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Ribonuclease
Kinds of Ribonuclease Terms modified by Ribonuclease Selected AbstractsGene expression of colony-stimulating factors and stem cell factor after myocardial infarction in the mouseACTA PHYSIOLOGICA, Issue 3 2002P. R. WOLDBAEK ABSTRACT Recent studies have suggested that cytokines such as macrophage colony-stimulating factor (M-CSF) might be involved in the pathogenesis of ischaemic heart disease. Macrophage colony-stimulating factor, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), stem cell factor (SCF), interleukin-3 (IL-3) and interleukin-7 (IL-7) are potent cytokines belonging to the same structual class that may affect function, growth and apoptosis both in the heart and other organs. The aims of the present study were to characterize a post-infarction model in the mouse and to examine mRNA expression of M-CSF, GM-CSF, SCF, IL-3 and IL-7 during the development of heart failure. Myocardial infarction (MI) was induced in mice by ligation of the left coronary artery. Average infarct size was 40% and the mice developed myocardial hypertrophy and pulmonary oedema. Ribonuclease (RNAase) protection assays showed abundant cardiac expression of M-CSF and SCF. After MI, we measured down-regulation of cytokine mRNA expression in the heart (M-CSF, SCF), lung (M-CSF), liver (M-CSF) and spleen (M-CSF) compared with sham. Cardiac G-CSF, GM-CSF and IL-7 mRNAs were not detected. In conclusion, abundant cardiac gene expression of M-CSF and SCF was found. In our mouse model of MI, M-CSF and SCF were down-regulated in the heart and several other organs suggesting specific roles for these cytokines during development of ischaemic heart failure. [source] Probing the unfolding region of ribonuclease A by site-directed mutagenesisFEBS JOURNAL, Issue 20 2004Jens Köditz Ribonuclease A contains two exposed loop regions, around Ala20 and Asn34. Only the loop around Ala20 is sufficiently flexible even under native conditions to allow cleavage by nonspecific proteases. In contrast, the loop around Asn34 (together with the adjacent ,-sheet around Thr45) is the first region of the ribonuclease A molecule that becomes susceptible to thermolysin and trypsin under unfolding conditions. This second region therefore has been suggested to be involved in early steps of unfolding and was designated as the unfolding region of the ribonuclease A molecule. Consequently, modifications in this region should have a great impact on the unfolding and, thus, on the thermodynamic stability. Also, if the Ala20 loop contributes to the stability of the ribonuclease A molecule, rigidification of this flexible region should stabilize the entire protein molecule. We substituted several residues in both regions without any dramatic effects on the native conformation and catalytic activity. As a result of their remarkably differing stability, the variants fell into two groups carrying the mutations: (a) A20P, S21P, A20P/S21P, S21L, or N34D; (b) L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/F46Y, or K31A/R33S/F46Y. The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, suggesting that the decrease in ,G can be mainly attributed to an increased unfolding rate. Although rigidification of the Ala20 loop by introduction of proline did not result in stabilization, disturbance of the network of hydrogen bonds and hydrophobic interactions that interlock the proposed unfolding region dramatically destabilized the ribonuclease A molecule. [source] Broadening the mission of an RNA enzymeJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Michael C. Marvin Abstract The "RNA World" hypothesis suggests that life developed from RNA enzymes termed ribozymes, which carry out reactions without assistance from proteins. Ribonuclease (RNase) P is one ribozyme that appears to have adapted these origins to modern cellular life by adding protein to the RNA core in order to broaden the potential functions. This RNA-protein complex plays diverse roles in processing RNA, but its best-understood reaction is pre-tRNA maturation, resulting in mature 5' ends of tRNAs. The core catalytic activity resides in the RNA subunit of almost all RNase P enzymes but broader substrate tolerance is required for recognizing not only the diverse sequences of tRNAs, but also additional cellular RNA substrates. This broader substrate tolerance is provided by the addition of protein to the RNA core and allows RNase P to selectively recognize different RNAs, and possibly ribonucleoprotein (RNP) substrates. Thus, increased protein content correlated with evolution from bacteria to eukaryotes has further enhanced substrate potential enabling the enzyme to function in a complex cellular environment. J. Cell. Biochem. 108: 1244,1251, 2009. © 2009 Wiley-Liss, Inc. [source] Structural properties of trimers and tetramers of ribonuclease APROTEIN SCIENCE, Issue 10 2001Arianna Nenci Abstract Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) , poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) , poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures co ld be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures. [source] Direct Refolding of Inclusion Bodies Using Reversed MicellesBIOTECHNOLOGY PROGRESS, Issue 6 2004Masafumi Sakono The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A. [source] Stability and Dynamics of Domain-Swapped Bovine-Seminal RibonucleaseCHEMISTRY & BIODIVERSITY, Issue 5 2004Kalyan The proteins of the ribonuclease-A (RNase-A) family are monomeric, with the exception of bovine-seminal ribonuclease (BS-RNase). BS-RNase is formed by swapping the N-terminal helices across the two monomeric units. A molecular-dynamics (MD) study has been performed on the protein for a simulation time of 5.5,ns to understand the factors responsible for the stability of the dimer. Essential dynamics analysis and motional correlation of the protein atoms yielded the picture of a stabilising, yet flexible, interface. We have investigated the role of intermolecular H-bonding, protein/water interaction, and protein/water networks in stabilising the dimer. The networks of interchain H-bonds involving side-chain/side-chain or side-chain/main-chain (ScHB) interactions between the two chains have also been studied. The ability of protein atoms in retaining particular H2O molecules was investigated as a function of the accessible surface area (ASA), depth, and hydration parameters, as well as their participation in protein/water networks. [source] Effect of sialic acid content on glycoprotein pI analyzed by two-dimensional electrophoresisELECTROPHORESIS, Issue 17 2010Sílvia Barrabés Abstract 2-DE is broadly used for quantitative analysis of differential protein expression in complex mixtures such as serum samples or cell lysates. PTMs directly influence the 2-DE pattern, and knowledge of the rules of protein separation is required in order to understand the protein distribution in a 2-DE gel. Glycosylation is the most common PTM and can modify both the molecular weight and the pI of a protein. In particular, the effect of charged monosaccharides (mainly sialic acids, SAs) on the 2-DE pattern of a protein is of major interest since changes in sialylation are regularly observed in comparative studies. Little is known about the pI shift of a glycoprotein induced by the presence of SAs, or whether this shift is the same for all glycoproteins. To address this issue, this study examined the influence of SA on the 2-DE pattern of three serum glycoproteins (haptoglobin, ,1-antitrypsin and ribonuclease 1), which N -glycan chains had been previously characterised, and reviewed existing bibliographic data. The SA content of the different glycoforms of a glycoprotein showed a negative linear correlation with the pI, although the slope varied among the studied glycoproteins. We also described a positive correlation between the protein pI and the pI decrease per SA molecule. [source] Protein separations using polyelectrolyte multilayer coatings with molecular micelles in open tubular capillary electrochromatographyELECTROPHORESIS, Issue 4 2008Candace A. Luces Abstract Novel polyelectrolyte multilayer (PEM) coatings for enhanced protein separations in open tubular CEC (OT-CEC) are reported. Use of four cationic polymers (poly- L -lysine, poly- L -ornithine, poly- L -lysine-serine, and poly- L -glutamic acid-lysine), and three anionic molecular micelles, sodium poly(N -undecanoyl- L -leucyl-alaninate) (poly- L -SULA), sodium poly(N -undecanoyl- L -leucyl-valinate) (poly- L -SULV), and sodium poly(undecylenic sulfate) (poly-SUS) were investigated in PEM coatings for protein separations. The simultaneous effects of cationic polymer concentration, number of bilayers, temperature, applied voltage, and pH of the BGE on the separation of four basic proteins (,-chymotrypsinogen A, lysozyme, ribonuclease A, and cytochrome c) were analyzed using a Box Behnken experimental design. The influence of NaCl on the run-to-run reproducibility was investigated for PEM coatings containing each cationic polymer. All coatings exhibited excellent reproducibilities with a %RSD of the EOF less than 1% in the presence of NaCl. Optimal conditions were dependent on both the cationic and anionic polymers used in the PEM coatings. Poly- L -glutamic acid-lysine produced the highest resolution and longest migration time. The use of molecular micelles to form PEM coatings resulted in better separations than single cationic coatings. Chiral poly- L -SULA and poly- L -SULV resulted in higher protein resolutions as compared to the achiral, poly-SUS. Furthermore, the use of poly- L -SULV reversed the elution order of lysozyme and cytochrome c when compared to poly- L -SULA and poly-SUS. [source] Conformational stability and multistate unfolding of poly(A)-specific ribonucleaseFEBS JOURNAL, Issue 10 2009Guang-Jun He Poly(A)-specific ribonuclease (PARN) specifically catalyzes the degradation of the poly(A) tails of single-stranded mRNAs in a highly processive mode. PARN participates in diverse and important intracellular processes by acting as a regulator of mRNA stability and translational efficiency. In this article, the equilibrium unfolding of PARN was studied using both guanidine hydrochloride and urea as chemical denaturants. The unfolding of PARN was characterized as a multistate process, but involving dissimilar equilibrium intermediates when denatured by the two denaturants. A comparison of the spectral characteristics of these intermediates indicated that the conformational changes at low concentrations of the chemical denaturants were more likely to be rearrangements of the tertiary and quaternary structures. In particular, an inactive molten globule-like intermediate was identified to exist as soluble non-native oligomers, and the formation of the oligomers was modulated by electrostatic interactions. An active dimeric intermediate unique to urea-induced unfolding was characterized to have increased regular secondary structures and modified tertiary structures, implying that additional regular structures could be induced by environmental stresses. The dissimilarity in the unfolding pathways induced by guanidine hydrochloride and urea suggest that electrostatic interactions play an important role in PARN stability and regulation. The appearance of multiple intermediates with distinct properties provides the structural basis for the multilevel regulation of PARN by conformational changes. [source] Solution structure of the bb, domains of human protein disulfide isomeraseFEBS JOURNAL, Issue 5 2009Alexey Y. Denisov Protein disulfide isomerase is the most abundant and best studied of the disulfide isomerases that catalyze disulfide bond formation in the endoplasmic reticulum, yet the specifics of how it binds substrate have been elusive. Protein disulfide isomerase is composed of four thioredoxin-like domains (abb,a,). Cross-linking studies with radiolabeled peptides and unfolded proteins have shown that it binds incompletely folded proteins primarily via its third domain, b,. Here, we determined the solution structure of the second and third domains of human protein disulfide isomerase (b and b,, respectively) by triple-resonance NMR spectroscopy and molecular modeling. NMR titrations identified a large hydrophobic surface within the b, domain that binds unfolded ribonuclease A and the peptides mastoparan and somatostatin. Protein disulfide isomerase-catalyzed refolding of reduced ribonuclease A in vitro was inhibited by these peptides at concentrations equal to their affinity to the bb, fragment. Our findings provide a structural basis for previous kinetic and cross-linking studies which have shown that protein disulfide isomerase exhibits a saturable, substrate-binding site. [source] Effect of the disease-causing mutations identified in human ribonuclease (RNase) H2 on the activities and stabilities of yeast RNase H2 and archaeal RNase HIIFEBS JOURNAL, Issue 19 2008Muhammad S. Rohman Eukaryotic ribonuclease (RNase) H2 consists of one catalytic and two accessory subunits. Several single mutations in any one of these subunits of human RNase H2 cause Aicardi,Goutičres syndrome. To examine whether these mutations affect the complex stability and activity of RNase H2, three mutant proteins of His-tagged Saccharomyces cerevisiae RNase H2 (Sc-RNase H2*) were constructed. Sc-G42S*, Sc-L52R*, and Sc-K46W* contain single mutations in Sc-Rnh2Ap*, Sc-Rnh2Bp*, and Sc-Rnh2Cp*, respectively. The genes encoding the three subunits were coexpressed in Escherichia coli, and Sc-RNase H2* and its derivatives were purified in a heterotrimeric form. All of these mutant proteins exhibited enzymatic activity. However, only the enzymatic activity of Sc-G42S* was greatly reduced compared to that of the wild-type protein. Gly42 is conserved as Gly10 in Thermococcus kodakareansis RNase HII. To analyze the role of this residue, four mutant proteins, Tk-G10S, Tk-G10A, Tk-G10L, and Tk-G10P, were constructed. All mutant proteins were less stable than the wild-type protein by 2.9,7.6 °C in Tm. A comparison of their enzymatic activities, substrate binding affinities, and CD spectra suggests that the introduction of a bulky side chain into this position induces a local conformational change, which is unfavorable for both activity and substrate binding. These results indicate that Gly10 is required to make the protein fully active and stable. [source] Protein disulfide isomerase family proteins involved in soybean protein biogenesisFEBS JOURNAL, Issue 3 2007Hiroyuki Wadahama Protein disulfide isomerase family proteins are known to play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill cv. Jack) mRNA by RT-PCR using forward and reverse primers designed from the expressed sequence tag clone sequences. The cDNA encodes a protein of either 364 or 362 amino acids, named GmPDIS-1 or GmPDIS-2, respectively. The nucleotide and amino acid sequence identities of GmPDIS-1 and GmPDIS-2 were 68% and 74%, respectively. Both proteins lack the C-terminal, endoplasmic reticulum-retrieval signal, KDEL. Recombinant proteins of both GmPDIS-1 and GmPDIS-2 were expressed in Escherichia coli as soluble folded proteins that showed both an oxidative refolding activity of denatured ribonuclease A and a chaperone activity. Their domain structures were identified as containing two thioredoxin-like domains, a and a,, and an ERp29c domain by peptide mapping with either trypsin or V8 protease. In cotyledon cells, both proteins were shown to distribute to the endoplasmic reticulum and protein storage vacuoles by confocal microscopy. Data from coimmunoprecipitation and crosslinking experiments suggested that GmPDIS-1 associates with proglycinin, a precursor of the seed storage protein glycinin, in the cotyledon. Levels of GmPDIS-1, but not of GmPDIS-2, were increased in cotyledons, where glycinin accumulates during seed development. GmPDIS-1, but not GmPDIS-2, was induced under endoplasmic reticulum-stress conditions. [source] Probing the unfolding region of ribonuclease A by site-directed mutagenesisFEBS JOURNAL, Issue 20 2004Jens Köditz Ribonuclease A contains two exposed loop regions, around Ala20 and Asn34. Only the loop around Ala20 is sufficiently flexible even under native conditions to allow cleavage by nonspecific proteases. In contrast, the loop around Asn34 (together with the adjacent ,-sheet around Thr45) is the first region of the ribonuclease A molecule that becomes susceptible to thermolysin and trypsin under unfolding conditions. This second region therefore has been suggested to be involved in early steps of unfolding and was designated as the unfolding region of the ribonuclease A molecule. Consequently, modifications in this region should have a great impact on the unfolding and, thus, on the thermodynamic stability. Also, if the Ala20 loop contributes to the stability of the ribonuclease A molecule, rigidification of this flexible region should stabilize the entire protein molecule. We substituted several residues in both regions without any dramatic effects on the native conformation and catalytic activity. As a result of their remarkably differing stability, the variants fell into two groups carrying the mutations: (a) A20P, S21P, A20P/S21P, S21L, or N34D; (b) L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/F46Y, or K31A/R33S/F46Y. The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, suggesting that the decrease in ,G can be mainly attributed to an increased unfolding rate. Although rigidification of the Ala20 loop by introduction of proline did not result in stabilization, disturbance of the network of hydrogen bonds and hydrophobic interactions that interlock the proposed unfolding region dramatically destabilized the ribonuclease A molecule. [source] Growth inhibition of mammalian cells by eosinophil cationic proteinFEBS JOURNAL, Issue 1 2002Takashi Maeda Eosinophil cationic protein (ECP), one of the major components of basic granules of eosinophils, is cytotoxic to tracheal epithelium. However, the extent of this effect on other cell types has not been evaluated in vitro. In this study, we evaluated the effect of ECP on 13 mammalian cell lines. ECP inhibited the growth of several cell lines including those derived from carcinoma and leukemia in a dose-dependent manner. The IC50 values on A431 cells, MDA-MB-453 cells, HL-60 cells and K562 cells were,estimated to be ,,1,5 µm. ECP significantly suppressed the size of colonies of A431 cells, and decreased K562 cells in G1/G0 phase. However, there was little evidence that ECP killed cells in either cell line. These effects of ECP were not enhanced by extending its N-terminus. Rhodamine B isothiocyanate-labeled ECP started to bind to A431 cells after 0.5 h and accumulated for up to 24 h, indicating that specific affinity for the cell surface may be important. The affinity of ECP for heparin was assessed and found to be reduced when tryptophan residues, one of which is located at a position in the catalytic subsite of ribonuclease in ECP, were modified. The growth-inhibitory effect was also attenuated by this modification. These results suggest that growth inhibition by ECP is dependent on cell type and is cytostatic. [source] Ribonucleases expressed by human pancreatic adenocarcinoma cell linesFEBS JOURNAL, Issue 5 2000Ester Fernández-Salas Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan-1, MDAPanc-3, IBF-CP3 and Panc-1, and the ampullary adenocarcinoma cell line MDAAmp-7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc-1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion-exchange and cation-exchange liquid chromatography and C4 RP-HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative-staining zymogram techniques. The results of N-terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory-type ribonuclease and that it is composed of several differently N -glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc-1 and MDAPanc-28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc-1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan-1 shows that it is highly glycosylated and its N -glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker. [source] Comparative analysis of the self-incompatibility (S -) locus region of Prunus mume: identification of a pollen-expressed F-box gene with allelic diversityGENES TO CELLS, Issue 3 2003Tetsuyuki Entani Background: Self-incompatibility (SI) in the Solanaceae, Rosaceae and Scrophulariaceae is gametophytically controlled by a single polymorphic locus, termed the S -locus. To date, the only known S -locus product is a polymorphic ribonuclease, termed S -RNase, which is secreted by stylar tissue and thought to act as a cytotoxin that degrades the RNA of incompatible pollen tubes. However, understanding how S -RNase causes S -haplotype specific inhibition of pollen tubes has been hampered by the lack of a cloned pollen S -determinant gene. Results: To identify the pollen S -determinant gene, we investigated the genomic structure of the S -locus region of the S1 - and S7 -haplotypes of Prunus mume (Japanese apricot), and identified 13 genes around the S-RNase gene. Among them, only one F-box gene, termed SLF (S -locus F-box), fulfilled the conditions for a pollen S -determinant gene: (i) together with the S-RNase gene, it is located within the highly divergent genomic region of the S -locus, (ii) it exhibits S -haplotype specific diversity among three analysed S -haplotypes, and (iii) it is specifically expressed in pollen, but not in the styles or leaves. Conclusion: The results indicate that SLF is a prime candidate for the pollen S -determinant gene of SI. [source] Peripheral myelin protein 22 is regulated post-transcriptionally by miRNA-29a,GLIA, Issue 12 2009Jonathan D. Verrier Abstract Peripheral myelin protein 22 (PMP22) is a dose-sensitive, disease-associated protein primarily expressed in myelinating Schwann cells. Either reduction or overproduction of PMP22 can result in hereditary neuropathy, suggesting a requirement for correct protein expression for peripheral nerve biology. PMP22 is post-transcriptionally regulated and the 3,untranslated region (3,UTR) of the gene exerts a negative effect on translation. MicroRNAs (miRNAs) are small regulatory molecules that function at a post-transcriptional level by targeting the 3,UTR in a reverse complementary manner. We used cultured Schwann cells to demonstrate that alterations in the miRNA biogenesis pathway affect PMP22 levels, and endogenous PMP22 is subjected to miRNA regulation. GW-body formation, the proposed cytoplasmic site for miRNA-mediated repression, and Dicer expression, an RNase III family ribonuclease involved in miRNA biogenesis, are co-regulated with the differentiation state of Schwann cells. Furthermore, the levels of Dicer inversely correlate with PMP22, while the inhibition of Dicer leads to elevated PMP22. Microarray analysis of actively proliferating and differentiated Schwann cells, in conjunction with bioinformatics programs, identified several candidate PMP22-targeting miRNAs. Here we demonstrate that miR-29a binds and inhibits PMP22 reporter expression through a specific miRNA seed binding region. Over-expression of miR-29a enhances the association of PMP22 RNA with Argonaute 2, a protein involved in miRNA function, and reduces the steady-state levels of PMP22. In contrast, inhibition of endogenous miR-29a relieves the miRNA-mediated repression of PMP22. Correlation analyses of miR-29 and PMP22 in sciatic nerves reveal an inverse relationship, both developmentally and in post-crush injury. These results identify PMP22 as a target of miRNAs and suggest that myelin gene expression by Schwann cells is regulated by miRNAs. © 2009 Wiley-Liss, Inc. [source] Protein identification via ion-trap collision-induced dissociation and examination of low-mass product ionsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2008Jeremiah J. Bowers Abstract A whole-protein tandem mass spectrometry approach for protein identification based on precursor ion charge state concentration via ion/ion reactions, ion-trap collisional activation, ion/ion proton-transfer reactions involving the product ions, and mass analysis over a narrow m/z range (up to m/z 2000) is described and evaluated. The experiments were carried out with a commercially available electrospray ion-trap instrument that has been modified to allow for ion/ion reactions. Reaction conditions and the approach to searching protein databases were developed with the assumption that the resolving power of the mass analyzer is insufficient to distinguish charge states on the basis of the isotope spacings. Ions derived from several charge states of cytochrome c, myoglobin, ribonuclease A, and ubiquitin were used to evaluate the approach for protein identification and to develop a two-step procedure to database searching to optimize specificity. The approach developed with the model proteins was then applied to whole cell lysate fractions of Saccharomyces cerevisiae. The results are illustrated with examples of assignments made for three a priori unknown proteins, each selected randomly from a lysate fraction. Two of the three proteins were assigned to species present in the database, whereas one did not match well any database entry. The combination of the mass measurement and the product ion masses suggested the possibility for the oxidation of two methionine residues of a protein in the database. The examples show that this limited whole-protein characterization approach can provide insights that might otherwise be lacking with approaches based on complete enzymatic digestion. Copyright © 2007 John Wiley & Sons, Ltd. [source] Peptide T revisited: conformational mimicry of epitopes of anti-HIV proteinsJOURNAL OF PEPTIDE SCIENCE, Issue 4 2001Delia Picone Abstract Peptide T (ASTTTNYT), a fragment corresponding to residues 185,192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C -terminal pentapeptide, and SAR studies showing that the C -terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations, albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N -terminus and a basic residue at the C -terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19,26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C -terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd. [source] Temperature scanning FTIR analysis of secondary structures of proteins embedded in amorphous sugar matrixJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2009Koreyoshi Imamura Abstract Heat-induced changes in secondary structures of five proteins (bovine serum albumin, BSA; human serum albumin, HSA; myoglobin; ribonuclease A, RNase A; and, ,-lactoglobulin, ,-Lg) in an amorphous sugar matrix were analyzed by temperature-scanning Fourier transform infrared spectroscopy to elucidate the mechanism of heat-induced conformational change of solid-phase proteins. Three sugars, trehalose, maltose, and dextran (MW 6000), were used. Loss of ,-helices due to increasing temperature was observed for BSA, HSA, and myoglobin, which are rich in ,-helices. RNase A showed a marked decrease in predominant secondary structural components (,-sheet) with increasing temperature. However, no noticeable changes in the content of secondary structures, except for a slight loss of ,-helices, were observed for ,-Lg, which is also ,-sheet-rich. These heat-induced conformational changes were significant at temperatures above the glass transition temperature. The heat-induced conformational change in BSA dried with sugar appeared time-independent and was clearly different from that due to dehydration and from the thermal conformational change for a solution of BSA. In particular, differences in secondary structural components that increased due to loss of ,-helices were noted. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3088,3098, 2009 [source] Performance of wide-pore monolithic silica column in protein separationJOURNAL OF SEPARATION SCIENCE, JSS, Issue 15-16 2009Hironobu Morisaka Abstract A monolithic wide-pore silica column was newly prepared for protein separation. The wide distribution of the pore sizes of monolithic columns was evaluated by mercury porosimetry. This column, as well as the conventional monolithic column, shows high permeability in the chromatographic separation of low-molecular-sized substances. In higher-molecular-sized protein separation, the wide-pore monolithic silica column shows better performance than that of the conventional monolithic column. Under optimized conditions, five different proteins , ribonuclease A, albumin, aldolase, catalase, and ferritin , were baseline-separated within 3 min, which is faster than that using the particle-packed columns. In addition, the monolithic wide-pore silica column could also be prepared in fused silica capillary (600 mm long, 0.2 mm i.d.) for highly efficient protein separation. The peak capacity of the wide-pore monolithic silica capillary column is estimated to be approximately 300 in the case of protein separation, which is a characteristic performance. [source] Capillary electrophoresis of intact basic proteins using noncovalently triple-layer coated capillariesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 14 2009Rob Haselberg Abstract The usefulness of a noncovalent, positively charged capillary coating for the efficient analysis of intact basic proteins with CE was studied. Capillaries were coated by subsequent flushing with solutions of 10% w/v Polybrene (PB), 3% w/v dextran sulfate (DS), and again 10% w/v PB. Coating characterization studies showed that stable coatings could be produced which exhibited a pH-independent and highly reproducible EOF. The PB,DS,PB coating was evaluated with Tris phosphate BGEs of various pH using the four basic model proteins: ,-chymotrypsinogen A, ribonuclease A, cytochrome c, and lysozyme. Typical migration time RSDs for the proteins were less than 0.85%, and apparent plate numbers were above 125 000 using a capillary length of 40 cm. The high separation efficiency allowed detection of several minor impurities in the model proteins. Using a BGE of medium pH, the CE system with triple-layer coating appeared to be useful for the repeatable profiling of recombinant humanized mouse monoclonal immunoglobulin G1 showing a characteristic pattern of glycoforms. The CE system was also applied to the characterization of two llama antibodies, which were produced in Saccharomyces cerevisiae, revealing the presence of a side product in one of the antibodies. The high migration time stability allowed the reliable determination of antibody,antigen binding by monitoring migration time shifts. Finally, the feasibility of using the PB,DS,PB coated capillaries for CE with mass spectrometric detection was shown by the characterization of the impure llama antibody sample. [source] Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variantPROTEIN SCIENCE, Issue 1 2009Antonello Merlino Abstract A specialized class of RNases shows a high cytotoxicity toward tumor cell lines, which is critically dependent on their ability to reach the cytosol and to evade the action of the ribonuclease inhibitor (RI). The cytotoxicity and antitumor activity of bovine seminal ribonuclease (BSRNase), which exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer, are peculiar properties of the swapped form. A dimeric variant (HHP2-RNase) of human pancreatic RNase, in which the enzyme has been engineered to reproduce the sequence of BSRNase helix-II (Gln28,Leu, Arg31,Cys, Arg32,Cys, and Asn34,Lys) and to eliminate a negative charge on the surface (Glu111,Gly), is also extremely cytotoxic. Surprisingly, this activity is associated also to the unswapped form of the protein. The crystal structure reveals that on this molecule the hinge regions, which are highly disordered in the unswapped form of BSRNase, adopt a very well-defined conformation in both subunits. The results suggest that the two hinge peptides and the two Leu28 side chains may provide an anchorage to a transient noncovalent dimer, which maintains Cys31 and Cys32 of the two subunits in proximity, thus stabilizing a quaternary structure, similar to that found for the noncovalent swapped dimer of BSRNase, that allows the molecule to escape RI and/or to enhance the formation of the interchain disulfides. [source] Structural properties of trimers and tetramers of ribonuclease APROTEIN SCIENCE, Issue 10 2001Arianna Nenci Abstract Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) , poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) , poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures co ld be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures. [source] An enigmatic peptide ligation reaction: Protease-catalyzed oligomerization of a native protein segment in neat aqueous solutionPROTEIN SCIENCE, Issue 4 2000Sangaralingam Kumaran Abstract We report an enigmatic peptide ligation reaction catalyzed by Glu-specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free ,-carboxyl and ,-amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 °C, producing a gamut of covalent oligomers (dimer through octamer or higher) of a native protein segment TAAAKFE (S39) derived from ribonuclease A (RNAse A). Size-exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length-dependent enhancement of secondary structure in the oligomers, suggesting that protease-catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the "conformational trapping" of the product. The ligation reaction proceeded in two phases,slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two-step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product-directed "conformational trap" as a mechanism to achieve peptide ligation of totally unprotected peptide fragments in neat aqueous solution. Further, the study projects the presence of considerable innate synthetic potential in V8 protease, baring rich possibilities of protein engineering of this enzyme to generate a "V8 peptide ligase." [source] Solution NMR evidence for a cis Tyr-Ala peptide group in the structure of [Pro93Ala] bovine pancreatic ribonuclease APROTEIN SCIENCE, Issue 2 2000Ying Xiong Abstract Proline peptide group isomerization can result in kinetic barriers in protein folding. In particular, the cis proline peptide conformation at Tyr92-Pro93 of bovine pancreatic ribonuclease A (RNase A) has been proposed to be crucial for chain folding initiation. Mutation of this proline-93 to alanine results in an RNase A molecule, P93A, that exhibits unfolding/refolding kinetics consistent with a cis Tyr92-Ala93 peptide group conformation in the folded structure (Dodge RW, Scheraga HA, 1996, Biochemistry 35:1548,1559). Here, we describe the analysis of backbone proton resonance assignments for P93 A together with nuclear Overhauser effect data that provide spectroscopic evidence for a type VI ,-bend conformation with a cis Tyr92-Ala93 peptide group in the folded structure. This is in contrast to the reported X-ray crystal structure of [Pro93Gly]-RNase A (Schultz LW, Hargraves SR, Klink TA, Raines RT, 1998, Protein Sci 7:1620,1625), in which Tyr92-Gly93 forms a type-II ,-bend with a trans peptide group conformation. While a glycine residue at position 93 accommodates a type-II bend (with a positive value of ř93), RNase A molecules with either proline or alanine residues at this position appear to require a cis peptide group with a type-VI ,-bend for proper folding. These results support the view that a cis Pro93 conformation is crucial for proper folding of wild-type RNase A. [source] Protein flexibility: coordinate uncertainties and interpretation of structural differencesACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2009Alexander A. Rashin Valid interpretations of conformational movements in protein structures determined by X-ray crystallography require that the movement magnitudes exceed their uncertainty threshold. Here, it is shown that such thresholds can be obtained from the distance difference matrices (DDMs) of 1014 pairs of independently determined structures of bovine ribonuclease A and sperm whale myoglobin, with no explanations provided for reportedly minor coordinate differences. The smallest magnitudes of reportedly functional motions are just above these thresholds. Uncertainty thresholds can provide objective criteria that distinguish between true conformational changes and apparent `noise', showing that some previous interpretations of protein coordinate changes attributed to external conditions or mutations may be doubtful or erroneous. The use of uncertainty thresholds, DDMs, the newly introduced CDDMs (contact distance difference matrices) and a novel simple rotation algorithm allows a more meaningful classification and description of protein motions, distinguishing between various rigid-fragment motions and nonrigid conformational deformations. It is also shown that half of 75 pairs of identical molecules, each from the same asymmetric crystallographic cell, exhibit coordinate differences that range from just outside the coordinate uncertainty threshold to the full magnitude of large functional movements. Thus, crystallization might often induce protein conformational changes that are comparable to those related to or induced by the protein function. [source] A neutron crystallographic analysis of phosphate-free ribonuclease A at 1.7,Ĺ resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2009Daichi Yagi A neutron crystallographic analysis of phosphate-free bovine pancreatic RNase A has been carried out at 1.7,Ĺ resolution using the BIX-4 single-crystal diffractometer at the JRR-3 reactor of the Japan Atomic Energy Agency. The high-resolution structural model allowed us to determine that His12 acts mainly as a general base in the catalytic process of RNase A. Numerous other distinctive structural features such as the hydrogen positions of methyl groups, hydroxyl groups, prolines, asparagines and glutamines were also determined at 1.7,Ĺ resolution. The protonation and deprotonation states of all of the charged amino-acid residues allowed us to provide a definitive description of the hydrogen-bonding network around the active site and the H atoms of the key His48 residue. Differences in hydrogen-bond strengths for the ,-helices and ,-sheets were inferred from determination of the hydrogen-bond lengths and the H/D-exchange ratios of the backbone amide H atoms. The correlation between the B factors and hydrogen-bond lengths of the hydration water molecules was also determined. [source] Structure of the newly found green turtle egg-white ribonucleaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Somporn Katekaew Marine green turtle (Chelonia mydas) egg-white ribonuclease (GTRNase) was crystallized from 1.1,M ammonium sulfate pH 5.5 and 30% glycerol using the sitting-drop vapour-diffusion method. The structure of GTRNase has been solved at 1.60,Ĺ resolution by the molecular-replacement technique using a model based on the structure of RNase 5 (murine angiogenin) from Mus musculus (46% identity). The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 86.271, b = 34.174, c = 39.738,Ĺ, , = 90, , = 102, , = 90°. GTRNase consists of three helices and seven ,-strands and displays the ,+, folding topology typical of a member of the RNase A superfamily. Superposition of the C, coordinates of GTRNase and RNase A superfamily members indicates that the overall structure is highly similar to that of angiogenin or RNase 5 from M. musculus (PDB code 2bwl) and RNase A from Bos taurus (PDB code 2blz), with root-mean-square deviations of 3.9 and 2.0,Ĺ, respectively. The catalytic residues are conserved with respect to the RNase A superfamily. The three disulfide bridges observed in the reptilian enzymes are conserved in GTRNase, while one further disulfide bond is required for the structural stability of mammalian RNases. GTRNase is expressed in egg white and the fact that its sequence has the highest similarity to that of snapping turtle pancreatic RNase suggests that the GTRNase secreted from oviduct cells to form egg white is probably the product of the same gene as activated in pancreatic cells. [source] Structure of bovine pancreatic ribonuclease complexed with uridine 5,-monophosphate at 1.60,Ĺ resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Steven B. Larson Bovine pancreatic ribonuclease A (RNase A) was crystallized from a mixture of small molecules containing basic fuchsin, tobramycin and uridine 5,-monophosphate (U5P). Solution of the crystal structure revealed that the enzyme was selectively bound to U5P, with the pyrimidine ring of U5P residing in the pyrimidine-binding site at Thr45. The structure was refined to an R factor of 0.197 and an Rfree of 0.253. [source] | ||||||||||||||||||||||||||||