strong activity as full-length CAS-2. Maltose-binding protein (MBP)-tagged CAS-2C is a dimer. However, MBP-CAS-2C with a truncation of either one or two C-terminal β-strands is monomeric. Truncations of the dimerization motif in MBP-CAS-2C nearly completely abolish its activity to sequester actin monomers from polymerization and enhance nucleotide exchange on actin monomers. As a result, these CAS-2C variants, also in the context of full-length CAS-2, fail to compete with ADF/cofilin to release actin monomers for polymerization. CAS-2C variants lacking the dimerization motif exhibit enhanced binding to actin filaments, which is mediated by WH2. Taken together, these results suggest that the evolutionarily conserved dimerization motif of CAP is essential for its C-terminal region to exert the actin monomer-specific regulatory function.

  12. Structural Basis for Toughness and Flexibility in the C-terminal Passenger Domain of an Acinetobacter Trimeric Autotransporter Adhesin*

    PubMed Central

    Koiwai, Kotaro; Hartmann, Marcus D.; Linke, Dirk; Lupas, Andrei N.; Hori, Katsutoshi

    2016-01-01

    Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three β-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner β-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions. PMID:26698633

  13. Collision-induced dissociation fragmentation inside disulfide C-terminal loops of natural non-tryptic peptides.

    PubMed

    Samgina, Tatiana Y; Vorontsov, Egor A; Gorshkov, Vladimir A; Artemenko, Konstantin A; Zubarev, Roman A; Ytterberg, Jimmy A; Lebedev, Albert T

    2013-07-01

    Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S-S bonds is often used in "bottom up" sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the "hidden" C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S-S bond rupture does not occur in this case.

  14. Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target.

    PubMed

    Inui, Ken; Zhao, Zongpei; Yuan, Juan; Jayaprakash, Sakthidasan; Le, Le T M; Drakulic, Srdja; Sander, Bjoern; Golas, Monika M

    2017-02-20

    In human cells, thousands of predominantly neuronal genes are regulated by the repressor element 1 (RE1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). REST/NRSF represses transcription of these genes in stem cells and non-neuronal cells by tethering corepressor complexes. Aberrant REST/NRSF expression and intracellular localization are associated with cancer and neurodegeneration in humans. To date, detailed molecular analyses of REST/NRSF and its C-terminal repressor complex have been hampered largely by the lack of sufficient amounts of purified REST/NRSF and its complexes. Therefore, the aim of this study was to express and purify human REST/NRSF and its C-terminal interactors in a baculovirus multiprotein expression system as individual proteins and coexpressed complexes. All proteins were enriched in the nucleus, and REST/NRSF was isolated as a slower migrating form, characteristic of nuclear REST/NRSF in mammalian cells. Both REST/NRSF alone and its C-terminal repressor complex were functionally active in histone deacetylation and histone demethylation and bound to RE1/neuron-restrictive silencer element (NRSE) sites. Additionally, the mechanisms of inhibition of the small-molecule drugs 4SC-202 and SP2509 were analyzed. These drugs interfered with the viability of medulloblastoma cells, where REST/NRSF has been implicated in cancer pathogenesis. Thus, a resource for molecular REST/NRSF studies and drug development has been established.

  15. NMR assignment and secondary structure of coiled coil domain of C-terminal myosin binding subunit of myosin phosphatase.

    PubMed

    Sharma, Alok K; Rigby, Alan C

    2014-07-01

    Protein-protein interactions between the C-terminal domain of Myosin Binding Subunit (MBS) of MLC Phosphatase (MBS(CT180); C-terminal 180 aa) and the N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKGIα, PKG-Iα(1-159) play an important role in the process of Smooth Muscle Cell relaxation. The paucity of three-dimensional structural information for MBS(CT180) prevents an atomic level understanding of the MBS-PKG contractile complex. MBS(CT180) is comprised of three structurally different sub-domains including a non-canonical CC, a CC, and a LZ. Recently we reported polypeptide purification and biophysical characterization of the CC domain and the LZ domain of MBS(CT180) (Sharma et al, Prot Expr Purif 2012). Here we report (1)H, (13)C, (15)N chemical shift assignments of homodimeric CC MBS domain encompassing amino acid residues Asp931-Leu980 using 2D and 3D heteronuclear NMR spectroscopy. Secondary structure analyses deduced from these NMR chemical shift data have identified a contiguous stretch of 36 residues from Phe932 to Ala967 that is involved in the formation of coiled coil α-helical region within CC MBS domain. The N-terminal residue Asp931 and the C-terminally positioned residues Thr968-Ala975, Arg977, and Ser978 adopt nonhelical loop conformations.

  16. Functions of the C-terminal domains of apoptosis-related proteins of the Bcl-2 family.

    PubMed

    Gómez-Fernández, Juan C

    2014-10-01

    Bcl-2 family proteins are involved in cell homeostasis, where they regulate cell death. Some of these proteins are pro-apoptotic and others pro-survival. Moreover, many of them share a similar domain composition with several of the so-called BH domains, although some only have a BH3 domain. A C-terminal domain is present in all the multi-BH domain proteins and in some of the BH3-only ones. This C-terminal domain is hydrophobic or amphipathic, for which reason it was thought when they were discovered that they were membrane anchors. Although this is indeed one of their functions, it has since been observed that they may also serve as regulators of the function of some members of this family, such as Bax. They may also serve to recognize the target membrane of some of these proteins, which only after an apoptotic signal, are incorporated into a membrane. It has been shown that peptides that imitate the sequence of C-terminal domains can form pores and may serve as a model to design cytotoxic molecules.

  17. Influence of C-terminal tail deletion on structure and stability of hyperthermophile Sulfolobus tokodaii RNase HI.

    PubMed

    Chen, Lin; Zhang, Ji-Long; Zheng, Qing-Chuan; Chu, Wen-Ting; Xue, Qiao; Zhang, Hong-Xing; Sun, Chia-Chung

    2013-06-01

    The C-terminus tail (G144-T149) of the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) plays an important role in this protein's hyperstabilization and may therefore be a good protein stability tag. Detailed understanding of the structural and dynamic effects of C-terminus tail deletion is required for gaining insights into the thermal stability mechanism of Sto-RNase HI. Focused on Sulfolobus tokodaii RNase HI (Sto-RNase HI) and its derivative lacking the C-terminal tail (ΔC6 Sto-RNase HI) (PDB codes: 2EHG and 3ALY), we applied molecular dynamics (MD) simulations at four different temperatures (300, 375, 475, and 500 K) to examine the effect of the C-terminal tail on the hyperstabilization of Sto-RNase HI and to investigate the unfolding process of Sto-RNase HI and ΔC6 Sto-RNase HI. The simulations suggest that the C-terminal tail has significant impact in hyperstabilization of Sto-RNase HI and the unfolding of these two proteins evolves along dissimilar pathways. Essential dynamics analysis indicates that the essential subspaces of the two proteins at different temperatures are non-overlapping within the trajectories and they exhibit different directions of motion. Our work can give important information to understand the three-state folding mechanism of Sto-RNase HI and to offer alternative strategies to improve the protein stability.

  18. Synthesis of histone proteins by CPE ligation using a recombinant peptide as the C-terminal building block.

    PubMed

    Kawakami, Toru; Yoshikawa, Ryo; Fujiyoshi, Yuki; Mishima, Yuichi; Hojo, Hironobu; Tajima, Shoji; Suetake, Isao

    2015-11-01

    The post-translational modification of histones plays an important role in gene expression. We report herein on a method for synthesizing such modified histones by ligating chemically prepared N-terminal peptides and C-terminal recombinant peptide building blocks. Based on their chemical synthesis, core histones can be categorized as two types; histones H2A, H2B and H4 which contain no Cys residues, and histone H3 which contains a Cys residue(s) in the C-terminal region. A combination of native chemical ligation and desulphurization can be simply used to prepare histones without Cys residues. For the synthesis of histone H3, the endogenous Cys residue(s) must be selectively protected, while keeping the N-terminal Cys residue of the C-terminal building block that is introduced for purposes of chemical ligation unprotected. To this end, a phenacyl group was successfully utilized to protect endogenous Cys residue(s), and the recombinant peptide was ligated with a peptide containing a Cys-Pro ester (CPE) sequence as a thioester precursor. Using this approach it was possible to prepare all of the core histones H2A, H2B, H3 and H4 with any modifications. The resulting proteins could then be used to prepare a core histone library of proteins that have been post-translationally modified.

  19. The C-terminal half of UvrC protein is sufficient to reconstitute (A)BC excinuclease

    SciTech Connect

    Lin, J.J.; Sancar, A. )

    1991-08-01

    The UvrC protein is one of three subunits of the Escherichia coli repair enzyme (A)BC excinuclease. This subunit is thought to have at least one of the active sites for nucleophilic attack on the phosphodiester bonds of damaged DNA. To localize the active site, mutant UvrC proteins were constructed by linker-scanning and deletion mutagenesis. In vivo studies revealed that the C-terminal 314 amino acids of the 610-amino acid UvrC protein were sufficient to confer UV resistance to cells lacking the uvrC gene. The portion of the uvrC gene encoding the C-terminal half of the protein was fused to the 3{prime} end of the E. coli malE gene (which encodes maltose binding protein), and the fusion protein MBP-C314C was purified and characterized. The fusion protein, in combination with UvrA and UvrB subunits, reconstituted the excinuclease activity that incised the eighth phosphodiester bond 5{prime} and the fourth phosphodiester bond 3{prime} to a psoralen-thymine adduct. These results suggest that the C-terminal 314 amino acids of UvrC constitute a functional domain capable of interacting with the UvrB-damaged DNA complex and of inducing the two phosphodiester bond incisions characteristic of (A)BC excinuclease.

  20. Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation.

    PubMed

    Bewley, Kathryn D; Bennallack, Philip R; Burlingame, Mark A; Robison, Richard A; Griffitts, Joel S; Miller, Susan M

    2016-11-01

    Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts.

  1. Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

    PubMed Central

    Bewley, Kathryn D.; Bennallack, Philip R.; Burlingame, Mark A.; Robison, Richard A.; Griffitts, Joel S.

    2016-01-01

    Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts. PMID:27791142

  2. The AtMYB12 activation domain maps to a short C-terminal region of the transcription factor.

    PubMed

    Stracke, Ralf; Turgut-Kara, Neslihan; Weisshaar, Bernd

    2017-03-11

    The Arabidopsis thaliana R2R3-MYB transcription factor MYB12 is a light-inducible, flavonol-specific activator of flavonoid biosynthesis. The transactivation activity of the AtMYB12 protein was analyzed using a C-terminal deletion series in a transient A. thaliana protoplast assay with the goal of mapping the activation domain (AD). Although the deletion of the last 46 C-terminal amino acids did not affect the activation capacity, the deletion of the last 98 amino acids almost totally abolished transactivation of two different target promoters. A domain swap experiment using the yeast GAL4 DNA-binding domain revealed that the region from positions 282 to 328 of AtMYB12 was sufficient for transactivation. In contrast to the R2R3-MYB ADs known thus far, that of AtMYB12 is not located at the rearmost C-terminal end of the protein. The AtMYB12 AD is conserved in other experimentally proven R2R3-MYB flavonol regulators from different species.

  3. Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

    PubMed Central

    Wiedemann, Christoph; Szambowska, Anna; Häfner, Sabine; Ohlenschläger, Oliver; Gührs, Karl-Heinz; Görlach, Matthias

    2015-01-01

    The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain. PMID:25712103

  4. Cholesterol dependent conformational exchange of the C-terminal domain of the influenza A M2 protein

    PubMed Central

    Kim, Sangwoo S.; Upshur, Mary Alice; Saotome, Kei; Sahu, Indra D.; McCarrick, Robert M.; Feix, Jimmy B.; Lorigan, Gary A.; Howard, Kathleen P.

    2016-01-01

    The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding. PMID:26569023

  5. Bacillus subtilis GlnR contains an autoinhibitory C-terminal domain required for the interaction with glutamine synthetase.

    PubMed

    Wray, Lewis V; Fisher, Susan H

    2008-04-01

    The Bacillus subtilis GlnR transcription factor regulates gene expression in response to changes in nitrogen availability. Glutamine synthetase transmits the nitrogen regulatory signal to GlnR. The DNA-binding activity of GlnR is activated by a transient protein-protein interaction with feedback-inhibited glutamine synthetase that stabilizes GlnR-DNA complexes. This signal transduction mechanism was analysed by creating mutant GlnR proteins with partial or complete truncations of their C-terminal domains. The truncated GlnR proteins were found to constitutively repress gene expression in vivo. This constitutive repression did not require glutamine synthetase. Purified mutant GlnR proteins bound DNA in vitro more tightly than wild-type GlnR protein and this binding was not activated by feedback-inhibited glutamine synthetase. While full-length GlnR is monomeric, the truncated GlnR proteins contained significant levels of dimers. These results indicate that the C-terminal region of GlnR acts as an autoinhibitory domain that prevents GlnR dimerization and thus impedes DNA binding. The GlnR C-terminal domain is also required for the interaction between GlnR and feedback-inhibited glutamine synthetase. Compared with the full-length GlnR protein, the truncated GlnR proteins were defective in their interaction with feedback-inhibited glutamine synthetase in cross-linking experiments.

  6. Kar1 binding to Sfi1 C-terminal regions anchors the SPB bridge to the nuclear envelope

    PubMed Central

    Seybold, Christian; Elserafy, Menattallah; Rüthnick, Diana; Ozboyaci, Musa; Neuner, Annett; Flottmann, Benjamin; Heilemann, Mike; Wade, Rebecca C.

    2015-01-01

    The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1’s function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31–Cdc31 interactions between Sfi1–Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation. PMID:26076691

  7. The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions.

    PubMed

    Vedovato, Natascia; Gadsby, David C

    2010-07-01

    Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K-adenosine triphosphatase (ATPase) alpha subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane's electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis alpha1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain-sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 microM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump-induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the

  8. Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation

    PubMed Central

    Ludgate, Laurie; Liu, Kuancheng; Luckenbaugh, Laurie; Streck, Nicholas; Eng, Stacey; Voitenleitner, Christian; Delaney, William E.

    2016-01-01

    ABSTRACT Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro. Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors. IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancer worldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have

  9. Urinary alpha and beta C-telopeptides of collagen I: clinical implications in bone remodeling in patients with anorexia nervosa.

    PubMed

    de la Piedra, C; Calero, J A; Traba, M L; Asensio, M D; Argente, J; Muñoz, M T

    1999-01-01

    Fragments derived from degradation of type I collagen C-telopeptide (CTX) can be nonisomerized (alpha) or beta-isomerized (beta) depending on the age of bone; i.e., mainly the alpha form is derived from new bone and the beta form from old bone. We have studied 41 female patients with anorexia nervosa (AN), aged 18.5 +/- 2.2 years (range 16-24 years), and with an evolution time between 1.5 and 11 years, and 31 healthy control females (C), with a mean age of 19 +/- 2.3 years (range 16-24 years). The AN patients showed a significant decrease in bone mass, with a mean Z-score of bone mineral density (BMD) of -3.2 +/- 0.8 (range -0.9 to -5.4). The aim of our study was to determine the levels of urinary alpha- and beta-CTX markers of bone resorption, the alpha/beta ratio (alpha/beta), and the level of bone alkaline phosphatase (bAP), a biochemical marker of bone formation, in order to relate them to the degree of osteopenia and the status of bone remodeling. Statistical analysis was by the Mann-Whitney test. The degree of osteopenia correlated with bAP levels (p = 0.0027) but not with the other parameters. Patients with AN were divided into three groups according to their levels of bAP: high (H), normal (N) or low (L). We found that BMD was significantly lower, and alpha- and beta-CTX were significantly higher, in groups H and N than in group L. Bone AP correlated significantly with alpha-CTX (p = 0.0042) and alpha/beta (0.0095) in the controls, but not with beta-CTX, while in AN patients bAP correlated with beta-CTX (p = 0.0000) and with alpha-CTX (p = 0.022) but not with the alpha/beta ratio. The ratio CTX/bAP (resorption/formation) was similar in AN patients and controls. It is concluded that: (1) patients with AN have a high degree of osteopenia which correlated with bAP levels; (2) urinary CTX fragments found in AN patients seem to come mainly from old bone (beta-CTX), while CTX found in healthy adolescent control females come from new bone (alpha-CTX). For this

  10. Biased Signaling Favoring Gi over β-Arrestin Promoted by an Apelin Fragment Lacking the C-terminal Phenylalanine*

    PubMed Central

    Ceraudo, Emilie; Galanth, Cécile; Carpentier, Eric; Banegas-Font, Inmaculada; Schonegge, Anne-Marie; Alvear-Perez, Rodrigo; Iturrioz, Xavier; Bouvier, Michel; Llorens-Cortes, Catherine

    2014-01-01

    Apelin plays a prominent role in body fluid and cardiovascular homeostasis. We previously showed that the C-terminal Phe of apelin 17 (K17F) is crucial for triggering apelin receptor internalization and decreasing blood pressure (BP) but is not required for apelin binding or Gi protein coupling. Based on these findings, we hypothesized that the important role of the C-terminal Phe in BP decrease may be as a Gi-independent but β-arrestin-dependent signaling pathway that could involve MAPKs. For this purpose, we have used apelin fragments K17F and K16P (K17F with the C-terminal Phe deleted), which exhibit opposite profiles on apelin receptor internalization and BP. Using BRET-based biosensors, we showed that whereas K17F activates Gi and promotes β-arrestin recruitment to the receptor, K16P had a much reduced ability to promote β-arrestin recruitment while maintaining its Gi activating property, revealing the biased agonist character of K16P. We further show that both β-arrestin recruitment and apelin receptor internalization contribute to the K17F-stimulated ERK1/2 activity, whereas the K16P-promoted ERK1/2 activity is entirely Gi-dependent. In addition to providing new insights on the structural basis underlying the functional selectivity of apelin peptides, our study indicates that the β-arrestin-dependent ERK1/2 activation and not the Gi-dependent signaling may participate in K17F-induced BP decrease. PMID:25012663

  11. The C-terminal extension of bacterial flavodoxin-reductases: involvement in the hydride transfer mechanism from the coenzyme.

    PubMed

    Bortolotti, Ana; Sánchez-Azqueta, Ana; Maya, Celia M; Velázquez-Campoy, Adrián; Hermoso, Juan A; Medina, Milagros; Cortez, Néstor

    2014-01-01

    To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD:NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP(+) than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the C-terminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding.

  12. Skin-Derived C-Terminal Filaggrin-2 Fragments Are Pseudomonas aeruginosa-Directed Antimicrobials Targeting Bacterial Replication

    PubMed Central

    Hansmann, Britta; Schröder, Jens-Michael; Gerstel, Ulrich

    2015-01-01

    Soil- and waterborne bacteria such as Pseudomonas aeruginosa are constantly challenging body surfaces. Since infections of healthy skin are unexpectedly rare, we hypothesized that the outermost epidermis, the stratum corneum, and sweat glands directly control the growth of P. aeruginosa by surface-provided antimicrobials. Due to its high abundance in the upper epidermis and eccrine sweat glands, filaggrin-2 (FLG2), a water-insoluble 248 kDa S100 fused-type protein, might possess these innate effector functions. Indeed, recombinant FLG2 C-terminal protein fragments display potent antimicrobial activity against P. aeruginosa and other Pseudomonads. Moreover, upon cultivation on stratum corneum, P. aeruginosa release FLG2 C-terminus-containing FLG2 fragments from insoluble material, indicating liberation of antimicrobially active FLG2 fragments by the bacteria themselves. Analyses of the underlying antimicrobial mechanism reveal that FLG2 C-terminal fragments do not induce pore formation, as known for many other antimicrobial peptides, but membrane blebbing, suggesting an alternative mode of action. The association of the FLG2 fragment with the inner membrane of treated bacteria and its DNA-binding implicated an interference with the bacterial replication that was confirmed by in vitro and in vivo replication assays. Probably through in situ-activation by soil- and waterborne bacteria such as Pseudomonads, FLG2 interferes with the bacterial replication, terminates their growth on skin surface and thus may contributes to the skin’s antimicrobial defense shield. The apparent absence of FLG2 at certain body surfaces, as in the lung or of burned skin, would explain their higher susceptibility towards Pseudomonas infections and make FLG2 C-terminal fragments and their derivatives candidates for new Pseudomonas-targeting antimicrobials. PMID:26371476

  13. Contribution of N- and C-terminal Kv4.2 channel domains to KChIP interaction [corrected].

    PubMed

    Callsen, Britta; Isbrandt, Dirk; Sauter, Kathrin; Hartmann, L Sven; Pongs, Olaf; Bähring, Robert

    2005-10-15

    Association of Shal gene-related voltage-gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C-terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine-scanning mutagenesis within the proximal 40 amino acid portion and a structure-based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C-terminus was truncated at various positions. Wild-type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2-induced modulation of Kv4.2 currents was studied with whole-cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N-terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C-terminal deletions also had marked effects on KChIP2-dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C-terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose 'anchoring' at T1- and C-terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.

  14. Crystal Structures of the S. cerevisiae Spt6 Core and C-Terminal Tandem SH2 Domain

    SciTech Connect

    Close, D.; Robinson, H.; Johnson, S. J.; Sdano, M. A.; McDonald, S. M.; Formosa, T.; Hill, C. P.

    2011-05-13

    The conserved and essential eukaryotic protein Spt6 functions in transcription elongation, chromatin maintenance, and RNA processing. Spt6 has three characterized functions. It is a histone chaperone capable of reassembling nucleosomes, a central component of transcription elongation complexes, and is required for recruitment of RNA processing factors to elongating RNA polymerase II (RNAPII). Here, we report multiple crystal structures of the 168-kDa Spt6 protein from Saccharomyces cerevisiae that together represent essentially all of the ordered sequence. Our two structures of the {approx} 900-residue core region reveal a series of putative nucleic acid and protein-protein interaction domains that fold into an elongated form that resembles the bacterial protein Tex. The similarity to a bacterial transcription factor suggests that the core domain performs nucleosome-independent activities, and as with Tex, we find that Spt6 binds DNA. Unlike Tex, however, the Spt6 S1 domain does not contribute to this activity. Crystal structures of the Spt6 C-terminal region reveal a tandem SH2 domain structure composed of two closely associated SH2 folds. One of these SH2 folds is cryptic, while the other shares striking structural similarity with metazoan SH2 domains and possesses structural features associated with the ability to bind phosphorylated substrates including phosphotyrosine. Binding studies with phosphopeptides that mimic the RNAPII C-terminal domain revealed affinities typical of other RNAPII C-terminal domain-binding proteins but did not indicate a specific interaction. Overall, these findings provide a structural foundation for understanding how Spt6 encodes several distinct functions within a single polypeptide chain.

  15. Crystal Structures of the S. cerevisiae Spt6 Core and C-terminal Tandem SH2 Domain

    SciTech Connect

    D Close; S Johnson; M Sdano; S McDonald; H Robinson; T Formosa; C Hill

    2011-12-31

    The conserved and essential eukaryotic protein Spt6 functions in transcription elongation, chromatin maintenance, and RNA processing. Spt6 has three characterized functions. It is a histone chaperone capable of reassembling nucleosomes, a central component of transcription elongation complexes, and is required for recruitment of RNA processing factors to elongating RNA polymerase II (RNAPII). Here, we report multiple crystal structures of the 168-kDa Spt6 protein from Saccharomyces cerevisiae that together represent essentially all of the ordered sequence. Our two structures of the {approx} 900-residue core region reveal a series of putative nucleic acid and protein-protein interaction domains that fold into an elongated form that resembles the bacterial protein Tex. The similarity to a bacterial transcription factor suggests that the core domain performs nucleosome-independent activities, and as with Tex, we find that Spt6 binds DNA. Unlike Tex, however, the Spt6 S1 domain does not contribute to this activity. Crystal structures of the Spt6 C-terminal region reveal a tandem SH2 domain structure composed of two closely associated SH2 folds. One of these SH2 folds is cryptic, while the other shares striking structural similarity with metazoan SH2 domains and possesses structural features associated with the ability to bind phosphorylated substrates including phosphotyrosine. Binding studies with phosphopeptides that mimic the RNAPII C-terminal domain revealed affinities typical of other RNAPII C-terminal domain-binding proteins but did not indicate a specific interaction. Overall, these findings provide a structural foundation for understanding how Spt6 encodes several distinct functions within a single polypeptide chain.

  16. Role of the C-Terminal Region of Vervet Monkey Polyomavirus 1 VP1 in Virion Formation

    PubMed Central

    YAMAGUCHI, Hiroki; KOBAYASHI, Shintaro; MARUYAMA, Junki; SASAKI, Michihito; TAKADA, Ayato; KIMURA, Takashi; SAWA, Hirofumi; ORBA, Yasuko

    2014-01-01

    ABSTRACT Recently, we detected novel vervet monkey polyomavirus 1 (VmPyV) in a vervet monkey. Among amino acid sequences of major capsid protein VP1s of other polyomaviruses, VmPyV VP1 is the longest with additional amino acid residues in the C-terminal region. To examine the role of VmPyV VP1 in virion formation, we generated virus-like particles (VLPs) of VmPyV VP1, because VLP is a useful tool for the investigation of the morphological characters of polyomavirus virions. After the full-length VmPyV VP1 was subcloned into a mammalian expression plasmid, the plasmid was transfected into human embryonic kidney 293T (HEK293T) cells. Thereafter, VmPyV VLPs were purified from the cell lysates of the transfected cells via sucrose gradient sedimentation. Electron microscopic analyses revealed that VmPyV VP1 forms VLPs with a diameter of approximately 50 nm that are exclusively localized in cell nuclei. Furthermore, we generated VLPs consisting of the deletion mutant VmPyV VP1 (ΔC VP1) lacking the C-terminal 116 amino acid residues and compared its VLP formation efficiency and morphology to those of VLPs from wild-type VmPyV VP1 (WT VP1). WT and ΔC VP1 VLPs were similar in size, but the number of ΔC VP1 VLPs was much lower than that of WT VP1 VLPs in VP1-expressing HEK293T cells. These results suggest that the length of VP1 is unrelated to virion morphology; however, the C-terminal region of VmPyV VP1 affects the efficiency of its VLP formation. PMID:24419975

  17. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

    SciTech Connect

    Fernández-Fueyo, Elena; Acebes, Sandra; Ruiz-Dueñas, Francisco J.; Martínez, María Jesús; Romero, Antonio; Medrano, Francisco Javier; Guallar, Victor; Martínez, Angel T.

    2014-11-22

    The genome ofCeriporiopsis subvermisporaincludes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. We expressed short, long and extralong MnPs heterologously and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+oxidation by the internal propionate, but prevents the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. Furthermore, the tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of anin silicoshortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

  18. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

    DOE PAGES

    Fernández-Fueyo, Elena; Acebes, Sandra; Ruiz-Dueñas, Francisco J.; ...

    2014-11-22

    The genome ofCeriporiopsis subvermisporaincludes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. We expressed short, long and extralong MnPs heterologously and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+oxidation by the internal propionate, but prevents the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. Furthermore, the tail, which is anchored by numerous contacts, not only affectsmore » the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of anin silicoshortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.« less

  19. Endomorphin-2 analogs with C-terminal esterification produce potent systemic antinociception with reduced tolerance and gastrointestinal side effects.

    PubMed

    Wang, Chang-Lin; Qiu, Ting-Ting; Yang, Dai-Jun; Yuan, Bi-Yu; Han, Feng-Tong; Li, Li; Gu, Ning

    2017-04-01

    C-terminal esterification of opioid peptides may change their opioid activities due to the modified physicochemical properties. In the present study, the pharmacological activities of C-terminal esterified endomorphin-2 (EM-2) analogs 1-3 were characterized by in vitro metabolic stability and octanol/buffer distribution assays. Also, the antinociceptive profiles in the radiant heat paw withdrawal test and related side effects of these analogs were determined. Our results showed that all three analogs significantly increased the metabolic stability and lipophilicity. Moreover, analogs 1-3 displayed potent antinociceptive activities after intracerebroventricular (i.c.v.) administration. Analogs 1 and 3 exhibited about 2-fold higher antinociception than EM-2, and differential opioid mechanisms were involved. In addition, EM-2 at 50 μmol/kg failed to produce any significant antinociceptive activity after subcutaneous (s.c.) administration, whereas equimolar dose of analogs 1-3 produced significant analgesic effects. Analog 3 showed the highest antinociceptive activity after systemic administration, which was consistent with its in vitro stability and lipophilicity. We further evaluated the antinociceptive tolerance of analogs 1-3. In acute tolerance test, analogs 1-3 shifted the dose-response curves rightward by only 1.4-3.2 fold as determined by tolerance ratio, whereas EM-2 by 5.6-fold, demonstrating reduced antinociceptive tolerance. Also, analogs 1 and 2 decreased chronic antinociceptive tolerance by central and peripheral administration of drugs. In particular, analogs 3 displayed insignificant chronic antinociceptive tolerance. Furthermore, analogs 1-3 were less prone to induce gastrointestinal side effects at analgesic doses. The present investigation gave the evidence that C-terminal esterified modifications of EM-2 will facilitate the development of novel opioid analgesics with reduced side effects.

  20. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi.

    PubMed

    Fernández-Fueyo, Elena; Acebes, Sandra; Ruiz-Dueñas, Francisco J; Martínez, María Jesús; Romero, Antonio; Medrano, Francisco Javier; Guallar, Victor; Martínez, Angel T

    2014-12-01

    The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn(2+)-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn(2+) oxidation by the internal propionate, but prevents the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd(2+) binds at the Mn(2+)-oxidation site and competitively inhibits oxidation of both Mn(2+) and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

  1. C-terminal phosphorylation regulates the kinetics of a subset of melanopsin-mediated behaviors in mice.

    PubMed

    Somasundaram, Preethi; Wyrick, Glenn R; Fernandez, Diego Carlos; Ghahari, Alireza; Pinhal, Cindy M; Simmonds Richardson, Melissa; Rupp, Alan C; Cui, Lihong; Wu, Zhijian; Brown, R Lane; Badea, Tudor Constantin; Hattar, Samer; Robinson, Phyllis R

    2017-03-07

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and mediate several non-image-forming visual functions, including circadian photoentrainment and the pupillary light reflex (PLR). ipRGCs act as autonomous photoreceptors via the intrinsic melanopsin-based phototransduction pathway and as a relay for rod/cone input via synaptically driven responses. Under low light intensities, where only synaptically driven rod/cone input activates ipRGCs, the duration of the ipRGC response will be determined by the termination kinetics of the rod/cone circuits. Little is known, however, about the termination kinetics of the intrinsic melanopsin-based phototransduction pathway and its contribution to several melanopsin-mediated behaviors. Here, we show that C-terminal phosphorylation of melanopsin determines the recovery kinetics of the intrinsic melanopsin-based photoresponse in ipRGCs, the duration of the PLR, and the speed of reentrainment. In contrast, circadian phase alignment and direct effects of light on activity (masking) are not influenced by C-terminal phosphorylation of melanopsin. Electrophysiological measurements demonstrate that expression of a virally encoded melanopsin lacking all C-terminal phosphorylation sites (C terminus phosphonull) leads to a prolonged intrinsic light response. In addition, mice expressing the C terminus phosphonull in ipRGCs reentrain faster to a delayed light/dark cycle compared with mice expressing virally encoded WT melanopsin; however, the phase angle of entrainment and masking were indistinguishable. Importantly, a sustained PLR in the phosphonull animals is only observed at brighter light intensities that activate melanopsin phototransduction, but not at dimmer light intensities that activate only the rod/cone pathway. Taken together, our results highlight how the kinetics of the melanopsin photoresponse differentially regulate distinct light-mediated behaviors.

  2. C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

    SciTech Connect

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

    2013-09-20

    Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  3. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

    PubMed Central

    Fernández-Fueyo, Elena; Acebes, Sandra; Ruiz-Dueñas, Francisco J.; Martínez, María Jesús; Romero, Antonio; Medrano, Francisco Javier; Guallar, Victor; Martínez, Angel T.

    2014-01-01

    The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+ oxidation by the internal propionate, but prevents the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+ binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+ and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences. PMID:25478843

  4. Membrane binding properties of EBV gp110 C-terminal domain; evidences for structural transition in the membrane environment

    SciTech Connect

    Park, Sung Jean; Seo, Min-Duk; Lee, Suk Kyeong; Lee, Bong Jin

    2008-09-30

    Gp110 of Epstein-Barr virus (EBV) mainly localizes on nuclear/ER membranes and plays a role in the assembly of EBV nucleocapsid. The C-terminal tail domain (gp110 CTD) is essential for the function of gp110 and the nuclear/ER membranes localization of gp110 is ruled by its C-terminal unique nuclear localization signal (NLS), consecutive four arginines. In the present study, the structural properties of gp110 CTD in membrane mimics were investigated using CD, size-exclusion chromatography, and NMR, to elucidate the effect of membrane environment on the structural transition and to compare the structural feature of the protein in the solution state with that of the membrane-bound form. CD and NMR analysis showed that gp110 CTD in a buffer solution appears to adopt a stable folding intermediate which lacks compactness, and a highly helical structure is formed only in membrane environments. The helical content of gp110 CTD was significantly affected by the negative charge as well as the size of membrane mimics. Based on the elution profiles of the size-exclusion chromatography, we found that gp110 CTD intrinsically forms a trimer, revealing that a trimerization region may exist in the C-terminal domain of gp110 like the ectodomain of gp110. The mutation of NLS (RRRR) to RTTR does not affect the overall structure of gp110 CTD in membrane mimics, while the helical propensity in a buffer solution was slightly different between the wild-type and the mutant proteins. This result suggests that not only the helicity induced in membrane environment but also the local structure around NLS may be related to trafficking to the nuclear membrane. More detailed structural difference between the wild-type and the mutant in membrane environment was examined using synthetic two peptides including the wild-type NLS and the mutant NLS.

  5. An Autoinhibitory Helix in the C-Terminal Region of Phospholipase C-β Mediates Gαq Activation

    PubMed Central

    Lyon, Angeline M.; Tesmer, Valerie M.; Dhamsania, Vishan D.; Thal, David M.; Gutierrez, Joanne; Chowdhury, Shoaib; Suddala, Krishna C.; Northup, John K.; Tesmer, John J. G.

    2011-01-01

    Phospholipase C-β (PLCβ) is a key regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to Gq. We have determined atomic structures of two invertebrate homologs of PLCβ (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCβ3 dramatically increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCβ. PMID:21822282

  6. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

    SciTech Connect

    Fernández-Fueyo, Elena; Acebes, Sandra; Ruiz-Dueñas, Francisco J.; Martínez, María Jesús; Romero, Antonio; Medrano, Francisco Javier; Guallar, Victor; Martínez, Angel T.

    2014-12-01

    The variable C-terminal tail of manganese peroxidases, a group of enzymes involved in lignin degradation, is implicated in their catalytic and stability properties, as shown by new crystal structures, molecular-simulation and directed-mutagenesis data. Based on this structural–functional evaluation, short and long/extralong manganese peroxidase subfamilies have been accepted; the latter are characterized by exceptional stability, while it is shown for the first time that the former are able to oxidize other substrates at the same site where manganese(II) is oxidized. The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn{sup 2+}-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn{sup 2+} oxidation by the internal propionate, but prevents the oxidation of 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd{sup 2+} binds at the Mn{sup 2+}-oxidation site and competitively inhibits oxidation of both Mn{sup 2+} and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their

  7. Silyl-based alkyne-modifying linker for the preparation of C-terminal acetylene-derivatized protected peptides.

    PubMed

    Strack, Martin; Langklotz, Sina; Bandow, Julia E; Metzler-Nolte, Nils; Albada, H Bauke

    2012-11-16

    A novel linker for the synthesis of C-terminal acetylene-functionalized protected peptides is described. This SAM1 linker is applied in the manual Fmoc-based solid-phase peptide synthesis of Leu-enkephalin and in microwave-assisted automated synthesis of Maculatin 2.1, an antibacterial peptide that contains 18 amino acid residues. For the cleavage, treatment with tetramethylammonium fluoride results in protected acetylene-derivatized peptides. Alternatively, a one-pot cleavage-click procedure affords the protected 1,2,3-triazole conjugate in high yields after purification.

  8. Asparagine 326 in the extremely C-terminal region of XRCC4 is essential for the cell survival after irradiation

    SciTech Connect

    Wanotayan, Rujira; Fukuchi, Mikoto; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa

    2015-02-20

    XRCC4 is one of the crucial proteins in the repair of DNA double-strand break (DSB) through non-homologous end-joining (NHEJ). As XRCC4 consists of 336 amino acids, N-terminal 200 amino acids include domains for dimerization and for association with DNA ligase IV and XLF and shown to be essential for XRCC4 function in DSB repair and V(D)J recombination. On the other hand, the role of the remaining C-terminal region of XRCC4 is not well understood. In the present study, we noticed that a stretch of ∼20 amino acids located at the extreme C-terminus of XRCC4 is highly conserved among vertebrate species. To explore its possible importance, series of mutants in this region were constructed and assessed for the functionality in terms of ability to rescue radiosensitivity of M10 cells lacking XRCC4. Among 13 mutants, M10 transfectant with N326L mutant (M10-XRCC4{sup N326L}) showed elevated radiosensitivity. N326L protein showed defective nuclear localization. N326L sequence matched the consensus sequence of nuclear export signal. Leptomycin B treatment accumulated XRCC4{sup N326L} in the nucleus but only partially rescued radiosensitivity of M10-XRCC4{sup N326L}. These results collectively indicated that the functional defects of XRCC4{sup N326L} might be partially, but not solely, due to its exclusion from nucleus by synthetic nuclear export signal. Further mutation of XRCC4 Asn326 to other amino acids, i.e., alanine, aspartic acid or glutamine did not affect the nuclear localization but still exhibited radiosensitivity. The present results indicated the importance of the extremely C-terminal region of XRCC4 and, especially, Asn326 therein. - Highlights: • Extremely C-terminal region of XRCC4 is highly conserved among vertebrate species. • XRCC4 C-terminal point mutants, R325F and N326L, are functionally deficient in terms of survival after irradiation. • N326L localizes to the cytoplasm because of synthetic nuclear export signal. • Leptomycin B restores the

  9. FLO11 is essential for flor formation caused by the C-terminal deletion of NRG1 in Saccharomyces cerevisiae.

    PubMed

    Ishigami, Mari; Nakagawa, Youji; Hayakawa, Masayuki; Iimura, Yuzuru

    2004-08-15

    The flor strains of Saccharomyces cerevisiae form a flor on the surface of wine after alcoholic fermentation. High hydrophobicity of the cell surface is suggested to be important for flor formation by the flor wine yeasts. However, the molecular mechanism of flor formation is not clear. We found that expression of C-terminal deleted NRG1 lacking its two C2H2 zinc finger motifs (NRG1(1-470)) on the multicopy plasmid conferred the ability to form a flor to a non-flor laboratory strain. The cell surface hydrophobicity of NRG1(1-470) was higher than of the non-flor strain. Disruption of the Nrg1p-repressed gene FLO11, which encodes a cell surface glycoprotein that functions as a flocculin or an adhesin, abolished flor formation. Moreover, expression of FLO11 on a multicopy plasmid could also cause flor formation. These results indicate that FLO11 is essential for flor formation by NRG1(1-470). In addition, the results suggest that the C-terminal truncated form of Nrg1p exerts a dominant negative effect on FLO11 repression, resulting in FLO11 expression and, thus, flor formation.

  10. Functional Role of the C-Terminal Amphipathic Helix 8 of Olfactory Receptors and Other G Protein-Coupled Receptors

    PubMed Central

    Sato, Takaaki; Kawasaki, Takashi; Mine, Shouhei; Matsumura, Hiroyoshi

    2016-01-01

    G protein-coupled receptors (GPCRs) transduce various extracellular signals, such as neurotransmitters, hormones, light, and odorous chemicals, into intracellular signals via G protein activation during neurological, cardiovascular, sensory and reproductive signaling. Common and unique features of interactions between GPCRs and specific G proteins are important for structure-based design of drugs in order to treat GPCR-related diseases. Atomic resolution structures of GPCR complexes with G proteins have revealed shared and extensive interactions between the conserved DRY motif and other residues in transmembrane domains 3 (TM3), 5 and 6, and the target G protein C-terminal region. However, the initial interactions formed between GPCRs and their specific G proteins remain unclear. Alanine scanning mutagenesis of the murine olfactory receptor S6 (mOR-S6) indicated that the N-terminal acidic residue of helix 8 of mOR-S6 is responsible for initial transient and specific interactions with chimeric Gα15_olf, resulting in a response that is 2.2-fold more rapid and 1.7-fold more robust than the interaction with Gα15. Our mutagenesis analysis indicates that the hydrophobic core buried between helix 8 and TM1–2 of mOR-S6 is important for the activation of both Gα15_olf and Gα15. This review focuses on the functional role of the C-terminal amphipathic helix 8 based on several recent GPCR studies. PMID:27869740

  11. Dual chaperone role of the C-terminal propeptide in folding and oligomerization of the pore-forming toxin aerolysin.

    PubMed

    Iacovache, Ioan; Degiacomi, Matteo T; Pernot, Lucile; Ho, Sylvia; Schiltz, Marc; Dal Peraro, Matteo; van der Goot, F Gisou

    2011-07-01

    Throughout evolution, one of the most ancient forms of aggression between cells or organisms has been the production of proteins or peptides affecting the permeability of the target cell membrane. This class of virulence factors includes the largest family of bacterial toxins, the pore-forming toxins (PFTs). PFTs are bistable structures that can exist in a soluble and a transmembrane state. It is unclear what drives biosynthetic folding towards the soluble state, a requirement that is essential to protect the PFT-producing cell. Here we have investigated the folding of aerolysin, produced by the human pathogen Aeromonas hydrophila, and more specifically the role of the C-terminal propeptide (CTP). By combining the predictive power of computational techniques with experimental validation using both structural and functional approaches, we show that the CTP prevents aggregation during biosynthetic folding. We identified specific residues that mediate binding of the CTP to the toxin. We show that the CTP is crucial for the control of the aerolysin activity, since it protects individual subunits from aggregation within the bacterium and later controls assembly of the quaternary pore-forming complex at the surface of the target host cell. The CTP is the first example of a C-terminal chain-linked chaperone with dual function.

  12. VIPP1 Has a Disordered C-Terminal Tail Necessary for Protecting Photosynthetic Membranes against Stress1[OPEN

    PubMed Central

    Zhang, Lingang; Kondo, Hideki

    2016-01-01

    Integrity of biomembranes is vital to living organisms. In bacteria, PspA is considered to act as repairing damaged membrane by forming large supercomplexes in Arabidopsis (Arabidopsis thaliana). Vulnerable to oxidative stress, photosynthetic organisms also contain a PspA ortholog called VIPP1, which has an additional C-terminal tail (Vc). In this study, Vc was shown to coincide with an intrinsically disordered region, and the role of VIPP1 in membrane protection against stress was investigated. We visualized VIPP1 by fusing it to GFP (VIPP1-GFP that fully complemented lethal vipp1 mutations), and investigated its behavior in vivo with live imaging. The intrinsically disordered nature of Vc enabled VIPP1 to form what appeared to be functional particles along envelopes, whereas the deletion of Vc caused excessive association of the VIPP1 particles, preventing their active movement for membrane protection. Expression of VIPP1 lacking Vc complemented vipp1 mutation, but exhibited sensitivity to heat shock stress. Conversely, transgenic plants over-expressing VIPP1 showed enhanced tolerance against heat shock, suggesting that Vc negatively regulates VIPP1 particle association and acts in maintaining membrane integrity. Our data thus indicate that VIPP1 is involved in the maintenance of photosynthetic membranes. During evolution, chloroplasts have acquired enhanced tolerance against membrane stress by incorporating a disordered C-terminal tail into VIPP1. PMID:27208228

  13. Charge neutralization and collapse of the C-terminal tail of alpha-synuclein at low pH

    PubMed Central

    McClendon, Sebastian; Rospigliosi, Carla C; Eliezer, David

    2009-01-01

    Alpha-synuclein (αS) is the primary component of Lewy bodies, the pathological hallmark of Parkinson's Disease. Aggregation of αS is thought to proceed from a primarily disordered state with nascent secondary structure through intermediate conformations to oligomeric forms and finally to mature amyloid fibrils. Low pH conditions lead to conformational changes associated with increased αS fibril formation. Here we characterize these structural and dynamic changes using solution state NMR measurements of secondary chemical shifts, relaxation parameters, residual dipolar couplings, and paramagnetic relaxation enhancement. We find that the neutralization of negatively charged side-chains eliminates electrostatic repulsion in the C-terminal tail of αS and leads to a collapse of this region at low pH. Hydrophobic contacts between the compact C-terminal tail and the NAC (non-amyloid-β component) region are maintained and may lead to the formation of a globular domain. Transient long-range contacts between the C-terminus of the protein and regions N-terminal to the NAC region are also preserved. Thus, the release of long-range contacts does not play a role in the increased aggregation of αS at low pH, which we instead attribute to the increased hydrophobicity of the protein. PMID:19475665

  14. Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension.

    PubMed

    Kataoka, T; Holler, N; Micheau, O; Martinon, F; Tinel, A; Hofmann, K; Tschopp, J

    2001-06-01

    The Bcl-2 family of proteins plays a central regulatory role in apoptosis. We have identified a novel, widely expressed Bcl-2 member which we have named Bcl-rambo. Bcl-rambo shows overall structural homology to the anti-apoptotic Bcl-2 members containing conserved Bcl-2 homology (BH) motifs 1, 2, 3, and 4. Unlike Bcl-2, however, the C-terminal membrane anchor region is preceded by a unique 250 amino acid insertion containing two tandem repeats. No interaction of Bcl-rambo with either anti-apoptotic (Bcl-2, Bcl-x(L), Bcl-w, A1, MCL-1, E1B-19K, and BHRF1) or pro-apoptotic (Bax, Bak, Bik, Bid, Bim, and Bad) members of the Bcl-2 family was observed. In mammalian cells, Bcl-rambo was localized to mitochondria, and its overexpression induces apoptosis that is specifically blocked by the caspase inhibitors, IAPs, whereas inhibitors controlling upstream events of either the 'death receptor' (FLIP, FADD-DN) or the 'mitochondrial' pro-apoptotic pathway (Bcl-x(L)) had no effect. Surprisingly, the Bcl-rambo cell death activity was induced by its membrane-anchored C-terminal domain and not by the Bcl-2 homology region. Thus, Bcl-rambo constitutes a novel type of pro-apoptotic Bcl-2 member that triggers cell death independently of its BH motifs.

  15. PCSK9-mediated degradation of the LDL receptor generates a 17 kDa C-terminal LDL receptor fragment.

    PubMed

    Tveten, Kristian; Strøm, Thea Bismo; Berge, Knut Erik; Leren, Trond P

    2013-06-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the LDL receptor (LDLR) at the cell surface and reroutes the internalized LDLR to intracellular degradation. In this study, we have shown that PCSK9-mediated degradation of the full-length 160 kDa LDLR generates a 17 kDa C-terminal LDLR fragment. This fragment was not generated from mutant LDLRs resistant to PCSK9-mediated degradation or when degradation was prevented by chemicals such as ammonium chloride or the cysteine cathepsin inhibitor E64d. The observation that the 17 kDa fragment was only detected when the cells were cultured in the presence of the γ-secretase inhibitor DAPT indicates that this 17 kDa fragment undergoes γ-secretase cleavage within the transmembrane domain. The failure to detect the complementary 143 kDa ectodomain fragment is likely to be due to its rapid degradation in the endosomal lumen. The 17 kDa C-terminal LDLR fragment was also generated from a Class 5 mutant LDLR undergoing intracellular degradation. Thus, one may speculate that an LDLR with bound PCSK9 and a Class 5 LDLR with bound LDL are degraded by a similar mechanism that could involve ectodomain cleavage in the endosome.

  16. C-Terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin.

    PubMed

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

    2013-09-20

    DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  17. Evaluation of Heavy-Chain C-Terminal Deletion on Product Quality and Pharmacokinetics of Monoclonal Antibodies.

    PubMed

    Jiang, Guoying; Yu, Christopher; Yadav, Daniela B; Hu, Zhilan; Amurao, Annamarie; Duenas, Eileen; Wong, Marc; Iverson, Mark; Zheng, Kai; Lam, Xanthe; Chen, Jia; Vega, Roxanne; Ulufatu, Sheila; Leddy, Cecilia; Davis, Helen; Shen, Amy; Wong, Pin Y; Harris, Reed; Wang, Y John; Li, Dongwei

    2016-07-01

    Due to their potential influence on stability, pharmacokinetics, and product consistency, antibody charge variants have attracted considerable attention in the biotechnology industry. Subtle to significant differences in the level of charge variants and new charge variants under various cell culture conditions are often observed during routine manufacturing or process changes and pose a challenge when demonstrating product comparability. To explore potential solutions to control charge heterogeneity, monoclonal antibodies (mAbs) with native, wild-type C-termini, and mutants with C-terminal deletions of either lysine or lysine and glycine were constructed, expressed, purified, and characterized in vitro and in vivo. Analytical and physiological characterization demonstrated that the mAb mutants had greatly reduced levels of basic variants without decreasing antibody biologic activity, structural stability, pharmacokinetics, or subcutaneous bioavailability in rats. This study provides a possible solution to mitigate mAb heterogeneity in C-terminal processing, improve batch-to-batch consistency, and facilitate the comparability study during process changes.

  18. C-terminal truncation of a bovine B(12) trafficking chaperone enhances the sensitivity of the glutathione-regulated thermostability.

    PubMed

    Jeong, Jinju; Park, Jihyun; Lee, Dong-Yeon; Kim, Jihoe

    2013-03-01

    The human B(12) trafficking chaperone hCblC is well conserved in mammals and non-mammalian eukaryotes. However, the C-terminal ~40 amino acids of hCblC vary significantly and are predicted to be deleted by alternative splicing of the encoding gene. In this study, we examined the thermostability of the bovine CblC truncated at the C-terminal variable region (t-bCblC) and its regulation by glutathione. t-bCblC is highly thermolabile (T(m) = ~42(o)C) similar to the full-length protein (f-bCblC). However, t-bCblC is stabilized to a greater extent than f-bCblC by binding of reduced glutathione (GSH) with increased sensitivity to GSH. In addition, binding of oxidized glutathione (GSSG) destabilizes t-bCblC to a greater extent and with increased sensitivity as compared to f-bCblC. These results indicate that t-bCblC is a more sensitive form to be regulated by glutathione than the full-length form of the protein.

  19. Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

    SciTech Connect

    Serek, Robert; Forwood, Jade K.; Hume, David A.; Martin, Jennifer L.; Kobe, Bostjan

    2006-02-01

    The C-terminal domain of the mouse long-chain acyl-CoA thioesterase has been expressed in bacteria and crystallized by vapour diffusion. The crystals diffract to 2.4 Å resolution. The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination.

  20. Membrane tethering of APP c-terminal fragments is a prerequisite for T668 phosphorylation preventing nuclear sphere generation.

    PubMed

    Bukhari, Hassan; Kolbe, Katharina; Leonhardt, Gregor; Loosse, Christina; Schröder, Elisabeth; Knauer, Shirley; Marcus, Katrin; Müller, Thorsten

    2016-11-01

    A central molecular hallmark of Alzheimer's disease (AD) is the β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP), which causes the generation of different c-terminal fragments like C99, AICD57, or AICD50 that fully or in part contain the APP transmembrane domain. In this study, we demonstrate that membrane-tethered C99 is phosphorylated by JNK3A at residue T668 (APP695 numbering) to a higher extent than AICD57, whereas AICD50 is not capable of being phosphorylated. The modification decreases the turnover of APP, while the blockade of APP cleavage increases APP phosphorylation. Generation of nuclear spheres, complexes consisting of the translocated AICD, FE65 and other proteins, is significantly reduced as soon as APP c-terminal fragments are accessible for phosphorylation. This APP modification, which we identified as significantly reduced in high plaque-load areas of the human brain, is linearly dependent on the level of APP expression. Accordingly, we show that APP abundance is likewise capable of modulating nuclear sphere generation. Thus, the precise and complex regulation of APP phosphorylation, abundance, and cleavage impacts the generation of nuclear spheres, which are under discussion of being of relevance in neurodegeneration and dementia. Future pharmacological manipulation of nuclear sphere generation may be a promising approach for AD treatment.

  1. Function and Control of RNA Polymerase II C-Terminal Domain Phosphorylation in Vertebrate Transcription and RNA Processing

    PubMed Central

    Hsin, Jing-Ping; Xiang, Kehui

    2014-01-01

    The C-terminal domain of the RNA polymerase II largest subunit (the Rpb1 CTD) is composed of tandem heptad repeats of the consensus sequence Y1S2P3T4S5P6S7. We reported previously that Thr 4 is phosphorylated and functions in histone mRNA 3′-end formation in chicken DT40 cells. Here, we have extended our studies on Thr 4 and to other CTD mutations by using these cells. We found that an Rpb1 derivative containing only the N-terminal half of the CTD, as well as a similar derivative containing all-consensus repeats (26r), conferred full viability, while the C-terminal half, with more-divergent repeats, did not, reflecting a strong and specific defect in snRNA 3′-end formation. Mutation in 26r of all Ser 2 (S2A) or Ser 5 (S5A) residues resulted in lethality, while Ser 7 (S7A) mutants were fully viable. While S2A and S5A cells displayed defects in transcription and RNA processing, S7A cells behaved identically to 26r cells in all respects. Finally, we found that Thr 4 was phosphorylated by cyclin-dependent kinase 9 in cells and dephosphorylated both in vitro and in vivo by the phosphatase Fcp1. PMID:24752900

  2. Alteration of the C-terminal ligand specificity of the erbin PDZ domain by allosteric mutational effects.

    PubMed

    Murciano-Calles, Javier; McLaughlin, Megan E; Erijman, Ariel; Hooda, Yogesh; Chakravorty, Nishant; Martinez, Jose C; Shifman, Julia M; Sidhu, Sachdev S

    2014-10-23

    Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity.

  3. Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes.

    PubMed

    Win, Joe; Morgan, William; Bos, Jorunn; Krasileva, Ksenia V; Cano, Liliana M; Chaparro-Garcia, Angela; Ammar, Randa; Staskawicz, Brian J; Kamoun, Sophien

    2007-08-01

    Oomycete plant pathogens deliver effector proteins inside host cells to modulate plant defense circuitry and to enable parasitic colonization. These effectors are defined by a conserved motif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens, Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution. We obtained robust evidence of positive selection in more than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistent with the view that RXLR effectors are modular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.

  4. Disruption of C-Terminal Cytoplasmic Domain of βPS Integrin Subunit Has Dominant Negative Properties in Developing Drosophila

    PubMed Central

    Jannuzi, Alison L.; Bunch, Thomas A.; Brabant, Marc C.; Miller, Steven W.; Mukai, Leona; Zavortink, Michael; Brower, Danny L.

    2002-01-01

    We have analyzed a set of new and existing strong mutations in the myospheroid gene, which encodes the βPS integrin subunit of Drosophila. In addition to missense and other null mutations, three mutants behave as antimorphic alleles, indicative of dominant negative properties. Unlike null alleles, the three antimorphic mutants are synthetically lethal in double heterozygotes with an inflated (αPS2) null allele, and they fail to complement very weak, otherwise viable alleles of myospheroid. Two of the antimorphs result from identical splice site lesions, which create a frameshift in the C-terminal half of the cytoplasmic domain of βPS. The third antimorphic mutation is caused by a stop codon just before the cytoplasmic splice site. These mutant βPS proteins can support cell spreading in culture, especially under conditions that appear to promote integrin activation. Analyses of developing animals indicate that the dominant negative properties are not a result of inefficient surface expression, or simple competition between functional and nonfunctional proteins. These data indicate that mutations disrupting the C-terminal cytoplasmic domain of integrin β subunits can have dominant negative effects in situ, at normal levels of expression, and that this property does not necessarily depend on a specific new protein sequence or structure. The results are discussed with respect to similar vertebrate β subunit cytoplasmic mutations. PMID:11950944

  5. Characterizing substrate selectivity of ubiquitin C-terminal hydrolase-L3 using engineered α-linked ubiquitin substrates.

    PubMed

    Navarro, Mario F; Carmody, Lisa; Romo-Fewell, Octavio; Lokensgard, Melissa E; Love, John J

    2014-12-30

    The ubiquitin-proteasome system (UPS) is highly complex and entails the concerted actions of many enzymes that function to ubiquitinate proteins targeted to the proteasome as well as enzymes that remove and recycle ubiquitin for additional rounds of proteolysis. Ubiquitin C-terminal hydrolase-L3 (UCH-L3) is a human cytosolic deubiquitinase whose precise biological function is not known. It is believed to hydrolyze small peptides or chemical adducts from the C-terminus of ubiquitin that may be remnant from proteasomal processing. In addition, UCH-L3 is a highly effective biotechnological tool that is used to produce small or unstable peptides/proteins recalcitrant to production in Escherichia coli expression systems. Previous research, which explored the substrate selectivity of UCH-L3, demonstrated a substrate size limitation for proteins/peptides expressed as α-linked C-terminal fusions to ubiquitin and also suggested that an additional substrate property may affect UCH-L3 hydrolysis [ Larsen , C. N. et al. (1998) Biochemistry 37 , 3358 - 3368 ]. Using a series of engineered protein substrates, which are similar in size yet differ in secondary structure, we demonstrate that thermal stability is a key factor that significantly affects UCH-L3 hydrolysis. In addition, we show that the thermal stabilities of the engineered substrates are not altered by fusion to ubiquitin and offer a possible mechanism as to how ubiquitin affects the structural and unfolding properties of natural in vivo targets.

  6. PS1 N- and C-terminal fragments form a complex that functions in APP processing and Notch signaling

    PubMed Central

    Levitan, Diane; Lee, Julie; Song, Lixin; Manning, Ron; Wong, Gwen; Parker, Eric; Zhang, Lili

    2001-01-01

    Presenilin proteins play critical roles in the proteolytic processing of both Notch and amyloid precursor protein (APP). Presenilin itself undergoes endoproteolytic processing to generate an N-terminal and C-terminal fragment. As demonstrated previously, overexpression of presenilin 1 (PS1) holoprotein does not change the levels of the N-terminal and C-terminal fragments (NTF and CTF). When we coexpress the PS1 NTF and CTF, marked increases in the cellular levels of these fragments are seen. By coexpressing the PS1 NTF and CTF, we demonstrate conclusively that a noncovalent complex of the NTF and CTF is the active species of presenilin. However, although the PS1 NTF/CTF complex is necessary for γ-secretase activity, it is not sufficient. Independent overexpression of the PS1 NTF and CTF was also used to show that the Asp-257 and Asp-385 mutations in PS1 decrease Aβ production by a direct effect on γ-secretase activity and not by the inhibition of PS1 endoproteolysis. PMID:11593035

  7. Octaarginine-modified liposomes enhance cross-presentation by promoting the C-terminal trimming of antigen peptide.

    PubMed

    Nakamura, Takashi; Ono, Kouhei; Suzuki, Yoshiteru; Moriguchi, Rumiko; Kogure, Kentaro; Harashima, Hideyoshi

    2014-08-04

    Exogenous antigen proteolysis by proteasomes and amino peptidases is essential for the production of mature major histocompatibility complex class I (MHC-I) peptides to induce cross-presentation. We report here that when liposomes are modified with octaarginine (R8-Lip), a type of cell-penetrating peptide, the production of the mature MHC-I peptide is enhanced by promoting the C-terminal trimming of the antigen peptide. The efficiency of cross-presentation of ovalbumin (OVA) using the R8-Lip was dramatically higher than that by octalysine modified liposomes (K8-Lip) in mouse bone-marrow derived dendritic cells (BMDCs), although the physical characters of both liposomes were comparable. In this study, we investigated the mechanism responsible for the enhancement in cross-presentation by R8-Lip. Although the efficiencies of cellular uptake, endosomal escape, proteolysis of OVA and DC maturation between the two systems were essentially the same, an analysis of peptide trimming to SIINFEKL (mature MHC-I peptide of OVA) by using R8-Lip and K8-Lip encapsulating peptides of various length clearly indicates that the use of R8-Lip enhances the efficiency of the C-terminal cleavage of antigen-derived peptides. This finding provides a new strategy for achieving efficient cross-presentation by using R8 peptide and arginine-rich peptides. Moreover, this result may contribute to the development of a new paradigm regarding the machinery associated with antigen peptide production.

  8. Three conserved C-terminal residues of influenza fusion peptide alter its behavior at the membrane interface.

    PubMed

    Worch, Remigiusz; Krupa, Joanna; Filipek, Alicja; Szymaniec, Anna; Setny, Piotr

    2017-02-01

    The N-terminal fragment of the viral hemagglutinin HA2 subunit is termed a fusion peptide (HAfp). The 23-amino acid peptide (HAfp1-23) contains three C-terminal W21-Y22-G23 residues which are highly conserved among serotypes of influenza A and has been shown to form a tight helical hairpin very distinct from the boomerang structure of HAfp1-20. We studied the effect of peptide length on fusion properties, structural dynamics, and binding to the membrane interface. We developed a novel fusion visualization assay based on FLIM microscopy on giant unilamellar vesicles (GUV). By means of molecular dynamics simulations and spectroscopic measurements, we show that the presence of the three C-terminal W21-Y22-G23 residues promotes the hairpin formation, which orients perpendicularly to the membrane plane and induces more disorder in the surrounding lipids than the less structured HAfp1-20. Moreover, we report cholesterol-enriched domain formation induced exclusively by the longer fusion peptide.

  9. Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

    PubMed Central

    De Bock, Marijke; Wang, Nan; Decrock, Elke; Bultynck, Geert; Leybaert, Luc

    2015-01-01

    The coordination of tissue function is mediated by gap junctions (GJs) that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx) proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs) in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs) are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury. PMID:26424967

  10. The vacuolar targeting signal of the 2S albumin from Brazil nut resides at the C terminus and involves the C-terminal propeptide as an essential element.

    PubMed

    Saalbach, G; Rosso, M; Schumann, U

    1996-11-01

    Genetic constructs in which different N- and C-terminal segments of Brazil nut (Bertholletia excelsa H.B.K.) 2S albumin were fused to secretory yeast invertase were transformed into tobacco (Nicotiana tabacum) plants to investigate the vacuolar targeting signal of the 2S albumin. None of the N-terminal segments, including the complete precursor containing all propeptides, was able to direct the invertase to the vacuoles. However, a short C-terminal segment comprising the last 20 amino acids of the precursor was sufficient for efficient targeting of yeast invertase to the vacuoles of the transformed tobacco plants. Further analyses showed that peptides of 16 and 13 amino acids of the C-terminal segment were still sufficient, although they had slightly lower efficiency. When segments of 9 amino acids or shorter were analyzed, a decrease to approximately 30% was observed. These segments included the C-terminal propeptide of four amino acids (Ile-Ala-Gly-Phe). When the 2S albumin was expressed in tobacco, it was also localized to the vacuoles of mesophyll cells. If the C-terminal propeptide was deleted from the 2S albumin precursor, all of this truncated 2S albumin was secreted from the tobacco cells. These results indicate that the C-terminal propeptide is necessary but not sufficient for vacuolar targeting. In addition, an adjacent segment of at least 12 amino acids of the mature protein is needed to form the complete signal for efficient targeting.

  11. Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1

    PubMed Central

    2009-01-01

    Background Antibacterial activity is a novel function of high-mobility group box 1 (HMGB1). However, the functional site for this new effect is presently unknown. Methods and Results In this study, recombinant human HMGB1 A box and B box (rHMGB1 A box, rHMGB1 B box), recombinant human HMGB1 (rHMGB1) and the truncated C-terminal acidic tail mutant (tHMGB1) were prepared by the prokaryotic expression system. The C-terminal acidic tail (C peptide) was synthesized, which was composed of 30 amino acid residues. Antibacterial assays showed that both the full length rHMGB1 and the synthetic C peptide alone could efficiently inhibit bacteria proliferation, but rHMGB1 A box and B box, and tHMGB1 lacking the C-terminal acidic tail had no antibacterial function. These results suggest that C-terminal acidic tail is the key region for the antibacterial activity of HMGB1. Furthermore, we prepared eleven different deleted mutants lacking several amino acid residues in C-terminal acidic tail of HMGB1. Antibacterial assays of these mutants demonstrate that the amino acid residues 201-205 in C-terminal acidic tail region is the core functional site for the antibacterial activity of the molecule. Conclusion In sum, these results define the key region and the crucial site in HMGB1 for its antibacterial function, which is helpful to illustrating the antibacterial mechanisms of HMGB1. PMID:19751520

  12. The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium.

    PubMed

    Hill, Kristina E; Zhou, Jiadong; Austin, Lori M; Motley, Amy K; Ham, Amy-Joan L; Olson, Gary E; Atkins, John F; Gesteland, Raymond F; Burk, Raymond F

    2007-04-13

    Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1-/- and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Delta240-361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Delta240-361 accounted for over 90% of the selenium in the plasma of Sepp1Delta240-361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Delta240-361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1-/- mice but they better maintained their whole body selenium. Sepp1Delta240-361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Delta240-361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1-/- mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.

  13. Accelerating the clinical development of protein-based vaccines for malaria by efficient purification using a four amino acid C-terminal 'C-tag'.

    PubMed

    Jin, Jing; Hjerrild, Kathryn A; Silk, Sarah E; Brown, Rebecca E; Labbé, Geneviève M; Marshall, Jennifer M; Wright, Katherine E; Bezemer, Sandra; Clemmensen, Stine B; Biswas, Sumi; Li, Yuanyuan; El-Turabi, Aadil; Douglas, Alexander D; Hermans, Pim; Detmers, Frank J; de Jongh, Willem A; Higgins, Matthew K; Ashfield, Rebecca; Draper, Simon J

    2017-01-30

    Development of bespoke biomanufacturing processes remains a critical bottleneck for translational studies, in particular when modest quantities of a novel product are required for proof-of-concept Phase I/II clinical trials. In these instances the ability to develop a biomanufacturing process quickly and relatively cheaply, without risk to product quality or safety, provides a great advantage by allowing new antigens or concepts in immunogen design to more rapidly enter human testing. These challenges with production and purification are particularly apparent when developing recombinant protein-based vaccines for difficult parasitic diseases, with Plasmodium falciparum malaria being a prime example. To that end, we have previously reported the expression of a novel protein vaccine for malaria using the ExpreS(2)Drosophila melanogaster Schneider 2 stable cell line system, however, a very low overall process yield (typically <5% recovery of hexa-histidine-tagged protein) meant the initial purification strategy was not suitable for scale-up and clinical biomanufacture of such a vaccine. Here we describe a newly available affinity purification method that was ideally suited to purification of the same protein which encodes the P. falciparum reticulocyte-binding protein homolog 5 - currently the leading antigen for assessment in next generation vaccines aiming to prevent red blood cell invasion by the blood-stage parasite. This purification system makes use of a C-terminal tag known as 'C-tag', composed of the four amino acids, glutamic acid - proline - glutamic acid - alanine (E-P-E-A), which is selectively purified on a CaptureSelect™ affinity resin coupled to a camelid single chain antibody, called NbSyn2. The C-terminal fusion of this short C-tag to P. falciparum reticulocyte-binding protein homolog 5 achieved >85% recovery and >70% purity in a single step purification directly from clarified, concentrated Schneider 2 cell supernatant under mild conditions

  14. Conformation and Lipid Binding of a C-Terminal (198-243) Peptide of Human Apolipoprotein A-I (apoA-I)†

    PubMed Central

    Zhu, Hongli L.; Atkinson, David

    2008-01-01

    Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on [1-44]apoA-I demonstrated that this N-terminal region is unstructured and folds into ~ 60% α-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46 residue peptide, [198-243]apoA-I, encompassing putative helix repeats 10, 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far ultraviolet circular dichroism spectra show that [198-243] apoA-I is also unfolded in aqueous solution. However, self-association induces ~ 50% α-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid mimicking detergents, above their CMC, ~ 60% α-helix was induced in the peptide. In contrast, SDS, an anionic lipid mimicking detergent, induced helical folding in the peptide at a concentration of ~ 0.003% (~ 100 μM), ~ 70 fold below its typical CMC (0.17–0.23% or 6–8 mM). Both monomeric and tetrameric peptide can solublize dimyristoyl phosphatidyl choline (DMPC) liposomes and fold into ~ 60% α-helix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy, demonstrated that the peptide binds to DMPC with high affinity to form at least two sizes of relatively homogenous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide w/w, diameter and density) of both complexes are similar to those of

  15. Effects of endocrine-disrupting chemicals on expression of ubiquitin C-terminal hydrolase mRNA in testis and brain of the Japanese common goby.

    PubMed

    Mochida, Kazuhiko; Ohkubo, Nobuyuki; Matsubara, Takahiro; Ito, Katsutoshi; Kakuno, Akira; Fujii, Kazunori

    2004-11-18

    We investigated the effects of endocrine-disrupting chemicals (EDCs) on the expression of ubiquitin C-terminal hydrolase (UCH) mRNA in the testis and brain of the Japanese common goby, Acanthogobius flavimanus. The cDNA sequence of goby UCH contained an open reading frame encoding 220 amino acid residues (M(r)=24,223) with 51.3% overall sequence identity with human and mouse UCHL1. A competitive PCR assay was used to quantify the levels of UCH mRNA in the testis and brain of male gobies after exposure to bisphenol A, nonylphenol, or estradiol-17beta for 3 weeks. Exposure to estradiol-17beta at a nominal concentration of 100 ng/L induced significant increase in UCH mRNA levels in both testis and brain (P<0.05), whereas exposure to nonylphenol induced a significant decrease in UCH mRNA levels in the testis (P<0.01). These results suggest that EDCs can either positively or negatively regulate UCH mRNA levels.

  16. The functional roles of the unstructured N- and C-terminal regions in αB-crystallin and other mammalian small heat-shock proteins.

    PubMed

    Carver, John A; Grosas, Aidan B; Ecroyd, Heath; Quinlan, Roy A

    2017-04-08

    Small heat-shock proteins (sHsps), such as αB-crystallin, are one of the major classes of molecular chaperone proteins. In vivo, under conditions of cellular stress, sHsps are the principal defence proteins that prevent large-scale protein aggregation. Progress in determining the structure of sHsps has been significant recently, particularly in relation to the conserved, central and β-sheet structured α-crystallin domain (ACD). However, an understanding of the structure and functional roles of the N- and C-terminal flanking regions has proved elusive mainly because of their unstructured and dynamic nature. In this paper, we propose functional roles for both flanking regions, based around three properties: (i) they act in a localised crowding manner to regulate interactions with target proteins during chaperone action, (ii) they protect the ACD from deleterious amyloid fibril formation and (iii) the flexibility of these regions, particularly at the extreme C-terminus in mammalian sHsps, provides solubility for sHsps under chaperone and non-chaperone conditions. In the eye lens, these properties are highly relevant as the crystallin proteins, in particular the two sHsps αA- and αB-crystallin, are present at very high concentrations.

  17. Mutational and Functional Analysis of the C-Terminal Region of the C3H Mouse Mammary Tumor Virus Superantigen

    PubMed Central

    Wrona, Thomas J.; Lozano, Mary; Binhazim, Awadh A.; Dudley, Jaquelin P.

    1998-01-01

    The mouse mammary tumor virus (MMTV) encodes within the U3 region of the long terminal repeat (LTR) a protein known as the superantigen (Sag). Sag is needed for the efficient transmission of milk-borne virus from the gut to target tissue in the mammary gland. MMTV-infected B cells in the gut express Sag as a type II transmembrane protein that is recognized by the variable region of particular beta chains (Vβ) of the T-cell receptor (TCR) on the surface of T cells. Recognition of Sag by particular TCRs results in T-cell stimulation, release of cytokines, and amplification of MMTV infection in lymphoid cells that are needed for infection of adolescent mammary tissue. Because the C-terminal 30 to 40 amino acids of Sag are variable and correlate with recognition of particular TCR Vβ chains, we prepared a series of C-terminal Sag mutations that were introduced into a cloned infectious MMTV provirus. Virus-producing XC rat cells were used for injection of susceptible BALB/c mice, and these mice were monitored for functional Sag activity by the deletion of C3H MMTV Sag-reactive (CD4+ Vβ14+) T cells. Injected mice also were analyzed for mutant infection and tumor formation in mammary glands as well as milk-borne transmission of MMTV to offspring. Most mutations abrogated Sag function, although one mutation (HPA242) that changed the negative charge of the extreme C terminus to a positive charge created a weaker Sag that slowed the kinetics of Sag-mediated T-cell deletion. Despite the lack of Sag activity, many of the sag mutant viruses were capable of sporadic infections of the mammary glands of injected mice but not of offspring mice, indicating that functional Sag increases the probability of milk-borne MMTV infection. Furthermore, although most viruses encoding nonfunctional Sags were unable to cause mammary tumors, tumors were induced by such viruses carrying mutations in a negative regulatory element that overlaps the sag gene within the LTR, suggesting that loss of

  18. A novel family of ubiquitin-specific proteases in chick skeletal muscle with distinct N- and C-terminal extensions.

    PubMed Central

    Baek, S H; Park, K C; Lee, J I; Kim, K I; Yoo, Y J; Tanaka, K; Baker, R T; Chung, C H

    1998-01-01

    We have recently identified a cDNA for a ubiquitin-specific protease (UBP), UBP41, that encodes the smallest functional UBP identified to date, using an Escherichia coli-based in vivo screening method. In the present study we isolated highly related cDNAs encoding a new family of UBP enzymes, named UBP46, UBP52 and UBP66. These UBPs have virtually identical catalytic domains spanning the sequence of UBP41 between the active-site Cys and the His box (95% identity). However, they possess distinct N- and/or C-terminal extensions. Moreover, they are more closely related to each other than to any other members of the UBP family. Thus these chick UBPs must define a novel family of de-ubiquitinating enzymes and should represent the first example among the UBP family enzymes, whose multiplicity is achieved by variation in their N- and C-terminal extensions. The chick UBPs were expressed in E. coli, and purified from the cells to apparent homogeneity using 125I-labelled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate. Each of the purified UBP46, UBP52 and UBP66 enzymes behaved as proteins of similar sizes under both denaturing and non-denaturing conditions, suggesting that all of them consist of a single polypeptide chain. The UBP enzymes cleaved the C-terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes and thus are active against ubiquitin-beta-galactosidase as well as a ubiquitin C-terminal extension protein of 80 amino acids. All UBPs except UBP66 released free ubiquitin from poly-His-tagged di-ubiquitin. However, the isopeptidase activity for hydrolysing polyubiquitinated lysozyme conjugates was not detected from these UBPs, which makes these UBPs distinct from UBP41. These results suggest that the chick UBPs may play an important role in production of free ubiquitin from linear polyubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins. PMID:9729477

  19. Mutations in FKBP10, which result in Bruck syndrome and recessive forms of osteogenesis imperfecta, inhibit the hydroxylation of telopeptide lysines in bone collagen

    PubMed Central

    Schwarze, Ulrike; Cundy, Tim; Pyott, Shawna M.; Christiansen, Helena E.; Hegde, Madhuri R.; Bank, Ruud A.; Pals, Gerard; Ankala, Arunkanth; Conneely, Karen; Seaver, Laurie; Yandow, Suzanne M.; Raney, Ellen; Babovic-Vuksanovic, Dusica; Stoler, Joan; Ben-Neriah, Ziva; Segel, Reeval; Lieberman, Sari; Siderius, Liesbeth; Al-Aqeel, Aida; Hannibal, Mark; Hudgins, Louanne; McPherson, Elizabeth; Clemens, Michele; Sussman, Michael D.; Steiner, Robert D.; Mahan, John; Smith, Rosemarie; Anyane-Yeboa, Kwame; Wynn, Julia; Chong, Karen; Uster, Tami; Aftimos, Salim; Sutton, V. Reid; Davis, Elaine C.; Kim, Lammy S.; Weis, Mary Ann; Eyre, David; Byers, Peter H.

    2013-01-01

    Although biallelic mutations in non-collagen genes account for <10% of individuals with osteogenesis imperfecta, the characterization of these genes has identified new pathways and potential interventions that could benefit even those with mutations in type I collagen genes. We identified mutations in FKBP10, which encodes the 65 kDa prolyl cis–trans isomerase, FKBP65, in 38 members of 21 families with OI. These include 10 families from the Samoan Islands who share a founder mutation. Of the mutations, three are missense; the remainder either introduce premature termination codons or create frameshifts both of which result in mRNA instability. In four families missense mutations result in loss of most of the protein. The clinical effects of these mutations are short stature, a high incidence of joint contractures at birth and progressive scoliosis and fractures, but there is remarkable variability in phenotype even within families. The loss of the activity of FKBP65 has several effects: type I procollagen secretion is slightly delayed, the stabilization of the intact trimer is incomplete and there is diminished hydroxylation of the telopeptide lysyl residues involved in intermolecular cross-link formation in bone. The phenotype overlaps with that seen with mutations in PLOD2 (Bruck syndrome II), which encodes LH2, the enzyme that hydroxylates the telopeptide lysyl residues. These findings define a set of genes, FKBP10, PLOD2 and SERPINH1, that act during procollagen maturation to contribute to molecular stability and post-translational modification of type I procollagen, without which bone mass and quality are abnormal and fractures and contractures result. PMID:22949511

  20. Effects of carbamazepine on serum parathormone, 25- hydroxyvitamin D, bone specific alkaline phosphatase, C-telopeptide, and osteocalcin levels in healthy rats.

    PubMed

    Kir, Hale Maral; Garip, Sebnem; Sahin, Deniz; Öztaş, Berrin

    2012-11-01

    It is still not completely clear whether carbamazepine causes alterations in vitamin D status and in bone metabolism. The objective of this study was to investigate the effects of carbamazepine on serum levels of 25-hydroxyvitamin D and on biomarkers of bone formation and resorption in healthy rats. Levels of calcium, 25- hydroxyvitamin D, parathormone, C-telopeptide, bone specific alkaline phosphatase and osteocalcin were measured in 3 groups of rats consisting of controls (n=10), isotonic saline solution group (n=10) and carbamazepine group (n=10). Mean calcium levels were found to be significantly lower in healthy controls in comparison to isotonic saline solution and carbamazepine groups (10.0±0.24, 10.81±0.16, 10.93±0.22 mg/dL, respectively, p<0.05). Mean levels of 25- hydroxyvitamin D, were found to be significantly higher in control group compared to isotonic saline solution group (25- hydroxyvitamin D; 25.91±1.12, 19.99±0.99 ng/mL, respectively, p<0.01). Mean levels of parathormone and osteocalcin were found to be significantly higher in control group compared to isotonic saline solution group and carbamazepine group. Parathormone levels were measured as 3.46±0.83, 1.08±0.08, 0.94±0.02 pg/mL, respectively (p<0.01). Osteocalcine levels were measured as 1.66±0.001, 1.32±0.002, 1.32±0.001 ng/mL, respectively (p<0.001). A significant difference in terms of mean serum bone specific alkaline phosphatase and C-telopeptide levels among groups was not observed. The main outcome of this prospective study in healthy rats showed no change in biochemical parameters of bone turnover during treatment with carbamazepine.

  1. Triptonide Effectively Inhibits Wnt/β-Catenin Signaling via C-terminal Transactivation Domain of β-catenin

    PubMed Central

    Chinison, Jessica; Aguilar, Jose S.; Avalos, Alan; Huang, Ying; Wang, Zhijun; Cameron, D. Joshua; Hao, Jijun

    2016-01-01

    Abnormal activation of canonical Wnt/β-catenin signaling is implicated in many diseases including cancer. As a result, therapeutic agents that disrupt this signaling pathway have been highly sought after. Triptonide is a key bioactive small molecule identified in a traditional Chinese medicine named Tripterygium wilfordii Hook F., and it has a broad spectrum of biological functions. Here we show that triptonide can effectively inhibit canonical Wnt/β-catenin signaling by targeting the downstream C-terminal transcription domain of β-catenin or a nuclear component associated with β-catenin. In addition, triptonide treatment robustly rescued the zebrafish “eyeless” phenotype induced by GSK-3β antagonist 6-bromoindirubin-30-oxime (BIO) for Wnt signaling activation during embryonic gastrulation. Finally, triptonide effectively induced apoptosis of Wnt-dependent cancer cells, supporting the therapeutic potential of triptonide. PMID:27596363

  2. The multiple forms of bovine seminal ribonuclease: structure and stability of a C-terminal swapped dimer.

    PubMed

    Sica, Filomena; Pica, Andrea; Merlino, Antonello; Russo Krauss, Irene; Ercole, Carmine; Picone, Delia

    2013-11-29

    Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111-116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed.

  3. C-terminal domain of SMYD3 serves as a unique HSP90-regulated motif in oncogenesis

    PubMed Central

    Harriss, June; Das, Chhaya; Zhu, Li; Edwards, Melissa; Shaaban, Salam; Tucker, Haley

    2015-01-01

    The SMYD3 histone methyl transferase (HMTase) and the nuclear chaperone, HSP90, have been independently implicated as proto-oncogenes in several human malignancies. We show that a degenerate tetratricopeptide repeat (TPR)-like domain encoded in the SMYD3 C-terminal domain (CTD) mediates physical interaction with HSP90. We further demonstrate that the CTD of SMYD3 is essential for its basal HMTase activity and that the TPR-like structure is required for HSP90-enhanced enzyme activity. Loss of SMYD3-HSP90 interaction leads to SMYD3 mislocalization within the nucleus, thereby losing its chromatin association. This results in reduction of SMYD3-mediated cell proliferation and, potentially, impairment of SMYD3′s oncogenic activity. These results suggest a novel approach for blocking HSP90-driven malignancy in SMYD3-overexpressing cells with a reduced toxicity profile over current HSP90 inhibitors. PMID:25738358

  4. α-Helical to β-Helical Conformation Change in the C-Terminal of the Mammalian Prion Protein

    NASA Astrophysics Data System (ADS)

    Singh, Jesse; Whitford, Paul; Hayre, Natha; Cox, Daniel; Onuchic, José.

    2011-03-01

    We employ all-atom structure-based models with mixed basis contact maps to explore whether there are any significant geometric or energetic constraints limiting conjectured conformational transitions between the alpha-helical (α H) and the left handed beta helical (LHBH) conformations for the C-terminal (residues 166-226) of the mammalian prion protein. The LHBH structure has been proposed to describe infectious oligomers and one class of in vitro grown fibrils, as well as possibly self- templating the conversion of normal cellular prion protein to the infectious form. Our results confirm that the kinetics of the conformation change are not strongely limited by large scale geometry modification and there exists an overall preference for the LHBH conformation.

  5. Peptides containing acylated C-terminal gem diamines: novel irreversible inactivators of the cysteine and serine proteinases.

    PubMed

    Gilmore, B F; Lynas, J F; Harriott, P; Healy, A; Walker, B

    2006-05-01

    This study reports on the synthesis of peptides containing C-terminal acylated gem-diamines and their utilization for the preparation of irreversible inactivators of the serine and cysteine proteinases. We have succeeded in obtaining an inhibitor Acetyl-Val-Pro-g-Val-CO-O-C(6)H(4)-NO(2) of neutrophil and pancreatic elastases that functions in a time-dependent manner, indicative of the action of an irreversible inactivator, functioning, most probably, through the formation of a long-lived acyl enzyme intermediate. In addition, we have demonstrated the irreversible inhibition of the cysteine proteinase bovine cathepsin B, by chloroacetyl and bromoacetyl derivatives of a dipeptide gem-diamine, Cbz-Phe-g-Ala-CO-CH(2)Hal (Hal = Br, Cl).

  6. Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

    PubMed Central

    Serek, Robert; Forwood, Jade K.; Hume, David A.; Martin, Jennifer L.; Kobe, Bostjan

    2006-01-01

    The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-­hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination. PMID:16511283

  7. The new function of two ubiquitin C-terminal hydrolase isozymes as reciprocal modulators of germ cell apoptosis.

    PubMed

    Kwon, Jungkee

    2007-04-01

    Ubiquitination is required throughout all developmental stages of mammalian spermatogenesis. The two ubiquitin C-terminal hydrolase (UCH) enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. These two UCH isozymes have 52% amino acid identity and share significant structural similarity. A new function of these two closely related UCH enzymes during spermatogenesis which is associated with germ cell apoptosis has been analyzed. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. During spermatogenesis, apoptosis controls germ cell numbers and eliminates defective germ cells to facilitate testicular homeostasis. In this paper, I review the distinct function of the two UCH isozymes in the testis of gad and Uchl3 knockout mice, which are strongly but reciprocally expressed during spermatogenesis. In addition, the importance of UCHL1-dependent apoptosis for normal spermatogenesis and sperm quality control is discussed.

  8. Lipoprotein lipase deficiency leads to α-synuclein aggregation and ubiquitin C-terminal hydrolase L1 reduction.

    PubMed

    Yang, H; Zhou, T; Wang, H; Liu, T; Ueda, K; Zhan, R; Zhao, L; Tong, Y; Tian, X; Zhang, T; Jin, Y; Han, X; Li, Z; Zhao, Y; Guo, X; Xiao, W; Fan, D; Liu, G; Chui, D

    2015-04-02

    We have previously reported that presynaptic dysfunction and cognitive decline have been found in lipoprotein lipase (LPL) deficient mice, but the mechanism remains to be elucidated. Accumulating evidence supported that α-synuclein (α-syn) and ubiquitin C-terminal hydrolase L1 (UCHL1) are required for normal synaptic and cognitive function. In this study, we found that α-syn aggregated and the expression of UCHL1 decreased in the brain of LPL deficient mice. Reduction of UCHL1 was resulted from nuclear retention of DNA cytosine-5-methyltransferase 1 in LPL knockout mice. Reverse changes were found in cultured cells overexpressing LPL. Furthermore, deficiency of LPL increased ubiquitination of α-syn. These results indicated that aggregation of α-syn and reduction of UCHL1 expression in LPL-deficient mice may affect synaptic function.

  9. [Domain organization of the ORF2 C-terminal region of the German cockroach retroposon R1].

    PubMed

    Kagramanova, A S; Kapelinskaia, T V; Korolev, A L; Mukha, D V

    2010-08-01

    Using cosmid vector, a gene library of German cockroach Blattella germanica was constructed. From this library, clones containing full-length copies of two subfamilies of R1 retroposons were selected. Retroposons R1 of German cockroach belonging to different subfamilies were shown to be different in domain organization of the ORF2 C-terminal region. For the first time, retroposons transmembrane domains were identified in the sequences of R1. It was demonstrated that two retroposon R1 subfamilies of German cockroach arose as a result of intragenomic divergence rather than via horizontal transfer of alien mobile element into cockroach genome. The differences in domain organization appeared not as a result of saltatory recombination processes, but as a consequence of gradual mutation accumulation, which led to either degeneration, or to domain formation.

  10. The fnr Gene of Bacillus licheniformis and the Cysteine Ligands of the C-Terminal FeS Cluster

    PubMed Central

    Klinger, Anette; Schirawski, Jan; Glaser, Philippe; Unden, Gottfried

    1998-01-01

    In the facultatively anaerobic bacterium Bacillus licheniformis a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated. Unlike Fnr proteins from gram-negative bacteria, but like Fnr from Bacillus subtilis, the protein contained a C-terminal cluster of cysteine residues. Unlike in Fnr from B. subtilis, this cluster (Cys226-X2-Cys229-X4-Cys234) is composed of only three Cys residues, which are supposed to serve together with an internal residue (Cys71) as the ligands for an FeS center. Transfer of the B. licheniformis gene to an fnr mutant of B. subtilis complemented the ability for synthesis of nitrate reductase during anaerobic growth. PMID:9642208

  11. The fnr gene of Bacillus licheniformis and the cysteine ligands of the C-terminal FeS cluster.

    PubMed

    Klinger, A; Schirawski, J; Glaser, P; Unden, G

    1998-07-01

    In the facultatively anaerobic bacterium Bacillus licheniformis a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated. Unlike Fnr proteins from gram-negative bacteria, but like Fnr from Bacillus subtilis, the protein contained a C-terminal cluster of cysteine residues. Unlike in Fnr from B. subtilis, this cluster (Cys226-X2-Cys229-X4-Cys234) is composed of only three Cys residues, which are supposed to serve together with an internal residue (Cys71) as the ligands for an FeS center. Transfer of the B. licheniformis gene to an fnr mutant of B. subtilis complemented the ability for synthesis of nitrate reductase during anaerobic growth.

  12. Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain.

    PubMed

    Gardezi, Sabiha R; Li, Qi; Stanley, Elise F

    2013-05-15

    Calcium entry through CaV2.2 calcium channels clustered at the active zone (AZ) of the presynaptic nerve terminal gates synaptic vesicle (SV) fusion and the discharge of neurotransmitters, but the mechanism of channel scaffolding remains poorly understood. Recent studies have implicated the binding of a PDZ ligand domain (PDZ-LD) at the tip of the channel C terminal to a partner PDZ domain on RIM1/2, a synaptic vesicle-associated protein. To explore CaV2.2 scaffolding, we created intracellular region fusion proteins and used these to test for binding by 'fishing' for native CaV2.2 channels from cell lysates. Fusion proteins mimicking the distal half of the channel C terminal (C3strep) reliably captured CaV2.2 from whole brain crude membrane or purified synaptosome membrane lysates, whereas channel I-II loop or the distal half of the II-III loop proteins were negative. This capture could be replicated in a non-synaptic environment using CaV2.2 expressed in a cell line. The distal tip PDZ-LD, DDWC-COOH, was confirmed as the critical binding site by block of pull-down with mimetic peptides. Pull-down experiments using brain crude membrane lysates confirmed that RIM1/2 can bind to the DDWC PDZ-LD. However, robust CaV2.2 capture was observed from synaptosome membrane or in the cell line expression system with little or no RIM1/2 co-capture. Thus, we conclude that CaV2.2 channels can scaffold to each other via an interaction that involves the PDZ-LD by an inter-channel linkage bridged by an unknown protein.

  13. Conformational instability of the N- and C-terminal lobes of porcine pepsin in neutral and alkaline solutions.

    PubMed Central

    Lin, X.; Loy, J. A.; Sussman, F.; Tang, J.

    1993-01-01

    Pepsin contains, in a single chain, two conformationally homologous lobes that are thought to have been evolutionarily derived by gene duplication and fusion. We have demonstrated that the individual recombinant lobes are capable of independent folding and reconstitution into a two-chain pepsin or a two-chain pepsinogen (Lin, X., et al., 1992, J. Biol. Chem. 267, 17257-17263). Pepsin spontaneously inactivates in neutral or alkaline solutions. We have shown in this study that the enzymic activity of the alkaline-inactivated pepsin was regenerated by the addition of the recombinant N-terminal lobe but not by the C-terminal lobe. These results indicate that alkaline inactivation of pepsin is due to a selective denaturation of its N-terminal lobe. A complex between recombinant N-terminal lobe of pepsinogen and alkaline-denatured pepsin has been isolated. This complex is structurally similar to a two-chain pepsinogen, but it contains an extension of a denatured pepsin N-terminal lobe. Acidification of the complex is accompanied by a cleavage in the pro region and proteolysis of the denatured N-terminal lobe. The structural components that are responsible for the alkaline instability of the N-terminal lobe are likely to be carboxyl groups with abnormally high pKa values. The electrostatic potentials of 23 net carboxyl groups in the N-terminal domain (as compared to 19 in the C-terminal domain) of pepsin were calculated based on the energetics of interacting charges in the tertiary structure of the domain. The groups most probably causing the alkaline denaturation are Asp11, Asp159, Glu4, Glu13, and Asp118.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8401224

  14. Structure, Orientation and Dynamics of the C-Terminal Hexapeptide of LRAP Determined Using Solid State NMR

    SciTech Connect

    Shaw, Wendy J.; Ferris, Kim F.

    2008-12-25

    Amelogenin is the predominant protein found during enamel development and has been shown to be essential to proper enamel formation. LRAP is a naturally occurring splice variant that preserves the charged N- and C-termini of full length amelogenin, regions thought to be crucial in interacting with hydroxyapatite. Particularly, the highly charged C-terminal hexapeptide (KREEVD) is thought to be the region most intimately interacting with HAP. We investigated the structure of this charged region, as well as the proximity to the surface and the mobility of two of the residues. We found the structure to be consistent with a random coil or more extended structure, as has been found for more internalized residues in the C-terminus. The backbone K54(13C’), V58(13C’) and V58(15N) were all found to be very close to the surface of HAP, ~ 6.0 Å, suggesting a strong interaction and emphasizing the importance of these residues in interacting with HAP. However, both ends of the hexapeptide, at residues K54 and V58, experience significant mobility under hydrated conditions, implying that another portion of the protein helps to stabilize the strong LRAP-HAP interaction. Interestingly, the backbone of the C-terminal third of the protein is consistently 6.0 Å from the HAP surface, suggesting that this region of the protein is laying flat on the surface with no 3-dimensional folding. The combination of these features, i.e., a random coil structure, a significant mobility and a lack of three-dimensional folding in this region of the protein may be important in a functional role, allowing the C-terminus to effectively interact with HAP while at the same time allowing maximum crystal inhibition. The Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.

  15. Biochemical characterization of Yarrowia lipolytica LIP8, a secreted lipase with a cleavable C-terminal region.

    PubMed

    Kamoun, Jannet; Schué, Mathieu; Messaoud, Wala; Baignol, Justine; Point, Vanessa; Mateos-Diaz, Eduardo; Mansuelle, Pascal; Gargouri, Youssef; Parsiegla, Goetz; Cavalier, Jean-François; Carrière, Frédéric; Aloulou, Ahmed

    2015-02-01

    Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.

  16. Erythrocytosis-associated HIF-2α Mutations Demonstrate a Critical Role for Residues C-terminal to the Hydroxylacceptor Proline*

    PubMed Central

    Furlow, Paul W.; Percy, Melanie J.; Sutherland, Scott; Bierl, Charlene; McMullin, Mary Frances; Master, Stephen R.; Lappin, Terence R. J.; Lee, Frank S.

    2009-01-01

    A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an α subunit (HIF-α) and a β subunit (HIF-β). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-α in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-α for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-α and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosis-associated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2α. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2α in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-α. PMID:19208626

  17. Erythrocytosis-associated HIF-2alpha mutations demonstrate a critical role for residues C-terminal to the hydroxylacceptor proline.

    PubMed

    Furlow, Paul W; Percy, Melanie J; Sutherland, Scott; Bierl, Charlene; McMullin, Mary Frances; Master, Stephen R; Lappin, Terence R J; Lee, Frank S

    2009-04-03

    A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an alpha subunit (HIF-alpha) and a beta subunit (HIF-beta). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-alpha in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-alpha for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-alpha and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosis-associated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2alpha. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2alpha in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-alpha.

  18. Chemokine-Like Factor 1-Derived C-Terminal Peptides Induce the Proliferation of Dermal Microvascular Endothelial Cells in Psoriasis

    PubMed Central

    Tan, Yaqi; Wang, Yixuan; Li, Li; Xia, Jinyu; Peng, Shiguang; He, Yanling

    2015-01-01

    Psoriasis is an inflammatory disease characterized by the abnormal proliferation of skin cells, including dermal microvascular endothelial cells. Recently, chemokine-like factor 1 (CKLF1) was found to participate in the local inflammation and cell proliferation. To explore its role in the pathogenesis of psoriasis, the expression of both CKLF1 and its receptor (CCR4) was determined in the psoriatic lesions. Also, the effect of the C-terminal peptides (C19 and C27) of CKLF1 on the proliferation of human umbilical vein endothelial cells was studied in vitro. By immunohistochemistry and immunofluorescence, the expression of both CKLF1 and CCR4 was determined in the psoriatic lesions. The effect of C-terminal peptides on human umbilical vein endothelial cells (HUVECs) was studied in vitro by the evaluation of cell proliferation and apoptosis. The in vivo assessment was performed accordingly through the subcutaneous injection peptides on BALB/c mice. The results showed that, by immunohistochemistry, both CKLF1 and CCR4 were increasingly expressed in psoriatic lesions as compared to normal skins. Moreover, the primary umbilical vein endothelial cells exhibited higher proliferation ratio under the C19 or C27 stimulation, which was even enhanced by the addition of psoriatic sera or TNF-α. Furthermore, the enhancement of peptide simulation was accompanied with the activation of ERK1/2-MAPKs pathway. In addition, such effect of C19 and C27 was mirrored by the hyperproliferation of cutaneous microvessels in BALB/c mice that were subcutaneously injected with the two peptides. Therefore, we concluded that CKLF1 plays a role in the pathogenesis of psoriasis by promoting the proliferation of microvascular endothelial cells that possibly correlates with ERK1/2-MAPKs activation. PMID:25915746

  19. Compaction and binding properties of the intrinsically disordered C-terminal domain of Henipavirus nucleoprotein as unveiled by deletion studies.

    PubMed

    Blocquel, David; Habchi, Johnny; Gruet, Antoine; Blangy, Stéphanie; Longhi, Sonia

    2012-01-01

    Henipaviruses are recently emerged severe human pathogens within the Paramyxoviridae family. Their genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). We have previously shown that in Henipaviruses the N protein possesses an intrinsically disordered C-terminal domain, N(TAIL), which undergoes α-helical induced folding in the presence of the C-terminal domain (P(XD)) of the P protein. Using computational approaches, we previously identified within N(TAIL) four putative molecular recognition elements (MoREs) with different structural propensities, and proposed a structural model for the N(TAIL)-P(XD) complex where the MoRE encompassing residues 473-493 adopt an α-helical conformation at the P(XD) surface. In this work, for each N(TAIL) protein, we designed four deletion constructs bearing different combinations of the predicted MoREs. Following purification of the N(TAIL) truncated proteins from the soluble fraction of E. coli, we characterized them in terms of their conformational, spectroscopic and binding properties. These studies provided direct experimental evidence for the structural state of the four predicted MoREs, and showed that two of them have clear α-helical propensities, with the one spanning residues 473-493 being strictly required for binding to P(XD). We also showed that Henipavirus N(TAIL) and P(XD) form heterologous complexes, indicating that the P(XD) binding regions are functionally interchangeable between the two viruses. By combining spectroscopic and conformational analyses, we showed that the content in regular secondary structure is not a major determinant of protein compaction.

  20. Dual-tagged amyloid-β precursor protein reveals distinct transport pathways of its N- and C-terminal fragments.

    PubMed

    Villegas, Christine; Muresan, Virgil; Ladescu Muresan, Zoia

    2014-03-15

    The amyloid-β precursor protein (APP), a type I transmembrane protein genetically associated with Alzheimer's disease, has a complex biology that includes proteolytic processing into potentially toxic fragments, extensive trafficking and multiple, yet poorly-defined functions. We recently proposed that a significant fraction of APP is proteolytically cleaved in the neuronal soma into N- and C-terminal fragments (NTFs and CTFs), which then target independently of each other to separate destinations in the cell. Here, we prove this concept with live imaging and immunolocalization of two dual, N- and C-termini-tagged APP constructs: CFP-APP-YFP [containing the fluorescent tags, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)] and FLAG-APP-Myc. When expressed at low levels in neuronal cells, these constructs are processed into differently tagged NTFs and CTFs that reveal distinct distributions and characteristics of transport. Like the endogenous N- and C-terminal epitopes of APP, the FLAG-tagged NTFs are present in trains of vesicles and tubules that localize to short filaments, which often immunostain for acetylated tubulin, whereas the Myc-tagged CTFs are detected on randomly distributed vesicle-like structures. The experimental treatments that selectively destabilize the acetylated microtubules abrogate the distribution of NTFs along filaments, without altering the random distribution of CTFs. These results indicate that the NTFs and CTFs are recruited to distinct transport pathways and reach separate destinations in neurons, where they likely accomplish functions independent of the parental, full-length APP. They also point to a compartment associated with acetylated microtubules in the neuronal soma--not the neurite terminals--as a major site of APP cleavage, and segregation of NTFs from CTFs.

  1. The nine C-terminal residues of the grapevine fanleaf nepovirus movement protein are critical for systemic virus spread.

    PubMed

    Belin, C; Schmitt, C; Gaire, F; Walter, B; Demangeat, G; Pinck, L

    1999-06-01

    The grapevine fanleaf virus (GFLV) RNA2-encoded polyprotein P2 is proteolytically cleaved by the RNA1-encoded proteinase to yield protein 2A, 2B(MP) movement protein and 2C(CP) coat protein. To further investigate the role of the 2B(MP) and 2C(CP) proteins in virus movement, RNA2 was engineered by alternatively replacing the GFLV 2B(MP) and 2C(CP) genes with their counterparts from the closely related Arabis mosaic virus (ArMV). Transcripts of all chimeric RNA2s were able to replicate in Chenopodium quinoa protoplasts and form tubules in tobacco BY-2 protoplasts in the presence of the infectious transcript of GFLV RNA1. Virus particles were produced when the GFLV 2C(CP) gene was replaced with its ArMV counterpart, but systemic virus spread did not occur in C. quinoa plants. In addition, chimeric RNA2 containing the complete ArMV 2B(MP) gene was neither encapsidated nor infectious on plants, probably because polyprotein P2 was incompletely processed. However, chimeric RNA2 encoding ArMV 2B(MP), in which the nine C-terminal residues were those of GFLV 2B(MP), formed virus particles and were infectious in the presence of GFLV but not ArMV 2C(CP). These results suggest that the nine C-terminal residues of 2B(MP) must be of the same virus origin as the proteinase for efficient proteolytic processing of polyprotein P2 and from the same virus origin as the 2C(CP) for systemic virus spread.

  2. PAAR-Rhs proteins harbor various C-terminal toxins to diversify the antibacterial pathways of type VI secretion systems.

    PubMed

    Ma, Jiale; Sun, Min; Dong, Wenyang; Pan, Zihao; Lu, Chengping; Yao, Huochun

    2017-01-01

    The type VI secretion system (T6SS) of bacteria plays a key role in competing for specific niches by the contact-dependent killing of competitors. Recently, Rhs proteins with polymorphic C-terminal toxin-domains that inhibit or kill neighboring cells were identified. In this report, we identified a novel Rhs with an MPTase4 (Metallopeptidase-4) domain (designated as Rhs-CT1) that showed an antibacterial effect via T6SS in Escherichia coli. We managed to develop a specific strategy by matching the diagnostic domain-architecture of Rhs-CT1 (Rhs with an N-terminal PAAR-motif and a C-terminal toxin domain) for effector retrieval and discovered a series of Rhs-CTs in E. coli. Indeed, the screened Rhs-CT3 with a REase-3 (Restriction endonuclease-3) domain also mediated interbacterial antagonism. Further analysis revealed that vgrGO1 and eagR/DUF1795 (upstream of rhs-ct) were required for the delivery of Rhs-CTs, suggesting eagR as a potential T6SS chaperone. In addition to chaperoned Rhs-CTs, neighborless Rhs-CTs could be classified into a distinct family (Rhs-Nb) sharing close evolutionary relationship with T6SS2-Rhs (encoded in the T6SS2 cluster of E. coli). Notably, the Rhs-Nb-CT5 was confirmed bioinformatically and experimentally to mediate interbacterial antagonism via Hcp2B-VgrG2 module. In a further retrieval analysis, we discovered various toxin/immunity pairs in extensive bacterial species that could be systematically classified into eight referential clans, suggesting that Rhs-CTs greatly diversify the antibacterial pathways of T6SS.

  3. A salt-bridge stabilized C-terminal hook is critical for the dimerization of a Bowman Birk inhibitor.

    PubMed

    Kumar, Vinod; Murugeson, Saravanan; Vithani, Neha; Prakash, Balaji; Gowda, Lalitha R

    2015-01-15

    Legume Bowman-Birk inhibitors (BBIs) that inhibit mammalian proteases exist as dimers in solution. The structural basis governing dimerization of HGI-III (horsegram seed BBI) was investigated. An intra-monomer salt bridge (D76-K71) stabilizes an atypical hook-like conformation at the C-terminus. We postulate that this hook, positions D75 to enable an inter-monomer salt-bridge D75(a)-K24(b), which results in dimerization. We verify this by K71A and D76A mutations of HGI-III. The mutants were both monomers, likely due to destabilization of the C-terminal hook. Dimerization was sustained in a double mutant K71D/D76K that was anticipated to form a similar hook critical for dimerization. Conversely, K24(b) that interacts with D75(a) of the loop is the specificity determining residue that interacts with trypsin to inhibit its activity. The inter-monomer salt bridge D75(a)-K24(b) must be disrupted for the inhibition of trypsin, requiring HGI-III to transition into a monomer. Size exclusion studies and a model of HGI-III-trypsin complex support this notion. Interestingly, isoforms of the inhibitor present in germinated seeds (HGGIs) are monomers; and most strikingly, the C-termini of these inhibitors are truncated with the loss the C-terminal hook critical for dimerization. The tendency of HGI-III to self-associate seems to relate to its physiological function of a storage protein.

  4. Regulation of ABCC6 trafficking and stability by a conserved C-terminal PDZ-like sequence.

    PubMed

    Xue, Peng; Crum, Chelsea M; Thibodeau, Patrick H

    2014-01-01

    Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE). The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein.

  5. Aph-1 associates directly with full-length and C-terminal fragments of gamma-secretase substrates.

    PubMed

    Chen, Allen C; Guo, Lucie Y; Ostaszewski, Beth L; Selkoe, Dennis J; LaVoie, Matthew J

    2010-04-09

    Gamma-secretase is a ubiquitous, multiprotein enzyme composed of presenilin, nicastrin, Aph-1, and Pen-2. It mediates the intramembrane proteolysis of many type 1 proteins, plays an essential role in numerous signaling pathways, and helps drive the pathogenesis of Alzheimer disease by excising the hydrophobic, aggregation-prone amyloid beta-peptide from the beta-amyloid precursor protein. A central unresolved question is how its many substrates bind and enter the gamma-secretase complex. Here, we provide evidence that both the beta-amyloid precursor protein holoprotein and its C-terminal fragments, the immediate substrates of gamma-secretase, can associate with Aph-1 at overexpressed as well as endogenous protein levels. This association was observed using bi-directional co-immunoprecipitation in multiple systems and detergent conditions, and an beta-amyloid precursor protein-Aph-1 complex was specifically isolated following in situ cross-linking in living cells. In addition, another endogenous canonical gamma-substrate, Jagged, showed association of both its full-length and C-terminal fragment forms with Aph-1. We were also able to demonstrate that this interaction with substrates was conserved across the multiple isoforms of Aph-1 (beta, alphaS, and alphaL), as they were all able to bind beta-amyloid precursor protein with similar affinity. Finally, two highly conserved intramembrane histidines (His-171 and His-197) within Aph-1, which were recently shown to be important for gamma-secretase activity, are required for efficient binding of substrates. Taken together, our data suggest a dominant role for Aph-1 in interacting with gamma-secretase substrates prior to their processing by the proteolytic complex.

  6. Human aldolase A natural mutants: relationship between flexibility of the C-terminal region and enzyme function.

    PubMed Central

    Esposito, Gabriella; Vitagliano, Luigi; Costanzo, Paola; Borrelli, Loredana; Barone, Rita; Pavone, Lorenzo; Izzo, Paola; Zagari, Adriana; Salvatore, Francesco

    2004-01-01

    We have identified a new mutation in the FBP (fructose 1,6-bisphosphate) aldolase A gene in a child with suspected haemolytic anaemia associated with myopathic symptoms at birth and with a subsequent diagnosis of arthrogryposis multiplex congenita and pituitary ectopia. Sequence analysis of the whole gene, also performed on the patient's full-length cDNA, revealed only a Gly346-->Ser substitution in the heterozygous state. We expressed in a bacterial system the new aldolase A Gly346-->Ser mutant, and the Glu206-->Lys mutant identified by others, in a patient with an aldolase A deficit. Analysis of their functional profiles showed that the Gly346Ser mutant had the same Km as the wild-type enzyme, but a 4-fold lower kcat. The Glu206-->Lys mutant had a Km approx. 2-fold higher than that of both the Gly346-->Ser mutant and the wild-type enzyme, and a kcat value 40% less than the wild-type. The Gly346-->Ser and wild-type enzymes had the same Tm (melting temperature), which was approx. 6-7 degrees C higher than that of the Glu206-->Lys enzyme. An extensive molecular graphic analysis of the mutated enzymes, using human and rabbit aldolase A crystallographic structures, suggests that the Glu206-->Lys mutation destabilizes the aldolase A tetramer at the subunit interface, and highlights the fact that the glycine-to-serine substitution at position 346 limits the flexibility of the C-terminal region. These results also provide the first evidence that Gly346 is crucial for the correct conformation and function of aldolase A, because it governs the entry/release of the substrates into/from the enzyme cleft, and/or allows important C-terminal residues to approach the active site. PMID:14766013

  7. NMR Determines Transient Structure and Dynamics in the Disordered C-Terminal Domain of WASp Interacting Protein

    PubMed Central

    Haba, Noam Y.; Gross, Renana; Novacek, Jiri; Shaked, Hadassa; Zidek, Lukas; Barda-Saad, Mira; Chill, Jordan H.

    2013-01-01

    WASp-interacting protein (WIP) is a 503-residue proline-rich polypeptide expressed in human T cells. The WIP C-terminal domain binds to Wiskott-Aldrich syndrome protein (WASp) and regulates its activation and degradation, and the WIP-WASp interaction has been shown to be critical for actin polymerization and implicated in the onset of WAS and X-linked thrombocytopenia. WIP is predicted to be an intrinsically disordered protein, a class of polypeptides that are of great interest because they violate the traditional structure-function paradigm. In this first (to our knowledge) study of WIP in its unbound state, we used NMR to investigate the biophysical behavior of WIPC, a C-terminal domain fragment of WIP that includes residues 407–503 and contains the WASp-binding site. In light of the poor spectral dispersion exhibited by WIPC and the high occurrence (25%) of proline residues, we employed 5D-NMR13C-detected NMR experiments with nonuniform sampling to accomplish full resonance assignment. Secondary chemical-shift analysis, 15N relaxation rates, and protection from solvent exchange all concurred in detecting transient structure located in motifs that span the WASp-binding site. Residues 446–456 exhibited a propensity for helical conformation, and an extended conformation followed by a short, capped helix was observed for residues 468–478. The 13C-detected approach allows chemical-shift assignment in the WIPC polyproline stretches and thus sheds light on their conformation and dynamics. The effects of temperature on chemical shifts referenced to a denatured sample of the polypeptide demonstrate that heating reduces the structural character of WIPC. Thus, we conclude that the disordered WIPC fragment is comprised of regions with latent structure connected by flexible loops, an architecture with implications for binding affinity and function. PMID:23870269

  8. The pH-sensitive structure of the C-terminal domain of voltage-gated proton channel and the thermodynamic characteristics of Zn{sup 2+} binding to this domain

    SciTech Connect

    Zhao, Qing; Li, Chuanyong; Li, Shu Jie

    2015-01-02

    Highlights: • The α-helical content of the C-terminus is decreased with a pH increase. • The thermostability of the C-terminus is decreased with a pH increase. • Zn{sup 2+} binds to His{sup 244} and His{sup 266} residues within the C-terminal domain. • The binding of Zn{sup 2+} to His{sup 244} residue is an endothermic heat reaction. • The binding of Zn{sup 2+} to His{sup 266} residue is an exothermic heat reaction. - Abstract: The voltage-gated proton channel Hv1 is strongly sensitive to Zn{sup 2+}. The H{sup +} conduction is decreased at a high concentration of Zn{sup 2+} and Hv1 channel closing is slowed by the internal application of Zn{sup 2+}. Although the recent studies demonstrated that Zn{sup 2+} interacts with the intracellular C-terminal domain, the binding sites and details of the interaction remain unknown. Here, we studied the pH-dependent structural stability of the intracellular C-terminal domain of human Hv1 and showed that Zn{sup 2+} binds to His{sup 244} and His{sup 266} residues. The thermodynamics signature of Zn{sup 2+} binding to the two sites was investigated by isothermal titration calorimetry. The binding of Zn{sup 2+} to His{sup 244} (mutant H266A) and His{sup 266} (mutant H244A) were an endothermic heat reaction and an exothermic heat reaction, respectively.

  9. The retroviral hypermutation specificity of APOBEC3F and APOBEC3G is governed by the C-terminal DNA cytosine deaminase domain.

    PubMed

    Haché, Guylaine; Liddament, Mark T; Harris, Reuben S

    2005-03-25

    The human proteins APOBEC3F and APOBEC3G restrict retroviral infection by deaminating cytosine residues in the first cDNA strand of a replicating virus. These proteins have two putative deaminase domains, and it is unclear whether one or both catalyze deamination, unlike their homologs, AID and APOBEC1, which are well characterized single domain deaminases. Here, we show that only the C-terminal cytosine deaminase domain of APOBEC3F and -3G governs retroviral hypermutation. A chimeric protein with the N-terminal cytosine deaminase domain from APOBEC3G and the C-terminal cytosine deaminase domain from APOBEC3F elicited a dinucleotide hypermutation preference nearly indistinguishable from that of APOBEC3F. This 5'-TC-->TT mutational specificity was confirmed in a heterologous Escherichia coli-based mutation assay, in which the 5'-CC-->CT dinucleotide hypermutation preference of APOBEC3G also mapped to the C-terminal deaminase domain. An N-terminal APOBEC3G deletion mutant displayed a preference indistinguishable from that of the full-length protein, and replacing the C-terminal deaminase domain of APOBEC3F with AID resulted in an AID-like mutational signature. Together, these data indicate that only the C-terminal domain of APOBEC3F and -3G dictates the retroviral minus strand 5'-TC and 5'-CC dinucleotide hypermutation preferences, respectively, leaving the N-terminal domain to perform other aspects of retroviral restriction.

  10. Removal of the four C-terminal glycine-rich repeats enhances the thermostability and substrate binding affinity of barley beta-amylase.

    PubMed

    Ma, Y F; Eglinton, J K; Evans, D E; Logue, S J; Langridge, P

    2000-11-07

    Barley beta-amylase undergoes proteolytic cleavage in the C-terminal region after germination. The implication of the cleavage in the enzyme's characteristics is unclear. With purified native beta-amylases from both mature barley grain and germinated barley, we found that the beta-amylase from germinated barley had significantly higher thermostability and substrate binding affinity for starch than that from mature barley grain. To better understand the effect of the proteolytic cleavage on the enzyme's thermostability and substrate binding affinity for starch, recombinant barley beta-amylases with specific deletions at the C-terminal tail were generated. The complete deletion of the four C-terminal glycine-rich repeats significantly increased the enzyme's thermostability, but an incomplete deletion with one repeat remaining did not change the thermostability. Although different C-terminal deletions affect the thermostability differently, they all increased the enzyme's affinity for starch. The possible reasons for the increased thermostability and substrate binding affinity, due to the removal of the four C-terminal glycine-rich repeats, are discussed in terms of the three-dimensional structure of beta-amylase.

  11. C-terminal domain on the outer surface of the Macrobrachium rosenbergii nodavirus capsid is required for Sf9 cell binding and internalization.

    PubMed

    Somrit, Monsicha; Watthammawut, Atthaboon; Chotwiwatthanakun, Charoonroj; Ounjai, Puey; Suntimanawong, Wanida; Weerachatyanukul, Wattana

    2017-01-02

    We have shown that Macrobrachium rosenbergii nodavirus (MrNV) was able to infect Sf9 cells and that MrNV virus-like particles (MrNV-VLPs) were capable nanocontainers for delivering nucleic acid-based materials. Here, we demonstrated that chymotryptic removal of a C-terminal peptide and its truncated variant (F344-MrNV-VLPs) exhibited a drastically reduced ability to interact and internalize into Sf9 cells. Electron microscopic observations revealed that the loss of C-terminal domain either from enzyme hydrolysis or genetic truncation did not affect the generated MrNV-VLPs' icosahedral conformation, but did drastically affect the VLPs' internalization ability into Sf9 cells. Homology-based modelling of the MrNV capsid with other icosahedral capsid models revealed that this chymotrypsin-sensitive C-terminal domain was not only exposed on the capsid surface, but also constituted the core of the viral capsid protrusion. These results therefore suggest the importance of the C-terminal domain as a structure for targeted cell interaction which is presumably localized at the protruding domain. This work thus provided the functional insights into the role of the MrNV C-terminal domain in viral entry into Sf9 cells and lead to the development of strategies in combatting MrNV infection in susceptible cells.

  12. Role of the C-terminal extension peptide of plastid located glutamine synthetase from Medicago truncatula: Crucial for enzyme activity and needless for protein import into the plastids.

    PubMed

    Ferreira, Maria João; Vale, Diogo; Cunha, Luis; Melo, Paula

    2017-02-01

    Glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is encoded by a small family of highly homologous nuclear genes that produce cytosolic (GS1) and plastidic (GS2) isoforms. Compared to GS1, GS2 proteins have two extension peptides, one at the N- and the other at the C-terminus, which show a high degree of conservation among plant species. It has long been known that the N-terminal peptide acts as a transit peptide, targeting the protein to the plastids however, the function of the C-terminal extension is still unknown. To investigate whether the C-terminal extension influences the activity of the enzyme, we produced a C-terminal truncated version of Medicago truncatula GS2a in Escherechia coli and studied its catalytic properties. The activity of the truncated protein was found to be lower than that of MtGS2a and with less affinity for glutamate. The importance of the C-terminal extension for the protein import into the chloroplast was also assessed by transient expression of fluorescently-tagged MtGS2a truncated at the C-terminus, which was correctly detected in the chloroplast. The results obtained in this work demonstrate that the C-terminal extension of M. truncatula GS2a is important for the activity of the enzyme and does not contain crucial information for the import process.

  13. Intrinsic disorder in the C-terminal domain of the Shaker voltage-activated K+ channel modulates its interaction with scaffold proteins

    PubMed Central

    Magidovich, Elhanan; Orr, Irit; Fass, Deborah; Abdu, Uri; Yifrach, Ofer

    2007-01-01

    The interaction of membrane-embedded voltage-activated potassium channels (Kv) with intracellular scaffold proteins, such as the postsynaptic density 95 (PSD-95) protein, is mediated by the channel C-terminal segment. This interaction underlies Kv channel clustering at unique membrane sites and is important for the proper assembly and functioning of the synapse. In the current study, we address the molecular mechanism underlying Kv/PSD-95 interaction. We provide experimental evidence, based on hydrodynamic and spectroscopic analyses, indicating that the isolated C-terminal segment of the archetypical Shaker Kv channel (ShB-C) is a random coil, suggesting that ShB-C belongs to the recently defined class of intrinsically disordered proteins. We show that isolated ShB-C is still able to bind its scaffold protein partner and support protein clustering in vivo, indicating that unfoldedness is compatible with ShB-C activity. Pulldown experiments involving C-terminal chains differing in flexibility or length further demonstrate that intrinsic disorder in the C-terminal segment of the Shaker channel modulates its interaction with the PSD-95 protein. Our results thus suggest that the C-terminal domain of the Shaker Kv channel behaves as an entropic chain and support a “fishing rod” molecular mechanism for Kv channel binding to scaffold proteins. The importance of intrinsically disordered protein segments to the complex processes of synapse assembly, maintenance, and function is discussed. PMID:17666528

  14. Distributed computing and NMR constraint-based high-resolution structure determination: applied for bioactive Peptide endothelin-1 to determine C-terminal folding.

    PubMed

    Takashima, Hiroyuki; Mimura, Norio; Ohkubo, Tadayasu; Yoshida, Takuya; Tamaoki, Haruhiko; Kobayashi, Yuji

    2004-04-14

    Distributed computing has been implemented to the solution structure determination of endothelin-1 to evaluate efficiency of the method for NMR constraint-based structure calculations. A key target of the investigation was determination of the C-terminal folding of the peptide, which had been dispersed in previous studies of NMR, despite its pharmacological significances. With use of tens of thousands of random initial structures to explore the conformational space comprehensively, we determined high-resolution structures with good convergences of C-terminal as well as previously defined N-terminal structures. The previous studies had missed the C-terminal convergence because of initial structure dependencies trapped in localized folding of the N-terminal region, which are strongly constricted by two disulfide bonds.

  15. Extrusion of the C-terminal Helix in Navel Orangeworm Moth Pheromone-Binding Protein (AtraPBP1) Controls Pheromone Binding†

    PubMed Central

    Xu, Wei; Xu, Xianzhong; Leal, Walter S.; Ames, James B.

    2011-01-01

    The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present a mutational analysis on a PBP from Amyelois transitella (AtraPBP1) to evaluate how the C-terminal helix in this protein controls pheromone binding as a function of pH. Pheromone binds tightly to AtraPBP1 at neutral pH, but the binding is much weaker at pH below 5. Deletion of the entire C-terminal helix (residues 129–142) causes more than 100-fold increase in pheromone binding affinity at pH 5 and only a 1.5-fold increase at pH 7. A similar pH-dependent increase in pheromone binding is also seen for the H80A/H95A double mutant that promotes extrusion of the C-terminal helix by disabling salt bridges at each end of the helix. The single mutants (H80A and H95A) also exhibit pheromone binding at pH below 5, but with ~2-fold weaker affinity. NMR and circular dichroism data demonstrate a large overall structural change in each of these mutants at pH 4.5, indicating an extrusion of the C-terminal helix that profoundly affects the overall structure of the low pH form. Our results confirm that sequestration of the C-terminal helix at low pH as seen in the recent NMR structure may serve to block pheromone binding. We propose that extrusion of these C-terminal residues at neutral pH (or by the mutations in this study) exposes a hydrophobic cleft that promotes high affinity pheromone binding. PMID:21130734

  16. Extrusion of the C-terminal helix in navel orangeworm moth pheromone-binding protein (AtraPBP1) controls pheromone binding.

    PubMed

    Xu, Wei; Xu, Xianzhong; Leal, Walter S; Ames, James B

    2011-01-07

    The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present a mutational analysis on a PBP from A. transitella (AtraPBP1) to evaluate how the C-terminal helix in this protein controls pheromone binding as a function of pH. Pheromone binds tightly to AtraPBP1 at neutral pH, but the binding is much weaker at pH below 5. Deletion of the entire C-terminal helix (residues 129-142) causes more than 100-fold increase in pheromone-binding affinity at pH 5 and only a 1.5-fold increase at pH 7. A similar pH-dependent increase in pheromone binding is also seen for the H80A/H95A double mutant that promotes extrusion of the C-terminal helix by disabling salt bridges at each end of the helix. The single mutants (H80A and H95A) also exhibit pheromone binding at pH below 5, but with ∼2-fold weaker affinity. NMR and circular dichroism data demonstrate a large overall structural change in each of these mutants at pH 4.5, indicating an extrusion of the C-terminal helix that profoundly affects the overall structure of the low pH form. Our results confirm that sequestration of the C-terminal helix at low pH as seen in the recent NMR structure may serve to block pheromone binding. We propose that extrusion of these C-terminal residues at neutral pH (or by the mutations in this study) exposes a hydrophobic cleft that promotes high affinity pheromone binding.

  17. Specific sequences determine the stability and cooperativity of folding of the C-terminal half of tropomyosin.

    PubMed

    Paulucci, Adriana A; Hicks, Leslie; Machado, Alessandra; Miranda, M Teresa M; Kay, Cyril M; Farah, Chuck S

    2002-10-18

    Tropomyosin is a flexible 410 A coiled-coil protein in which the relative stabilities of specific regions may be important for its proper function in the control of muscle contraction. In addition, tropomyosin can be used as a simple model of natural occurrence to understand the inter- and intramolecular interactions that govern the stability of coiled-coils. We have produced eight recombinant tropomyosin fragments (Tm(143-284(5OHW),) Tm(189-284(5OHW)), Tm(189-284), Tm(220-284(5OHW)), Tm(220-284), Tm(143-235), Tm(167-260), and Tm(143-260)) and one synthetic peptide (Ac-Tm(215-235)) to investigate the relative conformational stability of different regions derived from the C-terminal region of the protein, which is known to interact with the troponin complex. Analytical ultracentrifugation experiments show that the fragments that include the last 24 residues of the molecule (Tm(143-284(5OHW)), Tm(189-284(5OHW)), Tm(220-284(5OHW)), Tm(220-284)) are completely dimerized at 10 microm dimer (50 mm phosphate, 100 mm NaCl, 1.0 mm dithiothreitol, and 0.5 mm EDTA, 10 degrees C), whereas fragments that lack the native C terminus (Tm(143-235),Tm(167-260), and Tm(143-260)) are in a monomer-dimer equilibrium under these conditions. The presence of trifluoroethanol resulted in a reduction in the [theta](222)/[theta](208) circular dichroism ratio in all of the fragments and induced stable trimer formation only in those containing residues 261-284. Urea denaturation monitored by circular dichroism and fluorescence revealed that residues 261-284 of tropomyosin are very important for the stability of the C-terminal half of the molecule as a whole. Furthermore, the absence of this region greatly increases the cooperativity of urea-induced unfolding. Temperature and urea denaturation experiments show that Tm(143-235) is less stable than other fragments of the same size. We have identified a number of factors that may contribute to this particular instability, including an interhelix

  18. Helicity of alpha(404-451) and beta(394-445) tubulin C-terminal recombinant peptides.

    PubMed Central

    Jimenez, M. A.; Evangelio, J. A.; Aranda, C.; Lopez-Brauet, A.; Andreu, D.; Rico, M.; Lagos, R.; Andreu, J. M.; Monasterio, O.

    1999-01-01

    We have investigated the solution conformation of the functionally relevant C-terminal extremes of alpha- and beta-tubulin, employing the model recombinant peptides RL52alpha3 and RL33beta6, which correspond to the amino acid sequences 404-451(end) and 394-445(end) of the main vertebrate isotypes of alpha- and beta-tubulin, respectively, and synthetic peptides with the alpha-tubulin(430-443) and beta-tubulin(412-431) internal sequences. Alpha(404-451) and beta(394-445) are monomeric in neutral aqueous solution (as indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of beta(394-445) with subtilisin, instead of giving extensive degradation, resulted in main cleavages at positions Thr409-Glu410 and Tyr422-Gln423-Gln424, defining the proteolysis resistant segment 410-422, which corresponds to the central part of the predicted beta-tubulin C-terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol (TFE)-induced markedly helical CD spectra in alpha(404-451) and beta(394-445). A substantial part of the helicity of beta(394-445) was found to be in the CD spectrum of the shorter peptide beta(412-431) with TFE. Two-dimensional 1H-NMR parameters (nonsequential nuclear Overhauser effects (NOE) and conformational C alphaH shifts) in 30% TFE permitted to conclude that about 25% of alpha(404-451) and 40% of beta(394-451) form well-defined helices encompassing residues 418-432 and 408-431, respectively, flanked by disordered N- and C-segments. The side chains of beta(394-451) residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both alpha and beta chains share a characteristic sequence of

  19. Five glutamic acid residues in the C-terminal domain of the ChlD subunit play a major role in conferring Mg(2+) cooperativity upon magnesium chelatase.

    PubMed

    Brindley, Amanda A; Adams, Nathan B P; Hunter, C Neil; Reid, James D

    2015-11-10

    Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis by inserting a Mg(2+) ion into protoporphyrin IX in an ATP-dependent manner. The cyanobacterial (Synechocystis) and higher-plant chelatases exhibit a complex cooperative response to free magnesium, while the chelatases from Thermosynechococcus elongatus and photosynthetic bacteria do not. To investigate the basis for this cooperativity, we constructed a series of chimeric ChlD proteins using N-terminal, central, and C-terminal domains from Synechocystis and Thermosynechococcus. We show that five glutamic acid residues in the C-terminal domain play a major role in this process.

  20. Identification of the in vivo truncation sites at the C-terminal region of alpha-A crystallin from aged bovine and human lens

    NASA Technical Reports Server (NTRS)

    Takemoto, L. J.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Total alpha-A crystallin was purified from young versus old lens, followed by digestion with cyanogen bromide. Laser desorption mass spectrometry of the C-terminal fragment demonstrated age-dependent loss of one and five amino acids from the C-terminus of alpha-A crystallin from both bovine and human lens. These results demonstrate specific peptide bonds of alpha-A crystallin are cleaved during the aging process of the normal lens. The C-terminal region is cleaved in two places between the two hydroxyl-containing amino acids present in the sequence -P-S(T)-S-.

  1. Native Chemical Ligation Strategy to Overcome Side Reactions during Fmoc-Based Synthesis of C-Terminal Cysteine-Containing Peptides.

    PubMed

    Lelièvre, Dominique; Terrier, Victor P; Delmas, Agnès F; Aucagne, Vincent

    2016-03-04

    The Fmoc-based solid phase synthesis of C-terminal cysteine-containing peptides is problematic, due to side reactions provoked by the pronounced acidity of the Cα proton of cysteine esters. We herein describe a general strategy consisting of the postsynthetic introduction of the C-terminal Cys through a key chemoselective native chemical ligation reaction with N-Hnb-Cys peptide crypto-thioesters. This method was successfully applied to the demanding peptide sequences of two natural products of biological interest, giving remarkably high overall yields compared to that of a state of the art strategy.

  2. Loss of autoinhibition of the plasma membrane Ca(2+) pump by substitution of aspartic 170 by asparagin. A ctivation of plasma membrane calcium ATPase 4 without disruption of the interaction between the catalytic core and the C-terminal regulatory domain.

    PubMed

    Bredeston, Luis M; Adamo, Hugo P

    2004-10-01

    The plasma membrane calcium ATPase (PMCA) actively transports Ca(2+) from the cytosol to the extra cellular space. The C-terminal segment of the PMCA functions as an inhibitory domain by interacting with the catalytic core. Ca(2+)-calmodulin binds to the C-terminal segment and stops inhibition. Here we showed that residue Asp(170), in the putative "A" domain of human PMCA isoform 4xb, plays a critical role in autoinhibition. In the absence of calmodulin a PMCA containing a site-specific mutation of D170N had 80% of the maximum activity of the calmodulin-activated PMCA and a similar high affinity for Ca(2+). The mutation did not change the activation of the PMCA by ATP. Deletion of the C-terminal segment further downstream of the calmodulin-binding site led to an additional increase in the maximal activity of the mutant, which suggests that the mutation did not affect the inhibition because of this portion of the C-terminal segment. The calmodulin-activated PMCA was more sensitive to vanadate inhibition than the autoinhibited enzyme. In contrast, inhibition of the D170N mutant required higher concentrations of vanadate and was not affected by calmodulin. Despite its higher basal activity, the mutant had an apparent affinity for calmodulin similar to that of the wild type enzyme, and its rate of proteolysis at the C-terminal segment was still calmodulin-dependent. Altogether these results suggest that activation by mutation D170N does not involve the displacement of the calmodulin-binding autoinhibitory domain from the catalytic core and may arise directly from changes in the accessibility to the calcium-binding residues of the pump.

  3. A C-Terminal Coiled-Coil Region of CagL is Responsible for Helicobacter Pylori-Induced Il-8 Expression.

    PubMed

    Wiedemann, Tobias; Hofbaur, Stefan; Loell, Eva; Rieder, Gabriele

    2016-09-29

    Interleukin-8 (IL-8) is a potent neutrophil-activating chemokine which triggers the infiltration and migration of neutrophils into areas of bacterial infection. Helicobacter pylori-infected patient studies as well as animal models have revealed that H. pylori type I strains carrying an intact cytotoxin-associated gene pathogenicity island (cag-PAI) with a functional type IV secretion system (T4SS) induce IL-8 expression and secretion in gastric mucosa. This gastric mucosal IL-8 expression correlates with severe histological changes due to H. pylori infection. In the present study, we explored a new recognition pattern on the bacterial adhesion protein CagL inducing IL-8 expression in H. pylori-infected host cells. To analyze the secreted IL-8 concentration, we performed IL-8 enzyme-linked immunosorbent assay (ELISA). To investigate the H. pylori-induced IL-8 expression on the transcriptional level, we transiently transfected gastric epithelial cells (AGS) with a human IL-8 luciferase reporter construct. The results of this study demonstrate that specifically the C-terminal coiled-coil region of the H. pylori CagL protein, a protein described to be located on the tip of the T4SS-pilus, is responsible for several in vitro observations: 1) H. pylori-induced IL-8 secretion via the transforming growth factor (TGF)-α activated epidermal growth factor-receptor (EGF-R) signaling pathway; 2) H. pylori-induced elongation of the cells, a typical CagA-induced phenotype; and 3) the bridging of the T4SS to its human target cells. This novel bacterial-host recognition sequence allows a new insight into how H. pylori induces the inflammatory response in gastric epithelial cells and facilitates the development of precancerous conditions.

  4. A C-Terminal Coiled-Coil Region of CagL is Responsible for Helicobacter Pylori-Induced Il-8 Expression

    PubMed Central

    Wiedemann, Tobias; Hofbaur, Stefan; Loell, Eva; Rieder, Gabriele

    2016-01-01

    Interleukin-8 (IL-8) is a potent neutrophil-activating chemokine which triggers the infiltration and migration of neutrophils into areas of bacterial infection. Helicobacter pylori-infected patient studies as well as animal models have revealed that H. pylori type I strains carrying an intact cytotoxin-associated gene pathogenicity island (cag-PAI) with a functional type IV secretion system (T4SS) induce IL-8 expression and secretion in gastric mucosa. This gastric mucosal IL-8 expression correlates with severe histological changes due to H. pylori infection. In the present study, we explored a new recognition pattern on the bacterial adhesion protein CagL inducing IL-8 expression in H. pylori-infected host cells. To analyze the secreted IL-8 concentration, we performed IL-8 enzyme-linked immunosorbent assay (ELISA). To investigate the H. pylori-induced IL-8 expression on the transcriptional level, we transiently transfected gastric epithelial cells (AGS) with a human IL-8 luciferase reporter construct. The results of this study demonstrate that specifically the C-terminal coiled-coil region of the H. pylori CagL protein, a protein described to be located on the tip of the T4SS-pilus, is responsible for several in vitro observations: 1) H. pylori-induced IL-8 secretion via the transforming growth factor (TGF)-α activated epidermal growth factor-receptor (EGF-R) signaling pathway; 2) H. pylori-induced elongation of the cells, a typical CagA-induced phenotype; and 3) the bridging of the T4SS to its human target cells. This novel bacterial-host recognition sequence allows a new insight into how H. pylori induces the inflammatory response in gastric epithelial cells and facilitates the development of precancerous conditions. PMID:27766167

  5. Expression, purification and reconstitution of the C-terminal transmembrane domain of scavenger receptor BI into detergent micelles for NMR analysis.

    PubMed

    Chadwick, Alexandra C; Jensen, Davin R; Peterson, Francis C; Volkman, Brian F; Sahoo, Daisy

    2015-03-01

    Scavenger receptor class B type I (SR-BI), the high density lipoprotein (HDL) receptor, is important for the delivery of HDL-cholesteryl esters to the liver for excretion via bile formation. The focus on therapeutic strategies aimed at reducing cholesterol levels highlights the critical need to understand the structural features of SR-BI that drive cholesterol removal. Yet, in the absence of a high-resolution structure of SR-BI, our understanding of how SR-BI interacts with HDL is limited. In this study, we have optimized the NMR solution conditions for the structural analysis of the C-terminal transmembrane domain of SR-BI that harbors putative domains required for receptor oligomerization. An isotopically-labeled SR-BI peptide encompassing residues 405-475 was bacterially-expressed and purified. [U-(15)N]-SR-BI(405-475) was incorporated into different detergent micelles and assessed by (1)H-(15)N-HSQC in order to determine which detergent micelle best maintained SR-BI(405-475) in a folded, native conformation for subsequent NMR analyses. We also determined the optimal detergent concentration used in micelles, as well as temperature, solution buffer and pH conditions. Based on (1)H-(15)N-HSQC peak dispersion, intensity, and uniformity, we determined that [U-(15)N]-SR-BI(405-475) should be incorporated into 5% detergent micelles consisting of 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-[1'-rac-glycerol] (LPPG) and data collected at 40°C in a non-buffered solution at pH 6.8. Furthermore, we demonstrate the ability of SR-BI(405-475) to form dimers upon chemical crosslinking. These studies represent the first steps in obtaining high-resolution structural information by NMR for the HDL receptor that plays a critical role in regulating whole body cholesterol removal.

  6. A new role for the neuronal ubiquitin C-terminal hydrolase-L1 (UCH-L1) in podocyte process formation and podocyte injury in human glomerulopathies.

    PubMed

    Meyer-Schwesinger, C; Meyer, T N; Münster, S; Klug, P; Saleem, M; Helmchen, U; Stahl, R A K

    2009-02-01

    Glomerular epithelial cell (podocyte) injury is characterized by foot process retraction, slit diaphragm reorganization, and degradation of podocyte-specific proteins. However, the mechanisms underlying podocyte injury are largely unknown. The ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a key modulator of ubiquitin modification in neurons. Like neurons, UCH-L1 expression was associated with an undifferentiated status in cultured human podocytes, whereas differentiation and arborization decreased UCH-L1 and monoUb expression. Inhibition of UCH-L1 induced time and concentration-dependent process formation with alpha-actinin-4 distribution to the cell membrane and processes. An immunohistochemical approach was used to evaluate whether UCH-L1 expression was associated with podocyte injury in 15 different human glomerular diseases. Whereas normal kidneys expressed no UCH-L1 and little ubiquitin, a subset of human glomerulopathies associated with podocyte foot process effacement (membranous nephropathy, SLE class V, FSGS) de novo expressed UCH-L1 in podocyte cell bodies, nuclei, and processes. Interestingly, UCH-L1 expression correlated with podocyte ubiquitin content and internalization of the podocyte-specific proteins nephrin and alpha-actinin-4. In contrast, minimal change glomerulonephritis, a reversible disease, demonstrated minimal UCH-L1 and ubiquitin expression with intact alpha-actinin-4 but internalized nephrin. Glomerular kidney diseases typically not associated with foot process effacement (SLE class IV, ANCA+ necrotizing GN, amyloidosis, IgA nephritis) expressed intermediate to no UCH-L1 and ubiquitin. These studies show a role for UCH-L1 and ubiquitin modification in podocyte differentiation and injury.

  7. Parallel N- and C-Terminal Truncations Facilitate Purification and Analysis of a 155-kDa Cold-Adapted Type-I Pullulanase.

    PubMed

    Elleuche, Skander; Krull, Alina; Lorenz, Ute; Antranikian, Garabed

    2017-02-01

    The cold-adapted pullulanase Pul13A is an industrial useful amylolytic enzyme, but its low solubility is the major bottleneck to produce the protein in recombinant form. In a previous approach, a complex and time-consuming purification strategy including a step-wise dialysis procedure using decreasing concentrations of urea to renature the insoluble protein from inclusion bodies had been established. In this study, a truncation strategy was developed to facilitate the purification and handling of the type-I pullulanase. Pul13A has a size of 155-kDa with a multidomain architecture that is composed of the following predicted modules: CBM41/E-set/Amy-Pul/DUF3372/E-set/E-set/E-set, with CBM and E-set domains being putative carbohydrate-binding modules, Amy-Pul is the catalytic region and DUF is a domain of unknown function. Consecutive N- and C-terminal deletions of domains were applied to construct minimized enzyme variants retaining pullulanase activity and exhibiting improved renaturation efficiencies. A total of seven truncation constructs were generated and tested, which still led to the production of inclusion bodies. However, the parallel deletion of the exterior CBM41 and E-set domain enabled the direct refolding of active enzymes during one-step dialysis in urea-free buffer. Catalytic properties of truncation construct Pul13A-N1/C1 were not impaired indicating that this enzyme variant may be superior for industrial applications over the full-length pullulanase.

  8. Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

    SciTech Connect

    Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2014-11-15

    The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

  9. Characterization of β-domains in C-terminal fragments of TDP-43 by scanning tunneling microscopy.

    PubMed

    Xu, Meng; Zhu, Li; Liu, Jianghong; Yang, Yanlian; Wu, Jane Y; Wang, Chen

    2013-01-01

    The TAR DNA-binding protein 43 (TDP-43) has been identified as a critical player in a range of neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Recent discoveries demonstrate the important role of carboxyl-terminal fragments of TDP-43 in its proteinopathy. Herein, we report the characterization of β-domains in the C-terminal fragments of TDP-43 using scanning tunneling microscopy (STM). Careful comparison of the wild-type TDP-43 (Wt) and the three mutant TDP-43 peptides: an ALS-related mutant peptide: phosphorylated A315T mutant TDP-43 (A315T(p)) and two model peptides: A315T mutant TDP-43 (A315T), A315E mutant TDP-43 (A315E) reveals that A315T(p) has a longer core region of the β-domain than Wt. A315E possesses the longest core region of the β-domain and A315T(p) mutant TDP-43 has the second longest core region of the β-domain. The core regions of the β-domains for A315T and Wt TDP-43 have the same length. This observation provides a supportive evidence of a higher tendency in beta-sheet formation of A315T(p) containing TDP-43 fragment, and structural mechanism for the higher cytotoxicity and accelerated fibril formation of the A315T(p) mutation-containing TDP-43 peptide as compared with Wt TDP-43.

  10. Cloning, purification and preliminary X-ray analysis of the C-terminal domain of Helicobacter pylori MotB

    SciTech Connect

    Roujeinikova, Anna

    2008-04-01

    The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of a putative peptidoglycan-binding domain of H. pylori MotB, a stator component of the bacterial flagellar motor, are reported. The C-terminal domain of MotB (MotB-C) contains a putative peptidoglycan-binding motif and is believed to anchor the MotA/MotB stator unit of the bacterial flagellar motor to the cell wall. Crystals of Helicobacter pylori MotB-C (138 amino-acid residues) were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. These crystals belong to space group P2{sub 1}, with unit-cell parameters a = 50.8, b = 89.5, c = 66.3 Å, β = 112.5°. The crystals diffract X-rays to at least 1.6 Å resolution using a synchrotron-radiation source. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains one tetramer with 222 point-group symmetry. The anomalous difference Patterson maps calculated for an ytterbium-derivative crystal using diffraction data at a wavelength of 1.38 Å showed significant peaks on the v = 1/2 Harker section, suggesting that ab initio phase information could be derived from the MAD data.

  11. A molecular dynamics study of Fe2S2 putidaredoxin: multiple conformations of the C-terminal region.

    PubMed Central

    Roitberg, A E

    1997-01-01

    Putidaredoxin (Pdx) plays an essential role as an electron donor and effector in the biochemical cycle involving cytochrome P450cam. Only recently has an NMR-derived structure for this protein been published, but because of the presence of a paramagnetic Fe2S2 center, the NMR assignment could not be completed for residues within a region of 8 A around the active site. That region was modeled by homology with a related protein. The structural refinement for those experiments was done in vacuum, without the use of electrostatic terms in the force field. The present manuscript will describe and discuss a series of long-time, unrestrained, solution molecular dynamic runs for this system. Results will be presented that construct a molecular-level picture that rationalizes experimental results concerning the conformation and mobility of the C-terminal residue Trp106. At least two different conformers are found for this residue during the simulations. The time scale for interconversion between them is found to be in the subnanosecond regime. The results presented here open the possibility for studying binding and electron transfer between Pdx and P450cam, in a framework that allows for dynamical information to be used during the computational process, instead of the single structures deposited on the protein data base. PMID:9336209

  12. Ubiquitin C-terminal hydrolase37 regulates Tcf7 DNA binding for the activation of Wnt signalling

    PubMed Central

    Han, Wonhee; Lee, Hyeyoon; Han, Jin-Kwan

    2017-01-01

    The Tcf/Lef family of transcription factors mediates the Wnt/β-catenin pathway that is involved in a wide range of biological processes, including vertebrate embryogenesis and diverse pathogenesis. Post-translational modifications, including phosphorylation, sumoylation and acetylation, are known to be important for the regulation of Tcf/Lef proteins. However, the importance of ubiquitination and ubiquitin-mediated regulatory mechanisms for Tcf/Lef activity are still unclear. Here, we newly show that ubiquitin C-terminal hydrolase 37 (Uch37), a deubiquitinase, interacts with Tcf7 (formerly named Tcf1) to activate Wnt signalling. Biochemical analyses demonstrated that deubiquitinating activity of Uch37 is not involved in Tcf7 protein stability but is required for the association of Tcf7 to target gene promoter in both Xenopus embryo and human liver cancer cells. In vivo analyses further revealed that Uch37 functions as a positive regulator of the Wnt/β-catenin pathway downstream of β-catenin stabilization that is required for the expression of ventrolateral mesoderm genes during Xenopus gastrulation. Our study provides a new mechanism for chromatin occupancy of Tcf7 and uncovers the physiological significance of Uch37 during early vertebrate development by regulating the Wnt/β-catenin pathway. PMID:28198400

  13. Recombinant expression, purification and preliminary biophysical and structural studies of C-terminal carbohydrate recognition domain from human galectin-4.

    PubMed

    Rustiguel, Joane K; Kumagai, Patricia S; Dias-Baruffi, Marcelo; Costa-Filho, Antonio J; Nonato, Maria Cristina

    2016-02-01

    Galectin-4 (Gal4), a tandem-repeat type galectin, is expressed in healthy epithelium of the gastrointestinal tract. Altered levels of Gal4 expression are associated with different types of cancer, suggesting its usage as a diagnostic marker as well as target for drug development. The functional data available for this class of proteins suggest that the wide spectrum of cellular activities reported for Gal4 relies on distinct glycan specificity and structural characteristics of its two carbohydrate recognition domains. In the present work, two independent constructs for recombinant expression of the C-terminal domain of human galectin-4 (hGal4-CRD2) were developed. His6-tagged and untagged recombinant proteins were overexpressed in Escherichia coli, and purified by affinity chromatography followed by gel filtration. Correct folding and activity of hGal4-CRD2 were assessed by circular dichroism and fluorescence spectroscopies, respectively. Diffraction quality crystals were obtained by vapor-diffusion sitting drop setup and the crystal structure of CRD2 was solved by molecular replacement techniques at 1.78 Å resolution. Our work describes the development of important experimental tools that will allow further studies in order to correlate structure and binding properties of hGal4-CRD2 and human galectin-4 functional activities.

  14. C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

    PubMed Central

    Santiago-Frangos, Andrew; Kavita, Kumari; Schu, Daniel J.; Gottesman, Susan

    2016-01-01

    The bacterial Sm protein and RNA chaperone Hfq stabilizes small noncoding RNAs (sRNAs) and facilitates their annealing to mRNA targets involved in stress tolerance and virulence. Although an arginine patch on the Sm core is needed for Hfq’s RNA chaperone activity, the function of Hfq’s intrinsically disordered C-terminal domain (CTD) has remained unclear. Here, we use stopped flow spectroscopy to show that the CTD of Escherichia coli Hfq is not needed to accelerate RNA base pairing but is required for the release of dsRNA. The Hfq CTD also mediates competition between sRNAs, offering a kinetic advantage to sRNAs that contact both the proximal and distal faces of the Hfq hexamer. The change in sRNA hierarchy caused by deletion of the Hfq CTD in E. coli alters the sRNA accumulation and the kinetics of sRNA regulation in vivo. We propose that the Hfq CTD displaces sRNAs and annealed sRNA⋅mRNA complexes from the Sm core, enabling Hfq to chaperone sRNA–mRNA interactions and rapidly cycle between competing targets in the cell. PMID:27681631

  15. The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum.

    PubMed

    Nakayama, Yoshitaka; Becker, Michael; Ebrahimian, Haleh; Konishi, Tomoyuki; Kawasaki, Hisashi; Krämer, Reinhard; Martinac, Boris

    2016-01-01

    The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes.

  16. Enzyme Activities of the Ceramide Synthases CERS2–6 Are Regulated by Phosphorylation in the C-terminal Region*

    PubMed Central

    Sassa, Takayuki; Hirayama, Taisuke; Kihara, Akio

    2016-01-01

    Ceramide and complex sphingolipids regulate important cellular functions including cell growth, apoptosis, and signaling. Dysregulation of sphingolipid metabolism leads to pathological consequences such as sphingolipidoses and insulin resistance. Ceramides in mammals vary greatly in their acyl-chain composition: six different ceramide synthase isozymes (CERS1–6) that exhibit distinct substrate specificity and tissue distribution account for this diversity. In the present study, we demonstrated that CERS2–6 were phosphorylated at the cytoplasmic C-terminal regions. Most of the phosphorylated residues conformed to a consensus motif for phosphorylation by casein kinase 2 (CK2), and treatment of cells with the CK2-specific inhibitor CX-4945 lowered the phosphorylation levels of CERS2, -4, -5, and -6. Phosphorylation of CERS2 was especially important for its catalytic activity, acting mainly by increasing its Vmax value. Phosphorylation modestly increased the catalytic activities of CERS4 and -5 and mildly increased those of CERS3 and -6. Dephosphorylation of endogenous ceramide synthases in the mouse brain led to severely reduced activity toward the Cers2 substrates C22:0/C24:0-CoAs and modestly reduced activity toward the Cers5/6 substrate C16:0-CoA. These results suggest that the phosphorylation of ceramide synthases may be a key regulatory point in the control of the distribution and levels of sphingolipids of various acyl-chain lengths. PMID:26887952

  17. The C-terminal extension of PrhG impairs its activation of hrp expression and virulence in Ralstonia solanacearum.

    PubMed

    Zhang, Yong; Luo, Feng; Hikichi, Yasufumi; Kiba, Akinori; Yasuo, Igarashi; Ohnishi, Kouhei

    2015-04-01

    Ralstonia solanacearum is the second most destructive bacterial plant pathogens worldwide and HrpG is the master regulator of its pathogenicity. PrhG is a close paralogue of HrpG and both belong to OmpR/PhoB family of two-component response regulators. Despite a high similarity (72% global identity and 96% similarity in helix-loop-helix domain), they display distinct roles in pathogenicity. HrpG is necessary for the bacterial growth in planta and pathogenicity, while PrhG is dispensable for bacterial growth in planta and contributes little to pathogenicity. The main difference between HrpG and PrhG is the 50-amino-acid-long C-terminal extension in PrhG (amino-acid residues 230-283), which is absent in HrpG. When this extension is deleted, truncated PrhGs (under the control of its native promoter) allowed complete recovery of bacterial growth in planta and wild-type virulence of hrpG mutant. This novel finding demonstrates that the extension region in PrhG is responsible for the functional difference between HrpG and PrhG, which may block the binding of PrhG to target promoters and result in impaired activation of hrp expression by PrhG and reduced virulence of R. solanacearum.

  18. Neuroblastoma tumorigenesis is regulated through the Nm23-H1/h-Prune C-terminal interaction

    PubMed Central

    Carotenuto, Marianeve; Pedone, Emilia; Diana, Donatella; de Antonellis, Pasqualino; Džeroski, Sašo; Marino, Natascia; Navas, Luigi; Di Dato, Valeria; Scoppettuolo, Maria Nunzia; Cimmino, Flora; Correale, Stefania; Pirone, Luciano; Monti, Simona Maria; Bruder, Elisabeth; Ženko, Bernard; Slavkov, Ivica; Pastorino, Fabio; Ponzoni, Mirco; Schulte, Johannes H.; Schramm, Alexander; Eggert, Angelika; Westermann, Frank; Arrigoni, Gianluigi; Accordi, Benedetta; Basso, Giuseppe; Saviano, Michele; Fattorusso, Roberto; Zollo, Massimo

    2013-01-01

    Nm23-H1 is one of the most interesting candidate genes for a relevant role in Neuroblastoma pathogenesis. H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers. Our study focuses on the role of the Nm23-H1/h-Prune protein complex in Neuroblastoma. Using NMR spectroscopy, we performed a conformational analysis of the h-Prune C-terminal to identify the amino acids involved in the interaction with Nm23-H1. We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation. Meta-analysis performed on three Neuroblastoma cohorts showed Nm23-H1 as the gene highly associated to Neuroblastoma aggressiveness. We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP. These data suggest a new avenue for potential clinical application of CPP in Neuroblastoma treatment. PMID:23448979

  19. Design, synthesis, and biological evaluation of substrate-competitive inhibitors of C-terminal Binding Protein (CtBP).

    PubMed

    Korwar, Sudha; Morris, Benjamin L; Parikh, Hardik I; Coover, Robert A; Doughty, Tyler W; Love, Ian M; Hilbert, Brendan J; Royer, William E; Kellogg, Glen E; Grossman, Steven R; Ellis, Keith C

    2016-06-15

    C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24μM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18μM) and 3-chloro- (IC50=0.17μM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer.

  20. Unwinding of the C-Terminal Residues of Neuropeptide Y is critical for Y₂ Receptor Binding and Activation.

    PubMed

    Kaiser, Anette; Müller, Paul; Zellmann, Tristan; Scheidt, Holger A; Thomas, Lars; Bosse, Mathias; Meier, Rene; Meiler, Jens; Huster, Daniel; Beck-Sickinger, Annette G; Schmidt, Peter

    2015-06-15

    Despite recent breakthroughs in the structural characterization of G-protein-coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double-cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G-protein-coupled Y2 receptor (Y2R). Solid-state NMR measurements of specific isotope-labeled NPY in complex with in vitro folded Y2R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C-terminal α-helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T(32) to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y2R.

  1. Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex

    SciTech Connect

    Sampathkumar, Parthasarathy; Gheyi, Tarun; Miller, Stacy A.; Bain, Kevin T.; Dickey, Mark; Bonanno, Jeffrey B.; Kim, Seung Joong; Phillips, Jeremy; Pieper, Ursula; Fernandez-Martinez, Javier; Franke, Josef D.; Martel, Anne; Tsuruta, Hiro; Atwell, Shane; Thompson, Devon A.; Emtage, J. Spencer; Wasserman, Stephen R.; Rout, Michael P.; Sali, Andrej; Sauder, J. Michael; Burley, Stephen K.

    2012-10-23

    Nuclear pore complexes (NPCs), responsible for the nucleo-cytoplasmic exchange of proteins and nucleic acids, are dynamic macromolecular assemblies forming an eight-fold symmetric co-axial ring structure. Yeast (Saccharomyces cerevisiae) NPCs are made up of at least 456 polypeptide chains of {approx}30 distinct sequences. Many of these components (nucleoporins, Nups) share similar structural motifs and form stable subcomplexes. We have determined a high-resolution crystal structure of the C-terminal domain of yeast Nup133 (ScNup133), a component of the heptameric Nup84 subcomplex. Expression tests yielded ScNup133(944-1157) that produced crystals diffracting to 1.9{angstrom} resolution. ScNup133(944-1157) adopts essentially an all {alpha}-helical fold, with a short two stranded {beta}-sheet at the C-terminus. The 11 {alpha}-helices of ScNup133(944-1157) form a compact fold. In contrast, the previously determined structure of human Nup133(934-1156) bound to a fragment of human Nup107 has its constituent {alpha}-helices are arranged in two globular blocks. These differences may reflect structural divergence among homologous nucleoporins.

  2. Analysis of the Role of the C-Terminal Tail in the Regulation of the Epidermal Growth Factor Receptor

    PubMed Central

    Kovacs, Erika; Das, Rahul; Wang, Qi; Collier, Timothy S.; Cantor, Aaron; Huang, Yongjian; Wong, Kathryn; Mirza, Amar; Barros, Tiago; Grob, Patricia; Jura, Natalia; Bose, Ron

    2015-01-01

    The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ∼80 residues of the tail are inhibitory, as demonstrated previously. The entire ∼80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer. PMID:26124280

  3. The C-terminal helices of heat shock protein 70 are essential for J-domain binding and ATPase activation.

    PubMed

    Gao, Xue-Chao; Zhou, Chen-Jie; Zhou, Zi-Ren; Wu, Meng; Cao, Chun-Yang; Hu, Hong-Yu

    2012-02-17

    The J-domain co-chaperones work together with the heat shock protein 70 (HSP70) chaperone to regulate many cellular events, but the mechanism underlying the J-domain-mediated HSP70 function remains elusive. We studied the interaction between human-inducible HSP70 and Homo sapiens J-domain protein (HSJ1a), a J domain and UIM motif-containing co-chaperone. The J domain of HSJ1a shares a conserved structure with other J domains from both eukaryotic and prokaryotic species, and it mediates the interaction with and the ATPase cycle of HSP70. Our in vitro study corroborates that the N terminus of HSP70 including the ATPase domain and the substrate-binding β-subdomain is not sufficient to bind with the J domain of HSJ1a. The C-terminal helical α-subdomain of HSP70, which was considered to function as a lid of the substrate-binding domain, is crucial for binding with the J domain of HSJ1a and stimulating the ATPase activity of HSP70. These fluctuating helices are likely to contribute to a proper conformation of HSP70 for J-domain binding other than directly bind with the J domain. Our findings provide an alternative mechanism of allosteric activation for functional regulation of HSP70 by its J-domain co-chaperones.

  4. RNA polymerase II C-terminal heptarepeat domain Ser-7 phosphorylation is established in a mediator-dependent fashion.

    PubMed

    Boeing, Stefan; Rigault, Caroline; Heidemann, Martin; Eick, Dirk; Meisterernst, Michael

    2010-01-01

    The largest subunit of RNA polymerase II (RNAPII) C-terminal heptarepeat domain (CTD) is subject to phosphorylation during initiation and elongation of transcription by RNA polymerase II. Here we study the molecular mechanisms leading to phosphorylation of Ser-7 in the human enzyme. Ser-7 becomes phosphorylated before initiation of transcription at promoter regions. We identify cyclin-dependent kinase 7 (CDK7) as one responsible kinase. Phosphorylation of both Ser-5 and Ser-7 is fully dependent on the cofactor complex Mediator. A subform of Mediator associated with an active RNAPII is critical for preinitiation complex formation and CTD phosphorylation. The Mediator-RNAPII complex independently recruits TFIIB and CDK7 to core promoter regions. CDK7 phosphorylates Ser-7 selectively in the context of an intact preinitiation complex. CDK7 is not the only kinase that can modify Ser-7 of the CTD. ChIP experiments with chemical inhibitors provide evidence that other yet to be identified kinases further phosphorylate Ser-7 in coding regions.

  5. Solution structure of the C-terminal domain of Ole e 9, a major allergen of olive pollen

    PubMed Central

    Treviño, Miguel Á.; Palomares, Oscar; Castrillo, Inés; Villalba, Mayte; Rodríguez, Rosalía; Rico, Manuel; Santoro, Jorge; Bruix, Marta

    2008-01-01

    Ole e 9 is an olive pollen allergen belonging to group 2 of pathogenesis-related proteins. The protein is composed of two immunological independent domains: an N-terminal domain (NtD) with 1,3-β-glucanase activity, and a C-terminal domain (CtD) that binds 1,3-β-glucans. We have determined the three-dimensional structure of CtD-Ole e 9 (101 amino acids), which consists of two parallel α-helices forming an angle of ∼55°, a small antiparallel β-sheet with two short strands, and a 3–10 helix turn, all connected by long coil segments, resembling a novel type of folding among allergens. Two regions surrounded by aromatic residues (F49, Y60, F96, Y91 and Y31, H68, Y65, F78) have been localized on the protein surface, and a role for sugar binding is suggested. The epitope mapping of CtD-Ole e 9 shows that B-cell epitopes are mainly located on loops, although some of them are contained in secondary structural elements. Interestingly, the IgG and IgE epitopes are contiguous or overlapped, rather than coincident. The three-dimensional structure of CtD-Ole e 9 might help to understand the underlying mechanism of its biochemical function and to determine possible structure–allergenicity relationships. PMID:18096638

  6. C-terminal calcitonin gene-related peptide fragments and vasopressin but not somatostatin-28 induce miosis in monkeys.

    PubMed

    Almegård, B; Bill, A

    1993-11-30

    The miotic effects of C-terminal calcitonin gene-related peptide (CGRP) fragments, somatostatin-28 and vasopressin have been evaluated with special attention being paid to possible interactions with cholecystokinin (CCK)A receptors. The peptides were injected intracamerally to anesthetized monkeys pretreated with indomethacin and atropine. CGRP-(32-37) induced a miosis with a potency 1000 times lower than that previously found with sulphated CCK-8. Two other fragments, CGRP-(30-37) and CGRP-(31-37), also had miotic properties. The CGRP-(32-37)-induced miosis was antagonized by the CCKA receptor antagonist loxiglumide. No contractile effect was elicited by 67 pmol-7.4 nmol somatostatin-28. Vasopressin (360 pmol) caused a small reduction in pupil size. Loxiglumide pretreatment did not affect the reduction in pupil size but a vasopressin receptor antagonist partly inhibited the response. The results indicate that CGRP-(32-37) is a miotic with low potency but high efficacy in the monkey eye, probably interacting with CCKA receptors, and that vasopressin is a mitotic with low potency and efficacy, probably acting via vasopressin receptors.

  7. De-bugging and maximizing plant cytochrome P450 production in Escherichia coli with C-terminal GFP fusions.

    PubMed

    Christensen, Ulla; Vazquez-Albacete, Dario; Søgaard, Karina M; Hobel, Tonja; Nielsen, Morten T; Harrison, Scott James; Hansen, Anders Holmgaard; Møller, Birger Lindberg; Seppälä, Susanna; Nørholm, Morten H H

    2017-02-15

    Cytochromes P450 (CYP) are attractive enzyme targets in biotechnology as they catalyze stereospecific C-hydroxylations of complex core skeletons at positions that typically are difficult to access by chemical synthesis. Membrane bound CYPs are involved in nearly all plant pathways leading to the formation of high-value compounds. In the present study, we systematically maximize the heterologous expression of six different plant-derived CYP genes in Escherichia coli, using a workflow based on C-terminal fusions to the green fluorescent protein. The six genes can be over-expressed in both K- and B-type E. coli strains using standard growth media. Furthermore, sequences encoding a small synthetic peptide and a small bacterial membrane anchor markedly enhance the expression of all six genes. For one of the CYPs, the length of the linker region between the predicted N-terminal transmembrane segment and the soluble domain is modified, in order to verify the importance of this region for enzymatic activity. The work describes how membrane bound CYPs are optimally produced in E. coli and thus adds this plant multi-membered key enzyme family to the toolbox for bacterial cell factory design.

  8. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

    PubMed Central

    Lübker, Carolin; Dove, Stefan; Tang, Wei-Jen; Urbauer, Ramona J. Bieber; Moskovitz, Jackob; Urbauer, Jeffrey L.; Seifert, Roland

    2015-01-01

    Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils. PMID:26184312

  9. Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II.

    PubMed

    Voss, Kirsten; Forné, Ignasi; Descostes, Nicolas; Hintermair, Corinna; Schüller, Roland; Maqbool, Muhammad Ahmad; Heidemann, Martin; Flatley, Andrew; Imhof, Axel; Gut, Marta; Gut, Ivo; Kremmer, Elisabeth; Andrau, Jean-Christophe; Eick, Dirk

    2015-01-01

    Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression.

  10. Haemophilus influenzae protein E recognizes the C-terminal domain of vitronectin and modulates the membrane attack complex.

    PubMed

    Singh, Birendra; Jalalvand, Farshid; Mörgelin, Matthias; Zipfel, Peter; Blom, Anna M; Riesbeck, Kristian

    2011-07-01

    Haemophilus influenzae protein E (PE) is a 16 kDa adhesin that induces a pro-inflammatory immune response in lung epithelial cells. The active epithelial binding region comprising amino acids PE 84-108 also interferes with complement-mediated bacterial killing by capturing vitronectin (Vn) that prevents complement deposition and formation of the membrane attack complex (MAC). Here, the interaction between PE and Vn was characterized using site-directed mutagenesis. Protein E variants were produced both in soluble forms and in surface-expressed molecules on Escherichia coli. Mutations within PE(84-108) in the full-length molecule revealed that K85 and R86 residues were important for the Vn binding. Bactericidal activity against H. influenzae was higher in human serum pre-treated with full-length PE as compared with serum incubated with PE(K85E, R86D) , suggesting that PE quenched Vn. A series of truncated Vn molecules revealed that the C-terminal domain comprising Vn(353-363) harboured the major binding region for PE. Interestingly, MAC deposition was significantly higher on mutants devoid of PE due to a decreased Vn-binding capacity when compared with wild-type H. influenzae. Our results define a fine-tuned interaction between H. influenzae and the innate immune system, and identify the mode of control of the MAC that is important for pathogen complement evasion.

  11. The structure of Aβ42 C-terminal fragments probed by a combined experimental and theoretical study

    PubMed Central

    Wu, Chun; Murray, Megan M.; Bernstein, Summer L.; Condron, Margaret M.; Bitan, Gal; Shea, Joan-Emma; Bowers, Michael T.

    2009-01-01

    The C-terminus of Aβ42 plays an important role in this protein's oligomerization and may therefore be a good therapeutic target for treatment of Alzheimer's disease. Certain C-terminal fragments (CTFs) of Aβ42 have been shown to disrupt oligomerization and strongly inhibit Aβ42-induced neurotoxicity. Here we study the structures of selected CTFs (Aβ(x-42), x=29-31, 39) using replica exchange molecular dynamics (REMD) simulations and ion mobility mass spectrometry (IM-MS). Our simulations in explicit solvent reveal that the CTFs adopt a metastable β-structure: β-hairpin for Aβ(x-42), x=29-31 and extended β-strand for Aβ(39-42). The β-hairpin of Aβ(30-42) is converted into a turn-coil conformation when the last two hydrophobic residues are removed, suggesting that I41 and A42 are critical in stabilizing the β-hairpin in the Aβ42-derived CTFs. The importance of solvent in determining the structure of the CTFs is further highlighted in the IM-MS experiments and solvent free REMD simulations. A comparison between the structures with and without solvent reveals that hydrophobic interactions are critical for the formation of β-hairpin. The possible role played by the CTFs in disrupting oligomerization is discussed. PMID:19356595

  12. Intracellular repair of oxidation-damaged α-synuclein fails to target C-terminal modification sites

    PubMed Central

    Binolfi, Andres; Limatola, Antonio; Verzini, Silvia; Kosten, Jonas; Theillet, Francois-Xavier; May Rose, Honor; Bekei, Beata; Stuiver, Marchel; van Rossum, Marleen; Selenko, Philipp

    2016-01-01

    Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson's disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson's disease protein alpha-synuclein (α-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched α-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal α-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of α-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered α-Syn in cells. PMID:26807843

  13. Structure-Activity Relationship Studies of N- and C-Terminally Modified Secretin Analogs for the Human Secretin Receptor

    PubMed Central

    Arokiaraj, Aloysius Wilfred Raj; Leprince, Jérôme; Lefranc, Benjamin; Vaudry, David; Allam, Ahmed A.; Ajarem, Jamaan; Chow, Billy K. C.

    2016-01-01

    The pleiotropic role of human secretin (hSCT) validates its potential use as a therapeutic agent. Nevertheless, the structure of secretin in complex with its receptor is necessary to develop a suitable therapeutic agent. Therefore, in an effort to design a three-dimensional virtual homology model and identify a peptide agonist and/or antagonist for the human secretin receptor (hSR), the significance of the primary sequence of secretin peptides in allosteric binding and activation was elucidated using virtual docking, FRET competitive binding and assessment of the cAMP response. Secretin analogs containing various N- or C-terminal modifications were prepared based on previous findings of the role of these domains in receptor binding and activation. These analogs exhibited very low or no binding affinity in a virtual model, and were found to neither exhibit in vitro binding nor agonistic or antagonistic properties. A parallel analysis of the analogs in the virtual model and in vitro studies revealed instability of these peptide analogs to bind and activate the receptor. PMID:26930505

  14. A Novel Fold in the Tral Relaxase-Helicase C-Terminal Domain Is Essential for Conjugative DNA Transfer

    SciTech Connect

    Guogas, Laura M.; Kennedy, Sarah A.; Lee, Jin-Hyup; Redinbo, Matthew R.

    2009-06-04

    TraI relaxase-helicase is the central catalytic component of the multiprotein relaxosome complex responsible for conjugative DNA transfer (CDT) between bacterial cells. CDT is a primary mechanism for the lateral propagation of microbial genetic material, including the spread of antibiotic resistance genes. The 2.4-{angstrom} resolution crystal structure of the C-terminal domain of the multifunctional Escherichia coli F (fertility) plasmid TraI protein is presented, and specific structural regions essential for CDT are identified. The crystal structure reveals a novel fold composed of a 28-residue N-terminal {alpha}-domain connected by a proline-rich loop to a compact {alpha}/{beta}-domain. Both the globular nature of the {alpha}/{beta}-domain and the presence as well as rigidity of the proline-rich loop are required for DNA transfer and single-stranded DNA binding. Taken together, these data establish the specific structural features of this noncatalytic domain that are essential to DNA conjugation.

  15. Disulphide bond restrains the C-terminal region of thermostable direct hemolysin during folding to promote oligomerization.

    PubMed

    Kundu, Nidhi; Tichkule, Swapnil; Pandit, Shashi Bhushan; Chattopadhyay, Kausik

    2017-01-15

    Pore-forming toxins (PFTs) are typically produced as water-soluble monomers, which upon interacting with target cells assemble into transmembrane oligomeric pores. Vibrio parahaemolyticus thermostable direct hemolysin (TDH) is an atypical PFT that exists as a tetramer in solution, prior to membrane binding. The TDH structure highlights a core β-sandwich domain similar to those found in the eukaryotic actinoporin family of PFTs. However, the TDH structure harbors an extended C-terminal region (CTR) that is not documented in the actinoporins. This CTR remains tethered to the β-sandwich domain through an intra-molecular disulphide bond. Part of the CTR is positioned at the inter-protomer interface in the TDH tetramer. Here we show that the truncation, as well as mutation, of the CTR compromise tetrameric assembly, and the membrane-damaging activity of TDH. Our study also reveals that intra-protomer disulphide bond formation during the folding/assembly process of TDH restrains the CTR to mediate its participation in the formation of inter-protomer contact, thus facilitating TDH oligomerization. However, once tetramerization is achieved, disruption of the disulphide bond does not affect oligomeric assembly. Our study provides critical insights regarding the regulation of the oligomerization mechanism of TDH, which has not been previously documented in the PFT family.

  16. C-terminal domain (CTD) phosphatase links Rho GTPase signaling to Pol II CTD phosphorylation in Arabidopsis and yeast.

    PubMed

    Zhang, Bo; Yang, Guohua; Chen, Yu; Zhao, Yihong; Gao, Peng; Liu, Bo; Wang, Haiyang; Zheng, Zhi-Liang

    2016-12-13

    Rho GTPases, including the Rho, Cdc42, Rac, and ROP subfamilies, act as pivotal signaling switches in various growth and developmental processes. Compared with the well-defined role of cytoskeletal organization in Rho signaling, much less is known regarding transcriptional regulation. In a mutant screen for phenotypic enhancers of transgenic Arabidopsis plants expressing a constitutively active form of ROP2 (designated CA1-1), we identified RNA polymerase II (Pol II) C-terminal domain (CTD) phosphatase-like 1 (CPL1) as a transcriptional regulator of ROP2 signaling. We show that ROP2 activation inhibits CPL1 activity by promoting its degradation, leading to an increase in CTD Ser5 and Ser2 phosphorylation. We also observed similar modulation of CTD phosphorylation by yeast Cdc42 GTPase and enhanced degradation of the yeast CTD phosphatase Fcp1 by activated ROP2 signaling. Taken together, our results suggest that modulation of the Pol II CTD code by Rho GTPase signaling represents an evolutionarily conserved mechanism in both unicellular and multicellular eukaryotes.

  17. Cooperative and acute inhibition by multiple C-terminal motifs of L-type Ca2+ channels

    PubMed Central

    Liu, Nan; Yang, Yaxiong; Ge, Lin; Liu, Min; Colecraft, Henry M; Liu, Xiaodong

    2017-01-01

    Inhibitions and antagonists of L-type Ca2+ channels are important to both research and therapeutics. Here, we report C-terminus mediated inhibition (CMI) for CaV1.3 that multiple motifs coordinate to tune down Ca2+ current and Ca2+ influx toward the lower limits determined by end-stage CDI (Ca2+-dependent inactivation). Among IQV (preIQ3-IQ domain), PCRD and DCRD (proximal or distal C-terminal regulatory domain), spatial closeness of any two modules, e.g., by constitutive fusion, facilitates the trio to form the complex, compete against calmodulin, and alter the gating. Acute CMI by rapamycin-inducible heterodimerization helps reconcile the concurrent activation/inactivation attenuations to ensure Ca2+ influx is reduced, in that Ca2+ current activated by depolarization is potently (~65%) inhibited at the peak (full activation), but not later on (end-stage inactivation, ~300 ms). Meanwhile, CMI provides a new paradigm to develop CaV1 inhibitors, the therapeutic potential of which is implied by computational modeling of CaV1.3 dysregulations related to Parkinson’s disease. DOI: http://dx.doi.org/10.7554/eLife.21989.001 PMID:28059704

  18. Solution structure of a hydrocarbon stapled peptide inhibitor in complex with monomeric C-terminal domain of HIV-1 capsid.

    PubMed

    Bhattacharya, Shibani; Zhang, Hongtao; Debnath, Asim K; Cowburn, David

    2008-06-13

    The human immunodeficiency virus type 1 (HIV-1) capsid protein plays a critical role in virus core particle assembly and is an important target for novel therapeutic strategies. In a previous study, we characterized the binding affinity of a hydrocarbon stapled helical peptide, NYAD-1, for the capsid protein (K(d) approximately 1 mum) and demonstrated its ability to penetrate the cell membrane (Zhang, H., Zhao, Q., Bhattacharya, S., Waheed, A. A., Tong, X., Hong, A., Heck, S., Goger, M., Cowburn, D., Freed, E. O., and Debnath, A. K. (2008) J. Mol. Biol. 378, 565-580). In cell-based assays, NYAD-1 colocalized with the Gag polyprotein during traffic to the plasma membrane and disrupted the formation of mature and immature virus particles in vitro systems. Here, we complement the cellular and biochemical data with structural characterization of the interactions between the capsid and a soluble peptide analogue, NYAD-13. Solution NMR methods were used to determine a high resolution structure of the complex between the inhibitor and a monomeric form of the C-terminal domain of the capsid protein (mCA-CTD). The intermolecular interactions are mediated by the packing of hydrophobic side chains at the buried interface and unperturbed by the presence of the olefinic chain on the solvent-exposed surface of the peptide. The results of the structural analysis provide valuable insight into the determinants for high affinity and selective inhibitors for HIV-1 particle assembly.

  19. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

    PubMed

    Selzer, Lisa; Kant, Ravi; Wang, Joseph C-Y; Bothner, Brian; Zlotnick, Adam

    2015-11-20

    Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle.

  20. A Novel Bmal1 Mutant Mouse Reveals Essential Roles of the C-Terminal Domain on Circadian Rhythms.

    PubMed

    Park, Noheon; Kim, Hee-Dae; Cheon, Solmi; Row, Hansang; Lee, Jiyeon; Han, Dong-Hee; Cho, Sehyung; Kim, Kyungjin

    2015-01-01

    The mammalian circadian clock is an endogenous biological timer comprised of transcriptional/translational feedback loops of clock genes. Bmal1 encodes an indispensable transcription factor for the generation of circadian rhythms. Here, we report a new circadian mutant mouse from gene-trapped embryonic stem cells harboring a C-terminus truncated Bmal1 (Bmal1GTΔC) allele. The homozygous mutant (Bmal1GTΔC/GTΔC) mice immediately lost circadian behavioral rhythms under constant darkness. The heterozygous (Bmal1+/GTΔC) mice displayed a gradual loss of rhythms, in contrast to Bmal1+/- mice where rhythms were sustained. Bmal1GTΔC/GTΔC mice also showed arrhythmic mRNA and protein expression in the SCN and liver. Lack of circadian reporter oscillation was also observed in cultured fibroblast cells, indicating that the arrhythmicity of Bmal1GTΔC/GTΔC mice resulted from impaired molecular clock machinery. Expression of clock genes exhibited distinct responses to the mutant allele in Bmal1+/GTΔC and Bmal1GTΔC/GTΔC mice. Despite normal cellular localization and heterodimerization with CLOCK, overexpressed BMAL1GTΔC was unable to activate transcription of Per1 promoter and BMAL1-dependent CLOCK degradation. These results indicate that the C-terminal region of Bmal1 has pivotal roles in the regulation of circadian rhythms and the Bmal1GTΔC mice constitute a novel model system to evaluate circadian functional mechanism of BMAL1.

  1. The Stability Enhancement of Nitrile Hydratase from Bordetella petrii by Swapping the C-terminal Domain of β subunit.

    PubMed

    Sun, Weifeng; Zhu, Longbao; Chen, Xianggui; Wu, Lunjie; Zhou, Zhemin; Liu, Yi

    2016-04-01

    The thermal stability of most nitrile hydratases (NHase) is poor, which has been enhanced to some extent by molecular modifications in several specific regions of the C-terminal domain (C-domain) of β subunit of NHase. Since the C-domain could be present as a naturally separate domain in a few NHases, the whole C-domain is proposed to be related to the NHase stability. The chimeric NHase (SBpNHase) from the thermal-sensitive BpNHase (NHase from Bordetella petrii) and the relatively thermal-stable PtNHase (NHase from Pseudonocardia thermophila) was constructed by swapping the corresponding C-domains. After 30 min incubation at 50 °C, the original BpNHase nearly lost its activity, while the SBpNHase retained 50 % residual activity, compared with the melting temperature (Tm) (50 °C) of the original BpNHase, that of the SBpNHase was 55 °C. The SBpNHase with higher thermal stability would be useful for the thermal stability enhancement of NHase and for the understanding of the relationship between the stability of NHase and its structure.

  2. Structural insights into POT1-TPP1 interaction and POT1 C-terminal mutations in human cancer.

    PubMed

    Chen, Cong; Gu, Peili; Wu, Jian; Chen, Xianyun; Niu, Shuangshuang; Sun, Hong; Wu, Lijie; Li, Na; Peng, Junhui; Shi, Shaohua; Fan, Cuiying; Huang, Min; Wong, Catherine C L; Gong, Qingguo; Kumar-Sinha, Chandan; Zhang, Rongguang; Pusztai, Lajos; Rai, Rekha; Chang, Sandy; Lei, Ming

    2017-04-10

    Mammalian shelterin proteins POT1 and TPP1 form a stable heterodimer that protects chromosome ends and regulates telomerase-mediated telomere extension. However, how POT1 interacts with TPP1 remains unknown. Here we present the crystal structure of the C-terminal portion of human POT1 (POT1C) complexed with the POT1-binding motif of TPP1. The structure shows that POT1C contains two domains, a third OB fold and a Holliday junction resolvase-like domain. Both domains are essential for binding to TPP1. Notably, unlike the heart-shaped structure of ciliated protozoan Oxytricha nova TEBPα-β complex, POT1-TPP1 adopts an elongated V-shaped conformation. In addition, we identify several missense mutations in human cancers that disrupt the POT1C-TPP1 interaction, resulting in POT1 instability. POT1C mutants that bind TPP1 localize to telomeres but fail to repress a DNA damage response and inappropriate repair by A-NHEJ. Our results reveal that POT1 C terminus is essential to prevent initiation of genome instability permissive for tumorigenesis.

  3. Synapse associated protein 102 (SAP102) binds the C-terminal part of the scaffolding protein neurobeachin.

    PubMed

    Lauks, Juliane; Klemmer, Patricia; Farzana, Fatima; Karupothula, Ramesh; Zalm, Robbert; Cooke, Nancy E; Li, Ka Wan; Smit, August B; Toonen, Ruud; Verhage, Matthijs

    2012-01-01

    Neurobeachin (Nbea) is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea's PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system.

  4. Synapse Associated Protein 102 (SAP102) Binds the C-Terminal Part of the Scaffolding Protein Neurobeachin

    PubMed Central

    Farzana, Fatima; Karupothula, Ramesh; Zalm, Robbert; Cooke, Nancy E.; Li, Ka Wan; Smit, August B.; Toonen, Ruud; Verhage, Matthijs

    2012-01-01

    Neurobeachin (Nbea) is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea’s PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system. PMID:22745750

  5. Mice that lack the C-terminal region of Reelin exhibit behavioral abnormalities related to neuropsychiatric disorders

    PubMed Central

    Sakai, Kaori; Shoji, Hirotaka; Kohno, Takao; Miyakawa, Tsuyoshi; Hattori, Mitsuharu

    2016-01-01

    The secreted glycoprotein Reelin is believed to play critical roles in the pathogenesis of several neuropsychiatric disorders. The highly basic C-terminal region (CTR) of Reelin is necessary for efficient activation of its downstream signaling, and the brain structure of knock-in mice that lack the CTR (ΔC-KI mice) is impaired. Here, we performed a comprehensive behavioral test battery on ΔC-KI mice, in order to evaluate the effects of partial loss-of-function of Reelin on brain functions. The ΔC-KI mice were hyperactive and exhibited reduced anxiety-like and social behaviors. The working memory in ΔC-KI mice was impaired in a T-maze test. There was little difference in spatial reference memory, depression-like behavior, prepulse inhibition, or fear memory between ΔC-KI and wild-type mice. These results suggest that CTR-dependent Reelin functions are required for some specific normal brain functions and that ΔC-KI mice recapitulate some aspects of neuropsychiatric disorders, such as schizophrenia, bipolar disorder, and autism spectrum disorder. PMID:27346785

  6. The methylation of the C-terminal region of hnRNPQ (NSAP1) is important for its nuclear localization

    SciTech Connect

    Passos, Dario O.; Quaresma, Alexandre J.C.; Kobarg, Joerg . E-mail: jkobarg@lnls.br

    2006-07-28

    Protein arginine methylation is an irreversible post-translational protein modification catalyzed by a family of at least nine different enzymes entitled PRMTs (protein arginine methyl transferases). Although PRMT1 is responsible for 85% of the protein methylation in human cells, its substrate spectrum has not yet been fully characterized nor are the functional consequences of methylation for the protein substrates well understood. Therefore, we set out to employ the yeast two-hybrid system in order to identify new substrate proteins for human PRMT1. We were able to identify nine different PRMT1 interacting proteins involved in different aspects of RNA metabolism, five of which had been previously described either as substrates for PRMT1 or as functionally associated with PRMT1. Among the four new identified possible protein substrates was hnRNPQ3 (NSAP1), a protein whose function has been implicated in diverse steps of mRNA maturation, including splicing, editing, and degradation. By in vitro methylation assays we were able to show that hnRNPQ3 is a substrate for PRMT1 and that its C-terminal RGG box domain is the sole target for methylation. By further studies with the inhibitor of methylation Adox we provide evidence that hnRNPQ1-3 are methylated in vivo. Finally, we demonstrate by immunofluorescence analysis of HeLa cells that the methylation of hnRNPQ is important for its nuclear localization, since Adox treatment causes its re-distribution from the nucleus to the cytoplasm.

  7. Direct analysis of phosphorylation sites on the Rpb1 C-terminal domain of RNA polymerase II

    PubMed Central

    Suh, Hyunsuk; Ficarro, Scott B.; Kang, Un-Beom; Chun, Yujin; Marto, Jarrod A.; Buratowski, Stephen

    2015-01-01

    Summary Dynamic interactions between RNA polymerase II and various mRNA processing and chromatin modifying enzymes are mediated by the changing phosphorylation pattern on the C-terminal domain (CTD) of polymerase subunit Rpb1 during different stages of transcription. Phosphorylations within the repetitive heptamer sequence (YSPTSPS) of CTD have primarily been defined using antibodies, but these do not distinguish different repeats or allow comparative quantitation. Using a CTD modified for mass spectrometry (msCTD), we show that Ser5-P and Ser2-P occur throughout the length of CTD and are far more abundant than other phosphorylation sites. msCTD extracted from cells mutated in several CTD kinases or phosphatases showed the expected changes in phosphorylation. Furthermore, msCTD associated with capping enzyme was enriched for Ser5-P while that bound to the transcription termination factor Rtt103 had higher levels of Ser2-P. These results suggest a relatively sparse and simple "CTD code". PMID:26799764

  8. Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II

    PubMed Central

    Voss, Kirsten; Forné, Ignasi; Descostes, Nicolas; Hintermair, Corinna; Schüller, Roland; Maqbool, Muhammad Ahmad; Heidemann, Martin; Flatley, Andrew; Imhof, Axel; Gut, Marta; Gut, Ivo; Kremmer, Elisabeth; Andrau, Jean-Christophe; Eick, Dirk

    2015-01-01

    Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression. PMID:26566685

  9. Human thrombospondin's (TSP-1) C-terminal domain opens to interact with the CD-47 receptor: a molecular modeling study.

    PubMed

    Floquet, Nicolas; Dedieu, Stéphane; Martiny, Laurent; Dauchez, Manuel; Perahia, David

    2008-10-01

    Thrombospondin-1 (TSP-1) interaction with the membranous receptor CD-47 involves the peptide RFYVVMWK (4N-1) located in its C-terminal domain. However, the available X-ray structure of TSP-1 describes this peptide as completely buried into a hydrophobic pocket, preventing any interaction. Where classical standard methods failed, an appropriate approach combining normal mode analysis and an adapted protocol of energy minimization identified the large amplitude motions responsible of the partial solvent exposure of 4N-1. In agreement, the obtained model of the open TSP-1 was further used for protein-protein docking experiments against a homology model generated for CD-47. Considering the multiple applications of the CD-47 receptor as a target, our results open new pharmacological perspectives for the design of TSP-1:CD-47 inhibitors and CD-47 antagonists. We also suggest a common opening mechanism for proteins sharing the same fold as TSP-1. This work also suggests the usefulness of our approach in other topics in which predictions of protein-protein interactions are of importance.

  10. Interactions stabilizing the C-terminal helix of human phospholipid scramblase 1 in lipid bilayers: A computational study.

    PubMed

    Venken, Tom; Schillinger, Anne-Sophie; Fuglebakk, Edvin; Reuter, Nathalie

    2017-03-31

    The human phospholipid scramblase 1 (SCR) distributes lipids non-selectively between the cellular membrane leaflets. SCR has long been thought to be mostly localized in the cytoplasm (amino acids 1-287) and anchored to the membrane via the insertion of a 19 amino acid long transmembrane C-terminal helix (CTH, 288-306), which further extends to the exoplasmic side with a 12 amino acid long tail (307-318). Little is known about the structure of this protein, but recent experimental data on two CTH peptides (288-306 and 288-318) show that they insert through phospholipid bilayers and that the presence of cholesterol improves their affinity for lipid vesicles. Yet the sequence of the CTH ((288)KMKAVMIGACFLIDFMFFE(306)) contains an aspartic acid (D301), which is not exactly a prototypical amino acid for single-pass transmembrane helices. In this study, we investigate how the polar aspartate residue is accommodated in lipid bilayers containing POPC with and without cholesterol, using all-atom molecular dynamics simulations. We identify two cholesterol-binding sites: (i) A291, F298 and L299 and (ii) L299, F302 and E306 and suggest that cholesterol plays a role in stabilizing the helix in a transmembrane position. We suggest that the presence of the aspartate could be functionally relevant for the scramblase protein activity.

  11. C-Terminal Region of Sulfite Reductase Is Important to Localize to Chloroplast Nucleoids in Land Plants

    PubMed Central

    Kobayashi, Yusuke; Otani, Takuto; Ishibashi, Kota; Shikanai, Toshiharu; Nishimura, Yoshiki

    2016-01-01

    Chloroplast (cp) DNA is compacted into cpDNA-protein complexes, called cp nucleoids. An abundant and extensively studied component of cp nucleoids is the bifunctional protein sulfite reductase (SiR). The preconceived role of SiR as the core cp nucleoid protein, however, is becoming less likely because of the recent findings that SiRs do not associate with cp nucleoids in some plant species, such as Zea mays and Arabidopsis thaliana. To address this discrepancy, we have performed a detailed phylogenetic analysis of SiRs, which shows that cp nucleoid-type SiRs share conserved C-terminally encoded peptides (CEPs). The CEPs are likely to form a bacterial ribbon–helix–helix DNA-binding motif, implying a potential role in attaching SiRs onto cp nucleoids. A proof-of-concept experiment was conducted by fusing the nonnucleoid-type SiR from A. thaliana (AtSiR) with the CEP from the cp nucleoid-type SiR of Phaseolus vulgaris. The addition of the CEP drastically altered the intra-cp localization of AtSiR to cp nucleoids. Our analysis supports the possible functions of CEPs in determining the localization of SiRs to cp nucleoids and illuminates a possible evolutionary scenario for SiR as a cp nucleoid protein. PMID:27189994

  12. Targeting of C-Terminal Binding Protein (CtBP) by ARF Results in p53-Independent Apoptosis

    PubMed Central

    Paliwal, Seema; Pande, Sandhya; Kovi, Ramesh C.; Sharpless, Norman E.; Bardeesy, Nabeel; Grossman, Steven R.

    2006-01-01

    ARF encodes a potent tumor suppressor that antagonizes MDM2, a negative regulator of p53. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF or p53 singly null mice, results in a broadened tumor spectrum and decreased tumor latency. To investigate the mechanism of p53-independent tumor suppression by ARF, potential interacting proteins were identified by yeast two-hybrid screen. The antiapoptotic transcriptional corepressor C-terminal binding protein 2 (CtBP2) was identified, and ARF interactions with both CtBP1 and CtBP2 were confirmed in vitro and in vivo. Interaction with ARF resulted in proteasome-dependent CtBP degradation. Both ARF-induced CtBP degradation and CtBP small interfering RNA led to p53-independent apoptosis in colon cancer cells. ARF induction of apoptosis was dependent on its ability to interact with CtBP, and reversal of ARF-induced CtBP depletion by CtBP overexpression abrogated ARF-induced apoptosis. CtBP proteins represent putative targets for p53-independent tumor suppression by ARF. PMID:16508011

  13. P13, an Integral Membrane Protein of Borrelia burgdorferi, Is C-Terminally Processed and Contains Surface-Exposed Domains

    PubMed Central

    Noppa, Laila; Östberg, Yngve; Lavrinovicha, Marija; Bergström, Sven

    2001-01-01

    To elucidate antigens present on the bacterial surface of Borrelia burgdorferi sensu lato that may be involved in pathogenesis, we characterized a protein, P13, with an apparent molecular mass of 13 kDa. The protein was immunogenic and was expressed in large amounts during in vitro cultivation compared to other known antigens. An immunofluorescence assay, immunoelectron microscopy, and protease sensitivity assays indicated that P13 is surface exposed. The deduced sequence of the P13 peptide revealed a possible signal peptidase type I cleavage site, and computer analysis predicted that P13 is an integral membrane protein with three transmembrane-spanning domains. Mass spectrometry, in vitro translation, and N- and C-terminal amino acid sequencing analyses indicated that P13 was posttranslationally processed at both ends and modified by an unknown mechanism. Furthermore, p13 belongs to a gene family with five additional members in B. burgdorferi sensu stricto. The p13 gene is located on the linear chromosome of the bacterium, in contrast to five paralogous genes, which are located on extrachromosomal plasmids. The size of the p13 transcript was consistent with a monocistronic transcript. This new gene family may be involved in functions that are specific for this spirochete and its pathogenesis. PMID:11292755

  14. A Superhelical Spiral in the Escherichia coli DNA Gyrase A C-terminal Domain Imparts Unidirectional Supercoiling Bias

    SciTech Connect

    Ruthenburg,A.; Graybosch, D.; Huetsch, J.; Verdine, G.

    2005-01-01

    DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped {beta}-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (-) DNA supercoiling.

  15. Ubiquitin C-terminal hydrolase L1 (UCH-L1): structure, distribution and roles in brain function and dysfunction.

    PubMed

    Bishop, Paul; Rocca, Dan; Henley, Jeremy M

    2016-08-15

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is an extremely abundant protein in the brain where, remarkably, it is estimated to make up 1-5% of total neuronal protein. Although it comprises only 223 amino acids it has one of the most complicated 3D knotted structures yet discovered. Beyond its expression in neurons UCH-L1 has only very limited expression in other healthy tissues but it is highly expressed in several forms of cancer. Although UCH-L1 is classed as a deubiquitinating enzyme (DUB) the direct functions of UCH-L1 remain enigmatic and a wide array of alternative functions has been proposed. UCH-L1 is not essential for neuronal development but it is absolutely required for the maintenance of axonal integrity and UCH-L1 dysfunction is implicated in neurodegenerative disease. Here we review the properties of UCH-L1, and how understanding its complex structure can provide new insights into its roles in neuronal function and pathology.

  16. Ubiquitin C-terminal hydrolase L1 deletion ameliorates glomerular injury in mice with ACTN4-associated focal segmental glomerulosclerosis.

    PubMed

    Read, Naomi C; Gutsol, Alex; Holterman, Chet E; Carter, Anthony; Coulombe, Josée; Gray, Douglas A; Kennedy, Chris R J

    2014-07-01

    Renal ubiquitin C-terminal hydrolase L1 (UCHL1) is upregulated in a subset of human glomerulopathies, including focal segmental glomerulosclerosis (FSGS), where it may serve to promote ubiquitin pools for degradation of cytotoxic proteins. In the present study, we tested whether UCHL1 is expressed in podocytes of a mouse model of ACTN4-associated FSGS. Podocyte UCHL1 protein was detected in glomeruli of K256E-ACTN4(pod+)/UCHL1+/+ mice. UCHL1+/- mice were intercrossed with K256E-ACTN4(pod+) mice and monitored for features of glomerular disease. 10-week-old K256E-ACTN4(pod+)/UCHL1-/- mice exhibited significantly ameliorated albuminuria, glomerulosclerosis, tubular pathology and blood pressure. Interestingly, while UCHL1 deletion diminished both tubular and glomerular apoptosis, WT1-positive nuclei were unchanged. Finally, UCHL1 levels correlated positively with poly-ubiquitinated proteins but negatively with K256E-α-actinin-4 levels, implying reduced K256E-α-actinin-4 proteolysis in the absence of UCHL1. Our data suggest that UCHL1 upregulation in ACTN4-associated FSGS fuels the proteasome and that UCHL1 deletion may impair proteolysis and thereby preserve K256E/wt-α-actinin-4 heterodimers, maintaining podocyte cytoskeletal integrity and protecting the glomerular filtration barrier.

  17. Control of mRNA decapping by positive and negative regulatory elements in the Dcp2 C-terminal domain

    PubMed Central

    He, Feng; Jacobson, Allan

    2015-01-01

    Decapping commits an mRNA to complete degradation and promotes general 5′ to 3′ decay, nonsense-mediated decay (NMD), and transcript-specific degradation. In Saccharomyces cerevisiae, a single decapping enzyme composed of a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2) targets thousands of distinct substrate mRNAs. However, the mechanisms controlling this enzyme's in vivo activity and substrate specificity remain elusive. Here, using a genetic approach, we show that the large C-terminal domain of Dcp2 includes a set of conserved negative and positive regulatory elements. A single negative element inhibits enzymatic activity and controls the downstream functions of several positive elements. The positive elements recruit the specific decapping activators Edc3, Pat1, and Upf1 to form distinct decapping complexes and control the enzyme's substrate specificity and final activation. Our results reveal unforeseen regulatory mechanisms that control decapping enzyme activity and function in vivo, and define roles for several decapping activators in the regulation of mRNA decapping. PMID:26184073

  18. Bio-molecular architects: a scaffold provided by the C-terminal domain of eukaryotic RNA polymerase II.

    PubMed

    Zhang, Mengmeng; Gill, Gordon N; Zhang, Yan

    2010-01-01

    In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD). The CTD provides a dynamic platform to recruit different regulators of the transcription apparatus. Different posttranslational modifications are precisely applied to specific sites of the CTD to coordinate transcription process. Regulators of the RNA polymerase II must identify specific sites in the CTD for cellular survival, metabolism, and development. Even though the CTD is disordered in the eukaryotic RNA polymerase II crystal structures due to its intrinsic flexibility, recent advances in the complex structural analysis of the CTD with its binding partners provide essential clues for understanding how selectivity is achieved for individual site recognition. The recent discoveries of the interactions between the CTD and histone modification enzymes disclose an important role of the CTD in epigenetic control of the eukaryotic gene expression. The intersection of the CTD code with the histone code discloses an intriguing yet complicated network for eukaryotic transcriptional regulation.

  19. The Conformational Stability of Nonfibrillar Amyloid-β Peptide Oligomers Critically Depends on the C-Terminal Peptide Length

    PubMed Central

    2014-01-01

    The amyloid-β (Aβ) peptide is one key molecule in the pathogenesis of Alzheimer’s disease. We investigated the conformational stability of a nonfibrillar tetrameric Aβ structure by molecular dynamics (MD) simulations revealing that the stability of the Aβ tetramer depends critically on the C-terminal length. In contrast to the Aβ17–40 tetramer, which proved to be instable, the simulations demonstrate structural integrity of the Aβ17–42 and Aβ17–43 tetramers. These differences in stability can be attributed to an extension of the middle strand of a three-stranded antiparallel β sheet through residues 41–43, only present in the longer Aβ species that aggregate faster and are more neurotoxic. Additional MD simulations demonstrate that this higher stability is also present in the monomers forming the tetramer. In conclusion, our findings suggest the existence of a nonfibrillar oligomer topology that is significantly more stable for the longer Aβ species, thus offering a structural explanation for their higher neurotoxicity. PMID:24494584

  20. CK2-dependent C-terminal phosphorylation at T{sup 30} directs the nuclear transport of TSPY protein

    SciTech Connect

    Krick, Roswitha; Aschrafi, Amaz; Hasguen, Dilek; Arnemann, Joachim |. E-mail: Joachim_Arnemann@web.de

    2006-03-10

    TSPY (testis-specific protein, Y-encoded) is a member of the greater SET/NAP family of molecules with various functions, e.g., in chromatin remodeling, regulation of gene expression, and has been implicated to play a role in the malignant development of gonadoblastoma, testicular and prostate cancer. Here we demonstrate that the C-terminus has a functional role for the nucleo-cytoplasmatic shuttling of the TSPY protein. Using various combinations of in vitro mutagenesis and enhanced green fluorescent protein reporter gene-expression experiments we were able to show that while the deletion of C-terminus leads to a decreased stability and enhanced degradation of the protein, the selective mutation of a C-terminal CK2 phosphorylation site (T{sup 30}) prevents the TSPY protein from entering the nucleus. We conclude that phosphorylation of the (T{sup 30}) residue is a necessary and functional prerequisite for TSPY's transport into the nucleus reminding of comparable data from a related Drosophila molecule, NAP1.

  1. The BARD1 C-Terminal Domain Structure and Interactions with Polyadenylation Factor CstF-50

    SciTech Connect

    Edwards, Ross A.; Lee, Megan S.; Tsutakawa, Susan E.; Williams, R. Scott; Tainer, John A.; Glover, J. N. Mark

    2009-07-13

    The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF- 50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins.Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

  2. A C-terminal PDZ domain binding sequence is required for striatal distribution of the dopamine transporter

    PubMed Central

    Rickhag, Mattias; Hansen, Freja Herborg; Sørensen, Gunnar; Strandfelt, Kristine Nørgaard; Andresen, Bjørn; Gotfryd, Kamil; Madsen, Kenneth L.; Vestergaard-Klewe, Ib; Ammendrup-Johnsen, Ina; Eriksen, Jacob; Füchtbauer, Ernst-Martin; Gomeza, Jesus; Woldbye, David P.D.; Wörtwein, Gitta; Gether, Ulrik

    2013-01-01

    The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft. The cellular mechanisms controlling DAT levels in striatal nerve terminals remain poorly understood. DAT contains a C-terminal PDZ (PSD-95/Discs-large/ZO-1) domain binding sequence believed to bind synaptic scaffolding proteins, but its functional significance is uncertain. Here we demonstrate that two different DAT knock-in mice with disrupted PDZ-binding motifs (DAT-AAA and DAT+Ala) are characterized by dramatic loss of DAT expression in the striatum, causing hyperlocomotion and attenuated response to amphetamine. In cultured dopaminergic neurons and striatal slices from DAT-AAA mice, we find markedly reduced DAT surface levels and evidence for enhanced constitutive internalization. In DAT-AAA neurons, but not in wild type neurons, surface levels are rescued in part by expression of a dominant-negative dynamin mutation (K44A). Our findings suggest that PDZ domain interactions are critical for synaptic distribution of DAT in vivo and thereby for proper maintenance of dopamine homeostasis. PMID:23481388

  3. Fission yeast Cdk7 controls gene expression through both its CAK and C-terminal domain kinase activities.

    PubMed

    Devos, Maxime; Mommaerts, Elise; Migeot, Valerie; van Bakel, Harm; Hermand, Damien

    2015-05-01

    Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity.

  4. Fission Yeast Cdk7 Controls Gene Expression through both Its CAK and C-Terminal Domain Kinase Activities

    PubMed Central

    Devos, Maxime; Mommaerts, Elise; Migeot, Valerie; van Bakel, Harm

    2015-01-01

    Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity. PMID:25691663

  5. Glutathione-conjugating and membrane-remodeling activity of GDAP1 relies on amphipathic C-terminal domain

    PubMed Central

    Huber, Nina; Bieniossek, Christoph; Wagner, Konstanze Marion; Elsässer, Hans-Peter; Suter, Ueli; Berger, Imre; Niemann, Axel

    2016-01-01

    Mutations in the ganglioside-induced differentiation associated protein 1 (GDAP1) cause severe peripheral motor and sensory neuropathies called Charcot-Marie-Tooth disease. GDAP1 expression induces fission of mitochondria and peroxisomes by a currently elusive mechanism, while disease causing mutations in GDAP1 impede the protein’s role in mitochondrial dynamics. In silico analysis reveals sequence similarities of GDAP1 to glutathione S-transferases (GSTs). However, a proof of GST activity and its possible impact on membrane dynamics are lacking to date. Using recombinant protein, we demonstrate for the first time theta-class-like GST activity for GDAP1, and it’s activity being regulated by the C-terminal hydrophobic domain 1 (HD1) of GDAP1 in an autoinhibitory manner. Moreover, we show that the HD1 amphipathic pattern is required to induce membrane dynamics by GDAP1. As both, fission and GST activities of GDAP1, are critically dependent on HD1, we propose that GDAP1 undergoes a molecular switch, turning from a pro-fission active to an auto-inhibited inactive conformation. PMID:27841286

  6. C-terminal domain small phosphatase 1 and MAP kinase reciprocally control REST stability and neuronal differentiation

    PubMed Central

    Corson, Glen M.; McCleskey, Maxwell; Oyer, Jon A.; Mandel, Gail

    2014-01-01

    The repressor element 1 (RE1) silencing transcription factor (REST) in stem cells represses hundreds of genes essential to neuronal function. During neurogenesis, REST is degraded in neural progenitors to promote subsequent elaboration of a mature neuronal phenotype. Prior studies indicate that part of the degradation mechanism involves phosphorylation of two sites in the C terminus of REST that require activity of beta-transducin repeat containing E3 ubiquitin protein ligase, βTrCP. We identify a proline-directed phosphorylation motif, at serines 861/864 upstream of these sites, which is a substrate for the peptidylprolyl cis/trans isomerase, Pin1, as well as the ERK1/2 kinases. Mutation at S861/864 stabilizes REST, as does inhibition of Pin1 activity. Interestingly, we find that C-terminal domain small phosphatase 1 (CTDSP1), which is recruited by REST to neuronal genes, is present in REST immunocomplexes, dephosphorylates S861/864, and stabilizes REST. Expression of a REST peptide containing S861/864 in neural progenitors inhibits terminal neuronal differentiation. Together with previous work indicating that both REST and CTDSP1 are expressed to high levels in stem cells and down-regulated during neurogenesis, our results suggest that CTDSP1 activity stabilizes REST in stem cells and that ERK-dependent phosphorylation combined with Pin1 activity promotes REST degradation in neural progenitors. PMID:25197063

  7. Structure of FIV capsid C-terminal domain demonstrates lentiviral evasion of genetic fragility by coevolved substitutions

    PubMed Central

    Khwaja, Aya; Galilee, Meytal; Marx, Ailie; Alian, Akram

    2016-01-01

    Viruses use a strategy of high mutational rates to adapt to environmental and therapeutic pressures, circumventing the deleterious effects of random single-point mutations by coevolved compensatory mutations, which restore protein fold, function or interactions damaged by initial ones. This mechanism has been identified as contributing to drug resistance in the HIV-1 Gag polyprotein and especially its capsid proteolytic product, which forms the viral capsid core and plays multifaceted roles in the viral life cycle. Here, we determined the X-ray crystal structure of C-terminal domain of the feline immunodeficiency virus (FIV) capsid and through interspecies analysis elucidate the structural basis of co-evolutionarily and spatially correlated substitutions in capsid sequences, which when otherwise uncoupled and individually substituted into HIV-1 capsid impair virion assembly and infectivity. The ability to circumvent the deleterious effects of single amino acid substitutions by cooperative secondary substitutions allows mutational flexibility that may afford viruses an important survival advantage. The potential of such interspecies structural analysis for preempting viral resistance by identifying such alternative but functionally equivalent patterns is discussed. PMID:27102180

  8. Plasmids for C-terminal tagging in Saccharomyces cerevisiae that contain improved GFP proteins, Envy and Ivy.

    PubMed

    Slubowski, Christian J; Funk, Alyssa D; Roesner, Joseph M; Paulissen, Scott M; Huang, Linda S

    2015-04-01

    Green fluorescent protein (GFP) has become an invaluable tool in biological research. Many GFP variants have been created that differ in brightness, photostability, and folding robustness. We have created two hybrid GFP variants, Envy and Ivy, which we placed in a vector for the C-terminal tagging of yeast proteins by PCR-mediated recombination. The Envy GFP variant combines mutations found in the robustly folding SuperfolderGFP and GFPγ, while the Ivy GFP variant is a hybrid of GFPγ and the yellow-green GFP variant, Clover. We compared Envy and Ivy to EGFP, SuperfolderGFP and GFPγ and found that Envy is brighter than the other GFP variants at both 30°C and 37°C, while Ivy is the most photostable. Envy and Ivy are recognized by a commonly used anti-GFP antibody, and both variants can be immunoprecipitated using the GFP TRAP Camelidae antibody nanotrap technology. Because Envy is brighter than the other GFP variants and is as photostable as GFPγ, we suggest that Envy should be the preferred GFP variant, while Ivy may be used in cases where photostability is of the utmost importance.

  9. C-terminal functional unit of Rapana thomasiana (marine snail, gastropod) hemocyanin isoform RtH1: isolation and characterization.

    PubMed

    Parvanova, Katja; Idakieva, Krassimira; Todinova, Svetla; Genov, Nicolay

    2003-09-23

    Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin (Hc) isoforms termed RtH1 and RtH2. Both subunit types are built up of eight functional units (FUs). The C-terminal functional unit (RtH1-h) of the Rapana Hc subunit 1 has been isolated by limited trypsinolysis of the subunit polypeptide chain. The oxy- and apo-forms of the unit are characterized by fluorescence spectroscopy. Upon excitation of RtH1-h at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein globule determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the indole emission of the oxy-RtH1-h. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-RtH1-h. The thermal stability of the apo-RtH1-h is characterized fluorimetrically by the "melting" temperature T(m) (65 degrees C) and by the transition temperature T(m) (83 degrees C) obtained by differential scanning calorimetry for oxy-RtH1-h. The results confirm the role of the copper-dioxygen complex for the stabilization of the Hc structure in solution.

  10. Ubiquitin C-terminal hydrolase37 regulates Tcf7 DNA binding for the activation of Wnt signalling.

    PubMed

    Han, Wonhee; Lee, Hyeyoon; Han, Jin-Kwan

    2017-02-15

    The Tcf/Lef family of transcription factors mediates the Wnt/β-catenin pathway that is involved in a wide range of biological processes, including vertebrate embryogenesis and diverse pathogenesis. Post-translational modifications, including phosphorylation, sumoylation and acetylation, are known to be important for the regulation of Tcf/Lef proteins. However, the importance of ubiquitination and ubiquitin-mediated regulatory mechanisms for Tcf/Lef activity are still unclear. Here, we newly show that ubiquitin C-terminal hydrolase 37 (Uch37), a deubiquitinase, interacts with Tcf7 (formerly named Tcf1) to activate Wnt signalling. Biochemical analyses demonstrated that deubiquitinating activity of Uch37 is not involved in Tcf7 protein stability but is required for the association of Tcf7 to target gene promoter in both Xenopus embryo and human liver cancer cells. In vivo analyses further revealed that Uch37 functions as a positive regulator of the Wnt/β-catenin pathway downstream of β-catenin stabilization that is required for the expression of ventrolateral mesoderm genes during Xenopus gastrulation. Our study provides a new mechanism for chromatin occupancy of Tcf7 and uncovers the physiological significance of Uch37 during early vertebrate development by regulating the Wnt/β-catenin pathway.

  11. The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C-terminal cysteine of the CXXC motif.

    PubMed

    Davey, Lauren; Cohen, Alejandro; LeBlanc, Jason; Halperin, Scott A; Lee, Song F

    2016-01-01

    Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N-terminal cysteine interacts with substrates, whereas the C-terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C(86) P(87) D(88) C(89) catalytic motif. In vitro, SdbA single cysteine variants at the N or C-terminal position (SdbAC86P and SdbAC89A ) were active but displayed different susceptibility to oxidation, and N-terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N-terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C-terminal cysteine alone (in the SdbAC86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C-terminal cysteine.

  12. ATG4B contains a C-terminal LIR motif important for binding and efficient cleavage of mammalian orthologs of yeast Atg8.

    PubMed

    Skytte Rasmussen, Mads; Mouilleron, Stéphane; Kumar Shrestha, Birendra; Wirth, Martina; Lee, Rebecca; Bowitz Larsen, Kenneth; Abudu Princely, Yakubu; O'Reilly, Nicola; Sjøttem, Eva; Tooze, Sharon A; Lamark, Trond; Johansen, Terje

    2017-02-15

    The cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.

  13. Three-dimensional structure of a Streptomyces sviceus GNAT acetyltransferase with similarity to the C-terminal domain of the human GH84 O-GlcNAcase.

    PubMed

    He, Yuan; Roth, Christian; Turkenburg, Johan P; Davies, Gideon J

    2014-01-01

    The mammalian O-GlcNAc hydrolysing enzyme O-GlcNAcase (OGA) is a multi-domain protein with glycoside hydrolase activity in the N-terminus and with a C-terminal domain that has low sequence similarity to known acetyltransferases, prompting speculation, albeit controversial, that the C-terminal domain may function as a histone acetyltransferase (HAT). There are currently scarce data available regarding the structure and function of this C-terminal region. Here, a bacterial homologue of the human OGA C-terminal domain, an acetyltransferase protein (accession No. ZP_05014886) from Streptomyces sviceus (SsAT), was cloned and its crystal structure was solved to high resolution. The structure reveals a conserved protein core that has considerable structural homology to the acetyl-CoA (AcCoA) binding site of GCN5-related acetyltransferases (GNATs). Calorimetric data further confirm that SsAT is indeed able to bind AcCoA in solution with micromolar affinity. Detailed structural analysis provided insight into the binding of AcCoA. An acceptor-binding cavity was identified, indicating that the physiological substrate of SsAT may be a small molecule. Consistent with recently published work, the SsAT structure further questions a HAT function for the human OGA domain.

  14. Evaluation of Heavy Chain C-terminal Deletions on Productivity and Product Quality of Monoclonal Antibodies in Chinese Hamster Ovary (CHO) Cells.

    PubMed

    Hu, Zhilan; Tang, Danming; Misaghi, Shahram; Jiang, Guoying; Yu, Christopher; Yim, Mandy; Shaw, David; Snedecor, Brad; Laird, Michael; Shen, Amy

    2017-02-11

    Monoclonal antibodies (mAbs) have been well established as potent therapeutic agents and are used to treat many different diseases. During cell culture production, antibody charge variants can be generated by cleavage of heavy chain (HC) C-terminal lysine and proline amidation. Differences in levels of charge variants during manufacturing process changes make it challenging to demonstrate process comparability. In order to reduce heterogeneity and achieve consistent product quality, we generated and expressed antibodies with deletion of either HC C-terminal lysine (-K) or lysine and glycine (-GK). Interestingly, clones that express antibodies lacking HC C-terminal lysine (-K) had considerably lower specific productivities compared to clones that expressed either wild type antibodies (WT) or antibodies lacking HC glycine and lysine (-GK). While no measurable differences in antibody HC and LC mRNA levels, glycosylation and secretion were observed, our analysis suggests that the lower specific productivity of clones expressing antibody lacking HC C-terminal lysine was due to slower antibody HC synthesis and faster antibody degradation. This article is protected by copyright. All rights reserved.

  15. Differential cellulolytic activity of native-form and C-terminal tagged-form cellulase derived from coptotermes formosanus and expressed in E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The endogenous cellulase gene (CfEG3a) of Coptotermes formosanus, an economically important pest termite, was cloned and overexpressed in both native form (nCfEG) and C-terminal His-tagged form (tCfEG) in E.coli. Both forms of recombinant cellulases showed hydrolytic activity on cellulosic substrate...

  16. Artemis C-terminal region facilitates V(D)J recombination through its interactions with DNA Ligase IV and DNA-PKcs

    PubMed Central

    Malu, Shruti; De Ioannes, Pablo; Kozlov, Mikhail; Greene, Marsha; Francis, Dailia; Hanna, Mary; Pena, Jesse; Escalante, Carlos R.; Kurosawa, Aya; Erdjument-Bromage, Hediye; Tempst, Paul; Adachi, Noritaka; Vezzoni, Paolo; Villa, Anna; Aggarwal, Aneel K.

    2012-01-01

    Artemis is an endonuclease that opens coding hairpin ends during V(D)J recombination and has critical roles in postirradiation cell survival. A direct role for the C-terminal region of Artemis in V(D)J recombination has not been defined, despite the presence of immunodeficiency and lymphoma development in patients with deletions in this region. Here, we report that the Artemis C-terminal region directly interacts with the DNA-binding domain of Ligase IV, a DNA Ligase which plays essential roles in DNA repair and V(D)J recombination. The Artemis–Ligase IV interaction is specific and occurs independently of the presence of DNA and DNA–protein kinase catalytic subunit (DNA-PKcs), another protein known to interact with the Artemis C-terminal region. Point mutations in Artemis that disrupt its interaction with Ligase IV or DNA-PKcs reduce V(D)J recombination, and Artemis mutations that affect interactions with Ligase IV and DNA-PKcs show additive detrimental effects on coding joint formation. Signal joint formation remains unaffected. Our data reveal that the C-terminal region of Artemis influences V(D)J recombination through its interaction with both Ligase IV and DNA-PKcs. PMID:22529269

  17. C-terminal Pro-Gly-Pro tripeptide in contrast to full-length neuropeptide semax exhibits no neuroprotective effect in experimental cerebral ischemia.

    PubMed

    Fadyukova, O E; Kadi, A; Bai, Ou; Andzhusheva, G M; Koshelev, V B

    2005-04-01

    The C-terminal fragment Pro-Gly-Pro of semax does not modulate the development of symptoms of neurological deficiency and mortality in rats with incomplete global cerebral ischemia. Hence, previously revealed neuroprotective effects of semax are mainly determined by corticotropin ACTH4-7 fragment.

  18. The activity of protein phosphatase 5 towards native clients is modulated by the middle- and C-terminal domains of Hsp90

    PubMed Central

    Haslbeck, Veronika; Eckl, Julia M.; Drazic, Adrian; Rutz, Daniel A.; Lorenz, Oliver R.; Zimmermann, Kerstin; Kriehuber, Thomas; Lindemann, Claudia; Madl, Tobias; Richter, Klaus

    2015-01-01

    Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex. PMID:26593036

  19. Two Distinct Binding Modes Define the Interaction of Brox with the C-Terminal Tails of CHMP5 and CHMP4B

    SciTech Connect

    Mu, Ruiling; Dussupt, Vincent; Jiang, Jiansheng; Sette, Paola; Rudd, Victoria; Chuenchor, Watchalee; Bello, Nana F.; Bouamr, Fadila; Xiao, Tsan Sam

    2012-05-21

    Interactions of the CHMP protein carboxyl terminal tails with effector proteins play important roles in retroviral budding, cytokinesis, and multivesicular body biogenesis. Here we demonstrate that hydrophobic residues at the CHMP4B C-terminal amphipathic {alpha} helix bind a concave surface of Brox, a mammalian paralog of Alix. Unexpectedly, CHMP5 was also found to bind Brox and specifically recruit endogenous Brox to detergent-resistant membrane fractions through its C-terminal 20 residues. Instead of an {alpha} helix, the CHMP5 C-terminal tail adopts a tandem {beta}-hairpin structure that binds Brox at the same site as CHMP4B. Additional Brox:CHMP5 interface is furnished by a unique CHMP5 hydrophobic pocket engaging the Brox residue Y348 that is not conserved among the Bro1 domains. Our studies thus unveil a {beta}-hairpin conformation of the CHMP5 protein C-terminal tail, and provide insights into the overlapping but distinct binding profiles of ESCRT-III and the Bro1 domain proteins.

  20. Biophysical characterization of the C-terminal region of FliG, an essential rotor component of the Na+-driven flagellar motor.

    PubMed

    Gohara, Mizuki; Kobayashi, Shiori; Abe-Yoshizumi, Rei; Nonoyama, Natsumi; Kojima, Seiji; Asami, Yasuo; Homma, Michio

    2014-02-01

    The bacterial flagellar motor generates a rotational force by the flow of ions through the membrane. The rotational force is generated by the interaction between the cytoplasmic regions of the rotor and the stator. FliG is directly involved in the torque generation of the rotor protein by its interaction. FliG is composed of three domains: the N-terminal, Middle and C-terminal domains, based on its structure. The C-terminal domain of FliG is assumed to be important for the interaction with the stator that generates torque. In this study, using CD spectra, gel filtration chromatography and DSC (differential scanning calorimetry), we characterized the physical properties of the C-terminal domain (G214-Stop) of wild-type (WT) FliG and its non-motile phenotype mutant derivatives (L259Q, L270R and L271P), which were derived from the sodium-driven motor of Vibrio. The CD spectra and gel filtration chromatography revealed a slight difference between the WT and the mutant FliG proteins, but the DSC results suggested a large difference in their stabilities. That structural difference was confirmed by differences in protease sensitivity. Based on these results, we conclude that mutations which confer the non-motile phenotype destabilize the C-terminal domain of FliG.

  1. A K+ channel splice variant common in human heart lacks a C-terminal domain required for expression of rapidly activating delayed rectifier current.

    PubMed

    Kupershmidt, S; Snyders, D J; Raes, A; Roden, D M

    1998-10-16

    We have cloned HERG USO, a C-terminal splice variant of the human ether-à-go-go-related gene (HERG), the gene encoding the rapid component of the delayed rectifier (IKr), from human heart, and we find that its mRNA is approximately 2-fold more abundant than that for HERG1 (the originally described cDNA). After transfection of HERG USO in Ltk- cells, no current was observed. However, coexpression of HERG USO with HERG1 modified IKr by decreasing its amplitude, accelerating its activation, and shifting the voltage dependence of activation 8.8 mV negative. As with HERG USO, HERGDeltaC (a HERG1 construct lacking the C-terminal 462 amino acids) also produced no current in transfected cells. However, IKr was rescued by ligation of 104 amino acids from the C terminus of HERG1 to the C terminus of HERGDeltaC, indicating that the C terminus of HERG1 includes a domain (C-terminal domain not only explains the finding that HERG USO does not generate IKr but also indicates a similar mechanism for hitherto-uncharacterized long QT syndrome HERG mutations that disrupt the splice site or the C-terminal. We suggest that the amplitude and gating of cardiac IKr depends on expression of both HERG1 and HERG USO.

  2. Presence and in vivo biosynthesis of fragments of CPP (the C-terminal glycopeptide of the rat vasopressin precursor) in the hypothalamo-neurohypophyseal system

    SciTech Connect

    Seger, M.A.; Burbach, J.P.

    1987-09-01

    The existence and rate of formation of fragments of the 39-residue C-terminal glycopeptide of propressophysin (CPP1-39) was investigated in the hypothalamo-neurohypophyseal system. Newly-prepared antisera to CPP were used to confirm the existence of smaller C-terminal fragments derived from CPP1-39. Radiolabelled fucose was injected into rats in vivo into the area of the supraoptic nucleus, and the labelled peptides formed in the neurohypophysis were examined at various time intervals up to five weeks after the injection. The products derived from the neurohypophyseal hormone precursors were separated by high-performance liquid chromatography. The level of the major immunoreactive C-terminal fragment (CPP22-39) was constant and represented about 5% of the intact CPP1-39 in the neurohypophysis. The appearance of newly-synthesized N-terminal fragment of CPP1-39 occurred only after 3 or 4 days. This fucose labelled fragment increased slowly thereafter until it reached the same level as the CPP C-terminal fragment immunoreactivity between 21 and 28 days after injection. The results suggest that CPP1-39 is extremely stable in the hypothalamo-neurohypophyseal neurons, and that the cleavage at Arg21-Leu22 is a delayed proteolytic event in the magnocellular neurons of the SON.

  3. Structural and Biochemical Studies of the C-Terminal Domain of Mouse Peptide-N-glycanase Identify it as a Mannose-Binding Module

    SciTech Connect

    Zhou,X.; Zhao, G.; Truglio, J.; Wang, L.; Li, G.; Lennarz, W.; Schindelin, H.

    2006-01-01

    The inability of certain N-linked glycoproteins to adopt their native conformation in the endoplasmic reticulum (ER) leads to their retrotranslocation into the cytosol and subsequent degradation by the proteasome. In this pathway the cytosolic peptide-N-glycanase (PNGase) cleaves the N-linked glycan chains off denatured glycoproteins. PNGase is highly conserved in eukaryotes and plays an important role in ER-associated protein degradation. In higher eukaryotes, PNGase has an N-terminal and a C-terminal extension in addition to its central catalytic domain, which is structurally and functionally related to transglutaminases. Although the N-terminal domain of PNGase is involved in protein-protein interactions, the function of the C-terminal domain has not previously been characterized. Here, we describe biophysical, biochemical, and crystallographic studies of the mouse PNGase C-terminal domain, including visualization of a complex between this domain and mannopentaose. These studies demonstrate that the C-terminal domain binds to the mannose moieties of N-linked oligosaccharide chains, and we further show that it enhances the activity of the mouse PNGase core domain, presumably by increasing the affinity of mouse PNGase for the glycan chains of misfolded glycoproteins.

  4. Chimeras between single-stranded DNA-binding proteins from Escherichia coli and Mycobacterium tuberculosis reveal that their C-terminal domains interact with uracil DNA glycosylases.

    PubMed

    Handa, P; Acharya, N; Varshney, U

    2001-05-18

    Uracil, a promutagenic base in DNA can arise by spontaneous deamination of cytosine or incorporation of dUMP by DNA polymerase. Uracil is removed from DNA by uracil DNA glycosylase (UDG), the first enzyme in the uracil excision repair pathway. We recently reported that the Escherichia coli single-stranded DNA binding protein (SSB) facilitated uracil excision from certain structured substrates by E. coli UDG (EcoUDG) and suggested the existence of interaction between SSB and UDG. In this study, we have made use of the chimeric proteins obtained by fusion of N- and C-terminal domains of SSBs from E. coli and Mycobacterium tuberculosis to investigate interactions between SSBs and UDGs. The EcoSSB or a chimera containing its C-terminal domain interacts with EcoUDG in a binary (SSB-UDG) or a ternary (DNA-SSB-UDG) complex. However, the chimera containing the N-terminal domain from EcoSSB showed no interactions with EcoUDG. Thus, the C-terminal domain (48 amino acids) of EcoSSB is necessary and sufficient for interaction with EcoUDG. The data also suggest that the C-terminal domain (34 amino acids) of MtuSSB is a predominant determinant for mediating its interaction with MtuUDG. The mechanism of how the interactions between SSB and UDG could be important in uracil excision repair pathway has been discussed.

  5. Serum carboxy-terminal telopeptide of type I collagen (I-CTP) is predictive of clinical outcome in peripheral artery disease patients following endovascular therapy.

    PubMed

    Otaki, Yoichiro; Watanabe, Tetsu; Takahashi, Hiroki; Yamaura, Gensai; Nishiyama, Satoshi; Arimoto, Takanori; Shishido, Tetsuro; Miyamoto, Takuya; Kubota, Isao

    2017-02-01

    Peripheral artery disease (PAD) is a risk factor for the development of heart failure and mortality. The serum levels of carboxy-terminal telopeptide of type I collagen (I-CTP), a marker for collagen degradation derived from the extracellular matrix of vascular and myocardial tissue, is reportedly a useful marker for cardiac events in patients with heart disease. However, it remains unclear whether I-CTP can predict poor clinical outcome in patients with PAD. Serum I-CTP was measured in 246 consecutive PAD patients who underwent endovascular therapy. Patients were prospectively followed during a median follow-up period of 887 days with the end points of major adverse cardiovascular events (MACE). I-CTP was significantly higher in patients with critical limb ischemia than those without. A multivariate Cox proportional hazard analysis revealed that I-CTP was an independent predictor of MACE after adjusting for confounding factors. Patients were stratified into three groups based on I-CTP level tertile, and those with third tertile had higher levels of brain natriuretic peptide levels and high-sensitivity C-reactive protein compared to the other two groups. Kaplan-Meier analysis demonstrated that patients in the highest tertile of I-CTP also had the greatest risk of MACE. The net reclassification index significantly improved with the addition of I-CTP to basic predictors. I-CTP is a reliable marker and indicator for MACE in patients with PAD.

  6. Three-dimensional structure of a Streptomyces sviceus GNAT acetyltransferase with similarity to the C-terminal domain of the human GH84 O-GlcNAcase

    SciTech Connect

    He, Yuan; Roth, Christian; Turkenburg, Johan P.; Davies, Gideon J.

    2014-01-01

    The crystal structure of a bacterial acetyltransferase with 27% sequence identity to the C-terminal domain of human O-GlcNAcase has been solved at 1.5 Å resolution. This S. sviceus protein is compared with known GCN5-related acetyltransferases, adding to the diversity observed in this superfamily. The mammalian O-GlcNAc hydrolysing enzyme O-GlcNAcase (OGA) is a multi-domain protein with glycoside hydrolase activity in the N-terminus and with a C-terminal domain that has low sequence similarity to known acetyltransferases, prompting speculation, albeit controversial, that the C-terminal domain may function as a histone acetyltransferase (HAT). There are currently scarce data available regarding the structure and function of this C-terminal region. Here, a bacterial homologue of the human OGA C-terminal domain, an acetyltransferase protein (accession No. ZP-05014886) from Streptomyces sviceus (SsAT), was cloned and its crystal structure was solved to high resolution. The structure reveals a conserved protein core that has considerable structural homology to the acetyl-CoA (AcCoA) binding site of GCN5-related acetyltransferases (GNATs). Calorimetric data further confirm that SsAT is indeed able to bind AcCoA in solution with micromolar affinity. Detailed structural analysis provided insight into the binding of AcCoA. An acceptor-binding cavity was identified, indicating that the physiological substrate of SsAT may be a small molecule. Consistent with recently published work, the SsAT structure further questions a HAT function for the human OGA domain.

  7. Crystal structure of a crustacean hyperglycemic hormone (CHH) precursor suggests structural variety in the C-terminal regions of CHH superfamily members.

    PubMed

    Tsutsui, Naoaki; Sakamoto, Tatsuya; Arisaka, Fumio; Tanokura, Masaru; Nagasawa, Hiromichi; Nagata, Koji

    2016-12-01

    The crustacean hyperglycemic hormone (CHH) is one of the major hormones in crustaceans, and peptides belonging to the CHH superfamily have been found in diverse ecdysozoans. Although the basic function of CHH is to control energy metabolism, it also plays various roles in crustacean species, such as in molting and vitellogenesis. Here, we present the crystal structure of Pej-SGP-I-Gly, a partially active precursor of CHH from the kuruma prawn Marsupenaeus japonicus, which has an additional Gly residue in place of the C-terminal amide group of the mature Pej-SGP-I. The 1.6-angstrom crystal structure showed not only the common CHH superfamily scaffold comprising three α-helices, three disulfide bridges, and a hydrophobic core but also revealed that the C-terminal part has a variant backbone fold that is specific to Pej-SGP-I-Gly. The α-helix 4 of Pej-SGP-I-Gly was much longer than that of molt-inhibiting hormone (Pej-MIH) from the same species, and as a result, the following C-terminal helix, corresponding to α-helix 5 in MIH, was not formed. Unlike monomeric Pej-MIH, Pej-SGP-I-Gly forms a homodimer in the crystal structure via its unique α-helix 4. The unexpected dissimilar folds between Pej-SGP-I-Gly and Pej-MIH appear to be the result of their distinct C-terminal amino acid sequences. Variations in amino acid sequences and lengths and the resulting variety of backbone folds allow the C-terminal and sterically adjoining regions to confer different hormonal activities in diverse CHH superfamily members.

  8. C-terminal processing of reaction center protein D1 is essential for the function and assembly of photosystem II in Arabidopsis

    PubMed Central

    Che, Yufen; Fu, Aigen; Hou, Xin; McDonald, Kent; Buchanan, Bob B.; Huang, Weidong; Luan, Sheng

    2013-01-01

    Photosystem II (PSII) reaction center protein D1 is synthesized as a precursor (pD1) with a short C-terminal extension. The pD1 is processed to mature D1 by carboxyl-terminal peptidase A to remove the C-terminal extension and form active protein. Here we report functional characterization of the Arabidopsis gene encoding D1 C-terminal processing enzyme (AtCtpA) in the chloroplast thylakoid lumen. Recombinant AtCtpA converted pD1 to mature D1 and a mutant lacking AtCtpA retained all D1 in precursor form, confirming that AtCtpA is solely responsible for processing. As with cyanobacterial ctpa, a knockout Arabidopsis atctpa mutant was lethal under normal growth conditions but was viable with sucrose under low-light conditions. Viable plants, however, showed deficiencies in PSII and thylakoid stacking. Surprisingly, unlike its cyanobacterial counterpart, the Arabidopsis mutant retained both monomer and dimer forms of the PSII complexes that, although nonfunctional, contained both the core and extrinsic subunits. This mutant was also essentially devoid of PSII supercomplexes, providing an unexpected link between D1 maturation and supercomplex assembly. A knock-down mutant expressing about 2% wild-type level of AtCtpA showed normal growth under low light but was stunted and accumulated pD1 under high light, indicative of delayed C-terminal processing. Although demonstrating the functional significance of C-terminal D1 processing in PSII biogenesis, our study reveals an unsuspected link between D1 maturation and PSII supercomplex assembly in land plants, opening an avenue for exploring the mechanism for the association of light-harvesting complexes with the PSII core complexes. PMID:24043802

  9. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.

    PubMed

    Calver, A R; Robbins, M J; Cosio, C; Rice, S Q; Babbs, A J; Hirst, W D; Boyfield, I; Wood, M D; Russell, R B; Price, G W; Couve, A; Moss, S J; Pangalos, M N

    2001-02-15

    GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

  10. Alleles of the maize P gene with distinct tissue specificities encode Myb-homologous proteins with C-terminal replacements.

    PubMed Central

    Chopra, S; Athma, P; Peterson, T

    1996-01-01

    The maize P gene is a transcriptional regulator of genes encoding enzymes for flavonoid biosynthesis in the pathway leading to the production of a red phlobaphene pigment. Multiple alleles of the P gene confer distinct patterns of pigmentation to specific floral organs, such as the kernel pericarp and cob tissues. To determine the basis of allele-specific pigmentation, we have characterized the gene products and transcript accumulation patterns of the P-wr allele, which specifies colorless pericarps and red cob tissues. RNA transcripts of P-wr are present in colorless pericarps as well as in the colored cob tissues; however, the expression of P-wr in pericarp does not induce the accumulation of transcripts from the C2 and A1 genes, which encode enzymes for flavonoid pigment biosynthesis. The coding sequences of P-wr were compared with the P-rr allele, which specifies red pericarp and red cob. The P-wr and P-rr cDNA sequences are very similar in their 5' regions. There are only two nucleotide changes that result in amino acid differences; both are outside of the Myb-homologous DNA binding domain. In contrast, the 3' coding region of P-rr is replaced by a unique 210-bp sequence in P-wr. The predicted P-wr protein has a C-terminal sequence resembling a cysteine-containing metal binding domain that is not present in the P-rr protein. These results indicate that the differential pericarp pigmentation specified by the P-rr and P-wr alleles does not result from an absence of P-wr transcripts in pericarps. Rather, the allele-specific patterns of P-rr and P-wr pigmentation may be associated with structural differences in the proteins encoded by each allele. PMID:8768374

  11. Reducing C-terminal truncation mitigates synucleinopathy and neurodegeneration in a transgenic model of multiple system atrophy

    PubMed Central

    Bassil, Fares; Fernagut, Pierre-Olivier; Bezard, Erwan; Pruvost, Alain; Leste-Lasserre, Thierry; Hoang, Quyen Q.; Ringe, Dagmar; Petsko, Gregory A.; Meissner, Wassilios G.

    2016-01-01

    Multiple system atrophy (MSA) is a sporadic orphan neurodegenerative disorder. No treatment is currently available to slow down the aggressive neurodegenerative process, and patients die within a few years after disease onset. The cytopathological hallmark of MSA is the accumulation of alpha-synuclein (α-syn) aggregates in affected oligodendrocytes. Several studies point to α-syn oligomerization and aggregation as a mediator of neurotoxicity in synucleinopathies including MSA. C-terminal truncation by the inflammatory protease caspase-1 has recently been implicated in the mechanisms that promote aggregation of α-syn in vitro and in neuronal cell models of α-syn toxicity. We present here an in vivo proof of concept of the ability of the caspase-1 inhibitor prodrug VX-765 to mitigate α-syn pathology and to mediate neuroprotection in proteolipid protein α-syn (PLP-SYN) mice, a transgenic mouse model of MSA. PLP-SYN and age-matched wild-type mice were treated for a period of 11 wk with VX-765 or placebo. VX-765 prevented motor deficits in PLP-SYN mice compared with placebo controls. More importantly, VX-765 was able to limit the progressive toxicity of α-syn aggregation by reducing its load in the striatum of PLP-SYN mice. Not only did VX-765 reduce truncated α-syn, but it also decreased its monomeric and oligomeric forms. Finally, VX-765 showed neuroprotective effects by preserving tyrosine hydroxylase-positive neurons in the substantia nigra of PLP-SYN mice. In conclusion, our results suggest that VX-765, a drug that was well tolerated in a 6 wk-long phase II trial in patients with epilepsy, is a promising candidate to achieve disease modification in synucleinopathies by limiting α-syn accumulation. PMID:27482103

  12. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain.

    PubMed

    Poirier, Steve; Hamouda, Hocine Ait; Villeneuve, Louis; Demers, Annie; Mayer, Gaétan

    2016-01-01

    PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9.

  13. Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels.

    PubMed

    Mayer, Gaétan; Poirier, Steve; Seidah, Nabil G

    2008-11-14

    The proprotein convertase subtilisin/kexin-type 9 (PCSK9), which promotes degradation of the hepatic low density lipoprotein receptor (LDLR), is now recognized as a major player in plasma cholesterol metabolism. Several gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, and thus, inhibition of PCSK9-induced degradation of the LDLR may be used to treat this deadly disease. Herein, we discovered an endogenous PCSK9 binding partner by Far Western blotting, co-immunoprecipitation, and pull-down assays. Following two-dimensional gel electrophoresis and mass spectrometry analysis, we demonstrated that PCSK9 binds to a approximately 33-kDa protein identified as annexin A2 (AnxA2) but not to the closely related annexin A1. Furthermore, our functional LDLR assays and small hairpin RNA studies show that AnxA2 and the AnxA2.p11 complex could prevent PCSK9-directed LDLR degradation in HuH7, HepG2, and Chinese hamster ovary cells. Immunocytochemistry revealed that PCSK9 and AnxA2 co-localize at the cell surface, indicating a possible competition with the LDLR. Structure-function analyses demonstrated that the C-terminal cysteine-histidine-rich domain of PCSK9 interacts specifically with the N-terminal repeat R1 of AnxA2. Mutational analysis of this 70-amino acid-long repeat indicated that the RRTKK81 sequence of AnxA2 is implicated in this binding because its mutation to AATAA81 prevents its interaction with PCSK9. To our knowledge, this work constitutes the first to show that PCSK9 activity on LDLR can be regulated by an endogenous inhibitor. The identification of the minimal inhibitory sequence of AnxA2 should pave the way toward the development of PCSK9 inhibitory lead molecules for the treatment of hypercholesterolemia.

  14. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    PubMed Central

    Villeneuve, Louis; Demers, Annie; Mayer, Gaétan

    2016-01-01

    PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9. PMID:27280970

  15. An antibody against the C-terminal domain of PCSK9 lowers LDL cholesterol levels in vivo.

    PubMed

    Schiele, Felix; Park, John; Redemann, Norbert; Luippold, Gerd; Nar, Herbert

    2014-02-20

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.

  16. Crystal structure of the C-terminal globular domain of oligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 Å resolution.

    PubMed

    Matsumoto, Shunsuke; Igura, Mayumi; Nyirenda, James; Matsumoto, Masaki; Yuzawa, Satoru; Noda, Nobuo; Inagaki, Fuyuhiko; Kohda, Daisuke

    2012-05-22

    Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers glycan to asparagine in the N-glycosylation sequon. The catalytic subunit of OST is called STT3 in eukaryotes, AglB in archaea, and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three AglB paralogs. Two of them are the shortest AglBs across all domains of life. We determined the crystal structure of the C-terminal globular domain of the smallest AglB to identify the minimal structural unit. The Archaeoglobus AglB lacked a β-barrel-like structure, which had been found in other AglB and PglB structures. In agreement, the deletion in a larger Pyrococcus AglB confirmed its dispensability for the activity. By contrast, the Archaeoglobus AglB contains a kinked helix bearing a conserved motif, called DK/MI motif. The lysine and isoleucine residues in the motif participate in the Ser/Thr recognition in the sequon. The Archaeoglobus AglB structure revealed that the kinked helix contained an unexpected insertion. A revised sequence alignment based on this finding identified a variant type of the DK motif with the insertion. A mutagenesis study of the Archaeoglobus AglB confirmed the contribution of this particular type of the DK motif to the activity. When taken together with our previous results, this study defined the classification of OST: one group consisting of eukaryotes and most archaea possesses the DK-type Ser/Thr pocket, and the other group consisting of eubacteria and the remaining archaea possesses the MI-type Ser/Thr pocket. This classification provides a useful framework for OST studies.

  17. C-terminal Src Kinase Gates Homeostatic Synaptic Plasticity and Regulates Fasciclin II Expression at the Drosophila Neuromuscular Junction

    PubMed Central

    Spring, Ashlyn M.; Brusich, Douglas J.; Frank, C. Andrew

    2016-01-01

    Forms of homeostatic plasticity stabilize neuronal outputs and promote physiologically favorable synapse function. A well-studied homeostatic system operates at the Drosophila melanogaster larval neuromuscular junction (NMJ). At the NMJ, impairment of postsynaptic glutamate receptor activity is offset by a compensatory increase in presynaptic neurotransmitter release. We aim to elucidate how this process operates on a molecular level and is preserved throughout development. In this study, we identified a tyrosine kinase-driven signaling system that sustains homeostatic control of NMJ function. We identified C-terminal Src Kinase (Csk) as a potential regulator of synaptic homeostasis through an RNAi- and electrophysiology-based genetic screen. We found that Csk loss-of-function mutations impaired the sustained expression of homeostatic plasticity at the NMJ, without drastically altering synapse growth or baseline neurotransmission. Muscle-specific overexpression of Src Family Kinase (SFK) substrates that are negatively regulated by Csk also impaired NMJ homeostasis. Surprisingly, we found that transgenic Csk-YFP can support homeostatic plasticity at the NMJ when expressed either in the muscle or in the nerve. However, only muscle-expressed Csk-YFP was able to localize to NMJ structures. By immunostaining, we found that Csk mutant NMJs had dysregulated expression of the Neural Cell Adhesion Molecule homolog Fasciclin II (FasII). By immunoblotting, we found that levels of a specific isoform of FasII were decreased in homeostatically challenged GluRIIA mutant animals–but markedly increased in Csk mutant animals. Additionally, we found that postsynaptic overexpression of FasII from its endogenous locus was sufficient to impair synaptic homeostasis, and genetically reducing FasII levels in Csk mutants fully restored synaptic homeostasis. Based on these data, we propose that Csk and its SFK substrates impinge upon homeostatic control of NMJ function by regulating

  18. Multimodal Recognition of Diverse Peptides by the C-Terminal SH2 Domain of Phospholipase C-γ1 Protein.

    PubMed

    McKercher, Marissa A; Guan, Xiaoyang; Tan, Zhongping; Wuttke, Deborah S

    2017-04-11

    SH2 domains recognize phosphotyrosine (pY)-containing peptide ligands and play key roles in the regulation of receptor tyrosine kinase pathways. Each SH2 domain has individualized specificity, encoded in the amino acids neighboring the pY, for defined targets that convey their distinct functions. The C-terminal SH2 domain (PLCC) of the phospholipase C-γ1 full-length protein (PLCγ1) typically binds peptides containing small and hydrophobic amino acids adjacent to the pY, including a peptide derived from platelet-derived growth factor receptor B (PDGFRB) and an intraprotein recognition site (Y783 of PLCγ1) involved in the regulation of the protein's lipase activity. Remarkably, PLCC also recognizes unexpected peptides containing amino acids with polar or bulky side chains that deviate from this pattern. This versatility in recognition specificity may allow PLCγ1 to participate in diverse, previously unrecognized, signaling pathways in response to binding chemically dissimilar partners. We have used structural approaches, including nuclear magnetic resonance and X-ray crystallography, to elucidate the mechanisms of noncognate peptide binding to PLCC by ligands derived from receptor tyrosine kinase ErbB2 and from the insulin receptor. The high-resolution peptide-bound structures reveal that PLCC has a relatively static backbone but contains a chemically rich protein surface comprised of a combination of hydrophobic pockets and amino acids with charged side chains. We demonstrate that this expansive and chemically diverse PLCC interface, in addition to peptide conformational plasticity, permits PLCC to recognize specific noncognate peptide ligands with multimodal specificity.

  19. Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification

    PubMed Central

    Mbogning, Jean; Pagé, Viviane; Burston, Jillian; Schwenger, Emily; Fisher, Robert P.; Schwer, Beate; Shuman, Stewart; Tanny, Jason C.

    2015-01-01

    Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD ‘code’ for co-transcriptional histone modifications. PMID:26275777

  20. C-terminal, endoplasmic reticulum-lumenal domain of prosurfactant protein C - structural features and membrane interactions.

    PubMed

    Casals, Cristina; Johansson, Hanna; Saenz, Alejandra; Gustafsson, Magnus; Alfonso, Carlos; Nordling, Kerstin; Johansson, Jan

    2008-02-01

    Surfactant protein C (SP-C) constitutes the transmembrane part of prosurfactant protein C (proSP-C) and is alpha-helical in its native state. The C-terminal part of proSP-C (CTC) is localized in the endoplasmic reticulum lumen and binds to misfolded (beta-strand) SP-C, thereby preventing its aggregation and amyloid fibril formation. In this study, we investigated the structure of recombinant human CTC and the effects of CTC-membrane interaction on protein structure. CTC forms noncovalent trimers and supratrimeric oligomers. It contains two intrachain disulfide bridges, and its secondary structure is significantly affected by urea or heat only after disulfide reduction. The postulated Brichos domain of CTC, with homologs found in proteins associated with amyloid and proliferative disease, is up to 1000-fold more protected from limited proteolysis than the rest of CTC. The protein exposes hydrophobic surfaces, as determined by CTC binding to the environment-sensitive fluorescent probe 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate). Fluorescence energy transfer experiments further reveal close proximity between bound 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate) and tyrosine residues in CTC, some of which are conserved in all Brichos domains. CTC binds to unilamellar phospholipid vesicles with low micromolar dissociation constants, and differential scanning calorimetry and CD analyses indicate that membrane-bound CTC is less structurally ordered than the unbound protein. The exposed hydrophobic surfaces and the structural disordering that result from interactions with phospholipid membranes suggest a mechanism whereby CTC binds to misfolded SP-C in the endoplasmic reticulum membrane.

  1. Structural dynamics of native and V260E mutant C-terminal domain of HIV-1 integrase

    NASA Astrophysics Data System (ADS)

    Sangeetha, Balasubramanian; Muthukumaran, Rajagopalan; Amutha, Ramaswamy

    2015-04-01

    The C-terminal domain (CTD) of HIV-1 integrase is a five stranded β-barrel resembling an SH3 fold. Mutational studies on isolated CTD and full-length IN have reported V260E mutant as either homo-dimerization defective or affecting the stability and folding of CTD. In this study, molecular dynamics simulation techniques were used to unveil the effect of V260E mutation on isolated CTD monomer and dimer. Both monomeric and dimeric forms of wild type and V260E mutant are highly stable during the simulated period. However, the stabilizing π-stacking interaction between Trp243 and Trp243' at the dimer interface is highly disturbed in CTD-V260E (>6 Å apart). The loss in entropy for dimerization is -30 and -25 kcal/mol for CTD-wt and CTD-V260E respectively signifying a weak hydrophobic interaction and its perturbation in CTD-V260E. The mutant Glu260 exhibits strong attraction/repulsion with all the basic/acidic residues of CTD. In addition to this, the dynamics of CTD-wild type and V260E monomers at 498 K was analyzed to elucidate the effect of V260E mutation on CTD folding. Increase in SASA and reduction in the number of contacts in CTD-V260E during simulation highlights the instability caused by the mutation. In general, V260E mutation affects both multimerization and protein folding with a pronounced effect on protein folding rather than multimerization. This study emphasizes the importance of the hydrophobic nature and SH3 fold of CTD in proper functioning of HIV integrase and perturbing this nature would be a rational approach toward designing more selective and potent allosteric anti-HIV inhibitors.

  2. Involvement of Conserved Amino Acids in the C-Terminal Region of LINE-1 ORF2p in Retrotransposition.

    PubMed

    Christian, Claiborne M; Sokolowski, Mark; deHaro, Dawn; Kines, Kristine J; Belancio, Victoria P

    2017-03-01

    Long interspersed element 1 (L1) is the only currently active autonomous retroelement in the human genome. Along with the parasitic SVA and short interspersed element Alu, L1 is the source of DNA damage induced by retrotransposition: a copy-and-paste process that has the potential to disrupt gene function and cause human disease. The retrotransposition process is dependent upon the ORF2 protein (ORF2p). However, it is unknown whether most of the protein is important for retrotransposition. In particular, other than the Cys motif, the C terminus of the protein has not been intensely examined in the context of retrotransposition. Using evolutionary analysis and the Alu retrotransposition assay, we sought to identify additional amino acids in the C terminus important for retrotransposition. Here, we demonstrate that Gal4-tagged and untagged C-terminally truncated ORF2p fragments possess residual potential to drive Alu retrotransposition. Using sight-directed mutagenesis we identify that while the Y1180 amino acid is important for ORF2p- and L1-driven Alu retrotransposition, a mutation at this position improves L1 retrotransposition. Even though the mechanism of the contribution of Y1180 to Alu and L1 mobilization remains unknown, experimental evidence rules out its direct involvement in the ability of the ORF2p reverse transcriptase to generate complementary DNA. Additionally, our data support that ORF2p amino acids 1180 and 1250-1262 may be involved in the reported ORF1p-mediated increase in ORF2p-driven Alu retrotransposition.

  3. Reducing C-terminal truncation mitigates synucleinopathy and neurodegeneration in a transgenic model of multiple system atrophy.

    PubMed

    Bassil, Fares; Fernagut, Pierre-Olivier; Bezard, Erwan; Pruvost, Alain; Leste-Lasserre, Thierry; Hoang, Quyen Q; Ringe, Dagmar; Petsko, Gregory A; Meissner, Wassilios G

    2016-08-23

    Multiple system atrophy (MSA) is a sporadic orphan neurodegenerative disorder. No treatment is currently available to slow down the aggressive neurodegenerative process, and patients die within a few years after disease onset. The cytopathological hallmark of MSA is the accumulation of alpha-synuclein (α-syn) aggregates in affected oligodendrocytes. Several studies point to α-syn oligomerization and aggregation as a mediator of neurotoxicity in synucleinopathies including MSA. C-terminal truncation by the inflammatory protease caspase-1 has recently been implicated in the mechanisms that promote aggregation of α-syn in vitro and in neuronal cell models of α-syn toxicity. We present here an in vivo proof of concept of the ability of the caspase-1 inhibitor prodrug VX-765 to mitigate α-syn pathology and to mediate neuroprotection in proteolipid protein α-syn (PLP-SYN) mice, a transgenic mouse model of MSA. PLP-SYN and age-matched wild-type mice were treated for a period of 11 wk with VX-765 or placebo. VX-765 prevented motor deficits in PLP-SYN mice compared with placebo controls. More importantly, VX-765 was able to limit the progressive toxicity of α-syn aggregation by reducing its load in the striatum of PLP-SYN mice. Not only did VX-765 reduce truncated α-syn, but it also decreased its monomeric and oligomeric forms. Finally, VX-765 showed neuroprotective effects by preserving tyrosine hydroxylase-positive neurons in the substantia nigra of PLP-SYN mice. In conclusion, our results suggest that VX-765, a drug that was well tolerated in a 6 wk-long phase II trial in patients with epilepsy, is a promising candidate to achieve disease modification in synucleinopathies by limiting α-syn accumulation.

  4. Interactions of the C-terminal Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

    NASA Technical Reports Server (NTRS)

    Hu, Shaowen; Huff, Janice; Pluth, Janice M.; Cucinotta, Francis A.

    2007-01-01

    NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku(sub 70/80) complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The C-terminal domain of Ku70 (Ku70c, residues 559-609), contains an helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the Ku70c/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the subunit Ku70c and a 14 base pairs DNA duplex, whose starting structures are designed to be variable so as to mimic their different binding modes. By analyzing conformational changes and energetic properties of the complex during MD simulations, we found that interactions are preferred at DNA ends, and within the major groove, which is consistent with previous experimental investigations. In addition, the results indicate that cooperation of Ku70c with other subunits of Ku(sub 70/80) is necessary to explain the high affinity of binding as observed in experiments.

  5. Ubiquitin C-terminal hydrolase-L1 protects cystic fibrosis transmembrane conductance regulator from early stages of proteasomal degradation.

    PubMed

    Henderson, Mark J; Vij, Neeraj; Zeitlin, Pamela L

    2010-04-09

    DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) degradation involves ubiquitin modification and efficient proteasomal targeting of the nascent misfolded protein. We show that a deubiquitinating enzyme, ubiquitin C-terminal hydrolase-L1 (UCH-L1), is highly expressed in cystic fibrosis (CF) airway epithelial cells in vitro and in vivo. We hypothesized that the elevation in UCH-L1 in CF cells represents a cellular adaptation to counterbalance excessive proteasomal degradation. The bronchial epithelial cell lines IB3-1 (CF, high UCH-L1 expression) and S9 (non-CF, low UCH-L1 expression) were transiently transfected with wild type (WT) or DeltaF508 CFTR, WT UCH-L1 or small interfering RNA-UCH-L1, and a variety of ubiquitin mutants. We observed a positive correlation between UCH-L1 expression and steady state levels of WT- or DeltaF508-CFTR, and this stabilizing effect was confined to the early stages of CFTR synthesis. Immunolocalization of UCH-L1 by confocal microscopy revealed a partial co-localization with a ribosomal subunit and the endoplasmic reticulum. The UCH-L1-associated increase in CFTR levels was correlated with an increase in ubiquitinated CFTR (CFTR-Ub). Co-transfection with mutant ubiquitins and treatment with proteasome inhibitors suggested that UCH-L1 was reducing the proteasomal targeting of CFTR during synthesis by shortening conjugated polyubiquitin chains. Although not sufficient by itself to rescue mutant CFTR therapeutically, the elevation of UCH-L1 and its effect on CFTR processing provides insight into its potential roles in CF and other diseases.

  6. SUMOylation of the C-terminal domain of DNA topoisomerase IIα regulates the centromeric localization of Claspin

    PubMed Central

    Ryu, Hyunju; Yoshida, Makoto M; Sridharan, Vinidhra; Kumagai, Akiko; Dunphy, William G; Dasso, Mary; Azuma, Yoshiaki

    2015-01-01

    DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity. PMID:26131587

  7. Tissue distribution and safety evaluation of a claudin-targeting molecule, the C-terminal fragment of Clostridium perfringens enterotoxin.

    PubMed

    Li, Xiangru; Saeki, Rie; Watari, Akihiro; Yagi, Kiyohito; Kondoh, Masuo

    2014-02-14

    We previously found that claudin (CL) is a potent target for cancer therapy using a CL-3 and -4-targeting molecule, namely the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Although CL-3 and -4 are expressed in various normal tissues, the safety of this CL-targeting strategy has never been investigated. Here, we evaluated the tissue distribution of C-CPE in mice. Ten minutes after intravenous injection into mice, C-CPE was distributed to the liver and kidney (24.0% and 9.5% of the injected dose, respectively). The hepatic level gradually fell to 3.2% of the injected dose by 3 h post-injection, whereas the renal C-CPE level gradually rose to 46.5% of the injected dose by 6 h post-injection and then decreased. A C-CPE mutant protein lacking the ability to bind CL accumulated in the liver to a much lesser extent (2.0% of the dose at 10 min post-injection) than did C-CPE, but its renal profile was similar to that of C-CPE. To investigate the acute toxicity of CL-targeted toxin, we intravenously administered C-CPE-fused protein synthesis inhibitory factor to mice. The CL-targeted toxin dose-dependently increased the levels of serum biomarkers of liver injury, but not of kidney injury. Histological examination confirmed that injection of CL-targeted toxin injured the liver but not the kidney. These results indicate that potential adverse hepatic effects should be considered in C-CPE-based cancer therapy.

  8. Identification of a Ligand-Binding Site on the Staphylococcus aureus DnaG Primase C-Terminal Domain.

    PubMed

    Catazaro, Jonathan; Periago, Jessica; Shortridge, Matthew D; Worley, Bradley; Kirchner, Andrew; Powers, Robert; Griep, Mark A

    2017-02-21

    The interface between the DnaG primase C-terminal domain (CTD) and the N-terminal domain of DnaB helicase is essential for bacterial DNA replication because it allows coordinated priming of DNA synthesis at the replication fork while the DNA is being unwound. Because these two proteins are conserved in all bacteria and distinct from those in eukaryotes, their interface is an attractive antibiotic target. To learn more about this interface, we determined the solution structure and dynamics of the DnaG primase CTD from Staphylococcus aureus, a medically important bacterial species. Comparison with the known primase CTD structures shows there are two biologically relevant conformations, an open conformation that likely binds to DnaB helicase and a closed conformation that does not. The S. aureus primase CTD is in the closed conformation, but nuclear magnetic resonance (NMR) dynamic studies indicate there is considerable movement in the linker between the two subdomains and that N564 is the most dynamic residue within the linker. A high-throughput NMR ligand affinity screen identified potential binding compounds, among which were acycloguanosine and myricetin. Although the affinity for these compounds and adenosine was in the millimolar range, all three bind to a common pocket that is present only on the closed conformation of the CTD. This binding pocket is at the opposite end of helices 6 and 7 from N564, the key hinge residue. The identification of this binding pocket should allow the development of stronger-binding ligands that can prevent formation of the CTD open conformation that binds to DnaB helicase.

  9. Structure and interactions of the C-terminal metal binding domain of Archaeoglobus fulgidus CopA

    PubMed Central

    Agarwal, Sorabh; Hong, Deli; Desai, Nirav K.; Sazinsky, Matthew H.; Argüello, José M.; Rosenzweig, Amy C.

    2010-01-01

    The Cu+-ATPase CopA from Archaeoglobus fulgidus belongs to the P1B family of the P-type ATPases. These integral membrane proteins couple the energy of ATP hydrolysis to heavy metal ion translocation across membranes. A defining feature of P1B-1-type ATPases is the presence of soluble metal binding domains at the N-terminus (N-MBDs). The N-MBDs exhibit a conserved ferredoxin-like fold, similar to that of soluble copper chaperones, and bind metal ions via a conserved CXXC motif. The N-MBDs enable Cu+ regulation of turnover rates apparently through Cu-sensitive interactions with catalytic domains. A. fulgidus CopA is unusual in that it contains both an N-terminal MBD and a C-terminal MBD (C-MBD). The functional role of the unique C-MBD has not been established. Here, we report the crystal structure of the apo, oxidized C-MBD to 2.0 Å resolution. In the structure, two C-MBD monomers form a domain-swapped dimer, which has not been observed previously for similar domains. In addition, the interaction of the C-MBD with the other cytoplasmic domains of CopA, the ATP binding domain (ATPBD) and actuator domain (A-domain) has been investigated. Interestingly, the C-MBD interacts specifically with both of these domains, independent of the presence of Cu+ or nucleotides. These data reinforce the uniqueness of the C-MBD and suggest a distinct structural role for the C-MBD in CopA transport. PMID:20602459

  10. Translational Repression of a Splice Variant of Cynomolgus Macaque CXCL1L by Its C-Terminal Sequence.

    PubMed

    Nomiyama, Hisayuki; Osada, Naoki; Takahashi, Ichiro; Terao, Keiji; Yamagata, Kazuya; Yoshie, Osamu

    2017-03-01

    We previously isolated a cDNA clone from cynomolgus macaque encoding a novel CXC chemokine that we termed CXCL1L from its close similarity to CXCL1. However, the cDNA consisted of 3 exons instead of 4 exons that were typically seen in other CXC chemokines. Here, we isolated a cDNA encoding the full-length variant of CXCL1L that we termed CXCL1Lβ. CXCL1Lβ is 50 amino acids longer than the original CXCL1L, which we now term CXCL1Lα. The CXCL1Lβ mRNA is much more abundantly expressed in the cynomolgus macaque tissues than CXCL1Lα mRNA. However, CXCL1Lβ protein was poorly produced by transfected cells compared with that of CXCL1Lα. When the coding region of the fourth exon was fused to the C-terminus of CXCL1 or even to a nonsecretory protein firefly luciferase, the fused proteins were also barely produced, although the mRNAs were abundantly expressed. The polysome profiling analysis suggested that the inhibition was mainly at the translational level. Furthermore, we demonstrated that the C-terminal 5 amino acids of CXCL1Lβ were critical for the translational repression. The present study, thus, reveals a unique translational regulation controlling the production of a splicing variant of CXCL1L. Since the CXCL1L gene is functional only in the Old World monkeys, we also discuss possible reasons for the conservation of the active CXCL1L gene in these monkeys during the primate evolution.

  11. Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in Its Subcellular Localization and Shutoff Activity

    PubMed Central

    Hayashi, Tsuyoshi; Chaimayo, Chutikarn; McGuinness, James

    2016-01-01

    ABSTRACT PA-X is a recently identified influenza virus protein that is composed of the PA N-terminal 191 amino acids and unique C-terminal 41 or 61 residues. We and others showed that PA-X has a strong ability to suppress host protein synthesis via host mRNA decay, which is mediated by endonuclease activity in its N-terminal domain (B. W. Jagger, H. M. Wise, J. C. Kash, K. A. Walters, N. M. Wills, Y. L. Xiao, R. L. Dunfee, L. M. Schwartzman, A. Ozinsky, G. L. Bell, R. M. Dalton, A. Lo, S. Efstathiou, J. F. Atkins, A. E. Firth, J. K. Taubenberger, and P. Digard, 2012, Science 337:199–204, http://dx.doi.org/10.1126/science.1222213, and E. A. Desmet, K. A. Bussey, R. Stone, and T. Takimoto, 2013, J Virol 87:3108–3118, http://dx.doi.org/10.1128/JVI.02826-12). However, the mechanism of host mRNA degradation, especially where and how PA-X targets mRNAs, has not been analyzed. In this study, we determined the localization of PA-X and the role of the C-terminal unique region in shutoff activity. Quantitative subcellular localization analysis revealed that PA-X was located equally in both cytoplasm and nucleus. By characterizing a series of PA-X C-terminal deletion mutants, we found that the first 9 amino acids were sufficient for nuclear localization, but an additional 6 residues were required to induce the maximum shutoff activity observed with intact PA-X. Importantly, forced nuclear localization of the PA-X C-terminal deletion mutant enhanced shutoff activity, highlighting the ability of nuclear PA-X to degrade host mRNAs more efficiently. However, PA-X also inhibited luciferase expression from transfected mRNAs synthesized in vitro, suggesting that PA-X also degrades mRNAs in the cytoplasm. Among the basic amino acids in the PA-X C-terminal region, 3 residues, 195K, 198K, and 199R, were identified as key residues for inducing host shutoff and nuclear localization. Overall, our data indicate a critical role for the 15 residues in the PA-X C-terminal domain in

  12. C-Terminal Domain Residues Important for Secretion and Attachment of RgpB in Porphyromonas gingivalis▿

    PubMed Central

    Slakeski, Nada; Seers, Christine A.; Ng, Kaiting; Moore, Caroline; Cleal, Steven M.; Veith, Paul D.; Lo, Alvin W.; Reynolds, Eric C.

    2011-01-01

    Porphyromonas gingivalis, a periodontal pathogen, expresses a group of surface proteins with a common C-terminal domain (CTD) that are exported by a novel secretion system to the surface, where they are covalently attached. Using RgpB as a model CTD protein, we have produced a series of site-directed mutations in the CTD sequence at conserved residues and at residues that may be modified and, hence, surface attached. The mutant RgpB proteins were expressed in a P. gingivalis host lacking functional RgpB and RgpA Arg-specific proteases. The RgpB mutants produced were Y674F, Y674F Y718F, T675Q S679Q T682Q T684Q, T693Q, F695A, D696A, N698A, G699P, G716P, T724Q, T728Q T730Q, and K732Q and a protein with a deletion of residues 692 to 702 (Δ692-702). The mutants were characterized for cell-associated Arg-specific protease activity and for cellular distribution using anti-Rgp antibodies and Western blotting of culture fractions. All the mutants exhibited cell-associated Arg-specific activity similar to that of the positive control except for the D696A and Δ692-702 mutants. For all mutants, except D696A and Δ692-702, the RgpB proteins were found modified and attached to the cell surface, which was the same profile found in the positive-control strain. Only trace amounts of the precursor form of the Δ692-702 mutant were detected in the outer membrane, with none detected in the periplasm or culture fluid although cell transcript levels were normal. The results suggest that residues 692 to 702 of the CTD, in particular, residue D696, have an important role in the attachment of RgpB at the cell surface and that without attachment secretion does not occur. PMID:20971915

  13. Roles of the N- and C-terminal domains of mammalian mitochondrial initiation factor 3 in protein biosynthesis.

    PubMed

    Haque, Md Emdadul; Spremulli, Linda L

    2008-12-26

    Bacterial initiation factor 3 (IF3) is organized into N- and C-domains separated by a linker. Mitochondrial IF3 (IF3(mt)) has a similar domain organization, although both domains have extensions not found in the bacterial factors. Constructs of the N- and C-domains of IF3(mt) with and without the connecting linker were prepared. The K(d) values for the binding of full-length IF3(mt) and its C-domain with and without the linker to mitochondrial 28S subunits are 30, 60, and 95 nM, respectively, indicating that much of the ribosome binding interactions are mediated by the C-domain. However, the N-domain binds to 28S subunits with only a 10-fold lower affinity than full-length IF3(mt). This observation indicates that the N-domain of IF3(mt) has significant contacts with the protein-rich small subunit of mammalian mitochondrial ribosomes. The linker also plays a role in modulating the interactions between the 28S subunit and the factor; it is not just a physical connector between the two domains. The presence of the two domains and the linker may optimize the overall affinity of IF3(mt) for the ribosome. These results are in sharp contrast to observations with Escherichia coli IF3. Removal of the N-domain drastically reduces the activity of IF3(mt) in the dissociation of mitochondrial 55S ribosomes, although the C-domain itself retains some activity. This residual activity depends significantly on the linker region. The N-domain alone has no effect on the dissociation of ribosomes. Full-length IF3(mt) reduces the binding of fMet-tRNA to the 28S subunit in the absence of mRNA. Both the C-terminal extension and the linker are required for this effect. IF3(mt) promotes the formation of a binary complex between IF2(mt) and fMet-tRNA that may play an important role in mitochondrial protein synthesis. Both domains play a role promoting the formation of this complex.

  14. Urinary levels of cross-linked N-terminal telopeptide of type I collagen and nutritional status in Japanese professional baseball players.

    PubMed

    Iwamoto, Jun; Takeda, Tsuyoshi; Uenishi, Kazuhiro; Ishida, Hiromi; Sato, Yoshihiro; Matsumoto, Hideo

    2010-09-01

    The objective of the present study was to investigate the nutritional status from the aspect of bone metabolism in Japanese elite male athletes with increased bone resorption. Urinary levels of a bone resorption marker, cross-linked N-terminal telopeptide of type 1 collagen (NTX), were measured in 71 professional baseball players (age, 18-39 years); the mean urinary NTX level was 65.6 (range, 17.5-269.0) nM BCE/mM Cr. Of 71 athletes, 9 with high levels of urinary NTX (greater than mean + 1 SD) were examined by measuring serum biochemical markers and nutritional assessment (simple food frequency questionnaire). Serum biochemical marker analysis showed that 7 of these 9 athletes had vitamin D insufficiency, as indicated by low serum levels of 25-hydroxyvitamin D, and that all 9 athletes showed vitamin K insufficiency as indicated by low levels of vitamins K(1) and K(2). Nutritional assessment revealed high intakes of protein and low intakes of calcium and vitamin D based on adequate intake (AI). However, daily vitamin K intake achieved the AI. These results suggest that there exist elite male athletes who show increased bone resorption and calcium and vitamin D insufficiency. However, there was a discrepancy between vitamin K intake and serum levels of vitamins K(1) and K(2). The present study raised an issue regarding the nutritional status from the point of view of bone metabolism in elite male athletes such as professional baseball players.

  15. The Cold Denatured State of the C-terminal Domain of Protein L9 Is Compact and Contains Both Native and Non-native Structure

    PubMed Central

    Shan, Bing; McClendon, Sebastian; Rospigliosi, Carla; Eliezer, David; Raleigh, Daniel P

    2011-01-01

    Cold denaturation is a general property of globular proteins, and the process provides insight into the origins of the cooperativity of protein folding and the nature of partially folded states. Unfortunately, studies of protein cold denaturation have been hindered by the fact that the cold denatured state is normally difficult to access experimentally. Special conditions such as addition of high concentrations of denaturant, encapsulation into reverse micelles, the formation of emulsified solutions, high pressure or extremes of pHs, have been applied, but these can perturb the unfolded state of proteins. The cold denatured state of the C-terminal domain of the ribosomal protein L9 can be populated under native like conditions by taking advantage of a destabilizing point mutation which leads to cold denaturation at temperatures above zero °C. This state is in slow exchange with the native state on the NMR time scale. Virtually complete backbone 15N, 13C and 1H as well as side-chain 13Cβ and 1Hβ chemical shift assignments were obtained for the cold denatured state at pH 5.7, 12 °C. Chemical shift analysis, backbone N-H residual dipolar couplings, amide proton NOEs and R2 relaxation rates all indicate that the cold denatured state of CTL9 contains significant native like secondary structure, but also contains non-native structure. The regions corresponding to the two native α-helices show a strong tendency to populate helical Φ and Ψ angles. The segment which connects α-helix 2 and β-strand 2 (residues 107–124) in the native state exhibits a significant preference to form non-native helical structure in the cold denatured state. The structure observed in the cold denatured state of the I98A mutant is similar to that observed in the pH 3.8 unfolded state of wild type CTL9 at 25 °C, suggesting that it is a robust feature of the denatured state ensemble of this protein. The implications for protein folding and for studies of cold denatured states are

  16. Prolonged activation of phospholipase D in Chinese hamster ovary cells expressing platelet-activating-factor receptor lacking cytoplasmic C-terminal tail.

    PubMed Central

    Liu, B; Nakashima, S; Adachi, T; Ito, Y; Takano, T; Shimizu, T; Nozawa, Y

    1997-01-01

    The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Calpha (PKCalpha) to membrane was similar to that of DG formation. In WT-H cells, PKCalpha was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCalpha was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCalpha. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that

  17. Protective immunity against Eimeria tenella infection in chickens induced by immunization with a recombinant C-terminal derivative of EtIMP1.

    PubMed

    Yin, Guangwen; Lin, Qian; Wei, Wenjun; Qin, Mei; Liu, Xianyong; Suo, Xun; Huang, Zhijian

    2014-12-15

    Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. Cloning and sequence analysis has predicted the antigen to be a novel membrane protein of apicomplexan parasites. In order to assess the immunogenicity of EtIMP1, a C-terminal derivative of EtIMP1 was expressed in a bacterial host system and was used to immunize chickens. The protective efficacy against a homologous challenge was evaluated by body weight gains, lesion scores and fecal oocyst shedding. The results showed that the subunit vaccine can improve weight gains, reduced cecal pathology and lower oocyst fecal shedding compared with non immunized controls. The results suggested that the C-terminal derivative of EtIMP1 might be considered as a candidate in the development of subunit vaccines against Eimeria infection.

  18. In-Frame Deletion and Missense Mutations of the C-Terminal Helicase Domain of SMARCA2 in Three Patients with Nicolaides-Baraitser Syndrome

    PubMed Central

    Wolff, D.; Endele, S.; Azzarello-Burri, S.; Hoyer, J.; Zweier, M.; Schanze, I.; Schmitt, B.; Rauch, A.; Reis, A.; Zweier, C.

    2012-01-01

    Using high-resolution molecular karyotyping with SNP arrays to identify candidate genes for etiologically unexplained intellectual disability, we identified a 32-kb de novo in-frame deletion of the C-terminal helicase domain of the SMARCA2 gene in a patient with severe intellectual disability, epilepsy, sparse hair, prominent joints, and distinct facial anomalies. Sequencing of the gene in patients with a similar phenotype revealed de novo missense mutations in this domain in 2 further patients, pointing to a crucial role of the SMARCA2 C-terminal helicase domain. The clinical features observed in all 3 patients are typical of Nicolaides-Baraitser syndrome, an only rarely reported syndrome with mainly moderate to severe intellectual disability. Notably, one of our patients with a p.Gly1132Asp mutation showed typical morphological features but an exceptional good development with borderline overall IQ and learning difficulties, thus expanding the phenotypic spectrum of Nicolaides-Baraitser syndrome. PMID:22822383

  19. Glucagon of caviomorphs and other tetrapods immunohistochemically investigated with two antisera against the N- and C-terminal portions of the molecule.

    PubMed

    Iturriza, F C; Verzi, D H; Di Maggio, L A

    1995-08-01

    Pancreatic sections from diverse tetrapods, including various species of caviomorph rodents, were immunohistochemically investigated using two antisera which reacted with the N- and C-terminal portions of the glucagon molecule. While the antiserum against the N-terminal portion stained alpha cells in all the species studied, the antiserum against the C-portion failed to stain alpha cells in two caviomorphs of the Caviidae family (guinea pig and cuis) and in one of the Octodontidae family (degu). The observations in guinea pig and degu were expected, since their glucagons differ from those of many other tetrapods in the C-terminal portion of the molecule. In this paper, the cuis was added to these two species. It is noteworthy that among the caviomorphs studied herein (nine species), immunohistochemical differences were detected only in the three above-mentioned species and did not involve higher taxa, thus suggesting that these modifications are relatively recent in the evolution of this group of rodents.

  20. Expression, Purification And Preliminary X-Ray Analysis of the C-Terminal Domain of An Arginine Repressor Protein From Mycobacterium Tuberculosis

    SciTech Connect

    Lu, G.J.; Garen, C.R.; Cherney, M.M.; Cherney, L.T.; Lee, C.; James, M.N.J.

    2009-06-03

    The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 {angstrom}. The crystals belong to space group P1 and the Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of six molecules per unit cell.

  1. Expression, purification, crystallization and preliminary X-ray analysis of a C-terminal fragment of the Epstein–Barr virus ZEBRA protein

    SciTech Connect

    Morand, Patrice; Budayova-Spano, Monika; Perrissin, Monique; Müller, Christoph W. Petosa, Carlo

    2006-03-01

    A C-terminal fragment of the Epstein–Barr virus lytic switch protein ZEBRA has been crystallized in complex with DNA. A C-terminal fragment of the Epstein–Barr virus immediate-early transcription factor ZEBRA has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. The fragment behaves as a dimer in solution, consistent with the presence of a basic region leucine-zipper (bZIP) domain. Crystals of the fragment in complex with a DNA duplex were grown by the hanging-drop vapour-diffusion technique using polyethylene glycol 4000 and magnesium acetate as crystallization agents. Crystals diffract to better than 2.5 Å resolution using synchrotron radiation (λ = 0.976 Å). Crystals belong to space group C2, with unit-cell parameters a = 94.2, b = 26.5, c = 98.1 Å, β = 103.9°.

  2. Mutant ubiquitin (UBB+1) associated with neurodegenerative disorders is hydrolyzed by ubiquitin C-terminal hydrolase L3 (UCH-L3).

    PubMed

    Dennissen, Frank J A; Kholod, Natalia; Hermes, Denise J H P; Kemmerling, Nadja; Steinbusch, Harry W M; Dantuma, Nico P; van Leeuwen, Fred W

    2011-08-19

    Mutant ubiquitin (UBB(+1)) accumulates in the hallmarks of tauopathies and polyglutamine diseases. We show that the deubiquitinating enzyme YUH1 of Saccharomyces cerevisiae and its mouse and human ortholog UCH-L3 are able to hydrolyze the C-terminal extension of UBB(+1). This yields another dysfunctional ubiquitin molecule (UB(G76Y)) with biochemical properties similar to full length UBB(+1). UBB(+1) may be detected in post-mortem tissue due to impaired C-terminal truncation of UBB(+1). Although the level of UCH-L3 protein in several neurodegenerative diseases is unchanged, we show that in vitro oxidation of recombinant UCH-L3 impairs its deubiquitinating activity. We postulate that impaired UCH-L3 function may contribute to the accumulation of full length UBB(+1) in various pathologies.

  3. The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions

    PubMed Central

    Buensuceso, Ryan N. C.; Nguyen, Ylan; Zhang, Kun; Daniel-Ivad, Martin; Sugiman-Marangos, Seiji N.; Fleetwood, Aaron D.; Junop, Murray S.

    2016-01-01

    ABSTRACT FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels—and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)—by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the “FimV C-terminal domain,” which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861–919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCE FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa. FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures

  4. IN VITRO EVOLUTION OF AN HIV INTEGRASE BINDING PROTEIN FROM A LIBRARY OF C-TERMINAL DOMAIN γS-CRYSTALLIN VARIANTS

    PubMed Central

    Moody, Issa S.; Verde, Shawn C.; Overstreet, Cathie M.; Robinson, W. Edward; Weiss, Gregory A.

    2014-01-01

    A protein without natural binding functions was engineered to bind HIV-1 integrase. Phage display selections applied a library of variants based on the C-terminal domain of the eye lens protein human γS-crystallin. Multiple loop regions were altered to encode libraries with ≈3.6×1011 different variants. A crystallin variant, termed Integrase-Binding Protein-10 (IBP-10), inhibits integrase catalysis with nanomolar Ki values. IBP-10 interacts with the integrase C-terminal domain and inhibits integrase substrate affinity. This allosteric mechanism allows IBP-10 to inhibit drug resistant integrase variants. The results demonstrate the applicability of the crystallin scaffold for the discovery of binding partners and enzyme inhibitors. PMID:22858140

  5. Circulating concentrations of vitamin E isomers: Association with bone turnover and arterial stiffness in post-menopausal women.

    PubMed

    Hampson, G; Edwards, S; Sankaralingam, A; Harrington, D J; Voong, K; Fogelman, I; Frost, M L

    2015-12-01

    The effects of vitamin E on cardiovascular and bone health are conflicting with beneficial and detrimental findings reported. To investigate this further, we carried out a cross-sectional study to determine the relationship between circulating concentrations of the 2 vitamin E isomers, α- and γ-tocopherol (TP) with bone turnover and arterial stiffness. Two hundred and seventy eight post-menopausal women with mean age [SD] 60.9 [6.0] years were studied. Fasting serum α-TP and γ-TP, bone turnover markers; procollagen type 1 amino-terminal propeptide (P1NP) and C-terminal telopeptide of type 1 collagen (CTX), parathyroid hormone (PTH), total cholesterol (TC) and triglycerides (TG) were measured. Pulse wave velocity (PWV) and central augmentation index (AI) as markers of arterial stiffness were also determined. A positive correlation was observed between α-TP and γ-TP (r=0.14, p=0.022). A significant negative association between α-TP and P1NP only was seen in multiple linear regression analysis following adjustment for serum TC and TG (p=0.016). In a full multi-linear regression model, following correction for age, years since menopause, smoking habits, alcohol intake, use of calcium supplements, BMI, PTH, serum calcium, and estimated glomerular filtration rate (eGFR), the association between α-TP and P1NP remained significant (p=0.011). We did not observe any significant association between γ-TP or α-TP/γ-TP ratio with P1NP or CTX. P1NP was significantly lower in subjects with α-TP concentrations of >30 μmol/L (α-TP >30 μmol/L; P1NP: 57.5 [20.7], α-TP<30 μmol/L; P1NP: 65.7 [24.9] μg/L, p=0.005). PWV was significantly associated with α-TP/γ-TP ratio (p=0.04) but not with serum α-TP or γ-TP in a full multi-linear regression model adjusting for serum lipids, age, and blood pressure. The data suggest that high serum concentrations of α-TP may have a negative effect on bone formation. The balance of α-TP and γ-TP may be important in maintaining

  6. The narrow active-site cleft of O-acetylserine sulfhydrylase from Leishmania donovani allows complex formation with serine acetyltransferases with a range of C-terminal sequences.

    PubMed

    Raj, Isha; Kumar, Sudhir; Gourinath, Samudrala

    2012-08-01

    Cysteine is a crucial substrate for the synthesis of glutathione and trypanothione, which in turn maintain intracellular redox homeostasis and defend against oxidative stress in the pathogen Leishmania donovani. Here, the identification, sequencing, characterization and crystal structure at 1.79 Å resolution of O-acetylserine sulfhydrylase (OASS), a cysteine-biosynthetic pathway enzyme from L. donovani (LdOASS), are reported. It shows binding to the serine acetyltransferase (SAT) C-terminal peptide, indicating that OASS and SAT interact with each other to form a cysteine synthase complex, further confirmed by the structure of LdOASS in complex with SAT C-terminal octapeptide at 1.68 Å resolution. Docking and fluorescence binding studies show that almost all SAT C-terminus mimicking tetrapeptides can bind to LdOASS. Some peptides had a higher binding affinity than the native peptide, indicating that SAT-OASS interactions are not sequence-specific. The structure of LdOASS with a designed peptide (DWSI) revealed that LdOASS makes more interactions with the designed peptide than with the native peptide. In almost all known SAT-OASS interactions the SAT C-terminal sequence was shown to contain amino acids with large side chains. Structural comparison with other OASSs revealed that LdOASS has a relatively less open active-site cleft, which may be responsible for its interaction with the smaller-amino-acid-containing C-terminal LdSAT peptide. Biochemical studies confirmed that LdOASS interacts with SATs from Entamoeba histolytica and Brucella abortus, further displaying its sequence-independent and versatile mode of interaction with SATs. This implicates a critical role of the size of the active-site cleft opening in OASS for SAT-OASS interaction and thus cysteine synthase complex formation.

  7. Deletion of the C-terminal 33 amino acids of cucumber mosaic virus movement protein enables a chimeric brome mosaic virus to move from cell to cell.

    PubMed Central

    Nagano, H; Okuno, T; Mise, K; Furusawa, I

    1997-01-01

    The movement protein (MP) gene of brome mosaic virus (BMV) was precisely replaced with that of cucumber mosaic virus (CMV). Infectivity tests of the chimeric BMV on Chenopodium quinoa, a permissive host for cell-to-cell movement of both BMV and CMV, showed that the chimeric BMV failed to move from cell to cell even though it replicated in protoplasts. A spontaneous mutant of the chimeric BMV that displayed cell-to-cell movement was subsequently obtained from a local lesion during one of the experiments. A cloned cDNA representing the genomic RNA encoding the MP of the chimeric BMV mutant was analyzed and found to contain a mutation in the CMV MP gene resulting in deletion of the C-terminal 33 amino acids of the MP. Directed mutagenesis of the CMV MP gene showed that the C-terminal deletion was responsible for the movement capability of the mutant. When the mutation was introduced into CMV, the CMV mutant moved from cell to cell in C. quinoa, though the movement was less efficient than that of the wild-type CMV. These results indicate that the CMV MP, except the C-terminal 33 amino acids, potentiates cell-to-cell movement of both BMV and CMV in C. quinoa. In addition, since C. quinoa is a common host for both BMV and CMV, these results suggest that the CMV MP has specificity for the viral genomes during cell-to-cell movement of the virus and that the C-terminal 33 amino acids of the CMV MP are involved in that specificity. PMID:9032362

  8. Sucrose phosphate phosphatase in the green alga Klebsormidium flaccidum (Streptophyta) lacks an extensive C-terminal domain and differs from that of land plants.

    PubMed

    Nagao, Manabu; Uemura, Matsuo

    2012-04-01

    Previously, it was reported that like land plants, the green alga Klebsormidium flaccidum (Streptophyta) accumulates sucrose during cold acclimation (Nagao et al. Plant Cell Environ 31:872-885, 2008), suggesting that synthesis of sucrose could enhance the freezing tolerance of this alga. Because sucrose phosphate phosphatase (SPP; EC 3.1.3.24) is a key enzyme in the sucrose synthesis pathway in plants, we analyzed the SPP gene in K. flaccidum (KfSPP, GenBank accession number AB669024) to clarify its role in sucrose accumulation. As determined from its deduced amino acid sequence, KfSPP contains the N-terminal domain that is characteristic of the L-2-haloacid-dehalogenase family of phosphatases/hydrolases (the HAD phosphatase domain). However, it lacks the extensive C-terminal domain found in SPPs of land plants. Database searches revealed that the SPPs in cyanobacteria also lack the C-terminal domain. In addition, the green alga Coccomyxa (Chlorophyta) and K. flaccidum, which are closely related to land plants, have cyanobacterial-type SPPs, while Chlorella (Chlorophyta) has a land plant-type SPP. These results demonstrate that even K. flaccidum (Streptophyta), as a recent ancestor of land plants, has the cyanobacterial-type SPP lacking the C-terminal domain. Because SPP and sucrose phosphate synthase (SPS) catalyze sequential reactions in sucrose synthesis in green plant cells and the lack of the C-terminal domain in KfSPP is predicted to decrease its activity, the interaction between decreased KfSPP activity and SPS activity may alter sucrose synthesis during cold acclimation in K. flaccidum.

  9. PDZ Domain Dependent Regulation of NHE3 Occurs by Both Internal Class II and C-terminal Class I PDZ Binding Motifs.

    PubMed

    Cha, Boyoung; Yang, Jianbo; Singh, Varsha; Zachos, Nicholas C; Sarker, Rafiquel I; Chen, Tian-E; Chakraborty, Molee; Tse, Chung-Ming; Donowitz, Mark

    2017-03-10

    NHE3 directly binds NHERF family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (aa. 586-660) of the NHE3 C-terminus and to the NHE3 C-terminal four amino acids. The internal NHERF binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ binding motifs (PBM). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing: 1) Interaction between these binding sites since mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression were reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP and Ca2+-mediated inhibition of NHE3 were impaired both in the internal and in the C-terminal PBM mutations. 4) A significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) Some difference in NHE3 associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II and C-terminal Class I PBM, which interact. The former appears to determine NHE3 stability of a pool in the PM and the letter determines total expression and percent PM expression.

  10. Productive association between MHC class I and tapasin requires the tapasin transmembrane/cytosolic region and the tapasin C-terminal Ig-like domain

    PubMed Central

    Simone, Laura C.; Georgesen, Corey J.; Simone, Peter D.; Wang, Xiaojian; Solheim, Joyce C.

    2011-01-01

    The current model of antigen assembly with major histocompatibility complex (MHC) class I molecules posits that interactions between the tapasin N-terminal immunoglobulin (Ig)-like domain and the MHC class I peptide-binding groove permit tapasin to regulate antigen selection. Much less is known regarding interactions that might involve the tapasin C-terminal Ig-like domain. Additionally, the tapasin transmembrane/cytoplasmic region enables tapasin to bridge the MHC class I molecule to the transporter associated with antigen processing (TAP). In this investigation, we made use of two tapasin mutants to determine the relative contribution of the tapasin C-terminal Ig-like domain and the tapasin transmembrane/cytoplasmic region to the assembly of MHC class I molecules. Deletion of a loop within the tapasin C-terminal Ig-like domain (Δ334-342) prevented tapasin association with the MHC class I molecule Kd. Although tapasin Δ334-342 did not increase the efficiency of Kd folding, Kd surface expression was enhanced on cells expressing this mutant relative to tapasin-deficient cells. In contrast to tapasin Δ334-342, a soluble tapasin mutant lacking the transmembrane/cytoplasmic region retained the ability to bind to Kd molecules, but did not facilitate Kd surface expression. Furthermore, when soluble tapasin and tapasin Δ334-342 were co-expressed, soluble tapasin had a dominant negative effect on the folding and surface expression of not only Kd, but also Db and Kb. In addition, our molecular modeling of the MHC class I-tapasin interface revealed novel potential interactions involving tapasin residues 334-342. Together, these findings demonstrate that the tapasin C-terminal and transmembrane/cytoplasmic regions are critical to tapasin's capacity to associate effectively with the MHC class I molecule. PMID:22169163

  11. Crystal Structure of the C-terminal Region of Streptococcus mutans Antigen I/II and Characterization of Salivary Agglutinin Adherence Domains

    SciTech Connect

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Crowley, Paula J.; Kelly, Charles; Mitchell, Tim J.; Brady, L. Jeannine; Deivanayagam, Champion

    2012-05-29

    The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 {angstrom} resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C{sub 1}, C{sub 2}, and C{sub 3}. Each domain adopts a DE-variant IgG fold, with two {beta}-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C{sub 1} and C{sub 2}) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C{sub 1} and C{sub 2} domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C{sub 1} and C{sub 2} domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.

  12. Differential DNA binding by the androgen and glucocorticoid receptors involves the second Zn-finger and a C-terminal extension of the DNA-binding domains.

    PubMed Central

    Schoenmakers, E; Alen, P; Verrijdt, G; Peeters, B; Verhoeven, G; Rombauts, W; Claessens, F

    1999-01-01

    The androgen and glucocorticoid hormones evoke specific in vivo responses by activating different sets of responsive genes. Although the consensus sequences of the glucocorticoid and androgen response elements are very similar, this in vivo specificity can in some cases be explained by differences in DNA recognition between both receptors. This has clearly been demonstrated for the androgen response element PB-ARE-2 described in the promoter of the rat probasin gene. Swapping of different fragments between the androgen- and glucocorticoid-receptor DNA-binding domains demonstrates that (i) the first Zn-finger module is not involved in this sequence selectivity and (ii) that residues in the second Zn-finger as well as a C-terminal extension of the DNA-binding domain from the androgen receptor are required. For specific and high-affinity binding to response elements, the DNA-binding domains of the androgen and glucocorticoid receptors need a different C-terminal extension. The glucocorticoid receptor requires 12 C-terminal amino acids for high affinity DNA binding, while the androgen receptor only involves four residues. However, for specific recognition of the PB-ARE-2, the androgen receptor also requires 12 C-terminal residues. Our data demonstrate that the mechanism by which the androgen receptor binds selectively to the PB-ARE-2 is different from that used by the glucocorticoid receptor to bind a consensus response element. We would like to suggest that the androgen receptor recognizes response elements as a direct repeat rather than the classical inverted repeat. PMID:10417312

  13. Structural and Functional Modularity of the Orange Carotenoid Protein: Distinct Roles for the N- and C-Terminal Domains in Cyanobacterial Photoprotection[C][W

    PubMed Central

    Leverenz, Ryan L.; Jallet, Denis; Li, Ming-De; Mathies, Richard A.; Kirilovsky, Diana; Kerfeld, Cheryl A.

    2014-01-01

    The orange carotenoid protein (OCP) serves as a sensor of light intensity and an effector of phycobilisome (PB)–associated photoprotection in cyanobacteria. Structurally, the OCP is composed of two distinct domains spanned by a single carotenoid chromophore. Functionally, in response to high light, the OCP converts from a dark-stable orange form, OCPO, to an active red form, OCPR. The C-terminal domain of the OCP has been implicated in the dynamic response to light intensity and plays a role in switching off the OCP’s photoprotective response through its interaction with the fluorescence recovery protein. The function of the N-terminal domain, which is uniquely found in cyanobacteria, is unclear. To investigate its function, we isolated the N-terminal domain in vitro using limited proteolysis of native OCP. The N-terminal domain retains the carotenoid chromophore; this red carotenoid protein (RCP) has constitutive PB fluorescence quenching activity comparable in magnitude to that of active, full-length OCPR. A comparison of the spectroscopic properties of the RCP with OCPR indicates that critical protein–chromophore interactions within the C-terminal domain are weakened in the OCPR form. These results suggest that the C-terminal domain dynamically regulates the photoprotective activity of an otherwise constitutively active carotenoid binding N-terminal domain. PMID:24399299

  14. The Biological Function of DMP-1 in Osteocyte Maturation Is Mediated by Its 57-kDa C-terminal Fragment

    PubMed Central

    Lu, Yongbo; Yuan, Baozhi; Qin, Chunlin; Cao, Zhengguo; Xie, Yixia; Dallas, Sarah L; McKee, Marc D; Drezner, Marc K; Bonewald, Lynda F; Feng, Jian Q

    2011-01-01

    Dentin matrix protein 1 (DMP-1) is a key molecule in controlling osteocyte formation and phosphate homeostasis. Based on observations that full-length DMP-1 is not found in bone, but only cleaved fragments of 37 and 57 kDa are present, and in view of the finding that mutations in the 57-kDa fragment result in disease, we hypothesized that the 57-kDa C-terminal fragment is the functional domain of DMP-1. To test this hypothesis, a 3.6-kb type I collagen promoter was used to express this 57-kDa C-terminal fragment for comparison with full-length DMP-1 in Dmp1 null osteoblasts/osteocytes. Not only did expression of the full-length DMP-1 in bone cells fully rescue the skeletal abnormalities of Dmp1 null mice, but the 57-kDa fragment also had similar results. This included rescue of growth plate defects, osteomalacia, abnormal osteocyte maturation, and the abnormal osteocyte lacunocanalicular system. In addition, the abnormal fibroblast growth factor 23 (FGF-23) expression in osteocytes, elevated circulating FGF-23 levels, and hypophosphatemia were rescued. These results show that the 57-kDa C-terminal fragment is the functional domain of DMP-1 that controls osteocyte maturation and phosphate metabolism. © 2011 American Society for Bone and Mineral Research. PMID:20734454

  15. Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein

    PubMed Central

    Gayen, Nilanjan; Mroue, Kamal H.; Kar, Rajiv K.; Mandal, Atin K.; Bhunia, Anirban

    2015-01-01

    Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3ζ to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3ζ protein and determined the possible structural adaptation of this region. Biophysical elucidation of the interaction was carried out using phosphopeptide (residue number 615–630) in the presence of 14-3-3ζ protein. Using isothermal titration calorimetry (ITC), a high binding affinity with micro-molar range was found to exist between the peptide and 14-3-3ζ protein, whereas the non-phosphorylated peptide did not show any appreciable binding affinity. Further interaction details were investigated using several biophysical techniques such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, in addition to molecular modeling. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3ζ protein as well as structural insights responsible for phosphorylated S621-mediated 14-3-3ζ binding at an atomic resolution. PMID:26295714

  16. A Fmoc-compatible Method for the Solid-Phase Synthesis of Peptide C-Terminal (alpha)-Thioesters based on the Safety-Catch Hydrazine Linker

    SciTech Connect

    Camarero, J A; Hackel, B J; de Yoreo, J J; Mitchell, A R

    2003-11-22

    C-terminal peptide thioesters are key intermediates for the synthesis/semisynthesis of proteins and for the production of cyclic peptides by native chemical ligation. They can be synthetically prepared by solid-phase peptide synthesis (SPPS) methods or biosynthetically by protein splicing techniques. Until recently, the chemical synthesis of C-terminal a-thioester peptides by SPPS was largely restricted to the Boc/Benzyl methodology because of the poor stability of the thioester bond to the basic conditions employed for the deprotection of the N{sup {alpha}}-Fmoc group. In the present work, we describe a new method for the SPPS of C-terminal thioesters by Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazide linker, which is totally stable to the Fmoc-SPPS conditions. Once the peptide synthesis has been completed, activation of the linker can be achieved by mild oxidation. This step transforms the hydrazide group into a highly reactive diazene intermediate which can react with different H-AA-SEt to yield the corresponding {alpha}-thioester peptide in good yields. This method has been successfully used for the generation of different thioester peptides, circular peptides and a fully functional SH3 protein domain.

  17. Characterization of glutamate decarboxylase from Lactobacillus plantarum and its C-terminal function for the pH dependence of activity.

    PubMed

    Shin, Sun-Mi; Kim, Hana; Joo, Yunhye; Lee, Sang-Jae; Lee, Yong-Jik; Lee, Sang Jun; Lee, Dong-Woo

    2014-12-17

    The gadB gene encoding glutamate decarboxylase (GAD) from Lactobacillus plantarum was cloned and expressed in Escherichia coli. The recombinant enzyme exhibited maximal activity at 40 °C and pH 5.0. The 3D model structure of L. plantarum GAD proposed that its C-terminal region (Ile454-Thr468) may play an important role in the pH dependence of catalysis. Accordingly, C-terminally truncated (Δ3 and Δ11 residues) mutants were generated and their enzyme activities compared with that of the wild-type enzyme at different pH values. Unlike the wild-type GAD, the mutants showed pronounced catalytic activity in a broad pH range of 4.0-8.0, suggesting that the C-terminal region is involved in the pH dependence of GAD activity. Therefore, this study may provide effective target regions for engineering pH dependence of GAD activity, thereby meeting industrial demands for the production of γ-aminobutyrate in a broad range of pH values.

  18. Quantum mechanical investigations on the role of C-terminal residue in influencing the structural features of dipeptides containing N-terminal proline.

    PubMed

    Das, Gunajyoti; Mandal, Shilpi

    2014-04-01

    This study investigates the influence of the side chain moiety of C-terminal residue on the structural and molecular properties of seven dipeptides having proline at their N-terminal positions. The C-terminal component of the dipeptides is varied with seven different combinations viz. Ala, Leu, Asp, Thr, Asn, Arg and Sec. The calculations are carried out using B3LYP/6-311++G(d,p) level of theory in gas and implicit aqueous phase. Effects of explicit aqueous environment on the dipeptide structures are also investigated for two systems. The results furnished by this DFT study provide valuable information regarding the role of the side chain groups of C-terminal residues in determining the structural features of the amide planes, values of the ψ and ф dihedrals, geometry about the α-carbon atoms, theoretical IR spectra as well as the number and type of intramolecular H-bond interactions existing in the dipeptides, and extend a fine corroboration to the earlier theoretical and experimental observations. In aqueous phase the dipeptide geometries exhibit larger values of total dipole moments, greater HOMO-LUMO energy gaps and enhanced thermodynamic stability than those in gas phase. The explicit water molecules are found to modify the geometrical parameters related to the amide planes and vibrational spectra of the dipeptides.

  19. Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

    SciTech Connect

    Pazehoski, Kristina O.; Cobine, Paul A.; Winzor, Donald J.; Dameron, Charles T.

    2011-03-11

    Research highlights: {yields} A metal-binding protein domain is directly involved in protein dimerization. {yields} Fusing the metal-binding domain to a monomeric protein induces dimerization. {yields} Frontal size-exclusion chromatography measures the strength of dimer interaction. {yields} Ultracentrifugation studies confirm the influence of metal binding on dimerization. -- Abstract: Metal binding to the C-terminal region of the copper-responsive repressor protein CopY is responsible for homodimerization and the regulation of the copper homeostasis pathway in Enterococcus hirae. Specific involvement of the 38 C-terminal residues of CopY in dimerization is indicated by zonal and frontal (large zone) size-exclusion chromatography studies. The studies demonstrate that the attachment of these CopY residues to the immunoglobulin-binding domain of streptococcal protein G (GB1) promotes dimerization of the monomeric protein. Although sensitivity of dimerization to removal of metal from the fusion protein is smaller than that found for CopY (as measured by ultracentrifugation studies), the demonstration that an unrelated protein (GB1) can be induced to dimerize by extending its sequence with the C-terminal portion of CopY confirms the involvement of this region in CopY homodimerization.

  20. Amyloidogenic properties of a D/N mutated 12 amino acid fragment of the C-terminal domain of the Cholesteryl-Ester Transfer Protein (CETP).

    PubMed

    García-González, Victor; Mas-Oliva, Jaime

    2011-01-01

    The cholesteryl-ester transfer protein (CETP) facilitates the transfer of cholesterol esters and triglycerid