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Proline Residue (proline + residue)
Selected AbstractsCharacterization of membrane-bound prolyl endopeptidase from brainFEBS JOURNAL, Issue 17 2008Jofre Tenorio-Laranga Prolyl oligopeptidase (POP) is a serine protease that cleaves small peptides at the carboxyl side of an internal proline residue. Substance P, arginine,vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. POP has been implicated in a variety of brain processes, including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension, and psychiatric disorders. Although POP has been considered to be a soluble cytoplasmic peptidase, significant levels of activity have been detected in membranes and in extracellular fluids such as serum, cerebrospinal fluid, seminal fluid, and urine, suggesting the existence of noncytoplasmic forms. Furthermore, a closely associated membrane prolyl endopeptidase (PE) activity has been previously detected in synaptosomes and shown to be different from the cytoplasmic POP activity. Here we isolated, purified and characterized this membrane-bound PE, herein referred to as mPOP. Although, when attached to membranes, mPOP presents certain features that distinguish it from the classical POP, our results indicate that this protein has the same amino acid sequence as POP except for the possible addition of a hydrophobic membrane anchor. The kinetic properties of detergent-soluble mPOP are fully comparable to those of POP; however, when attached to the membranes in its natural conformation, mPOP is significantly less active and, moreover, it migrates anomalously in SDS/PAGE. Our results are the first to show that membrane-bound and cytoplasmic POP are encoded by variants of the same gene. [source] Specific Ser-Pro phosphorylation by the RNA-recognition motif containing kinase KISFEBS JOURNAL, Issue 14 2000Alexandre Maucuer We present here a first appraisal of the phosphorylation site specificity of KIS (for ,kinase interacting with stathmin'), a novel mammalian kinase that has the unique feature among kinases to possess an RNP type RNA-recognition motif (RRM). In vitro kinase assays using various standard substrates revealed that KIS has a narrow specificity, with myelin basic protein (MBP) and synapsin I being the best in vitro substrates among those tested. Mass spectrometry and peptide sequencing allowed us to identify serine 164 of MBP as the unique site phosphorylated by KIS. Phosphorylation of synthetic peptides indicated the importance of the proline residue at position +1. We also identified a tryptic peptide of synapsin I phosphorylated by KIS and containing a phosphorylatable Ser-Pro motif. Altogether, our results suggest that KIS preferentially phosphorylates proline directed residues but has a specificity different from that of MAP kinases and cdks. [source] Exclusive expression of a membrane-bound Spink3-interacting serine protease-like protein TESPL in mouse testisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2010Chung-Mao Ou Abstract We identified a testis-specific protease-like protein tentatively named TESPL and a pancreatic trypsinogen Prss2 from the clones of a yeast two-hybrid screen against a mouse testicular cDNA library using the trypsin inhibitor Spink3 from male accessory sexual glands as bait. The enzymatic motifs and the cysteine patterns in serine proteases are highly conserved in these two proteins. Based on the phylogenetic analysis, Prss2 duplicated recently and TESPL underwent distant evolution without gene duplication from the progenitor of trypsin-like and chymotrypsin-like proteases. We found that TESPL transcription was restricted to the testis and that the level of transcription was positively correlated with animal maturation. In contrast, Prss2 was constitutively expressed in many tissues including testis. Alignment of the cDNA-deduced sequences of serine proteases showed the replacement of an essential serine residue in the catalytic triad of serine proteases by a proline residue in TESPL, which was demonstrated to be a membrane-bound protein devoid of proteolytic activity. The immunohistochemical staining patterns of seminiferous tubules in the testis revealed TESPL mainly on postmeiotic cells such as spermatids and spermatozoa. On the mouse sperm from caudal epididymis, TESPL was localized mainly on the plasma membrane overlaying the acrosomal region. Further, orthology group for mouse TESPL was identified in the conserved gene family of eutherian testis serine protease 5. J. Cell. Biochem. 110: 620,629, 2010. © 2010 Wiley-Liss, Inc. [source] New antitumour cyclic astin analogues: synthesis, conformation and bioactivityJOURNAL OF PEPTIDE SCIENCE, Issue 2 2004Dr Filomena Rossi Abstract Astins, antitumour cyclic pentapeptides, were isolated from the Aster tataricus. Their chemical structures, consist of a 16-membered ring system containing a unique ,,,-dichlorinated proline [Pro(Cl)2], other non-coded amino acid residues and a cis conformation in one of the peptide bonds. The astin backbone conformation, along with the cis peptide bond in which the ,,,-dichlorinated proline residue is involved, was considered to play an important role in their antineoplastic activities on sarcoma 180A and P388 lymphocytic leukaemia in mice, but the scope and potential applications of this activity remain unclear. With the aim at improving our knowledge of the conformational properties influencing the bioactivity in this class of compounds, new astin-related cyclopeptides were synthesized differing from the natural products by the presence of some non-proteinogenic amino acid residues: Aib, Abu, -(S),3 -hPhe and a peptide bond surrogate (-SO2 -NH-). The analogues prepared c(-Pro-Thr-Aib-,3 -Phe-Abu-), c[Pro-Thr-Aib-(S),3 -hPhe-Abu], c[Pro-Abu-Ser-(S),3 -hPhe,(CH2 -SO2 -NH)-Abu] and c[Pro-Thr-Aib-(S),3 -hPhe,(CH2 -SO2 -NH)-Abu] were synthesized by classical methods in solution and tested for their antitumour effect. These molecules were studied by crystal-state x-ray diffraction analysis and/or solution NMR and MD techniques. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] A missense mutation in the vacuolar protein GOLD36 causes organizational defects in the ER and aberrant protein trafficking in the plant secretory pathwayTHE PLANT JOURNAL, Issue 6 2010Lucia Marti Summary A central question in cell biology is how the identity of organelles is established and maintained. Here, we report on GOLD36, an EMS mutant identified through a screen for partial displacement of the Golgi marker, ST-GFP, to other organelles. GOLD36 showed partial distribution of ST-GFP into a modified endoplasmic reticulum (ER) network, which formed bulges and large skein-like structures entangling Golgi stacks. GOLD36 showed defects in ER protein export as evidenced by our observations that, besides the partial retention of Golgi markers in the ER, the trafficking of a soluble bulk-flow marker to the cell surface was also compromised. Using a combination of classical mapping and next-generation DNA sequencing approaches, we linked the mutant phenotype to a missense mutation of a proline residue in position 80 to a leucine residue in a small endomembrane protein encoded by the gold36 locus (At1g54030). Subcellular localization analyses indicated that GOLD36 is a vacuolar protein and that its mutated form is retained in the ER. Interestingly also, a gold36 knock-out mutant mirrored the GOLD36 subcellular phenotype. These data indicate that GOLD36 is a protein destined to post-ER compartments and suggest that its export from the ER is a requirement to ensure steady-state maintenance of the organelle's organization and functional activity in relation to other secretory compartments. We speculate that GOLD36 may be a factor that is necessary for ER integrity because of its ability to limit deleterious effects of other secretory proteins on the ER. [source] Characterization of a prolyl endoprotease from Eurygaster integriceps puton (Sunn pest) infested wheatARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2010Charles Darkoh Abstract Sunn pest, Eurygaster integriceps, Puton, infested and uninfested wheat seeds were obtained from the International Center for Agriculture Research in the Dry Areas (ICARDA), Aleppo, Syria, with the primary objective to identify the type of enzyme deposited by the Sunn pest on the wheat responsible for the gluten degradation. Enzyme levels were extremely low due to the enzyme being secreted by the insect in localized areas on the seed. Only extract from the infested wheat contained glutenase activity. Anion exchange, Cu2+ sepharose, and gel filtration chromatography were used to partially purify and enrich protein samples from both infested wheat and uninfested wheat. An SDS-gluten assay was used to show gluten specificity while a commercially available chromogenic proline peptide, benzyloxycarbonyl-Gly-Pro-p-nitroanalide (ZGPpNA), was utilized to identify fractions containing the active proline specific enzyme activity and to determine Michaelis-Menten kinetics. Despite low levels of enzyme on the infested wheat, the enzyme was partially purified and enriched exhibiting a specific activity of 4.5,U/mg of total protein for gluten in a SDS gluten assay (1,U of enzyme activity was defined as the decrease in gel height in millimeters in 1,h) and exhibited a high-affinity Km of 65,µM for ZGPpNA, cleaving at the carboxy terminus of the proline residue. The enzyme exhibited optimal activity between pH 8 and 10.0 at temperatures between 20° and 35°C. The enzyme was identified to be a prolyl endoprotease. © 2010 Wiley Periodicals, Inc. [source] The effect of a proline residue on the rate of growth and the space group of ,-spectrin SH3-domain crystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Ana Cámara-Artigas ,-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a proline residue that participates in a crystal contact mimicking the binding of proline-rich sequences to SH3 domains. This residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical proline residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P41212, with unit-cell parameters a = b = 42.231, c = 93.655,Å, and that diffracts to 1.45,Å resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2,M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 residue plays a key role in the rate of crystal growth. [source] A C-terminal disulfide bond in the copper-containing amine oxidase from pea seedlings violates the twofold symmetry of the molecular dimerACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2006Anthony P. Duff The structure of a newly crystallized form of the copper-dependent amine oxidase from pea seedlings has been refined at a resolution of 2.2,Å to a final R factor of 0.181. The structure (form II) was originally discovered during a study of xenon binding to copper-dependent amine oxidases as a probe for dioxygen-binding sites [Duff et al. (2004), J. Mol. Biol.344, 599,607]. The form II crystals belong to space group P21, with two dimers in the asymmetric unit. The overall structure is very similar to the crystals of form I in space group P212121 with a dimer in the asymmetric unit [Kumar et al. (1996), Structure, 4, 943,955]. In form I the last three residues (644,647) observable in the two subunits were apparently splayed apart. It was noted that the absence of a disulfide bond between the Cys647 residues of the two subunits was inconsistent with chemical evidence for the absence of free sulfhydryl groups. In both of the crystallographically independent dimers of form II the two subunits are clearly joined by a disulfide bridge between the C-terminal cysteine residues. This is only possible if the two polypeptide chains in the dimer adopt different conformations near the C-terminus so that the twofold symmetry is lost. A proline residue (645) two residues before the cysteine has a cis conformation in one chain and a trans conformation in the other. As a result, the disulfide bond lies more than 5,Å from the twofold axis. The loss of local twofold symmetry in form II can be explained by intermolecular contacts, which provide an asymmetric environment. [source] Melectin: A Novel Antimicrobial Peptide from the Venom of the Cleptoparasitic Bee Melecta albifronsCHEMBIOCHEM, Issue 17 2008Václav, ovský Dr. Abstract A novel antimicrobial peptide designated melectin was isolated from the venom of the cleptoparasitic bee Melecta albifrons. Its primary sequence was established as H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Ala-His-Met-Lys-NH2 by Edman degradation and ESI-QTOF mass spectrometry. Synthetic melectin exhibited antimicrobial activity against both Gram-positive and -negative bacteria and it degranulated rat peritoneal mast cells, but its hemolytic activity was low. The CD spectra of melectin measured in the presence of trifluoroethanol and sodium dodecyl sulfate showed a high content ,-helices, which indicates that melectin can adopt an amphipathic ,-helical secondary structure in an anisotropic environment such as the bacterial cell membrane. To envisage the role of the proline residue located in the middle of the peptide chain on biological activity and secondary structure, we prepared several melectin analogues in which the Pro11 residue was either replaced by other amino acid residues or was omitted. The results of biological testing suggest that a Pro kink in the ,-helical structure of melectin plays an important role in selectivity for bacterial cells. In addition, a series of N- and C-terminal-shortened analogues was synthesized to examine which region of the peptide is related to antimicrobial activity. [source] Stylissamides A,D , New Proline-Containing Cyclic Heptapeptides from the Marine Sponge Stylissa caribicaEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 24 2007Gesine Schmidt Abstract Four new cyclic heptapeptides, stylissamides A,D (1,4), were isolated from the Caribbean sponge Stylissa caribica. The structures of these metabolites were elucidated by NMR and MS/MS methods. The peptides contain three and, in one case, four proline residues. The sequence assignment of 1,4 by NMR was supported by fragmentations in HR-MS/MS measurements. The absolute configuration of all amino acid residues was assigned as L using Marfey's method and the OPA method.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] The periplasmic peptidyl prolyl cis,trans isomerases PpiD and SurA have partially overlapping substrate specificitiesFEBS JOURNAL, Issue 13 2008Krista H. Stymest One of the rate-limiting steps in protein folding has been shown to be the cis,trans isomerization of proline residues, catalysed by a range of peptidyl prolyl cis,trans isomerases (PPIases). In the periplasmic space of Escherichia coli and other Gram-negative bacteria, two PPIases, SurA and PpiD, have been identified, which show high sequence similarity to the catalytic domain of the small PPIase parvulin. This observation raises a question regarding the biological significance of two apparently similar enzymes present in the same cellular compartment: do they interact with different substrates or do they catalyse different reactions? The substrate-binding motif of PpiD has not been characterized so far, and no biochemical data were available on how this folding catalyst recognizes and interacts with substrates. To characterize the interaction between model peptides and the periplasmic PPIase PpiD from E. coli, we employed a chemical crosslinking strategy that has been used previously to elucidate the interaction of substrates with SurA. We found that PpiD interacted with a range of model peptides independently of whether they contained proline residues or not. We further demonstrate here that PpiD and SurA interact with similar model peptides, and therefore must have partially overlapping substrate specificities. However, the binding motif of PpiD appears to be less specific than that of SurA, indicating that the two PPIases might interact with different substrates. We therefore propose that, although PpiD and SurA have partially overlapping substrate specificities, they fulfil different functions in the cell. [source] Influence of an extract from kudzu symbiosomes containing leghemoglobin on in vitro cutaneous procollagen productionINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 3 2010J. V. Gruber J. Cosmet. Sci., 60, 475,484 (September/October 2009) Synopsis Cytoglobin is a hexacoordinateglobin protein that was recently discovered in mammals. Interestingly, of the four human globin proteins that are now known, hemoglobin, myoglobin, neuroglobin and cytoglobin, the latter appears to have the closest resemblance to strikingly similar proteins expressed in plants. In legumes, these proteins accumulate in symbiosomes (root nodules) of various legumes and are called leghemoglobin. The paper will discuss the ability of an aqueous extract from Pueraria lobata (kudzu) symbiosomes that contains leghemoglobin to stimulate procollagen production in human dermal fibroblasts. This effect may be partly due to the possibility that leghemoglobin may mimic the function of cytoglobin by shuttling oxygen to prolyl-4-hydroxylase, the enzyme responsible for oxidizing proline residues in procollagen bundles. This hypothesis is supported by DNA microarray sequencing data that demonstrate that treatment of normal human dermal fibroblasts (NHDF) with highly purified cytoglobin or leghemoglobin upregulates a number of key collagen-related genes including COL1A1 and COL1A2. [source] Gas-phase basicities for ions from bradykinin and its des-arginine analoguesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 8 2001Nigel P. Ewing Abstract Apparent gas-phase basicities (GBapps) for [M + H]+ of bradykinin, des-Arg1 -bradykinin and des-Arg9 -bradykinin have been assigned by deprotonation reactions of [M + 2H]2+ in a Fourier transform ion cyclotron resonance mass spectrometer. With a GBapp of 225.8 ± 4.2 kcal mol,1, bradykinin [M + H]+ is the most basic of the ions studied. Ions from des-Arg1 -bradykinin and des-Arg9 -bradykinin have GBapp values of 222.8 ± 4.3 kcal mol,1 and 214.9 ± 2.3 kcal mol,1, respectively. One purpose of this work was to determine a suitable reaction efficiency ,break point' for assigning GBapp values to peptide ions using the bracketing method. An efficiency value of 0.1 (i.e. approximately 10% of all collisions resulting in a deprotonation reaction) was used to assign GBapps. Support for this criterion is provided by the fact that our GBapp values for des-Arg1 -bradykinin and des-Arg9 -bradykinin are identical, within experimental error, to literature values obtained using a modified kinetic method. However, the GBapps for bradykinin ions from the two studies differ by 10.3 kcal mol,1. The reason for this is not clear, but may involve conformation differences produced by experimental conditions. The results may be influenced by salt-bridge conformers and/or by conformational changes caused by the use of a proton-bound heterodimer in the kinetic method. Factors affecting the basicities of these peptide ions are also discussed, and molecular modeling is used to provide information on protonation sites and conformations. The presence of two highly basic arginine residues on bradykinin results in its high GBapp, while the basicity of des-Arg1 -bradykinin ions is increased by the presence of two proline residues at the N-terminus. The proline residue in the second position folds the peptide chain in a manner that increases intramolecular hydrogen bonding to the protonated N-terminal amino group of the proline at the first position. Copyright © 2001 John Wiley & Sons, Ltd. [source] Primary sequence and site-selective hydroxylation of prolines in isoforms of a major peanut allergen protein Ara h 2PROTEIN SCIENCE, Issue 1 2010Jinxi Li Abstract The Ara h 2 proteins are major determinants of peanut allergens. These proteins have not been fully studied at the molecular level. It has been previously proposed that there are two isoforms of Ara h 2, based on primary structures that were deduced from two reported cDNA sequences. In this report, four isoforms have been purified and characterized individually. Mass spectrometric methods have been used to determine the protein sequences and to define post-translational modifications for all four isoforms. Two pairs of isoforms have been identified, corresponding to a long-chain form and a form that is shorter by 12 amino acids. Each pair is further differentiated by the presence or absence of a two amino acid sequence at the carboxyl terminus of the protein. Modifications that were characterized include site-specific hydroxylation of proline residues, but no glycosylation was found, in contrast to previous reports. [source] Localization of the mRNA encoding prolyl endopeptidase in the rat brain and pituitaryTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2004Gaelle Bellemère Abstract Prolyl endopeptidase (EC 3.4.21.26, PEP), a serine protease that hydrolyzes peptides at the carboxyl side of proline residues, is involved in the breakdown of several proline-containing neuropeptides and, thus, may contribute to the regulation of behavioral activities. In this study, the distribution of PEP mRNA was investigated in the central nervous system and pituitary of rat by means of quantitative reverse transcriptase-polymerase chain reaction analysis and in situ hybridization histochemistry. High densities of PEP transcripts were found in cerebellar Purkinje and granule cells, within most hypothalamic nuclei, in pyramidal neurons of the Ammon's horn, in granule cells of the dentate gyrus, and within the basolateral complex of the amygdala. Moderate levels of PEP mRNA were observed in layers 3,5 of the cerebral cortex, the anterior thalamic group, the septal region, the substantia nigra, the magnocellular neurons of the red nucleus, and the motor nuclei of the cranial nerves. Low concentrations of PEP mRNA were detected in the deep mesencephalic nuclei, the reticular formation, the pretectum, and the tectum. A high density of PEP mRNA was found in the intermediate and the anterior lobes of the pituitary, while the neural lobe was devoid of labeling. In several brain regions, the distribution pattern of PEP mRNA overlapped that of various neuropeptide receptors, suggesting that PEP is actually involved in the inactivation of regulatory neuropeptides. J. Comp. Neurol. 471:128,143, 2004. © 2004 Wiley-Liss, Inc. [source] Partitioning and Characterization of Tyrosine-Tagged Green Fluorescent Proteins in Aqueous Two-Phase SystemsBIOTECHNOLOGY PROGRESS, Issue 3 2004Sara Fexby The green fluorescent protein GFPuv has been genetically engineered to investigate the influence of N-terminal tyrosine extensions in aqueous two-phase systems. Fusions in the N-terminus affected the protein expression, and tags containing three tyrosines and prolines influenced the expression favorably. This effect is probably due to changes in mRNA stability, because the amounts of corresponding mRNAs correlated with the amounts of GFPuv proteins. The partitioning was investigated in two different aqueous two-phase systems, a two-polymer system composed of EO30PO70/dextran and a PEG/salt system with potassium phosphate. Partitioning in the PEG/salt system generally was more favorable than in the EO30PO70/dextran system. Tags with three tyrosines resulted in higher partitioning toward the EO30PO70- and PEG-rich phases, respectively. The effect of adding proline residues to the tag was also investigated, and the partitioning effect of the tag was enhanced when prolines were included in the tags with three tyrosines. The best tyrosine tag, Y3P2, increased the partition coefficient 5 times in the PEG/salt system. Thermoseparation of the EO30PO70 phase allowed recovery of 83% Y3P2-GFPuv protein in a water phase. [source] | |||||||||||||