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C-terminal Half (c-terminal + half)
Selected AbstractsFunctional properties of the protein disulfide oxidoreductase from the archaeon Pyrococcus furiosusFEBS JOURNAL, Issue 16 2004A member of a novel protein family related to protein disulfide-isomerase Protein disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol,disulfide exchange reactions with a CXXC sequence motif at their active site. A disulfide oxidoreductase, a highly thermostable protein, was isolated from Pyrococcus furiosus (PfPDO), which is characterized by two redox sites (CXXC) and an unusual molecular mass. Its 3D structure at high resolution suggests that it may be related to the multidomain protein disulfide-isomerase (PDI), which is currently known only in eukaryotes. This work focuses on the functional characterization of PfPDO as well as its relation to the eukaryotic PDIs. Assays of oxidative, reductive, and isomerase activities of PfPDO were performed, which revealed that the archaeal protein not only has oxidative and reductive activity, but also isomerase activity. On the basis of structural data, two single mutants (C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites. The results indicate that the CPYC site in the C-terminal half of the protein is fundamental to reductive/oxidative activity, whereas isomerase activity requires both active sites. In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and an optimum temperature of 90 °C. These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel protein disulfide oxidoreductase family. [source] Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricinFEBS JOURNAL, Issue 17 2002Goran Kragol Members of the proline-rich antibacterial peptide family, pyrrhocoricin, apidaecin and drosocin appear to kill responsive bacterial species by binding to the multihelical lid region of the bacterial DnaK protein. Pyrrhocoricin, the most potent among these peptides, is nontoxic to healthy mice, and can protect these animals from bacterial challenge. A structure,antibacterial activity study of pyrrhocoricin against Escherichia coli and Agrobacterium tumefaciens identified the N-terminal half, residues 2,10, the region responsible for inhibition of the ATPase activity, as the fragment that contains the active segment. While fluorescein-labeled versions of the native peptides entered E. coli cells, deletion of the C-terminal half of pyrrhocoricin significantly reduced the peptide's ability to enter bacterial or mammalian cells. These findings highlighted pyrrhocoricin's suitability for combating intracellular pathogens and raised the possibility that the proline-rich antibacterial peptides can deliver drug leads into mammalian cells. By observing strong relationships between the binding to a synthetic fragment of the target protein and antibacterial activities of pyrrhocoricin analogs modified at strategic positions, we further verified that DnaK was the bacterial target macromolecule. Inaddition, the antimicrobial activity spectrum of native pyrrhocoricin against 11 bacterial and fungal strains and the binding of labeled pyrrhocoricin to synthetic DnaK D-E helix fragments of the appropriate species could be correlated. Mutational analysis on a synthetic E. coli DnaK fragment identified a possible binding surface for pyrrhocoricin. [source] Characterization of a novel silkworm (Bombyx mori) phenol UDP-glucosyltransferaseFEBS JOURNAL, Issue 3 2002Teresa Luque Sugar conjugation is a major pathway for the inactivation and excretion of both endogenous and exogenous compounds. We report here the molecular cloning and functional characterization of a phenol UDP-glucosyltransferase (UGT) from the silkworm, Bombyx mori, which was named BmUGT1. The complete cDNA clone is 1.6 kb, and the gene is expressed in several tissues of fifth-instar larvae, including fat body, midgut, integument, testis, silk gland and haemocytes. The predicted protein comprises 520 amino acids and has ,,30% overall amino-acid identity with other members of the UGT family. The most conserved region of the protein is the C-terminal half, which has been implicated in binding the UDP-sugar. BmUGT1 was expressed in insect cells using the baculovirus expression system, and a range of compounds belonging to diverse chemical groups were assessed as potential substrates for the enzyme. The expressed enzyme had a wide substrate specificity, showing activity with flavonoids, coumarins, terpenoids and simple phenols. These results support a role for the enzyme in detoxication processes, such as minimizing the harmful effects of ingested plant allelochemicals. This work represents the first instance where an insect ugt gene has been associated with a specific enzyme activity. [source] Functional interaction sites of OprM with MexAB in the Pseudomonas aeruginosa multidrug efflux pumpFEMS MICROBIOLOGY LETTERS, Issue 2 2009Eisaku Yoshihara Abstract Subunit-swapping between Pseudomonas aeruginosa MexAB-OprM and MexEF-OprN efflux pumps has shown that OprM can interact with MexEF to produce a functional efflux pump, but that OprN cannot functionally interact with MexAB. Taking advantage of this subunit selectivity, we carried out experiments using chimeric proteins composed of OprM and OprN to determine which regions of OprM are necessary for functional interaction with MexAB. We constructed two types of chimeric proteins: one with the N-terminal half of OprM and the C-terminal half of OprN (OprMN), and the second with these halves reversed (OprNM). Introduction of either of the chimeric protein genes into a mutant expressing MexEF alone restored the functionality of the efflux pump. However, expression of OprMN or OprNM in the presence of MexAB did not restore the pump functionality, indicating that the both the N- and C-terminal halves of OprM are necessary for a functional interaction with MexAB. [source] Ebp2p, yeast homologue of a human protein that interacts with Epstein,Barr virus Nuclear Antigen 1, is required for pre-rRNA processing and ribosomal subunit assemblyGENES TO CELLS, Issue 7 2000Rota Tsujii Background A defect in the secretory pathway causes the transcriptional repression of both rRNA and ribosomal protein genes in Saccharomyces cerevisiae, suggesting a coupling of ribosome synthesis and plasma membrane synthesis. Rrs1p, an essential nuclear protein, is required for the secretory response. Results EBP2, encoding the yeast homologue of a human protein that interacts with Epstein,Barr virus Nuclear Antigen 1, was cloned in a two-hybrid screen using RRS1 as a bait. The rrs1-1 mutation, which produces Rrs1p without the C-terminal half and causes a defect in the secretory response, almost abolished the interaction with Ebp2p. Ebp2p is essential for growth and is mainly localized in the nucleolus. The effects of Ebp2p depletion on ribosome biogenesis is quite similar to that of Rrs1p depletion; in the Ebp2p-depleted cells, the rate of pre-rRNA processing is slower, and significantly less mature 25S rRNA is produced compared to those in wild-type cells. The polysome pattern indicates that Ebp2p-depletion causes a decrease of 80S monosomes and polysomes, an accumulation of 40S subunits, and the appearance of half-mer polysomes. Conclusions Ebp2p is required for the maturation of 25S rRNA and 60S subunit assembly. Ebp2p may be one of the target proteins of Rrs1p for executing the signal to regulate ribosome biogenesis. [source] CCN2, connective tissue growth factor, stimulates collagen deposition by gingival fibroblasts via module 3 and ,6- and ,1 integrinsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Edwin C.K. Heng Abstract CCN2, (connective tissue growth factor, CTGF) is a matricellular factor associated with fibrosis that plays an important role in the production and maintenance of fibrotic lesions. Increased collagen deposition and accumulation is a common feature of fibrotic tissues. The mechanisms by which CCN2/CTGF contributes to fibrosis are not well understood. Previous studies suggest that CTGF exerts some of its biological effects at least in part by integrin binding, though this mechanism has not been previously shown to contribute to fibrosis. Utilizing full length CCN2/CTGF, CCN2/CTGF fragments, and integrin neutralizing antibodies, we provide evidence that the effects of CCN2/CTGF to stimulate extracellular matrix deposition by gingival fibroblasts are mediated by the C-terminal half of CCN2/CTGF, and by ,6 and ,1 integrins. In addition, a synthetic peptide corresponding to a region of CCN2/CTGF domain 3 that binds ,6,1 inhibits the collagen-deposition assay. These studies employed a new and relatively rapid assay for CCN2/CTGF-stimulated collagen deposition based on Sirius Red staining of cell layers. Data obtained support a pathway in which CCN2/CTGF could bind to ,6,1 integrin and stimulate collagen deposition. These findings provide new experimental methodologies applicable to uncovering the mechanism and signal transduction pathways of CCN2/CTGF-mediated collagen deposition, and may provide insights into potential therapeutic strategies to treat gingival fibrosis and other fibrotic conditions. J. Cell. Biochem. 98: 409,420, 2006. © 2006 Wiley-Liss, Inc. [source] Rho exchange factor ECT2 is induced by growth factors and regulates cytokinesis through the N-terminal cell cycle regulator-related domains,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2003Shin'ichi Saito Abstract The ECT2 protooncogene plays a critical role in cytokinesis, and its C-terminal half encodes a Dbl homology-pleckstrin homology module, which catalyzes guanine nucleotide exchange on the Rho family of small GTPases. The N-terminal half of ECT2 (ECT2-N) contains domains related to the cell cycle regulator/checkpoint control proteins including human XRCC1, budding yeast CLB6, and fission yeast Cut5. The Cut5-related domain consists of two BRCT repeats, which are widespread to repair/checkpoint control proteins. ECT2 is ubiquitously expressed in various tissues and cell lines, but elevated levels of ECT2 expression were found in various tumor cell lines and rapidly developing tissues in mouse embryos. Consistent with these findings, induction of ECT2 expression was observed upon stimulation by serum or various growth factors. In contrast to other oncogenes whose expression is induced early in G1, ECT2 expression was induced later, coinciding with the initiation of DNA synthesis. To test the role of the cell cycle regulator/checkpoint control protein-related domains of ECT2 in cytokinesis, we expressed various ECT2 derivatives in U2OS cells, and analyzed their DNA content by flow cytometry. Expression of the N-terminal half of ECT2, which lacks the catalytic domain, generated cells with more than 4N DNA content, suggesting that cytokinesis was inhibited in these cells. Interestingly, ECT2-N lacking the nuclear localization signals inhibited cytokinesis more strongly than the derivatives containing these signals. Mutational analyses revealed that the XRCC1, CLB6, and BRCT domains in ECT2-N are all essential for the cytokinesis inhibition by ECT2-N. These results suggest that the XRCC1, CLB6, and BRCT domains of ECT2 play a critical role in regulating cytokinesis. Published 2003 Wiley-Liss, Inc. [source] Phototropins and Their LOV Domains: Versatile Plant Blue-Light ReceptorsJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 1 2007Winslow R. Briggs Abstract The phototropins phot1 and phot2 are plant blue-light receptors that mediate phototropism, chloroplast movements, stomatal opening, leaf expansion, the rapid inhibition of hypocotyl growth in etiolated seedlings, and possibly solar tracking by leaves in those species in which it occurs. The phototropins are plasma membrane-associated hydrophilic proteins with two chromophore domains (designated LOV1 and LOV2 for their resemblance to domains in other signaling proteins that detect light, oxygen, or voltage) in their N-terminal half and a classic serine/threonine kinase domain in their C-terminal half. Both chromophore domains bind flavin mononucleotide (FMN) and both undergo light-activated formation of a covalent bond between a nearby cysteine and the C(4a) carbon of the FMN to form the signaling state. LOV2-cysteinyl adduct formation leads to the release downstream of a tightly bound amphipathic ,-helix, a step required for activation of the kinase function. This cysteinyl adduct then slowly decays over a matter of seconds or minutes to return the photoreceptor chromophore modules to their ground state. Functional LOV2 is required for light-activated phosphorylation and for various blue-light responses mediated by the phototropins. The function of LOV1 is still unknown, although it may serve to modulate the signal generated by LOV2. The LOV domain is an ancient chromophore module found in a wide range of otherwise unrelated proteins in fungi and prokaryotes, the latter including cyanobacteria, eubacteria, and archaea. Further general reviews on the phototropins are those by Celaya and Liscum (2005) and Christie and Briggs (2005). [source] The R1162 relaxase/primase contains two, type IV transport signals that require the small plasmid protein MobBMOLECULAR MICROBIOLOGY, Issue 1 2007Christopher Parker Summary The relaxase of the plasmid R1162 is a large protein essential for conjugative transfer and containing two different and physically separate catalytic activities. The N-terminal half cleaves one of the DNA strands at the origin of transfer (oriT) and becomes covalently linked to the 5, terminal phosphate; the C-terminal half is a primase essential for initiation of plasmid vegetative replication. We show here that the two parts of the protein are independently transported by the type IV pathway. Part of the domain containing the catalytic activity, as well as an adjacent region, is required in each case, but the required regions do not physically overlap. Both transport systems contribute to the overall frequency of conjugative transfer. MobB is a small protein, encoded within mobA but in a different reading frame, that stabilizes the relaxase at oriT. MobB is required for efficient type IV transport of both the complete relaxase and its two, separate functional halves. MobB inserts into the membrane and could thus stabilize the association between the relaxase and the type IV transfer apparatus. [source] The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamianaTHE PLANT JOURNAL, Issue 2 2006Jorunn I. B. Bos Summary The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a,R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure,function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions. [source] The structure of uracil-DNA glycosylase from Atlantic cod (Gadus morhua) reveals cold-adaptation featuresACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2003Ingar Leiros Uracil-DNA glycosylase (UDG; EC 3.2.2.3) is a DNA-repair protein that catalyses the hydrolysis of promutagenic uracil residues from single- or double-stranded DNA, generating free uracil and abasic DNA. The crystal structure of the catalytic domain of cod uracil-DNA glycosylase (cUDG) has been determined to 1.9,Å resolution, with final R factors of 18.61 and 20.57% for the working and test sets of reflections, respectively. This is the first crystal structure of a uracil-DNA glycosylase from a cold-adapted species and a detailed comparison with the human enzyme is performed in order to rationalize the cold-adapted behaviour of the cod enzyme at the structural level. The catalytic domain of cUDG comprises 223 residues, with a sequence identity to the human UDG of 75%. The tertiary structures of the two enzymes are also similar, with an overall displacement in main-chain atomic positions of 0.63,Å. The amino-acid substitutions and the differences in intramolecular hydrogen bonds, hydrophobic interactions, ion-pair interactions and electrostatic potentials are compared and discussed in order to gain insight into the factors that cause the increased activity and reduced thermostability of the cod enzyme. In particular, the reduced number of strong ion-pair interactions in the C-terminal half of cUDG is believed to greatly affect the flexibility and/or stability. Increased positive electrostatic surface potential on the DNA-facing side of cUDG seems to be responsible for increasing the affinity for the negatively charged DNA compared with that of hUDG. [source] A strained DNA binding helix is conserved for site recognition, folding nucleation, and conformational modulation,BIOPOLYMERS, Issue 6 2009Diana E. Wetzler Abstract Nucleic acid recognition is often mediated by ,-helices or disordered regions that fold into ,-helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical ,-helix is stabilized at the N-terminus, while the PII forms at the C-terminus half of the peptide. Re-examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N-terminus half is a canonical ,-helix, while the C-terminal half adopts a 310 helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 104 -fold lower affinity, and 500-fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained ,-helix conformation, "presented" by this unique architecture. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 432,443, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Decreased serum dependence in the growth of NIH3T3 cells from the overexpression of human nuclear receptor-binding SET-domain-containing protein 1 (NSD1) or fission yeast su(var)3-9, enhancer-of-zeste, trithorax 2 (SET2)CELL BIOCHEMISTRY AND FUNCTION, Issue 2 2008Toshiko Yamada-Okabe Abstract Nuclear receptor-binding SET-domain-containing protein 1 (NSD1), a culprit gene for Sotos syndrome, contains a su(var)3-9, enhancer-of-zeste, trithorax (SET) domain that is responsible for histone methyltransferase activity and other domains such as plant homeodomain (PHD) and proline-tryptophan-tryptophan-proline (PWWP) involved in protein,protein interactions in the C-terminal half of NSD1. To elucidate the function of NSD1 on cell growth, we overexpressed NSD1 in NIH3T3 cells. Cells overexpressing NSD1 grew in the presence of 2% serum, whereas vector transfected cells did not. Overexpression of the C-terminal half of NSD1 but not the N-terminal half of NSD1 also produced cell growth under low serum concentration. Furthermore, overexpression in NIH3T3 of Schizosaccharomyces pombe SET2 which has a SET domain but not PHD or PWWP domains conferred the reduced serum dependence. Thus, the SET domain of NSD1 is involved in cell growth by modulating serum dependence. Copyright © 2007 John Wiley & Sons, Ltd. [source] Functional interaction sites of OprM with MexAB in the Pseudomonas aeruginosa multidrug efflux pumpFEMS MICROBIOLOGY LETTERS, Issue 2 2009Eisaku Yoshihara Abstract Subunit-swapping between Pseudomonas aeruginosa MexAB-OprM and MexEF-OprN efflux pumps has shown that OprM can interact with MexEF to produce a functional efflux pump, but that OprN cannot functionally interact with MexAB. Taking advantage of this subunit selectivity, we carried out experiments using chimeric proteins composed of OprM and OprN to determine which regions of OprM are necessary for functional interaction with MexAB. We constructed two types of chimeric proteins: one with the N-terminal half of OprM and the C-terminal half of OprN (OprMN), and the second with these halves reversed (OprNM). Introduction of either of the chimeric protein genes into a mutant expressing MexEF alone restored the functionality of the efflux pump. However, expression of OprMN or OprNM in the presence of MexAB did not restore the pump functionality, indicating that the both the N- and C-terminal halves of OprM are necessary for a functional interaction with MexAB. [source] Bacterial cell death induced by human pro-apoptotic Bax is blocked by an RNase E mutant that functions in an anti-oxidant pathwayGENES TO CELLS, Issue 3 2000Rika Nanbu-Wakao Background Bax is a member of the Bcl-2 family and induces apoptosis of mammalian cells. We have shown that a trace amount of human Bax induces the cell death of Escherichia coli, accompanied by damage to DNA, and that the region of Bax which is lethal to E. coli is also responsible for apoptosis-inducing activity in the mammalian cells. Results We isolated a Bax-resistant mutant from E. coli cells that survive in the presence of paraquat, a generator of superoxide, by screening a library constructed from the random insertion of a transposon. Psb1 (paraquat-resistant, suppressor of Bax-1) mutant had a Tn 10 transposon inserted in the rne gene of E. coli, splitting the RNase E gene (rne) into N- and C-terminal halves. The introduction of the truncated 5, end of rne specifically enhanced resistance to paraquat, prevented cell death induced by Bax and decreased the intracellular H2O2 concentration. The region responsible for the paraquat- and Bax-resistance was not the catalytic site for the endoribonuclease activity of RNase E. Conclusions The N-terminal region of the RNase E protein inhibits bacterial death induced by human Bax as well as paraquat through a unique mechanism that is distinct from RNA digestion. This study implies that the protection of bacterial death induced by Bax is associated with an anti-oxidant pathway and that a mutant RNase E has a novel function as an anti-oxidant. [source] | |||||||||||||